Acta okl1-2014.cdr - Polskie Towarzystwo Farmaceutyczne

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ACTA POLONIAE
PHARMACEUTICA
VOL. 73 No. 4 July/August 2016
Drug Research
ISSN 2353-5288
EDITOR
Aleksander P. Mazurek
National Medicines Institute, The Medical University of Warsaw
ASSISTANT EDITOR
Jacek Bojarski
Medical College, Jagiellonian University, KrakÛw
EXECUTIVE EDITORIAL BOARD
Boøenna Gutkowska
Roman Kaliszan
Jan Pachecka
Jan Pawlaczyk
Janusz Pluta
Witold Wieniawski
Pavel Komarek
Henry Ostrowski-Meissner
Erhard Rˆder
Phil Skolnick
Zolt·n Vincze
The Medical University of Warsaw
The Medical University of GdaÒsk
The Medical University of Warsaw
K. Marcinkowski University of Medical Sciences, PoznaÒ
The Medical University of Wroc≥aw
Polish Pharmaceutical Society, Warsaw
Czech Pharmaceutical Society
Charles Sturt University, Sydney
Pharmazeutisches Institut der Universit‰t, Bonn
DOV Pharmaceutical, Inc.
Semmelweis University of Medicine, Budapest
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Acta Poloniae Pharmaceutica ñ Drug Research
Volume 73, Number 4
July/August 2016
CONTENTS
REVIEW
811.
Tomasz Urbaniak, Witold Musia≥
Methods for obtaining poly-ε-caprolactone for producing microand nanoscale drug carriers.
827.
Md. Shahidul Islam, Vijayalakshmi Venkatesan
Experimentally-induced animal models of prediabetes and
insulin resistance: a review.
835.
Chenglong Zheng, Yue Lan, Jian Zhang, Lu Zhang,
Jiani Wu, Shuwen Guo
Potential approaches for reducing amyloid β production.
843.
Krystyna Cegielska-Perun, Ewa Marczuk, Magdalena
Bujalska-Zadroøny
Inhibitors of leukotrienes synthesis: novel agents and their
implementation.
851.
Aneela Maalik, Syed Majid Bukhari, Asma Zaidi, Kausar
Hussain Shah, Farhan A. Khan
Chlorogenic acid: a pharmacologically potent molecule.
ANALYSIS
855.
Fangzhou Yin, Lin Li, Tuling Lu, Weidong Li,
Baochang Cai, Wu Yin of Yi-Huang
Decoction process optimization and quality evaluation
decoction by HPLC fingerprint analysis.
865.
Anna ZieliÒska, S≥awek Wicherkiewicz, Wojciech
£uniewski, Katarzyna Sidoryk, Edyta Pesta,
Andrzej Kutner
The comparison of the stability indicating two HPLC methods
and their application for determination of bosentan in coated
tablets.
875.
Kamila Osadnik, Joanna Jaworska
Analysis of ω-3 fatty acid content of Polish fish oil drug and
dietary supplements.
885.
Loretta Poblocka-Olech, Daniel GlÛd, Barbara KrÛl-Kogus,
Miroslawa Krauze-Baranowska
2D LC heart cutting on-line of phenolic compounds from three
species of the genus Salix.
DRUG BIOCHEMISTRY
895.
Jin Zhang
Blockade of large conductance Ca2+ activated K+ channel may
protect neuronal cells from hypoxia mimetic insult and
oxidative stress.
903.
Micha≥ OtrÍba, Dorota Wrzeúniok, Artur Beberok,
Jakub Rok, Ewa Buszman
Fluphenazine and perphenazine impact on melanogenesis
and antioxidant enzymes activity in normal human melanocytes.
DRUG SYNTHESIS
913.
Sujeet Kumar, Basavaraj Metikurki, Vivek Singh Bhadauria,
Erik De Clercq, Dominique Schols, Harukuni Tokuda,
Subhas S. Karki
Synthesis of imidazo[2,1-b][1,3,4]thiadiazole derivatives as
possible biologically active agents.
931.
Magdalena PakosiÒska-Parys, Andrzej Zimniak, Anna
Chodkowska, Ewa Jagie≥≥o-WÛjtowicz, Marek G≥owala,
Marta Struga
Synthesis, structure and pharmacological evaluation of
1-(1H-pyrrol-1-ylmethyl)-4-azatricyclo[5.2.1.02,6]dec8-ene-3,5-dione.
937.
Arpit Katiyar, Basavaraj Metikurki, Sarala Prafulla, Sujeet
Kumar, Satyaprakash Kushwaha, Dominique Schols,
Erik De Clercq, Subhas S. Karki
Synthesis and pharmacological activity of
imidazo[2,1-b][1,3,4]thiadiazole derivatives.
APPHAX 73 (4) 809 ñ 1098 (2016)
NATURAL DRUGS
949.
Alamgeer, Abdul Qayum Khan, Taseer Ahmad,
Muhammad Naveed Mushtaq, Muhammad Nasir
Hayat Malik, Huma Naz, Haseb Ahsan, Hira Asif,
Nabeela Noor, Muhammad Shafiq Ur Rahman, Umair Dar,
Muhammad Rashid
Phytochemical analysis and cardiotonic activity of methanolic
extract of Ranunculus muricatus Linn. in isolated rabbit heart.
955.
Ada Stelmakiene, Kristina Ramanauskiene, Vilma
Petrikaite, Valdas Jakstas, Vitalis Briedis
Application of dry hawthorn (Crataegus oxycantha L.)
extract in natural topical formulations.
967.
Alamgeer, Muhammad Numan, Sayed Atif Raza,
Muhammad Naveed Mushtaq, Zahid Khan,
Taseer Ahmad, Haseeb Ahsan, Hira Asif, Nabeela Noor,
Ambreen Malik Uttra, Laiba Arshad
Evaluation of anti-diabetic effects of poly-herbal product
ìdiabetic balî in alloxan-induced diabetic rabbits.
975.
Danuta Trojanowska, Paulina Paluchowska, £ukasz Soja,
Alicja Budak
Activity of thyme oil (Oleum Thymi) against multidrugresistant Acinetobacter baumannii and Pseudomonas
aeruginosa.
983.
Bushra Akhtar, Muhammad Ashraf, Aqeel Javeed,
Ali Sharif, Muhammad Furqan Akhtar, Ammara Saleem,
Irfan Hamid, Sadia Alvi, Ghulam Murtaza
Analgesic, antipyretic and anti-inflammatory activities of
Grewia asiatica fruit extracts in albino mice.
991.
Mohammad Taher Boroushaki, Azar Hosseini, Karim
Dolati, Hamid Mollazadeh, Arezoo Rajabian
Protective effect of pomegranate seed oil against mercuric
chloride-induced hepatotoxicity in rat.
PHARMACEUTICAL TECHNOLOGY
999.
Humayun Riaz, Brian Goodman, Sajid Bashir, Shahzad
Hussain, Sidra Mahmood, Farnaz Malik, Durana Waseem,
Syed Atif Raza, Wajahat Mahmood, Alamgeer
Comparative bioavailability analysis of oral alendronate
sodium formulations in Pakistan.
1009. Ume Ruqia Tulain, Mahmood Ahmad, Ayesha Rashid,
Furqan Muhammad Iqbal
Development and characterization of smart drug delivery
system.
1023. Majeed Ullah, Ghulam Murtaza, Izhar Hussain
Relative bioavailability study of succinic acid cocrystal tablet
and marketed conventional immediate release tablet formulation
of carbamazepine 200 mg in rabbits.
1029. Edyta Mazurek-Wπdo≥kowska, Katarzyna Winnicka,
Urszula Czyøewska, Wojciech Miltyk
Effect of simultaneously silicified microcrystalline cellulose
and pregelatinized starch on the theophylline tablets stability.
1037. Aamna Shah, Gul M. Khan, Hanif Ullah, Kamran Ahmad
Khan, Kaleem Ullah, Shujaat A. Khan
Formulation, evaluation and in vitro dissolution performance of
enalapril maleate sustained release matrices: effect of polymer
composition and viscosity grade.
1045. Zermina Rashid, Nazar Muhammad Ranjha, Hina Raza,
Rabia Razzaq, Asif Mehmood
Preparation and evaluation of pH responsive poly(2hydroxyethyl methacrylate-co-itaconic acid) microgels for
controlled drug delivery.
PHARMACOLOGY
1057. Magdalena Bamburowicz-Klimkowska, Tadeusz Szost,
Anna Ma≥kowska, Miros≥aw Szutowski
Quinidine and domperidone interactions in the rat
experimental model of repeated administration.
1067. Anna Ma≥kowska, Miros≥aw Szutowski, Wanda Dyr,
Magdalena Bamburowicz-Klimkowska
Excretion of ethyl glucuronide in the urine of Warsaw
high preferring rats depends on the concentration of ingested
ethanol.
1073. Ilona Kaczmarczyk-Sedlak, Weronika Wojnar, Maria Zych,
Ewa Ozimina-KamiÒska, Anna BoÒka
Effect of dietary flavonoid naringenin on bones in rats with
ovariectomy-induced osteoporosis.
GENERAL
1083. Ewa Wiúniewska, Aleksandra Czerw, Marta Makowska,
Adam Fronczak
Television advertising of selected medicinal products in Poland
and in The United States ñ a comparative analysis of selected
elevision commercials.
Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 73 No. 4 pp. 811ñ825, 2016
ISSN 0001-6837
Polish Pharmaceutical Society
REVIEW
METHODS FOR OBTAINING POLY-ε-CAPROLACTONE FOR PRODUCING
MICRO- AND NANOSCALE DRUG CARRIERS
TOMASZ URBANIAK and WITOLD MUSIA£*
Department of Physical Chemistry, Faculty of Pharmacy, Wroclaw Medical University,
Borowska 211A, 50-556 Wroc≥aw, Poland
Abstract: Poly-ε-caprolactone due to favorable properties including biocompatibility, miscibility and controlled degradation is promising material for long term drug-delivery devices preparation, especially in blend
with other polymers. Commercially available polymer is synthesized without considering this way of utilization, and may contain potentially toxic residues of compounds used during polymerization process. Therefore,
we attempt to summarize in this work available methods for synthesis of poly-ε-caprolactone using nontoxic
reactants. The product properties important for polymer drug delivery system such as number average molecular weight and polydyspersity index (PDI) were included. Also we present known methods for purification of
the polymer from catalyst residues, and other methods that may improve process of polymerization catalyzed
by less active compounds.
Keywords: poly-ε-caprolactone, polymer, polymerization, drug delivery
20 ppm of tin in materials in contact with blood or
fluids administered parenterally. Although there are
no official guidelines for tin content in the forms of
the drug, it is reasonable to search for alternative,
biocompatible catalysts for this reaction. It should
be noted that the problem also applies to other polyesters used in pharmacy, obtained by means of the
catalyst, among others, the polylactic acid. Apart
from biocompatibility, catalysts should provide a
controlled reaction, giving the product of suitable
molecular weight and low polydispersity. In the case
of the drug form based on polymeric compounds,
the release rate of the drug depends on these particular parameters. An additional advantage of the
sought catalysts is the capacity to dissolve directly
in the monomer, which avoids the use of solvents.
This publication provides an overview of the current
state of knowledge on methods for the preparation
of poly-ε-caprolactone using catalysts based on the
elements involved in the metabolism of metal, such
as iron, zinc, calcium and magnesium. In addition,
the enzymatic biocatalysts are discussed, as well as
the nontoxic organic compounds capable of catalyzing the ring-opening polymerization. Citations
include such polymer properties like number average molecular weight and polydispersity index
(PDI).There were also described the methods for the
purification of the polymer from the residue of
organotin catalysts, as well as methods allowing the
Biodegradable polyesters, including poly-εcaprolactone and its copolymers, are used in pharmacy as carriers of the medicinal substance, in the
form used orally and parenterally. On its basis we
can form microspheres and nanospheres characterized by the ability of the sustained release of the
medicinal substance. Moreover, poly-ε-caprolactone due to its long resorption time is used in medicine to form implants, surgical suture or scaffolding
for the regenerating tissues (1). This polymer is
characterized by biocompatibility, ability to degrade
by hydrolysis and cellular mechanisms, and the ability to leave the body through the implementation of
the degradation products to the citric acid cycle (2).
It can be formed at low temperatures, and may form
copolymers with other polyesters, which allows to
control the process of degradation and release of
drug substance (3). Poly-ε-caprolactone is obtained
in the reaction of polymerization with ring-opening
from the monomer of ε-caprolactone. This reaction
can be catalyzed by a number of substances. In the
industrial production of a polymer, the most commonly used catalyst is 2-ethyl hexanoate of tin (II)
(4). While it has been approved by the FDA as a
food additive with antimicrobial properties, reports
on the toxicity of organotin compounds (5, 6) may
raise doubts about the legitimacy of its use as a catalyst for preparing polymers for pharmaceutical use.
European Pharmacopoeia VIII allows the content of
* Corresponding author: e-mail: witold.musial@umed.wroc.pl
811
812
TOMASZ URBANIAK and WITOLD MUSIA£
improvement of reactions catalyzed by the less
active, biocompatible catalysts.
species which are capable to attack cyclic monomer
molecules (Fig. 2).
Ring opening polymerization mechanism
Polylactones, i.e., poly-ε-caprolactone are
formed during ring opening polymerization in result
of reaction of cyclic monomer, and catalyst or initiator. Generally, each formed macromolecule is a
chain with two end groups, first one is determined
by the type of termination reaction, second terminus
is end-capped with end group originating from initiator. Nature of end groups can vary properties of
product, allowing to customize polymer to specific
needs. Depending on catalytic system the polymerization proceeds according to three different major
reaction mechanisms: cationic, anionic, or ìcoordination-insertionî mechanism.
ìCoordination-insertionî ring-opening polymerization
In ìcoordination-insertionî mechanism chain
propagation proceeds by coordination of monomer
to active species, and then insertion of the monomer
into the metal-oxygen bond. Until the termination
reaction, growing chain remains attached to metal
through alkoxide bond (8) (Fig. 3).
Cationic ring-opening polymerization
The cationic ring-opening polymerization
involves the formation of a positively charged
species which are attacked by a monomer. Attack
results in opening of positively charged monomer
particle, and addition of another monomer molecule
in SN2 substitution mechanism (7) (Fig. 1).
Anionic ring-opening polymerization
The anionic ring-opening polymerization
mechanism takes place by the nucleophilic attack of
a negatively charged initiator on the carbonyl carbon. This process results in cleavage of acyl-oxygen
bond and formation of negatively charged chain
The catalysts based on metallic elements
These catalysts are the largest group among the
compounds used for catalyzing the ring-opening
polymerization of ε-caprolactone. The catalytic
activity of metalorganic compounds of tin, aluminum, iron, titanium, zinc, sodium, zirconium,
scandium, calcium, magnesium, and many others
was confirmed in numerous experimental works (4).
In practice, the most commonly used substances are
the organotin derivatives. The polymer obtained by
the reactions catalyzed by these compounds even
after the purification of the crude product is not free
of catalyst residues. Therefore, especially in parenteral administration, it is appropriate to use metal
derivatives containing elements, which are naturally
present in the human body. The following describes
catalysts containing in its structure metals, such as
iron, calcium, magnesium and zinc. The selected
elements take part in the metabolism and do not
pose a threat to the organism, even in the case of the
Figure 1. Cationic ring-opening polymerization mechanism
Figure 2. Anionic ring-opening polymerization mechanism
Figure 3. Coordination-insertion ring opening polymerization mechanism
Methods for obtaining poly-ε-caprolactone for
813
Figure 4. Single-site organocalcium ring-opening polymerization catalysts synthesized by Zhong et al. (11)
accumulation associated with the long-term use of
the drug carrier containing the contaminant.
Calcium-organic catalysts
Hundred years after Victor Grignardís discoveries in the field of organomagnesium chemistry,
research over organocalcium compounds started to
grow faster. A variety of calcium derivatives were
recently utilized as polymeryzation catalysts. Due to
lack of toxicity of these compounds works are
focused on receiving polymers used in biomedical
application, such as polylactic acid, trimethylene
carbonate, and also poly-ε-caprolactone (9). Zhong
et al. (10) conducted the polymerizations of ε-caprolactone catalyzed by bis(tetrahydrofuran)calcium
bis[bis(trimethylsilyl)amide]. The reaction was conducted in tetrahydrofuran at room temperature. It
was carried out without the addition of alcohol as
the initiator and led to the formation of a product
with a high PDI equal to 2.39 and the average
molecular weight of 11.8 kDa. The addition of
methanol or ethanol, led to a greater control of the
reaction and obtaining of the products, successively,
with PDI 1.29 and 1.24 and the average molecular
weights of 9.0 and 6.2 kDa. The same catalyst is also
used successfully in the reaction of ring-opening
polymerization of lactide of the lactic acid, which is
also used in pharmacy. In another work (11) these
researchers have subjected the catalyst to modification by using chelating agent 2,2,6,6-tetramethylheptane-3,5-dione ligand as well as the 2-hydroxyphenylethyl group (Fig. 4).
Reactions were carried out at the room temperature, in tetrahydrofuran as the solvent. The first of
these components has demonstrated the superior
performance catalyzing a product of high molecular
weight of 16.7 kDa and a high level of PDI of 1.80.
The second one showed a high ability to initiate the
reaction, which in turn gave the product with the
molecular weight of 1.5 kDa and low PDI equal to
1.13. For the initiating functions was responsible the
2-hydroxy-2-phenylethyl group that is found at the
end of the obtained polymer chains. The same group
received a poly-ε-caprolactone catalyzing the reaction of commercially available calcium methoxide
(12). The reaction carried out in bulk at the temperature of 120OC for 10 min allowed to get a product
having an average molecular weight of 22.2 kDa and
a PDI of 1.25. Piao et al. (13), for the polymerization
of ε-caprolactone used catalysts such as calcium
aminoisopropoxide and calcium aminoethoxyloxide. The reaction carried out at the temperature of
70OC in xylene has led to obtain a product having an
average molecular weight of 8.5 kDa for the catalyst
with an isopropoxyl substituent and the 10.0 kDa for
the ethoxyl substituent. In both cases, the NMR
spectra showed the presence of isopropoxyl and
ethoxyl groups at the ends of polymer chains. The
molecular weight of the products increased linearly
with the progress of the reaction, which indicates the
good control of the reaction. Khan et al. (14)
attempted to catalyze the reaction for producing
poly-ε-caprolactone using the calcium oxide immobilized on silica. The catalyst was obtained by the
reactions of carbide with silica. In addition to the
version in which the silicon atoms were attached to
calcium through oxygen atom, the alternative catalyst was silica having functional groups of 3-glycidoxytrimethyloxysilane additionally binding the
calcium atoms. In the case of the catalyst free of
functional groups, after a reaction lasting for 216 h,
the product had a molecular weight of 6.34 kDa,
characterized by PDI at 1.12. In addition, 10% of the
obtained polymer was associated with silica. In the
case of the catalyst enriched with functional groups,
as a result of reaction lasting for 100 h, a polymer
was obtained having a molecular weight of 4.22 kDa
and PDI = 1.10. The silica was connected with 30%
polymer chains, which was most likely related to the
presence of functional groups rich with hydroxyl
groups. A linear increase in the degree of polymerization with the progress of the reaction indicates the
ìlivingî character of the reaction. Huang et al. (15)
attempted to carry out the reaction using a catalyst
based on oxamidine ligands (Fig. 5).
As a result of a series of reactions using several variations of a catalyst, there was obtained, among
others, polymer with a molecular weight of 140 kDa
and PDI equal to 1.13. The reaction was carried out
at 0OC, in benzene, in the presence of benzyl alcohol
814
Iron-organic catalysts
Iron based catalysts can participate in multiple
polymerization processes including atom transfer
radical polymerization, organometallic radical polymerization, catalytic chain transfer and ring opening
polymerization. Low toxicity of these compounds
encourages to use them on field of green and biomedical chemistry. Hege and Schiller (18) undertook the investigation of the activity of iron(III)
chloride, bromide and perchlorate as catalysts of the
ring-opening polymerization. The reaction was conducted at the room temperature with different concentrations of the catalyst and in the presence of four
different initiators: isopropyl alcohol, benzyl alcohol, 2-allylphenol and water. The highest obtained
molecular weights were: 23.15 kDa when the PDI
was equal to 1.52 in the reaction catalyzed by the
ferric chloride and initiated by 2-allylphenol in ratio
of monomer to the catalyst and initiator equal to 200
: 1 : 5, and 20.43 kDa when the PDI was 1.88 in the
reaction catalyzed with iron perchlorate, initiated by
2-allylphenol in ratio of 1200 : 1 : 5. The polymer
with the lowest PDI equal to 1.3 characterized by a
molecular weight of 11.6 kDa was obtained by using
ferric chloride and isopropyl alcohol in ratios of 200
: 1 : 5. Gowda and Chakraborty (19) analyzed the
catalytic activity of the hydrated iron chlorides on II
and III degree of oxidation. Reactions were conducted at the temperature of 27OC in the bulk, using
the initiators of ethylene glycol and ethyl, benzyl or
isopropyl alcohol. Reactions were also conducted
without the addition of the initiating agent. The
highest molecular weights obtained in the over 13 h
reaction catalyzed by the ferric(III) chloride hexahydrate in the ratio of 1200 : 1 was 12.52 kDa with PDI
equal to 1.28. In the case of ferric(II) chloride
tetrahydrate in a ratio of 1200 : 1 a 31-h reaction
gave a product with a molecular weight of 10.36
kDa and a PDI of 1.25, which was also the lowest of
all the samples. Gibson et al. (20) studied the activity of three-coordinate iron(II) complex containing a
diketamine ligand. In the case of this catalyst
already the 5-min reaction yielded a molecular
weight of 86.2 kDa and a PDI value of 1.38. Arbaoui
et al. (21) used the calixarene derivative in the polymerization reaction of ε-caprolactone initiated with
benzyl alcohol. Reactions were carried out for 40 h
at the room temperature. The ratio of reactants 700 :
1 : 1 allowed to obtain a product having an average
molecular weight of 2.38 kDa level, and a PDI of
1.9, which indicates a number of transesterification
reactions and provides poor control of the reaction.
Iron N-heterocyclic complexes (Fig. 6) in the
role of catalysts were used by Wang et al. (22).
Reactions were carried out in toluene at the temperature of 80OC. The product after the 2-h reaction has
a molecular weight of 6.2 kDa with PDI equal to 3.1,
the further course of the reaction resulted in shortening of the average length of the chains.
Figure 5. Organocalcium ring-opening polymerization reaction
catalyst investigated by Huang et al. (15)
Magnesium-organic catalysts
Known for a long time organomagnesium
compounds found use in synthesis of polymers such
as polylactic acid, β-butyrolactone, β-valerolactone
or 1,5-dioxepan-2-one (23). All of them, like poly-εcaprolactone, can be used in medicine, which
encourages further research on non-toxic organomagnesium compounds. Fang et al. (24) studied the
activity of the derivative of magnesium bis(2,6-ditert-butyl-4-methylphenoxylane) (Fig. 7).
The reaction was carried out in tetrahydrofuran
in the presence of benzyl alcohol as an initiator at the
room temperature. The process was conducted in two
as an initiator. With an increase of its concentration,
the molecular weight of the product increased in a
linear manner. Also the attempts to synthesize the
poly-ε-caprolactone with other polymers were carried out, which gives the ability to modify the profile
to release the drug substance from the carrier.
Sarazin et al. (16) synthesized the multidentate calcium-organic catalyst with complex substituents.
Reactions were carried out in toluene at the temperature of 60OC for 12 h, the product has a molecular
weight of 26 kDa when the PDI was 1.3, while
40.1% of monomer underwent the conversion.
Cowell (17) proposed the method of synthesis of the
three-block copolymer of the polyethylene glycol
(PEG) and poly-ε-caprolactone described by the formula PCLx/PEG45/PCLy. The reaction was carried
out using the initiator system obtained in the reaction
of glycol polyethylene with a carbide. The product of
the longest chains was characterized by a molecular
weight equal to 55.08 kDa, and PDI equal to 1.48.
Figure 6. N-heterocyclic complex of iron used by Wang et al. (22)
as ring-opening polymerization catalyst
Figure 7. Metallorganic magnesium catalyst investigated by Fang
et al. (24)
variants: a nitrogen gas environment, and in an open
reactor. In the first case, the molecular weight of the
obtained products, depending on the molar ratio of
monomer to initiator and catalyst ranged from 7.4 kDa
to 16.3 kDa and the PDI ranged from 1.08 to 1.14. A
similar reaction carried out in an air atmosphere gave
products with molecular weights of 12.5 kDa to 28.5
kDa and PDI from 1.2 to 1.33. The interaction
between the molecular weight of the product and the
concentration of the initiator showed the linear character, which indicates the good control of the reaction.
Moreover, successfully the same catalyst was used
with a polyethylene glycol with a molecular weight of
2 kDa as an initiator to obtain a PEG-PCL copolymer.
Kong and Wang (25) synthesized the magnesium
organic catalyst based on the bis(iminopyrrolide) ligands (Fig. 8), which showed activity in the reaction of
ring-opening polymerization.
The reactions were carried out in toluene at
35OC. A reaction with benzyl alcohol as the initiator
yielded a molecular weight of 2.72 kDa and a high
PDI equal to 1.68. A similar reaction performed
without the addition of the initiator yielded a molecular weight of 5.75 kDa and a PDI of 1.70, but the
level of monomer conversion was noticeably lower
than in the case of reaction carried out with the initiator. The linear decrease in the concentration of the
monomer with the progress of the reaction indicates
a good control of the reaction. Wang et al. (26) used
the N,O-bidentate pyridyl functionalized alkoxy ligands to obtain magnesium chelate (Fig. 9) with the
catalytic activity in the reaction of ring-opening
polymerization of ε-caprolactone.
815
The reaction was conducted in tetrahydrofuran
at the room temperature, with benzyl alcohol as the
initiating agent. Reactions were also performed without the initiator, but the product was characterized by
a high PDI. In the synthesis carried out in the presence of benzyl alcohol the obtained product was
characterized with the highest molecular weight
equal to 46.4 kDa, but simultaneously the PDI was
1.77. The products of lower PDI were characterized
by molecular weights from 0.76 to 1.74 kDa. The
catalyst was also used to obtain polylactic acid. Shen
et al. (27) studied the activity of the magnesiumorganic catalyst based on biphenyl ligands (Fig. 10).
Reactions were carried out in dichloromethane at
the room temperature. The product of the reaction conducted for an hour was a polymer having a molecular
weight of 23.5 kDa and PDI values qual to 1.08. With
the decrease of the used concentration of the catalyst,
the value of the molecular weight value increased linearly. The same catalyst was active in the polymerization reaction of lactide. Chen et al. (28) studied the
activity of the catalyst synthesized by them containing
the novel sulfonate phenol ligands (Fig. 11).
Reactions were carried out at the temperature of
25OC in toluene in the presence of 9-hydroxyantracene
as an initiator. After the synthesis lasting 15 min, the
obtained product was characterized with a molecular
weight of 23.9 kDa and PDI at 1.14. The same paper
also describes the successful process of polymerization of trimethylene carbonate (TMC) and PCL-TMC
copolymer, which are used pharmaceutically.
DobrzyÒski et al. (29) used magnesium butoxide and
magnesium acetoacetate to synthesise the copolymer
of polyglycolide and poly-ε-caprolactone. The reaction catalyzed by magnesium butoxide, carried out in
bulk at a temperature of 150OC for 240 h gave a product having an average molecular weight of 38.0 kDa at
the level of the PDI of 1.5. This copolymer demonstrated the biocompatibility with the cerebral tissue.
Wei et al. (30) used the magnesium 2-ethylhexanoate
in the copolymerization reaction of lactic acid and εcaprolactone. The reaction carried out in bulk at a temperature of 130OC gave a product with molecular
weight of 18.7 kDa at the level of the PDI of 1.74.
Zinc-organic catalysts
Diversity of zinc-based polymerization catalysts resulted in many works dealing with the problem of high purity polymers for medical use. Among
monomers such as lactic acid, α-methyl-β-pentyl-βpropiolactone, β-butyrolactone, β-valerolactone,
described here ε-caprolactone got a lot of attention
from researchers. Barakat et al. (31) investigated the
catalytic activity of the alkoxy derivatives of zinc.
816
The reactions catalyzed by zinc bis(2-bromoethoxide) in ratio of 45 : 1 and ethyl zinc 2-bromoethoxide in ratio of 53 : 1 were carried out at the temperature of 25OC in toluene as the solvent. The first catalyst allowed to obtain a product with a molecular
weight of 3.1 kDa and PDI equal to 1.05, the reaction catalyzed by the second one gave the product
having a molecular weight of 5.2 kDa and PDI of
1.07. The study also showed a linear relationship
between the degree of monomer conversion and
molecular weight. Hao et al. (32) decided to examine the activity of zinc undecylenate. Reactions were
carried out in bulk, in the presence of benzyl alcohol
as an initiator, at a temperature of 90OC at a ratio of
reactants 50 : 1 : 1 and at a temperature of 110OC in
ratios of 50 : 1 : 1 and 200 : 1 : 1. The first variant
has allowed to obtain a product with the molecular
weight of 5.04 kDa, the PDI of 1.2. With the reaction temperature set at 110OC and the ratio of reactants 50 : 1 : 1 the product has a molecular weight of
5.76 kDa, the PDI equal to 1.2. With a ratio of 200 :
1 : 1, molecular weight of 17.45 kDa and PDI of 1.2
were achieved. The conducted reactions had the
nature of the ìlivingî polymerization. Gallaway et
al. (33) described the activity of tridentate zinc complexes with the Schiff bases. The synthesized catalysts were different in terms of substituents attached
to the imino group, these were the pyridine, piperi-
Figure 8. Dinuclear magnesium complex supported by bis(iminopyrrolide) ligands synthesized by Kong and Wang (25)
Figure 9. Bidentate magnesium chelate investigated by Wang et
al. (26)
dine, morpholine, diisopropylamine, dimethylamine
and quinoline groups. Reactions were carried out at
room temperature in toluene as solvent, the ratio of
monomer to catalyst was 100 : 1. The catalytic activity for the reaction of ring-opening polymerization
were shown by compounds having the substituents:
dimethylamine, piperidine and morpholine, giving
products with molecular weight, successively, kDa
37.8, 25.2 and 51.7 kDa and PDI values, successively, 1.71, 1.72, and 1.79. Other types of Schiff
bases were used by Kong et al. (34) obtaining
N,N,O-tridentate zinc atom chelate (Fig. 12).
Reactions were carried out at 80OC in toluene
in the presence of benzyl alcohol as an initiator;
monomer, catalyst and initiator were mixed in a
ratio of 400 : 1 : 2. The reaction conducted for 45
min gave a product with a molecular weight of 1.9
kDa and PDI of 1.13, a similar one catalyzed by the
second derivative lasted 85 min and gave a product
with a molecular weight of 1.08 kDa and PDI equal
to 1.14. Low values of PDI and a linear increase in
the average molecular weight with the progress of
the reaction indicates the ìlivingî nature of the reaction. Another catalytic agent, β-ketiminate pyrazolonate zinc complex (Fig. 13) was obtained and
investigated by Chen et al. (35).
A 4-h reaction at the temperature of 30OC in
dichloromethane as a solvent for the monomer and catalyst mixture in a ratio of 300 : 1 gave the product having a molecular weight of 15.9 kDa and PDI equal to
1.1. The series of reactions carried out with the increasing molar ratio of reactants showed a linear dependence between the molecular weight and the concentration of the catalyst. Pandey (36) used the zinc prolinate
to catalyze the polymerization reaction of ε-caprolactone. The reaction was carried out at the temperature of
195OC for 8 h, its product was a polymer having a
molecular weight of 85 kDa and PDI equal to 1.9.
Gowda et al. (37) conducted a polymerization reaction
catalyzed by a zinc acetate dihydrate at the temperature
of 100OC. The reaction without the addition of the initiating agent was carried out in the proportion of a
monomer to a catalyst ranging, in order, 200 : 1, 400 :
1, 800 : 1 and 1000 : 1. The highest molecular weight
of 79.66 kDa, and the lowest level of PDI of 1.18 was
obtained in the last of the mentioned variants as a result
of a reaction lasting for 36 h. Also, there were conducted two alternative 8 h long reactions initiated by
benzyl alcohol and isopropyl alcohol, both in a ratio of
monomer to catalyst and to initiator equal 200 : 1 : 5.
The resulting products were characterized by consecutively molecular weights of 34.27 kDa and 32.29 kDa,
and PDI at 1.21 and 1.19. The average molecular
weights increased linearly with the decrease in the con-
centration of the catalyst. Naumann et al. (38) used as
catalysts the imidazolidine and tetrahydropyrimidine
zinc derivatives (Fig. 14).
The monomer mixture of the catalyst and benzyl alcohol in a ratio of 280 : 1 : 2 was subjected to
the reaction at the room temperature, 70 and 130OC.
In the case of the imidazolidine derivative, the reaction took place at temperatures of 70 and 130OC giving products having a molecular weight of, in order,
12 kDa and 16 kDa, and PDI equal to 1.68 in both
cases. The catalyst being the derivative of tetrahydropyrimidine allowed the occurrence of the 15-min
reaction at the temperature of 130OC to give the
product having a molecular weight of 16.5 kDa and
PDI equal to 1.64. Chen et al. (39) synthesized the
sugar complexes containing pyrazolyl-phenolate
ligands (Fig. 15), and examined their ability to catalyze the polymerization of ε-caprolactone.
The activity showed two distinct complexes,
different in terms of the presence of methyl groups on
the imidazole rings. Reactions were carried out in the
presence of benzyl alcohol in toluene. At the room
temperature, with the reactant ratio of 50 : 1 : 1 the
catalyst containing methyl groups gave the product
with a molecular weight of 5.24 kDa and PDI equal
to 1.14, the catalyst devoid of methyl substituents
gave a product with a molecular weight of 6.61 kDa
and PDI of 1.07. The highest molecular weight was
31.89 kDa at the PDI level of 1.1 at a ratio of reactants
200 : 1 : 1 at the temperature of 50OC. Also, there was
examined the effect of the initiator to the other
reagents on the molecular weight of the product,
which allowed the statement of a linear relationship
between these variables, and points to the ìlivingî
nature of the polymerization.
Tai et al. (40) received the zinc complexes with
benzotriazole ligands. Reactions were catalyzed by
five variants of the catalyst in toluene at the temperature of 30OC in the presence of 9-hydroxyanthracene. The highest activity was shown by the catalyst with the tert-butyl substituents. The highest
achieved molecular weight of 65.6 kDa with PDI 1.3
characterized the reaction product of reactants in the
ratio 1000 : 1 : 1 (Fig 16, compound 2). The most
preferred PDI equal to 1.03 with a molecular weight
of 6.7 kDa was achieved in the reaction with a ratio
of the reactants 50 : 1 : 1 (Fig. 16, compound 1).
And in this case, a linear relationship between the
concentration of monomer and initiator indicates the
ìlivingî character of the reaction. Arbaoui et al.
(21), for the catalyst synthesis of the ring-opening
polymerization used the calixarene derivatives.
Among the tested variants of the reaction, the most
preferred parameters was the reaction product con-
817
Figure 10. Magnesium organic derivative of 2,2-ethylidenebis(4,6-di-tert-butylphenol) investigated by Shen et al. (27)
Figure 11. Sulfonate phenoxide magnesium complex synthesized
by Chen et al. (28)
ducted in toluene for 24 h, using benzyl alcohol. The
ratio of the reactants was 700 : 1 : 1, the reactions
were performed at the temperature of 60OC. The
product was characterized by a molecular weight
equal to 9.02 kDa, and PDI equal to 1.1. Longer
chains were obtained in the reaction without the use
of an initiator, but the resulting sample was characterized by high PDI. Darensbourg and Karroonnirun
(41) used to obtain poly-ε-caprolactone a complex
of zinc based on the Schiff base, derivative of
phenylalanine (Fig. 17).
The product of an hour-long polymerization at
the temperature of 110OC was characterized by the
molecular weight of 19.6 kDa and PDI equal to 1.23.
A series of reactions of various mole ratios of reactants showed a linear relationship between the catalyst concentration and molecular weight of the
obtained polymers.
Enzymatic catalysts
The catalytic activity in ε-caprolactone polymerization reaction also showed macromolecules of
an enzyme nature. These processes, as compared to
the previously discussed, are long-lasting and give
products having a relatively high PDI. At the same
time, due to the macromolecular nature, they allow
in a simple manner, by filtration, to separate the
final product and often reuse the catalyst.
Furthermore, processes of this type are considered
818
environmentally friendly on the industrial scale. The
publications listed below discussed factors essential
for this type of reaction, such as the origin and type
of enzyme, reaction medium, temperature or water
content in the system. Determination of the optimal
environment for the reaction results in the proper
control of the polymerization and desired properties
and purity of the product. The authors of one of the
first reports on the use of enzymes in the reaction of
ring-opening polymerization of ε-caprolactone are
MacDonald et al. (42). They used the lipase enzyme
obtained from pig pancreas. Reactions were carried
out at the temperature of 65OC in heptane as a solvent, with the addition of water and butyl alcohol.
The product of the four-day reaction was a polymer
having a molecular weight equal to 2.7 kDa and PDI
of 1.9. A more often used enzyme is lipase B
obtained from fungus Candida albicans, also immo-
Figure 12. N,N,O-tridentate zinc chelate investigated by Kong et
al. (34)
Figure 13. Metallorganic zinc catalyst based on derivatives of
iminopyrazolonone ligands, investigated by Chen et al. (35)
bilized on a porous acrylic resin, known inter alia
under the trade name of Novozym 435. Castano et
al. (43) used this enzyme in combination with
propargyl alcohol as the initiator for the polymerization of ε-caprolactone. The reaction was carried out
in toluene at the temperature of 70O C for 12 h. The
reaction product wherein the ratio of monomer to
initiator was 300 : 1 was characterized by a molecular weight of 22.8 kDa and a PDI of 1.58.
Arumugasamy and Ahmad (44) studied the effect of
temperature and type of solvent on the molecular
weight of the reaction product with the same catalyst. The highest obtained value was 86 kDa, the
most appropriate of the three used solvents was
toluene. Inprakhon et al. (45) attempted to use this
enzyme in an aqueous environment, in an immobilized form. The reaction mixture had the nature of
dispersion of the enzyme in a mixture of monomer
and water. After a 15 min reaction at 60OC, a product with a molecular weight of 3.7 kDa was,
obtained with the increase of the reaction time the
molecular weight decreased, which explains the
process of hydrolysis of poly-ε-caprolactone catalyzed by a lipase. Wu et al. (46) used the immobilized lipase of Candida albicans in an ionic liquid
environment (Fig. 18).
Reactions were carried out in two ways, in four
ionic liquids as solvents, or coated with a thin layer
of immobilized enzyme of the same ionic liquids,
and the reaction was performed in bulk. In both
cases, the process temperature was 60OC and it lasted 48 h. Higher molecular weights and comparable
PDI were obtained in the case of enzymes coated
with ionic liquids, the highest obtained value of the
molecular weight was 35.6 kDa, while the PDI 1.64.
Thurecht et al. (47) studied the activity of the same
enzyme in a super-critical carbon dioxide with addition of water as initiator. Reactions were carried out
in an autoclave at 35OC for 24 h. The product with
minimal water concentration of 0.004% has reached
a molecular weight of 110 kDa, but the achieved
PDI values cantered around the value of 2, suggesting the occurrence of a number of transesterification
reactions. Gumel et al. (48) dealt with the issue of
increase in the viscosity of the reaction mixture with
the progress of the reaction, which is believed to
negatively affect the amount of molecular weights.
The reaction was conducted in the ionic liquid at
50OC, the sample prior to the ongoing 85 h the reaction subjected to ultrasounds for 20 min. In one of
the samples they achieved the molecular weight of
20.6 kDa, and with the similarly conducted reaction
without using ultrasounds the value of 9.8 kDa was
achieved. Kundu et al. (49), in order to obtain high-
Figure 14. Zinc organic catalysts based on tetrahydropyrimidine
(left) and imidazolidine (right) ligands synthesized by Naumann et
al. (38)
Figure 15. Zinc complex containing pendant pyrazolyl-phenolate
ligands used as catalyst by Chen et al. (39)
er molecular weights, studied the effect of water
contents acting as the initiator on the course of the
reaction. The water conditioning the start of the
reaction, at later stages gives rise to a number of
centers of growth of polymer chains leading to a
decrease of the average length. In order to slowly
reduce the water concentration in the reaction medium there was applied the addition of molecular
sieves slowly adsorbing water. Reactions were carried out in toluene at 70OC. The difference in the
increase in average molecular weight in the time
interval from 45 to 180 min in both cases was significant. In the presence of molecular sieves the
increase was 52 ± 18%, for the control reaction the
increase was 10%. Ma et al. (50), to catalyze the
reaction of the ring-opening polymeryzation, used
the esterase enzyme derived from a thermophilic
species Archaeoglobus fulgidus, resistant to higher
reaction temperature. Based on numerous trials,
these researchers found the optimal conditions of
reaction for the enzyme, such as temperature, reaction time, type of solvent, concentration of the
enzyme or the water content. With conditions
deemed to be the most beneficial (lasting 72 h, the
819
reaction in toluene at 80OC) product with a molecular weight of 1.4 kDa with PDI of 1.21 was obtained.
Li et al. (51) studied the lipase derived from bacteria, Fervidobacterium nodosum. The thermophilic
nature of the enzyme allowed to carry out the reaction at high temperatures. The conducted experiments have helped to determine the optimum reaction conditions. The highest molecular weight of
2.34 kDa at the PDI level of 1.34 was obtained in a
reaction carried out for 72 h at 90OC in toluene as the
solvent. The same group of researchers in other
work (52) introduced the gene encoding this enzyme
into the genome of Escherichia coli. The recombinant cell thus serves the catalysis of the ring-opening polymerization of ε-caprolactone. In the study, it
was selected the most favorable temperature and a
suitable solvent for this reaction. The highest molecular weight has been reached in the 72-h reaction
carried out in cyclohexane at 70OC and was 2 kDa at
the PDI level of 1.47. Furthermore, it has confirmed
the possibility of the repeated use of the catalyst.
Dong et al. (53) decided to investigate lipase derived
from Pseudomonas bacteria. In order to optimize the
reaction, the researchers studied the effect on the
reaction course, of such factors as the nature of the
solvent and water content. The product with the
highest molecular weight of 14.5 kDa with PDI of
1.23 was obtained in a reaction carried out at 45OC
in 1,2-dichloroethane as a solvent in the presence of
molecular sieves. Barrera-Rivera et al. (54) analyzed
the activity of lipase derived from the fungus
Yarrowia lipolytica. Reactions were carried out in nheptane at a temperature range of 50-70OC with different reaction times. The highest achieved value of
the molecular weight was 0.977 kDa, obtained in the
15 days reaction at 55OC. In another study (55) they
investigated the activity of the same enzyme in ionic
liquids. There were used five types of ionic-type solvents, and in addition, the effect of reaction temperature and monomer on the properties of the product
was studied. The highest molecular weight equal to
8.158 kDa with PDI at 1.6 was obtained in the reaction conducted in 1-butylpyridinium trifluoroacetate, at 60OC for 60 h. Hunsen et al. (56) used the
enzyme of cutinase belonging to the group of hydrolases. The enzyme showed a greater activity in
toluene as compared to the reaction carried out in
bulk. The reaction carried out at 70OC gave a product with a molecular weight of 24.9 kDa equal to the
level of PDI 1.7.
Non-metallic catalysts
As catalysts of the ring-opening polymerization there were also used the organic molecules,
820
Table 1. Selected catalysts applied in the course of polymerization of poly-ε-caprolactone, with respective initiators and solvents. Used
concentrations (M) are also presented, as well as resulting polydispersity index (PDI) of the obtained polymer, and publication source
(Ref). THF ñ tetrahydrofuran.
Catalyst
Initiator
Solvent
M (g/mol)
PDI
Ref.
Bis(tetrahydrofuran)calcium
bis[bis(trimethylsilyl)amide]
none
THF
11.8
2.39
10
methanol
THF
9.0
1.29
10
ethanol
THF
6.2
1.24
10
[(THF)Ca(tmhd)]2[pN(SiMe3)2](p-tmhd)
none
THF
16.7
1.8
11
[(THF)Ca(tmhd)]z[pOCH(Me)Ph](µ-tmhd)
none
THF
1.5
1.13
11
Calcium methoxide
none
none
22,2
1.25
12
Calcium aminoisopropoxide
none
xylene
8,5
-
13
Calcium aminoetoxyoxide
none
xylene
10
-
13
Silica-Ca alkoxide
none
-
6.34
1.12
14
GPS-silica-Ca alkoxide
none
-
4.22
1,10
14
[Ca{N(SiMe3)2}(THF)(PhTriMeN)C{N(CH2)2OMe}ñ]2
benzyl alcohol
benzene
140
1.13
15
[Ca{Me2NC2H4N(CH2-3,5-Bu2t
C6H2O-2)2}]2
toluene
none
26
1.3
16
Ferric (III) chloride
2-allylphenol
none
23.15
1.52
18
Ferric (III) perchlorate
2-allylphenol
none
20.43
1.88
18
Ferric (III) chloride
isopropyl alcohol
none
11.6
1.3
18
Ferric (III) chloride hexahydrate
none
none
12.52
1.12
19
Ferric (II) chloride tetrahydrate
none
none
10.36
1.25
19
(But-BDI)FeOBut
none
toluene
86.2
1.38
20
[Na4(CH3CN)8(L1Fe)2(µ-O)]
7∑4CH3CN
benzyl alcohol
toluene
2.38
1.9
21
C30H34FeN4O4∑C7H8
none
toluene
6.2
3.1
22
BHT2Mg∑THF2
benzyl alcohol
tetrahydrofuran
16.3
1.14
24
[(µ-OBn)2Mg2LBu]
none
toluene
5.75
1.7
25
[L2MgnBu]2
benzyl alcohol
tetrahydrofuran
46.4
1.77
26
[(EDBP-Me)Mg(m-OBn)]2
none
dichloromethane
23.5
1.08
27
(LCF3)2Mg
9-hydroxyantracene
toluene
23.9
1.14
28
Zinc bis(2-bromoethoxide)
none
toluene
3.1
1.05
31
Ethyl zinc 2-bromoethoxide
none
toluene
5.2
1.07
31
Zinc undecanoate
benzyl alcohol
none
17.45
1.2
32
Mor[ONN]ZnN(SiMe3)2
none
toluene
51.7
1.79
33
Pip[ONN]ZnN(SiMe3)2
none
toluene
25.2
1.72
33
NMe2 [ONN]ZnN(SiMe3)2Me2
none
toluene
37.8
1.71
33
[Zn(Et){2-{OC(Ph)=CH}-6(3,5-Me2C3HN2)C5H3N}]2
benzyl alcohol
toluene
1.9
1.13
34
[Zn(Et){2-{OC(But)=CH}-6(3,5-Me2C3HN2)C5H3N}]2
benzyl alcohol
toluene
1.08
1.14
34
[L4-ZnOBn]2
none
dichloromethane
15.9
1.1
35
Zinc prolinate
none
none
85.0
1.9
36
Zinc acetate dihydrate
none
none
79.66
1.18
37
821
Table 1. cont.
Catalyst
Initiator
Solvent
M (g/mol)
PDI
Ref.
Zinc acetate dihydrate
benzyl alcohol
none
32.29
1.19
37
5s-Mes-ZnCl2
benzyl alcohol
none
16.0
1.68
38
6-Mes-ZnCl2
benzyl alcohol
none
16.5
1.64
38
(EtZnOPhtBu(CH2)Pz)2
benzyl alcohol
toluene
6.61
1.07
39
(EtZnOPhtBu(CH2)PzMe)2
benzyl alcohol
toluene
5.24
1.14
39
[(t-BuBTP)2Zn]
9-hydroxyanthracene
toluene
6.7
1.03
40
[(l-TMClBTP)ZnEt]2
9-hydroxyanthracene
toluene
65.6
1.3
40
[L4(ZnEt)4Zn2(CH3CN)4
(µ-OEt)2] 10∑2(CH3CN)
benzyl alcohol
toluene
9.02
1.1
21
[L1 ZnN(SiMe3)2]
none
none
19.6
1.23
41
Pig pancreas lipase
butyl alcohol
heptane
2.7
1.9
42
Lipase B from C. albicans
propargyl alcohol
toluene
22.8
1.58
43
none
toluene
86.0
-
44
none
water
3.7
-
45
none
[C4(C6Im)2][NTf2]2
35.6
1.64
46
water
supercritical CO2
110.0
2
47
none
[Emim][BF4]
20,6
-
48
Esterase from A. fulgidus
none
toluene
1.4
1.21
50
Lipase from F. nodosum
none
toluene
2.34
1.34
51
Recombinant E. coli
none
cyclohexane
2
1.47
52
Lipase from Pseudomonas
none
1,2-dichloroethane
14.5
1.23
53
Lipase from Y. lipolytica
none
n-heptane
0.977
-
54
Lipase from Y. lipolytica
none
1-butylpyridinium
trifluoroacetate
8.158
1.6
55
Cutinase
none
toluene
24.9
1.7
56
Succinic acid
threo-9.10dihydroxyoctadecanoic
acid
none
11.5
1.48
57
Fumaric acid
threo-9.10dihydroxyoctadecanoic
acid
none
15.2
1.23
57
Tartaric acid
benzyl alcohol
none
2.73
1,3
58
Lactic acid
methyl β-D-glucopyranoside
none
6.5
1.5
59
(3,3í,5,5í-Tetra-tertbutylbiphenyl-2,2í-diol)
phosphoric acid
none
none
8.4
1.13
60
[2,2í-ethylidene-bis (4,6-di-tertbutylphenol)] phosphoric acid
benzyl alcohol
none
5.28
1.19
60
Nonafluorobutanesulfonimide
ethyl alcohol
none
13.3
1.25
61
1,5,7-Triazabicyklo[4.4.0]dec5-ene
pyrenebutanol
benzene-d6
20.8
1.16
62
mainly of the acidic nature. These compounds, or
their derivatives, because of their natural origin do
not exhibit toxicity to the human body. Oledzka and
Narine (57) carried out the synthesis of poly-ε-
caprolactone having functional groups of the octadecanoic acid derivative, which was used as a reaction
initiator. In the role of catalysts there were used fatty
acids, fumaric acid and succinic acid. The NMR
822
Figure 16. Zinc based benzotriazole derivatives synthesized by Tai et al. (40)
Figure 17. Metallorganic catalyst based on phenylalanine derivative investigated by Darensbourg and Karroonnirun (41)
analysis showed the presence of acidic functional
groups at the ends of polymer chains. In the case of
a reaction catalyzed by succinic acid, with a ratio of
50 : 5 : 1 in the 24-h reaction product was obtained
with a molecular weight of 11.5 kDa at the level of
the PDI of 1.48. In the case of fumaric acid at the
same ratio of the reactants, the reaction temperature
of 105OC, the polymer has a molecular weight of
15.2 kDa with PDI equal to 1.23. Casas et al. (58)
investigated the catalytic activity of various organic
acids in the presence of benzyl alcohol as an initiator. The highest average molecular weight was
obtained in a reaction catalyzed with the tartaric acid
in a 4-h reaction carried out at a temperature of
120OC. The obtained product was characterized by a
molecular weight equal to 2.73 kDa and PDI of 1.3.
Persson et al. (59) synthesized poly-ε-aprolactone
containing sugar groups. Reactions were catalyzed
with the lactic acid and initiated with sugars such as
sucrose, β-D-glucopyranoside, or raffinose. The
product with the highest molecular weight of 6.5
kDa with PDI of 1.5 was obtained in a reaction time
of 21 h, carried out in bulk at a temperature of
120OC. An analysis of the product structure showed
the presence of the sugar groups at the ends of polymer chains. Yao et al. (60) synthesized two new
Brønsted acids derived from phosphoric acid (Fig
19).
Reactions were carried out in bulk at a temperature of 110OC. The first acid after a reaction time of
two hours has allowed to obtain a product having an
average molecular weight of 8.4 kDa PDI equal to
1.13. The second derivative of the two-hour reaction, in the presence of benzyl alcohol as an initiator
allowed to obtain a polymer having a molecular
weight of 5.28 kDa and PDI at the level of 1.19.
Oshimura et al. (61) studied the activity of a number
of Brønsted acids being the derivatives of perfluoroalkanesulfonates and perfluoroalkanesulfonimides. The most preferred results were obtained in
the reaction catalyzed by nonafluorobutanesulfonimide in a reaction lasting eight hours in the presence of ethyl alcohol as an initiator. The product was
characterized by a molecular weight of 13.3 kDa and
a PDI of 1.25. Lohmeije et al. (62) examined the
activity of catalysts which are derivatives of guanidine and amidines. The product with the highest
molecular weight of 20.8 kDa and PDI equal to 1.16
was obtained in a reaction catalyzed by 1,5,7-triazabicyklo[4.4.0]dec-5-ene. The eight-hour-long reaction was initiated by pyrenebutanol.
Alternative methods of obtaining poly-εε-caprolactone for pharmaceutical use
In order to achieve a higher polymer purity and
other properties relevant to the materials used for
obtaining nanocarriers of medicinal substances, different methods, mainly physical ones, were used.
Sobczak et al. (63) developed a method for the
purification of poly-ε-caprolactone produced in the
reaction catalyzed by tin 2-ethylhexanoate. This
entailed the dissolving of the crude product in methylene chloride, and then precipitated it with distilled
water acidified with concentrated HCl to 5%. This
process was repeated four times in four samples of
the polymer, which allowed for the reduction of pollution of the tin to a value of 4 ppm. A single cleaning process with drying of the precipitated polymer
lasted about a week. Stjerndahl et al. (64) aimed at
reducing the residues of tin in the resulting product,
thus in the process used a minimum amount of catalyst, of the ratio of 1 : 10 000. The obtained polymer
after triple precipitation with the chilled methanol
and hexane from the chloroform solution was characterized by the contents at the level of 5 ppm. The
resulting polymers were used in the form of films as
a scaffold for proliferating murine stem cells. It has
been shown that the process proceeds in a similar or
greater intensity as compared to the control utilizing
the conventional polymer used for cell proliferation.
Stjerndahl et al. (65) conducted a synthesis of polyε-caprolactone at a larger scale catalyzed with 1-din-butyl-1-tin-2,5-dioxocyclopentane. After completion of the reaction and evaporation of the solvent,
the crude product was reacted with 1,2-ethanedithiol. After completion of reaction, the resulting compound is characterized by good solubility in organic
compounds, and was removed in the single precipitation process with hexane from the chloroform
solution of the polymer. The obtained product was
characterized by a tin content of 23 ppm. In order to
avoid contamination of the product with the remains
823
of the solvent, the most preferred type of reaction is
the reaction carried out in bulk. However, in many
cases, the insolubility of the catalyst in the monomer
leads to poor control of the reaction, requiring the
use of solvents such as toluene. F. Stassin et al. (66)
demonstrated on the example of the reaction catalyzed with a tin-organic compound that it is possible to use supercritical carbon dioxide as a solvent.
The solvent is non-toxic, and easy to remove from
the reaction medium. Supercritical carbon dioxide in
the reaction with the most frequently used tin-organic catalyst of 2-ethylhexanoate was also used by
Bratton et al. (67) They have shown that the process
occurs in the same mechanism as in conventional
solvents, maintaining the linear increase in the average molecular weight with monomer conversion,
but with less efficiency. In order to improve the
reaction yield and elongate the obtained chains there
were also used physical methods not requiring the
introduction of additional substances into the reaction mixture. Price et al. (68) used the operation of
the ultrasound in the polymerization reaction of
cyclic lactones including ε-caprolactone. The ultrasonic cavitation, or formation and collapse of microscopic bubbles in the mixture under the influence of
ultrasound, provides a large amount of energy to the
system, and provides a uniform mixing during the
reaction. Researchers have shown a slight increase
in the average molecular weight in the application of
Figure 18. Ionic liquids used as solvents by Wu et al. (46)
Figure 19. Novel Brønsted acid catalysts ivestigated by Yao et al. (60)
824
this method in the polymerization reaction of εcaprolactone catalyzed by the tin-organic compound. Another factor which may preferably affect
the ring-opening polymerization are microwaves. In
addition to ensuring a uniform heating of the mixture throughout the volume, there is suggested the
additional effect of the microwave radiation not connected with heating. Dipole molecules rotate under
the influence of the alternating magnetic field,
which can lead to the increased incidence of collisions between the reacting particles and affect the
course of the polymerization process (69).
Microwave radiation in the polymerization process
of ε-caprolactone was used by Barbier-Baudry et al.
(70). The reaction catalyzed with lanthanide halides
occurred faster under the influence of microwaves
than in the case of standard heating, further there
were observed higher molecular weight and lower
rates of PDI in the case of products obtained in the
microwave reactors.
SUMMARY
The listed publications illustrate the number
and variety of potential possibilities of obtaining
poly-ε-caprolactone for pharmaceutical uses, but
there are no studies discussing the synthesis of polymer to a larger scale, which would constitute a step
towards the introduction of these methods to industrial production. Among the discussed enzyme catalysts, despite the simplicity of the product purification process, due to the high coefficient of PDI characterizing the products and the long reaction times
seem to be the least promising for the purposes of
pharmacy. A relatively poorly studied group are the
non-metallic organic catalysts, which stand out due
to the non-toxicity and often the low cost of obtaining, which favors the further research on this variety
of catalysts.
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Received: 27. 04. 2015
ISSN 0001-6837
EXPERIMENTALLY-INDUCED ANIMAL MODELS OF PREDIABETES
AND INSULIN RESISTANCE: A REVIEW
MD. SHAHIDUL ISLAM1,2* and VIJAYALAKSHMI VENKATESAN2
1
Department of Biochemistry, School of Life Sciences, University of KwaZulu-Natal (Westville Campus),
Durban 4000, South Africa
2
Department of Biochemistry/Stem Cell Research, National Institute of Nutrition (ICMR),
Tarnaka, Hyderabad 500007, India
Abstract: Animal models are very common in diabetes research when rodents are mostly used in this regard.
Although a number of animal models of type 1 and type 2 diabetes (T2D) are developed in last few decades,
the numbers of animal models of prediabetes and insulin resistance are very scanty. Due to the rapidly changing pathogenesis of the disease and preventive study, the popularity of the prediabetic and insulin resistance
models are largely increased in the recent years. Some genetically or spontaneously induced models of diabetes
are used as models for prediabetes and insulin resistance in the early stage of their lives such as prediabetic
SHROB rats, Zucker Diabetic Fatty (ZDF) rats, Goto Kakizaki rats, Otsuka Long Evan Tokushima Fatty Rats
(OLETF), prediabetic BB-DP rats and prediabetic Chinese hamster (non-genetic model), however, these models are relatively expensive, not widely available compared experimentally-induced non-genetic models hence
not suitable for routine pharmacological screening of anti-diabetic agents. In the present review, we carefully
discussed the induction method, induction period, advantages, disadvantages and suitability of various nongenetic or experimentally-induced animal models of prediabetes and insulin resistance. We have also summarized the key factors of different models in a couple of tables to give a quick overview to the diabetes
researchers in order to more appropriately select an authentic animal model of prediabetes and/or insulin resistance to achieve their specific research outcomes.
Keywords: animal model, high-fat diet, high-fructose diet, insulin resistance, prediabetes
number of studies done on normal glucose-tolerant
individuals with a first degree NIDDM relative where
pancreatic β-cell dysfunction was found as a primary
genetic lesion rather than insulin resistance with no
evident insulin sensitivity (5). On the other hand, from
the results of completed United Kingdom Prospective
Diabetes Study (UKPDS), it has been suggested that
the declining β-cell function is the major cause for the
progression of T2D (6) when in more recent studies,
the insulin resistance has been reported as a primary
cause for T2D. It has been also mentioned that T2D
develops as the compensation of insulin production is
failed by the pancreatic β-cell due to defective β-cell
function and impaired β-cell mass (7, 8). Although the
first appearance of ëinsulin resistanceí or ëβ-cell failureí is an ongoing controversy till today, there is no
doubt that both of these two factors play major role in
the progression of T2D.
Although there is no officially approved definition of prediabetes, it is an existence of one or more
of the following conditions such as: impaired fasting
glucose (IFG), impaired glucose tolerance (IGT),
Diabetes is a multi-factorial disease with diverse
pathogenesis affecting millions people in the world.
Although it is mainly classified in two major classes:
(a) type 1 or insulin dependent diabetes mellitus
(IDDM) and (b) type 2 or non-insulin dependent diabetes mellitus (NIDDM), the types of diabetes is
expanding due to its rapidly changing pathogenesis,
particularly for type 2 diabetes (T2D). T2D is a heterogeneous disorder characterized by insulin resistance followed by inability of pancreatic β-cells to
compensate for insulin resistance or partial pancreatic
β-cell dysfunction (1). Although these are considered
as two major factors for the development of T2D, the
order of their existence on the development of T2D is
still controversial. The question has been arisen in
1970 whether the diabetes begins with insulin resistance (2) or β-cell failure (3), when the answer of the
question is still not clear and it has been considered as
an unresolved controversy (3). Further question has
been raised that whether insulin resistance or β-cell
failure comes first in NIDDM or not (4). This question
has been apparently answered from the results of a
* Corresponding author: e-mail: islamd@ukzn.ac.za or sislam1974@yahoo.com; phone: +27 31 260 8717, fax: +27 31 260 7942
827
828
MD. SHAHIDUL ISLAM and VIJAYALAKSHMI VENKATESAN
insulin resistance (IR) and/or partial pancreatic β-cell
dysfunction/failure. Fasting blood glucose (FBG)
<100 mg/dL is considered as normoglycemia in
humans (9). On the other hand, according to the definition of American Diabetes Association (ADA),
fasting plasma glucose (FPG) >100 mg/dL but <126
mg/dL is considered as IFG, blood glucose >140
mg/dL but < 200 mg/dL at 2 h post-glucose load is
considered as IGT, when FPG >140 mg/dL and/or 2
h post-glucose load >200 mg/dL is considered as diabetes (10). So the existence of any of the above-mentioned diabetes associated conditions before the confirmation of frank diabetes can be considered as prediabetes. However, in a recent study, Buysschaert
and Bergman (11) defined prediabetes as the existence of IFG and/or IGT. They have also added that
an increased risk of developing diabetes have been
observed in the normoglycemic individuals - those
who have the history of IFG and/or IGT. Although
IFG and/or IGT are considered as the major conditions as the signs of prediabetes, the insulin resistance and partial pancreatic β-cell failure are considered as the major pathogenesis of T2D. Hence, prevention of insulin resistance and/or pancreatic β-cell
failure could be an excellent alternative on the way
of the development of T2D. To know more about the
origination of all of the above-mentioned factors
such IFG, IGT, insulin resistance and partial pancreatic β-cell failure more authentic animal models with
all features of human prediabetes and insulin resistance are very crucial.
Although a number of animal models of type 1
and T2D are available in the market, the numbers of
animal models of prediabetes and/or insulin resistance are very scanty. Some genetically or spontaneously induced model of diabetes are used as a
model for prediabetes and insulin resistance in the
early stage of their lives such as prediabetic SHROB
rats (12), Zucker Diabetic Fatty (ZDF) rats (13, 14),
Goto Kakizaki rats (15), Otsuka Long Evan
Tokushima Fatty Rats (OLETF) (16, 17), non-obese
prediabetic model (18), prediabetic BB-DP rats (19)
and prediabetic Chinese hamster (non-genetic
model) (20, 21), however these models are relatively expensive, not widely available compared experimentally-induced non-genetic models, hence not
suitable for routine pharmacological screening of
anti-diabetic agents. In order to understand the origin of the disease animal models of prediabetes
and/or insulin resistance can be the better models
compared to frank hyperglycemic T2D model. Since
the prediabetic stage is a relatively milder stage of
diabetes compared to frank diabetic stage, so prediabetic or insulin resistance model can also be used to
study the disease reversal effects of various antidiabetic materials. Recently, no review has been published on this particular topic. Although Velez et al.
(22) recently published a review on animal models
of insulin resistance and heart failure, there focus
was not generalized but completely on the association between insulin resistance and heart failure. In
the present review, we carefully discussed the
induction method, advantages, disadvantages and
suitability of various non-genetic or experimentallyinduced animal models prediabetes and insulin
resistance. We have also summarized the key factors
of different models to give a quick overview to the
diabetes researchers in order to more appropriately
select an authentic animal model of prediabetes
and/or insulin resistance to achieve their specific
research outcomes (Table 1).
LITERATURE SEARCH METHOD
A systematic review of the published literature
has been conducted using key words: prediabetes,
pre-diabetes, pre diabetes, insulin resistance along
with or without animal model in Pubmed, Google
Scholar, Science Direct, Scopus and other relevant
databases. The reference lists of the selected articles
have also been scrutinized to retrieve additional articles in the area of our review.
PREDIABETES MODELS
High-fat/high-calorie diet-fed rodent models
In 2007, high-fat diet induced prediabetes as
well as prediabetic neuropathy has been induced in
C57BL/6J mice by feeding high-fat diet for a 16week period (23). The prediabetes was characterized
by obesity, increased plasma free fatty acids (FFA)
and insulin concentrations, and IGT. The prediabetic neuropathy was characterized by motor and sensory nerve conduction deficit, tractile allodynia, and
thermal hypoalgesia with the absence of intraepidermal nerve fiber loss or axonal atrophy, which have
been further confirmed by another subsequent study
in the same animal strain with same experimental
setup (24). Subsequently, Shevalye et al. (25) fed
high-calorie/high-fat diet (58% calorie from fat) to
C57BL/6J mice for a 16-week period to develop a
prediabetic model. At the end of the experimental
period, the model was characterized by increased
body weight, IGT, hyperinsulinemia and polyuria.
Prediabetic nephropathy has also been confirmed by
2.7 fold increase in 24-h urinary albumin excretion,
20% increase in renal glomerular volume, 18%
increase in renal collagen deposition, and 8%
829
Experimentally-induced animal models of prediabetes and insulin...
decrease in glomerular podocytes. Although some
other diabetic nephropathy and neuropathy related
parameters were measured in the above-mentioned
experiments, the model induction time was relatively long (16 weeks) and this model has not been evaluated by using any anti-diabetic, anti-nephropathic
or anti-neuropathic drugs.
Jin et al. (26) developed a prediabetic model
using high fat diet (24.5% lard +.2.5% soybean oil)
in 4-week old C57BL/6J mice. After 12 week feeding, prediabetes was characterized by non-significantly higher blood glucose, glucose intolerance,
but significantly higher serum triglyceride, total
cholesterol and lower HDL-cholesterol concentrations in high-fat diet-fed mice. Serum insulin,
insulin resistance or β-cell functions were not
measured in this model and this model was not
evaluated by using any anti-diabetic drugs.
Although this model has been evaluated by using
anti-diabetic plant extract, which was significantly
effective in improving glucose tolerance level, the
other parameters were not influenced at all. Hence,
more detail study is needed to confirm the efficacy
of this model for prediabetes.
High-fat diet-fed streptozotocin-injected canine
models
Ionut et al. (27) developed a canine model of
obese prediabetes with mild type 2 diabetes by feeding a high-fat diet containing 27% protein, 38% carbohydrate and 35% fat for 10 weeks followed by various low dose streptozotocin (STZ) injections (30-15
mg/kg b.w.) to lean dogs with body weight 29.0 ± 0.9
kg. The same diet has been continuously fed for the
Table 1. List of prediabetes models with their method of characterization, advantages and disadvantages.
Mode of induction/
induction time
References/
induction time
High-fat/high-calorie
diet-fed rodent
models
Obrosova
et al. (23)
Watcho et
al. (24)/
16-week
●
Shevalye et
al. (25)/
16-week
●
Characterizations
●
●
●
●
●
Jin et al.
(26)/
4-week
●
●
●
High-fat diet-fed
STZ-injected canine
models
Ionut et al.
(27)/
10-week
●
●
●
Sucrose-fed
rodent models
Soares et al.
(28) /
9-week
●
Nunes et al.
(29) /
16-week
●
●
●
●
●
●
●
●
High saturated
fat/cholesterol/
sugar-fed swine
model
Te Pas et al.
(30) /
10-week
●
●
Advantages
Disadvantages
Obesity
Increased plasma free
fatty acids and insulin
Impaired glucose
tolerance (IGT)
Can be used for
prediabetic
neuropathy.
Relatively longer
induction period.
Not validated by any
antidiabetic drug.
Increased body weight
Polyuria
IGT
Hyperinsulinemia
Can be used for
prediabetic
neuropathy and
nephropathy.
Relatively longer
induction time.
Not validated by any
relevant drug.
Non significantly higher
blood glucose
Glucose intolerance
Dyslipidemia
Evaluated by
anti-diabetic plant
extract and found
effective for
improving glucose
tolerance.
Serum insulin,
insulin resistance
and β-cell functions
were not measured.
Not evaluated by any
anti-diabetic drug.
Increased visceral and
subcutaneous fat
Reduced insulin
sensitivity
Impaired fasting glucose
Can be a proper
model for mild
T2D and IGT.
Not validated using
anti-diabetic drug.
Bigger body size
of the animal.
Hyperinsulinemia
Hypertriglyceridemia
with normoglycemia
Can be used
as a model
for prediabetic
neuropathy.
Not validated by using
any anti-diabetic or
anti-neuropathic drug.
Hyperinsulinemia
Insulin resistance
IGT
with normoglycemia
Obesity
Hypertension
Can be used
as a model
for prediabetic
neuropathy and
cardiomyopathy.
Not validated using
any relevant drug.
Dyslipidemia
Hyperglycemia
Can be used
as a model
for metabolic
syndrome.
Insulin resistance or
glucose intolerance
has not been analyzed.
Not validated by using
any relevant drug.
830
entire 22 weeks experimental period including 12
weeks after the STZ injection. The model has been
characterized by significantly increased visceral and
subcutaneous fat, reduced insulin sensitivity. The animals injected with moderate dose (22.5 mg/kg b.w.)
of STZ had mild T2D with normal or IFG when prediabetes with normal FBG was observed in the low
STZ (15 mg/kg b.w.) injected group. Animals with no
frank hyperglycemia had significantly higher body
fat, lower β-cell function and serum insulin level even
after 12-week experimental period. Although from
the data of this study it has been suggested that the
feeding high-fat diet followed by the injection of low
to moderation dosages of STZ may induced proper
models of mild T2D and IGT, the models have not
been evaluated using any anti-diabetic drugs. The relatively higher body size compared to small rodents
like rats and mice will make it less popular to the scientists for routine pharmacological screening of antidiabetic drugs due to higher maintenance cost.
Sucrose-fed rodent models
Soares et al. (28) developed a prediabetic
model in adult Wistar rats by feeding them 35%
sucrose solution ad libitum for 9 weeks. The prediabetes was characterized by hyperinsulinemia and
hypertriglyceridemia with normoglycemia at fed
state. Although some prediabetic neuropathy related
parameters were measured in this model, the induction time of model was relatively higher (9 weeks)
and it has not been evaluated by using any anti-diabetic or anti-neuropathic drugs (28). Using exactly
the same approach, Nunes et al. (29) developed a
prediabetic model in 16-week-old Wistar rats and
the model has been characterized by hyperinsulinemia, insulin resistance, impaired glucose tolerance,
hypertriglyceridemia with the absence of hyperglycemia, obesity and hypertension. Additionally,
the elevated levels of liver weight/body weight ratio
and brain natriuretic peptide (BNP) mRNA expression along with upregulation of fibrosis, hypertrophy, angiogenesis and endothelial lesions and oxidative stress suggest this as a better model to evaluation the cardiac issues in prediabetic condition.
However, this model has not been evaluated using
any anti-diabetic or anti-cardiomyopathic drug.
High saturated fat/cholesterol/sugar (cafeteria
diet) fed swine model
It has been reported in many studies that westernized and cafeteria diets are responsible for the
induction of insulin resistance, prediabetes as well
as metabolic syndrome. In a recent study, Te Pas et
al. (30) fed either high unsaturated fat containing
Mediterranean diet or high saturated fat/cholesterol/sugar containing diet to 11 weeks old pigs (BW
30 kg) for 10 weeks as two one-hour-long ad libitum
meals per day in the morning (08:00 ñ 09:00) and
afternoon (15:00 ñ 16:00). The prediabetic condition
was characterized by the overexpression of proteins
of several prediabetes related parameters such as
total cholesterol, VLDL-cholesterol, LDL-cholesterol, non-esterified fatty acids and glucose in high
saturated fat/cholesterol/sugar fed pigs compared to
Mediterranean diet fed pigs. At the end of the study,
high saturated fat/cholesterol/sugar fed pigs were
recommended as a better model of prediabetes or
metabolic syndrome. However, this model has not
been evaluated by using any anti-diabetic drugs.
INSULIN RESISTANCE MODELS
High-fat diet-fed rat models
Ai et al. (31) developed an insulin resistance
model by oral ingestion of a high fat emulsion in
180-220 g weighing Wistar rats for a 10 days period. The 100 mL emulsion was prepared by emulsifying 20 g lard, 1 g thyreostat, 5 g cholesterol, 1 g
sodium glutamate, 5 g sucrose, 5 g saccharose, 20
mL Tween 80, 30 mL propylene glycol when the
final volume was made up by distilled water. The
model has been characterized by insulin resistance,
insulin tolerance test, larger adipocyte and pancreatic islets, increased GLUT2 and α-glucosidase
mRNA expression in high fat emulsion ingested
group. Although no lipidogenic parameters (except
adipocyte) were measured and the model has not
been evaluated by using any relevant drug, this can
be suitable and cost effective model for its very
short induction time. However, researchers must be
careful about the resemblance of the pathogenesis of
this quickly induced model with the slowly induced
insulin resistance of humans.
In another study, Viswanad et al. (32) fed highfat diet ad libitum to Spargue-Dawley rats for a period of 4 weeks and the insulin resistance was characterized by obesity, hyperinsulinemia, mild hyperglycemia, hypertriglyceridemia, hypercholesterimia, glucose intolerance and hypertension. Although a
number of vascular related parameters were studied
in the isolated thoracic aorta of high-fat diet-fed
insulin resistance rats and evaluated by relevant
drug (Tempol 30-300 mM), the model has not been
evaluated by using any anti-diabetic drug in this
study. In a subsequent study by the same research
group, the efficacy of this insulin resistance model
has been evaluated by using two but similar anti-diabetic drugs (pioglitazone and rosiglitazone) (33).
831
Since this model developed almost all the symptoms
of insulin resistance and prediabetes and evaluated
by relevant anti-diabetic drugs so it can be a suitable
model to study the anti-prediabetic and insulin sensitizing effects of various synthetic or natural antidiabetic agents.
Table 2. List of insulin resistance models with their method of characterization, advantages and disadvantages.
Mode of induction/
induction time
References/
induction time
High-fat diet-fed
rat models
Ai et al. (31)/
10 days
Characterizations
●
●
●
●
Viswanad et
al. (32),
Gaikwad et al.
(33)/ 4-week
●
●
●
●
●
●
●
High fructosefed models
Pooranaperundevi
et al. (38)/
30 days
●
Bremer et al.
(39)/ 12 months
●
●
●
●
●
Amin and Gilani
(40)/ 8-week
●
●
●
●
●
High-fat highfructose diet-fed
rodent models
Zaman et al.
(41)/
10-week
●
●
●
Charlton et al.
(42) / 6 months
●
●
●
●
●
●
Munshi et al.
(43)/ 6-week
●
●
●
Dexamethasoneinduced
rat model
Severino et al.
(44) / 4-week
●
●
●
●
Advantages
Insulin resistance
Glucose intolerance
Larger adipocyte and
pancreatic islets
Increased GLUT2 and
α-glucosidase mRNA
expression
Very short
induction period
(10 days) so it will
be cost effective.
Obesity
Hyperinsulinemia
Mild hyperglycemia
Hypercholesterimia
Glucose intolerance
Hypertensiona
Can be used as
model for
prediabetic
cardiomyopathy.
Insulin resistance
Increased lipid
peroxidation
Disadvantages
No lipid related
parameters were
analyzed.
Not validated
using any antidiabetic drug.
Not validated by
Insulin resistance
Central obesity
Dyslipidemia
Inflammation
Very similar
model to human
(rhesus monkey).
Not validated by
any anti-diabetic
drug.
Bigger body size.
Hyperglycemia
Hyperinsulinemia
Hypertension
Dyslipidemia
Endothelial
dysfunction
Relatively shorter
induction period
(8 weeks).
Not validated
using any
relevant drug.
Higher body weight
and body fat mass
Lower insulin
sensitivity
Dyslipidemia
Suggested that
high-fat model is
better than highfructose fed model.
Not validated by
Obesity
Insulin resistance
Liver fibrosis
Inflammation
Endoplasmic
reticulum stress
Lipoapotosis
Data suggested
that combination
of high-fat and
high-fructose diet
may be suitable to
induce insulin
resistance.
Originally
developed for
NASH but not
for insulin
resistance.
Not validated
using relevant
drug.
Hyperglycemia
Hyperinsulinemia
Dyslipidemia
Shorter induction
time (6 weeks)
so cost effective.
Suggested as
a better model
to study dyslipidemia
and insulin resistance.
Lower insulin
sensitivity or higher
insulin resistance
Hyperinsulinemia
Dyslipidemia
Hypertension
Evaluated by
using relevant
drugs.
Shorter induction
time (4 weeks).
832
High-fructose fed models
A number of previous studies reported that
high-fructose diet is one of the major contributors for
the development of overweight, obesity, insulin
resistance, type 2 diabetes as well as metabolic syndrome (34-37). Pooranaperundevi et al. (38) developed an insulin resistance model by feeding 60%
(w/w) high fructose diet for 30 days in rats. The
model has been characterized by insulin resistance,
declined insulin resistance status and increased lipid
peroxidation. Although the suitability of this model
has been tested by using hepatotoxic drug (thioacetamide), this model has not been evaluated by using
any anti-diabetic drugs. Due to the metabolic difference between rodents and humans, Bremer et al. (39)
developed a high fructose (15% fructose containing
500 mL/monkey/day) fed primate (rhesus monkey,
12-20 years old, 16.3 ± 0.4 kg) model which has
been characterized by insulin resistance along with
central obesity, dyslipidemia, and inflammation in a
period of 12 months. The fructose containing beverage was supplied along with diet contained 30%
energy as protein, 11% energy as fat, and 59% energy as carbohydrate. Although this model has not
been evaluated by using any relevant drugs and body
size of the animals are significantly larger than
rodents, it can be a better animal model of insulin
resistance due to its more similarity with humans.
In a more recent study, Amin and Gilani (40)
developed a rat model of metabolic syndrome along
with insulin resistance by dietary manipulation. A
60% fructose containing diet has been provided to
Sprague-Dawley rats for an 8-week experimental
period along with fiber free refined wheat flower. The
model was characterized by hyperglycemia, hyperinsulinemia, hypertension, reduced HDL-cholesterol
level at 4-week period hypertriglyceridemia when
endothelial dysfunction was observed at 8-week period. Although this model has not been evaluated using
any anti-diabetic or relevant drugs, the model induction time was relatively shorter than many other models and can be suitable for routine pharmacological
screening of newly developed anti-diabetic drugs,
functional and medicinal foods and natural products.
High-fat high-fructose diet-fed rodent models
Although both high-fat and high-fructose diets
are usually used for the development of insulin
resistance in animals, it could be better to know,
which dietary components are more effective for the
development of insulin resistance in animals. To
answer this question, Zaman et al. (41) conducted a
comparative study in rats by feeding either a high-fat
(65% calorie from fat) or high-fructose (65% calorie
from fructose) diet for a 10-week period. Each model
has been characterized by higher body weight, body
fat mass, lower insulin sensitivity and dyslipidemia
after 10-week feeding period. Although these models
have not been evaluated by using any relevant drugs,
from the data of this study, it has been concluded that
high-fat diet-fed rats can be better than high-fructose
fed rats as an animal model of insulin resistance.
The high-fat high-fructose diet-fed approach of
the induction of insulin resistance is further supported by the study conducted by Charlton et al. (42)
where they fed either high fat (60% calorie from fat)
or fast food (40% calorie from fat including 12%
saturated fatty acids and 2% cholesterol) diet along
with high fructose containing drinking water (23.1
g/L) to mice for a 6 months period. In both cases,
animals become obese and insulin resistance was
evident. Apart from obesity and insulin resistance,
the combination of high fat and high fructose diet
induced liver fibrosis, inflammation, endoplasmic
reticulum stress and lipoapoptosis when inflammation was minimal and no fibrosis was observed in
the high fat diet fed animals. Although this model
has been originally developed for the nonalcoholic
steatohepatitis (NASH) and not validated by any relevant drugs, the combination of high fat and high
fructose diet can be an excellent approach for the
development of insulin resistance model with some
other associated features of metabolic syndrome.
In a very recent study, Munshi et al. (43) developed a rat model of insulin resistance along with
hyperlipidemia in 200-270 g weighing male Wistar
rats by feeding 3 : 1 ratio of animal fat : coconut oil
containing diet along with 25% fructose in drinking
water for a 6 weeks period. The model was characterized by hyperglycemia, hyperinsulinemia,
increased total cholesterol, LDL-cholesterol and
triglycerides with decreased HDL-cholesterol. This
model has been further evaluated by using relevant
drugs and suggested as a cost-effective model to
study at least two cardiovascular biomarkers such as
dyslipidemia and insulin resistance. Due to the
shorter induction time and better response to relevant test drug with the existence of all major symptoms this model can be a suitable tool for routine
pharmacological screening of anti-dyslipidemic and
insulin sensitizing drugs.
Dexamethasone induced rat model
Severino et al. (44) developed a model of
insulin resistance by injecting (s.c.) a low dose (2
mg/day) of dexamethasone for 4 weeks in Wistar
rats. This model was characterized by lower insulin
sensitivity or higher insulin resistance, high blood
pressure, higher serum triglyceride, insulin and
hematocrit level. This model has also been evaluated by using three relevant drugs, two of which have
been successfully reduced dexamethasone-induced
insulin resistance and related abnormalities in rats.
The induction time of this model was also relatively
shorter compared to many other models so it can be
a cost-effective model of insulin resistance for routine pharmacological screening of anti-diabetic
drugs and natural products.
Zymosan-induced mice model
Wang and colleagues (45) used zymosan, a
mixture of cell-wall particles from the yeast named
Saccharomyces cerevisiae, to induce zymosan in
mice. Although this model has been found as a better insulin resistance model compared to high-fructose diet-fed insulin resistance model, it has been
reported that this model is not sustainable without
the zymosan treatment. Hence, it cannot be used as
proper animal model of insulin resistance.
CONCLUSIONS
According to our review, a number of different
approaches have been used to develop the animal
models of prediabetes and insulin resistance such as
high-fat or high-calorie diet-fed models, high-fat
diet-fed STZ-injected models, high sucrose-fed
model, high saturated fat/ cholesterol/ sugar-fed
models, high fructose-fed models, high-fat highfructose diet-fed models, dexamethsone-induced
models and zymosan-induced models. Although
many of these models have been successfully developed either for prediabetes or insulin resistance or
both, all of them did not receive similar appreciation
for several reasons. Some of them showed very similar pathogenesis of prediabetes or insulin resistance
but took very long time to develop. Although some
of them developed in a very short period of time but
they did not completely reproduce the pathogenesis
of either prediabetes or insulin resistance or both.
Some models did analyze very fewer parameters
from which no conclusion can be drawn regarding
their suitability as a model for either prediabetes or
insulin resistance. Finally, most of these models
have not been validated by any anti-diabetic or relevant drugs which reduced the suitability of these
models. Although high-fat diet-fed STZ-injected
model has been found suitable to study prediabetes,
it has been developed in bigger rodent (dog) hence it
may not be popular to scientist not only due to bigger body size but also due to higher housing and
maintenance costs. On the other hand, although
833
most of them are not validated by relevant drugs, the
high-fat/ high-calorie/high sucrose diet fed models
were found to be most suitable to study the prediabetes and associated complications such as prediabetic neuropathy and nephropathy and cardiomyopathy with almost similar induction period, when
all of these approaches almost required similar
induction time (9-16 weeks). Although a number of
approaches have been used for the development of
insulin resistance model, either high-fat or highfructose or both fed was found to meet the most
major pathogenesis of insulin resistance model.
Adding high-fructose with high-fat diet did not
make any significant difference in terms of pathogenesis or model induction time when feeding highfat diet alone has been recommend as a better
approach for the development of insulin resistance
model rather than with high fructose diet or with the
combination of them (41). Although most of the
insulin resistance models have been developed in 412 weeks of time, a couple of models have been
taken significantly longer time (6-12 months or
24ñ48 weeks) (39, 42) and another model taken only
10 days to induce insulin resistance (31). Since naturally it takes considerably longer period of time to
induce insulin resistance in humans, it is not clear
how similar will be the quickly developed model
with the human pathogenesis of insulin resistance.
Hence, high-fat diet fed insulin resistance models
developed in 4-12 weeks period could be considered
as the models of choice for insulin resistance.
Acknowledgment
This work was supported jointly by the
Department of Science and Technology (DST) and
Federation of Indian Chambers of Commerce and
Industries (FICCI), New Delhi, India and Research
Reward from the University of KwaZulu-Natal,
Westville Campus, Durban 4000, South Africa. First
author received CV Raman Visiting Research
Fellowship for African Researchers during the period of this work.
Disclosure
There is no conflict of interest within this article.
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Received: 13. 06. 2015
ISSN 0001-6837
POTENTIAL APPROACHES FOR REDUCING AMYLOID β PRODUCTION
CHENGLONG ZHENG1,2,#, YUE LAN3,#, JIAN ZHANG2, LU ZHANG2, JIANI WU2
and SHUWEN GUO2,*
1
Beijing Gulou Hospital of Traditional Chinese Medicine, 13 DouFuChi Hutong, Dongcheng District,
Beijing 100009, P.R. China
2
Beijing University of Chinese Medicine, 11 Bei San Huan Dong Lu, Chaoyang District,
Beijing 100029, P.R. China
3
Fuwai Hospital CAMS & PUMC, 167 Bei Li Shi Lu, Xicheng District, Beijing 100037, P.R. China
Abstract: Alzheimerís disease (AD), a neurodegenerative disorder, is associated with the mitochondrial dysfunction, defective synapses, and impaired cognition in elderly patients. The accumulation of amyloid β (Aβ)
in synapses and synaptic mitochondria is thought one of the critical pathological events of synaptic defect and
impaired mitochondrial dynamics in AD neurons. In order to understand disease progression and designing
therapeutic agents using available molecular targets, extensive research is in progress throughout the world.
However, no drug has been reported, up to now, as effectively preventing and treating moiety for AD, due to
hidden knowledge about exact mode of AD pathogenesis. However, some hypotheses based-drugs, possessing
capability of regulating amyloid β precursor protein, have been indicated for alleviation of psychological and
behavioral symptoms of AD patients. This review article briefly describes the recent developments made for
exploring the Aβ induced-mitochondrial defects in AD and some treatment possibilities through Aβ-targeting
approaches for AD therapy.
Keywords: Alzheimerís disease, cognitive defects, reactive oxygen species, oxidative stress, postmortem brain,
transgenic mice, amyloid β, amyloid β precursor protein
leading to production of H2O2 and oxygen.
Glutathione peroxidase, the endogenous antioxidant, converts some H2O2 to H2O (4). The remaining
H2O2, superoxide radicals, and other radicals are
transported to the cytoplasm, where these entities
take part in peroxidation of lipid as well as in oxidation of protein and DNA (Fig. 1). Briefly, there is an
excellent balance between the oxidants and antioxidants in the mitochondria; this balance is required to
protect cells against oxidant toxicity (5).
Conversely, in comparison to limited antioxidant
levels, the oxidants are produced comparatively in
larger amounts in pyramidal neurons in cortex and
hippocampus in AD brain. This imbalance condition
leads to production of oxidative stress in neurons of
AD brain (6).
The number of mitochondria varies from cell
to cell depending on need of ATP. In addition to
nuclear genome, mitochondrial genome also plays a
significant role in controlling mitochondrial function, principally oxidative phosphorylation in cells.
Oxidative phosphorylation generates ATP and by-
Life depends on mitochondria, a cytoplasmic
organelle (1). Mitochondria are double-membrane
structures having a central compartment (namely,
matrix) surrounded by two lipid membranes, namely, the inner and the outer mitochondrial membrane.
The outer and inner mitochondrial membranes are
separated by the inter-membrane space (2). These
two membranes surround the core (cytosol) of mitochondria (Fig. 1). The outer mitochondrial membrane does not act as barrier to transfer of low
molecular-weight substances; however, restricted
ionic movement is observed across the inner mitochondrial membrane, which surrounds the mitochondrial matrix and hosts electron transport chain
(ETC). β-Oxidation and tricarboxylic acid (TCA)
cycle occur in the mitochondrial matrix. In the ETC,
oxygen molecules, present in mitochondria, receive
the electrons, released from complex I (NADH
dehydrogenase) and complexes III (cytochrome C
oxidoreductase), generating free radicals, mainly
superoxide radicals (3). Superoxide radicals undergo dismutation by manganese superoxide dismutase,
* Corresponding author: e-mail: shuwenguobucm@gmail.com
#
Equal contribution
835
836
CHENGLONG ZHENG et al.
products (free radicals) within the inner mitochondrial membrane (Fig. 1) (7).
Mitochondrial DNA resembles in structure
nuclear DNA (8). Mitochondrial DNA has the genes
needed for ETC. Mitochondrial genome is capable
of synthesizing specific proteins through its own
transcription and translation systems (9). The
remaining mitochondrial proteins, including metabolic enzymes and DNA and RNA polymerases, are
fabricated in the cytoplasm under the influence of
nuclear genes followed by transportation of these
proteins into mitochondria (10).
The mitochondrial matrix contains many proteins, such as amyloid β (Aβ) (11). Aβ, a c-terminal
part of Aβ precursor protein (AβPP), consists of 99
amino acid residues having molecular mass of 4
kDa. The Aβ generation occurs via proteolysis of
AβPP. There are many studies which reveal the
development of senile plaques through deposition of
Aβ in the hippocampus (12).
Mitochondrial half-life in neuron cells is
approximately 30 days (13). Mitochondria in
healthy cells possess necessarily the balanced mitochondrial dynamics, i.e., there is balance between
destruction (fission) of old mitochondria and synthesis (fusion) of new one, without disturbing normal mitochondrial function in all cells (1, 14).
However, the mitochondrial dynamics balance does
not prevail in diseased cells.
Atypical elevated level of intracellular calcium, over-production of the reactive oxygen species
(ROS), and diminished mitochondrial ATP play a
role in mitochondrial dysfunction. Mitochondrial
dysfunction arises when mitochondrial dynamics is
disturbed, i.e., suppressed mitochondrial fision and
enhanced fussion (15). In addition to aging, mitochondrial dysfunction produces many diseases pertaining to various body systems such as nervous and
circulatory systems (16). Alzheimerís disease (AD)
is one of the nervous system related-disease that
develops in elderly people due to gradual buildup of
somatic mitochondrial DNA modifications (17).
The genetic, pathological, and functional evidences have revealed that the delicate balance
between generation and degeneration of Aβ in the
AD brain is disturbed, leading to abnormal accumulation of toxic Aβ. It causes the formation of amyloid plaques causing injury to synapses, resulting in
the development of neurodegeneration and dementia
due to the presence of microtubule-associated protein t and its hyperphosphorylation and the Aβ cascade (18). Protein τ is generally a soluble protein,
however its aggregates, formed during neurofibrillary tangles formation, are insoluble. The mechanisms responsible for the conversion of a normally
soluble monomeric protein into the insoluble filamentous aggregates have been the subject of intense
study and the target for some drug development.
Moreover, the onset of AD depends on the overproduction of Aβ due to mutations in various genes
including APP, presenilin 1 or presenilin 2 genes.
These genes are integral parts of the β- and γ-secretases complexes that are responsible for generation
of Aβ (19). Out of several risk factor genes for AD,
the strongest one is the ε4 allele of the apolipoprotein E (APOE) gene, which exists as three polymor-
Figure 1. Mitochondrial structure, locations of free radical and Aβ production in mitochondria
Potential approaches for reducing amyloid β production
phic alleles - 2, ε3 and ε4 (20). The molecular mass
of Apo≠E is approximately 34 kDa cocnsisting of
299 amino acids. The production of Apo-E generally occurs by the liver and the astrocytes. Moreover,
Apo-E is involved in lipid metabolism (21). In addition, decreased mitochondrial activity, respiratory
function, and cytochrome C oxidase activity as well
as increased mitochondrial oxidative damage in Aβinsulted synaptic mitochondria has been observed
(22).
Due to growing number of elderly patients suffering from AD, massive attention has been focused
to explore mitochondrial therapeutics in human
patients and animal models (23). Due to undiscovered knowledge about exact mode of AD pathogenesis, no drug has been reported, up to now, as effectively preventing and treating moiety for AD.
However, some hypotheses have been proposed for
alleviation of psychological and behavioral symptoms of AD patients. Regardingly, the Aβ cascade
and the τ hyperphosphorylation are two commonly
accepted hypotheses (24).
The objective of this review article is to prepare a comprehensive summary of recent studies on
Aβ induced-mitochondrial defects in Alzheimerís
disease and its treatment through Aβ-targeting
strategies.
Mitochondrial impairment and Alzheimerís disease
The factors responsible for late-onset of
Alzheimerís disease (AD) include various genetic
variants, food, lifestyle, and environmental factors.
The clinical characteristics of this mental disorder
include its progressiveness, cognitive impairment,
memory loss, age-dependence, and behavioral
changes (25, 26). In older people, AD is associated
to neurodegeneration, manifested with oxidative
stress induced-synaptic and mitochondrial dysfunctions (27, 28). The pathogenesis of mitochondria
may occur through various ways, as discussed
below.
Genetic variants
Lin et al. reported the abnormal changes in
mitochondrial DNA in postmortem brains (PmB)
from AD patients in comparison to that of control
group, comprising of age-matched healthy, young
subjects. This observation supports that defective
mitochondrial DNA is responsible for AD pathogenesis in age-dependent manner (29, 30). In other
study, the possible association between the mitochondrial DNA copy number and AD progression
was investigated in AD patients and control subjects
837
employing molecular approaches. Resultantly, an
inverse relationship was noted, i.e., there was a
decrease in mitochondrial DNA copy number with
an increase in mitochondrial DNA changes in PmB
from AD patients, possibly due to mitochondrial
DNA mutations (31). In another study, the
Caucasian AD subjects were used to examine the
mitochondrial haplotypes (mitochondrial DNA variations). The results showed that the ApoE4 allele
had no involvement in conferring genetic susceptibility to AD through the mitochondrial ëhaplo group
UKí (32).
Another genetic variant is abnormal mitochondrial gene expression in AD (33-37). The investigators used Tg2576 mice to conduct gene expression
study and reported that both, the impaired mitochondrial metabolism and the up-regulation of mitochondrial genes, were associated to mutant AβPP
and Aβ (34). Another study states similar finding
that the initial step in AD progression is mitochondrial metabolic defect (35). Conclusively, mitochondrial impairment is caused by age-dependent production of AβPP and Aβ. Alternatively, mitochondrial impairment is responsible for overexpression
of mitochondrial-encoded genes as a compensatory
mechanism.
Oxidative stress
The association between AD pathogenesis and
oxidative stress induced-mitochondrial dysfunction is
supported by many studies involving PmB of AD
patients and AD transgenic mice lines. In PmB of AD
patients, elevated levels of free radicals, oxidative
stress induced-damage of mitochondrial DNA and
proteins, lipid peroxidation, suppressed production of
ATP, and higher cell death have been observed in
comparison to PmB from age-matched healthy subjects (28, 38-41). On the other hand, various studies
on AD transgenic mice lines have also shown suppressed cytochrome C oxidase activity, elevated levels of free radicals, lipid peroxidation, and suppressed
production of ATP (12, 27, 34, 42-46).
A feature of intrinsic aging is the generation
and deposition of mitochondrial-reactive oxygen
species (mtROS) in neurons. These mtROS are
involved in activation of β- and γ-secretases, the
proteolytic proteins, leading to fragmentation of the
AβPP moiety resulting in production of Aβ, an
AβPP fragment (Fig. 1). Thereafter, Aβ further
induces elevated levels of free radicals, oxidative
stress induced-damage of mitochondrial DNA and
proteins, lipid peroxidation, suppressed cytochrome
C oxidase activity, increased carbonyl proteins, and
suppressed production of ATP leading to neurode-
838
generation in AD elderly patients. The mutation in
AβPP further potentiate this neurodegenerative
effect (13, 25).
Normally, mitochondrial membrane hosts the
AβPP and Aβ in neuronal cells as well (40, 47).
After cleavage from AβPP, Aβ moves to mitochondrial matrix and interacts there with Aβ-binding
alcohol dehydrogenase and cyclophilin D, the mitochondrial matrix proteins. This interaction results in
mitochondrial dysfunction (12, 45, 48). In short, the
linkage of AβPP and Aβ with mitochondria has a
role in the induction of mitochondrial dysfunction in
AD neurons (45, 48-51).
Synaptic and axonal impairment
Many studies have reported that the increased
degeneration of AD neuron synapses with decrease
in anterograde transport of mitochondria in AD
transgenic mice neurons indicates impairment in
mitochondria at synapses (45, 52). The studies have
concluded that Aβ is a neurotoxin owing to its role
in reducing length and impairing motion of mitochondria as well as degenerating the AD neurons
synapses. Moreover, Aβ is also involved in mitochondrial cleavage in neuronal cells leading to
defective axonal transport and distribution of mitochondria, which results in degeneration of synapses
(53, 54).
Many authors have demonstrated that accumulation of Aβ in synapses and synaptic mitochondria
is not a normal condition because it may cause
synaptic degeneration (13, 45, 49, 55). In this context, the investigations on AD mouse have reported
the association of mitochondrial Aβ levels, the
degree of cognitive impairment, and the extent of
mitochondrial dysfunction. This finding explored
the possible contribution of mitochondrial Aβ-provoked signaling cascade to cognitive defect (55, 56).
Moreover, a greater extant of age-regarded, Aβ
buildup and mitochondrial changes in synaptic mitochondria has been found in comparison to nonsynaptic mitochondria in AD transgenic mouse
brain. Moreover, in comparison to non-synaptic
mitochondria in AD transgenic mouse brain, synaptic mitochondria are studied to have a greater
buildup of age-related Aβ and mitochondrial
changes including increase in both, mitochondrial
oxidative damage and mitochondrial permeability
transition, and suppression of both, respiratory function and cytochrome C oxidase (45). Conclusively,
synaptic mitochondria amassed with Aβ are more
prone to Aβ-induced impairment, which may lead to
development of synaptic degeneration and cognitive
defects in AD.
Imbalanced mitochondrial dynamics
It has been studied using human neuroblastoma
(M17) that AD neuronal mitochondria have
impaired dynamics induced by AβPP and Aβ (49).
Another study proposes that Aβ treated-neurons
contain over-expressed mitochondrial fission genes
(Drp1 and Fis1) with concomitant decreased expressions of fusion genes (Mfn1, Mfn2, and Opa1). This
difference in expression of both, fission and fusion
genes, is responsible for impaired mitochondrial
dynamics in AD neurons. Moreover, suppressed
neurite outgrowth, elevated level of mitochondrial
fragments, and reduced mitochondrial activity has
been observed in Aβ treated-neurons indicating
impaired mitochondrial dynamics, which results in
mitochondrial impairment (27, 57). Another group
of authors describes the synaptic alterations and
mitochondrial activity in Tg2576 mouse line, also
termed as the AβPP transgenic mice, using transmission electron microscopy. Resultantly, beside an
elevated level of Aβ and neuronal apoptosis, they
observed fragmented cristae containing-mitochondria, which were significantly smaller in size than
that of normal but greater in number. In short,
buildup of Aβ in AβPP neurons results in synaptic
defects, which lead to neurodegenerative disorder
(52). By and large, after entering into AD neuronal
mitochondria, Aβ demolish mitochondrial dynamics
leading to mitochondrial dysfunction and atypical
mitochondrial activity.
Decreased levels of mitochondrial enzymes
In comparison to that of age-matched healthy
subjects, the PmB from AD patients are found to
have suppressed functions of mitochondrial
enzymes including cytochrome oxidase function in
fibroblasts, pyruvate dehydrogenase activity in lymphoblasts, and α-ketodehydrogenase function (13).
In some biochemical studies, the impaired glucose
metabolism in PmB from AD patients is reported,
which demonstrate the impaired glucose consumption in AD (58, 59). Additionally, the impaired glucose consumption is absolutely associated to ApoE4
genotype.
β induced-mitochondrial defects
Treatment of Aβ
β-targeting
in Alzheimerís disease through Aβ
strategies
Since the published data exhibits association of
various genetic variants, oxidative stress, synaptic
and axonal impairment, imbalanced mitochondrial
dynamics, and decreased levels of mitochondrial
enzymes with mitochondrial and cognitive impairments in AD patients, the development of new mol-
ecules against these pathologic factors is critically
needed. Thus, AD development may be prevented
by decreased Aβ generation, enhanced Aβ clearance, or retarded aggregation of Aβ into amyloid
plaques. On the basis of these considerations and
corresponding molecular targets, some remedial
strategies, as discussed below in text and Figure 1,
are designed and tested in PmB cells from AD
patients and AD transgenic mouse.
β- and γ-secretase inhibitors
Many studies have suggested the therapeutic
potential of β-secretase (or β-site APP cleaving
enzyme 1) and γ-secretase inhibitors. In this context,
normal growth and significant similarity in the phenotypes of β-secretase-knockout mice and their wildtype littermates has been reported. Moreover, the production of Aβ from AβPP was much lesser in the former mice than the later one (60-62). On the other
hand, significantly reduced levels of Aβ was
observed in plasma and cerebrospinal fluid (CSF) of
AD mice treated with γ-secretase inhibitors, DAPT,
BMS-299897 and MRK-560. LY450139 dihydrate
(63). In addition, HMG-CoA reductase inhibitors,
also known as statins, are found to reduce Aβ production through inhibiting β-secretase. The epidemiological studies have shown that atorvastatin and
lovastatin use is clinically beneficial against AD (64).
Muscarinic receptor activators
Beside regulating the secretase function, M1
muscarinic receptors are involved the linkage
between the Aβ and the impaired cholinergics. It has
been reported that talsaclidine induced-activation of
muscarinic M1 receptors inhibits γ-secretase function leading to reduction in Aβ level (65-68).
β-aggregation inhibitors
Aβ
The neurotoxicity of Aβ may be reduced by
suppressing its aggregation (69). It has been reported that injection of iAβ5p, a β-sheet breaker, into
hippocampus reduces the aggregation of Aβ to amyloid plaque (68, 70, 71).
Vaccination and monoclonal antibodies
There are several studies about the role of vaccination and monoclonal antibodies in AD treatment. Vaccination is the active mode of
immunotherapy, while monoclonal antibodies are
passive in this regard. In a publication, authors have
claimed that there was a decrease in plaque formation and improvement in cognitive activity in transgenic mice after vaccination against Aβ42 (72).
Moreover, AN1792/QS-21 antibody has been found
839
effective for improving cognitive activity in AD
patients (73-75).
β-degradation enhancers
Aβ
Aβ-degrading enzymes exert protease like
effect resulting in Aβ degradation (76). Neprilysin,
plasmin, endothelin converting enzyme 1 and 2,
insulin degrading enzyme, and angiotensin-converting enzyme are some representative compounds
with Aβ-degrading activity. Beside the improvement in spatial memory, an improvement in Aβdegrading activity is observed when neprilysin is
overexpressed in neprilysin-knockout mice (77).
Moreover, the elevated level of neprilysin has been
observed with concomitant intrahippocampical
injection of some drugs including imatinib, a tyrosine kinase inhibitor (78), and valproic acid, an
antiepileptic drug and a histone deacetylase inhibitor
(79).
Sporadic AD is characterized by the presence
of apolipoprotein E (ApoE) 4 allele as well as the
reduced clearance of Aβ (80, 81). Mechanistically,
ApoE is involved in the enhanced clearance of Aβ
resulting in reduced buildup of plaque in AD transgenic mice brain (82, 83). One of the tested ApoE
activators is bexarotene (84, 85).
β-blood-brain barrier transport inhibitors
Aβ
Brain blood vessels carry many receptors
including the RAGE (advanced glycation end-products) and LRP (low-density lipoprotein receptorrelated protein)-1 receptor; the former are involved
in transport of Aβ across the blood brain barrier
from blood to brain, while reverse movement is regulated by LRP-1 (86). Therefore, the levels of
RAGE and LRP-1 are increased and decreased,
respectively, in AD patients. Conclusively, AD may
be treated by designing RAGE inhibitors or LRP-1
activators leading to decreased buildup of Aβ in
brain (64, 87).
Metal chelators
The AβPP processing in the pathogenesis of
AD involves metabolism of various metals including Fe, Cu, and Zn (86). Their chelators are capable
of inhibiting the buildup of Aβ. Similarly, the reduction in plasma Aβ42 level has been observed after
administering clioquinol and its 8-OH quinoline
derivative, known as PBT2 (88).
CONCLUSIONS
Cell viability, aging, and many age-dependent
human diseases essentially depend on mitochondria.
840
The age-dependent buildup of mitochondrial DNA
alterations are intensively involved in the atypical
elevated level of intracellular calcium, over-production of ROS, diminished mitochondrial ATP, mitochondrial dysfunction, and impaired neurons causing many neurodegenerative diseases such as AD.
Moreover, synaptic buildup of Aβ in AD neurons
results in defective mitochondrial dynamic activity,
impaired axonal transport, and degenerated synapses demonstrating that mitochondrial and synaptic
defects develop from the accumulation of Aβ. Due
to growing number of elderly patients suffering
from AD, massive attention has been focused to
explore mitochondrial therapeutics in human
patients and animal models. In order to understand
disease progression and designing therapeutic
agents using available molecular targets, extensive
research is in progress throughout the world. Due to
undiscovered knowledge about exact mode of AD
pathogenesis, no drug has been reported, up to now,
as effectively preventing and treating moiety for
AD. However, some hypotheses have been proposed
for alleviation of psychological and behavioral
symptoms of AD patients.
Acknowledgment
This research was supported by the National
Natural Science Foundation of China (No: 307 ñ
2850 and 81173142), the Talents Training &
Supporting Project at Dongcheng District (hold by
Chenglong Zheng) and the International &
Cooperation Program of China (No: 2011 DFA33040).
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Received: 15. 06. 2015
ISSN 0001-6837
INHIBITORS OF LEUKOTRIENES SYNTHESIS: NOVEL AGENTS
AND THEIR IMPLEMENTATION
KRYSTYNA CEGIELSKA-PERUN, EWA MARCZUK and MAGDALENA BUJALSKA-ZADROØNY*
Department of Pharmacodynamics, Centre for Preclinical Research and Technology (CePT) Laboratory,
Medical University of Warsaw, , Banacha 1b St., 02-097 Warszawa, Poland
Abstract: Leukotrienes (LTs) belong to pro-inflammatory mediators that are biosynthesized from arachidonic
acid (AA), inter alia, by 5-lipoxygenase (5-LOX) enzyme in association with 5-LOX-activating protein
(FLAP). An activation of LTs synthesis pathway occurs during the development and maintenance of numerous
diseases such as asthma, anaphylactic shock, allergic rhinitis, psoriasis, rheumatoid arthritis, osteoporosis, as
well as cardiovascular diseases, neurodegenerative diseases and certain types of cancer. The main goal of this
review was to present recent advances on the new compounds influencing the LOX pathway, which are undergoing clinical studies. The mechanisms of action and possible implementations of these molecules in a treatment of asthma, cancer and cardiovascular diseases are discussed.
Keywords: leukotriene, lipoxygenase inhibitor, 5-lipoxygenase-activating inhibitor, novel agent
LTD4 and less affinity to LTC4 or LTE4. These
receptors are involved in bronchoconstriction,
mucus secretion and edema in the airways.
Therefore, selective CysLT1 antagonists, such as
zafirlukast, montelukast and pranlukast, block the
proasthmatic action of the CysLT1. On the other
hand, CysLT2 receptors contribute to inflammation,
vascular permeability as well as tissue fibrosis.
Specific antagonists of CysLT2 receptors have not
been known so far, but these receptors bind with
equal affinity to LTC4 and LTD4 and with less
affinity to LTE4. BLT1 receptors mediate of its
chemoattractant and proinflammatory action.
However, little is known about BLT2 physiological
function (4, 8).
Efforts of creating new drugs inhibiting LOX
pathway are focused on three targets: inhibition of
enzyme, blocking of leukotrienes receptors or inhibition of FLAP, a protein involved into activation
and/or presentation of AA to the enzyme. This
review summarizes the current knowledge on the
new LTs inhibitors and possibility of their implementation in the selected diseases (Fig. 1).
Leukotrienes (LTs) are considered to play a
significant role in the pathomechanism in plenty of
diseases, such as bronchial asthma, allergic rhinitis,
psoriasis, rheumatoid arthritis, osteoporosis, as well
as cardiovascular diseases, neurodegenerative diseases and certain types of cancer (1-3). It is commonly known that one of the substantial pathways of
LTs production is associated with the oxidation of
arachidonic acid (AA) by 5-lipoxygenase (LOX)
using 5-lipoxygenase-activating protein (FLAP)
which increases the affinity of 5-LOX to AA (4). 5LOX converts AA to leukotriene A4 (LTA4), which
is further enzymatically transformed into leukotrienes C4 (LTC4), D4 (LTD4) and E4 (LTE4). This
group of LTs is called cysteinyl leukotrienes, in contrast to leukotriene B4 (LTB4) which is formed from
LTA4 by LTA4 hydrolase (5). There are two types
of cysteinyl leukotrienes receptors: CysLT1 (located
in leucocytes, airway smooth muscles, spleen), and
CysLT2 (heart, brain, central nervous system, placenta, spleen, leukocytes) and two types of receptors
for LTB4: BLT1 (located on leukocytes) and BLT2
(leukocytes, spleen, liver, ovary) (6, 7). The latest
studies suggest the existence of the third type of cysteinyl LT receptor (8). The diversity of LT receptors
occurrence may indicate a role of leukotrienes in
numerous physiological and pathological conditions. CysLT1 receptors bind with high affinity to
Novel inhibitors of leukotrienes synthesis in asthma treatment
In asthma process a notable increase in inflammatory mediators and multiple cytokines is observed,
* Corresponding author: e-mail: mbujalska@gmail.com; phone/fax: +48 (22) 116 61 26
843
844
KRYSTYNA CEGIELSKA-PERUN et al.
as well as the up-regulation of 5-LOX activity and
FLAP expression is well-documented (9).
Furthermore, the latest studies have shown that an
increasing production of the CysLTs in asthma contributes significantly to exacerbations of asthma
symptoms (8). Nowadays, two drugs blocking LOX
pathway in asthma treatment: zileuton, a 5-LOX
inhibitor, and a group of CysLT1 receptor blockers
named lukasts (zafirlukast, montelukast, pranlukast
etc.) are available. Unfortunately, these medicaments
AM-803 (GSK 2190915)
Auranofin
Curcumin
Atreleuton (Via-2291)
possess numerous drawbacks. Montelukast has limited mechanism of action as it blocks only CysLT1
receptor. In turn, zileuton possesses adverse pharmacodynamics effects, it may produce aminotransferase
elevations and numerous interactions with other
drugs, inter alia, theophylline. Moreover, zileuton
immediate-release form was withdrawn in 2008,
while the modified-release form is still available (outside Poland). Additionally, it is difficult to predict
responsiveness to antileukotriene therapy in the indi-
Zileuton
NDGA (nordihydroguaiaretic acid)
CDC (cinamyl-3,4-dihydroxy-a-cyanocinnamat)
Veliflapon (DG-031)
Figure 1. Chemical structures of leukotrienes synthesis inhibitors
Inhibitors of leukotrienes synthesis: novel agents and their implementation
vidual patients because the clinical effect depends on
such factors as: age of patient, body-mass index and
smoking. It was also reported that in less than 10% of
asthma patients the genetic polymorphism of 5-LOX
gene (ALOX5) promoter occurs that could explain the
diminished efficacy of antileukotriene therapy (10).
Therefore, the great interest in asthma therapy is associated with new FLAP inhibitors compounds.
AM-803 (GSK 2190915)
AM-803 (GSK 2190915) is a selective FLAP
inhibitor, in a less extend it also influences the production of 12- and 15-LOXs, COX enzymes, LTA4
hydrolase and LTC4 synthase (9). In the preclinical
studies, it was revealed that AM-803 possessed
dose-dependent anti-inflammatory activities in animal models of inflammation by reduction LTB4
level, CysLTs synthesis, protein extravasation and
neutrophil accumulation (11). In clinical studies,
this compound exhibited an excellent tolerance,
good pharmacokinetic and pharmacodynamics profile in healthy adults (12). Further trials have shown
that GSK 2190915 significantly reduced the early
asthma response to inhaled allergen, but addition of
the test compound to various asthma regular therapeutic medicines had no encouraging effect in the
asthma patients (9). In a recently published study, it
was concluded that using GSK 2190915 may be
beneficial in exercise-induced bronchoconstriction
as the study compound at 200 and 100 mg dose significantly attenuated the response to exercise in 2
and 9.5 hour, respectively, compared with placebo
(13). Currently, this compound has passed successfully the II phase clinical trial in asthma patients.
Novel 5-LOX inhibitors in cancer treatment
Carcinogenesis is a complex process which
takes place on molecular, cellular and systemic level
and involves wide range of exogenous and endogenous substances. Recent studies put huge emphasis
on a role of inflammation in creation and development of conditions such as colorectal cancer (14),
breast, lungs and prostate cancer (15). Epidemiological data suggest that uncontrolled inflammation
and chronic infection is a cause of 20% deaths in
cancer patients (16). Increased level of COX-2 has
been found in more than 85% of colorectal cancers,
moreover, its overexpression is a hallmark of poor
prognosis (17). Recent findings show that expression
and balance between different LOX isoforms play an
important role in inflammation-related carcinogenesis (18). Numerous studies revealed that 5-LOX and
its metabolites promote cancer cell proliferation (19).
Furthermore, a product of 5-LOX activity, 5-hydrox-
845
yeicosatetraenoic acid (5-HETE), plays also a role in
angiogenesis by an induction of vascular endothelial
growth factor (VEGF) expression in colon cancer.
(20). VEGF is considered to be the most potent
tumor angiogenic factor (21). Some reports suggest
that elevated 5-LOX expression results in disturbance in metalloproteinase activity, which leads to
extracellular matrix destruction and enhances metastasis. This phenomenon reduces survival in animal
models of cancer and has shown to increase the invasiveness of many cancer cell types in human (22), for
example, head and neck cancer (23). Moreover, suppression of 5-LOX in some studies increased
chemosensitivity of cancer cells, which means that it
may condition tumor response to chemotherapeutic
agents (18). All those factors together contribute to
an important role of LOX in human prostate, pancreatic, colon, bladder, testicular, esophageal, renal,
hepatoma, lung and breast cancers (21). While the
pro-carcinogenic result of 5-LOX overexpression is
well documented, the role of 15-LOX seems to be
controversial. In some animal models and in human
colorectal and prostate cancer cells 15-LOX was
found to suppress tumor growth (24), however data
still remain discrepant. Therefore, in the article we
present recent developments in anti-LOX strategy in
the cancer therapy. Furthermore, dual COX/LOX
inhibitors are in particular interest of scientist, since
it is known that they act synergistically in inflammation-related cancer prevention and treatment.
Zileuton + imatinib
Zileuton was introduced in USA in 1996 by
Abbott Laboratories for asthma treatment and is
now marketed by Cornerstone Therapeutics Inc.
under the brand name ZYFLO. Recently, efficacy of
zieluton in other diseases than asthma is being
investigated. It is in the II phase of clinical trials for
acne vulgaris treatment conducted by Clinical
Therapeutics (25). Emerging evidence suggests that
it may be effective in chronic myelogenous
leukemia (CML) in co-treatment with imatinib, a
tyrosine-kinase inhibitor. Imatinib has been used in
treatment of multiple cancers, especially Philadelphia chromosome-positive (Ph+) CML (26).
Although imatinib proved to be an excellent treatment option for patients with CML, it was found that
about one-third of patients remain resistant or intolerant (27). In animal studies, it has been revealed
that in knockout mice without Alox5 (5-lipoxygenase coding gene) in leukemia-stem cells CML did
not develop, which suggests 5-LOX significance in
leukemia genesis. Therefore, treatment of CML
mice with zileuton reduced CML stem cells and pro-
846
longed the survival of CML mice (28). Furthermore,
combined use of zileuton with imatinib appeared to
be more effective than each drug in monotherapy.
Zileuton and imatinib combination in CML treatment is currently in a phase I of clinical study conducted by University of Massachusetts, Worcester.
Auranofin
Auranofin (AUR) is a gold complex used in
rheumatoid arthritis treatment. However, the exact
mechanism of action of gold compounds still
remains unknown; it is believed that they act
through modulation of autoimmune system (29, 30).
In an in vitro study it was shown that AUR acted as
a 5-LOX inhibitor in human polymorphonuclear
cells (PMNs). It also reduced chemotaxis of PMNs
towards LTB4 (31). This activity may contribute to
efficacy of auranofin in OA treatment. Recently, its
activity and efficacy is being examined in few clinical trials: in chronic lymphocytic leukemia patients
(CLL) and in patients with recurrent epithelial ovarian, primary peritoneal or fallopian tube cancer.
Another studies evaluated the effectiveness of AUR
in HIV patients, in paclitaxel-induced pain syndrome and in squamous cell lung cancer (in coadministration with sirolimus, an immunosuppressive drug). These studies are in recruitment phase,
while the clinical trial in which the combined
administration of AUR with sirolimus in patients
with advanced solid tumors was planned (data
obtained from www.clinicaltrials.gov).
NDGA
In the study of Tong et al. (32) the efficacy of
NDGA (nordihydroguaiaretic acid), a 5-LOX inhibitor, was tested in two breast cancer cell lines:
MCF-7 (estrogen receptor positive, ER+) and
MDA-MB-231 (estrogen receptor negative, ER-).
The influence on breast cancer cells proliferation as
well as mechanism of action were investigated. The
study revealed that NDGA inhibited growth and
induced apoptosis in both cell lines, suggesting that
its activity is independent from estrogen receptor
presence. Inhibition of LOX pathway led to induction of intrinsic apoptosis through cytochrome C
release from mitochondrial membrane and subsequent activation of caspase cascade. Moreover,
NDGA decreased the level of Bcl-2 and Mcl-1 proteins, known as the anti-apoptotic factors, and
increased the level of pro-apoptotic bax protein in
both cell lines. It is known that carcinogenesis
depends on pro- and anti-apoptotic proteins balance
(33). Therefore, NDGA leads to cancer cells death
by acting on LOX, Bcl-2, Mcl-1 and bax proteins.
Nowadays, NDGA has been examined in clinical
trials in other cancer types. In II phase study conducted by University of California, San Francisco,
NDGA in oral daily dose 2000 mg did not decline
the specific antigen (PSA) level in 12 non-metastatic prostate cancer patients. The study has been terminated after 12 weeks of the observation due to
lack of the desired effect. In other I/II phase clinical
study conducted by Sidney Kimmel Comprehensive
Cancer Center the activity of tetra-O-methyl nordihydroguaiaretic acid (terameprocol/EM-1421), an
inhibitor of Sp-1 mediated surviving transcription,
after intravenous (i.v.) administration has been
examined in 35 patients with recurrent high-grade
glioma. However, the results of the study have not
been published. EM-1421 has also been investigated
in recurrent or refractory solid tumors, in cervical
intraepithelial neoplasia, in malignant tumors of
head and neck and in leukemia treatment by Erimos
Pharmaceuticals (data obtained from www.clinicaltrials.gov).
Curcumin
Curcumin is a natural phenol compound
derived from Curcuma longa (Zingiberaceae) rhizoma powder, which is also known as turmeric. It
has been used in traditional Asian cuisine as a spice
and medicine for thousands of years. Due to its antiinflammatory, analgetic and anti-microbial activity
it has been used in traditional Chinese and Indian
medicine in treatment of disorders such as anorexia,
biliary and hepatic disorders, cough and sore throat,
diabetic wounds, rheumatism and sinusitis (34).
Over the last decades, curcumin properties were
extensively investigated in the in vitro and in vivo
animal models, as well as in clinical trials. It has
been proven that it reduces blood cholesterol, prevents LDL oxidation, prevents aggregation of
platelets, thrombosis and myocardial infarction,
reduces symptoms of type II diabetes, rheumatoid
arthritis, Alzheimerís disease and inhibits HIV replication (35). Moreover, its anti-cancer activity seems
to be well documented in different types of cancer
models.
Such a wide range of curcumin therapeutic
properties results from diversified biochemical activity. It is known that curcumin blocks arachidonic acid
metabolism by COX-2 and 5-LOX inhibition (36,
37), and as a result prevents subsequent inflammatory state development. Moreover, in numerous in vitro
models of cancer it proved to have antiproliferative,
apoptotic, antimetastatic and antiangiogenic properties. Antitumor activity results from regulation of
many transcription factors (for instance, NF-κB,
STAT-3, PPAR-γ), TNF, inflammatory cytokines and
protein kinases (EGFR/HER2, MAPK) (35).
Therefore, curcumin is being tested in numerous clinical trials in different medical conditions, especially
in cancer. However, in vivo trials showed that curcumin has poor bioavailability, so it must be administrated in large oral dose in order to receive therapeutic blood concentration. Nowadays, research is ongoing on the creation of a new form of the drug, for
example nanoforms, conjugates or liposomes (38).
In clinical trial conducted by University of
Rochester Medical Center & Wilmot Cancer Center
curcumin was tested in 35 breast cancer patients in
order to assess the prevention of radiation-induced
dermatitis. In this study, patients were taking curcumin at the dose 2.0 grams three times daily p.o.,
for 4-7 weeks. It was found that the severity of dermatitis in radiation treatment site in breast cancer
patient was slightly reduced in curcumin group in
comparison to control group. No adverse events in
active treatment group were detected. However, the
primary limitation of this clinical study is the small
sample size (data obtained from www.clinicaltrials.gov). In numerous II phase clinical studies, efficacy of curcumin was evaluated in chemotherapyinduced oral mucositis in children, in advanced pancreatic cancer patients or curcumin in co-administration with gemcitabine (a nucleoside analog), in
pancreatic cancer treatment. However, the results of
these studies have not been published so far.
Nowadays, numerous clinical studies are performed
to estimate therapeutic efficacy of curcumin in prevention of colon cancer, subclinical neoplastic
lesions, in multiple myeloma (alone or in co-treatment with bioperine, an extract obtained from the
black pepper fruit) and in glioblastoma, as well as in
obese women with high risk of breast cancer, in
breast cancer patients undergoing radiotherapy, in
solid tumors in co-administration with irinotekan
(an inhibitor of topoisomerase), in prostate cancer,
in patients with inoperable colorectal cancer in combination with FOLFOX regimen, in epidermal grow
factor receptor (EGFR)-mutant non-small cell lung
cancer with tyrosine kinase inhibitors, in endometrial carcinoma, chronic lymphocytic leukemia, familial adenomatous polyposis and osteosarcoma (data
obtained from www.clinicaltrials.gov).
Other compounds that can inhibit LOX are also
tested as anti-cancer agents in different kind of cancer in vivo. In I phase (adenocarcinoma) and II phase
(renal cell cancer and kidney cancer) of clinical trials CDC (cinamyl-3,4-dihydroxy-a-cyanocinnamat,
a metabolite of ciprofloxacin), a non-selective 5-,
12- and 15-LOX inhibitor, has been assessed (21).
847
Based on the results of the studies, 5-LOX
inhibitors may represent a promising group of drugs
in complementing cancer polytherapy. Furthermore,
they seem to be a good strategy in cancer prevention
in the high-risk patients.
Leukotriene modifiers in cardiovascular diseases
treatment
Cardiovascular disease (CD) is one of the main
causes of death worldwide. Epidemiological data
show that in USA heart disease (620 000 of 2.4 million total deaths) and stroke (135 000 deaths) are the
first and third leading causes of death (39). In spite
of the fact that medical progress of last 50 years in
CD prevention, diagnostic methods and pharmacological treatment led to decrease of mortality rate, it
still remains a burning issue.
Invention of statins is certainly a milestone in
cardiology as it prevents from atherosclerosis and its
complications. Atherogenesis is a chronic process
which includes arterial wall injury, lipid accumulation
and oxidation, aberrant immune and inflammatory
reactions. A role of inflammation and lipoxygenase
pathway in atherogenesis has been investigated in
numerous studies. It is well established that LT mediators play a significant role in vascular inflammation.
LTB4 is a strong chemoattractant of neutrophils and T
cells, it promotes adhesion of leucocytes to vascular
endothelium, increases vascular permeability and promotes smooth muscle cells proliferation and migration
(40, 41). CysLTs are micro vessels constrictors, they
enhance permeability, reduce myocardial contractility
and blood flow (42), they also play role in ischemia
and shock (43). The influence of leukotriene pathway
on atherogenesis was proven in arterial walls of
patients with atherosclerosis (44). Clinical trial on
asthmatic patients taking montelucast, a CysLT receptor antagonist, showed that LT receptor blocking
results in decrease of c-reactive protein (CRP), total
cholesterol, LDL, HDL and triglycerides (45).
Moreover, there are some reports from epidemiological studies on FLAP single nucleotide polymorphism
(SNP) that indicate the role of different FLAP haplotypes in cardiovascular risk in Chinese (46) and
Icelanders (47), but another study on large European
population finds no association (48). The exact mechanism of leukotriene action in atherogenesis and other
cardiovascular diseases seems to leave many to discover, nevertheless yet it became a subject of interest
in cardiologic drug research.
Atreleuton (Via-2291)
Atreleuton is a 5-LOX inhibitor introduced by
VIA Pharmaceuticals and has completed the II
848
phase of clinical trials in vascular inflammation in
patients after acute coronary syndrome event. In
randomized, placebo trial in 56 post-acute coronary
syndrome patients, after 6 months of administration,
it significantly decreased the LTE4, high-sensitivity
CRP (hs-CRP) levels, coronary plaque volume (PV)
and increased left ventricular ejection fraction
(LVEF). The study revealed that the decrease in
necrotic core PV and increase of LVEF were correlated with decrease of LTE4 level (49).
Veliflapon (DG-031)
Introduced by deCode genetics, veliflapon, a
FLAP inhibitor, has successfully Phase I and Phase
II of clinical trials in myocardial infarction and
stroke patients. The results of these studies demonstrated that veliflapon was well-tolerated and considerably reduced production of LTB4 level in a
dose-depended manner. The Phase III study has
been conducted on African-American patients with
acute coronary syndrome, as this population has the
highest risk for myocardial infarction developing
due to unfavorable FLAP and leukotriene A4 hydrolase (LTA4H) genes haplotypes. Unfortunately, in
2006 deCode genetics suspended trial due to unexpected tablet formulation problem since too slow
dissolution of tablets could adversely influence drug
blood concentration and therapeutic effect.
In randomized, placebo, cross-over trial in 191
patients carrying at-risk genes of FLAP/LTA4H
treated with DG-031 for 4 weeks (at the doses 250,
500 or 750 mg/day) the 750 mg dose resulted in
decrease of biomarkers associated with myocardial
infarction (26% LTB4 reduction and 12% myeloperoxidase reduction). Furthermore, two doses 500 and
750 mg also resulted in long-term (in 4th week of
wash-out) decrease of CRP (50).
CONCLUSIONS
Inflammation is known to be involved in
pathogenesis and maintenance of numerous diseases, such as pain, osteoarthritis, bronchial asthma,
psoriasis, ulcerative colitis, atherosclerosis, cancer,
neurodegenerative diseases and ischemic reperfusion injury. A recent approach to many disease
treatment includes multi-target pharmacotherapy
and blocking of leukotriene pathway, which seems
to be a promising one. In spite of the fact that
leukotriene biochemistry leaves much to discover,
two medications - zileuton and a group of lukasts are already in use in asthma treatment, and others
are about to finish clinical trials. Not only 5-LOX
inhibitors, but also many of FLAP inhibitors, for
instance veliflapon, are being in different stages of
development, suggesting that FLAP may be a beneficial target for new pharmaceuticals. Future will
show how many of todayís ideas for leukotriene
antagonists find a practical use.
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Received: 19. 06. 2015
ISSN 0001-6837
CHLOROGENIC ACID: A PHARMACOLOGICALLY POTENT MOLECULE
ANEELA MAALIK1, SYED MAJID BUKHARI1*, ASMA ZAIDI1, KAUSAR HUSSAIN SHAH2
and FARHAN A. KHAN1**
1
Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad-22060, Pakistan
Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan-60800-Multan, Pakistan
2
Abstract: Chlorogenic acid (CGA; (1S,3R,4R,5R)-3-{[(2Z)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,4,5trihydroxycyclohexanecarboxylic acid) is a naturally occurring polyphenol mostly present in vegetables and
fruits. CGA is a prominent component of Traditional Chinese Medicines and is known for various pharmacological activities such as antioxidant, antimicrobial, anti-inflammatory and hepatoprotective etc. This minireview is an attempt to summarize the available literature in the last decade and to point out future perspectives
in this area of research.
Keywords: chlorogenic acid, oxidative stress, antioxidant, inflammation
Pharmacological importance of CGA
CGA is known to exhibit a range of pharmacological activities such as anticancer, antioxidant,
anti-inflammatory, cardiovascular, hepatoprotective, renoprotective, anti-diabetic and anti-lipidemic. Pharmacological properties exhibited by CGA
are summarized in Table 1.
Chlorogenic acid (CGA) (Fig. 1) is a biologically active polyphenol which is soluble in ethanol
and acetone and is marketed as svetol. It is generally used as an ingredient in chewing gum and mints
and is naturally present in different fruits such as
peach and prunes (1, 2) and vegetables such as potato (3). CGA has also been found as a phenolic component of bamboo (4), green coffee bean extract (5)
and tobacco (6). A family of esters of hydroxycinnamic acids (including ferulic acid, caffeic acid and
p-coumaric acid) with quinic acid is also some times
refered as chlorogenic acids (7, 8).
Encapsulated CGA possesses remarkable antioxidant activity
Although CGA is a good antioxidant agent (8,
20); its unstable nature, on exposure to light and
heat, limits its industrial applications, especially in
the food industry. However, recently, this problem
has been eradicated by the use of encapsulation
technology. A study conducted on the determination
Synthesis of CGA
The esterification of caffeic acid with L-quinic
acid yields CGA (Fig. 1) (9).
Figure 1. Synthesis of chlorogenic acid
Corresponding authors:
* e-mail: majidbukhari@ciit.net.pk, tel. +92 332 466 3334, fax +92 992 383 441
** e-mail: farhankhan@ciit.net.pk, tel: +92 334 728 6986, fax: +92 992 383 441
851
852
ANEELA MAALIK et al.
of antioxidant activity of CGA encapsulated with βcyclodextrin (β-CD) (inclusion ratio = 79.86 ±
1.92%) and hydroxypropyl-β-cyclodextrin (HB-βCD) reported that the encapsulated CGA possesses
an efficient antioxidant activity in grape juice. The
complex of CGA/HB-β-CD is described to possess
better antioxidant activity compared to CGA/β-CD
complex and CGA alone; this enhanced activity can
be attributed to the interactions and stabilization of
radical species with the guest molecule (scavenger).
The technological modifications in the formation of
CGA/β-CD inclusion complex results in the reduced
freedom of rotation for quinic acid moiety, which is
due to the penetration into the cavity of β-CD; this
was evident by the reported downfield shift of protons of quinic acid moiety in proton NMR of the
CGA/β-CD complex formed. On the other hand, a
little to no change in the NMR data was reported for
caffeic acid moiety, present in chlorogenic acid,
before and after complexation with β-CD. From the
reported NMR it can be stated that quinic acid moiety was included in the β-CD cavity. No new peaks
were reported in the proton NMR of formed complex, which signifies that chlorogenic acid was in a
state of rapid exchange among free state and complex state. Also, storage stability was reported to be
increased in an inclusion complex of β-CD and
CGA (10).
Another major hurdle in the use of CGA for
certain applications is its hydrophobic nature. This
problem has been solved in a research where the
structure of CGA was modified and converted to
chlorogenic laurate (CGL) in order to improve its
liposolubility profile. Interestingly, this structural
modification was reported to enhance antioxidant
activity (EC50 for CGA = 112.3 µg/mL and for CGL
= 70.5 µg/mL) (11). In another study, a dose of CGA
(100 mg/kg body weight) was given to mice for 8
days; it is reported that CGA reversed lipid peroxidation, inactivation of cytochrome P450 and
increased cellular defense (21).
CGA suppresses DSS-induced colitis
It has been reported that a compound possessing good antioxidant activity normally shows good
anti-inflammatory activity as well (22). Recently, in
an in vivo study conducted on C57BL/6 mice, an
anti-inflammatory activity of CGA was reported on
dextran sulfate sodium-induced (DSS-induced) colitis. CGA was stated more potent to suppress DSSinduced colitis than its hydrolyzed derivative (caffeic acid) which has well established anti-inflammatory activity; in vitro, CGA was reported to inhibit
H2O2- and tumor necrosis factor (TNF-α)- induced
interlukin-8 (IL-8) production (12). A water extract
of Trachelospermum jasminoides was studied in λcarrageenan-induced paw edema in ICR mice and an
anti-inflammatory response has been reported;
besides other phenolic compounds, CGA has also
been found in the extract analyzed by HPLC analytical plot and is a possible inhibitor of inflammatory
mediators, TNF-α and NO production (13). Apart
from these, the inhibition of cyclooxygenase-2
(COX-2), inducible nitric oxide synthase (iNOS)
with lack of cytotoxic effect, attenuation of IL-1β
and IL-6 along with TNF-α in a dose dependent
manner and inhibition of nuclear factor-κB (NF-κB)
by CGA is reported in a recent studies (23, 24).
CGA is an active agent against cardiovascular
problems
CGA is a biologically effective component of
Chinese folk medicine used against cardiovascular
disorders as it reduces heart triglyceride levels (25).
Cardiac hypertrophy is considered as one of the
major reasons for heart failure. In a recent study,
CGA has been found effective against isoproterenol
(Iso)-induced hypertrophy in cardiomyocytes; the
number of reactive oxygen species (ROS) increases
in the Iso-induced cardiac hypertrophy and CGA has
the ability to reduce this pathological condition by
scavenging ROS (14). Increased cholesterol level
(hypercholesterolemia) is considered as one of the
Table 1. Pharmacological properties of chlorogenic acid.
No.
Pharmacological properties
References
1.
Antioxidant activity
(1, 10, 11)
2.
Anti-inflammatory activity
(12, 13)
3.
Cardiovascular activity
(14)
4.
Hepatoprotective activity
(15, 16)
5.
Renoprotective activity
(17, 18)
6.
Anti-diabetic and anti-lipidemic activity
(19)
Chlorogenic acid: a pharmacologically potent molecule
853
major reasons for heart diseases as well; CGA is
well known to reduce heart cholesterol (25); in this
regard a pre-clinical study was conducted on rats for
28 days; this study reports that CGA has the ability
to control elevated levels of cholesterol significantly if taken up as a dietary ingredient (26).
In contrast to above reports, an increase in
plasma concentration of homocysteine is reported in
the blood with increased intake of coffee and black
tea containing CGA; high concentration of homocysteine in plasma may cause cardiovascular disease
(27).
studies conducted on Leprdb/db mice, CGA has been
reported to inhibit gluconeogenesis by affecting
expression and activity of enzyme glucose-6-phosphatase (G6Pase), moreover, it improves skeletal
muscle glucose uptake by increasing expression and
translocation of glucose transporter type 4 (GLUT
4); a 2.5-fold increase in glucose transport was
described as an additive action of CGA with insulin;
on treatment with CGA a vacuolar degeneration has
been reported in Hep G2 cells (human liver carcinoma cell line) resulting in reduction of lipid accumulation (19).
Hepatoprotective nature of CGA
CGA possesses hepatoprotective nature (26).
In a recent study conducted on tetrachlorobenzoquinone (TCBQ, a metabolite of an environmental
pollutant pentachlorophenol)-induced liver damage
in mice; the CGA pretreatment was reported to be
effective in suppressing TCBQ-induced oxidative
stress and therefore, possessing hepatoprotective
nature (15). Moreover, in vitro a protecting property
of CGA was reported at a dose of 300 or 500 mg/kg
against CCl4-induced acute liver injury in male
Sprague Dawley rats (16).
CGA reduces body weight
According to another study, performed at a
clinical trial level on 12 subjects, the CGA plays a
significant role in body weight reduction. The coffee
products were tested on volunteers and it was
reported that coffee containing enriched CGA
caused 6.9% reduction in glucose absorption compared to control (29). The reduction in glucose
absorption is attributed with alteration in glucose
uptake pattern in small intestine, which is caused by
antagonistic effect of CGA on glucose transport
(30). CGA has been reported as a specific inhibitor
of G6Pase translocase (an enzyme which regulates
homeostasis of glucose in blood) in microsomes of
rats (31). Another aspect studied was the effect of
enriched coffee with CGA on 30 overweight individuals. The reported reduction in body mass was
3.17 times higher in individuals who used CGA
enriched coffee compared to control (29).
CGA can alter body fat in high-fat diet; this has
been reported in a study conducted on mice. The
mice were given 0.02% (w/w) dose of CGA, which
resulted in a pronounced body fat reduction,
decrease in weight and decrease in plasma leptin as
well as insulin levels compared to control. Plasma
adiponectin elevation, fatty acid β-oxidation activity increment and peroxisome proliferator activated
receptors an expression enhancement in liver were
caused by CGA. Further, the reduction in synthesis
of fatty acids, 3-hydroxy-3-methylglutaryl CoA
reductase, acetyl CoA and cholesterol acyltransferase was reported (25).
Renoprotective activity of CGA
Cisplatin (CP), an antineoplastic drug, is
famous for various solid tumor treatments and is
known to cause nephrotoxicity through various
mechanisms, including inflammation, necrosis and
apoptosis (17). Recently, in vivo study was conducted on intraperitoneally administrated with CP male
BALB/cN mice; CGA pretreatment was reported to
attenuate 4-hydroxynonenal expression, which is an
indicator of renal oxidative stress, moreover, CGA
was also described to attenuate heme oxygenase 1
and cytochrome P450 E1 overexpression as a result
of CP administration; also CGA has been reported to
inhibit advanced glycation end products (AGEs)
with an IC50 value of 148.32 µM; these AGEs play a
vital role in the development of chronic diabetic
complications (28), these results established renoprotective activity of CGA (18). CGA is known to
reduce triglyceride levels in the liver as well (25).
CGA holds anti-diabetic and anti-lipidemic activity
Diabetes is a common disease of the modern
age and its major possible causes are obesity and
lifestyle. CGA is abundantly found in our daily food
like coffee and its consumption is advantageous
because it can help in lowering the blood sugar level
as well as to improve the lipid profile. Recently, in
CONCLUSION
The literature reveals the biological applications of CGA, however, some adverse effects have
also been reported in the case of high dose administration of CGA, like its role as an anti-inflammatory
agent is pre-established but simultaneously, it can
cause inflammation and behaves as a double-edge
854
ANEELA MAALIK et al.
sword; CGA is present in ample amount in Chinese
herbal injections and medicines, therefore, a careful
dose selection is required while prescribing it (32).
Moreover, an increased level of plasma homocystine is reported in the users of coffee and black
tea containing CGA as a major polyphenol, this
increased level is thought to be a risk factor in various cardiovascular diseases (33). Therefore, a careful study to investigate the amount of CGA which is
useful for the humans is required.
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Received: 15. 12. 2015
ISSN 0001-6837
ANALYSIS
DECOCTION PROCESS OPTIMIZATION AND QUALITY EVALUATION OF
YI-HUANG DECOCTION BY HPLC FINGERPRINT ANALYSIS
FANGZHOU YIN1, LIN LI1, XIN LI2, TULING LU1, WEIDONG LI1, BAOCHANG CAI1*
and WU YIN1,2*
1
College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China, 210023
The State Key Lab of Pharmaceutical Biotechnology, College of Life Sciences, Nanjing University,
Nanjing, China, 210023
2
Abstract: Yi Huang decoction (YHD) has been used as one of famous traditional formula because of its unique
effectiveness against gynecological diseases. YHD is composed of five herbs, including the rootstock of
Dioscorea opposita Thunb. (Dioscoreaceae), the kernel of Euryale ferox Salisb. (Nymphaeaceae), the bark of
Phellodendron chinense Schneid. (Rutaceae), the seed of Plantago asiatica L. (Plantaginaceae), and the seed of
Ginkgo biloba L. (Ginkgoaceae). To effectively control the quality, the processing method for YHD was optimized by means of single factor test as well as orthogonal test in this study. A completely validated method
based on HPLC coupled with diode array detector was performed on a Kromasil C18 column at 30O with mobile
phase of 0.1% aqueous phosphoric acid and acetonitrile. As a result, HPLC fingerprint on the basis of the chromatographic data from 32 batches of samples was obtained, which contained 44 common peaks. Among these
common peaks, 6 peaks were identified as geniposidic acid, berberine hydrochloride, palmatine hydrochloride,
phellodendrine chloride, magnoflorine, and verbascoside, respectively, based on their retention time relative to
the standards. Meanwhile, the contents of these 6 compounds were also simultaneously examined. In sum, this
study offered valuable information for the proper processing and quality control for YHD.
Keywords: Yi-Huang decoction, Dioscorea opposita Thunb., Euryale ferox Salisb., Phellodendron chinense
Schneid., Plantago asiatica L., Ginkgo biloba L., HPLC, content determination, quality control
used in the field of drug analysis, is often used
because of its simplicity and stability (3-5).
Yi Huang decoction (YHD) is a famous traditional formula because it demonstrates unique effectiveness against gynecological diseases (6, 7). By
taking this decoction, the function of kidney and
spleen could be strengthened, leucorrhea could be
reduced, lumbar aching and limp could be lessened
(8). It was originally recorded in ìFu-Qing-Zhu-NuKe,î written by the famous doctor, FuShan, in the
17th century. YHD is composed of five herbs,
including the rootstock of Dioscorea opposita
Thunb. (Dioscoreaceae), the kernel of Euryale ferox
Salisb. (Nymphaeaceae), the bark of Phellodendron
chinense Schneid. (Rutaceae), the seed of Plantago
asiatica L. (Plantaginaceae) and the seed of Ginkgo
biloba L. (Ginkgoaceae). The relevant study has
shown that berberine hydrochloride (9, 10), palmatine hydrochloride, phellodendrine chloride (11),
Because of their relatively minor side effects
and high efficacy, herbal medicines and relevant
prescriptions have gained increasing attention.
However, lots of components are present in a single
herbal medicine or herbal medicine prescriptions,
which makes the quality control rather complex and
difficult (1). Herbal medicine preparations involve
combinations of herbs in which the quality may be
variable with components from different producing
environments, different preprocessing methods, or
chemical changes of the active components.
Nevertheless, the relevant studies on the processing
and quality control of herbal preparations are insufficient (2). At present, the method of determining
one or several marker components is popular, but it
obviously can not represent the whole quality of a
specified herbal medicine preparation. Among the
analytical technologies, high-performance liquid
chromatography (HPLC), which has been widely
* Corresponding authors: Baochang Cai: e-mail: bccai2012@163.com; phone: +86 025 85811513; Yin Wu: e-mail: wyin2003@163.com;
phone: 0086-25-66099006
855
856
FANGZHOU YIN et al.
and magnoflorine all contribute to the therapeutic
effects of YHD. However, these components are all
from Phellodendron chinense Schneid., which is one
of the therapeutic herbs in YHD rather than whole of
YHD, and its content does not completely represent
the inherent quality of YHD. Meanwhile, the appropriate components for the quality control of
Dioscorea opposita Thunb. and Euryale ferox Salisb.
have not been identified yet.
To date, few literature sources were documented to investigate the processing method for YHD
and its quality control. In this study, we optimized
the processing method of YHD for the first time
using single factor test and orthogonal test. HPLC
fingerprint was generated to evaluate the quality of
YHD. Additionally, six main compounds were
simultaneously determined in this decoction by
using the optimized method.
National Institute for the Control of Pharmaceutical
and Biological Products (Beijing, China).
Phellodendrine chloride, magnoflorine, and verbascoside were isolated from the crude drugs in our laboratory and were characterized by the methods of
UV, IR, NMR and MS, and the purity of these compounds was > 98.0%, as measured by HPLC-PDA.
The chemical structures of these 6 standards are
shown in Figure 1.
HPLC-grade acetonitrile was purchased from
Merck Company (Darmstadt, Germany). High-purity deionized water (18.2 MΩ cm) was obtained from
a Milli-Q3 water purification system (Millipore
Bedford, MA, USA). Methanol used for sample
extraction and phosphoric acid used for preparation
of the mobile phase were all of analytical grade and
from Shanghai Chemical Corp. (Shanghai, China).
Sodium dodecyl sulfate (SDS) of analytical grade
were from Sigma Co. (St. Louis, MO, USA).
EXPERIMENTAL
Chemical and reagents
The chemical standards (purity > 99%) of
geniposidic acid, berberine hydrochloride and
palmatine hydrochloride were purchased from
Plant material
All the crude herbs were purchased from
HaiChang Pharmaceutical Group (Nanjing, China).
The two batches of the rootstock of Dioscorea
opposita Thunb. were collected from Jiaozuo of
Figure 1. Chemical structure of geniposidic acid, verbascoside, phellodendrine chloride, magnoflorine, berberine hydrochloride, and
palmatine hydrochloride
Decoction process optimization and quality evaluation of Yi-Huang...
Hennan (S1, S2). The two batches of the kernel of
Euryale ferox Salisb. were collected from Dongpin
of Shandong (Q1) and Suzhou of Jiangsu (Q2),
respectively. The two batches of the bark of
Phellodendron chinense Schneid. were collected
from Yaan (H1) and Guangyuan (H2) of Sichuan,
respectively. The two batches of the seeds of
Plantago asiatica L. were collected from Jian of
Jiangxi (C1) and Dexiang of Sichuang (C2), respectively. The two batches of the seed of Ginkgo biloba
L. were collected from Xianan of Guangxi (B1) and
Taizhou of Jiangsu (B2), respectively. All the crude
plants were authenticated by Professor Tulin Lu of
Nanjing University of Chinese Medicine. The
voucher specimens were deposited at College of
Pharmacy of the Nanjing University of Chinese
Medicine.
Preparation of YHD
YHD was prepared by the method recorded in
ìFuqingzhunvkeî and the optimization process was
provided in the Section ìOptimization of the processing method for YHDî. In detail, 30 g of the
rootstock of Dioscorea opposita Thunb., 30 g of the
kernel of Euryale ferox Salisb., 6 g of the bark of
Phellodendron chinense Schneid., 3 g of the seeds of
Plantago asiatica L. and 12 g of the seeds of Ginkgo
biloba L. were mixed together and immersed in
water for a designated period of time, then the mixture was heated and kept boiling for another designated period of time, and this process was repeated
for several times. At last, all the water extractions
were mixed and centrifuged at 10000 rpm for 10
min, and the supernatants were collected for analysis.
Preparation of standard and analytical sample
The supernatant of YHD was concentrated to
dryness, then was ultrasonic extracted for 30 min in
50% methanol solution. The extracts were filtered,
evaporated, and the residues were dissolved in 20
mL of 50% methanol solution. The solutions were
filtered through a 0.45 µm membrane before analysis.
The 6 standards were accurately weighed and
dissolved in methanol to prepare the mixed stock
solution consisting of geniposidic acid (0.714
mg/mL), verbascoside (0.404 mg/mL), phellodendrine chloride (0.344 mg/mL), magnoflorine (0.442
mg/mL) berberine hydrochloride (0.708 mg/mL)
and palmatine hydrochloride (0.450 mg/mL). A set
of standard solutions with six different concentration levels were prepared by further dilution of the
stock solution with methanol for assessment of lin-
857
earity. All standard solutions were kept at 4OC and
filtered through a 0.45 µm membrane before injection.
HPLC apparatus and conditions
The HPLC-DAD analysis was performed on
Agilent series 1100 equipment comprising a
G1311A quanternary pump, a D1313A automatic
sampler, a G1315B diode-array detector (DAD) and
a G1316A column compartment. Samples were separated on a Kromasil C18 column (4.6 ◊ 250 mm, 5
µm) and an Agilent Zorbax C18 guard column (4.6 ◊
12.5 mm, 5 µm). The mobile phase was a gradient
prepared from solvent A (0.1% aqueous phosphoric
acid, in which 0.1 g of SDS was added per 100 mL
of 0.1% aqueous phosphoric acid) and solvent B
(acetonitrile), and the conditions used for gradient
elution were: 0-10 min, 2% B; 10-20 min, 2-10% B;
20-35 min, 10-20% B; 35-60 min, 20-45% B; 60-75
min, 45% B; 75-80 min, 45-60% B; 80-85 min, 60100% B; 85-95 min, 100% B. The separation was
conducted at 30O with a flow rate of 1.0 mL/min.
The injection volume was 5 µL. The detection
wavelength was 275 nm for fingerprint, 265 nm for
determination of berberine hydrochloride, 285 nm
for determination of phellodendrine chloride, 275
nm for determination of palmatine hydrochloride
and magnoflorine, 240 nm for determination of
geniposidic acid and 330 nm for determination of
verbascoside, respectively.
Development of HPLC fingerprint chromatogram and the methods for simultaneous
determination of the six constituents
Thirty two batches of YHD were prepared and
analyzed under the chromatographic condition to
establish HPLC fingerprint. Similarity analysis of
YHD was performed by ìSimilarity Evaluation
System for Chromatographic Fingerprint of
Traditional Chinese Medicineî (Version 2004A), as
recommended by Chinese Pharmacopoeia
Commission. The contents of 6 analytes , including
geniposidic acid, berberine hydrochloride, palmatine hydrochloride, phellodendrine chloride, magnoflorine, and verbascoside in 32 batches of YHD
were simultaneously determined.
RESULTS AND DISCUSSION
Optimization of the processing method for YHD
The decoction process by single factor test was
first investigated. The variables involved in the
decoction procedure including solvent volume,
immersion time, boiling duration, and number of
858
FANGZHOU YIN et al.
boiling times were optimized. The volume of water
that was used at a designated multiplication of the
weight of the drug (12, 14, 16, 18, and 20 times) was
also inspected. The immersion duration (0, 15, 30,
45, and 60 min), boiling duration (20, 30, 40, 50,
and 60 min), and extraction cycles (1, 2, and 3) were
optimized. Finally, the optimal conditions for the
processing of YHD were confirmed. In Figure 2, as
the solvent volume, immersion time, boiling time,
and boiling frequency increased, the contents of 5
analytes (geniposidic acid, verbascoside, phellodendrine chloride, magnoflorine, and palmatine
hydrochloride) in the YHD were also accordingly
elevated. However, when the volume of solvent
reached 18 times the weight of the drug, the immersion time reached at 45 min, and the boiling duration
reached at 50 min, the contents of the main components failed to further increase. Similar results also
occurred for berberine hydrochloride. Notably, most
of the constituents can be effectively extracted after
two cycles of extraction.
In order to examine the relationship between
the above mentioned experimental parameters, the
volume of water used (14, 16, 18 times the weight of
the drug), the immersion duration (15, 30, 45 min),
and boiling duration (30, 40, 50 min) were further
investigated by orthogonal test. All the samples
were extracted twice. An orthogonal design table of
L9 (34) was generated and the contents of 6 analytes
were used as the indicators, as shown in Table 1.
Based on the results given by the contents of 5 analytes, the best method for the preparation of YHD
included the following steps: (1) immersion of all
herbs in water at a volume 10 times the total weight
of herbal medicine for 30 min, followed by boiling
for 40 min; (2) boiling of the herb residues in water
Figure 2. Effects of immersion time, solvent volume, boiling duration and boiling frequency on the contents of geniposidic acid, verbascoside, phellodendrine chloride, magnoflorine, berberine hydrochloride, and palmatine hydrochloride in YHD.
A: The influence of immersion time on the contents of 5 analytes. All herbs were immersed in water at 12 times the volume of medicine
for 0, 15, 30, 45 and 60 min, respectively, followed by boiling procedure for an additional 40 min. B: The influence of solvent volume on
the contents of 5 analytes. All herbs were immersed in water at 7, 8, 10, 11, 12 times the volume of herbal medicine for 30 min, respectively, followed by boiling procedure for an additional 40 min; the herb residues were further extracted in boiling water at 5, 6, 6, 7, 8 times
the volume of herbal medicine for 30 min, respectively. The water extracts from these two steps were combined. C: The influence of boiling duration on the contents of 5 analytes. All herbs were immersed in water at 10 times the volume of herbal medicine for 30 min, followed by boiling procedure for 20, 30, 40, 50 and 60 min, respectively; The herb residues were further extracted in water at 6 times the
volume of herbal medicine for 20, 30, 40, 50 and 60 min, respectively, the water extract from these two steps were combined. D: The influence of boiling frequency on the contents of 5 analytes. All herbs were immersed in water at 8 times the volume of herbal medicine for 30
min, followed by boiling procedure for another 40 min. The boiling procedure was performed at 1, 2 and 3 times, the filtrate of each extraction was stored separately
859
Table 1. The project of orthogonal test.
Factors
Sample-solvent
ratio
Immersion
duration (min)
Boiling
duration (min)
1
1 : 14
15
30
2
1 : 14
30
40
3
1 : 14
45
50
4
1 : 16
15
40
5
1 : 16
30
50
6
1 : 16
45
30
7
1 : 18
15
50
8
1 : 18
30
30
9
1 : 18
45
40
Table 2. Linearity calibration curves of 13 components.
RT
(min)
Regression
equation
Geniposidic acid
23.9
y = 2482x + 5.593
0.9994
14.3-178.5
1.4
4.7
Verbascoside
39.1
y = 1319x - 1.989
0.9994
8.08-101.0
0.8
2.7
LOD
(µg/mL)
LOQ
(µg/mL)
Compound
1
2
3
Phellodendrine chloride
66.7
y = 3207x - 11.21
0.9995
34.4-430.0
2.6
8.7
4
Magnoflorine
67.2
y = 6666.4x - 24.49
0.9995
44.2-552.5
1.5
4.9
5
Palmatine hydrochloride
83.2
y = 16235x - 13.11
0.9996
9.0-45.0
0.4
1.2
6
Berberine hydrochloride
83.8
y = 18922x + 0.370
0.9997
70.8-885.0
0.4
1.2
at a volume 6 times the total weight of herbal medicine for 30 min, followed by mixing of the solution.
The three factors of water volume, immersion time,
and boiling time had no significant influence on the
final decoction result. Therefore, the ideal processing conditions for YHD were established.
Optimization of sample extraction and HPLC
condition
To achieve maximum recovery of the components of YHD, the extraction conditions including
extracted solvent, extracted method, solvent volume, and extraction time were optimized. Methanol
: water (50%, v/v) was shown to be the best solvent
because it could be used to extract most of the constituents in YHD. Extraction methods include ultrasonication and heat reflux. Although similar recovery was achieved by use of these two methods, ultrasonication was selected because of high extraction
efficiency. Finally, the extraction duration (20, 30,
40 min) and extraction times (1, 2) were also optimized.
For good peak resolution, chromatographic conditions were optimized, and acetonitrile ñ 0.1% aque-
R
Linear ranges
(µg/mL)
No.
ous phosphoric acid was confirmed to be the best
mobile phase system. Because many alkaloids are
present in the decoction, SDS was added to the
mobile phase to improve the peak shape. The gradient
elution was applied to improve peak resolution.
Based on the 3D chromatograms, 275 nm was chosen
as detection wavelength for fingerprint chromatogram, 265 nm for berberine hydrochloride, 285
nm for phellodendrine chloride, 275 nm for palmatine
hydrochloride and magnoflorine, 240 nm for geniposidic acid, and 330 nm for verbascoside, respectively.
Method validation of quantitative analysis
Six concentrations of the standard solutions
were analyzed in triplicate and the calibration curves
were constructed from peak areas of standards versus their concentrations. The standard solutions
were diluted to a series of appropriate concentrations with methanol, the limits of determinations
(LODs) (S/N ≈ 3) and limits of quantifications
(LOQs) (S/N ≈ 10) under the present conditions
were determined, respectively.
The calibration data, linear ranges, R, LOD and
LOQ are listed in Table 2. The data revealed that a
860
FANGZHOU YIN et al.
A
B
C
D
E
F
G
Figure 3. The HPLC chromatograms for YHD extraction, herbs extraction and the standards. A: YHD extraction, B: the standard, C: the
rootstock of Dioscorea opposita Thunb. (Dioscoreaceae), D: the kernel of Euryale ferox Salisb. (Nymphaeaceae), E: the bark of
Phellodendron chinense Schneid. (Rutaceae), F: the seed of Plantago asiatica L., G: the seed of Ginkgo biloba L. (Ginkgoaceae). Peaks:
10, 23, 36, 37, 43 and 44 were identified as geniposidic acid, verbascoside, phellodendrine chloride, magnoflorine, palmatine hydrochloride, berberine hydrochloride, respectively
861
Table 3. Precision, repeatability, stability and accuracy data of the content determination.
Precision
Reproducibility
Stability
Recovery
No.
Compound
Intra-day
RSD (%)
Inter-day
RSD (%)
RSD (%)
RSD (%)
Mean
RSD (%)
1
Geniposidic acid
1.45
1.52
2.02
1.68
101.33
1.19
2
Verbascoside
1.80
1.99
2.40
2.48
100.76
1.59
3
Phellodendrine chloride
0.39
1.01
1.14
0.55
102.03
0.86
4
Magnoflorine
0.80
0.95
1.76
1.03
100.49
1.64
5
Palmatine hydrochloride
1.78
1.88
2.81
1.53
101.37
0.97
6
Berberine hydrochloride
0.06
0.12
2.26
0.11
101.13
1.12
Recovery (%) = (amount found ñ original amount)/ amount spiked ◊100%; RSD (%) = (S.D./mean) ◊ 100%.
good linear relationship existed between the concentrations of the six compounds measured and their
peak areas within the test range (R > 0.9994).
To assess the intraday and interday precisions
of the method, the standard stock was analyzed during a single day or three consecutive days. The same
sample solution was also analyzed at different time
(0, 5, 10, 15, 24, 36, and 48 h) to test its stability.
The reproducibility of the method was evaluated by
analysis of six replicates of the same sample.
Accuracy was determined in recovery experiments
in which the standard compounds of 80, 100 and
120% of the sample content were added to 5 mL of
YHD. As shown in Table 3, the relative standard
deviations (RSD) of intraday precision, interday
precision, reproducibility, stability for all the components under the established method were < 3%.
All results of recovery were within the usually
required recovery range of 101.13-102.03%. The
above result suggested that the method was effective
for simultaneous determination of the 6 compounds
in YHD.
Method validation of fingerprinting
Data analysis of the chromatographic fingerprint was performed by use of SES software, and
similarities between chromatograms were used to
evaluate fingerprint quality. The same solution was
analyzed during a single day or three consecutive
days, respectively, to assess the intraday and interday precisions of the method. The same sample
solution was also analyzed at different times (0, 5,
10, 15, 24, 36 and 48 h) after preparation to test its
stability. The reproducibility of the method was
evaluated by analysis of six replicates of the same
sample. In the precision and stability test, the average similarity of the different chromatograms was
0.999. The results from the reproducibility test
revealed that the average similarity of the chromatograms obtained from six replicate analyses was
0.999. All the RSDs were < 3%. Therefore, the analytical method of fingerprint used in this study is
precise, reproducible, and samples are stable during
the test period.
The result of a fingerprint chromatogram and
simultaneous determination of six analytes
Chromatograms from 32 batches of YHD of
different regions were obtained. The result demonstrated that more than 84 peaks were separated at
baseline. The common pattern of the HPLC fingerprint of YHD was constructed using the SES software. Based on the principle of fingerprinting, when
the relative standard deviation (RSD) of the peaksí
relative retention times for all batches of samples is
less than 1%, these peaks belong to the same substance and can be assigned as a ìcommon peak.î In
this study, 44 peaks, of which the relative peak areas
were more than 1%, separated in all batches of samples and were regarded as common peaks, accounting for over 93.56% of the total peak areas for individual samples, as detected at 275 nm. In the test,
the HPLC chromatograms of the five herbal drugs
alone and the preparations with only four herbal
drugs involved were also obtained. Comparing the
chromatograms of these herbal drugs and preparations (Fig. 3), it was shown that 10 peaks were from
the rootstock of Dioscorea opposita Thunb. (peaks
1, 2, 4, 6, 7, 8, 26, 29, 30, and 32), 5 of the peaks
were from the kernel of Euryale ferox Salisb. (peaks
5, 7, 28, 29, and 32), 18 peaks were from the bark of
Phellodendron chinense Schneid. (peaks 4, 7, 11,
13, 14, 16, 20, 21, 22, 25, 32, 36, 37, 40, 41, 42, 43,
and 44), 4 peaks were from the seed of Plantago asiatica L. (peaks 10, 23, 29, and 32), and 10 peaks
were from the seed of Ginkgo biloba L. (peaks 1, 12,
0.0211 ± 0.0003
0.0221 ± 0.0002
0.0224 ± 0.0004
0.0223 ± 0.0003
0.0198 ± 0.0002
0.0175 ± 0.0003
0.0203 ± 0.0003
0.0197 ± 0.0003
0.0237 ± 0.0003
0.0271 ± 0.0004
0.0187 ± 0.0003
0.0177 ± 0.0003
0.0178 ± 0.0003
0.0184 ± 0.0003
0.0193 ± 0.0003
0.0221 ± 0.0004
0.0270 ± 0.0003
0.0299 ± 0.0003
0.0209 ± 0.0004
0.0311 ± 0.0005
0.0245 ± 0.0005
0.0345 ± 0.0004
0.0214 ± 0.0004
0.0245 ± 0.0003
0.0189 ± 0.0003
0.0223 ± 0.0005
YHD 2 (S1 + Q1 + H1 + C1 + B2)
YHD 3 (S1 + Q1 + H1 + C2 + B1)
YHD 4 (S1 + Q1 + H1 + C2 + B2)
YHD 5 (S1 + Q1 + H2 + C1 + B1)
YHD 6 (S1 + Q1 + H2 + C1 + B2)
YHD 7 (S1 + Q1 + H2 + C2 + B1)
YHD 8 (S1 + Q1 + H2 + C2 + B2)
YHD 9 (S1 + Q2 + H1 + C1 + B1)
YHD 10 (S1 + Q2 + H1 + C1 + B2)
YHD 11 (S1 + Q2 + H1 + C2 + B1)
YHD 12 (S1 + Q2 + H1 + C2 + B2)
YHD 13 (S1 + Q2 + H2 + C1 + B1)
YHD 14 (S1 + Q2 + H2 + C1 + B2)
YHD 15 (S1 + Q2 + H2 + C2 + B1)
YHD 16 (S1 + Q2 + H2 + C2 + B2)
YHD 17 (S2 + Q1 + H1 + C1 + B1)
YHD 18 (S2 + Q1 + H1 + C1 + B2)
YHD 19 (S2 + Q1 + H1 + C2 + B1)
YHD 20 (S2 + Q1 + H1 + C2 + B2)
YHD 21 (S2 + Q1 + H2 + C1 + B1)
YHD 22 (S2 + Q1 + H2 + C1 + B2)
YHD 23 (S2 + Q1 + H2 + C2 + B1)
YHD 24 (S2 + Q1 + H2 + C2 + B2)
YHD 25 (S2 + Q2 + H1 + C1 + B1)
YHD 26 (S2 + Q2 + H1 + C1 + B2)
Compound 1
YHD 1 (S1 + Q1 + H1 + C1 + B1)1
Sample
Compound 3
0.0535 ± 0.008
0.0329 ± 0.0008 0.0650 ± 0.0011
0.0358 ± 0.0007 0.0631 ± 0.0012
0.0423 ± 0.0008 0.0623 ± 0.0011
0.0363 ± 0.0007 0.0557 ± 0.0010
0.0333 ± 0.0006 0.0556 ± 0.0011
0.0293 ± 0.0006 0.0527 ± 0.0008
0.0347 ± 0.0005 0.0623 ± 0.0011
0.0316 ± 0.0006 0.0514 ± 0.0008
0.0420 ± 0.0008 0.0652 ± 0.0005
0.0422 ± 0.0008 0.0652 ± 0.0007
0.0335 ± 0.0006 0.0632 ± 0.0007
0.0383 ± 0.0006 0.0613 ± 0.0006
0.0325 ± 0.0007 0.0538 ± 0.0009
0.0334 ± 0.0006
0.0304 ± 0.0006 0.0556 ± 0.0013
0.0290 ± 0.0004 0.0576 ± 0.0013
0.0306 ± 0.0004 0.0523 ± 0.0011
0.0436 ± 0.0006 0.0538 ± 0.0008
0.0431 ± 0.0009 0.0652 ± 0.0009
0.0422 ± 0.0008 0.0634 ± 0.0010
0.0357 ± 0.0007 0.0572 ± 0.0012
0.0334 ± 0.0007 0.0556 ± 0.0010
0.0378 ± 0.0006 0.0623 ± 0.0012
0.0402 ± 0.0008 0.0533 ± 0.0008
0.0316 ± 0.0007 0.0583 ± 0.0007
0.0321 ± 0.0005 0.0534 ± 0.0006
Compound 2
Compound 5
0.0432 ± 0.0004 0.0059 ± 0.0001
0.0564 ± 0.0002 0.0065 ± 0.0002
0.0523 ± 0.0004 0.0066 ± 0.0002
0.0435 ± 0.0003 0.0076 ± 0.0002
0.0482 ± 0.0004 0.0068 ± 0.0001
0.0461 ± 0.0008 0.0088 ± 0.0002
0.0444 ± 0.0009 0.0073 ± 0.0002
0.0401 ± 0.0008 0.0063 ± 0.0002
0.0532 ± 0.0009 0.0065 ± 0.0001
0.0459 ± 0.0008 0.0065 ± 0.0001
0.0486 ± 0.0011 0.0054 ± 0.0002
0.0542 ± 0.0009 0.0069 ± 0.0002
0.0553 ± 0.0011 0.0068 ± 0.0002
0.0512 ± 0.0012 0.0046 ± 0.0001
0.0492 ± 0.0010 0.0054 ± 0.0001
0.0473 ± 0.0009 0.0063 ± 0.0002
0.0468 ± 0.0008 0.0061 ± 0.0002
0.0567 ± 0.0014 0.0082 ± 0.0001
0.0522 ± 0.0009 0.0077 ± 0.0002
0.0487 ± 0.0009 0.0066 ± 0.0002
0.0532 ± 0.0010 0.0064 ± 0.0002
0.0524 ± 0.0011 0.0065 ± 0.0001
0.0487 ± 0.0006 0.0064 ± 0.0001
0.0454 ± 0.0006 0.0053 ± 0.0002
0.0455 ± 0.0005 0.0062 ± 0.0001
0.0478 ± 0.0006 0.0073 ± 0.0002
Compound 4
Contents (mg/g)2
Table 4. The contents of 6 investigated components and similarity value for 32 batches of YHD (n = 3).
0.2345 ± 0.0043
0.2244 ± 0.0032
0.2323 ± 0.0031
0.2132 ± 0.0017
0.1993 ± 0.0011
0.2142 ± 0.0041
0.2245 ± 0.0034
0.1892 ± 0.0031
0.2145 ± 0.0029
0.2234 ± 0.0032
0.2089 ± 0.0018
0.1765 ± 0.0028
0.1843 ± 0.0033
0.1956 ± 0.0039
0.2216 ± 0.0037
0.2234 ± 0.0042
0.1784 ± 0.0033
0.1878 ± 0.0019
0.2133 ± 0.0033
0.2213 ± 0.0032
0.1934 ± 0.0022
0.1877 ± 0.0033
0.1992 ± 0.0024
0.2088 ± 0.0018
0.2021 ± 0.0022
0.2134 ± 0.0032
Compound 6
0.4029 ± 0.0022
0.4046 ± 0.0029
0.4197 ± 0.0077
0.3771 ± 0.0068
0.3769 ± 0.0055
0.3748 ± 0.0065
0.4040 ± 0.0081
0.3392 ± 0.0063
0.4108 ± 0.0075
0.4097 ± 0.0077
0.3813 ± 0.0064
0.3556 ± 0.0058
0.3503 ± 0.0067
0.3555 ± 0.0032
0.3795 ± 0.0027
0.3820 ± 0.0033
0.3412 ± 0.0041
0.3736 ± 0.0032
0.4005 ± 0.0041
0.4019 ± 0.0031
0.3630 ± 0.0027
0.3549 ± 0.0033
0.3763 ± 0.0032
0.3751 ± 0.0043
0.3656 ± 0.0025
0.3748 ± 0.0041
Total
0.935
0.929
0.932
0.921
0.909
0.923
0.932
0.921
0.932
0.941
0.945
0.954
0.976
0.957
0.923
0.931
0.923
0.901
0.913
0.943
0.923
0.912
0.973
0.953
0.934
0.965
Similarity
value
862
FANGZHOU YIN et al.
0.943
0.921
S1, S2: the rootstock of Dioscorea opposita Thunb.(Dioscoreaceae) were collected from Jiaozuo of Hennan. Q1, Q2: the kernel of Euryale ferox Salisb. (Nymphaeaceae) were collected from Dongpin of Shandong
and Suzhou of Jiangsu, respectively. H1, H2: the bark of Phellodendron chinense Schneid. (Rutaceae) were collected from Yaan, Guangyuan of Sichuan, respectively. C1, C2: the seed of Plantago asiatica L.
(Plantaginaceae) were collected from Jian of Jiangxi and Dexiang of Sichuang, respectively. B1, B2: the seed of Ginkgo biloba L. (Ginkgoaceae) were collected from Xianan of Guangxi and Taizhou of Jiangsu,
respectively. 2 Compounds 1-6 refer to geniposidic acid, verbascoside, phellodendrine chloride, magnoflorine, palmatine hydrochloride, and berberine hydrochloride, respectively.
1
0.4156 ± 0.0051
0.3890 ± 0.0053
0.2221 ± 0.0032
0.2342 ± 0.0022
0.0501 ± 0.0009 0.0068 ± 0.0002
0.0553 ± 0.0008 0.0066 ± 0.0002
0.0316 ± 0.0006
YHD 32 (S2 + Q2 + H2 + C2 + B2)
0.0332 ± 0.0006 0.0605 ± 0.0008
0.0179 ± 0.0004
YHD 31 (S2 + Q2 + H2 + C2 + B1)
0.0343 ± 0.0008 0.0534 ± 0.0011
0.921
0.943
0.3813 ± 0.0062
0.3671 ± 0.0055
0.1892 ± 0.0026
0.1998 ± 0.0018
0.0578 ± 0.0003 0.0065 ± 0.0002
0.0546 ± 0.0004 0.0054 ± 0.0001
0.0232 ± 0.0005
YHD 30 (S2 + Q2 + H2 + C1 + B2)
0.0332 ± 0.0006 0.0613 ± 0.0012
0.0243 ± 0.0005
YHD 29 (S2 + Q2 + H2 + C1 + B1)
0.0367 ± 0.0007 0.0573 ± 0.0005
0.961
0.941
0.3668 ± 0.0044
0.3476 ± 0.0031
0.1988 ± 0.0035
0.2034 ± 0.0031
0.0473 ± 0.0002 0.0062 ± 0.0002
0.0408 ± 0.0002 0.0062 ± 0.0002
0.0173 ± 0.0005
YHD 28 (S2 + Q2 + H1 + C2 + B2)
0.0345 ± 0.0007 0.0434 ± 0.0008
0.0241 ± 0.0004
YHD 27 (S2 + Q2 + H1 + C2 + B1)
Total
Compound 6
Compound 5
Compound 4
Compound 3
Compound 1
Compound 2
Contents (mg/g)2
Sample
Table 4. Cont.
0.0404 ± 0.0007 0.0524 ± 0.0011
Similarity
value
863
13, 15, 17, 27, 29, 30, 32, and 35). Meanwhile,
among them, peaks 10, 23, 36, 37, 43, and 44 were
identified as geniposidic acid, verbascoside, phellodendrine chloride, magnoflorine, palmatine
hydrochloride, and berberine hydrochloride by comparison with the retention times and UV absorption
of the standards. Among these peaks, peaks 2, 6, 8,
and 26 were the distinctive peaks from Dioscorea
opposita Thunb. (Dioscoreaceae), peaks 5 and 28
were the distinctive peaks from the kernel of
Euryale ferox Salisb., peaks 11, 14, 16, 20, 21, 22,
25, 36, 37, 40, 41, 42, 43, and 44 were the distinctive peaks from Phellodendron chinense Schneid.
and peaks 10 and 23 were the distinctive peaks from
Plantago asiatica L., peaks 12, 15, 17, 27 and 35
were the distinctive peaks from Ginkgo biloba L.
and peaks 1, 4, 7, 13, 29, 30 and 32 were from several herbal drugs. Nevertheless, peaks 3, 9, 18, 19,
24, 31, 33, 34, 38 and 39 were not found in the
chromatograph of the herbal drugs alone, perhaps
because they were newly produced as a result of the
heating decocting of several drugs. There were several peaks, including peak 5 from the kernel of
Euryale ferox Salisb. and peak 40 from
Phellodendron chinense Schneid., for which the
peak areas were increased. The possible explanation
for this phenomenon might be that the solubility of
those components was improved after addition of
other drugs.
Simultaneously, the similar values of the fingerprints of 32 batches of YHD were calculated
using SES software, which were more than 0.901.
These results suggest that the established fingerprint
method used in the experiment was validated in the
quality control of YHD. In addition, six main components in 32 batches of YHD were simultaneously
determined using the HPLC-DAD method. The
detailed results of the similar values and content of
these six compounds are shown in Table 4. As
shown there, berberine hydrochloride was the most
abundant component with a varied content ranging
from 0.1765 to 0.2345 mg/g and accounting for
49.6~58.5% of the total content of the six analytes,
followed by phellodendrine chloride, magnoflorine,
verbascoside, geniposidic acid and palmatine
hydrochloride.
CONCLUSION
This is the first report on the optimization of
YHD processing and the quality assessment of 32
batches of YHD, and on the use of the fingerprinting
method for the simultaneous determination of 6 analytes in YHD. The preparation method that gives the
864
FANGZHOU YIN et al.
highest yield of YHD included the following steps:
(1) All herbs are immersed in water at 10 times the
volume of medicine for 30 min, followed by boiling
extraction for an additional 40 min. (2) After collection of water extract from Step 1, the herbal residues
were further extracted in boiling water at 6 times the
volume of medicine for 30 min. (3) The water
extracts from Step 1 and Step 2 were combined. This
proposed method is simple, reliable, with high precision, stability, and repeatability, and thus could be
used for the proper quality control for YHD.
Acknowledgments
We appreciate the financial supports from Key
Projects of Natural Science Foundation of Jiangsu
Province for University (13KJA360003), National
Natural Science Foundation (No. 30701108,
31071250, 81473293), the Open Project Program of
Jiangsu Key Laboratory of Pediatric Respiratory
Disease, Nanjing University of Chinese Medicine
(No. JKLPRD201403), a project funded by the
Priority Academic Program Development of Jiangsu
Higher Education Institutions (PAPD), Preliminary
Research Foundation of Nanjing University of
Chinese Medicine (No. 12XYY06). the specific
research program for the industry of Chinese medicine (201007010).
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21, 52 (2012).
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Immunopharmacol. 24, 128 (2015).
10. Feng A.W., Yu C., Mao Q., Li N., Li Q.R., Li
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Received: 21. 05. 2015
ISSN 0001-6837
THE COMPARISON OF THE STABILITY INDICATING TWO HPLC
METHODS AND THEIR APPLICATION FOR THE DETERMINATION
OF BOSENTAN IN COATED TABLETS
ANNA ZIELI—SKA*, S£AWEK WICHERKIEWICZ, WOJCIECH £UNIEWSKI,
KATARZYNA SIDORYK, EDYTA PESTA and ANDRZEJ KUTNER
Pharmaceutical Research Institute, 8 Rydygiera St., 01-793 Warszawa, Poland
Abstract: Due to the raising requirements of drug quality, there is an increasing need for fast liquid separations
of pharmaceutical substances with high efficiency and good resolution. The ultra-high pressure liquid chromatography (UHPLC) has been considered to meet this challenge. However, it was found that this fast method
has also serious disadvantages. The range of applications of the UHPLC in the analysis of pharmaceutical substances and dosage forms is currently extensively discussed. In this study we investigated the consequences of
the shortening of the analysis time of the liquid chromatographic method. Bosentan, a non-peptide antagonist
of human endothelin receptors, was chosen as an example in this study, due to its therapeutic importance and
lack of the reported analytical methods of the drug product. Two high-performance, reversed phase liquid chromatography methods with UV detection at 220 nm were developed for this purpose. Both methods were validated and the resulting performance characteristics were compared. The first separation (method A) was
achieved on Kinetex column, (2.6 µ C18 100A, 150 ◊ 4.6 mm), the second ñ fast (method B) employed Kinetex
column, (1.7 µ XB-C18 100A 50 ◊ 3.0 mm). Both methods were performed with a buffered mobile phase containing 0.1% of triethylamine in water brought to the pH 2.5 with phosphoric acid and methanol as the solvent
A and acetonitrile as the solvent B. Gradient program was used and flow rate of 0.8 mL/min and 0.4 mL/min,
for the methods A and B, respectively. The methods were validated according to the ICH guidelines for specificity, precision on the specified and LOQ limits, intermediate precision, accuracy, linearity (correlation coefficient = 0.999) and robustness. The robustness was confirmed using four factors: the mobile phase pH, the flow
rate of the mobile phase, column temperature and the second column of the same kind. The limits of detection
and quantification were established as 0.0132 and 0.1505 µg/mL for methods A and B, respectively. Both validated methods complied with the acceptance criteria. The method B was 3.5 times faster than the method A,
but the method A showed much better sensitivity. The resolution between compound B and bosentan was 3.39
and 1.75 for methods A and B, respectively. The lower sensitivity limits the use of Method B, especially in the
analyses at low levels of active substances (e.g., bioanalysis, validation of the cleaning procedures) and makes
method A more suitable for this purpose.
Keywords: bosentan monohydrate, validation, HPLC, UHPLC, impurities
GP120 (2). Bosentan affects vasoconstricting,
hypertrophic and fibrotic effects by blocking the
receptors ETA and ETB.
The recommended maintenance dose of bosentan is 125 mg, twice daily (3). The pharmacological
properties and the potential use of bosentan, in the
management of clinical disorders associated with
vasoconstriction, were described by Clozel et al. (4).
Bosentan was chosen for our investigation because
of its therapeutical importance. It is also an interesting analytical object, because of its numerous
known impurities. Determination of bosentan and its
metabolites in plasma and other biological matrices
Bosentan is a non-peptide antagonist of human
endothelin receptors (ETA and ETB) and it is used for
the treatment of pulmonary arterial hypertension
(PAH), chronic heart failure and Raynaudís syndrome. Endothelin (ET) levels in the lungs are significantly increased in patients with the PAH and
they correlate with the disease severity and prognosis. PAH can occur without an obvious cause, as in
primary pulmonary hypertension (PPH) or secondary to the systematic disease or congenital heart disease (1). It is observed in 0.5% of patients with an
HIV infection, where endothelin-1 synthesis is stimulated by the HIV type-1 envelope glycoprotein
* Corresponding author: e-mail: anna.zielinska@ifarm.eu; phone.:+48 22 456 38 08; fax:+48 22 456 38 38
865
866
ANNA ZIELI—SKA et al.
Table 1. List of known USP bosentan related impurities.
Structure
IUPAC name, symbol and CAS number
4-(t-butyl)-N-(6-chloro-5-(2-methoxyphenoxy[2,2í-bipyrimidin]-4-yl)benzenesulfonamide
Compound A [150727-06-3]
4-(t-butyl)-N-(6-hydroxy-5-(2-methoxyphenoxy)[2,2í-bipyrimidin]-4-yl)benzenesulfonamide
Compound B [174227-14-6]
N,Ní-(6,6íí-(ethane-1,2-diylbis(oxy))bis(5(2-methoxyphenoxy)-[2,2í-bipyrimidine]-6,4-diyl))
bis(4-(tert-butyl)benzenesulfonamide)
Compound C [1097263-60-9]
4,6-dichloro-5-(2-methoxyphenoxy)-2,2íbipyrimidine
Compound D [150728-13-5]
4-(t-butyl)benzenesulfonamide
Compound E [ 6292-59-7]
4-t-butyl-N-[6-(2-hydroxyethoxy)-5(2-methoxyphenoxy)-2-(pyrimidin-2-yl)pyrimidin4-yl]benzene-1-sulfonamide monohydrate
bosentan monohydrate [14536-97-8]
The comparison of the stability indicating two HPLC methods and...
were described. Most of the methods are based on
the HPLC with the LC-MS detection (5-9).
However, no liquid chromatographic determination
of bosentan in the pharmaceutical formulation was
reported. Only recently, Bansal et al., characterized
the forced degradation products of bosentan and
described the stability-indicating LC-UV method
for the stability testing of bosentan tablets (10).
There is no monograph of bosentan in any pharmacopeia. The draft under the USP`s Pending
Monographs Guideline (11) was based on HPLC
using a phenyl column.
To investigate the consequences of shortening
of the analysis time, two methods were developed
for the quantification of bosentan in coated tablets.
Both methods were validated according to the ICH
guidelines and pharmacopoeias requirements (12,
15, 16). Method A operated on the classical HPLC
apparatus, using 2.6 mm core-shell technology
columns. The method fulfilled the acceptable validation criteria, but the disadvantage was too long
analysis time. The method was transferred to the
UHPLC conditions (12, 19-21). For the method B, a
typical UHPLC apparatus was used, but the use of
the 1.7 mm particles, and 3 mm diameter column
makes the method B to be considered as the fast
HPLC. The validation parameters of method A and
method B were compared.
EXPERIMENTAL
Materials, reagents and chemicals
Bosentan monohydrate and related impurities
(Compounds A, B, C, D, E) were synthesized in the
Chemistry Department of the Pharmaceutical
Research Institute (PRI). Chemical structures,
names and formulas of the impurities are given in
Table 1. The placebo for model solutions was pre-
867
pared in the Analytical Department of the
Pharmaceutical Dosage Forms of PRI. The Tracleer Æ
coated tablets 62.5 mg and 125 mg (Actelion
Registration Ltd., London, UK) were from commercial source. The HPLC grade acetonitrile, methanol
and o-phosphoric acid were purchased from Avantor
Performance Materials Poland S.A., triethylamine
(TEA) was from Sigma Aldrich. Ultra pure water
was obtained from the Milli-Q water purification
system. All samples and solutions were filtered prior
to use through 0.2 mm PTFE filters (Supelco).
Apparatus
Method A
The separations were performed on the
Shimadzu LC-10 liquid chromatography system
equipped with two LC-10AT pumps, a UV-Vis
SPD-10A detector, an SIL 10-A XL auto injector, a
CTO-10A column oven and a CBM-10A communication bus module. Class LC-10 ver.1.64 software
was used for the instrument control and data collection.
Method B
The Shimadzu Nexera liquid chromatography
system equipped with two LC-30AD pumps, an
SPD-M20A Prominence diode array detector, an
SIL 30-AC Nexera autosampler, a CTO-20AC
Prominence column oven and a CBM-20A
Prominence communication bus module were used.
LC Solution software was used for the instrument
control and data collection.
Chromatographic separation conditions
Table 2 shows the chromatographic conditions
used for the method A and method B. Both methods
were based on the reversed phase chromatography
with gradient elution. The mobile phase contained
Table 2. The comparison of conditions of method A and method B.
Parameter
Method A
Method B
Column
Kinetex 2.6 m C18 100A 150 ◊ 4.60 mm,
Kinetex 1.7 m XB-C18 100A 50 ◊ 3.0 mm
Flow rate
0.8 mL/min
0.4 mL/min
Injection volume
10 µL
2 µL
Gradient profile
T0 (15%), T33 (35%), T34 (55%), T44
T0 (20%), T1 (20%), T7 (30%), T7.5 (55%),
T(time in min.) (%B)
(65%), T45 (15%), T50 Stop
T10 (65%), T11 (20%), T14 Stop
UV detection
λ = 220 nm
λ = 220 nm
Column temperature
35OC
35OC
Diluent
solvent A
solvent A
Analysis time
50 min
14 min
868
the mixture of methanol and buffered 0.1% of triethylamine (TEA) in water brought to pH 2.5 with
phosphoric acid (40 : 60 v/v) as the solvent A and
acetonitrile as the solvent B. To achieve the optimum resolution of bosentan and its impurities, a
number of gradient models were tested in both
methods. Because the injection volume was scaled
down from method A to method B, the concentrations of the samples were adjusted.
Preparation of the solutions of samples
Preparation of the solutions for the System
Suitability Test (SST)
The stock standard solutions of bosentan
monohydrate and USP bosentan related compounds
A-D (0.5 mg/mL) were prepared in the diluent. The
appropriate aliquots of these solutions were diluted
with the diluent to the final concentration of 20
µg/mL and 100 µg/mL, for method A and method B,
respectively (4% in comparison to the analyzed
solutions for both methods).
Preparation of the standard solution
The appropriate aliquots of the stock solution
of the standard of bosentan monohydrate (0.5
mg/mL) were diluted with the diluent to the concen-
tration of 0.5 µg/mL, and 0.25 µg/mL for method A
and method B, (0.1% in comparison to the analyzed
solutions for both methods), respectively.
Preparation of the analyzed solutions for method A
and method B
Twenty weighed TracleerÆ coated tablets
labelled 62.5 mg or 125 mg/tablet were powdered
and the equivalent of 62.5 mg of bosentan, calculated for the anhydrous substance, was weighed and
then suspended in the diluent to obtain the concentration of 0.5 mg/mL and 2.5 mg/mL, for method A
and method B, respectively. The samples were sonicated for 5 min and filtered prior to use through 0.2
µm PTFE filters.
Preparation of the model solutions for method A
The model solutions were prepared by diluting
the stock solution of bosentan in the diluent (0.01
mg/mL) to obtain the final concentrations in the
range of 0.052 to 0.624 µg/mL. (LOQ to 120% corresponded to the specified limit of unknown impurities 0.1% counted as bosentan). The adequate
amounts of the placebo solution, in the same concentration as in the analyzed solution, were also
added to the model solution.
Table 3. SST data for method A and method B.
SST data for the method A.
Compound
RT (min)
RRTa
(n = 6)b
Resolution Rsc
(n = 6)b
Tailing factor
(n = 6)b
E
8.814
0.430 ± 0.001
-
1.22 ± 0.01
D
11.833
0.456 ± 0.001
9.83 ± 0.14
1.15 ± 0.00
B
24.880
0.959 ± 0.004
42.42 ± 0.42
1.01 ± 0.00
bosentan
25.936
1.000 ± 0.000
3.39 ± 0.05
1.02 ± 0.01
A
34.276
1.322 ± 0.007
25.60 ± 0.08
1.00 ± 0.00
C
44.323
1.709 ± 0.004
38.90 ± 0.55
1.14 ± 0.01
Compound
RT (min)
RRTa
(n = 6)b
Resolution Rsc
(n = 6)b
Tailing factor
(n = 6)b
E
1.736
0.262 ± 0.001
-
1.11 ± 0.01
SST data for the method B.
a
D
2.243
0.338 ± 0.002
3.91 ± 0.02
1.15 ± 0.01
B
6.349
0.957 ± 0.003
17.55 ± 0.12
1.00 ± 0.00
bosentan
6.636
1.00 ± 0.000
1.75 ± 0.01
1.04 ± 0.00
A
8.451
1.234 ± 0.001
17.18 ± 0.08
1.19 ± 0.01
C
10.311
1.554 ± 0.001
31.87 ± 0.24
1.08 ± 0.00
b
c
RRT were calculated against the RT of the bosentan peak. Mean value ± confidence interval. Resolution Rs was calculated between two
neighboring peaks.
869
Figure 1. Method A chromatogram of the SST solution
Figure 2. Method B chromatogram of the SST solution
Preparation of the model solutions for method B
The model solutions were prepared by diluting
the stock solution of bosentan in the diluent (0.01
mg/mL) to obtain the final concentrations in the
range of 0.52 to 3.12 µg/mL (LOQ to 120% corresponded to the specified limit of unknown impurities 0.1% counted as bosentan). The adequate
amounts of the placebo solution in the same concentration as in the analyzed solution were also added to
the model solutions.
Methods validation
Method A and method B were validated
according to the ICH Guidelines and Pharmacopoeia
requirements (12, 15, 16) with reference to the following parameters: SST, specificity, precision on
the specified and LOQ limits, intermediate precision, accuracy, limit of quantitation (LOQ), limit of
detection (LOD), linearity and robustness.
Methods development and optimization
The main purpose to develop method A and
method B was to achieve the good separation of
bosentan and its related impurities (Table 1).
Compounds A, D, E are process impurities, and
compounds B and C are the degradation products.
The development of the method A, aimed to obtain
a good separation between bosentan and the degradation product (compound B), originated from
bosentan. This task was extremely difficult, because
in the initial chromatogram recorded according to
the USP`s Pending Monograph, the separation of
bosentan and Compound B was very weak (resolution Rs < 1.5) . Different chromatographic conditions were examined in order to optimize the separation. Various columns were tested with stationary
phases, such as C8, C18, phenyl and the mobile
870
phases with different buffers (e.g., acetate, phosphate, different pH). As a result of this study, the
resolution Rs of about 3.0 was obtained between
bosentan and compound B. However, despite
numerous efforts, the peak originated from compound C, could not have been eluted from the column. Many experiments with changing gradient
programs resulted in the optimized method A. This
method complied with the acceptance criteria and
gave the good separations of all peaks, including
compound C. However, the long analysis time still
remained as disadvantage of this method, especially
in the quality control of the drug.
The long time of the analysis encouraged us to
develop the method B ñ as a fast HPLC method and
transfer chromatographic conditions from method A to
method B. To check the effectiveness of the transfer
the resolution between bosentan and compound B was
monitored. In the first step, the analytical conditions
were transfered from method A to B using the calculator available on line (22). The satisfactory resolution
was not achieved, mostly because of the difference
between the stationary phases C18 and XB C18 used in
methods A and B, respectively. The basic chromatographic conditions, like solvents, UV detection, column temperature used in the method A were taken as
the starting conditions to transfer to the method B. The
flow of the mobile phase was lowered from 0.8
mL/min to 0.4 mL/min and the injection volume was
reduced from 10 µL to 2 µL. Several gradient programs were checked to obtain the optimum result.
Validation of the methods
System Suitability Tests (SST)
In order to perform the system suitability test
for both methods, the SST solutions were injected
six times after the equilibration of the system. The
chromatograms, retention times (RT), relative retention times (RRT) and widths of peaks were recorded. The same profiles of peaks occurred on the SST
solution chromatograms for both the methods. The
symmetry coefficients (tailing factors) of bosentan
peak and peaks of its related compounds were in the
range 0.8 ñ 1.5, and the resolution values Rs between
the nearest peaks were higher than 1.5, as required
(Table 3, Figs. 1 and 2).
Specificity
The specificity of both methods was tested
through the analysis of the diluent, the placebo solution containing the mixture of the tablet excipients,
prepared according to the sample preparation procedure, the SST solution containing the mixture of
bosentan and its impurities, standard solution containing bosentan at the concentration 0.1% in comparison to the analyzed solutions, and the analyzed
solution. All solutions were prepared according to
the described analytical procedure to evaluate possible interfering peaks. The examination of the solutions confirmed the lack of the matrix influence and
the lack of the interference of peaks originated from
bosentan and its impurities. The methods fulfilled
the acceptance criteria.
Limit of detection LOD and limit of quantitation
LOQ
The LOD and LOQ were estimated by the calibration curve method with the use of the model
solutions in the range from 5% in relation to the
specified level of unknown impurities equal to 0.1%
to 80% (two points above the reporting level of
unknown impurities 0.05%).
Table 4. Data obtained for the determination of LOD and LOQ for method A and method B.
Parameter
Method A
Method B
LOQ (µg/mL)
0.0132
0.1505
LOD (µg/mL)
0.0040
0.0452
Number of points
6
6
Concentration range (µg/mL)
0.052 - 0.416
0.26 - 2.08
Slope (b) ± confidence interval
54014 ± 608.2
8567.8 ± 219.5
Intercept (a) ± confidence interval
-28.992 ± 159.2
218.15 ± 287.3
Standard deviation Syx
71.45
128.95
Regression coefficient (r )
0.9999
0.9997
Precision on LOQ level (RSD%)a
3.29
1.16
Regression equation
2
a
each value is the mean of six determinations.
871
Table 5. Regression parameters of linearity for method A and method B.
Regression parameter
Method A
Method B
Number of points
8
7
Concentration range ( µg/mL)
0.052 - 0.624
0.52 - 3.12
Slope (b) ± confidence interval
53805 ± 471.5
8741.8 ± 214.5
Intercept (a) ± confidence interval
14.458 ± 172.5
29.964 ± 418.8
Regression coefficient (r2)
0.9999
0.9995
279.21 > tkr(a,f = n-2)
104.80 > tkr(a,f = n-2)
0.210 < tkr(a,f = n-2)
0.218 < tkr(a,f = n-2)
tkr(0.05;6) 2.447
tkr(0.05;5) 2.571
Regression equation
ta
a
tb a
tkr
a
a
value of the Studentís t-distribution
The following equations were applied (12):
3.3 ∑ Syx
10 ∑ Syx
LOD = ññññññ
LOQ = ñññññññ
a
a
where Syx is the standard deviation of y estimation in
relation to x and a is the slope of the regression
equation.
The estimated values of LOQ and LOD were
compared with the data obtained from the chromatograms by measuring of the signal-to-noise rate
equal to 3 : 1 and 10 : 1, respectively. After fixing
the LOQ, the precision was evaluated on that concentration levels for both methods. The precision of
both methods on the LOQ level was characterized
by the relative standard deviation (RSD) not greater
than 5.3% (13), as it was included in the acceptance
criteria (Table 4).
Linearity
The linearity of both methods was evaluated by
analyzing several model solutions in the range
between LOQ and 120% of the specified level of
unknown impurities (equal to 0.1%). The regression
analyses were performed, the correlation coefficients, slopes and y-intercepts of the calibration
curves were calculated (14). The Studentís t-distribution test was performed. Values ta and tb were estimated according to the following equations:
|a|
|b|
ta = ññññññññ ª tkr tb = ññññññ ≤ tkr
Sa
Sb
tkr(a,f = n ñ 2)
where a is the slope of the regression equitation, b ñ
intercept, Sa and Sb ñ standard deviation of a and b,
ta and tb - values of the Studentís t-distribution.
It was revealed that the response of bosentan
was linear for both methods. ta - Studentís t-distri-
bution value indicated that the results statistically
differ, so the methods were characterized as the
ones sensitive enough. tb - Studentís t-distribution
value indicated that the results did not differ statistically, so the methods showed no systematic error
(Table 5).
Precision
In order to evaluate the precision of both methods, six model solutions were prepared (M 100%) at
the concentration of 0.5 µg/mL, and 2.5 µg/mL for
HPLC and fast HPLC, respectively. The concentration of the solutions corresponded to the 0.1% of the
specified limit of unknown impurities counted as
bosentan monohydrate. The model solutions were
analyzed according to the analytical procedure
described above, the areas of bosentan peaks were
recorded. The procedures were repeated on the following day, as the intermediate precision, by another team of analysts, using different instruments. The
comparison of both series of results was made with
the Snedecorís F distribution test ñ the homogeneity
of variance.
To verify statistical hypothesis about the equality of variance of the two statistical populations, the
following values S22, S21 were determined for samples of sizes n1 = 6. The determined expression F =
S22/S21 for S22 > S21 and F = S21/S22 for S21 > S22 were
compared with the critical value F a; f1, f2, for f = n-1,
read from Snedecorís F distribution boards (14).
The precision of both methods was characterized by
the relative standard deviation RSD not greater than
2.7% (13). The calculated F values were lower than
the F a, f1, f2 value in this way that there is no reason
to reject the null hypothesis: H0; S22 = S21. Both
methods were found to be precise enough (Table 6).
872
Accuracy
The accuracy of both methods was examined
with the use of the model solutions on three levels:
80, 100 and 120%, according to the specified level
of unknown impurities equal to 0.1% counted as
bosentan monohydrate. Three samples were prepared for each level and analyzed according to the
analytical procedure. The recovery of bosentan
was calculated with the reference to the specified
level of unknown impurities equal to 0.1% on each
level.
The RSD value for the mean results from nine
determinations was calculated. The average bosentan recovery for both methods was within the range
of 97.0-103.0% and the RSD values were not greater
than 2.7%, as required (13) (Table 7).
Table 6. Precision and the intermediate precision tests for method A and method B.
Regression parameter
a
Method A
Method B
Number of determinations
6
6
Precision (RSD %)a
0.64
1.02
Intermediate precision (RSD %) a
0.33
1.14
Fα f1, f2b
5.05 (0.05;5)
5.05 (0.05;5)
Fcal b
3.8 < Fα; f1, f2
1.35
each value is the mean of six determinations. b value of the Snedecorís F distribution.
Table 7, Accuracy tests for method A and method B.
Method
Level (%)
Amount found (%)a
80
Method A
100
120
0.0795
99.44
0.0801
100.08
0.0792
98.96
0.1003
100.29
0.0997
99.66
0.1004
100.45
0.1187
98.92
0.1183
98.56
0.1189
0.67
Range
98.56 ñ 100.45% (1.89%)
Method B
100
120
0.0767
95.83
0.0789
98.60
0.0808
101.03
0.1006
100.55
0.1002
100.20
0.1010
101.03
0.1190
99.16
0.1194
99.49
0.1196
Mean recovery n = 9
99.63
99.50 % ± 1.24 α = 0.05
RSD (%)
1.62
Range
95.83 ñ 101.03% (5.20%)
ñ each value is the mean of three determinations
99.49
100.13
98.86
99.10
RSD (%)
80
Mean recovery (%)
99.50% ± 0.52 α = 0.05
Mean recovery n = 9
a
Recovery (%)
98.49
100.60
99.43
873
Table 8. Robustness for method A and method B.
Retention time of
bosentan (min)
Peak symmetry of
bosentan
Resolution Rs
between comp. B
and bosentan
Parameter
Fixed conditions
Mobile phase
pH
2.30 ± 0.02
2.70 ± 0.02
Method
A
Method
B
Method
A
Method
B
Method
A
Method
B
25.93
6.64
1.02
1.04
3.39
1.75
25.33
6.58
0.95
1.04
3.04
1.74
25.52
6.69
0.99
1.04
3.22
1.73
Mobile phase
0.7 mL/min 0.44 mL/min
24.24
6.17
1.04
1.04
2.89
1.72
flow speed
0.9 mL/min 0.36 mL/min
28.39
7.00
1.07
1.05
3.23
1.72
UV
215 nm
25.70
6.64
1.06
1.04
3.02
1.75
Detection, λ
225 nm
25.82
6.64
1.05
1.04
3.03
1.75
Column
O
30 C
27.45
6.74
0.86
1.04
2.75
1.73
temperature
40OC
24.93
6.31
0.92
1.04
3.57
1.73
26.36
6.60
0.92
1.04
3.00
1.76
Column
(other batch)
Method A
Method B
Robustness
The robustness of both methods was tested
using the SST solution. Chromatographic parameters and their influence on the SST results for both
methods are presented in Table 8.
The stability of the analyzed solution and the
standard solution was also tested. The samples were
analyzed according to the analytical procedure, right
after the preparation and after 24 h of storage at
room temperature. The difference in the bosentan
peak areas from the solutions analyzed right after
the preparation and after 24 h of storage was smaller than 2.0% indicating that the solution remained
stable.
Both methods were robust to the minor
changes of the analytical parameters, and complied
with the acceptance criteria.
CONCLUSIONS
As a result of the validation both methods
showed acceptable values for all the tested parameters. The validations confirmed the robustness and
specificity of both methods. The statistical analysis
of the validation parameters: precision, accuracy
and linearity, showed that both methods met the
acceptance criteria, however method A gave better
results. Method A showed better sensitivity and resolution than method B. Both methods showed much
better sensitivity (LOQ 0.0132 µg/mL and 0.1505
µg/mL, respectively) compared to the method
described by Bansal et al. (LOQ 0.7 µg/mL) (10).
As expected, the analysis time of method B was
reduced (from 50 to 14 min) as compared to method
A which is especially important in the quality control of drugs. Method B consumed much smaller
volume of solvents, thus making the method more
economic and environmentally friendly. Both methods can be successfully used in the evaluation and
stability testing of bosentan and its pharmaceutical
dosage forms. However, the reduced sensitivity of
method B, compared to method A, limits the analytical utility of this method, especially in analysis
of low levels of active substances e.g., in bioanalysis and in cleaning procedures of the production
lines.
Acknowledgment
The project was supported by The National
Centre for Research and Development, under the
contract No. UDA-POIG.01.03.01-14-062/09-00.
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Received: 19. 06. 2015
ISSN 0001-6837
ANALYSIS OF ω-3 FATTY ACID CONTENT OF POLISH FISH OIL DRUG
AND DIETARY SUPPLEMENTS
KAMILA OSADNIK1* and JOANNA JAWORSKAb
1
School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University
of Silesia, Katowice, Poland, Department of Biopharmacy, Jednoúci 8, Sosnowiec, Poland
2
Centre of Polymer and Carbon Materials, Polish Academy of Sciences,
34 M. Curie-Sk≥odowska St., 41-819 Zabrze, Poland
Abstract: Study results indicate that a diet rich in polyunsaturated fatty acids ω-3 (PUFA n-3) exerts favorable
effect on human health, accounting for reduced cardiovascular morbidity and mortality. PUFA n-3 contained in
marine fish oils, particularly eicosapentaenoic (EPA, 20:5 n-3) and docosahexaenoic (DHA, 22:6 n-3) acids, are
attributed antithrombotic, anti-inflammatory, anti-atherosclerotic and anti-arrhythmic effects. They have also
beneficial effects on cognitive functions and immunological mechanisms of an organism. Considering the fact
that marine fish are not abundant in Western diet, the pharmaceutical industry reacts with a broad selection of
PUFA n-3 containing dietary supplements and drugs. Increased consumersí interest with those products has
been observed recently. Therefore, their quality, understood as reliability of manufacturerís declaration of composition of offered dietary supplements, is highly important. We have tested 22 products available in pharmacies and supermarkets, manufacturers of which declared content of n-3 fatty acids (21 dietary supplements and
1 drug). Identity and content of DHA and EPA were assessed using 1H NMR spectroscopy, based on characteristic signals from protons in methylene groups. Almost one in five of the examined dietary supplements contains < 89% of the PUFA n-3 amount declared by its manufacturer. For a majority of tested products the manufacturer-declared information regarding DHA (58%) and EPA (74%) content was consistent with the actual
composition. It is notable that more cases of discrepancy between the declared and the actual content regarded
DHA than EPA, which indicates a less favorable balance, considering the pro-health effect of those acids. Over
a half of tested products provides the supplementary dose (250 mg/day) with one capsule taken daily, and in
27% of cases the daily dosage should be doubled. Only 10% of those products ensure the appropriate dose for
cardiovascular patients (1 g/day) with the use of 1 capsule a day. Correct information provided by a manufacturer on a label regarding the total amount of DHA and EPA is a basis for selection of an appropriate dosage.
Keywords: dietary supplements, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), fish oil
Polyunsaturated fatty acids ω-3 (PUFA n-3)
play numerous roles in the human organism, therefore they are an important compound of healthy diet.
PUFA n-3 belong to exogenous compounds, and
their availability is limited to fatty marine fish
(salmon, mackerel, tuna, sardines, herring). Two
most important acids belonging to the n-3 family
are: eicosapentaenoic acid (EPA, 20:5 n-3) and
docosahexaenoic acid (DHA, 22:6 n-3). DHA and
EPA may be also produced by conversion of the 18carbon α-linolenic acid (ALA), present in vegetable
products including soya beans, linen and nuts.
Conversion of ALA into EPA and DHA remains at
a very low level in humans, and therefore, those
fatty acids should be supplied with a diet, mostly by
consumption of fish (1-4).
PUFA n-3 (especially DHA and EPA) contained in marine fish oils are attributed numerous
favorable effects associated with reduction of cardiovascular morbidity and mortality. Studies indicate that PUFA n-3 rich diet, or a regular supplementation of those fatty acids, has an antithrombotic, anti-inflammatory, anti-atherosclerotic and antiarrhythmic effects (5-9). Scientific data indicate that
the use of PUFA n-3 by type 2 diabetes patients
largely supports the therapy aimed at reduction of
VLDL and triglyceride levels (10).
The growing awareness of health benefits associated with eating marine fish and substitution of
saturated fats with unsaturated ones in the daily diet
becomes more and more noticeable in the Polish
society (11). Unfortunately, statistics show that
* Corresponding author: e-mail: kamila.osadnik@onet.pl
875
876
KAMILA OSADNIK and JOANNA JAWORSKA
Poles, in general, do not comply with the guidelines
of the Polish Society of Family Medicine, and the
European Society of Cardiology that recommend
increasing quantity of fish in the weekly diet. This
resulted with a response of the pharmaceutical and
food industry and introducing multiple dietary supplements containing PUFA n-3 acids on the market
(12, 13). Availability of dietary supplements in various points of sale (supermarkets, drugstores, newsstands) beyond pharmacies causes a systematic
expansion of the group of consumers of that kind of
products, who want to completely replace a seabased fish diet by dietary supplements of n-3 fatty
acids. Currently, n-3 acids containing products
designed for all age groups are available in the
Polish market. They are recommended for healthy
individuals as supplements (for vegetarians, pregnant women, elderly) and for patients with cardiovascular diseases, vision problems, impaired immunity and memory disorders.
Taking PUFA n-3 in the form of dietary supplements seems to be equivalent to consumption of
fish. That assumption is supported by the fact that
dietary supplements contain fish oil from the same
natural source. It should be noted, however, that not
all products are marked with the information concerning fish species or fishing area. Question of the
supplementary dose of PUFA n-3 determined by the
European Food Safety Authority and the Food and
Agriculture Organization of the United Nations at
250 mg/day for a healthy adult, and of therapeutic
doses recommended by scientific societies to
patients with various conditions, used to be treated
rather subjectively by some manufacturers (14, 15).
Considering a very low consumption of marine fish
in the Polish population, the Polish Society of
Family Medicine stresses the urgent need for intensification of educational efforts aimed at increased
consumption of EPA and DHA in everyday diet, up
to the optimum level of 1 g a day. In risk groups of
cardiovascular diseases, cancer, rheumatoid and
neurodegenerative diseases the EPA and DHA content should be increased to 1.5 g a day, if possible.
Experts state that the basic source of both acids
should be marine fish: herring, mackerel, salmon,
cod, flounder, halibut, served twice a week. If meeting those recommendations proves impractical, EPA
and DHA supplements should be used, containing
the amount of acids consistent with the declaration
(12).
According to the Polish law, dietary supplements are treated as food, and thus they are exempt
from rigorous control applicable to drugs. As a
result, in terms of quality and purity of those products, consumers of dietary supplements have to
depend on declarations made by their manufactures.
Figure 1. Study design
Analysis of ω-3 fatty acid content of Polish fish oil drug a dietary supplements
877
Figure 2. Standard curves: a) DHA, b) EPA
Reliable label information is therefore of exceptionally high importance.
This paper presents results of identity and content analyses of two principal PUFA n-3: DHA and
EPA in selected dietary supplements available in the
Polish market, and in one registered medicinal product. Quality of selected products and correctness of
manufacturersí declarations were also assessed by
comparison of label information with results of the
analysis of liquid content of capsules. The paper
confronts also manufacturersí recommendations
regarding dosage of PUFA n-3 contained in their
products and recommendations of scientific societies issued in Poland and worldwide (15-17).
MATERIAL AND METHOD
Material
Study material was dietary supplements (n =
21) available in pharmacies and markets, and the
only one registered in Poland as a drug, available
only in pharmacies. Each product was accompanied
by a leaflet stating the declared n-3 acid content.
Among the selected product, three were tested only
for α-linolenic acid content, as they contained no
DHA and EPA (Fig. 1)
Methods
Total 19 products were tested using high definition nuclear magnetic resonance (NMR) for determination of identity and DHA and EPA content. The
testing procedure was developed and performed
according to methodology accepted for the given
drug form (18). In case of products containing any
additional ingredients (herbal extracts, vitamins) 0.5
mL of chloroform was added to 1 mL of liquid capsule content, and centrifuged. Identity and content of
capsules filled with liquid oil was tested directly.
Identity and content of manufacturer-declared DHA
and EPA were assayed for all products.
878
Figure 3. The percentage of the declared content of DHA and EPA
n = 19
n = 19
Figure 4. Necessary number of capsules of a PUFA n-3 product
ensuring a daily supplementation dose (250 mg/day)
Figure 5. Necessary number of capsules of a PUFA n-3 product
ensuring the daily therapeutic dose (1 g/day)
Identity testing
The identity was tested in order to confirm that
the product corresponds to its label description. The
test involved the comparison of characteristic signals of fatty acids present in 1H NMR proton spectra
of a tested product (dietary supplement or drug) with
1
H NMR spectra of purchased analytical standards:
docosahexaenoic acid analytical standard (SigmaAldrich), eicosapentaenoic acid analytical standard
(Sigma-Aldrich).
Chemical shifts characteristic for DHA and
EPA are related to protons of β-methylene groups,
present within the range of 2.30-2.34 ppm for DHA,
and 1.61-1.72 ppm for EPA.
Content verification
Verification of content consisted in quantitative determination of DHA and EPA in each tested
product. Calibration curves for each determined
fatty acid were developed to that purpose.
Standard curves
Contents of individual fatty acids in each product were calculated based on the measurement of
intensity of characteristic signals emitted by both n3 acids, based on the equation curve of relative
intensity of those groups in relation to the intensity
of the internal standard (methanol). Standard curves
for DHA and EPA are presented in Figure 2.
879
should fit within the ±5% range for capsules with
the declared active substance content of over 100
mg, and for capsules with the declared acid content
below 100 mg the range was ± 10% (18).
Solutions for each calibration curve were prepared by dilution of known amounts of DHA and
EPA analytic standards in sunflower oil. Prepared
solutions were mixed with the internal standard in
the amount corresponding to 1% concentration in
prepared dilutions. Obtained solutions were assayed
using NMR spectroscopy, according to the method
developed by Igarashi et al. (19).
1
H NMR spectroscopy
1
H NMR spectra of solutions prepared for
development of standard curves and of tested products were acquired from the Bruker-Avance II
Ultrashield Plus spectrometer, with frequency of
600 MHz, using deuterated chloroform as a diluent
and methanol as the internal standard. All spectra
were acquired after 64 passages, with pulse length of
11 µs and the acquisition time of 2.65 s. All determinations were performed at 23OC, considering perfect detection of fatty acids at higher temperatures
and storage conditions recommended by manufacturers.
Determination of DHA and EPA content in tested
products
Tested sample was drawn from a mixed content of 10 capsules of the particular product. Liquid
filling was collected with a syringe and needle into
a beaker. Representative sample (200 µL) of the tested product was transferred into a 2 mL disposable
test tube. Seven hundred fifty microliters of deuterated chloroform and 7.5 µL of methanol were added.
Content of the disposable test tube was mixed, and
300 µL of the solution was collected and transferred
into the NMR test tube.
All samples of tested products were assayed
twice. Mean values and deviations from the declared
n-3 acid content defined in requirements for that
form of drug were calculated. The admissible deviation from the declared DHA and EPA content
RESULTS
Comparison of identity of n-3 acids contained in
dietary supplements and a drug with manufacturersí declarations
The comparative analysis of 1H NMR spectra
of tested products with analytical standards demon-
Table 1. EPA to DHA ratio in Polish n-3 fish oil drug and dietary supplements.
Ratio
EPA:DHA (n)
EPA:DHA (%)
≤ 0.5 : 1
5
26
1.5 : 1
10
53
1.51-2.0 : 1
2
11
2.1-2.5 : 1
1
5
2.6-3.9 : 1
0
0
≥4:1
1
5
Figure 6. Cost of supplementation with PUFA n-3 products to achieve daily intake of 1 g/day
880
strated a complete consistency of DHA and EPA
identity from both the tested drug, and all dietary
supplements. It is worth noting that, regardless the
price, each product contained those n-3 acids that
had been declared on the information leaflet by a
manufacturer.
Comparison of n-3 acid content in dietary supplements and a medicinal product with the manufacturerís declaration
Based on the NMR spectroscopy it was found
that: in case of DHA 42% of products (n = 8) the
result of analysis was different from the declared
one; in case of EPA a discrepancy was found in 26%
of products (n = 5).
It is worth noting that in case of the medicinal
product registered as a drug and available in the
Polish market, the NMR analysis of DHA and EPA
content demonstrated a complete consistency with
the manufacturerís declarations.
In case of 21% (n = 4) of tested products the
amount of DHA and/or EPA determined experimentally was higher than declared by the manufacturer
(Fig. 3).
Necessary number of capsules of a PUFA n-3
product ensuring a daily supplementation dose
Sixty three percent of tested products (n = 12)
satisfy the dietetic demand with one capsule taken a
day, as they contain overall amount of 250 mg of
DHA and EPA. Twenty seven percent (n = 5) of
them require ordination of two capsules a day (Fig.
4).
Necessary number of capsules of a PUFA n-3
products allow the daily therapeutic dose
The number of capsules necessary to reach the
daily EPA + DHA consumption of 1 g/day recommended by the European Society of Cardiology
(ESC) and the European Athercoslerosis Society
(EAS) was summed up for tested products (16).
Only 21% of products allows realization of ESC recommendations with the use of 2 capsules a day, and
only 10% meet those requirements with the use of 1
capsule a day. Over a half of tested products require
taking at least 4 capsules a day. Results of the analysis are presented graphically in Figure 5.
Cost of supplementation with PUFA n-3 products
A majority of PUFA n-3 containing products
available in the Polish market is priced below 20
PLN per pack. However, the number of capsules in
a single pack, and n-3 acid content are highly variable. The analysis indicated that in one of three
cases a consumer who had bought a relatively cheap
product has to consider the necessity of taking more
capsules a day to meet the ESC guidelines regarding
consumption of 1 g of PUFA n-3/day. Increasing
price (as per 1 capsule) reduces the necessity of taking multiple daily doses, which accounts for convenient use of a product.
It is surprising that in the group of the most
expensive products there are dietary supplements
containing low amount of PUFA n-3. Figure 6 presents the listing.
EPA : DHA index
The relative EPA and DHA content in each
product was calculated based on experimental data.
In half of them (n = 10) the EPA : DHA index was
1.5 : 1. Every fifth product is characterized by the
EPA : DHA index higher than 1.5 : 1, but as much
as 26% of products contain more DHA than EPA
(Table 1).
DICUSSION
Nutritional habits of Polish people do not follow dietary recommendations of the Food and
Nutrition Institute regarding fish consumption.
According to recommendations, fish should be
served 2-3 times a week, whereas just over 40% of
Poles eat fish once a week, and 60% consumed fish
hardly ever (20). The consequence of depletion of
PUFA n-3 containing foods is abnormal proportion
of n-6 to n-3 acids of 10-20 : 1, whereas a correctly
balanced diet should provide the ratio at the level of
4 : 1 - 5 : 1. Increased cardiovascular morbidity and
mortality may be an effect of that nutritional error
(21).
Increasing awareness of healthy lifestyle is
reflected rather by growing demand on dietary supplements with PUFA n-3, than in changes of diet
(12, 13). Fortunately, a complete identity consistence of those acids was demonstrated in all tested
products, which means that oil contained in capsules
is of marine fish origin, as declared by their manufacturers.
Unfortunately, information regarding DHA
and EPA content provided for some products are
unreliable and misleading, which may lead to
improper PUFA n-3 levels in consumersí organisms. Kris-Etherton et al. and Piecyk et al. compared
EPA : DHA proportions declared by manufacturers
of dietary supplements. Their results indicate the
unfavorable prevalence of EPA over DHA in a
majority of tested products (22, 23). The problem
has been also discussed by Gorjão et al., who indi-
cated a higher DHA content compared to EPA in
cold-water fish, and the opposite relation of acids in
available dietary supplements (24). It has not been
determined which of n-3 acids is of a greater therapeutic value, because a majority of clinical trials use
a mixture of both acids. There are some single
reports indicating that only DHA is able to reduce
the arrhythmia-induced mortality (the risk reduced
by 45%), effectively reduce the total cholesterol
level, increase the HDL level, inhibit dementia and
depression (25-29). On the other hand, EPA is of
significance for reduction of myocardial infarctioninduced mortality (the risk reduced by 28%) (25).
Results of the EPA : DHA proportion analysis in 19
Polish products containing both acids are comparable to results reported by other authors. In case of
over a half of them the ratio is 1.5. Each fourth
product contains more DHA than EPA, which
stands the Polish market out in comparison to other
ones (22, 23, 30). Unfortunately, it is notable that a
more common discrepancy is related to the difference between the declared and the actual content of
DHA, than of EPA, which in consequence maintains
a less favorable balance, considering the pro-health
effects of those acids. Results of this study indicate
that not only the manufacturer-declared EPA : DHA
ratio is unfavorable, but also in 21% (n = 4) of tested product the situation is additionally made worse
by lower than declared actual DHA level.
Those fluctuations may be explained by a variable DHA and EPA content in marine fish, depending on their physiological condition, season of the
year, and especially the fishing area (31, 32). Fish
from northern seas are characterized by a higher
EPA content, while DHA content is higher in southern sea fish. The observation has been confirmed by
Opperman et al. for dietary supplements available in
the South African markets. Contrary to European
researchers, the authors indicate reduced EPA content in products available in South Africa (30).
Manufacturers of dietary supplements available in
the Polish market base on salmon, sardine,
anchovies and cod fished in the Atlantic Ocean, the
Northern Sea and the Baltic Sea, which could
explain inconsistent DHA contents in their products
(32).
There are also products available in the Polish
market, with α-linolenic acid (ALA) as the only
declared PUFA n-3. Three dietary supplements of
that kind were also analyzed for identity of DHA
and EPA. In case of two product, the 1H NMR spectrum demonstrated no signals characteristic for
DHA and/or EPA. One supplement contained minor
(at the border of the tolerated content deviation)
881
DHA content. According to scientific data, adult
human organism is able to convert ALA into longchain n-3 fatty acids. However, the process is inefficient and in men constitutes only a fraction of percent. Organism of a young healthy male converts
8% of dietary ALA into EPA, and only 0-4% of
ALA into DHA (3). In young healthy females
approx. 21% of dietary ALA is converted into EPA,
and 9% into DHA (4). The study by Plourde et al.
demonstrated that consumption of ALA has only a
minor effect on increased EPA and DHA levels in
serum or red blood cells. Moreover, it was demonstrated that increased consumption of EPA leads to
its increased serum level, but fails to increase the
DHA level, whereas the increased consumption of
DHA results in increased levels of DHA and EPA
(although to a lesser extent). The fact is a result of a
partial reconversion of DHA into EPA (33).
Considering a relatively low conversion of ALA
into long-chain n-3 fatty acids, especially DHA,
those fatty acids have to be supplied. A sale of
dietary supplements under the name of Omega-3,
although correct from the chemical point of view,
may have some measureable consequences for consumers. Therefore, precise information concerning
composition of dietary supplements is so important.
The importance of dietary supplements containing oil from crustaceans has been growing lately. One of the tested products contained PUFA n-3
from krill. It may be surprising that the supplement
is one of the most expensive ones, and its n-3 acid
content is low (32.5 mg DHA and 70 mg EPA).
High price of some products may be explained by its
association with a particular fish species, from
which the oil is pressed, extraordinary purity of
some water, where animals were fished, and a timeconsuming and expensive process of production
based on small crustaceans. One of the recent studies on bioavailability index of n-3 acids indicates,
that the highest index if found in products combining krill oil with phospholipids (34).
The question of consumption of PUFA n-3 is
regulated in Poland by recommendations of several
scientific and research organisations, including the
European Food Safety Authority and the Food and
Agriculture Organization of the United Nations, that
determine the minimum daily intake of DHA + EPA
by healthy adults at 250 mg (13, 14). The World
Health Organization recommends daily intake of
both EPA and DHA in amount of 250-300 mg plus
800-1100 mg of ALA (35). That problem seems to
be favorably reflected by dietary supplements present in the Polish market. A majority of tested products contain an appropriate amount of n-3 acids,
882
allowing provision of the supplementation dose by
consumption of 1 or 2 capsules a day. It should be
noted that the dose corresponds to an amount preventing PUFA n-3 deficiency in a healthy adult,
which means that 250 mg/day is a necessary dietetic minimum. It should be also noted that the supplementation dose is not a synonym of the therapeutic
dose ñ a dose appropriate for treatment of diseases
in which long-chain PUFA may lead to improvement of the health condition.
However, some producers of n-3 containing
dietary supplements offer much more than just a
nutritional supplementation. Approx. 63% (n = 13)
of tested dietary supplements (n = 22) clearly states
on the label or in the product leaflet (as a purchase
promoting campaign) that taking the product will
result in ìreduced cholesterol levelî, ìimprove the
function of vascular endotheliumî, ìreduce blood
pressureî, and ìreduce inflammationî. It should be
stated that, according to scientific research, those
effects are possible, but achievable only with doses
much higher than 250 mg/day (10, 35, 36).
Current guidelines of the European Society of
Cardiology (ESC) and the European Atherosclerosis
Society (EAS) recommend 1 g of PUFA n-3/day for
all patients with a history of myocardial infarction.
That dosage is difficult to achieve based on natural
food sources, but achievable with dietary supplements, nutraceuticals and/or medicinal products
(16). Polish Diabetes Association recommends type
1 diabetes patients using 2-4 g of n-3 acids/day (17).
Despite clear scientific recommendations, producers
of n-3 containing dietary supplements often suggest
the number of capsules to be taken daily, with no
information concerning the basis for that recommendation.
A majority of tested products requires taking at
least 4 capsules a day to reach the ESC-recommended therapeutic dose of 1 g and to allow health effects
promised in information materials. Unfortunately,
only 31% of dietary supplements dedicated to cardiovascular patients allows intake of the dose of 1
g/day with not more than 2 capsules a day. The
problem of incorrect dietary supplement dosage recommendations has been also described by Belgian
scientists. For a majority of dietary supplements
tested by them (n = 9) reaching the dose of 1 g
proved impossible, even with the use of the highest
dosage recommended by a manufacturer (37).
Unreliable label information are misleading for consumers, with a possible consequence of deterioration of their heath condition.
One have to remember that according to medical recommendation and legislation dietary supple-
ments should be used only to supplement but not
substitute a healthy diet that is recommended as a
base of cardiovascular disease prevention (class I,
ESC recommendation) (38).
CONCLUSIONS
1. All tested dietary supplements and the single
tested drug available in the Polish market demonstrate consistency in terms of identity of DHA and
EPA fatty acids, which means that they are based on
raw material obtained from marine fish.
2. For a majority of tested products, the information provided by a manufacturer on a label
regarding the total amount of DHA and EPA was
reliable. Correct information in that respect is a basis
for selection of an appropriate dosage.
3. Choosing n-3 acid containing products it is
reasonable to consider the content of individual
acids and their ratio, as those parameters decide on
pro-health effects of a product.
4. Patients with cardiovascular conditions or
diabetes should pay special attention to the number
of capsules to be taken to meet the DHA + EPA dose
recommended by scientific societies.
Acknowledgment
Kamila Osadnik is a scholarship holder within
the DoktoRIS project - Scholarship program for the
innovative Silesia, supported by the European
Community from the European Social Fund.
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Received: 30. 07. 2015
ISSN 0001-6837
2D LC HEART CUTTING ON-LINE OF PHENOLIC COMPOUNDS FROM
THREE SPECIES OF THE GENUS SALIX
LORETTA POB£OCKA-OLECH, DANIEL G£”D, BARBARA KR”L-KOGUS
and MIROS£AWA KRAUZE-BARANOWSKA*
Department of Pharmacognosy with Medicinal Plants Garden, Medical University of GdaÒsk,
Al. Gen. J. Hallera 107, 80-416 GdaÒsk, Poland
Abstract: The 2D LC heart-cutting on-line system was elaborated and employed to the analysis of simple phenols and polyphenols occurring in willow barks. Using the test-set of 52 compounds, the conditions of chromatographic separation in each dimension were optimized. The worked-up system was based on RP-separation
in both dimensions and the use of different elution profiles on the first- and second-dimensional columns: gradient and multistep gradient elution, respectively. In all analyses the UV detector was used. Under optimized
separation conditions slightly modified in respect to chemical composition of the each analyzed MeOH extracts
from three willow barks: Salix daphnoides, S. purpurea and S. sachalinensis ëSekkaí the differences in phenolic acid and flavonoid compositions were revealed.
Keywords: two-dimensional HPLC, polyphenols, heart-cutting technique, willow bark, Salix
anthocyanins (21), procyanidins (22, 23) and flavan3-ols (catechins) (27) were presented.
In LC◊LC effluent from the first dimensional
column, divided into small fractions, with proper
modulation time, is analyzed on the second dimensional column (1-5, 8, 9, 19, 28, 40). On the other
hand, in the LC-LC only the selected fractions of
interest are transferred from the first dimension to
the second one (7, 9, 19, 29, 40). Heart-cutting systems are easier to build, as each of the dimensions
may be optimized separately with the use of equipment usually available in every HPLC laboratory.
Moreover, in off-line mode LC-LC enables to detect
analytes appearing in the sample in small concentrations, since the fraction of interest may be evaporated and concentrated after collection (9, 40). Modern
2D LC systems are automated and operated by computer programs which enable the presentation of
two-dimensional separations in the form of 2D
plots.
It is described in literature (41-44), that composition of phenolic compounds, comprising simple
phenols and polyphenols, in bark of willows
evolved dependently on the species of Salix. In our
experiments, 2D LC was employed to compare the
chemical composition of three species from the
genus Salix, namely Salix purpurea L, S. daphnoides
Plant extracts are complex matrices, which
chromatographic analysis is very often difficult due
to the co-elution of analytes and the presence of frequently more than one compound in a single peak.
For their analysis, new chromatographic techniques
are developed. Multidimensional chromatographic
systems, particularly two dimensional liquid chromatography (2D LC) open a new perspective for the
analysis of chemical composition of plant material
(1-11). 2D LC is a powerful tool for the separation
of complex samples, as it offers higher peak capacity, selectivity and resolution power in comparison
with one-dimensional HPLC (1, 4, 7, 12-15). In
recent years, the number of publications concerning
2D LC separation of natural compounds or plant
extracts have significantly increased [1, 3, 8, 11, 1625). Among the two techniques used in various
experimental set-ups of 2D LC: heart-cutting (LCLC) and comprehensive (LC◊LC), the latter is more
popular (1-3, 8, 16, 26-28). However, several applications of heart-cutting technique have also been
described (11, 25, 29-33). In the last decade, special
attention was paid mainly to the polyphenolic plant
secondary metabolites. Researches were performed
on phenolic acids (2, 5, 26, 28, 29, 34-36) and different groups of flavonoids, (2, 5, 15, 16, 24, 25, 30,
31, 33, 35, 37-39). Also the 2D LC separations of
* Corresponding author: e-mail: krauze@gumed.edu.pl; phone: +48 58 3491960
885
886
LORETTA POB£OCKA-OLECH et al.
Vill. and S. sachalinensis Fr. Schmidt var. ëSekkaí.
The willow bark is herbal remedy used for centuries
as anti-inflammatory, antipyretic and also
antirheumatic (41). As a source of medicinal plant
material some species are used, including S. daphnoides, S. purpurea and S. alba (45). The extracts
from willow bark used in pharmaceutical industry
are standardized only on simple phenol ñ salicin
(45). Salicin and its derivatives decide about pharmacological activity of willow bark. However, also
the other phenols ñ flavonoids, phenolic acids and
catechins are considered as responsible for its
medicinal properties (46, 47). These compounds
possess differentiated antioxidant activity and may
probably participate in an anti-inflammatory activity of willow bark by synergistic effect (47, 48).
In this paper we report the use of elaborated
two-dimensional heart cutting on-line method (LCLC) for the analysis of natural compoundsí complexes present in the MeOH extracts from willow
barks of different origin.
EXPERIMENTAL
General
Standard compounds: kaempferol-3-O-glucoside (2), rutin (6), apigenin-7-O-glucoside (8), luteolin (9), luteolin-7-O-glucoside (10), naringenin-7-
Figure 1. Instrumentation
2D LC heart cutting on-line of phenolic compounds from...
O-glucoside (15), naringin (17), m-hydroxybenzoic
acid (25), isoferulic acid (41) were obtained from
Extrasynthese (Genay, France); kaempferol (1),
quercetin (4), quercetin-3-O-glucoside (5), apigenin
(7), mirycetin (13), catechin (22), salicylic acid (24),
p-hydroxybenzoic acid (26), β-resorcylic acid (28),
gentisinic acid (29), α-resorcylic acid (31), gallic
acid (32), 2,4-dimethoxybenzoic acid (33), veratric
acid (34), m-coumaric acid (38), o-coumaric acid
(39), ferulic acid (40), caffeic acid (42), 4methoxycinnamic acid (44), 3,4-dimethoxycinnamic acid (45), cinnamic acid (46), homovanillic acid
(48), homogentisinic acid (49), chlorogenic acid
(50), rosmarinic acid (51), salicin (52) from Fluka
(Buchs, Switzerland); naringenin (14) and pcoumaric acid (37) from Koch-Light (Colnbrook,
UK), and epicatechin (23), 2,3-dihydroxybenzoic
acid (27), protocatechuic acid (30), vanillic acid
(35), syringic acid (36), sinapinic acid (43), dihydrocaffeic acid (47) from Sigma (Steinheim,
Germany). Kaempferol-3-O-rhamnoside (3),
isorhamnetin (11), isorhamnetin-3-O-glucoside
(12), naringenin-5-O-glucoside (16), amentoflavone
(18), cupressuflavone (19), isosalipurposide (20),
isosalipurposide 6íí-p-coumaroyl ester (21) originated from the collection of standards of the
Department of Pharmacognosy of the Medical
University of GdaÒsk. Some of them ñ compounds
16, 20, 21 were isolated from the willow bark (49)
and their structures were elucidated on the basis of
spectroscopic methods (MS and NMR).
Acetonitrile (ACN) and methanol (MeOH) of
HPLC grade were obtained from Baker (J.T. Baker,
Deventer, The Netherlands). CH3COOH and H3PO4
of analytical grade were obtained from POCH
(Gliwice, Poland), while HCOOH of analytical
grade from Merck (Darmstadt, Germany). H2O was
prepared with a Millipore (Molsheim, France) MilliQ H2O-purification system.
Plant material and samples preparation
Plant material
The bark of Salix purpurea L. and S. daphnoides
Vill. originated from the willow collection of the
University of Warmia and Mazury from Olsztyn
(Poland). The bark of S. sachalinensis Fr. Schmidt var.
ëSekkaí was collected from the Medicinal Plants
Garden of the Medical University of GdaÒsk (Poland).
Sample preparation
Dried and pulverized willow bark (1.0 g) was
exhaustively extracted with MeOH (3 ◊ 30 mL,
60OC). The combined MeOH extracts were evaporated to dryness under reduced pressure. The dried
887
residue was dissolved in MeOH (5 mL) and after filtration through syringe filter (0.22 µm) submitted
for further HPLC analysis.
Chromatographic system
The used 2D LC system (Fig. 1) consisted of
two pumps model L-7100, two UV detectors model
L-7420 (Merck-Hitachi, Germany-Japan), two-position switching valve Model 7000 (Rheodyne, USA),
six-port switching valve Model 7725 (Rheodyne,
USA) and a column oven Jetstream 2 (MerckHitachi, Germany-Japan). The system was operated
under Eurochrom 2000 software (Knauer,
Germany).
2D LC separation of willow bark constituents
The separation in the first dimension (1D) was
performed on a Supelcosil LC-18 column (150 mm
◊ 3 mm, I.D. 3 µm) (Supelco) with linear gradient
elution from 0 to 150 min from 3% to 70% (v/v)
MeOH in H2O with 0.1% H3PO4 (v/v) (program gradient elution I). The flow rate was 0.4 mL/min and
UV detection was perfomed at λ = 280 nm. The volume of an injection loop was 2.5 mL. The flow was
split 1 : 4 by a T connector and 0.125 mm i.d. PEEK
(polyethertherketone) tubing to achieve a flow rate
of 80 µL/min towards the switching valve used as an
interface.
In the second dimension (2D) the Chromolith
Performance RP-18e column (100 mm ◊ 4.6 mm)
(Merck) and the multi-step gradient in mixtures of
ACN in H2O with 0.1% CH3COOH (v/v) (program
elution II), with increasing concentration of ACN:
S1 (5 + 95, v/v), S2 (10 + 90, v/v), S3 (15 : 85, v/v), S4
(20 : 80, v/v), S5 (30 : 70, v/v) at flow rate 1.0
mL/min were used. UV detection was performed at
λ = 210 nm. The separation on both columns was
carried out at ambient temperature. The proper fractions (Fr.) containing unresolved compounds (S.
daphnoides ñ 4 fractions: Fr. I D ñ IV D, S. purpurea
ñ 6 fractions: Fr. I P ñ VI P, S. sachalinensis ëSekkaí
ñ 5 fractions: Fr. I S ñ V S, the mixture of standards
ñ 6 fractions: Fr. I M ñ VI M) were manually transferred via switching valve from the first dimensional column directly on the second dimensional one
(Tab. 1). The volume of transferring fractions was
changeable due to their complexity and elution time
on the first dimensional column (from 128 µL to 744
µL).
However, the use of two different mechanisms
of separation in the first and second dimension is
888
Table 1. The characteristic of fractions (Fr.) and compounds transferring from first dimension (1D) and separated in second dimension
(2D) of the elaborated on-line heart-cutting LC-LC method.
Fraction
(Fr.) code
I M / S1
Transferring
time [min]
29.4-32.4
Effluent
volume [µl] *
240
II M / S3
46.5-50.1
288
III M / S2
64.1-67.6
280
IV M / S4
69.4-73.5
328
V M / S5
89.4-95.9
520
VI M / S5
115.0-117.4
192
I D / S1
II D / S2
11.3-14.6
46.0-48.7
264
216
III D / S3
80.7-84.3
288
IV D / S4
90.0-96.0
480
I P / S1
II P / S1
11.2-15.9
16.0-17.6
376
128
III P / S2
47.1-49.4
184
IV P / S2
V P / S3
50.6-54.7
89.4-97.6
328
656
VI P / S4
I S / S1
115.3-117.1
45.0-48.8
144
304
II S / S1
IV S / S3
V S / S4
50.0-53.1
80.1-89.4
89.5-95.6
248
744
488
Separated compounds (no.) (2D)
tR ± SD (2D)
2,3-Dihydroxybenzoic acid (27)
β-Resorcylic acid (28)
Syringic acid (36)
Chlorogenic acid (50)
2,4 -Dimethoxybenzoic acid (33)
m-Coumaric acid (38)
Ferulic acid (40)
o-Coumaric acid (39)
Isoferulic acid (41)
Sinapinic acid (43)
Quercetin-3-O-glucoside (5)
Rutin (6)
Apigenin-7-O-glucoside (8)
Mirycetin (13)
Isosalipurposide (20)
4-Methoxycinnamic acid (44)
Rosmarinic acid (51)
Apigenin (7)
Isorhamnetin (11)
Protocatechic acid (30)
Syringic acid (36)
Naringenin-7-O-glucoside (15)
Cinnamic acid (46)
Mirycetin (13)
Protocatechic acid (30)
α-Resorcylic acid (31)
Salicin (52)
Syringic acid (36)
Salicylic acid (24)
Rutin (6)
Mirycetin (13)
Apigenin (7)
Syringic acid (36)
Salicylic acid (24)
Cinnamic acid (46)
Rutin (6)
Mirycetin (13)
39.31 ± 0.17
38.84 ± 0.18
52.42 ± 0.34
51.71 ± 0.34
76.33 ± 0.61
4.45 ± 0.23
72.55 ± 0.23
90.82 ± 0.56
89.43 ± 0.56
94.71 ± 0.57
122.47 ± 0.53
121.52 ± 0.54
126.51 ± 0.53
129.08 ± 0.54
137.05 ± 0.55
142.71 ± 0.55
131.03 ± 0.54
123.62 ± 0.41
124.76 ± 0.41
18.41 ± 0.021
55.03 ± 0.28
54.04 ± 0.28
88.41 ± 0.15
96.93 ± 0.61
101.07 ± 0.28
106.92 ± 0.27
115.04 ± 0.27
18.73 ± 0.02
22.32 ± 0.02
21.61 ± 0.02
52.13 ± 0.32
51.25 ± 0.32
67.07 ± 0.24
102.02 ± 0.31
105.78 ± 0.31
108.19 ± 0.32
112.62 ± 0.30
123.93 ± 0.39
52.95 ± 0.44
52.17 ± 0.44
64.24 ± 0.33
108.31 ± 0.47
103.83 ± 0.47
101.85 ± 0.46
105.91 ± 0.44
108.47 ± 0.46
Abrreviations: The fractions (Fr.) transferring from first column (1D) to second column (2D): I M ñ VI M ñ standard mixture; I D ñ IV D
ñ Salix daphnoides 1095; I P ñ V P ñ Salix purpurea; I S ñ V S ñ Salix sachalinensis; S1 ñ S5 ñ mobile phases used in the second dimension; *effluent volume calculated as product of flow rate and time of fraction (Fr.) transferring.
generally preferred in 2D LC systems (1, 4, 7), for
our purposes the system consisting of two RP-18
columns was chosen. Such solution, although not
definitely orthogonal, enables significant increase of
2D LC system resolution power (33, 39). The
changes in each dimension selectivity may be performed not only by the choice of the type of RP-18
columns, but also by the use of different organic
modifiers in mobile phases (3, 7, 50). Large choice
of stationary phases in RP-LC gives a possibility to
create many configurations in RP-LC◊RP-LC (50).
Moreover, reverse phases are suitable for many different groups of analytes and therefore they can be
applied to the majority of samples (50).
In the developed 2D LC system three stages of
work can be distinguished, as it is showed on Figure
1. In the first step, the analyzed plant matrix was
separated on the first dimensional column and the
second one was simultaneously reequilibrated with
appropriate mobile phase. A stream of mobile phase
from the first dimension was split in ratio 1 : 4 and
directed in parallel to UV detector and to waste by
switching valve. Next, multi-component effluent
889
fractions were transferred manually by a change of a
position in switching valve. The six-port switching
valve without storage loop was used. A volume of
transferred fraction was changing dependently on its
complexity and elution time on first dimensional
column from 128 µL to 744 µL. However, it is well
known that in 2D LC systems injection of a large
fraction volume on the second-dimensional column
will implicate serious band broadening. This inconvenience can be minimized by employing in second
dimension the mobile phase of higher elution
strength (in comparison to the first dimension) (51).
The third step of elaborated 2D LC system comprised parallel separation processes on first- and second-dimensional columns.
It is stated, that to create a successful 2D LC
system, several parameters should be optimized,
among others: matched flow rates of mobile phases,
dimensions of columns, modulation time, miscibility
and eluent strength of mobile phases in both dimensions (1, 3-7, 20). An optimization process is simpler, when a test-set of compounds covers a wide
range of constituents expected in analyzed sample. In
Figure 2. HPLC chromatogram of heart cutting (LC-LC) separation of the 52-components test-set: I ñ the first dimension; Supelcosil LC18 column (150 ◊ 3 mm, I.D. 3 µm) gradient elution from 0 to 150 min increasing from 3% to 70% (v/v) MeOH in H2O with 0.1% H3PO4
(v/v); II ñ the second dimension; Chromolith Performance RP-18e column (100 ◊ 4.6 mm). Separations performed at differend concentration of ACN in water with 0.1% CH3COOH (v/v): S1 (5 : 95, v/v), S2 (10 : 90, v/v), S3 (15 : 85, v/v), S4 (20 : 80, v/v), S5 (30 : 70, v/v).
Experimental conditions and numbers of compounds, see text
890
Figure 3. HPLC chromatogram of heart cutting (LC-LC) separation of the MeOH extract from the bark of Salix daphnoides: I ñ the first
dimension Supelcosil LC-18 column (150 ◊ 3 mm, I.D. 3 µm), II ñ the second dimension Chromolith Performance RP-18e column (100 ◊
4.6 mm). Experimental conditions and numbers of compounds, see text
Figure 4. HPLC chromatogram of heart cutting (LC-LC) separation of the MeOH extract from the bark of S. purpurea: I ñ the first dimension Supelcosil LC-18 column (150 ◊ 3 mm, I.D. 3 µm), II ñ the second dimension Chromolith Performance RP-18e column (100 ◊ 4.6
mm). Experimental conditions and numbers of compounds, see text
891
Figure 5. HPLC chromatogram of heart cutting (LC-LC) separation of the MeOH extract from the bark of S. sachalinensis ëSekkaí: I ñ the
first dimension Supelcosil LC-18 column (150 ◊ 3 mm, I.D. 3 µm), II ñ the second dimension Chromolith Performance RP-18e column
(100 ◊ 4.6 mm). Experimental conditions and numbers of compounds, see text.
case of some plant extracts, the number of adjacent
and unrecognized peaks, which have to be resolved,
is theoretically unlimited. Regarding this fact, in
response to complication degree of plant matrices,
the use of heart-cutting technique is easier to perform, as both dimensions may be optimized separately and the equipment requirements make it possible to perform in almost every HPLC laboratory.
LC-LC separation of the extracts from willow
bark
To compare the chemical composition of three
willow barks, the 2D RP-LC system consisting of
Supelcosil LC-18 column (150 mm ◊ 3 mm) in the
first dimension (1D) and Chromolith Performance
RP-18e column (100 mm ◊ 4.6 mm) in the second
dimension (2D) was constructed. The separation conditions were optimized for the standard mixture containing 52 constituents, belonging to different groups
of compounds, isolated and identified previously in
willow bark (9-12, 41, 42): flavones (compounds 710), flavonols (1-6, 11-13), flavanones (14-17), chalcones (20, 21), biflavones (18, 19), phenolic acids
(24-51), catechins (22, 23) and salicin (52).
For the separation of standards mixture in the
first dimension on the used conventional porous particle RP-18 column, the program of gradient elution
with increasing concentration of MeOH (from 3 to
70%) in a mixture of MeOH and 0.1% solution
H3PO4 in H2O was adopted. As a result, the resolution of about 45 peaks, attributed to single compounds or co-eluting compounds was obtained at the
time tG = 150 min (Fig. 2).
Selected first-dimensional fractions (Tab. 1)
containing co-eluting compounds were on-line
directly transferred manually by the switching valve
onto the second-dimensional column.
Fractions due to their complexity were
resolved on the second column in different separation time, which varied from 3 min (Fr. VI M / S5)
to 40 min (Fr. V M / S5 (Figs. 3-5).
In two-dimensional LC systems an approach
used extensively for achieving a fast separation in
the second dimension is mainly the employment of
monolithic columns (51). This type of columns
allows very high flow rates without the problem of
back-pressure.
However, the higher flow rate
implies the higher fractions dilutions, what finally
results in worsening the sensitivity. For this reason
in the second dimension, UV detection at λ = 210
nm was used instead of λ = 280 nm as less selective
and specific.
Finally, among 52 analyzed standard compounds, 19 as single peaks were separated in the sec-
892
ond dimension (2D) on a monolithic column
employing multi-step gradient in mixtures of ACN
and 0.1% acetic acid solution in water, with increasing concentration of ACN from 5% to 30% as follows: Fr. I M / S1 (5 + 95, v/v), Fr. II M / S2 (10 +
90, v/v), Fr. III M / S3 (15 : 85, v/v), Fr. IV M / S4 (20
: 80, v/v), Fr. V M and VI M / S5 (30 : 70, v/v) (Fig.
2). After each second-dimensional separation, the
column was eluted with the next mixture of solvents, with one exception ñ the second-dimensional
separations of two fractions, Fr. V M and Fr. VI M,
containing compounds 5, 6, 8, 13, 20, 44, 51 (Fr. V
M) and 7, 11 (Fr. VI M), respectively, were performed with the same mobile phase S5 (Fig. 2). An
increase of the ACN concentration was connected
with the decrease of hydrophilic properties of compounds poorly resolved or co-eluting in the first
dimension. However, it was also important to perform fast separation in the second dimension to
make the next first-dimensional fraction transfer
possible.
Among analyzed constituents of the standards
mixture, the following phenolic acids, unresolved on
first-dimensional column, were separated in the second dimension: 2,3-dihydrobenzoic (compound 27)
and β-resorcylic (28) (Fr. I M / S1); syringic (36) and
chlorogenic (50) (Fr. II M / S2); m-coumaric (38),
2,4-dimethoxybenzoic (33) and ferulic (40) (Fr. III
M / S3), sinapinic (43), o-coumaric (39) and isoferulic (41) (Fr. IV M / S4) next to apigenin (7) and
isorhamnetin (11) (Fr. VI M / S5). The most complicated fraction from the first dimension, with volume
ca. 520 µL, contained seven compounds, namely:
rutin (6), quercetin-3-O-glucoside (5), apigenin-7O-glucoside (8), myricetin (13), rosmarinic acid
(51), isosalipurposide (20) and 4-methoxycinnamic
acid (44) (Fr. V M / S5) (Fig. 2). All these compounds were satisfactory resolved in the second
dimension on monolithic column by the use of a
mobile phase of lower or the same elution strength,
as compared to the first-dimension eluent, but possessing different selectivity (52) (Fig. 2). Moreover,
the retention times of some compounds were shorter, in comparison to their separation on the first
dimensional column, as a result of higher flow of
mobile phase and elution volume. The worked-up
system gave repeatable retention times of analyzed
peaks (Tab. 1)
One dimensional HPLC separation of MeOH
extracts of willow barks showed differences in their
chromatographic profiles dependently on the analyzed willow species, comprising the presence of
additional peaks besides peaks of compounds characteristic for this plant material.
As a consequence of this fact the different program
of multi-step gradients for the separation on the second dimension was used (Tab. 1).
Compounds, which were separated under the
same conditions of chromatographic analysis
revealed the repeatable retentions times, for example, protocatechuic acid (30) ñ in Fr. I D / S1 from
Salix daphnoides and Fr. I P / S1 from Salix purpurea
(Figs. 3 and 4). However, for the other compounds
they were different. Depending on matrices complexity some compounds were separated on first column or were carried out to the second column, for
example salicin. This compound was separated in
the Fr. II P / S1 in the extract of Salix purpurea, while
in the case of Salix daphnoides extract salicin was
separated from the other components of matrix on
the first column (Figs. 3 and 4).
The chemical composition of three willow
barks, namely S. purpurea, S. daphnoides and S.
sachalinensis ëSekkaí was compared with the use of
designed set-up of 2D LC system (Figs. 3-5). In all
extracts the presence of chlorogenic (50) and syringic (36) acids was confirmed besides unidentified
compounds, co-eluting with determined phenolic
acids in the first dimension (Figs. 3-5).
Consequently, in the extracts from S. purpurea and
S. sachalinensis ëSekkaí, the resolution on the second-dimensional column revealed the presence of
protocatechuic acid (30) next to the several unidentified peaks (Figs. 3 and 5). Furthermore, the separation of the extract from S. sachalinensis ëSekkaí in
the second dimension did not confirmed the presence of veratric acid (34), eluted as a single peak on
first dimensional chromatogram (Fig. 5). On the
other hand, analyses of the extract from S. purpurea
on the second-dimensional column, allowed to
obtain better separation of salicin (52) and α-resorcylic acid (31) from co-eluting compounds (Fig. 4).
Considering a high concentration of naringenin-7O-glucoside (15) in the extract of S. daphnoides, the
separation in the second dimension provided the resolution of cinnamic acid (46) from this flavanone
glucoside (Fig. 3).
As a result of separation in the second dimension in the extract from S. daphnoides bark
quercetin-3-O-glucoside (5), myricetin (13) and
isosalipurposide (20) as dominating compounds
were identified (Fig. 3). On the other hand, the same
fractions ñ Fr. V P / S3 from the extracts of S. purpurea and Fr. V S / S4 of S. sachalinensis ëSekkaí
which were separated on the second-dimensional
column contained respectively: rutin (6), apigenin7-O-glucoside (8) and myricetin (13) besides isosalipurposide (20), quercetin-3-O-glucoside (5), api-
genin-7-O-glucoside (8) and myricetin (13) (Figs. 4
and 5). Considering the first dimensional-separation
some differences in chromatographic profiles
between the analyzed willow barks were confirmed.
It concerns the presence of salicylic compound:
salicin (52), flavanones: naringenin-5-O-glucoside
(16), naringenin-7-O-glucoside (15) and chalcones:
isosalipurposide and its p-coumaric ester (20 and
21). These compounds were not displayed in the
extract from S. sachalinensis ëSekkaí bark, what
remains in agreement with literature data (11).
Furthermore, isosalipurposide p-coumaric ester (21)
showed to be a distinctive compound only for the
bark of S. daphnoides.
The elaborated system, regarding a wide range
of resolved compounds, may be applied in the quality control of willow bark extracts for medicinal purposes. Moreover, it makes the analysis of phenolic
acids possible without a purification step. Their
analysis in plant material requires mainly a sample
pre-treatment - purification by extraction with solvent or SPE (26, 27).
CONCLUSION
The 2D LC system was worked-up for analysis
of simple phenols and polyphenols occurring in
three willow barks: Salix daphnoides, S. purpurea
and S. sachalinensis ëSekkaí. The two-dimensional
chromatographic separations were performed with
the use of heart-cutting on-line technique. Fifty two
standard compounds were separated and their presence was analyzed in the investigated plant material
by comparison of the tR values.
Compounds belonging to the groups of simple
phenols (salicin derivatives, phenolic acids) and
polyphenols (flavanones, flavones, flavonols, chalcones, flavan-3-ols) were efficiently resolved
(Figs. 3-5). The earlier studies on the chemical
composition of willows with the use of HPLC (42,
43, 53) concerned only two groups of secondary
metabolites ñ salicin derivatives and flavonoids
(chalcones and flavanones). The elaborated 2D LC
heart-cutting on-line system enabled the chromatographic identification of several unrecognized earlier compounds in willow barks, namely apigenin,
luteolin 7-O-glucoside, quercetin, mirycetin,
quercetin 3-O-glucoside, catechin, epicatechin, αresorcylic acid, gallic acid in the Salix daphnoides
bark, quercetin, kaempferol 3-O-glucoside,
mirycetin, kaempferol 3-O-rhamnoside, rutin, apigenin 7-O-glucoside, α-resorcylic acid, gallic acid
in the S. purpurea bark, and naringenin, apigenin 7O-glucoside, luteolin 7-O-glucoside, quercetin 3-
893
O-glucoside, rutin, gallic acid in the S. sachalinensis ëSekkaí bark.
The elaborated 2D LC system can be used to
the routine analysis of the complex of biologically
active compounds present in other willows.
Moreover, the worked-up 2D LC heart-cutting system is simple and easy to construct practically in
each chromatographic laboratory.
Acknowledgments
We would like to give special thank to
Scientific Committee of the 6th Balaton Symposium
on High-Performance Separation Methods held in
Siofok (Hungary) in 2005 for distinction of the
results of this study by Best Poster Award.
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Received: 4. 08. 2015
ISSN 0001-6837
DRUG BIOCHEMISTRY
BLOCKADE OF LARGE CONDUCTANCE CA2+ ACTIVATED K+ CHANNEL
MAY PROTECT NEURONAL CELLS FROM HYPOXIA MIMETIC INSULT
AND OXIDATIVE STRESS
JIN ZHANG
School of Pharmacy and Pharmaceutical Science, Cardiff University, 2.33 Redwood Building,
King Edward VII Ave., Cathays, Cardiff, Wales, CF10 3NB, U.K.
Abstract: Previous studies have linked neuronal cell death with changes of intracellular Ca2+ ([Ca2+]i) homeostasis. Such changes of [Ca2+]i has been noticed in various neurodegenerative models. It has also been suggested that K+ channel, such as large conductance Ca2+ activated K+ channel (BK), might present a neuronal protective effect. Hence, this study has established two cell insult models, oxidative stress induced by H2O2 and
hypoxia mimetic induced by CoCl2, on a human neuronal cell line SH-SY5Y, since both insults are related to
neurodegeneration and are able to increase the [Ca2+]i. MTS assays were used to test the possible effect of BK
activators and blockers. According to the results, BK activators, NS1619 and isopimaric acid (IPA), would
potentiate toxicity to the cells under both of the two insults. But the K+ channel blockers, tetraethylammonium
(TEA) and tetrandrine, have presented the neuronal protective effect against CoCl2 insult. Considering that
[Ca2+]i, which would activate the BK channel, is the key issue in neurodegeneration, it would be suggested by
the results from this study that K+ channel blocker, rather than activator, would potentially present the neuronal
protective effect.
Keywords: oxidative stress, hypoxia mimetic, BK, MTS assay, neuroprotection
and Ca2+ influx through NMDA receptor (2). In
ALS, the Ca2+ content in motor neurones is significantly increased as well (2), and such an increase
has been linked with a copper (Cu)/zinc (Zn)-superoxide dismutase (SOD) mutation and with the death
of motor neurones (3). In AD, Ca2+ entering through
putative Ca2+ channels formed by β-amyloid (Aβ)
stemming from amyloid precursor protein (APP)
(4), and this certain Ca2+ current through an Aβ
channel has been recorded with electrophysiological
techniques previously (5). In PD, the death of
dopaminergic neurones is contributed by mitochondrial stress, with a possible role of perturbed [Ca2+]i
homeostasis downstream of the mitochondrial alterations (6). In HD, a polyglutamine expansion would
modify hunting to mutant huntingtin fragmentation,
and the latter could be switched to huntingtin
oligomers through polyglutamine expansion and/or
the increasing of hydrogen peroxide (H2O2). And the
huntingtin oligomers may then increase [Ca2+]i and
cause cell damage or death (7).
Previous studies have suggested the large conductance Ca2+ activated K+ channel (BK) as a neu-
Neurodegenerative diseases, including Alzheimerís disease (AD), Parkinsonís disease (PD),
ischemic stroke, Huntingdonís disease (HD), amyotrophic lateral sclerosis (ALS) and others are widely spread nowadays. Regarding the possible mechanism of neurodegenerative neuronal death, the overload of intracellular calcium (Ca2+) concentration is
a widely accepted hypothesis. Neurones must maintain ionic gradients and a membrane potential to
function properly in their signalling role, and even
small decrements in membrane potential can alter
firing properties and lead to significant brain dysfunction. Further, neurones need to maintain the
intracellular Ca2+ concentration ([Ca2+]i) low (~ 100
nM), hence Ca2+ entering into neurones from any
pathway could be a cause of neuronal death. In
stroke, compromised blood flow in vascular damage
results in a massive release of glutamate which can
activate the N-methyl-D-asparate (NMDA) receptor. The activation of NMDA receptor can cause a
rise in [Ca2+]i (1). In addition, membrane associated
oxidative stress can impair the function of glutamate
transporters, and promote membrane depolarization
* Corresponding author: e-mail: jin.zhang@dal.ca; phone: +1-902-4032378
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JIN ZHANG
ronal protective target (8, 9). The increasing of
[Ca2+]i would open the BK channel and cause the K+
releasing from the cells. Such K+ release would
change the membrane potential, and would spike
narrowing and thus limited Ca2+ entry through voltage gated Ca2+ channel (Cav) (10, 11). BK channel is
composed by 1 α subunit with 0ñ4 different β subunit(s). The channel area is located in α subunit, but
the modulator binding area might be located on
either α or some particular β subunit, and the later
would be related to the selectivity of BK modulators. For example, the BK channel composed with
β4 subunit would be resistant to iberiotoxin (IbTX)
which is a BK blocker (12).
Therefore, a human neuroblastoma cell line,
SH-SY5Y, which can be differentiated to human
neurone has been selected here. Whether the BK
channel is existing in neuronal cell line has been
studied first with polymerase chain reaction (PCR).
Then, the possible effect of BK channel in neuronal
cell death has been studied by testing BK activators,
NS1619 and isopimaric acid (IPA), and K+ channel
blockers, tetraethylammonium (TEA) and tetrandrine, in the cell proliferation and MTS assays with
two cell insult models. One is the oxidative stress
induced by hydrogen peroxide (H2O2) since previous studies have linked it with neurodegeneration
and suggested that H2O2 would increase the [Ca2+]i
(13). And the other one is hypoxia mimetic induced
by cobalt chloride (CoCl2) because it was related to
some neurodegenerative disease as well and was
proved to increase the generation of Aβ (14).
MATERIALS AND METHODS
Materials
SH-SY5Y cell line is from Sigma. PCR kit,
reverse transcription kit, and MTS [3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]
are from Promega. Fetal bovine serum (FBS), penicillin ñ streptomycin mixture (P/S), phosphatebuffered saline (PBS, pH = 7.4), TRIzol and
primers (designed on Primer III webpage) are from
Invitrogen. Minimum essential Eagle medium (EMEM), nutrient mixture F12 ham (F12), non
essential amino acid (NEAA), glutamine, retinoic
acid, CoCl2, H2O2, phenazine methosulfate (PMS),
chloroform (CHCl3), ethylenediaminetetraacetic
acid (EDTA), trypsin and nuclease free water
(H2O) were from Sigma-Aldrich. Ethanol
(C2H5OH) and isopropyl alcohol [(CH3)2CHOH]
were from Fisher. CoCl2 was dissolved in PBS as
10 mM for stock, and H2O2 and TEA were dis-
solved in PBS as 100 mM for stock, IbTX was dissolved in PBS as 100 µM for stock, NS1619 is dissolved in C2H5OH as 10 mM for stock, and isopimaric acid (IPA) and tetrandrine were dissolved in
DMSO as 10 mM for stock.
Cell culture
The cells are cultured in culture medium in
flasks (25 cm2 or 75 cm2) in an incubator kept at
37OC and 5% carbon dioxide (CO2). The prescription for SH-SY5Y culture is: E-MEM / F12 1 : 1,
plus 15% FBS, 2 mM glutamine, 1 ◊ NEAA and 100
u P/S. SH-SY5Y cells can be differentiated to
human neurones after 7 days of retinoic acid treatment, and the differentiation medium is the culture
medium plus 10 µM retinoic acid. The SH-SY5Y
cells with passage numbers higher than 22 were not
used in this study.
Reverse transcription ñ PCR (RT-PCR)
RNA was extracted from a confluent 75 cm2
flask of alive cells with TRIzol. The TRIzol was
removed with CHCl3 and the RNA sample was
washed twice with (CH3)2CHOH and C2H5OH,
respectively. The final RNA sample was dissolved
in H2O and its concentration was measured with a
UV spectrophotometer.
Reverse transcription has been taken to transfer
the extracted RNA into cDNA for PCR. Reverse
transcription has been done with reverse transcriptase and RNAsin kit and the same amount (1 µg) of
RNA has been used in each reaction of reverse transcription to make sure the PCR or Q-PCR results are
comparable. Negative control was run in every reaction and its prescription was the same as with the
sample only except the reverse transcriptase.
PCR has been done with Go Taq enzyme kit
and the PCR of β-actin, which is a housekeeping
gene, was applied as the positive control. The
primers for BK subunits are shown in Table 1.
The optimized PCR conditions are: 58OC as the
annealing temperature, 30 cycles.
Cell proliferation and MTS assay
Cells were taken out from a confluent 25 cm2
flask with EDTA/trypsin and seeded at a density of
15,000 per well in 100 µL of relevant culture medium without drug treatment. After the cells were
incubated at 37OC overnight, medium was aspirated
and 100 µL of insult medium (culture medium without FBS) containing insult drugs (such as H2O2 or
CoCl2) and/or modulators was added into each well.
Then, cells were incubated at 37OC for 24 h. After
the insult, 20 µL of MTS/PMS (20 : 1) were added
897
Blockade of large conductance Ca2+ activated K+ channel...
into each well. Then, cells were incubated at 37OC
for another 2 to 4 h. Finally, absorbance data were
acquired at a wavelength of 490 nm with a plate
reader.
Blank and control wells were run alongside
treatment wells. The blank is the absorbance of the
same treatments but without any cells, and the control is the absorbance of cells cultured in medium
without any drug. The absorbance of relevant blank
was subtracted from the relevant data during analysis. The final result of each sample was compared
with the one from control, and can present the relative cell number to control. Each cell proliferation
was at least in triplicate for statistics.
Statistics
In each group of results, outlier is determined
by both G test and Q test. If a datum is suggested as
an outlier by both of the two tests, it would be
removed from further statistics. T test was used for
two groupsí comparison, and ANOVA with posthoc test (Tukey, Bonferroni or Dunnett was used
under different circumstances) was used for the
comparison of three groups or more. The significance level was set as p < 0.05.
RESULTS
PCR
The PCR result (Fig. 1) has shown that all of
the 5 subunits are existing in both undifferentiated
and differentiated SH-SY5Y cells.
H2O2 insult to SH-SY5Y cell line and BK activatorís effect
A range of concentrations (100ñ600 µM) of
H2O2 have been applied on both undifferentiated and
differentiated SH-SY5Y cells in cell proliferation
and MTS assays to demonstrate the oxidative stress
insult to the cell line. Then, NS1619, which is a BK
activator targeting on α subunit (12), was combined
with the same range of H2O2 insult on both undifferentiated and differentiated SH-SY5Y cells to see
whether it would protect the cells or not. Since 3ñ30
µM of NS1619 was reported to active the BK channel in previous studies (12), the concentration 10
µM was used in this study. The results are shown in
Figure 2.
From the result above, it could be noticed that
the differentiated SH-SY5Y cells, which should be
the human neurones, were more sensitive to H2O2
Figure 1. PCR of BK subunits in SH-SY5Y cell line
Table 1. PCR primers for BK subunits.
Name
BKα, 3,537 bp mRNA
BKﬂ1, 1,518 bp mRNA
Sequence
Forward
5'-ACGCAATCTGCCTCGCAGAGTTG-3'
Reverse
5'-CATCATGACAGGCCTTGCAG-3'
Forward
5'-CTGTACCACACGGAGGACACT-3'
Reverse
5'-GTAGAGGCGCTGGAATAGGAC-3'
Forward
5'-CATGTCCCTGGTGAATGTTG-3'
Reverse
5'-TTGATCCGTTGGATCCTCTC-3'
Forward
5'-AACCCCCTTTTCATGCTTCT-3'
Reverse
5'-TCTTCCTTTGCTCCTCCTCA-3'
Forward
5'-GTTCGAGTGCACCTTCACCT-3'
Reverse
5'-TAAATGGCTGGGAACCAATC-3'
Location
1,639 - 2,047 bp
668 - 865 bp
808 - 1,044 bp
1,404 - 1,680 bp
648 - 892 bp
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JIN ZHANG
Figure 2. H2O2 insult to both undifferentiated and differentiated SH-SY5Y cells, and the effect of NS1619 against the oxidative stress on
both cells. *** p < 0.001, H2O2 insult between undifferentiated and differentiated cells; # p < 0.05, NS1619 effect to H2O2 insult on differentiated cells; n = 3
Figure 3. IPA effect to H2O2 insult on SH-SY5Y cell line (n = 3). a: on undifferentiated cells; b: on differentiated cells
insult. The difference of relative cell number at 300
µM was significant (p < 0.001) between undifferentiated and differentiated cells. It could also be found
that 10 µM NS1619 has failed to protect neither
cells from H2O2 insult, and somehow even would
aggravate the insult of oxidative stress on differentiated SH-SY5Y cells. On the differentiated cells,
10 µM of NS1619 has significantly decreased the
relative cell number from H2O2 insult at 150 µM (p
< 0.05).
Besides NS1619, the effect of IPA was tested
on the H2O2 insult model on both undifferentiated
and differentiated SH-SY5Y cells as well. IPA is
another BK activator which can active BK channel
consisting with only α subunit with EC50 at about 3
µM (12). The results are shown in Figure 3.
From Figure 3, it could be suggested that IPA
has not increased the relative cell numbers from
H2O2 insult on neither undifferentiated nor differentiated cells. On the differentiated cells, 10 µM IPA
would even present the trend to decrease the relative
cell number under high concentrations of H2O2 (the
difference was not significant in post-hoc of
ANOVA, but was significant in t-test at 450 µM, p
< 0.01). These results suggested that IPA has failed
to protect the cells from H2O2 insult neither, and
even has presented a trend to potentiate the toxic
effect on the differentiated cells.
CoCl2 insult to SH-SY5Y cell line
A range of concentrations (100 nM to 1 mM)
of CoCl2 insults have been taken on both undiffer-
entiated and differentiated SH-SY5Y cells to estimate the ED50. After the relative cell numbers under
different CoCl2 concentrations have been tested,
trend line was estimated, and the ED50 was calculated according to the trend line. The sample sizes are
3 to 9 according to different CoCl2 concentrations.
Figure 4 has demonstrated the dosage-effect curve
of CoCl2 insult to both undifferentiated and differentiated SH-SY5Y cells.
BK activators effect to CoCl2 insult on SH-SY5Y
cells
Based on the CoCl2 insult result above, BK
modulators, NS1619 and IPA as activators, as well
as K+ channel blockers, TEA and tetrandrine, have
been tested on the insult model on both undifferentiated and differentiated SH-SY5Y cells to find out
the effect of BK channel in hypoxia mimetic insult
on neurones.
Both NS1619 and IPA have been tested against
CoCl2 insult on both undifferentiated and differentiated SH-SY5Y cells. Figure 5 presents the effect of
BK activators.
From Figure 5, it would be noticed that both
NS1619 and IPA themselves would increase the cell
proliferation in both undifferentiated and differentiated SH-SY5Y cells, however both NS1619 and
IPA have aggravated the toxic effect of CoCl2.
899
K+ channel blocker effect against CoCl2 insult on
SH-SY5Y cells
Based on the results above which suggested the
potentiated toxic effect shown by BK activators, this
study has also tested some blockers of K+ channel to
see whether they would present any effect on the
opposite direction. TEA, which would inhibit K+
channel in the high-micromolar or millimolar range,
and IbTX, which would inhibit the BK channel with
IC50 at about 10 nM (12) have been tested. Besides,
a herbal source alkaloid, tetrandrine, which would
induce a flicker block to BK channel (15) has been
tested as well. Figure 6 has shown the effects of
those blockers.
From Figure 6, it could be noticed that both
TEA and tetrandrine, which are K+ channel blockers,
would protect the differentiated SH-SY5Y cells
from CoCl2 insult. The protective effect of TEA was
not significant on undifferentiated cells. But, BK
channel blocker, IbTX, has failed to present any protective effect on neither undifferentiated nor differentiated cells.
DISCUSSION
Cell insults
From Figures 3 and 4, it would be noticed that
the differentiated SH-SY5Y cells are more sensitive
Figure 4. Dosage-effect curve of CoCl2 insult to both undifferentiated and differentiated SH-SY5Y cells. It can be suggested that the differentiated cells are more sensitive to CoCl2 insult, and the differences are significant at 10 and 20 µM. The LD50 of CoCl2 to undifferentiated and differentiated cells were estimated at around 40 and 30 µM, respectively. * p < 0.05; n = 3
900
JIN ZHANG
than the undifferentiated ones to both oxidative
stress induced by H2O2 and hypoxia mimetic
induced by CoCl2. In H2O2 insult, the relative cell
number of differentiated cells was significantly
lower than the counterpart of undifferentiated cells
at 300 µM. In CoCl2 insult, the relative cell numbers
of differentiated cells were significantly lower as
well at 10 and 20 µM.
Since it is believed that both of the two
insults would increase the [Ca2+]i, according to
previous studies, it would be suggested that the
differentiated SH-SY5Y cells are more sensitive
to the [Ca2+]i. This kind of results would potentially suggest that some of the Ca2+ modulators in the
cells, such as some of the Ca2+ release channel on
the endoplasmic reticulum (ER) like ryanodine
receptor (RyR) or inositol trisphosphate (IP3),
would possibly have a higher expression in the
differentiated than in the undifferentiated SHSY5Y cells .
BK activator may aggravate the insults
From Figures 3 and 5, it could be noticed that
BK activators, NS1619 and IPA, have failed to presented any protective effect against neither oxidative
stress induced by H2O2 nor hypoxia mimetic
induced by CoCl2. And, except the negative results
shown by NS1619 and IPA on undifferentiated SHSY5Y cells with H2O2 insult, all of the other results
have indicated that the BK activators have even
aggravated the insult by significantly reduced the
relative cell numbers from insults.
Figure 5. BK activators effect to CoCl2 insult on SH-SY5Y cell line. a: NS1619 effect to CoCl2 insult on undifferentiated cells; b: IPA
effect to CoCl2 insult on undifferentiated cells; c: NS1619 effect to CoCl2 insult on differentiated cells; d: IPA effect to CoCl2 insult on differentiated cells. * p < 0.05; ** p < 0.01; ***: p < 0.001; n = 6 for control and IPA in b, and n = 3 for all the other groups
901
Figure 6. K+ channel blockers effect against CoCl2 insult on SH-SY5Y cell line. a: TEA effect on differentiated cells; b: IbTX effect on
differentiated cells; c: tetrandrine effect on differentiated cells; d: TEA effects on undifferentiated cells; e: IbTX effect on undifferentiated cells. * p < 0.05; ** p < 0.01; *** p < 0.001; n = 3
Actually, as reported, BK channel is activated
by intracellular Ca2+. Both of the two insults, H2O2
and CoCl2, would increase the [Ca2+]i and cause the
cell death, according to previous studies. In that case,
the BK channel on the cells should have been activated already when the cells were treated with
insults. Hence, if the BK channel were further activated with extra activators, the K+ efflux, and cell
shrinkage and death thereafter, might be caused (16).
Nevertheless, this result is not going against
with previous studies which suggested the neuronal
protective role of BK channel (9). Those studies
have pointed out that the activation of BK channel
would block the Ca2+ entry from extracellular sauce
by reducing the NMDA receptor mediated Ca2+
overload (11). However, obviously, the entry of
extracellular Ca2+ would not be the only cause to
increase the [Ca2+]i. Some of the intracellular Ca2+
modulators, such as RyR or IP3 on the ER, have
been proved to be involved in the anti-neurodegenerative treatment as well (17).
K+ blockers potentially have neuronal protective
effect
Since the BK activators would possibly aggravate the insults, it is reasonable to suggest that the K+
channel blocker, which acts in the opposite direction, might protect the cells from insult by avoiding
the possible K+ efflux. Actually, the data shown in
Figure 6a and 6c have suggested that both TEA and
tetrandrine, K+ channel blockers, would protect the
differentiated SH-SY5Y cells from CoCl2 insult by
significantly increasing the relative cell number.
This result consists with the findings from previous
studies. One study in 1998 has suggested that 100
µM to 5 mM of TEA would significantly reduce the
neuronal death induced by Aβ25-35 in 24ñ48 h (18).
For the negative result demonstrated by IbTX,
it is possible to be contributed by the existence of β4
subunit. Previous studies have suggested that β1
enhances the toxin binding but complexes of β4
with BK are resistant to IbTX (12). From the PCR
result shown in Fig. 1, it could be noticed that both
902
JIN ZHANG
β1 and β4 messages exist, which might be the reason for the negative results of IbTX in this study.
Society and Henry Lester Trust. Besides, I much
appreciate Prof. Kenneth T. Wann for his idea and
discussion on this study.
CONCLUSION AND FUTURE WORKS
REFERENCES
To summarize all of the results above, it could
be suggested that BK channel is existing and functional in SH-SY5Y cell line and the differentiated
SH-SY5Y cells are more sensitive to both oxidative
stress induced by H2O2 and hypoxia mimetic
induced by CoCl2.
More importantly, BK activators, NS1619 and
IPA, have failed to show any neuronal protective
effect on neither oxidative stress nor hypoxia
mimetic, and would somehow aggravate the insults
by reducing the relative cell numbers. That could be
explained by the overactivation of BK channel and
the K+ efflux caused thereafter.
In the contrast, K+ channel blockers, TEA and
tetrandrine, have demonstrated some protective
effect on differentiated SH-SY5Y cells against the
hypoxia mimetic induced by CoCl2. That consists
with previous studies and has suggested that the
blockade of K+ channel might be a new strategy for
neuronal protection. And the negative result shown
by IbTX, which could be contributed by the β4 subunit, has suggested the possible selectivity of BK
blockers. If BK channel in different organs would
have different β subunits expression, it would be
possible to find some selective BK blockers which
might target on the neurones only rather than some
other irrelative organs. This could possibly be the
future strategy of neurodegeneration treatment.
The future works would be to investigate the
expression of intracellular Ca2+ channels, such as
RyR and IP3, to see whether they would be higher
expressed in the differentiated SH-SY5Y cells. The
possible effects of K+ blockers, for example TEA
and tetrandrine, are going to be tested on the neuronal cell models with oxidative stress to see
whether they would present any protection or not. If
the K+ blockers would be neuroprotective, the β
subunits of BK channel in neurones will be studied
to develop selective BK blockers as the strategy of
neurodegeneration treatment.
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Acknowledgments
Received: 19. 06. 2015
This study is supported by School of Pharmacy
and Pharmaceutical Science, Great Britain ñ China
ISSN 0001-6837
FLUPHENAZINE AND PERPHENAZINE IMPACT ON MELANOGENESIS
AND ANTIOXIDANT ENZYMES ACTIVITY IN NORMAL HUMAN
MELANOCYTES
MICHA£ OTR BA, DOROTA WRZEåNIOK, ARTUR BEBEROK, JAKUB ROK
and EWA BUSZMAN*
School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec,
Medical University of Silesia in Katowice, Department of Pharmaceutical Chemistry,
JagielloÒska 4, 41-200 Sosnowiec, Poland
Abstract: Fluphenazine and perphenazine as a phenothiazine-class antipsychotic drugs are widely used to treat
psychoses and schizophrenia, however their use is associated with significant side effects such as extrapyramidal symptoms as well as ocular and skin disorders. The aim of this study was to examine the effect of
fluphenazine and perphenazine on cell viability, melanogenesis and antioxidant defense system in normal
human melanocytes. It has been shown that both phenothiazines induce concentration-dependent loss in cell
viability. The value of EC50 was calculated to be 1.24 and 2.76 µM for fluphenazine and perphenazine, respectively. Fluphenazine in concentration of 1.0 µM and perphenazine in concentrations of 1.0 and 3.0 µM inhibited melanogenesis and decreased microphthalmia-associated transcription factor content. To study the effect of
both analyzed drugs on antioxidant defense system in melanocytes, the level of hydrogen peroxide and the
activities of antioxidant enzymes: superoxide dismutase, catalase and glutathione peroxidase were determined.
Fluphenazine and perphenazine in higher analyzed concentrations caused depletion of melanocytes antioxidant
status, what indicated the induction of oxidative stress. The observed changes in melanization process and
antioxidant defense system in pigmented cells exposed to fluphenazine and perphenazine in vitro suggest a significant role of melanin and melanocytes in the mechanisms of undesirable side effects of these drugs in vivo,
especially directed to pigmented tissues. Moreover, the presented differences in modulation of biochemical
processes in melanocytes may be an explanation for various toxic activity of the analyzed phenothiazine derivatives in vivo.
Keywords: fluphenazine, perphenazine, melanocytes, melanogenesis, oxidative stress
Melanins are synthesized during multistep
complex process called melanogenesis. The process
is localized in specialized cytoplasmic organelles,
melanosomes, in the cytoplasm of melanocytes and
retinal pigment epithelial (RPE) cells. Disturbation
of melanogenesis may determine hypo- and/or
hyperpigmentation defects, which may occur with
or without an altered number of melanocytes (7-10).
Interestingly, fluphenazine therapy may rarely cause
vitiligoid depigmentation and chemical leukoderma,
which are an acquired, hypopigmented dermatosis
that result from repeated, cutaneous application of
an agent that destroys epidermal melanocytes (11,
12).
Specialized
pigment
dendritic
cells,
melanocytes, protect organism from ultraviolet
light, determine pigmentation of skin, hair and eyes,
Fluphenazine and perphenazine belong to the
high-potency antipsychotic drugs with a piperazinyl
structure, which are used for treatment of psychoses
and positive symptoms of schizophrenia, such as
hallucinations and delusions (1, 2). Perphenazine is
also indicated in the manic phases of bipolar disorders (2). The phenothiazine treatment is associated
with high extrapyramidal symptoms (EPS), such as
dystonia and/or akathisia, as well as with other
adverse side effects including movement disorders,
akinesia, dyskinesia, dizziness, drowsiness, sedation, severe toxicity, tachycardia, hypotension, ocular effects (e.g., blurred vision, maculopathy and
cataract) and skin disorders (e.g., rash, exfoliative
dermatitis, drug hypersensitivity syndrome, cutaneous photosensitivity and abnormal skin pigmentation) (1-6).
* Corresponding author: e-mail: ebuszman@sum.edu.pl; phone:+48-32-364-16-11
903
904
MICHA£ OTR BA et al.
reversibly bind metal ions, organic amines, cyclic
compounds and drugs, e.g., psychotropic agents or
fluoroquinolones, as well as protect from reactive
oxygen species (ROS) and oxidative stress (9, 1315). The binding of drugs to melanin probably protects organism against undesirable drug side effects,
but also may lead to accumulation of the drug in
melanin-containing tissues (especially in the eye,
ear, skin and brain) causing degeneration of pigmented cells (9, 16).
Cutaneous melanin is synthesized in melanocytes, in specialized organelles called melanosomes
during complex, multistage biochemical pathway
leading to brownish-black eumelanins and/or reddish-yellow pheomelanins. The melanogenesis
process is regulated by very important proteins like
tyrosinase, tyrosinase related proteins TRP1 and
TRP2 as well as transcription factor MITF, which is
also responsible for proliferation, dendrite formation
and induction of antiapoptotic genes expression.
Synthesized melanin is moved from melanocytes to
neighboring epidermal keratinocytes where is
responsible for skin pigmentation, UV light absorption, scavenging free radicals, chelating metal ions
and binding a variety of drugs and other xenobiotics
(7, 9, 17).
To prevent the damage of melanocytes by ROS
induced by endogenous and exogenous sources
(e.g., melanogenesis, mitochondria, lipoxygenases,
peroxisomes, ultraviolet radiation, toxins, chemicals, ionizing radiation and inflammation), cellular
antioxidant enzymes: superoxide dismutase (SOD),
catalase (CAT), glutathione peroxidase (GPx), are
activated to restore normal redox state in cells (18).
Melanin biosynthesis involves oxidation reactions
as well as superoxide anion (O2.-) and hydrogen peroxide (H2O2) generation, which subject melanocytes
to oxidative stress (18). On the other hand, melanin
acts as a free radicals scavenger and possesses
superoxide dismutase activity, what may reduce
ROS content and thus protect pigmented tissues
against cellular damage induced by oxidative stress
(7, 19).
Previously, we have documented that aminoglycoside antibiotics (20, 21) and fluoroquinolones
(22, 23), induce inhibition of cell viability and
melanogenesis in normal human epidermal
melanocytes light pigmented (HEMa-LP). We have
also demonstrated the inhibitory effect of ketoprofen
on melanization process (24), as well as modulation
of melanin biosynthesis and alterations in antioxidant defense system after nicotine treatment (25) in
normal human epidermal melanocytes dark pigmented (HEMn-DP).
The aim of this study was to estimate the effect
of fluphenazine and perphenazine on cell viability,
melanogenesis and antioxidant defense system in
normal human melanocytes dark pigmented
(HEMn-DP).
EXPERIMENTAL
Chemicals
Fluphenazine dihydrochloride, perphenazine,
phosphated-buffered saline (PBS), 3,4-dihydroxyL-phenylalanine (L-DOPA) and amphotericin B
were purchased from Sigma-Aldrich Inc. (USA).
Neomycin sulfate was obtained from Amara
(Poland). Penicillin was acquired from Polfa
Tarchomin (Poland). Growth medium M-254 and
human melanocyte growth supplement-2 (HMGS-2)
were obtained from Cascade Biologics (UK).
Trypsin/EDTA was obtained from Cytogen
(Poland). Cell proliferation reagent WST-1 was purchased from Roche GmbH (Germany). The remaining chemicals were produced by POCH S.A.
(Poland).
Cell treatment
The normal human epidermal melanocytes
(HEMn-DP, Cascade Biologics) were cultured in
M-254 basal medium supplemented with HMGS-2,
penicillin (100 U/mL), neomycin (10 µg/mL) and
amphotericin B (0.25 µg/mL) at 37OC in 5% CO2.
Cells in the passages 6-9 were used in all performed
experiments.
Cell viability assay
The viability of melanocytes was evaluated by
the WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)2H-5-tetrazolio]-1,3-benzene disulfonate) colorimetric assay according to the method described earlier (22, 26).
Measurement of melanin content
Melanin content was measured in normal
human melanocytes cultured in T-25 flasks at a density of 100,000 cells/flask. Cells were incubated for
48 h after seeding and then began 24-h fluphenazine
or perphenazine treatment in a concentration range
from 0.0001 to 1.0 µM and from 0.0001 to 3.0 µM,
respectively. After the three times washings with
PBS, cells were detached with trypsin-EDTA. Cell
pellets were placed into Eppendorf tubes, dissolved
in 100 µL of 1 M NaOH at 80OC for 1 h, and then
centrifuged for 20 min at 16,000 ◊ g. The supernatants were placed into a 96-well microplate, and
absorbance was measured at 405 nm ñ a wavelength
Fluphenazine and perphenazine impact on melanogenesis and...
at which melanin absorbs light (27). A standard synthetic melanin curve (0 to 400 µg/mL) was performed in triplicate for each experiment. Melanin
content in fluphenazine or perphenazine treated cells
was expressed in pg/cell and finally as the percentage of the controls (untreated melanocytes).
Tyrosinase activity assay
Tyrosinase activity in melanocytes was determined by measuring the rate of oxidation of LDOPA to DOPAchrome, according to the method
described previously (22). The cells were cultured at
a density of 100,000 cells/flask for 48 h and then,
incubated with fluphenazine (concentration range
from 0.0001 to 1.0 µM) or perphenazine (concentration range from 0.0001 to 3.0 µM) for 24 h. Before
lysis and clarification (centrifugation at 10,000 ◊ g
for 5 min) cells were washed three times with PBS.
Tyrosinase activity assay was performed in 96-well
plate. A tyrosinase substrate L-DOPA (2 mg/mL)
was prepared in the same lysis phosphate buffer.
Hundred µL of each lysate and 40 µL of L-DOPA
solution were pipetted into each well and incubated
at 37OC for initiation of the enzymatic assay.
Absorbance was measured every 10 min for at least
1.5 h at 475 nm using a microplate reader.
Tyrosinase activity was expressed in µmol/min/mg
protein.
Microphthalmia-associated transcription factor
(MITF) assay
Microphthalmia-associated transcription factor
(MITF) content was measured using ELISA an
assay kit (USCN Life Science Inc, USA) according
905
to the manufacturerís instruction. This kit is a sandwich enzyme immunoassay for in vitro quantitative
measurement of MITF providing a 96-well
microplate pre-coated with a biotin-conjugated antibody specific for MITF. The color change of the
enzyme (horseradish peroxidase) ñ substrate (TMB)
reaction was measured spectrophotometrically at
450 nm using a microplate reader. MITF content in
the samples was expressed in ng/mg protein.
Cellular antioxidant status assay
Superoxide dismutase (SOD), catalase (CAT),
glutathione peroxidase (GPx) activities and hydrogen peroxide (H2O2) content were measured using
an assay kits (Cayman, MI, USA and Cell Biolabs,
Inc., USA) according to the manufacturerís instructions, after 24-h incubation of human melanocytes
HEMn-DP with fluphenazine (in concentration
range from 0.0001 to 1.0 µM) or perphenazine (in
concentration range from 0.0001 to 3.0 µM). The
SOD, CAT, GPx activity and H2O2 content were
measured spectrophotometrically at 441, 540, 340
and 595 nm, respectively, using a microplate reader.
Statistical analysis
In all experiments, mean values of at least three
separate experiments (n = 3) performed in triplicate
± standard deviation (SD) were calculated.
Statistical analysis was performed with one-way
ANOVA followed by Tukey post-hoc test using
GraphPad Prism 6.01 software. The significance
level was estabilished at value of p < 0.05 (*) or p <
0.01 (**), by comparing the data with those for control (cells without drug).
Figure 1. The effect of fluphenazine and perphenazine on viability of melanocytes. Cells were treated with various drug concentrations
(0.0001-10.0 µM) and examined by the WST-1 assay. Data are expressed as percent of the controls. Mean values ± SD from three independent experiments (n = 3) performed in triplicate are presented. * p < 0.05 vs. the control samples, ** p < 0.01 vs. the control samples
906
RESULTS
The effect of fluphenazine and perphenazine on
cell viability
The cell viability was determined by the WST1 test after 24-h incubation with fluphenazine or perphenazine in a concentrations range from 0.0001
µM to 10.0 µM. It has been demonstrated that the
analyzed drugs induce concentration-dependent loss
in cell viability (Fig. 1). Melanocytes treated with
fluphenazine in concentrations from 0.001 µM to 10
µM lost 11.3% to 98% in cell viability. After incubation of cells with perphenazine in concentrations
from 0.1 µM to 10 µM, the loss of cell viability was
19.1% to 98.5%. The value of EC50 (the concentration of a drug that produces loss in cell viability by
50%) was calculated to be 1.24 and 2.76 µM, respectively for fluphenazine and perphenazine. At lower
concentrations of fluphenazine (0.0001 µM) and
perphenazine (0.0001, 0.001 and 0.01 µM) the loss
in melanocytes viability was not observed.
melanization process in melanocytes
The effectiveness of melanization process was
estimated by measuring the melanin content, cellular tyrosinase activity and microphthalmia-associated transcription factor (MITF) content in
melanocytes treated with fluphenazine (concentration range from 0.0001 to 1.0 µM) or perphenazine
(concentration range from 0.0001 to 3.0 µM) for 24
h. The melanin content per cell was determined as
Figure 2. The effect of fluphenazine and perphenazine on melanin content (A), tyrosinase activity (B) and microphthalmia-associated transcription factor (MITF) content (C) in melanocytes. Cells were cultured with 0.0001, 0.001, 0.01, 0.1 and 1.0 µM of fluphenazine or
0.0001, 0.001, 0.01, 0.1, 1.0 and 3.0 µM of perphenazine for 24 h. Data are the mean ± SD of at least three independent experiments (n =
3) performed in triplicate. * p < 0.05 vs. the control samples, ** p < 0.01 vs. the control samples
907
Figure 3. The superoxide dismutase (SOD) (A), catalase (CAT) (B), glutathione peroxidase (GPx) (C) activity and hydrogen peroxide
(H2O2) content (D) in HEMn-DP cells after 24-h incubation with 0.0001, 0.001, 0.01, 0.1 and 1.0 µM of fluphenazine or 0.0001, 0.001,
0.01, 0.1, 1.0 and 3.0 µM of perphenazine. Data are the mean ± SD of at least three independent experiments (n = 3) performed in triplicate. * p < 0.05 vs. the control samples, ** p < 0.01 vs. the control samples
57.7 to 65.4 pg/cell and 53.9 to 63.3 pg/cell for
melanocytes treated with fluphenazine and perphenazine, respectively, while the values determined for the control samples of both drugs were
62.5 ± 1.82 pg/cell and 61.3 ± 1.34 pg/cell, respectively. The obtained results, recalculated for culture
(1 ◊ 105 cells), were finally expressed as a percentage of the controls (Fig. 2A). Treatment of HEMnDP cells with 1.0 µM of fluphenazine decreased
melanin content by 7.7%, and perphenazine in concentrations of 1.0 and 3.0 µM also decreased the pig-
ment content by 10.6 and 12.1%, respectively. Both
analyzed drugs in concentrations from 0.0001 to 0.1
µM had no impact on melanin content in
melanocytes.
Tyrosinase activity in melanocytes treated with
fluphenazine or perphenazine decreased in a manner
correlating well with the effect on melanin production (Fig. 2B). The enzyme activity was determined
as 0.93 to 1.06 µmol/min/mg protein and 0.95 to
1.11 µmol/min/mg protein for melanocytes treated
with fluphenazine and perphenazine, respectively,
908
while the values determined for the control samples
of both drugs were 1.00 ± 0.03 µmol/min/mg protein
and 1.07 ± 0.03 µmol/min/mg protein, respectively.
The tyrosinase activity decreased by 6.7% for cells
treated with fluphenazine in concentration of 1.0
µM, while perphenazine in concentrations of 1.0 and
3.0 µM decreased the enzyme activity by 10.1% and
11.9%, respectively, as compared with the controls.
Fluphenazine and perphenazine in concentrations
from 0.0001 to 0.1 µM had no impact on cellular
tyrosinase activity.
The MITF content was determined as 0.14 to
0.33 ng/mg protein and 0.20 to 0.29 ng/mg protein
for melanocytes treated with fluphenazine and perphenazine, respectively, while the values determined for the control samples of both drugs were
0.29 ± 0.02 ng/mg protein and 0.26 ± 0.02 ng/mg
protein, respectively (Fig. 2C). Treatment of
HEMn-DP cells with fluphenazine in concentration
of 0.0001 µM increased MITF content by 15.6%,
while the drug in concentrations of 0.01, 0.1 and
1.0 µM decreased this transcription factor content
by 16.9, 29.9 and 52.9%. In contrast to
fluphenazine, perphenazine only decreased the
MITF content by 15.5 and 23.5% in concentrations
of 1.0 and 3.0 µM, respectiviely. Fluphenazine in
concentration of 0.001 µM and perphenazine in
concentrations from 0.0001 to 0.1 µM had no
impact on the cellular MITF content in comparison
to the control cells.
antioxidant defense system in melanocytes
To study the effect of analyzed drugs on reactive oxygen species metabolism in melanocytes, the
activity of antioxidant enzymes and the content of
hydrogen peroxide were determined. Cells were
exposed to fluphenazine (concentration range from
0.0001 to 1.0 µM) or perphenazine (concentration
range from 0.0001 to 3.0 µM) for 24 h.
Both of the analyzed drugs rised SOD activity
(Fig. 3A). The SOD activity was determined as 0.88
to 1.26 U/mg protein and 0.84 to 1.08 U/mg protein
for melanocytes treated with fluphenazine and perphenazine, respectively, while the values determined for the control samples of both drugs were
0.90 ± 0.06 U/mg protein and 0.83 ± 0.03 U/mg protein, respectively. The treatment of cells with 0.1
and 1.0 µM of fluphenazine or 1.0 and 3.0 µM of
perphenazine increased the SOD activity by 21.1
and 40.1% or by 13.3 and 30.4%, respectively, as
compared with the controls. Both analyzed drugs in
concentration range from 0.0001 to 0.01 µM had no
impact on SOD activity.
After 24-h incubation with fluphenazine or
perphenazine the intracellular CAT activity
decreased (Fig. 3B). The CAT activity was determined as 21.60 to 26.83 nmol/min/mg protein and
20.28 to 25.93 nmol/min/mg protein for
melanocytes treated with fluphenazine and perphenazine, respectively, while the values determined for the control samples of both drugs were
26.75 ± 1.50 nmol/min/mg protein and 25.33 ± 1.91
nmol/min/mg protein, respectively. Treatment of
HEMn-DP cells with 0.1 and 1.0 µM of
fluphenazine or 1.0 and 3.0 µM of perphenazine
decreased the CAT activity by 14.1 and 19.2% or by
13.9 and 19.9%, respectively, as compared with the
controls. Both analyzed drugs in concentration
range from 0.0001 to 0.01 µM had no impact on
CAT activity.
Fluphenazine modified GPx activity, while
perphenazine decreased the enzyme activity in
melanocytes (Fig. 3C). The GPx activity was determined as 14.70 to 20.83 nmol/min/mg protein and
13.61 to 15.95 nmol/min/mg protein for cells treated with fluphenazine and perphenazine, respectively, while the values determined for the control
samples of both drugs were 17.84 ± 0.47
nmol/min/mg protein and 15.24 ± 1.10
nmol/min/mg protein, respectively. Treatment of
melanocytes with 0.1 µM of fluphenazine caused
an increase in GPx activity by 16.8%, while the
concentration 1.0 µM decreased the enzyme activity by 17.6%. Perphenazine in concentration of 3.0
µM decreased GPx activity by 10.7%. Both analyzed drugs in lower concentrations had no impact
on GPx activity.
After 24-h incubation of melanocytes with the
analyzed drugs, the hydrogen peroxide (H2O2) content increased in a concentration-dependent manner
(Fig. 3D). The H2O2 content was determined as
158.81 to 276.98 µmol/mg protein and 155.31 to
232.50 µmol/mg protein for melanocytes treated
with fluphenazine and perphenazine, respectively,
while the values determined for the control samples
of both drugs were 153.39 ± 2.09 µmol/mg protein
and 156.41 ± 9.37 µmol/mg protein, respectively.
The treatment of cells with fluphenazine in concentrations from 0.001 to 1.0 µM increased the H2O2
content by 9.2 to 80.6%, while perphenazine in concentrations from 0.01 to 3.0 µM increased the hydrogen peroxide content by 12.7 to 48.7%, as compared
with the controls. Fluphenazine in concentration of
0.0001 µM and perphenazine in concentrations
0.0001 and 0.001 µM had no impact on H2O2 content
in cells.
DISCUSSION
Like other phenothiazine derivatives,
fluphenazine and perphenazine have been reported
to exhibit a variety of new, important biological
effects such as anticancer, antibacterial, antiprotozoic, antiviral and antimultidrug resistance reversal
activity. The biological activities of phenothiazinebased compounds are strongly dependent on their
chemical structure, intrinsic physical properties and
their ability to aggregate with themselves and solvent molecules (28, 29).
Phenothiazine derivatives treatment may
potentially lead to harmful toxic effects.
Fluphenazine treatment is associated with extrapyramidal symptoms in 97% of patients, incident depression in 55% of patients, blurred vision, excess salivation as well as dose-related changes in prolactin
levels. Similar side effects like acute extrapyramidal
symptoms and hyperprolactinemia are observed
during perphenazine therapy (30).
Interestingly, in the treatment of psychotic disorders, the daily dosage of fluphenazine up to 40
mg/day (31) is relatively lower in comparison to
other phenothiazines: chlorpromazine more than
800 mg/day (32), perphenazine up to 64 mg/day
(33) and thioridazine up to 800 mg/day (34).
Because of the fact that long-term thioridazine therapy with a daily dose of 100 mg is required to trigger chorioretinal changes and the highest dose (800
mg/day) can induce a peripheral retinopathy, as well
as that fluphenazine therapy 2 mg/day may lead to
toxic retinopathy (maculopathy) ñ fluphenazine may
be considered to be more toxic drug than other phenothiazines (5, 35).
Our previous studies showed the ability of phenothiazine derivatives: chlorpromazine, fluphenazine, trifluoperazine and thioridazine to form stable
complexes with model synthetic as well as natural
melanin (14, 36), which may be responsible for
toxic effects of these drugs directed to pigmented
tissues.
In this study, we have used the culture of normal human epidermal melanocytes as an in vitro
experimental model system. It was observed that
fluphenazine in the lowest concentration (0.0001
µM) and perphenazine in concentration range from
0.0001 to 0.01 µM did not have any significant effect
on melanocytes viability. The treatment of cells with
both drugs in higher concentrations resulted in the
loss in cell viability in a concentration-dependent
manner (Fig. 1). The value of EC50 was calculated to
be 1.24 and 2.76 µM for fluphenazine and perphenazine, respectively. For the same cell line the
909
value of EC50 established for chlorpromazine was
2.53 µM (26), what indicates that fluphenazine is
more cytotoxic than chlorpromazine and perphenazine. Taking into account the value of EC50, the
order of drugs cytotoxicity would be as follows:
fluphenazine >> chlorpromazine > perphenazine.
The analysis of melanogenesis process in cells
cultured in the presence of fluphenazine showed that
the drug in concentration of 1.0 µM decreased
tyrosinase activity and melanin content.
Perphenazine in concentrations of 1.0 and 3.0 µM
also decreased the enzyme activity, as well as
melanin content (Fig. 2A and 2B). Our results
demonstrate that both drugs at higher studied concentrations (= 1.0 µM) suppress melanogenesis in
normal human melanocytes, probably due to their
inhibitory effect on tyrosinase activity.
Because of the fact that MITF is a major transcriptional regulator of melanogenesis, cell survival
and differentiation of melanocytes, we have examined the effect of fluphenazine and perphenazine on
the content of this protein. Fluphenazine in concentration of 0.0001 µM increased the MITF content,
while in the concentrations from 0.01 to 1.0 µM significantly decreased the content of this protein.
Perphenazine in concentrations of 1.0 and 3.0 µM
also decreased the content of MITF (Fig. 2C). The
obtained results indicate that fluphenazine possesses
greater inhibitory effect on MITF than perphenazine, what may explain that only during
fluphenazine therapy hypopigmentation disorders,
such as vitiligoid depigmentation and chemical
leukoderma, occur. Moreover, the observed inhibition of melanogenesis process and decrease in cellular viability in the presence of fluphenazine and perphenazine is probably caused by the suppression of
microphthalmia-associated transcription factor.
In contrast to fluphenazine and perphenazine,
chlorpromazine used in lower concentrations
(0.0001, 0.001 and 0.01 µM) increased melanin and
MITF content as well as tyrosinase activity in
HEMn-DP cells, without any impact on melanogenesis in higher drug concentrations (0.1 and 1.0 µM)
(26). This may explain the occurence of photodistributed hyperpigmentation in patients during chlorpromazine therapy.
Reactive oxygen species are produced during
normal cellular metabolic processes, under pathologic conditions as well as by many exogenous factors including UV radiation, xenobiotics and drugs.
The overproduction of pro-oxidant species in cells
and/or reduction of cellular antioxidant capacity
leads to oxidative stress and results in damage of
nucleic acids, lipids, and proteins causing cell muta-
910
genesis or death (18, 37, 38). The human body has a
complex antioxidant defense system which contains
small-molecule antioxidants (e.g., glutathione, uric
acid, vitamin C, vitamin E) as well as antioxidant
enzymes such as SOD, GPx and CAT (37).
In the present study, we have examined the
effect of fluphenazine and perphenazine on the
activity of antioxidant enzymes. Fluphenazine in
concentrations of 0.1 and 1.0 µM and perphenazine
in concentrations of 1.0 and 3.0 µM significantly
increased SOD activity, decreased CAT activity and
exerted different effect on GPx activity (Fig. 3A, 3B
and 3C). The observed changes in antioxidant
enzymes activity after exposure of melanocytes to
the analyzed drugs might be the main reason for
overproduction of superoxide anion and elevated
level of H2O2 (Fig. 3D) that cannot be eliminated.
This indicates that antioxidant defense system does
not work properly. The obtained results also indicate
that fluphenazine possesses greater stimulatory
effect on SOD formation, produces higher levels of
hydrogen peroxide and therefore induces oxidative
stress in melanocytes, what confirms that
fluphenazine is more toxic than perphenazine.
The fluphenazine and perphenazine induced
changes in SOD and GPx activities were also stated
by other authors. Abdalla et al. (39) showed the
increased SOD and decreased GPx activity as well
as an accumulation of H2O2 in red blood cells in
schizophrenic patients treated with fluphenazine and
chlorpromazine in combination with 8 different
antipsychotic drugs. Michel et al. (40) demonstrated
the increase of SOD activity in the brain of patients
with schizophrenic psychosis treated with
fluphenazine and Zhang et al. (41) suggested that
increased SOD level in schizophrenic patients, treated with fluphenazine, perphenazine, trifluperazine
and chlorpromazine in combination with 3 different
antipsychotic drugs, may be a response to increased
oxidative stress associated with neuroleptic treatment (40, 41).
One has to take into consideration that
fluphenazine and perphenazine concentrations
found to have a modulatory effect on melanogenesis
and antioxidant defense system in normal human
melanocytes are about 10- to 100-fold higher for
fluphenazine and 20- to 60-fold higher for perphenazine than the concentrations normally
observed in blood (42). However, we have previously demonstrated that phenothiazine derivatives
form complexes with melanin, what may lead to the
accumulation of these drugs in melanin reach tissues
(14, 36). Such accumulation increases the drug concentration and may induce some toxic effects on
melanin containing cells and surrounding tissues.
Thus, it is possible that fluphenazine and perphenazine concentrations in melanocytes may be
significantly higher than that in blood and therefore
the reduction of melanin content, the inhibition of
tyrosinase activity as well as the depletion of cellular antioxidant status in the presence of these drugs
could be observed in vivo.
CONCLUSION
Due to the fact that phenothiazine derivatives
are the subject of new researches, it is important to
explain molecular mechanisms underlying these
drugs toxic effects. The present work provides the
first in vitro study on mechanisms involved in
fluphenazine and perphenazine induced disorders in
HEMn-DP cells. The observed changes in melanization process and antioxidant defense system in pigmented cells exposed to the analyzed drugs in vitro
suggest a significant role of melanin and melanocytes
in the mechanisms of undesirable side effects of these
drugs in vivo. The presented differences in modulation of biochemical processes in melanocytes may be
an explanation for various toxic activity of the analyzed phenothiazine derivatives in vivo.
Declaration of interest
All authors declare that there are no conflicts
of interest.
Acknowledgment
This work was financially supported by the
Medical University of Silesia in Katowice, Poland
(Grants No. KNW-2-002/D/5/K and KNW-1041/N/5/0).
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Received: 20. 07. 2015
ISSN 0001-6837
DRUG SYNTHESIS
SYNTHESIS OF IMIDAZO[2,1-b][1,3,4]THIADIAZOLE DERIVATIVES
AS POSSIBLE BIOLOGICALLY ACTIVE AGENTS
SUJEET KUMAR1, BASAVARAJ METIKURKI2, VIVEK SINGH BHADAURIA1,
ERIK DE CLERCQ3, DOMINIQUE SCHOLS3, HARUKUNI TOKUDA4 and SUBHAS S. KARKI1*
1
Department of Pharmaceutical Chemistry, KLE Universityís College of Pharmacy,
Bengaluru 560010, India
2
Department of Pharmaceutical Chemistry, Vivekananda College of Pharmacy, Bengaluru 560010, India
3
Rega Institute for Medical Research, KU Leuven, B-3000 Leuven, Belgium
4
Department of Complementary and Alternative Medicine, R&D Graduate School of Medical Sciences,
Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8640, Japan
Abstract: A series of 2-(4-methylbenzyl)-5,6-substituted-imidazo[2,1-b][1,3,4]thiadiazole derivatives were
synthesized, characterized and evaluated for antiproliferative activity and cancer chemopreventive activity.
Results showed that molecules with formyl and thiocyanate substituents at the 5 position exhibited an increase
in activity against the full panel of 60 human tumor cell lines at a minimum of five concentrations at 10-fold
dilutions. Derivative 22 displayed significant in vitro anticancer activity against colon cancer (MID GI50 = 0.75
µM). The cancer chemopreventive effect of 19 (IC50 = 489 nM) was almost equipotent to standard oleanolic
acid (IC50 = 449 nM).
Keywords: synthesis, imidazo[2,1-b][1,3,4]thiadiazole, antiproliferative, in vivo two-stage mouse-skin carcinogenesis test, cancer chemopreventive agents
EXPERIMENTAL
An ongoing project of interest in our laboratories are imidazo[2,1-b][1,3,4]thiadiazoles [I],
which display anticancer activities and they act by
inducing apoptosis without arresting the cell cycle
(1-3). Levamisole [II] acts by stimulating the
responsiveness of lymphocytes to tumor antigen
and is most effective in patients with small tumor
burden (4). In addition, levamisole derivatives
such as imidazo[2,1-b]thiazoles [III] have been
reported as anticancer agents (5) (Fig. 1). Review
of the literature revealed that the imidazo[2,1b][1,3,4]thiadiazole scaffold possesses diverse
pharmacological properties such as antisecretory
(6), anti-inflammatory (7, 8), cardiotonic (9),
diuretic (10), antitubercular (11, 12), antibacterial
(13-17), antifungal (18, 19), anticonvulsant, analgesic (20), anticancer (21) and herbicidal activities (22, 23).
The objectives of the present investigations
were to develop analogs of levamisole [II] as possible biologically active agents [IV].
Chemistry
The melting points are uncorrected. Silica gel
plates were used for TLC by using mobile phases
CHCl3/MeOH in various proportions. The IR spectra were recorded in KBr discs on a Jasco 430+
apparatus; the 1H NMR spectra were recorded on a
Bruker (400 MHz), and J values are reported in
hertz (Hz). Mass spectra were recorded in triple
quadrapole LCMS-6410 from Agilent Technologies. Required substituted phenacyl bromides and
3-(bromoacetyl)-2H-chromen-2-one were prepared
according to literature (1-3). Compounds 4, 18, 19,
23 and 24 were prepared according to the literature
(3).
General procedure for the synthesis of imidazo[2,1-b][1,3,4]thiadiazoles (4-12)
The 2-amino-5-aralkyl-1,3,4-thiadiazole 1, or
2 (30 mmol) was treated with the respective
* Corresponding author: e-mail: subhasskarki@gmail.com
913
914
SUJEET KUMAR et al.
phenacyl bromide 3 (30 mmol), in ethanol (150
mL). The mixture was refluxed for 12 h. Excess of
solvent was removed under reduced pressure and the
solid hydrobromide was separated by filtration,
washed with cold ethanol and dried. Neutralization
of hydrobromide salts with cold aqueous solution of
sodium carbonate yielded the corresponding free
base which was filtered with good yield.
6-(4-Chlorophenyl)-2-(4-methylbenzyl)imidazo[2,1-b][1,3,4]thiadiazole (5)
Obtained according to the general procedure
for the synthesis of imidazo[2,1-b][1,3,4]thiadiazoles. IR (KBr, cm-1): 3026 (C-H), 2909-2737 (CH), 1525 (>C=C), 1470 (C=N); 1H-NMR (400 MHz,
DMSO-d6, δ, ppm): 8.66 (1H, s), 7.86 (2H, d, J = 8
Hz), 7.44 (2H, d, J = 8 Hz), 7.28 (2H, d, J = 8 Hz),
7.18 (2H, d, J = 8 Hz), 4.37 (2H, s, -CH2-), 2.28 (3H,
s, CH3). MS (ESI) m/z: 340.1 (339.8).
6-(4-Bromophenyl)-2-(4-methylbenzyl)imidazo[2,1-b][1,3,4]thiadiazole (6)
for the synthesis of the imidazo[2,1-b][1,3,4]thiadiazoles. IR (KBr, cm-1): 3029 (C-H), 2924-2875 (CH), 1609 (>C=C), 1505 (C=N). 1H-NMR (400 MHz,
DMSO-d6, δ, ppm): 7.80 (2H, d, J = 8 Hz), 7.58 (2H,
d, J = 8 Hz), 7.28 (2H, d, J = 8 Hz), 7.19 (2H, d, J =
8 Hz), 4.37 (2H, s, -CH2-), 2.28 (3H, s, CH3). MS
(ESI) m/z: 386.1 (384.3).
6-(4-Fluorophenyl)-2-(4-methylbenzyl)imidazo[2,1-b][1,3,4]thiadiazole (7)
for the synthesis of imidazo[2,1-b][1,3,4]thiadiazoles. IR (KBr, cm-1): 3025 (C-H), 2909-2733 (CH), 1522 (>C=C), 1478 (C=N). 1H-NMR (400 MHz,
DMSO-d6, δ, ppm): 8.60 (1H, s), 7.88-7.84 (2H, m,
arom.), 7.26 (2H, d, J = 8 Hz), 7.24 (4H, m, arom.),
4.37 (2H, s, -CH2-), 2.29 (3H, s, CH3). MS (ESI)
m/z: 324.0 (323.4).
2-(4-Methylbenzyl)-6-(4-nitrophenyl)imidazo[2,1-b][1,3,4]thiadiazole (8)
for the synthesis of imidazo[2,1-b][1,3,4]thiadiazoles. IR (KBr, cm-1): 3029 (C-H), 2922ó2833
(C-H), 1601 (>C=C), 1508 (C=N). 1H-NMR (400
MHz, DMSO-d6, δ, ppm): 8.92 (1H, s), 8.27 (2H, d,
J = 8 Hz), 8.10 (2H, d, J = 8 Hz), 7.30 (2H, d, J = 8
Hz), 7.20 (2H, d, J = 8 Hz), 4.40 (2H, s, -CH2-), 2.29
(3H, s, -CH3).
2-(4-Methylbenzyl)-6-(2,4-dichlorophenyl)imidazo[2,1-b][1,3,4]thiadiazole (9)
for the synthesis of the imidazo[2,1-b][1,3,4]thiadiazoles. IR (KBr, cm-1): 3022 (C-H), 2954-2851 (CH), 1518 (>C=C), 1482 (C=N). 1H-NMR (400 MHz,
DMSO-d6, δ, ppm): 8.66 (1H, s), 8.09 (1H, d, J = 8
Hz), 7.67 (1H, m, arom.), 7.50-7.48 (1H, m, arom.),
Figure 1. Chemical structures of levamisole and its analogs as cytotoxic and anticancer agents
Synthesis of imidazo[2,1-b][1,3,4]thiadiazole derivatives as...
7.29 (2H, d, J = 8 Hz), 7.19 (2H, d, J = 8 Hz), 4.40
(2H, s, -CH2-), 2.29 (3H, s, -CH3).
2-(4-Methylbenzyl)-6-(4-methylphenyl)imidazo[2,1-b][1,3,4]thiadiazole (10)
for the synthesis of the imidazo[2,1-b][1,3,4]thiadiazoles. IR (KBr, cm-1): 3024 (C-H), 2913-2730
(C-H), 1518 (>C=C), 1460 (C=N). 1H-NMR (400
MHz, DMSO-d6, δ, ppm): 8.54 (1H, s), 7.73 (2H, d,
J = 8 Hz), 7.28 (2H, d, J = 8 Hz), 7.20-7.17 (4H, dd,
J = 8, 8 Hz), 4.37 (2H, s, -CH2-), 2.30 (3H, s, -CH3),
2.28 (3H, s, -CH3).
6-(4-Methoxyphenyl)-2-(4-methylbenzyl)imidazo[2,1-b][1,3,4]thiadiazole (11)
Obtained according to the general procedure for
the synthesis of the imidazo[2,1-b][1,3,4]thiadiazoles.
IR (KBr, cm-1): 3011 (C-H), 2936-2732 (C-H), 1604
(>C=C), 1477 (C=N). 1H-NMR (400 MHz, DMSO-d6,
δ, ppm): 8.49 (1H, s), 7.76 (2H, d, J = 8 Hz), 7.28 (2H,
d, J = 8 Hz), 7.19 (2H, d, J = 8 Hz), 6.96 (2H, d, J = 8
Hz), 4.36 (2H, s, -CH2-), 3.76 (3H, s, CH3), 2.29 (3H,
s, CH3). MS (ESI) m/z: 336.2 (335.4).
3-[2-(4-Methylbenzyl)imidazo[2,1-b][1,3,4]thiadiazol-6-yl]-2H-chromen-2-one (12)
for the synthesis of the imidazo[2,1-b][1,3,4]thiadiazoles. IR (KBr, cm-1): 3025 (C-H), 2911-2778
(C-H),1711 (>C=O), 1518 (>C=C),1426 (C=N).
General procedure for the preparation of 5-bromoimidazo[2,1-b][1,3,4]thiadiazole derivatives (13-17)
To the respective imidazo[2,1-b][1,3,4]thiadiazole (5 mmol) 2, sodium acetate (10 mmol) and 10
mL of glacial acetic acid stirred together at room
temperature was added dropwise bromine (6 mmol).
After the addition, stirring was continued for 30
min. The mixture was poured into 100 mL of water
from which a solid was separated. The solid was collected by filtration and washed with water, dried and
crystallized from EtOH-CHCl3 with good yield.
5-Bromo-6-(4-chlorophenyl)-2-(4-methylbenzyl)imidazo[2,1-b][1,3,4]thiadiazole (13)
for the synthesis of 5-bromo-imidazo[2,1-b][1,3,4]thiadiazoles. IR (KBr, cm-1): 3030 (C-H), 2870-2771
(C-H),1617 (>C=C), 1443 (C=N). 1H-NMR (400
MHz, DMSO-d6, δ, ppm): 7.97 (2H, d, J = 8 Hz),
7.54 (2H, d, J = 8 Hz), 7.30 (2H, d, J = 8 Hz), 7.20
(2H, d, J = 8 Hz), 4.43 (2H, s, -CH2-), 2.29 (3H, s,
-CH3). MS (ESI) m/z: 418.0 (418.0).
915
5-Bromo-6-(4-bromophenyl)-2-(4-methylbenzyl)imidazo[2,1-b][1,3,4]thiadiazole (14)
for the synthesis of the 5-bromo-imidazo[2,1b][1,3,4]thiadiazoles. IR (KBr, cm-1): 3046 (C-H),
2871-2755 (C-H),1518 (>C=C), 1426 (C=N). 1HNMR (400 MHz, DMSO-d6, δ, ppm): 7.90 (2H, d, J
= 8 Hz), 7.68 (2H, d, J = 8 Hz), 7.30 (2H, d, J = 8
Hz), 7.20 (2H, d, J = 8 Hz), 4.43 (2H, s, -CH2-), 2.29
(3H, s, CH3). MS (ESI) m/z: 462.0 (463.2).
5-Bromo-6-(4-fluorophenyl)-2-(4-methylbenzyl)imidazo[2,1-b][1,3,4]thiadiazole (15)
2922-2726 (C-H), 1597 (>C=C), 1477 (C=N). 1HNMR (400 MHz, DMSO-d6, δ, ppm): 7.98-7.94
(2H, m, arom.), 7.33-7.28 (4H, m, arom.), 7.18 (2H,
d, J = 8 Hz) 4.43 (2H, s, -CH2-), 2.29 (3H, s, CH3).
MS (ESI) m/z: 403.2 (402.3).
5-Bromo-2-(4-methylbenzyl)-6-phenylimidazo[2,1-b][1,3,4]thiadiazole (16)
2850-2736 (C-H), 1613 (>C=C), 1448 (C=N). 1HNMR (400 MHz, DMSO-d6, δ, ppm): 7.95 (2H, d, J
= 8 Hz), 7.46 (2H, d, J = 8 Hz), 7.37-7.33 (1H, m,
arom.), 7.30 (2H, d, J = 8 Hz), 7.20 (2H, d, J = 8
Hz), 4.42 (2H, s, -CH2-), 2.29 (3H, s, -CH3). MS
(ESI) m/z: 386.1 (384).
5-Bromo-2-(4-methylbenzyl)-6-(4-methylphenyl)imidazo[2,1-b][1,3,4]thiadiazole (17)
the synthesis of the 5-bromo-imidazo[2,1-b][1,3,4]thiadiazoles. IR (KBr, cm-1): 3026 (C-H), 2921-2729 (C-H),
1515 (>C=C), 1472 (C=N). 1H-NMR (400 MHz,
DMSO-d6, δ, ppm): 8.34 (2H, d, J = 8 Hz), 8.24 (2H, d,
J = 8 Hz), 7.31 (2H, d, J = 8 Hz), 7.20 (2H, d, J = 8 Hz),
4.45 (2H, s, -CH2-), 2.29 (3H, s, -CH3).
General procedure for the preparation of the imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehydes (1822)
The Vilsmeier reagent was prepared at 0-5OC
by dropping POCl3 (2.3 g, 15 mmol) into a stirred
solution of DMF (10 mL). The respective imidazo[2,1-b][1,3,4]thiadiazole 2 (4 mmol) was added
slowly to the Vilsmeier reagent while maintaining
stirring and cooling for 2 h. Further stirring was continued for 6 h at 80-90OC. The resulting reaction
916
SUJEET KUMAR et al.
mixture was poured into 100 mL of water; the precipitate was filtered, pressed, suspended in water
and neutralized to pH 7 with cold aqueous solution
of sodium carbonate. The solid was separated by filtration, washed with water, dried and crystallized
from EtOH with a yield of 45-58%.
2-Benzyl-6-(4-methoxyphenyl)imidazo[2,1-b][1,
3,4]thiadiazole-5-carbaldehyde (19)
for the synthesis of the imidazo[2,1-b] [1,3,4]thiadiazole-5-carbaldehydes. IR (KBr, cm-1): 3072 (C-H),
2991-2875 (C-H), 1681 (>C=O), 1600 (>C=C),
1481 (C=N). 1H-NMR (400 MHz, DMSO-d6, δ,
ppm): 10.01 (1H, s, -CHO), 7.80 (2H, d, J = 8 Hz),
7.42-7.34 (5H, m, arom.), 7.04 (2H, d, J = 8 Hz),
4.45 (2H, s, -CH2-), 3.88 (3H, s, -CH3). MS (ESI)
m/z: 350.1 (349.41).
6-(4-Chlorophenyl)-2-(4-methylbenzyl)imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde (20)
for the synthesis of the imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehydes. IR (KBr, cm-1): 3040 (C-H),
2858-2714 (C-H) 1668 (>C=O), 1619 (>C=C), 1436
(C=N). 1H-NMR (400 MHz, DMSO-d6, δ, ppm):
9.99 (1H, s, CHO), 8.00 (2H, d, J = 8 Hz), 7.58 (2H,
d, J = 8 Hz), 7.31 (2H, d, J = 8 Hz), 7.20 (2H, d, J =
8 Hz), 4.49 (2H, s, -CH2-), 2.29 (3H, s, CH3). MS
(ESI) m/z: 368.1 (367.9).
6-(4-Fluorophenyl)-2-(4-methylbenzyl)imidazo[2,
1-b][1,3,4]thiadiazole-5-carbaldehyde (21)
for the synthesis of the imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehydes. IR (KBr, cm-1): 3035 (C-H),
2900-2736 (C-H), 1669 (>C=O), 1601 (>C=C),
1447 (C=N). 1H-NMR (400 MHz, DMSO-d6, δ,
ppm): 9.97 (1H, s, -CHO), 8.03-8.00 (2H, m, arom.),
7.36-7.32 (2H, m, arom.), 7.29 (2H, d, J = 8 Hz),
7.18 (2H, d, J = 8 Hz), 4.48 (2H, s, -CH2-), 2.29 (3H,
s, CH3). MS (ESI) m/z: 352.4 (351.4).
6-(4-Methoxyphenyl)-2-(4-methylbenzyl)imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde (22)
the synthesis of the imidazo[2,1-b][1,3,4]thiadiazole5-carbaldehydes. IR (KBr, cm-1): 3046 (C-H), 28762740 (C-H), 1678 (>C=O), 1598 (>C=C), 1464
9.95 (1H, s, -CHO), 7.92 (2H, d, J = 8 Hz), 7.31 (2H,
d, J = 8 Hz), 7.20 (2H, d, J = 8 Hz), 7.07 (2H, d, J =
8 Hz), 4.47 (2H, s, -CH2-), 3.82 (3H, s, OCH3), 2.29
(3H, s, CH3). MS (ESI) m/z: 364.2 (363.4).
General procedure for the preparation of the imidazo[2,1-b][1,3,4]thiadiazol-5-yl-thiocyanates (2331)
To a mixture of imidazo[2,1-b][1,3,4]thiadiazole 2 (10 mmol) and potassium thiocyanate (1.56
g,16 mmol) in glacial acetic acid (50 mL) was added
bromine (1.6 g, 10 mmol) in glacial acetic acid (20
mL) at 0-5OC, dropwise with stirring. Stirring was
continued for 30 min at 15-20OC and then at room
temperature for 1 h. The reaction mixture was
poured into ice water, filtered, washed with water
and crystallized from EtOH-CHCl3 with a yield of
50-57%.
6-(4-Chlorophenyl)-2-(4-methylbenzyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl-thiocyanate (25)
for the synthesis of the imidazo[2,1-b][1,3,4]thiadiazol-5-yl-thiocyanates. IR (KBr, cm-1): 3029 (C-H),
2924-2735 (C-H), 2161 (-C=N), 1598 (>C=C), 1467
(C=N). 1H-NMR (400 MHz, CDCl3, δ, ppm): 7.94
(2H, d, J = 8.8 Hz), 7.50 (2H, d, J = 8.8 Hz), 7.267.20 (4H, m, arom.), 4.38 (2H, s, -CH2-), 2.17 (3H,
s, CH3). MS (ESI) m/z: 395.0 (396.9).
6-(4-Bromophenyl)-2-(4-methylbenzyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl-thiocyanate (26)
the synthesis of the imidazo[2,1-b][1,3,4]thiadiazol5-yl-thiocyanates. IR (KBr, cm-1): 3028 (C-H), 29232735 (C-H), 2159 (-C=N), 1513 (>C=C), 1467
(2H, d, J = 8 Hz), 7.75 (2H, d, J = 8 Hz), 7.32 (2H, d,
J = 8 Hz), 7.21 (2H, d, J = 8 Hz), 4.51 (2H, s, -CH2), 2.29 (3H, s, CH3). MS (ESI) m/z: 441.0 (441.4).
6-(4-Fluorophenyl)-2-(4-methylbenzyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl-thiocyanate (27)
2919-2821 (C-H), 2162 (-C=N), 1603 (>C=C), 1467
8.01-7.97 (2H, m, arom.), 7.40-7.36 (2H, m, arom.),
7.30 (2H, d, J = 8 Hz), 7.19 (2H, d, J = 8 Hz), 4.50
(2H, s, -CH2-), 2.29 (3H, s, CH3). MS (ESI) m/z:
381.0 (380.5).
2-(4-Methylbenzyl)-6-(4-nitrophenyl)imidazo[2,
1-b][1,3,4]thiadiazol-5-yl-thiocyanate (28)
2854-2737 (C-H), 2124 (-C=N), 1605 (>C=C), 1473
(2H, d, J = 8 Hz), 8.21 (2H, d, J = 8 Hz), 7.26-7.19
(4H, m, arom.), 4.38 (2H, s, -CH2-), 2.38 (3H, s,
CH3). MS (ESI) m/z: 406.1 (407.5).
6-(4-Methoxyphenyl)-2-(4-methylbenzyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl-thiocyanate (29)
2912-2736 (C-H), 2135 (-C=N), 1599 (>C=C), 1423
(2H, d, J = 8 Hz), 7.32 (2H, d, J = 8 Hz, 7.21 (2H,
d, J = 8 Hz), 7.11 (2H, d, J = 8 Hz), 4.49 (2H, s,
-CH2-), 3.82 (3H, s, -OCH3), 2.30 (3H, s, CH3). MS
(ESI) m/z: 391.1 (392.5).
2-(4-Methylbenzyl)-6-(4-methylphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl-thiocyanate (30)
2917-2731 (C-H), 2157 (-C=N), 1515 (>C=C),1467
(C=N). 1H-NMR (400 MHz, CDCl3, δ, ppm): 7.887.82 (2H, m, arom.), 7.31-7.19 (6H, m, arom.), 4.36
(2H, s, -CH2-), 2.42 (3H, s, CH3), 2.37 (3H, s, CH3).
MS (ESI) m/z: 399.1 (M + Na).
2-(4-Methylbenzyl)-6-(2-oxo-2H-chromen-3-yl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl-thiocyanate
(31)
the synthesis of the imidazo[2,1-b][1,3,4]thiadiazol-5yl-thiocyanates. IR (KBr, cm-1): 3029 (C-H), 29202747 (C-H), 2160 (-C=N),1705 (>C=O),1607 (>C=C),
1470 C=N). 1H-NMR (400 MHz, CDCl3, δ, ppm): 8.23
(1H, s, arom.), 7.62-7.58 (2H, m, arom), 7.42-7.39
(1H, m, arom.), 7.36-7.32 (1H, m, arom.), 7.25-7.20
(4H, m, arom.), 4.39 (2H, s, -CH2-), 2.37 (3H, s, CH3).
Pharmacological screening
NCI 60 DTP human tumor cell line screening anticancer activity
The DTP anticancer drug discovery program is
a two stage process, beginning with the evaluation
of all selected compounds against the 60 cell lines at
a single dose of 10-5 M. Compounds which show significant growth inhibition are evaluated against the
60 cell line panel at five concentration levels.
The tumor growth inhibition properties of the
selected eleven compounds 11, 12, 13, 18, 20, 21,
22, 23, 27, 29 and 31 were screened on human tumor
cell lines at 10-5 M concentration at the NCI, NIH,
Bethesda, Maryland, under the drug discovery pro-
917
gram of the NCI. Among the tested compounds, six
compounds namely 11, 12, 20, 22 and 31 were further screened for 5-log dose molar range as they
showed prominent cell growth inhibition at 10-5 M
against variety of cell lines.
Methodology of the in vitro cancer screen
The human cancer cell lines of the cancer
screening panel are grown in RPMI 1640 medium
containing 5% fetal bovine serum at 2 mM L-glutamine. For a typical screening experiment, cells are
inoculated into 96 well microtiter plates in 100 mL
at plate densities ranging from 5000 to 40,000
cells/well depending upon the doubling time of individual cell lines. After cell inoculation, the
microtiter plates were incubated at 37OC, 5% CO2,
95% air and 100% relative humidity for 24 h prior to
addition of experimental drugs.
After 24 h, two plates of each cell lines were
fixed in situ with TCA, to represent a measurement
of the cell population for each cell line at the time of
drug addition (Tz). Experimental drugs were solubilized in dimethyl sulfoxide at 400-fold the desired
final maximum test concentration and stored frozen
prior to use. At the time of drug addition, an aliquot
of frozen concentrate was thawed and diluted to
twice the desired final maximum test concentration
with complete medium containing 50 mg/mL gentamicin. Additional four, 10-fold or 1/2 log serial
dilutions were made to provide a total of five drug
concentrations plus control. Aliquots of 100 mL of
these different drug dilutions were added to the
appropriate microtiter wells already containing 100
mL of medium, resulting in the required final drug
concentration.
Following drug addition, the plates are incubated for an additional 48 h at 37OC, 5% CO2, 95%
air and 100% relative humidity. For adherent cells,
the assay was terminated by the addition of cold
TCA. Cells were fixed in situ by the gentle addition
of 50 mL of cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 min at 4OC.
The supernatant was discarded and the plates were
washed five times with tap water and air dried.
Sulforhodamine (SRB) solution (100 mL) at 0.4%
(w/v) in 1% acetic acid was added to each well, and
plates were incubated for 10 min at room temperature. After staining, unbound dye was removed by
washing five times with 1% acetic acid and the
plates were air dried. Bound stain was subsequently
solubilized with 10 mM trizma base, and the
absorbance was read on an automated plate reader at
a wavelength of 515 nm. For suspension cells, the
methodology was the same except that the assay was
918
SUJEET KUMAR et al.
terminated by fixing settled cells at the bottom by
the wells by gentle adding 50 mL of 80% TCA (final
concentration 16% TCA). Using the seven
absorbance measurements [time zero, (Tz), control
growth, (C) and the test growth in the presence of
drug at five concentration levels (Ti)], the percentage growth was calculated at each of the drug concentrations levels. Percentage growth inhibition is
calculated as:
[(Ti - Tz)/ (C - Tz)] ◊ 100 for concentrations
for which Ti ≥ Tz
[(Ti ñ Tz)/Tz] ◊ 100 for concentrations for which
Ti < Tz
Three dose response parameters were calculated for each experimental agent. Growth inhibition of
50% (GI50) is calculated from [(Ti - Tz)/(C - Tz)] ◊
100 = 50, which is the drug concentration resulting
in a 50% reduction in the net protein increase (as
measured by SRB staining) in control cells during
the drug incubation.
The drug concentration resulting in total
growth inhibition (TGI) is calculated from Ti = Tz.
The LC50 concentration of the drug resulting in 50%
reduction in the measured protein at the end of the
drug treatment as compared from [(Ti - Tz)/Tz] ◊
100 = -50. Values are calculated for each of these
parameters if the level of activity is reached; however, if the effect is not reached or is exceeded, the
value for that parameter is expressed as greater or
less than the maximum or minimum concentration
tested (24-26).
Primary single high dose (10-5 M) full NCI 60 cell
panel in vitro assay
All the selected compounds were submitted to
the National Cancer Institute (NCI) for in vitro anticancer assay. They were evaluated for their anticancer activity primarily in an in vitro one dose anticancer assay in full NCI 60 cell panel representing
leukemia, melanoma, lung, colon, brain, breast,
ovary, kidney and prostate cancers in accordance
with the protocol of the NCI. The compounds were
added at a single concentration (10-5 M) and the culture was incubated for 48 h. End point determinations were made with a protein binding dye, sulforhodamine B. Results for each compound were
reported as a mean graph of the percent growth of
the treated cells when compared to the untreated
control cells. After the results were obtained for one
dose assay, analysis of historical Development
Therapeutics Programm (DTP) was performed and
compounds 11, 12, 20, 22, 29 and 31 which satisfied
pre-determined threshold inhibition criteria and
were selected for NCI full panel 5 dose assays.
Cancer chemoprevention activity-cells
EBV genome-carrying lymphoblastoid cells
(Raji cells derived from Burkittís lymphoma) were
cultured in 10% fetal bovine serum (FBS) in RPMI1640 under the conditions described previously
(27). Spontaneous activation of EBV-EA in our subline of Raji cells was less than 0.1%.
Inhibition of EBV-EA activation assay
EBV-EA-positive serum from a patient with
nasopharyngeal carcinoma (NPC) was a gift from
the Department of Biochemistry, Oita Medicinal
University. The EBV genome-carrying lymphoblastoid cells (Raji cells derived from Burkittís lymphoma) were cultured in 10% fetal bovine serum
(FBS) in RPMI-1640 medium (Nissui). Spontaneous activation of EBV-EA in our sub-line Raji
cells was less than 0.1%. The inhibition of EBV-EA
activation was assayed using Raji cells (virus nonproducer type) as described previously (28). The
indicator cells (Raji cells, 1-106/mL) were incubated at 37OC for 48 h in 1 mL of a medium containing
n-butyric acid (4 mmol), TPA (32 pmol = 20 ng in
dimethyl sulfoxide (DMSO), 2 mL) as an inducer
and various amounts of test compounds in 5 mL
DMSO. Smears were made from the cell suspension, and the activated cells that were stained by
EBV-EA-positive serum from NPC patients were
detected by an indirect immunofluorescence technique (29). In each assay, at least 500 cells were
counted, and the number of stained cells (positive
cells) present was recorded. Triplicate assays were
performed for each compound. The average EBVEA induction of the test compounds was expressed
as a relative ratio to the control experiment (100%)
which was carried out only with n-butyric acid (4
mmol) plus TPA (32 pmol). EBV-EA induction was
ordinarily around 35%. The viability of treated Raji
cells was assayed by the Trypan Blue staining
method.
Animals
Specific pathogen-free female ICR mice (6
weeks old, body weight approx. 30 g) were obtained
from Japan SLC Inc. (Shizuoka, Japan), and the animals were housed, five animals per polycarbonate
cage, in a temperature-controlled room at 24 ± 2OC
and given food and water ad libitum throughout the
experiment.
Two-stage mouse-skin carcinogenesis test
Animals were divided into three experimental
groups containing 15 mice each. The back (2 ◊ 8
cm2) of each mouse was shaved with surgical clip-
pers, and the mice were topically treated with 7,12dimethylbenz[a]anthracene (DMBA) (100 mg, 390
nmol) in acetone (0.1 mL) as the initial treatment.
One week after initiation, papilloma formation was
promoted twice weekly by the application of 12-Otetradecanoylphorbol-13-acetate (TPA) (1 mg, 1.7
nmol) in acetone (0.1 mL) to the skin. One hour
before each treatment with TPA, the mice were
treated with the samples (85 nmol) in acetone (0.1
mL). The incidence of papillomas was examined
weekly over a period of 20 weeks (28).
Cytotoxicity in human and murine tumor cell
lines
All the compounds in Scheme 1 were evaluated for their cytostatic activity against human HeLa
cervix carcinoma cells, human CEM CD4þ T-lymphocytes as well as murine L1210 cells (30). All
919
assays were performed in 96-well microtiter plates.
To each well were added (5-7.5) ◊ 104 tumor cells
and a given amount of the test compound. The cells
were allowed to proliferate for 48 h (murine
leukemia L1210 cells) or 72 h (human lymphocytic
CEM and human cervix carcinoma HeLa cells) at
37OC in a humidified CO2-controlled atmosphere. At
the end of the incubation period, the cells were
counted in a Coulter counter. The IC50 (50%
inhibitory concentration) was defined as the concentration of the compound that inhibited cell proliferation by 50%.
Chemistry
In order to determine the structure activity relationship, 28 derivatives were prepared according to
Scheme 1. Synthesis of 2,5,6-substituted imidazo[2,1-b][1,3,4]thiadiazole derivatives. Reagents and conditions: i) Br2, CH3COOH. ii)
DMF, POCl3, 80-90OC, Na2CO3, iii) KSCN, Br2, CH3COOH
920
SUJEET KUMAR et al.
Table 1. Physicochemical properties.
Comp.
No.
Molecular
formula
Molecular
weight
%
yield
M.p.
(OC)
Rf
value*
4
C17H12FN3S
309.36
59
152-155
0.62
5
C18H14ClN3S
339.84
60
226-28
0.62
6
C18H14BrN3S
384.29
65
152-56
0.74
7
C18H14FN3S
323.39
66
149-50
0.58
8
C18H14N4O2S
350.39
58
170-72
0.66
9
C18H13Cl2N3S
374.29
50
244-45
0.71
10
C19H17N3S
319.42
49
180-82
0.60
11
C19H17N3OS
335.42
55
138-40
0.70
12
C21H15N3O2S
373.43
62
172-74
0.48
13
C18H13BrClN3S
418.74
64
92-94
0.59
14
C18H13Br2N3S
463.19
68
128-30
0.76
15
C18H13BrFN3S
402.28
60
98-100
0.44
16
C18H14BrN3S
384.29
50
109-111
0.80
17
C19H16BrN3S
398.32
50
150-52
0.48
18
C18H12FN3OS
337.37
48
112-114
0.62
19
C19H15N3O2S
349.41
52
101-103
0.53
20
C19H14ClN3OS
367.85
58
218-19
0.45
21
C19H14FN3OS
351.40
48
202-04
0.55
22
C20H17N3O2S
363.43
62
50-52
0.66
23
C19H14N4OS2
378.47
55
120-122
0.64
24
C18H12N4S2
348.44
59
98-100
0.62
25
C19H13ClN4S2
396.92
50
156-58
0.88
26
C19H13BrN4S2
441.37
48
164-65
0.63
27
C19H13FN4S2
380.46
50
100-02
0.59
28
C19H13N5O2S2
407.47
65
68-70
0.51
29
C20H16N4OS2
392.50
60
157-59
0.59
30
C20H16N4S2
376.50
60
74-76
0.53
31
C22H14N4O2S2
430.50
40
120-122
0.69
*Mobile phase: chloroform : ethanol (0.95 : 0.05, v/v).
Elemental analysis calcd./found
(%)
C
H
N
66.00
3.91
13.58
59.98
3.90
13.55
63.62
4.15
12.36
63.57
4.14
12.34
56.26
3.67
10.93
56.21
3.51
11.01
66.85
4.36
12.99
66.81
4.25
13.01
61.70
4.03
15.99
61.65
4.00
16.05
57.76
3.50
11.23
57.81
3.52
11.15
71.44
5.36
13.15
71.25
5.28
13.24
68.03
5.11
12.53
67.94
5.00
12.61
67.54
4.05
11.25
67.41
3.98
11.32
51.63
3.13
10.03
51.25
3.11
10.10
46.67
2.83
9.07
46.49
2.78
9.11
53.74
3.26
10.45
53.59
3.21
10.51
56.26
3.67
10.93
56.19
3.61
11.00
57.29
4.05
10.55
57.15
4.06
10.49
64.08
3.59
12.46
63.97
3.55
12.51
65.31
4.33
12.03
65.02
4.24
12.11
62.04
3.84
11.42
61.98
3.75
11.59
64.94
4.02
11.96
65.00
4.00
11.91
66.10
4.71
11.56
65.99
4.65
11.71
60.30
3.73
14.80
60.18
3.66
14.91
62.05
3.47
16.08
61.95
3.41
16.21
57.49
3.30
14.12
57.12
3.28
14.15
51.70
2.97
12.69
51.55
2.91
12.75
59.98
3.44
14.73
59.77
3.39
14.81
56.01
3.22
17.19
55.95
3.15
17.25
61.20
4.11
14.27
61.15
4.05
14.31
63.80
4.28
14.88
63.71
4.31
14.91
61.38
3.28
13.01
61.15
3.25
13.09
the strategy outlined in Scheme 1 and Table 1. The
required α-bromoketones were used in the first step;
they were prepared according to the literature (2, 3)
by using bromine in acetic acid. Derivatives 2,6substituted imidazo[2,1-b][1,3,4]thiadiazoles (4-12)
were prepared by condensing 2-amino-1,3,4-thiadiazole (1 and 2) with α-bromoketones (3) in anhydrous ethanol in good yields. The bromination step
was carried out in glacial acetic acid by using
bromine in good yields (13-17). The formylation
was performed by standard Vilsmeier conditions to
give derivatives (18-22). The introduction of a thiocyanate was performed by treatment with bromine
and potassium thiocyanate to give derivatives (2331).
All the synthesized compounds exhibited
absorption bands ranging from 3059 to 3021 cm-1
for C-H aromatic stretching and 2954-2714 cm-1 for
C-H aliphatic stretching. Stretching vibration
bands of CHO for compounds 20-22 showed
between 1677 and 1668 cm-1. While derivatives 25
to 31 exhibited vibration bands for C≡N between
2165-2124 cm-1 in their respective IR spectra. In 1H
NMR, imidazole proton (C5-H) confirms the
cyclization of 2-amino-5-(4-methylbenzyl)-1,3,4thiadiazole 1 or 2 with respective phenacyl bromide 3 by the presence of singlet between δ 8.92
and 8.49 ppm. Bromination, formylation and thiocyanation reactions were performed on imidazo[2,1-b][1,3,4]thiadiazole derivatives 4-12, and
afforded the respected 5-substituted derivatives
(13-17, 18-22, 23-31). All these 5-substituted
derivatives showed the absence of C5-H in their
respective spectra and confirmed the substitution at
C-5 position. Derivatives 20-22 showed a singlet
between δ 9.99 to 9.95 ppm for the CHO proton.
Aromatic protons showed prominent signals
around δ 8.34-6.96 ppm. Bridge headed methylene
proton at C2 appeared between δ 4.52 to 4.36 ppm.
The presence of OCH3 protons in compounds 11,
22 and 29 exhibited as singlet between δ 3.82-3.76
ppm. Methyl proton appeared as singlet between δ
2.38-2.17 ppm. The structures of all the compounds were finally ascertained by mass spectra.
In vitro 5 dose full NCI 60 cell panel assay and
discussion
All the cell lines (about 60), representing nine
tumor subpanels, were incubated at five different
concentrations (0.01, 0.1, 1, 10 and 100 µM). The
outcomes were used to create log concentration vs. %
growth inhibition curves and three response parameters (GI50, TGI and LC50) were calculated for each
cell line. The GI50 value (growth inhibitory activity)
921
corresponds to the concentration of the compound
causing 50% decrease in net cell growth, the TGI
value (cytostatic activity) is the concentration of the
compound resulting in total growth inhibition and
LC50 value (cytotoxic activity) is the concentration of
the compound causing net 50% loss of initial cells at
the end of the incubation period of 48 h.
Derivative 20 (NSC: D753993) exhibited anticancer activity against Melanoma SK-MEL-2 cell
line with GI50: 0.77 µM, and derivative 12 (NSC:
D753984) was active against Colon Cancer HCT-15
cell line with GI50: 0.87 µM) and Melanoma Cancer
UACC-62 and M14 cell line with GI50: 0.54 and
0.96
µM,
respectively.
Compound
22
(NSC:D753991) exhibited significant anticancer
activity against most of the tested cell lines representing nine different subpanels with GI50 values
ranging between 0.23 and 11.7 µM and emerged as
potential candidate of the series (Table 2).
Compound 31 (NSC: D753989) showed potent anticancer activity against Leukemia K-562 and SR cell
lines with GI50: 0.43 and 0.47 µM, respectively,
Non-Small Cell Lung Cancer NCI-H522 cell line
(GI50: 0.60 µM) and Colon Cancer KM12 and HCC2998 cell line with GI50: 0.75 and 0.91 µM, respectively (Table 2).
The criterion for selectivity of a compound
depends upon the ratio obtained by dividing the full
panel MIDa (the average sensitivity of all cell lines
toward the test agent) by their individual subpanel MIDb
(the average sensitivity of all cell lines of a particular
subpanel toward the test agent). The ratios greater than
6 indicates high selectivity for the corresponding cell
line, while ratios between 3 and 6 referred as moderate
selectivity and not meeting either of these criteria rated
them as non-selective (26). According to these criterion,
compounds 29 and 22 exhibited moderate (MIDa/MIDb:
3.21 and 3.25, respectively) selectivity towards prostate
and colon cancer cell line, respectively, whereas compound 11 was found to be highly selectivity towards
Leukemia, Colon Cancer subpanel with selectivity ratio
of 8.35 and 6.08, respectively. Another compound, 12
was found to be moderately selective towards CNS and
Breast cancer cell line with selectivity ratio 5.61 and
5.87, respectively. Remaining derivatives were found to
be non-selective against remaining cell panel (Table 2).
Chemoprevention activity
The compounds (4-31) were subjected to in
vitro inhibition assay against EBV-EA activation.
The results of the in vitro and in vivo anti-tumor
promoting activities have been reported for the
newly synthesized analogs (4-31). EBV-EA is activated by tumor promoters, producing viral early
24.2
97.0
NCI-H226
NCI-H23
NA
4.29
SW-620
HCT-15
3.28
NA
HCT-116
KM12
>100
HCC-2998
HT29
NA
>100
COLO 205
8.88
17.0
HOP-92
NCI-H522
13.4
HOP-62
19.6
23.0
EKVX
>100
>100
A549/ATCC
NCI-H460
2.83
SR
NCI-H322M
NA
>100
RPMI-8226
K-562
MOLT-4
3.61
1.84
HL-60 (TB)
>100
11
CCRF-CEM
Panel/Cell Line
Compound
3.79
29.01
2.76
MIDb
6.08
0.79
8.35
MIDa/
MIDb
17.8
3.20
7.68
0.87
12.8
4.34
70.2
2.47
20.1
45.2
52.4
>100
3.64
>100
13.4
19.4
6.22
42.7
58.3
1.27
15.8
20.7
12
16.7
22.4
24.2
MIDb
1.21
0.91
0.84
MIDa/
MIDb
MIDa/
MIDb
2.68
1.27
Leukemia
MIDb
6.21
4.37
2.00
2.72
2.46
3.31
2.78
3.46
1.81
2.96
6.39
2.75
4.18
1.24
3.67
2.34
0.97
3.01
1.13
COLON Cancer
3.50
22
0.32
3.16
0.85
0.38
0.36
1.57
0.43
0.56
0.46
0.47
0.50
1.41
1.45
0.46
1.89
10.3
1.94
2.55
1.85
3.41
1.55
2.95
Non-Small Cell Lung Cancer
2.36
2.15
4.13
1.76
3.24
2.43
20
Table 2. Nine subpanels at five concentrations: growth inhibition (GI50) activity (µM) of selected compounds.
0.75
2.98
1.11
MIDb
3.25
0.82
2.20
MIDa/
MIDb
8.29
3.52
8.40
6.48
6.05
5.26
>100
3.80
3.88
19.5
7.19
3.45
6.36
5.41
3.82
6.34
2.98
4.42
4.86
8.17
3.06
3.93
29
6.33
6.63
4.57
1.92
1.83
2.66
MIDb MIDa/
MIDb
1.52
0.75
2.27
2.15
2.27
0.91
8.28
0.60
2.89
10.1
7.26
18.7
18.4
3.24
4.46
4.84
0.49
4.47
5.98
0.43
1.12
9.30
31
2.59
7.83
3.63
MIDb
2.81
0.93
2.01
MIDa/
MIDb
922
SUJEET KUMAR et al.
>100
83.1
7.73
17.2
OVCAR-5
OVCAR-8
NCI/ADRRES
SK-OV-3
29.9
IGROV1
62.2
6.47
UACC-62
>100
>100
UACC-257
OVCAR-4
>100
OVCAR-3
>100
39.4
MALME-3M
SK-MEL-5
8.01
LOX IMVI
SK-MEL-28
18.5
U251
8.91
13.5
SNB-75
MDA-MB435
23.6
SNB-19
NA
18.0
SF-539
1.41
8.14
SF-295
SK-MEL-2
26.3
SF-268
M14
11
Compound
Table 2. Cont.
40.02
12.84
18.00
MIDb
0.58
1.80
1.28
MIDa/
MIDb
>100
5.14
>100
>100
77.3
8.89
26.6
0.54
53.8
6.60
56.3
3.01
1.48
0.96
26.9
1.05
21.8
47.9
>100
30.9
7.23
>100
12
29.3
16.7
27.0
MIDb
0.69
1.21
0.75
MIDa/
MIDb
6.97
2.34
2.91
6.43
5.17
2.38
3.96
3.42
4.71
3.08
7.60
2.98
0.77
2.44
4.94
3.02
2.54
3.07
4.39
2.16
2.15
3.59
20
MIDa/
MIDb
1.14
0.93
4.30
0.79
Ovarian Cancer
3.66
Melanoma
2.98
CNS Cancer
MIDb
2.27
0.36
3.41
5.89
4.77
0.62
3.53
0.94
11.2
1.22
11.3
2.53
0.23
0.48
11.7
0.75
1.19
1.23
2.80
0.80
0.90
2.50
22
2.97
4.48
1.57
MIDb
0.82
0.54
1.55
MIDa/
MIDb
25.8
4.69
5.79
15.2
7.62
4.25
47.1
4.35
>100
11.0
90.3
4.67
5.54
5.67
7.83
3.78
3.71
6.76
6.40
7.18
2.38
5.78
29
15.77
16.64
5.37
0.77
0.73
2.26
MIDb MIDa/
MIDb
3.14
0.45
13.3
28.2
5.86
0.37
81.1
0.45
>100
0.48
13.8
10.1
0.26
0.36
10.7
0.66
1.54
0.55
1.39
0.78
0.63
2.89
31
18.91
4.60
1.30
MIDb
0.39
1.58
5.61
MIDa/
MIDb
923
15.7
>100
>100
23.4
>100
21.7
>100
>100
68.8
9.60
12.5
13.9
NT
84.8
2.71
23.1
ACHN
CAK-1
RXF 393
SN12C
TK-10
UO-31
PC-3
DU-145
MCF7
MDA-MB231/ATCC
HS 578T
BT-549
T-47D
MDA-MB468
MIDa
31.3
786-0
A498
11
Compound
Table 2. Cont.
24.70
68.8
23.05
MIDb
0.93
0.34
1.00
MIDa/
MIDb
20.3
7.22
21.0
>100
40.7
17.7
11.2
40.7
35.1
64.7
>100
>100
>100
1.46
1.70
45.1
>100
12
19.6
37.9
28.3
MIDb
1.04
0.54
0.72
MIDa/
MIDb
3.4
2.26
3.44
3.56
3.09
2.81
3.23
2.44
3.31
2.84
2.90
4.39
7.98
3.00
3.46
4.23
4.50
20
MIDa/
MIDb
0.82
1.18
3.06
1.11
Breast Cancer
2.88
Prostate Cancer
4.16
Renal Cancer
MIDb
2.4
0.33
1.98
NT
1.02
1.55
0.51
1.94
3.67
2.37
2.21
2.87
3.23
0.77
2.58
1.18
2.47
22
1.07
2.80
2.21
MIDb
2.28
0.87
1.10
MIDa/
MIDb
12.2
>100
>100
NT
3.57
5.25
>100
>100
3.78
2.95
>100
60.2
52.8
>100
22.5
11.9
55.0
29
4.41
3.78
34.22
2.75
3.21
0.35
MIDb MIDa/
MIDb
7.3
0.40
1.35
NA
0.32
3.14
0.99
7.56
2.31
8.34
18.3
80.4
6.36
1.96
3.00
0.22
0.80
31
1.25
4.93
14.92
MIDb
5.87
1.48
0.49
MIDa/
MIDb
924
SUJEET KUMAR et al.
925
antigen (EA), and the evaluation of its inhibitors is
used as a primary screen for in vivo anti-tumor promoting activities (27). The in vitro inhibitory activities of compounds 4-31 against EBV-EA activation
are shown in Table 3. Oleanolic acid was used as
standard to compare with the test compounds 4-31.
Their effects on the viability of Raji cells and their
50% inhibitory concentration (IC50) values are
shown in Table 3. The in vitro results showed moderate to mild cytotoxicity against cell line (Table 3).
The inhibitory activities were as follows: 19 (IC50
489 mol ratio/32 pmol/TPA), 11 (IC50 = 495), 10
(IC50 = 500), 23 (IC50 = 501) and 24 (IC50 = 505).
Compound 19 is a 5-formyl derivative, and the
EBV-EA activation was lower than those of the
other compounds. The relative rates of 19 with
respect to TPA (100%) were 10.5, 42.6, 74.6 and
100%, at concentrations of 1000, 500, 100 and 10
mol ratio/TPA (Table 3), showing 89.5, 47.4, 25.4,
(compound 19) inhibition of TPA-induced EBV-EA
Table 3. Relative ratioa of EBV-EA activation levels (%) in the presence of compounds 4-31 and oleanolic acid.
Comp.
No.
a
Concentration
(mol ratio / TPA)
IC50
(nM)
1000
500
100
10
4
14.3 (60) ± 0.3
49.6 ± 1.4
80.0 ± 1.7
100 ± 1.9
5
15.9 (60) ± 0.5
48.3 ± 1.3
79.1 ± 1.9
100 ± 1.9
519
6
17.9 (50) ± 0.5
54.6 ± 1.5
84.3 ± 1.8
100 ± 1.7
539
7
14.0 (60) ± 0.5
48.3 ± 1.3
79.3 ± 1.5
100 ± 1.6
509
8
16.1 (60) ± 0.6
53.0 ± 1.5
81.9 ± 1.6
100 ± 1.7
531
510
9
18.0 (60) ± 0.7
54.3 ± 1.6
83.9 ± 1.9
100 ± 1.9
542
10
12.1 (60) ± 0.3
43.6 ± 1.3
76.4 ± 1.6
100 ± 1.8
500
11
11.7 (60) ± 0.3
42.3 ± 1.2
75.3 ± 1.6
100 ± 1.7
495
12
--
--
--
--
NT
13
18.3 (50) ± 0.6
55.6 ± 1.4
84.8 ± 1.7
100 ± 1.9
548
14
19.9 (50) ± 0.8
58.7 ± 1.5
86.2 ± 2.0
100 ± 1.9
561
15
16.8 (60) ± 0.6
52.7 ± 1.5
82.0 ± 2.0
100 ± 1.9
534
16
17.4 (50) ± 0.5
53.8 ± 1.5
83.2 ± 2.0
100 ± 2.0
535
17
17.0 (50) ± 0.6
53.6 ± 1.6
83.1 ± 2.0
100 ± 1.9
535
18
13.8 (60) ± 0.4
48.7 ± 1.2
79.2 ± 2.0
100 ± 1.9
507
19
10.5 (60) ± 0.4
42.6 ± 1.1
74.6 ± 1.3
100 ± 1.7
489
20
15.3 (60) ± 0.4
49.7 ± 1.4
79.3 ± 1.7
100 ± 1.8
521
21
12.9 (60) ± 0.3
47.3 ± 1.4
78.1 ± 1.6
100 ± 1.7
508
22
--
--
--
--
NT
23
12.8 (60) ± 0.5
43.8 ± 1.2
76.6 ± 1.4
100 ± 1.9
501
24
13.9 (60) ± 0.5
44.9 ± 1.3
78.3 ± 1.6
100 ± 1.8
505
25
16.5 (60) ± 0.5
44.9 ± 1.3
78.3 ± 1.6
100 ± 1.8
529
26
18.1 (50) ± 0.7
56.2 ± 1.4
85.3 ± 1.7
100 ± 1.8
554
27
15.6 (60) ± 0.4
51.1 ± 1.4
81.3 ± 1.8
100 ± 1.7
523
28
17.0 (60) ± 0.6
53.9 ± 1.4
82.1 ± 1.8
100 ± 1.9
533
29
14.1 (60) ± 0.4
49.3 ± 1.2
80.3 ± 1.6
100 ± 1.8
511
30
14.9 (60) ± 0.4
51.2 ± 1.5
81.4 ± 1.8
100 ± 1.8
516
31
--
--
--
--
NT
Oleanolic acidc
12.7 (70)
30.0
80.0
100
449
The values obtained in the assay where the EBV-EA activation was performed by treatment with TPA (32 pmol) alone (without adding
any triterpenoids) was evaluated as 100%. bValues in parentheses are the percentage viability of Raji cells. cA standard sample to compare
the inhibitory activities of 1-28 against EBV-EA activation. NT = not tested.
926
SUJEET KUMAR et al.
activation. Compounds 10, 11, 23 and 24 exhibited
mild inhibitory activity. The other compounds did
not show preferable inhibitory activities.
Thus, we selected compounds 14 and 19
among the 28 compounds to examine their effects
on in vivo two-stage carcinogenesis using mouseskin papillomas induced by DMBA as an initiator
and TPA as a promoter. During the in vivo test, the
body weight gains of the mice were not influenced
by treatment with the test compounds and no toxic
effects, such as lesional damage and inflammation
(edema, erosion and ulcer), were observed in areas
of mouse skin topically treated with the test compounds.
Figure 2. Inhibitory effects of compound 14 & 19 on mouse-skin carcinogenesis induced by DMBA and TPA (after 20 weeks). a. Positive
control [DMBA (390 nmol) + TPA (1.7 nmol); b. TPA + 85 nmol of compound 14; c. TPA + 85 nmol of compound 19
927
Table 4. Inhibitory effects of compound 14 and 19 on two-stage mouse skin carcinogenesis.
Weeks of
treatment
DMBA (390 nmol) + TPA
(1.7 nmol)
TPA + (85 nmol) of
compound 14
TPA + (85 nmol) of
compound 19
Papillomas
(%)
Papillomas/
mouse
Papillomas
(%)
Papillomas/
mouse
Papillomas
(%)
Papillomas/
mouse
1
0
0
0
0
0
0
2
0
0
0
0
0
0
3
0
0
0
0
0
0
4
0
0
0
0
0
0
5
0
0
0
0
0
0
6
10
0.3
0
0
0
0
7
30
0.9
10
0.5
10
0.4
8
50
1.2
30
0.9
20
0.8
9
60
1.7
50
1.3
40
1.1
10
80
2.1
50
1.7
50
1.4
11
100
2.7
60
2.2
50
1.8
12
100
3.2
70
2.7
60
2.3
13
100
3.8
80
3.2
60
2.9
14
100
4.7
90
3.7
60
3.4
15
100
5.5
90
4.0
70
3.7
16
100
6.0
100
4.3
70
4.0
17
100
6.5
100
4.9
80
4.3
18
100
7.1
100
5.2
90
4.6
19
100
7.6
100
5.6
100
4.9
20
100
7.9
100
6.3
100
5.2
As shown in Table 4, papilloma-bearing mice
in the positive control group treated with DMBA
(390 nmol) and TPA (1.7 nmol, twice/week)
appeared as early as at week 6, and the percentage of
papillomas bearers increased rapidly to reach 100%
after week 11.
On the other hand, treatment with compound
19 (85 nmol) along with DMBA/TPA reduced the
percentage of papilloma-bearing mice to 10.060.0% during weeks 7-14, and thereafter 100.0%
during week 19. As shown in Table 4, in the positive
control group treated with DMBA/TPA, the number
of papillomas formed per mouse increased rapidly
after week 6 to reach 7.9 papillomas/mouse at week
20, whereas mice treated with 19 bore only 5.2
papillomas. As shown in Table 4, treatment with
compound 14 (85 nmol), along with DMBA/TPA
reduced the percentage of papilloma bearing mice to
10.0-50.0% during weeks 7-10, and thereafter 60.0
and 100% in 11-16 week.
As shown in Table 4, after treatment with
compound 14, the number of papillomas formed per
mouse increased rapidly after week 15 to reach 4.0
papillomas/mouse.
In the in vivo two-stage mouse-skin carcinogenesis test, 14 and 19 were found to delay papilloma formation (Fig. 2). The results of in vitro EBVEA induction and the in vivo two-stage mouse-skin
carcinogenesis test suggest that compound 19 is
mildly useful as cancer chemopreventive agent.
Cytostatic activity
The compounds were evaluated for their cytostatic activity against human Molt4/C8 and CEM Tlymphocytes as well as murine L1210 leukemia
cells. The data are summarized in Table 5.
Several compounds: 22, 27, 29 and 30 exhibited IC50 values in the low micromolar range. HeLa
cells were slightly more sensitive to the cytostatic
activity of the compounds than CEM or L1210 cells.
There was, in general, a strong correlation
between the three tumor cell lines regarding the
cytostatic activities of the compounds. The most
potent inhibitors of murine L1210 cell proliferation
928
SUJEET KUMAR et al.
Table 5. Inhibitory effects of compounds on the proliferation of murine leukemia cells (L1210) and human Tlymphocyte cells (CEM) and human cervix carcinoma cells (HeLa).
IC50* (µM)
Compound
L1210
CEM
HeLa
NT
NT
NT
5
137 ± 6
100 ± 8
147 ± 108
6
130 ± 30
112 ± 3
116 ± 1
7
141 ± 16
121 ± 8
129 ± 18
8
NT
NT
NT
4
9
162 ± 2
123 ± 40
185 ± 16
10
116 ± 2
123 ± 41
114 ± 11
11
181 ± 13
116 ± 8
101 ± 2
12
NT
NT
NT
13
206 ± 47
110 ± 2
196 ± 76
14
204 ± 56
229 ± 30
80 ± 4
15
163 ± 96
> 250
≥ 250
16
68 ± 40
59 ± 27
127 ± 13
17
106 ± 81
101 ± 43
125 ± 11
18
NT
NT
NT
19
NT
NT
NT
20
NT
NT
NT
21
NT
NT
NT
22
3.9 ± 0.0
2.6 ± 1.1
3.6 ± 1.2
23
NT
NT
NT
24
NT
NT
NT
25
83 ± 19
41 ± 24
24 ± 4
26
114 ± 18
79 ± 16
32 ± 7
27
17 ± 1
10 ± 2
23 ± 0
28
107 ± 11
63 ± 5
32 ± 5
29
11 ± 6
9.0 ± 2.9
14 ± 8
30
37 ± 20
18 ± 9
22 ± 2
31
NT
NT
NT
Levamisole
206 ± 6
> 250
≥ 250
5-Fluorouracil
0.33 ± 0.17
18 ± 5
0.54 ± 0.12
*50% inhibitory concentration. NT = not tested.
(i.e., 22 and 29) were also most inhibitory to human
T-lymphocyte CEM, and cervix carcinoma HeLa
cell proliferation.
CONCLUSION
A new series of substituted imidazo[2,1b][1,3,4]thiadiazoles (4-31) were synthesized by
condensing aminothiadiazole with various phenacyl
bromides in good yields. Derivatives 4, 5, 11, 12, 13,
16, 18-25, 27, 29 and 31 were tested at a single dose
of 10-5 concentration at the NCI over 60 cell line
panel, and derivatives 11, 12, 20, 22, 29 and 31 were
subsequently tested in 5 dose testing mode.
Derivative 12 was found to be highly selectivity
towards Leukemia, Colon cancer subpanels and
moderately selective in CNS and breast cancer subpanels. Other two compounds 22 and 29 were found
moderately selective in prostate and colon cancer
subpanels. The cancer chemopreventive effect of 19
was equipotent to standard oleanolic acid (IC50 = 449
nM). These preliminary results of biological screening of the tested derivatives could offer an excellent
framework that may lead to discovery of potent anticancer and cancer chemoprevention agents.
Acknowledgments
We gratefully acknowledge the support for this
research effort from All India Council for Technical
Education (AICTE), New Delhi (Ref. No.
8023/BOR/RID/RPS-169/2008-09) for SSK. The
cytotoxic activities were determined with financial
support of the KU Leuven (GOA 10/014). We
would also like to thank NMR facility, Indian
Institute of Science, Bangalore for characterization
of all the compounds. We thank Lizette van
Berckelaer for the cytostatic determinations.
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Received: 18. 07. 2015
ISSN 0001-6837
SYNTHESIS, STRUCTURE AND PHARMACOLOGICAL EVALUATION
OF 1-(1H-PYRROL-1-YLMETHYL)-4-AZATRICYCLO[5.2.1.02,6]
DEC-8-ENE-3,5-DIONE
MAGDALENA PAKOSI—SKA-PARYS1,*, ANDRZEJ ZIMNIAK2, ANNA CHODKOWSKA3,
EWA JAGIE££O-W”JTOWICZ3, MAREK G£OWALA4 and MARTA STRUGA4
Chair and Department of Biochemistry, 2 Department of Drug Technology and Pharmaceutical
Biotechnology, Medical University of Warsaw, 02-097 Warszawa, Poland
3
Department of Toxicology, Medical University, 20-059 Lublin, Poland
4
Department of Pharmacogenomics, Faculty of Pharmacy, Medical University, 02-097 Warszawa, Poland
1
Abstract: A set of arylpiperazine derivatives with imide fragments, 1-(1H-pyrrol-1-ylmethyl)-10-oxa-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5-dione connected by propyl and butyl linkers, were synthesized and tested for the
potential anxiolytic and antidepressant activities. Compounds 3a and 3b demonstrated antidepressant activity
in the forced swimming tests in mice and were devoid of neurotoxic effects (chimney test in mice).
Keywords: antidepressant activity, anxiolytic, 1-(1H-pyrrol-1-ylmethyl)-10-oxa-4-azatricyclo[5.2.1.02,6]dec-8ene-3,5-dione derivatives
With this background in mind, in the present
study, we report the synthesis and CNS-activity of
the new derivatives (2a-7a) and the crystal structure
of initial product (1).
The serotonergic system has been consistently
implicated in the pathophysiology of number of psychiatric disorders including depression and anxiety (1).
Long-chain arylpiperazine derivatives are one
of the most important classes of 5-HT1A ligands. In
general, the arylpiperazine moiety is a good template for drugs acting on many different biological
targets, especially CNS receptors (2ñ4). As a consequence, many compounds containing this scaffold
bind with high affinity at 5-HT1A receptors, but only
a few of them also show high selectivity for 5-HT1A
over other receptors (5). Some compounds of that
group reveal equal high affinity for the 5-HT2A
receptor (4). Structure-activity relationship (SAR)
studies, performed with numerous generation of
arylpiperazine derivatives, showed that CNS activity and both, receptor affinity and selectivity depend
basically on the N-1-aryl (pyrimidinyl, o-OCH3phenyl and 5 other classic aryl groups) substituent
and a terminal fragment bound to the N-4 atom of
the piperazine moiety by an alkyl chain. In these
studies, this alkyl spacer is frequently composed of
two to four metylene units (6, 7). On the other hand,
the imide substructure is the basic element on a large
number of biologically active synthetic products
(8ñ10).
EXPERIMENTAL
Chemistry
Melting points were determined in a capillary
Kofler`s apparatus and are uncorrected. The NMR
spectra were recorded on a Bruker AVANCE DMX
400 spectrometer (Rheinstetten, Germany), operating
at 400 or 300 MHz (1H NMR). The chemical shift values, expressed in ppm, were references downfield to
TMS at ambient temperature. All values of microanalysis were within ± 0.4% of the calculated compositions. Mass spectra ESI measurements were carried
out on Waters ZQ Micromass instrument with
quadrupol mass analyzer. The spectra were performed
in the positive ion mode at a declustering potential of
40-60 V. The sample was previously separated on a
UPLC column (C18) using UPLC ACQUITYTM system by Waters connected with DPA detector. Column
flash chromatography and TLC were performed on
silica gel 60 (Merck) using chloroform or chloroform/methanol (9:1, v/v) mixture as eluent.
* Corresponding author: e-mail: mpakosinska@wum.edu.pl
931
932
MAGDALENA PAKOSI—SKA-PARYS et al.
4-(3-Chloropropyl)-1-(1H-pyrrol-1-ylmethyl)-10oxa-4-azatricyclo-[5.2.1.02,6]dec-8-ene-3,5-dione (2a)
A mixture of imide 1-(1H-pyrrol-1-ylmethyl)10-oxa-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5-dione
(12) (0.5 g, 0.002 mol), 1-bromo-3-chloropropane
(0.96 g, 0.0061 mol) and anhydrous K2CO3 (0.5 g,
0.0036 mol) was stirred on magnetic stirrer at ambient temperature for 30 h in acetone (50 mL). When
reaction was completed (confirmed by TLC on silica gel, developing system: chloroform-methanol 9 :
1, v/v), the inorganic precipitate was filtered off and
the solvent was evaporated.
Yield 60%, m.p. oil; 1H NMR (300 MHz,
CDCl3, δ, ppm): 6.83 (m, 2H, C-Hpyrrole), 6.46 (d, J =
6.0 Hz, 1H, CH=), 6.17 (m, 3H, CH=, CHpyrrole), 5.26
(s, 1H, CH-O), 4.71 (d, J = 15.3 Hz, 1H, CHC=O),
4.33 (d, J = 15.6 Hz, 1H, CHC=O), 3.70 (m, 2H,
NCH2), 3.52 (t, J = 6.6 Hz, 2H, CH2Cl), 2.90 (dd, J1
= 6.6 Hz, J2 = 20.0 Hz, 2H, CH2), 1.75 (m, 2H,
-CH2-). For C16H17ClN2O3 (320.77) calc.: 59.91% C,
5.34% H, 8.73% N; found: 59.67% C, 5.48% H,
8.69% N.
General procedure for preparation of 1-(1Hpyrrol-1-ylmethyl)-10-oxa-4-azatricyclo[5.2.1.02,6]
dec-8-ene-3,5-dione derivatives (3a-7a)
A mixture of compound 2a (0.2 g, 0.00062
mol), anhydrous K2CO3 (0.2 g, 0.0014 mol), KI (0.2
g, 0.00012 mol) and the corresponding N-substituted piperazine (0.16 ñ0.23 g, 0.001 mol) was stirred
on magnetic stirrer at ambient temperature for 60-80
h in acetone (30 mL). When the reaction was completed (as found by TLC on silica gel, developing
system: chloroform-methanol 9 : 1, v/v) the mixture
was filtered off and the solvent was evaporated.
4-{3-[4-(2-Methoxyphenyl)piperazin-1-yl]propyl}
-1-(1H-pyrrol-1-ylmethyl)-10-oxa-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5-dione (3a)
Yield 58%, m.p. 178-179OC; 1H NMR (300
MHz, CDCl3, δ, ppm): 7.08 (m, 6H, CHarom.,
CHpyrrole), 6.68 (d, J = 5.2 Hz, 1H, CH=), 6.18 (d, J =
5.6 Hz, 3H, CH=, CHpyrrole), 5.27 (s, 1H, CH-O), 4.53
(d, J = 15.2 Hz, 1H, CHC=O), 4.04 (d, J = 15.6 Hz,
1H, CHC=O), 3.77 (s, 3H, O-CH3), 3.57 (t, J = 6.8
Hz, 2H, NCH2), 3.11 (s, 4H, -(CH2)2N), 2.98 (d, J =
6.4 Hz, 1H, CH2), 2.91 (d, J = 6.4 Hz, 1H, CH2),
2.61 (m, 4H, N(CH2-)2), 1.77 (m, 4H, CH2N,
-CH2-). EMI MS: m/z = 477.32 [M + H]+ 100%. Rf
= 0.426. 13C NMR (DMSO-d6, δ, ppm): 176.20,
175.03, 151.77, 139.21, 137.18, 136.58, 128.08
(2C), 123.51, 120.82, 118.23, 111.94, 90.43 (2C),
80.54 (2C), 61.22, 55.35, 53.3 (2C), 51.18 (2C),
48.54, 47.92, 46.76, 42.39, 26.17.
4-{3-[4-(Pyrimidin-2-yl)piperazin-1-yl]propyl}-1(1H-pyrrol-1-ylmethyl)-10-oxa-4-azatricyclo[5.2.
1.02,6]dec-8-ene-3,5-dione (4a)
Yield 61%, m.p. 172-173OC; 1H NMR (300
MHz, CDCl3, δ, ppm): 8.45 (m, 2H, CH· arom.), 7.33
(m, 2H, CHpyrrole), 6.93 (s, 1H, CH‚ arom.), 6.15 (m, 2H,
CHpyrrole), 5.78 (m, 2H, CH=), 5.38 (s, 1H, CH-O),
4.36 (d, J = 15.2 Hz, 1H, CHC=O), 4.11 (d, J = 15.5
Hz, 1H, CHC=O), 3.95 (br, s, 2H, NCH2), 3.86 (br.
s, 2H, CH2), 3.44 (m, 4H, -(CH2)2N), 3.15 (m, 4H,
N(CH2-), 2.46 (m, 2H, CH2N), 1.70 (m, 2H, -CH2-).
EMI MS: m/z = 485.5 [M + H]+ 100%. Rf = 0.296.
13
C NMR (DMSO-d6, δ, ppm): 176.81, 175.06,
161.56, 157.93 (2C), 139.22, 137.18, 136.58, 129.08
(2C), 115.34, 90.88 (2C), 80.64 (2C), 61.45, 55.48
(2C), 50.18 (2C), 48.56, 47.98, 46.71, 42.3, 26.45.
4-[3-(4-Phenylpiperazin-1-yl)propyl]-1-(1H-pyrrol-1-ylmethyl)-10-oxa-4-azatricyclo[5.2.1.0 2,6]
dec-8-ene-3,5-dione (5a)
Yield 67%, m.p. 157-158OC; 1H NMR (300
MHz, CDCl3, δ, ppm): 7.66 (m, 2H, CHpyrrole), 7.43
(m, 3H, CHarom.), 6.86 (s, 2H, CHpyrrole), 6.49 (m, 1H,
CHarom.), 6.17 (m, 3H, CH=, CHarom.), 5.28 (s, 1H,
CH-O), 4.72 (d, J = 15.2 Hz, 1H, CHC=O), 4.36 (d,
J = 15.5 Hz, 1H, CHC=O), 3.97 (br. s, 2H, NCH2),
3.70 (m, 8H, CHpyrrole), 3.20 (br. s, 2H, CH2N), 3.06
(m, 2H, CH2), 2.28 (br. s, 2H, -CH2-). EMI MS: m/z
= 447.28 [M + H]+ 100%. Rf = 0.407. 13C NMR
(DMSO-d6, δ, ppm): 176.20, 175.08, 149.76, 141.18,
137.1, 136.18, 128.08 (2C), 123.41, 121. 97, 120.82,
118.28, 111.94, 90.56 (2C), 80.87 (2C), 61.28, 53.3
(2C), 51.18 (2C), 48.54, 47.99, 46.74, 42.3, 26.12.
4-{3-[4-(Pyridin-2-yl)piperazin-1-yl]propyl}-1(1H-pyrrol-1-ylmethyl)-10-oxa-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5-dione (6a)
Yield 60%, m.p. 157-158OC; 1H NMR (400
MHz, CDCl3, δ, ppm): 8.24 (d, J = 5.6 Hz, 2H,
CHpyrrole), 8.04 (t, J = 7.6 Hz, 2H, CHarom.), 7.12 (t, J
= 6.4 Hz, 2H, CHpyrrole), 7.02 (m, 2H, CHarom.), 6.85
(m, 1H, CH=), 6.17 (m, 1H, CH=), 5.27 (m, 1H,
CH-O), 4.60 (m, 3H, CHC=O), 4.32 (m, 3H,
CHC=O, CH2N), 3.72 (m, 4H, -CH2)2N), 3.50 (m,
2H, CH2), 3.26 (m, 4H, N(CH2-)2, 2.50 (br. s, 2H,
-CH2-). EMI MS: m/z = 448.26 [M + H]+ 100%. Rf
= 0.278. 13C NMR (DMSO-d6, δ, ppm): 175.20,
174.03, 158.13, 148.18, 138.18, 136.58, 128.08 (2C),
117.23, 106.23, 90.43 (2C), 80.54 (2C), 61.22, 53.3
(2C), 51.18 (2C), 48.54, 47.92, 46.72, 42.35, 26.76.
4-{3-[4-(4-Fluorophenyl)piperazin-1-yl]propyl}1-(1H-pyrrol-1-ylmethyl)-10-oxa-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5-dione (7a)
933
Synthesis, structure and pharmacological evaluation of...
Yield 62%, m.p. 178-179OC; 1H NMR (400
MHz, CDCl3, δ, ppm): 13.27 (br. s, 1H, HCl), 7.05
(m, 4H, CHpyrrole, CHarom.), 6.84 (br. s, 2H, CHpyrrole),
6.35 (d, J = 4.2 Hz, 1H, CH=), 6.17 (br. s, 3H, CH=,
CHarom.), 5.26 (br. s, 1H, CH-O), 4.71 (d, J = 15.2
Hz, 1H, CHC=O), 4.34 (d, J = 15.2 Hz, 1H,
CHC=O), 4.09 (br. s, 4H, -CH2)2N),), 3.71 (t, J = 6.0
Hz, 2H, NCH2), 3.58 (m, 2H, N(CH2-), 3.05 (m, 2H,
CH2), 2.13 (m, 2H, -CH2-). EMI MS: m/z = 465.26
[M+ H]+ 100%. Rf = 0.426. 13C NMR (DMSO-d6, δ,
ppm): 176.20, 175.03, 158.26,155.12, 137.18,
136.58, 128.08 (2C), 118.0, 117.9, 115.69, 115.39,
90.47 (2C), 80.77 (2C), 61.22, 53.19 (2C), 50.66
(2C), 48.54, 47.92, 46.09, 42.45, 26.16.
The synthesis of the target compounds was
started with the preparation of appropriate imide in
the Diels-Alder reaction. The imide was obtained in
reaction of 1-(furan-2-ylmethyl)-1H-pyrrole with
1H-pyrrole-2,5-dione (12). In this reaction were
obtained two products ñ endo and exo as a mixture
(1-(1H-pyrrol-1-ylmethyl)-10-oxa-4-azatricyclo[5.
2.1.02,6]dec-8-ene-3,5-dione.
The product exo can be separated from the
product endo by manual separation. To obtain the
product exo 1H-pyrrole-2.5-dione must be added to
hot solvent together with the second substrate. It is
impossible to get only the endo form.
The molecular structure of exo ñ imide (1) was
confirmed by an X-ray crystallography (11). In the
present study, the nuclear magnetic resonance
results are shown, i. e., the full assignment of signals
in 1H and 13C NMR high resolution spectra taken in
solution is reported. The spectral investigations
were carried out using homo- and heteronuclear correlation techniques. Results obtained for both conformers exo and endo are collected in Table 1.
Table 1. 1H and 13C NMR assignments for exo and endo (1-(1H-pyrrol-1-ylmethyl)-10-oxa-4-azatricyclo[5.2.1.02,6]dec-8-ene3,5-dione (1). The spectra were taken in CDCl3 at r. t., chemical shifts are given in ppm (δ) from TMS.
Group
Exo
Endo
2-CH
3.01; d; J2H-6H = 6.6 Hz
C: 50.83
3.06; d; J2H-6H = 7.8 Hz
C: 48.82
6-CH
CH2
7-CH
pyrrole β-CH
9-CH
3
3
13
13
3
3.08; d; 3J2H-6H = 6.6 Hz;
J6H-7H ≈ 0 Hz
13
C: 52.83
3
HA: 4.33; d; 2J2HA-2HB = 15.0 Hz
HB: 4.78; d; 2J2HA-2HB = 15.0 Hz
13
C: 49.11
HA: 4.56; d; 2J2HA-2HB = 15.3 Hz
HB: 4.64; d; 2J2HA-2HB = 15.3 Hz
13
C: 50.47
5.15; d; 3J7H-8H = 1.5 Hz
C: 82.08
3.61; dd; 3J2H-6H = 7.8 Hz;
J6H-7H = 5.7 Hz
13
C: 50.51
5.22; dd; 3J6H-7H = 5.4 Hz;
J7H-8H = 1.6 Hz
13
C: 80.26
13
3
6.01; t (dis)
13
C: 108.79
6.03; t (dis)
13
C: 108.87
6.22; d; 3J8H-9H = 5.7 Hz
C: 137.57
6.40; d; 3J8H-9H = 6.0 Hz
C: 135.73
13
13
8-CH
6.49; dd (br)
3
J8H-9H = 5.7 Hz
13
C: 138.22
6.50; dd (br); 3J7H-8H = 1.6 Hz;
3
J8H-9H = 6.0 Hz
13
C: 136.90
pyrrole α-CH
6.85; t (dis)
13
C: 122.73
6.76; t (dis)
13
C: 122.75
NH
10.13; s (br)
9.70; s (br)
1-C
13
13
3 and 5-C=O
13
13
C: 91.79
C: 176.57; 177.34
C: 92.22
C: 176.08; 176.43
Denotation of signals: s ñ singlet, d ñ doublet, t ñ triplet, m ñ multiplet, br ñ broad, dis ñ distorted. The groups 3 and 5 were not discriminated. The sample of endo conformer contained ca. 10% of the exo form.
934
Scheme 1. Synthesis of compounds 1-10b
When comparing the NMR data obtained for
conformers exo and endo of (1) one can see noticable differentiation in the regions of 6-H, 9-H and
CH2 groups. Thus, a substantial downfield shift for
the proton 6-H has been observed for endo form in
respect to exo (ca. 0.5 ppm), similar effect was noted
for 9-H (0.2 ppm). Moreover, the geminal protons in
CH2 group are stronger differentiated in exo form as
compared with endo (Table 1). Such shifts usually
result from changes in anisotropic effects due to spatial dislocation of carbonyl groups, double bonds or
aromatic rings.
The standard alkylation procedure of imide
with 1,3-dibromopropane or 1,4-dibromobutane led
to 3-bromopropyl or 4-bromopropyl derivatives
which were then condensed with appropriate amines
to yield the final compounds (Scheme 1).
Derivatives (2b-10b) with four carbon linkers
have been synthesized as described previously (12).
New obtained compounds were purified by
flash chromatography. MS, 1H NMR spectra confirmed the identity of products (2a-7a).
The affinity to serotonergic receptors has not
been tested for the obtained products. Compounds
were selected for the pharmacological tests due to
the comparison of their chemical structure to the
substances, which had been examined in earlier
research (15).
The potential anxiolytic and antidepressant
activities were evaluated by a four-plate test (13)
and a forced swimming test (14) in mice, respectively. The effect of active compounds on the spontaneous locomotor activity of mice was also tested
(equivalent to 0.1 of LD50). No neurotoxic effects
were detected in the chimney test for any of the 14
investigated compounds, nor did any of them
(administrated in the screening dose) show anxiolytic properties in the four-plate test in mice.
Synthesis, structure and pharmacological evaluation of...
Strong antidepressant-like action was observed
for 3a and 3b (Fig. 2). These effects seem to be specific, since screened compounds did not affect the
locomotor activity with respect to the control group.
Furthermore, it was found that the antidepressant
effects produced by 3a and 3b were dose-dependent
up to 0.0125 of LD50, and when given in doses of 8,
10 and 20 mg/kg i.p. they revealed similar antidepressive profile to imipramine in a dose 15 mg/kg
i.p. (Fig. 2).
Compound 3a was arylpiperazine derivative
with propyl linker, whereas compound 3b is also
arylpiperazine derivative with butyl linker. Both the
compounds have methoxy group in position 2 at the
phenyl ring of piperazine.
Antidepressant activity of 3a and 3b derivatives is directly connected with the presence of
methoxy substituent.
Figure 1. Structure of compound 1.
935
The research presented is a part of major project. For the earlier examined group of compounds,
5-HT1A and 5-HT2A receptor affinities and in vitro
studies on basis of their ability to displace [3H]-8OH-DPAT [8-hydroxy-2-(di-n-propylamino)tetraline] and [3H]-ketanserin [3-{2-[4-(4-fluorobenzoyl)piperidino]ethyl}quinazoline-2,4(1H,3H)dione], respectively, were done (15).
The earlier research proved that relationship
with receptors 5-HT1A, 5-HT2A did not correlate with
antidepressant activity which was described with the
help of experiments on animals (15).
Indeed, many different receptors system can be
in antidepressant-like activity observed in forced
swimming test, and piperazine derivatives may produce their effect via e.g., other serotonin (16), σ 1
(17) and melanocortin-4 (18) receptors.
To know the mechanism of 3a and 3b compounds activity one should continue pharmacological research.
Pharmacology
The experiments were carried out on male
Albino Swiss mice (20-24 g) kept at a room temperature of 18-20OC on a natural day/night cycle, with
free access to food and water. Permission to carry
out animal tests and experiments was issued by the
Ethical Board at the Medical University of Lublin.
The investigated compounds were administered
intraperitoneally (i.p.) as suspensions in 1% Tween
80 at a constant volume of 0.1 mL/10 g body weight
of mice. Control animals received the same volume
of the solvent. The compounds were administered in
Figure 2. Antidepressive effect of compounds 3a and 3b in the ìforced swimmingî test in mice. Imipramine and investigated compounds were given 30 min before the test. The data represent the mean ± SEM
936
doses equivalent to 0.1 or 0.05 of their LD50. Each
experimental group consisted of eight animals.
Motor coordination in a ëëchimney testíí was
measured according to the method of Boissier et al.
(19), at 30 min after administration of the investigated compounds in a dose equivalent to 0.1 of their
LD50. The mice had to climb up backwards in a plastic tube (inner diameter: 3 cm, length: 25 cm). The
mice that were unable to perform the three tasks
within 60 s were considered to display motor
impairment. The motor impairment was quantified
as percentage of animals which failed to complete
the test.
Spontaneous locomotor activity
Locomotor activity of mice was measured by
automatic photoresistor actometers (DIGISCAN
Optical Animal Activity Monitoring System,
Omnitech Electronics, Inc.). Thirty minutes after the
administration of the investigated compounds in a
dose equivalent to 0.1 of their LD50, the animals
were placed in the actometers for 60 min and the
total distance, horizontal activity and vertical activity were recorded automatically. Anxiolytic activity
was assessed by a ëëfour-plateíí test in mice according to Aron et al. (13), at 30 min after injection of
the investigated compounds in a dose of 0.1 of their
LD50. The number of punished crossings was counted for 1 min. Antidepressant properties were
assessed by a ëëforced swimmingíí test according to
Porsolt et al. (14), at 30 min after administration of
investigated compounds in doses of 0.1e 0.00635 of
their LD50 or imipramine in a dose of 15 mg/kg i.p.
The mice were individually placed and forced to
swim in a glass cylinder (27 ◊ 16 cm) containing 15
cm of water (25OC). The mice were left in the cylinder for 6 min. After the first 2 min, the total duration
of immobility was measured during a 4-min test. A
mouse was judged to be immobile when it remained
floating passively, making slow movements to keep
its head above the water.
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Received: 19. 07. 2015
ISSN 0001-6837
SYNTHESIS AND PHARMACOLOGICAL ACTIVITY OF IMIDAZO[2,1b][1,3,4]THIADIAZOLE DERIVATIVES
ARPIT KATIYAR1, BASAVARAJ METIKURKI2, SARALA PRAFULLA1, SUJEET KUMAR1,
SATYAPRAKASH KUSHWAHA1, DOMINIQUE SCHOLS3, ERIK DE CLERCQ3
and SUBHAS S. KARKI1*
1
Department of Pharmaceutical Chemistry, KLE Universityís College of Pharmacy,
Rajajinagar, Bangalore, India
2
Department of Pharmaceutical Chemistry, Vivekananda College of Pharmacy, Bengaluru 560010, India
3
Rega Institute for Medical Research, KU Leuven, Leuven, Belgium
Abstract: In this paper, a series of imidazo[2,1-b][1,3,4]thiadiazoles have been prepared by reacting 2-amino1,3,4-thiadiazole with various phenacyl bromides in alcohol. The structures of all the derivatives were confirmed by IR, NMR and mass spectroscopy. All the derivatives have been tested for cytostatic activity against
human T-lymphocyte cells (CEM), human cervix carcinoma cells (HeLa), murine leukemia cell line (L1210),
and antiviral activity. Among the tested compounds, derivatives 5b, 5c, and 7a were cytostatic between 49-63
mM against Hela and 7g was cytotoxic at 23 mM against L1210 and CEM cell lines. Compounds 5h and 7h
emerged as antiviral agents with slight activity against influenza A and B.
Keywords: anti-influenza, imidazo[2,1-b][1,3,4]thiadiazole, cytotoxicity, antiviral
cies were found in those compounds with the presence of formyl and thiocyanate functional group at
the 5 position of imidazo[2,1-b][1,3,4]thiadiazole
nucleus (III). From these studies, two compounds
possessing 2H-chromen-2-one-3-yl at the 6 position
of imidazo[2,1-b][1,3,4]thiadiazole were identified
as the most promising cytotoxic agents and are
referred to as (IV) (2). In general, these compounds
are potent cytotoxic compounds having IC50 values
less than 1 µM and are substantially more potent
than melphalan and levamisole which are used clinically in treating cancers. These encouraging results
justify the further bioevaluation of the series 5a-i
and 7a-i. An examination of the effects of placing
different heteroaryl substituents instead of aralkyl
rings of imidazo[2,1-b][1,3,4]thiadiazole on cytotoxic potencies was proposed (V). The goal was to
gain some idea of the sensitivity of these compounds
towards human lymphomas and leukemia.
Imidazo[2,1-b][1,3,4]thiadiazole is an isoster
of biologically significant bridgeheaded heterocyclic imidazo[2,1-b][1,3]thiazole (e.g., levamisole)
and during the past decades, a significant development has been noted for imidazo[2,1-b][1,3,4]thiadiazoles in their chemistry and biology. The principal interest in our laboratories is the synthesis and
anticancer activity of imidazo[2,1-b][1,3,4]thiadiazoles (I). The perceived importance of these compounds is their ability to act as anticancer by inducing apoptosis without arresting the cell cycle (1-3).
Initially compounds containing 2-benzyl and 6
phenyl or substituted phenyl imidazo[2,1b][1,3,4]thiadiazole derivatives were prepared and
demonstrated cytotoxic and anticancer properties (1)
(II). Most of the compounds displayed micromolar
or submicromolar IC50 values against various human
and murine tumor cancer cell lines (2, 3).
In general, the greatest antiproliferative poten-
* Corresponding author: e-mail: subhasskarki@gmail.com
937
938
ARPIT KATIYAR et al.
EXPERIMENTAL
The melting points are uncorrected. Silica gel
plates were used for TLC using toluene and ethyl
acetate in various proportions as mobile phases. The
IR spectra were recorded in KBr on a Jasco 430+;
the 1H NMR spectra were recorded in CDCl3/DMSO
on a Bruker (400 MHz), and J values are reported in
hertz (Hz). Mass spectra were recorded in triple
quadrapole LCMS-6410 from Agilent Technologies. Required substituted phenacyl bromides and
3-(bromoacetyl)-2H-chromen-2-one were prepared
according to the literature (1-3).
Synthesis of 5-(thiophen-2-yl)-1,3,4-thiadiazol-2amine (4) (4)
The thiophen-2-carboxylic acid [1] (0.1 M),
POCl3 (20-30 mL) and thiosemicarbazide [3] (0.1
M) were treated in a round bottom flask. The contents of the reactions were heated to reflux for one
hour and cooled to room temperature. The contents
of the reaction mixtures were poured in ice cold
water and made basic with potassium hydroxide
solution. The products obtained were filtered and
washed with cold water and purified from ethyl
alcohol. Yield 50%. M.p. 135-138OC, IR (KBr, cm-1):
3285, 3097, 1621, 1513, 1464, 1420, 1328, 1045.
General procedure for the synthesis of 2-thiophen-2-yl-imidazo[2,1-b][1,3,4]thiadiazoles (5a-i)
The
5-(thiophen-2-yl)-1,3,4-thiadiazol-2amine 1, (30 mmol) was treated with the respective
phenacyl bromide 3 (30 mmol), in ethanol (150
bases, which were filtered with good yield.
6-Phenyl-2-(thio117,228 phen-2-yl)imidazo[2,1b][1,3,4] thiadiazole (5a)
for the synthesis of 2-thiophen-2-yl-imidazo[2,1b][1,3,4]thiadiazoles. IR (KBr, cm-1): 3075, 2930,
1602, 1540, 1493, 1469, 1254. 1H NMR (400 MHz,
DMSO-d6, δ, ppm): 7.27-7.30 (m, 2H), 7.41 (t, 2H,
J = 16 Hz), 7.84-7.88 (m, 3H), 7.92-7.93 (m, 1H),
8.69 (s, 1H, imidazole).
6-(4-Bromophenyl)-2-(thiophen-2-yl)imidazo[2,
1-b][1,3,4]thiadiazole (5b)
the synthesis of 2-thiophen-2-yl-Imidazo[2,1b][1,3,4]thiadiazoles. IR (KBr, cm-1): 3144, 3053,
2878, 1632, 1582, 1470, 1425, 1280. 1H NMR (400
MHz, DMSO-d6, δ, ppm): 7.26-7.28 (m, 1H), 7.62 (d,
2H, J = 8.4 Hz), 7.83 (d, 2H, J = 8.4 Hz), 7.86-7.87
(m, 1H), 7.93-7.94 (m, 1H), 8.72 (s, 1H, imidazole).
6-(4-Chlorophenyl)-2-(thiophen-2-yl)imidazo[2,
1-b][1,3,4]thiadiazole (5c)
the synthesis of 2-thiophen-2-yl-imidazo[2,1b][1,3,4]thiadiazoles. IR (KBr, cm-1): 3148, 3105,
3065, 2973, 1592, 1535, 1470, 1425, 1335, 1254. 1H
NMR (400 MHz, DMSO-d6, δ, ppm): 7.26-7.29 (m,
1H), 7.49 (d, 2H, J = 8.4 Hz), 7.86-7.90 (md, 3H, J =
8.4 Hz), 7.93-7.95 (m, 1H), 8.75 (s, 1H, imidazole).
Synthesis and pharmacological activity of...
6-(4-Nitrophenyl)-2-(thiophen-2-yl)imidazo[2,1b][1,3,4]thiadiazole (5d)
2933, 1599, 1546, 1510, 1460, 1255. 1H NMR (400
MHz, DMSO-d6, δ, ppm): 7.28-7.30 (m, 1H), 7.907.91 (m, 1H), 7.96-7.97 (m, 1H), 8.14 (d, 2H, J = 8.4
Hz), 8.30 (d, 2H, J = 8.4 Hz), 8.99 (s, 1H, imidazole). MS (ESI) m/z: 329 (M + H).
6-(4-Methylphenyl)-2-(thiophen-2-yl)imidazo[2,
1-b][1,3,4]thiadiazole (5e)
3029, 2911, 2851, 1604, 1543, 1494, 1471, 1427,
1332, 1254. 1H NMR (400 MHz, DMSO-d6, δ,
ppm): 2.32 (s, 3H, -CH3), 7.23 (d, 2H, J = 8 Hz),
7.26-7.28 (m, 1H), 7.76 (d, 2H, J = 8.4 Hz), 7.84-
939
7.86 (m, 1H), 7.92-7.93 (m, 1H), 8.63 (s, 1H, imidazole). MS (ESI) m/z: 298.1 (M + H).
6-(4-Methoxyphenyl)-2-(thiophen-2-yl)imidazo
[2,1-b][1,3,4]thiadiazole (5f)
3077, 2992, 2963, 2939, 2836, 1601, 1545, 1470,
1333, 1249, 1175. 1H NMR (400 MHz, DMSO-d6, δ,
ppm): 3.78 (s, 3H, -OCH3), 6.99 (d, 2H, J = 8.4 Hz),
7.26-7.28 (m, 1H), 7.80 (d, 2H, J = 8.4 Hz), 7.847.85 (m, 1H), 7.91ó7.93 (m, 1H), 8.57 (s, 1H, imidazole). MS (ESI) m/z: 314.1 (M + H).
3-[2-(Thiophen-2-yl)imidazo[2,1-b][1,3,4]thiadiazol-6-yl]-2H-chromen-2-one (5g)
for the synthesis of 2-thiophen-2-yl-imidazo[2,1b][1,3,4]thiadiazoles. IR (KBr, cm-1): 3059, 2967-
Scheme 1. Synthesis of title compounds 5a-i
Scheme 2. Synthesis of title compounds 7a-i
940
Table 1. Physicochemical properties.
f
Comp.
no.
Molecular
formula
Color
Molecular
weight
Yield
%
M.p. (OC)
Rf
value*
5a
C14H9N3S2
Pale yellow
283.37
59
110-113
0.53
5b
C14H8BrN3S2
Pale yellow
362.27
60
110-115
0.62
5c
C14H8ClN3S2
Green
317.82
65
160-165
0.54
5d
C14H8N4O2S2
Yellow
328.37
66
109-112
0.55
5e
C15H11N3S2
Yellow
297.40
58
135-138
0.62
5f
C15H11N3OS2
Pale yellow
313.40
50
125-128
0.61
5g
C17H9N3O2S2
Brown
351.40
49
> 300
0.54
5h
C14H10N4S2
Brown
298.39
55
120-124
0.61
5i
C14H8FN3S2
Pale green
301.36
62
160-164
0.53
7a
C15H11N3S2
Brown
297.04
64
120-122
0.56
7b
C15H10BrN3S2
Pale brown
376.29
68
170-173
0.64
7c
C15H10ClN3S2
Pale yellow
331.84
60
101-104
0.52
7d
C15H10N4O2S2
Pale yellow
342.40
50
135-138
0.53
7e
C16H13N3S2
Blackish green
311.42
50
153-157
0.54
7f
C16H13N3OS2
Blackish green
327.42
48
110-112
0.61
7g
C18H11N3O2S2
Brown
365.43
52
176-179
0.62
7h
C15H12N4S2
Brown
312.41
58
163-166
0.61
7i
C15H10FN3S2
Pale brown
315.39
48
130-132
0.55
*Mobile phase: toluene : ethyl acetate
2826, 1713, 1606, 1471. 1H NMR (400 MHz,
DMSO-d6, δ, ppm): 7.28-7.30 (1H, m, arom.), 7.40
(1H, t, J = 16 Hz), 7.48 (1H, d, J = 8 Hz), 7.60-7.64
(1H, m, arom.), 7.88-7.90 (2H, m, arom.), 7.95-7.97
(1H, m, ar), 8.63 (1H, s, ar), 8.70 (1H, s, arom.). MS
(ESI) m/z: 352.8 (M + H).
4-[2-(Thiophen-2-yl)imidazo[2,1-b][1,3,4]thiadiazol-6-yl]aniline (5h)
3065, 2930, 1610, 1509, 1310, 1258. MS (ESI) m/z:
299.5 (M + H).
6-(4-Fluorophenyl)-2-(thiophen-2-yl)imidazo[2,1b][1,3,4]thiadiazole (5i)
2925, 2881, 1599, 1544, 1491, 1426, 1340, 1225. 1H
NMR (400 MHz, DMSO-d6, δ, ppm): 7.25-7.28 (m,
3H), 7.85-7.94 (m, 4H), 8.69 (s, 1H, imidazole). MS
(ESI) m/z: 302.1 (M + H).
Synthesis of 5-(thiophen-2-ylmethyl)-1,3,4-thiadiazol-2-amine (6)
The mixture of thiophen-2-yl-acetic acid [2]
(0.1 M) and thiosemicarbazide [3] (0.15 M) was
added slowly to the round bottom flask containing
concentrated H2SO4 (30 mL) with constant stirring in
an ice bath. After the reaction was completed, the ice
bath was replaced by a water bath and slowly heated
to 70-80OC and maintained at that temperature for 7
h. After cooling to room temperature, the contents of
the reaction mixture were poured into an ice water
and made basic with ammonia, and the precipitate
was filtered, washed with water and recrystallized
from ethanol. Yield 40%. M.p. 183-187OC, IR (KBr,
cm-1): 3290, 3102, 2967, 2778, 1641, 1523, 1489,
1338, 1254, 1052. 1H NMR (400 MHz, DMSO-d6, δ,
ppm): 4.36 (s, 2H, -CH2-), 6.97-6.94 (m, 2H), 7.06 (s,
br, NH2), 7.41-7.39 (m, 1H, ar),
General procedure for the synthesis of the 6-aryl2-(thiophen-2-ylmethyl)imidazo[2,1-b][1,3,4]
thiadiazoles (7a-i)
The 5-(thiophen-2-ylmethyl)-1,3,4-thiadiazol2-amine 2, (30 mmol) was treated with the respective phenacyl bromide 3 (30 mmol), in ethanol (150
941
base which was filtered with good yield.
6-Phenyl-2-(thiophen-2-ylmethyl)imidazo[2,1b][1,3,4]thiadiazole (7a)
for the synthesis of 6-aryl-2-(thiophen-2-ylmethyl)imidazo[2,1-b][1,3,4]thiadiazoles. IR (KBr, cm-1):
3115, 3053, 2943, 1603, 1527, 1473, 1257. 1H NMR
(400 MHz, DMSO-d6, δ, ppm): 4.70 (s, 2H, -CH2-),
7.02-7.04 (m, 1H), 7.13-7.15 (m, 1H), 7.24-7.28 (m,
1H), 7.39 (t, 2H, J = 16 Hz), 7.49-7.50 (m, 1H),
7.82-7.85 (m, 2H), 8.64 (s, 1H, imidazole). MS
(ESI) m/z: 298.0 (M + H).
6-(4-Bromophenyl)-2-(thiophen-2-ylmethyl)imidazo[2,1-b][1,3,4]thiadiazole (7b)
for the synthesis of 6-aryl-2-(thiophen-2ylmethyl)imidazo[2,1-b][1,3,4]thiadiazoles. IR
(KBr, cm-1): 3141, 3073, 2933, 1526, 1475, 1399,
1285. 1H NMR (400 MHz, DMSO-d6, δ, ppm): 4.70
(s, 2H, -CH2-), 7.02-7.04 (m, 1H), 7.13-7.14 (m,
1H), 7.49-7.50 (m, 1H), 7.59 (d, 2H, J = 8.4 Hz),
7.80 (d, 2H, J = 8.4 Hz), 8.70 (s, 1H, imidazole).
6-(4-Chlorophenyl)-2-(thiophen-2-ylmethyl)imidazo[2,1-b][1,3,4]thiadiazole (7c)
3136, 3071, 2920, 1590, 1528, 1471, 1284, 1254. 1H
NMR (400 MHz, DMSO-d6, δ, ppm): 4.70 (s, 2H,
-CH2-), 7.02-7.04 (m, 1H), 7.13-7.14 (m, 1H), 7.46
(d, 2H, J = 8.4 Hz), 7.49-7.50 (m, 1H), 7.87 (d, 2H,
J = 8.4 Hz), 8.69 (s, 1H, imidazole). MS (ESI) m/z:
332.0 (M).
6-(4-Nitrophenyl)-2-(thiophen-2-ylmethyl)imidazo[2,1-b][1,3,4]thiadiazole (7d)
Table 2. Inhibitory effects of compounds on the proliferation of murine leukemia cells (L1210), human T-lymphocyte cells (CEM) and human cervix carcinoma cells (HeLa).
IC50* (µM)
Compound
L1210
CEM
HeLa
5a
5b
94 ± 0
94 ± 20
108 ± 0
82 ± 4
136 ± 19
63 ± 15
5c
68 ± 4
87 ± 0
52 ± 3
5d
136 ± 1
120 ± 4
131 ± 1
5e
152 ±40
> 250
157 ± 23
5f
118 ± 71
146 ± 71
102 ± 4
5g
112 ± 18
114 ± 18
77 ± 6
5h
129 ± 91
> 250
> 250
5i
130 ± 33
> 250
122 ± 14
7a
120 ± 24
≥ 250
49 ± 18
7b
> 250
> 250
> 250
7c
> 250
222 ± 40
≥ 250
7d
118 ± 3
101 ± 14
118 ± 3
7e
> 250
> 250
> 250
7f
> 250
> 250
> 250
7g
23 ± 1
24 ± 4
87 ± 6
7h
128 ± 71
> 250
> 250
7i
> 250
> 250
> 250
Levamisole
206 ± 6
> 250
≥ 250
Melphalan
2.13 ± 0.02
1.4 ± 0.4
NT
*50% inhibitory concentration. NT = Not tested.
942
3131, 2933, 1600, 1504, 1467, 1405, 1340, 1260. 1H
NMR (400 MHz, DMSO-d6, δ, ppm): 4.72 (s, 2H,
-CH2-), 7.03-7.05 (m, 1H), 7.14-7.15 (m, 1H), 7.497.51 (m, 1H), 8.11 (d, 2H, J = 8.8 Hz), 8.27 (d, 2H,
J = 8.8 Hz), 8.94 (s, 1H, imidazole). MS (ESI) m/z:
343.1 (M + H).
3071, 3029, 2903, 1550, 1524, 1470, 1346, 1296,
(s, 3H, -CH3), 4.69 (s, 2H, -CH2-), 7.02-7.04 (m, 1H),
7.13-7.14 (m, 1H), 7.19 (d, 2H, J = 8.4 Hz), 7.497.50 (m, 1H), 7.74 (d, 2H, J = 8.4 Hz), 8.57 (s, 1H,
imidazole). MS (ESI) m/z: 312.1 (M + H).
6-(4-Methylphenyl)-2-(thiophen-2-ylmethyl)imidazo[2,1-b][1,3,4]thiadiazole (7e)
the synthesis of 6-aryl-2-(thiophen-2-ylmethyl)-imidazo[2,1-b][1,3,4]thiadiazoles. IR (KBr, cm-1): 3133,
6-(4-Methoxyphenyl)-2-(thiophen-2-ylmethyl)
imidazo[2,1-b][1,3,4]thiadiazole (7f)
Table 3. Anti-influenza virus activity and cytotoxicity in MDCK cell cultures.
Antiviral EC50c
Cytotoxicity
Compound
a
Conc.
unit
CC50a
Minimum
cytotoxic
concentrationb
Influenza A
H1N1 subtype
Influenza A
H3N2 subtype
Influenza B
visual
CPE
score
MTS
visual
CPE
score
MTS
visual
CPE
score
MTS
5a
µM
>100
>100
>100
>100
>100
>100
>100
>100
5b
µM
>100
>100
>100
>100
>100
>100
>100
>100
5c
µM
>100
>100
>100
>100
>100
>100
>100
>100
5d
µM
>100
>100
>100
>100
>100
>100
>100
>100
5e
µM
>100
>100
>100
>100
>100
>100
>100
>100
5f
µM
>100
>100
>100
>100
>100
>100
>100
>100
5g
µM
>100
>100
>100
>100
>100
>100
>100
>100
5h
µM
>100
>100
100
>100
45
23.2
100
90.8
5i
µM
>100
>100
>100
>100
>100
>100
>100
>100
7a
µM
>100
>100
>100
>100
>100
>100
>100
>100
7b
µM
>100
>100
>100
>100
>100
>100
>100
>100
7c
µM
>100
>100
>100
>100
>100
>100
>100
>100
7d
µM
>100
>100
>100
>100
>100
>100
>100
>100
7e
µM
>100
>100
>100
>100
>100
>100
>100
>100
7f
µM
>100
>100
>100
>100
>100
>100
>100
>100
7g
µM
41.7
20
>4
>4
>4
>4
>4
>4
7h
µM
>100
>100
20
57.2
58
16.2
100
56.5
7i
µM
>100
>100
>100
>100
>100
>100
>100
>100
Levamisole
µM
>100
>100
>100
>100
>100
>100
>100
>100
Oseltamivir
carboxylate
µM
>100
>100
4
1.4
2
0.8
0.7
0.5
Ribavirin
µM
>100
>100
9
5.9
9
4.2
9
7.3
Amantadine
µM
>200
>200
40
45.0
8
10.8
>200
>200
Rimantadine
µM
>200
>200
90
60.5
1
1.1
>200
>200
b
50% Cytotoxic concentration, as determined by measuring the cell viability with the colorimetric formazan-based MTS assay. Minimum
compound concentration that causes a microscopically detectable alteration of normal cell morphology. c50% Effective concentration, or
concentration producing 50% inhibition of virus-induced cytopathic effect, as determined by visual scoring of the CPE, or by measuring
the cell viability with the colorimetric formazan-based MTS assay. MDCK cells: Madin Darby canine kidney cells.
943
Table 4. Cytotoxicity and antiviral activity of compounds in HEL cell cultures.
Compound
Minimum
cytotoxic
concentrationa (µM)
EC50b (µM)
Herpes
simplex
virus-1 (KOS)
Herpes
simplex
virus-2 (G)
Vaccinia
virus
Vesicular
stomatitis
virus
Herpes simplex
virus-1
TK- KOS ACVr
5a
100
>20
>20
>20
>20
>20
5b
100
>20
>20
>20
>20
>20
5c
>100
>100
>100
>100
>100
>100
5d
>100
>100
>100
>100
>100
>100
5e
>100
>100
>100
>100
>100
>100
5f
≥100
>100
>100
>100
>100
>100
5g
>100
>100
>100
>100
>100
>100
5h
>100
>100
>100
>100
>100
>100
5i
100
>20
>20
>20
>20
>20
7a
100
>20
>20
>20
>20
>20
7b
≥100
>100
>100
>100
>100
>100
7c
>100
>100
>100
>100
>100
>100
7d
>100
>100
>100
>100
>100
>100
7e
>100
>100
>100
>100
>100
>100
7f
>100
>100
>100
>100
>100
>100
7g
100
>20
>20
>20
>20
>20
7h
>100
>100
>100
>100
>100
>100
7i
>100
>100
>100
>100
>100
>100
Levamisole
>100
>100
>100
>100
>100
>100
Brivudin
>250
0.03
250
10
>250
250
Cidofovir
>250
5
2
50
>250
10
Acyclovir
>250
0.4
0.08
>250
>250
10
Ganciclovir
>100
0.08
0.03
>100
>100
4
a
b
Required to cause a microscopically detectable alteration of normal cell morphology. Required to reduce virus-induced cytopathogenicity by 50%.
3135, 2994, 2964, 2933, 1606, 1539, 1514, 1400,
(s, 3H, -OCH3), 4.68 (s, 2H, -CH2-), 6.97 (d, 2H, J
= 8.8 Hz), 7.02-7.04 (m, 1H), 7.13-7.14 (m, 1H),
7.48-7.50 (m, 1H), 7.77 (d, 2H, J = 8.8 Hz), 8.51 (s,
1H, imidazole). MS (ESI) m/z: 328.1 (M + H).
3-[2-(Thiophen-2-ylmethyl)imidazo[2,1-b][1,
3,4]thiadiazol-6-yl]-2H-chromen-2-one (7g)
3034, 2969-2912, 1716, 1605, 1480. 1H NMR (400
MHz, DMSO-d6, δ, ppm): 4.73 (2H, s, -CH2-), 7.037.05 (1H, m, ar), 7.15-7.16 (1H, m, arom.), 7.38
(1H, t, J = 16 Hz), 7.47-7.45 (1H, m, arom.), 7.527.50 (1H, m, arom.), 7.66-7.59 (1H, m, arom.), 7.93-
7.86 (1H, m, arom.), 8.58 (1H, s, arom.), 8.67 (1H,
s, arom.). MS (ESI) m/z: 366.5 (M + H).
4-[2-(Thiophen-2-ylmethyl)imidazo[2,1-b][1,3,4]
thiadiazol-6-yl]aniline (7h)
3473, 3292, 3109, 2970, 1640, 1606, 1521, 1479,
1328. MS (ESI) m/z: 313.5 (M + H).
6-(4-Fluorophenyl)-2-(thiophen-2-ylmethyl)imidazo[2,1-b][1,3,4]thiadiazole (7i)
3130, 3078, 2950, 1601, 1565, 1473, 1259. 1H NMR
944
(400 MHz, DMSO-d6, δ, ppm): 4.71 (s, 2H, -CH2-),
7.05-7.09 (m, 1H), 7.13-7.18 (m, 1H), 7.49-7.55 (m,
3H), 7.91 (d, 2H, J = 8.4 Hz), 8.72 (s, 1H, imidazole). MS (ESI) m/z: 316.4 (M + H).
the incubation period, the cells were counted in a
Coulter counter. The IC50 (50% inhibitory concentration) was defined as the concentration of the compound that inhibited cell proliferation by 50%.
Cytotoxicity in human and murine tumor cell
lines
All the compounds in Scheme 1 and 2 were
evaluated for their cytostatic activity against human
HeLa cervix carcinoma cells, human CEM CD4+ Tlymphocytes as well as murine L1210 cells (5). All
assays were performed in 96-well microtiter plates.
To each well were added (5 - 7.5) ◊ 104 tumor cells
and a given amount of the test compound. The cells
were allowed to proliferate for 48 h (murine leukemia
L1210 cells) or 72 h (human lymphocytic CEM and
human cervix carcinoma HeLa cells) at 37OC in a
humidified CO2-controlled atmosphere. At the end of
Antiviral assays
Antiviral activity
The synthesized compounds were evaluated for
antiviral activity (6, 7) by using the following viruses: human cytomegalovirus (HCMV) strains AD-169
and Davis, herpes simplex virus type 1 (HSV-1)
strain KOS, thymidine kinase deficient (TK-), HSV1 KOS strain resistant to ACV (ACVr), herpes simplex virus type 2 (HSV-2) strain G, vaccinia virus,
vesicular stomatitis virus (VSV), varicella-zoster
virus (VZV) strain Oka, TK- VZV strain 07-1, respiratory syncytial virus (RSV) strain Long, VSV,
Coxsackie B4, parainfluenza 3, Reovirus-1, Sindbis,
Table 5. Cytotoxicity and antiviral activity of compounds in HeLa cell cultures.
EC50b (µM)
Compound
Minimum
cytotoxic
Vesicular
stomatitis
virus (VSV)
Coxsackie
virus B4
Respiratory
syncytial
virus (RSV)
5a
≥20
>20
>20
>20
5b
≥100
>100
>100
>100
5c
≥100
>100
>100
>100
5d
>100
>100
>100
>100
5e
>100
>100
>100
>100
5f
100
>20
>20
>20
5g
>100
>100
>100
>100
5h
>100
>100
>100
>100
5i
>100
>100
>100
>100
7a
100
>20
>20
>20
7b
>100
>100
>100
>100
7c
>100
>100
>100
>100
7d
>100
>100
>100
>100
7e
100
>20
>20
>20
7f
100
>20
>20
>20
7g
100
>20
>20
>20
7h
>100
>100
>100
>100
7i
100
>20
>20
>20
Levamisole
>100
>100
>100
>100
DS-5000 (µg/mL)
>100
>100
>100
20
(S)-DHPA
>250
>250
>250
>250
Ribavirin
>250
50
250
10
aRequired to cause a microscopically detectable alteration of normal cell morphology.
genicity by 50%.
b
Required to reduce virus-induced cytopatho-
945
Table 6. Cytotoxicity and antiviral activity of compounds in Vero cell cultures.
Compound
Minimum
cytotoxic
EC50b (µM)
Para-influenza-3
virus
Reovirus-1
Sindbis
virus
Coxsackie
virus B4
Punta Toro
virus
5a
100
>20
>20
>20
>20
>20
5b
100
>20
>20
>20
>20
>20
5c
>100
>100
>100
>100
>100
>100
5d
>100
>100
>100
>100
>100
>100
5e
>100
>100
>100
>100
>100
>100
5f
100
>20
>20
>20
>20
>20
5g
>100
>100
>100
>100
>100
>100
5h
>100
>100
>100
>100
>100
>100
5i
>100
>100
>100
>100
>100
>100
7a
100
>20
>20
>20
>20
>20
7b
100
>20
>20
>20
>20
>20
7c
100
>20
>20
>20
>20
>20
7d
>100
>100
>100
>100
>100
>100
7e
100
>20
>20
>20
>20
>20
7f
100
>20
>20
>20
>20
>20
7g
≥100
>100
>100
>100
>100
>100
7h
>100
>100
>100
>100
>100
>100
7i
100
>20
>20
>20
>20
>20
Levamisole
>100
>100
>100
>100
>100
>100
DS-5000 (µg/mL)
>100
>100
>100
>100
>100
>100
(S)-DHPA
>250
>250
>250
>250
>250
>250
Ribavirin
>250
50
>250
>250
>250
112
a
Required to cause a microscopically detectable alteration of normal cell morphology. bRequired to reduce virus-induced cytopathogenicity by 50 %.
Punta Toro, feline coronavirus (FIPV: feline infectious peritonitis virus), influenza A virus subtypes
H1N1 and H3N2, and influenza B virus. Antiviral
assays were carried out in CrandellñRees feline kidney cells (feline corona virus and feline herpes
virus), HeLa cell cultures (vesicular stomatitis virus,
Coxsackie virus B4, and RSV), human embryonic
lung (HEL) cell cultures (HSV-1 (KOS), HSV-2 (G),
vaccinia virus, vesicular stomatitis virus and HSV-1
TK- KOS ACVr), Madin Darby canine kidney
(MDCK) cells (influenza A, H1N1 subtype, influenza A, H3N2 subtype, and influenza B) and Vero cell
cultures (parainfluenza-3 virus, reovirus-1, Sindbis
virus, Coxsackie virus B4, and Punta Toro virus).
The HEL, Vero, and HeLa cell lines used in this
study were monitored for mycoplasma contamination and were found to be mycoplasma-free.
Screening for inhibition of virus-induced cytopathic effect in vitro
Confluent cell cultures in microtiter 96-well
plates were inoculated with 100 CCID50 of virus (1
CCID50 being the virus dose to infect 50% of the
cell cultures) or with 20 plaque forming units
(PFU) (for VZV) and the cell cultures were incubated in the presence of varying concentrations of
the test compounds. Viral cytopathicity or plaque
formation (VZV) was recorded as soon as it
reached completion in the control virus-infected
cell cultures that were not treated with the test
compounds. Antiviral activity was expressed as the
EC50 or compound concentration required to reduce
virus-induced cytopathicity or viral plaque formation by 50%.
946
Cytotoxicity assays
Cytostatic activity was based on the inhibition
of HEL cell growth. HEL cells were seeded at 5 ◊
103 cells/well into 96-well microtiter plates and
allowed to proliferate for 24 h. Then, medium containing different concentrations of the test compounds was added. After 3 days of incubation at
37OC, the cell number was determined with a
Coulter counter. The cytostatic concentration was
calculated as CC50 (the compound concentration
required to reduce cell growth by 50% relative to the
number of cells in the untreated controls). CC50 values were estimated from graphic plots of the number
of cells (percentage of control) as a function of the
concentration of the test compounds. Cytotoxicity
was expressed as minimum cytotoxic concentration
(MCC) or the compound concentration that causes a
microscopically detectable alteration of cell morphology.
Anti-influenza virus activity
The synthesized compounds were evaluated for
their antiviral activity against three influenza virus
subtypes [A/Puerto Rico/8/34 (H1N1); A/Hong
Kong/7/87 (H3N2), and B/Hong Kong/5/72].
Antiviral activity was estimated from the inhibitory
effect on virus-induced cytopathic effect, as determined by microscopical examination and/or the formazan-based MTS cell viability test. The EC50 (50%
effective concentration), or concentration producing
50% inhibition of virus-induced cytopathic effect,
was determined by visual scoring of the CPE, or by
measuring the cell viability with the colorimetric formazan-based MTS assay. Cytotoxicity of the test
compounds was expressed as the compound concentration causing minimal changes in cell morphology
(MCC), or the concentration causing 50% cytotoxicity (CC50), as determined by the MTS assay.
Chemistry
Eighteen derivatives were prepared according
to the strategy outlined in Schemes 1 and 2 and Table
1. The required α-bromoketones were used in the
first step; they were prepared according to the literature by using bromine in acetic acid. Derivatives 2,6substituted imidazo[2,1-b][1,3,4] thiadiazoles (5a-i
and 7a-i) were prepared by condensing 2-amino1,3,4-thiadiazole (1 and 2) with α-bromoketones (3)
in anhydrous ethanol in good yields.
All the synthesized compounds exhibited
absorption bands ranging from 3059 to 3021 cm-1 for
C-H aromatic stretching and 2954-2714 cm-1 for C-H
aliphatic stretching. 1H NMR for imidazole proton
(C5-H) confirms the cyclization of 2-amino-5-substituted-1,3,4-thiadiazole 1 or 2 with respective
phenacyl bromide 3 by the presence of singlet
between δ 8.99 and 8.51 ppm. Aromatic protons
showed prominent signals around δ 8.30-6.97 ppm.
Bridge-headed methylene proton at C2 appeared
between δ 4.72 to 4.68 ppm. The presence of OCH3
proton in compounds 5f and 7f behaved as singlet
between δ 3.78 and 3.77 ppm, respectively. Methyl
proton in 5e and 7e appeared as singlet between δ
2.32 and 2.30 ppm, respectively. The structures of
compounds were finally ascertained by mass spectra.
Bioevaluations
Cytostatic activity
The compounds were evaluated for their cytostatic activity against human HeLa cervical carcinoma and CEM T-lymphocytes as well as murine
L1210 leukemia cells. The data are summarized in
Table 2.
Compounds 5b, 5c, and 7g exhibited IC50 values in the low micromolar range against both HeLa
cervical carcinoma and leukemia L1210 cell lines.
Compounds 5g and 7a were cytostatic only against
cervical carcinoma HeLa cells. The human T-lymphocyte CEM cell line was the least sensitive cell
line towards most of the tested compounds except
for 7g, which showed moderate cytostatic activity at
24 µM concentration.
There was, in general, a strong correlation
between the three tumor cell lines regarding the
cytostatic activities of the compounds. The moderate inhibitors of cervix carcinoma HeLa were also
moderately inhibitory to murine L1210 cell proliferation.
Anti-influenza virus activity
The anti-influenza virus activities of a series of
imidazo[2,1-b][1,3,4]thiadiazole derivatives were
determined by measuring their inhibitory effects on
virus replication in MDCK cells using three strains
of the influenza subtype. The antiviral data obtained
by microscopic evaluation of the virus-induced CPE
were confirmed by the MTS cell viability assay and
were expressed as the EC50. The cytotoxicity of the
compounds were expressed as the MCC (the compound concentration causing minimal alterations in
cell morphology as estimated by microscopy) or the
CC50 (the compound concentration causing 50%
reduction in cell viability based on the MTS assay).
Compounds 5h and 7h showed some activity against
the cytopathic effects of three influenza virus subtypes i.e., H1N1, H3N2, and influenza B (Table 3).
Antiviral activity
The selected compounds were evaluated
against various strains of DNA viruses HSV type 1
(HSV-1) and type 2 (HSV-2), vaccinia virus, and
acyclovir-resistant (ACVr) HSV (Table 4).
The test compounds were also evaluated
against several RNA viruses (i.e., VSV, Coxsackie
B4 virus, RSV, parainfluenza virus type 3, reovirus1, Sindbis virus and Punta Toro virus (Tables 5 and
6). The EC50 was calculated as the effective concentration of compound that decreased the percentage
of formazan production in compound treated, virusinfected cells to 50% of that produced by compound-free uninfected cells. The 50% cytostatic
concentration (CC50) was calculated as the concentration required to reduce cell growth by 50%. MCC
is the minimum cytotoxic concentration that causes
a microscopically detectable alteration of cell morphology. The HEL, Vero, and HeLa cell lines used
in this study were regularly monitored for
mycoplasma contamination and found to be
mycoplasma-free. The antiviral activity was measured for eighteen derivatives of imidazo[2,1b][1,3,4]thiadiazole using a cell-protection assay.
The data are recorded in Tables 4-6.
No specific antiviral effects were noted for
most of the compounds at concentrations close to
their toxicity levels against HSV-1 (strain KOS),
HSV-2 (strain G), HSV-1 (ACV resistant KOS
strain), vaccinia virus, vesicular stomatitis virus,
Coxsackie virus B4, respiratory syncytial virus,
parainfluenza-3 virus, reovirus-1, Sindbis virus, and
Punta Toro virus.
CONCLUSON
In this paper, we report synthesis of 18 derivatives of 2-heteroaryl-6-aryl-imidazo[2,1-b][1,3,4]
thiadiazole. All compounds were evaluated for cytostatic activity against human cervix carcinoma HeLa
and CEM T-lymphocytes and murine L1210
leukemia cells. The compounds 5b and 5c had IC50
values in the range of 52-136 µM. Only derivative
7g displayed moderate cytostatic activity against
both murine leukemia L1210 and T-lymphocytic
947
CEM cells between 23-24 µM. Among the tested
compounds, 5h and 7h showed some activity
against the cytopathic effects of three influenza
virus subtypes, namely Influenza A, H1N1, H3N2
and Influenza B.
Acknowledgments
We gratefully acknowledge the support for this
research effort from All India Council for Technical
Education (AICTE), New Delhi (Ref. No.
8023/BOR/RID/RPS-169/2008-09) for SSK. The
cytotoxic activities were determined by financial
support of the KU Leuven (GOA 10/014). We
would also like to thank NMR facility, Indian
Institute of Science, Bangalore for characterization
of all compounds. We would like to thank Mrs.
Lizette van Berckelaer and Mrs. Leentje Persoons
for the cytotoxicity and antiviral cellular evaluation
assays.
REFERENCES
1. Karki S.S., Panjamurthy K., Kumar S., Nambiar
M., Ramareddy S.A. et al.: Eur. J. Med. Chem.
46, 2109 (2011).
2. Kumar S., Hegde M., Gopalakrishnan V., 1.
Renuka V.K., Ramareddy S.A. et al.: Eur. J.
Med. Chem. 84, 687 (2014).
3. Kumar S., Gopalakrishnan V., Hegde M., Rana,
V., Dhepe S.S. et al.: Bioorg. Med. Chem. Lett.
24, 4682 (2014).
4. Kolavi G., Hegde V., Khazi I.A.M., Gadad P.:
Bioorg. Med. Chem. 14, 3069 (2006).
5. Baraldi P.B., Nunez M.DelC., Tabrizo M.A.,
De Clercq. E., Balzarini J. et al.: J. Med. Chem.
47, 2877 (2004).
6. De Clercq E., Descamps J., Verhelst G., Walker
R.T., Jones A.S. et al.: J. Infect. Dis. 141, 563
(1980).
7. De Clercq E., Hol˝ A., Rosenberg I., Sakuma
T., Balzarini J., Maudgal P.C.,: Nature 323, 464
(1986).
Received: 2. 09. 2015
ISSN 0001-6837
NATURAL DRUGS
PHYTOCHEMICAL ANALYSIS AND CARDIOTONIC ACTIVITY
OF METHANOLIC EXTRACT OF RANUNCULUS MURICATUS LINN.
IN ISOLATED RABBIT HEART
ALAMGEER1*, ABDUL QAYUM KHAN1, TASEER AHMAD2, MUHAMMAD NAVEED MUSHTAQ1,
MUHAMMAD NASIR HAYAT MALIK1, HUMA NAZ1, HASEB AHSAN1, HIRA ASIF1,
NABEELA NOOR1, MUHAMMAD SHAFIQ UR RAHMAN3, UMAIR DAR4
and MUHAMMAD RASHID1
1
Faculty of Pharmacy University of Sargodha, Sargodha, Pakistan
Shifa College of Pharmaceutical Sciences, Shifa Tameer-e-Millat University, Islamabad, Pakistan
3
University of Central Punjab, Lahore, Pakistan
4
Lahore College of Pharmaceutical Sciences, Lahore, Pakistan
2
Abstract: Ranunculus muricatus Linn. (RML) have been traditionally used for the treatment of various cardiovascular disorders. The aim of present study was to evaluate their cardiovascular effects in isolated perfused
rabbit heart. The methanolic extract of RML was prepared by cold maceration process. The methanolic extract
of RML (1 ng to 10 mg) was used to determine the percentage change in force of contraction (FC), heart rate
(HR) and perfusion pressure (PP) by using Langendorffís Perfused Heart Apparatus. The PP, FC and HR of
isolated rabbit heart were measured by power lab data acquisition system. Moreover, phytochemical analysis
and acute toxicity study were also performed. The methanolic extract at the doses from 1 ng to 10 mg exhibited a significant increase in perfusion pressure and force of contraction. Moreover, the crude extract of RML
revealed a significant increase in heart rate at doses from 1 ng to 1 µg. The maximum rise in all the three parameters was observed at 1 µg and 1 ng, respectively. In another study, the methanolic extract was tested in the presence of propranolol and verapamil on isolated perfused rabbit heart. The study shown that the increase in HR
and FC produced by the plant extracts was significantly reduced in the presence of propranolol whereas PP
remained significantly raised even in the presence of propranolol. However, in the presence of verapamil, this
increased PP was significantly reversed to a decrease while a significant positive inotropic and chronotropic
effects were observed. It is concluded that the cardiotonic activity of methanolic extract of RML might be due
certain cardio active chemical compounds. Further studies are needed to isolate these pharmacologically active
phytochemical constituents and elucidate their exact mechanism of action.
Keywords: Ranunculus muricatus Linn., cardiotonic, rabbit, Langendorffís isolated heart apparatus, phytochemical, acute toxicity
pharmacological activities are distributed throughout Pakistan. Out of these 5000 species, about 600 to
700 species are being used by local people for many
medicinal purposes (2). It is understood that synthetic medicines cause more side effects as compared to natural products; western countries are also
shifting to natural products (3). Finding healing
powers in plants is an ancient idea, in this respect
herbs have been used for medical treatment since the
beginning of human civilization (4).
The Ranunculaceae is a family with a large
number of plants. It has 50 genera and 2000 species
Cardiovascular diseases have been considered
a severe health problem around the globe. The major
risk factors for heart diseases include family history,
sex, hypercholesterolemia, hypertension, obesity,
and cigarette smoking. Most of these risk factors are
prevalent in developing countries because of the
absence of appropriate infrastructure. Therefore,
these diseases have become a very common problem
in the rich population of the developing countries
(1). Pakistan is enriched with large variety of medicinal plants. According to a survey report more than
5000 species of plants with probable potential of
* Corresponding author: e-mail: alam_yuchi@yahoo.com; phone: +92-343-7547785
949
950
ALAMGEER et al.
which are widely distributed throughout the northern hemisphere. It also found in southern temperature regions (5). One of Ranunculaceae important
member is RML traditionally used to regulate normal heart beat, body temperature and also beneficial
in fever and nausea (6).
MATERIAL AND METHODS
Plant material
RMLwas collected from marshy areas of
Talash district Dir (lower) Khyber Pukhtoon Khwa
(KPK) Pakistan in the month of May 2013. The
plant was identified by Dr. Ali Hazrat (taxonomist)
research officer of Shaheed Benazir Bhutto
University, Sheringle Upper Dir, KPK, Pakistan.
The specimen was deposited in the pharmacy
department herbarium.
Plant extraction
After collection the plant was dried under
shade at room temperature. The dried plantsí material was grounded into a coarse powder form by
china herbal grinder. Methanolic extract of the
plants was prepared using cold maceration process.
RML material (12 kg) was soaked in methanol 5
liters and 8 liters for 72 hours at room temperature,
respectively, with occasional shaking. After 3 days,
these materials were filtered using muslin cloth and
filter papers. The filtrates of extracts were evaporated under reduced pressure in rotary evaporator at
50OC. This methanolic extract was then air-dried to
obtain a solid mass (7). The crude extract was greenish black in color and was also soluble in distilled
water.
Animals and housing conditions
Rabbits (1000-1500 g) of either sex were used.
All the animals were housed in controlled environment (23-25OC) at animal house of University of
Sargodha, Sargodha. The study protocol was
approved by the Institutional Animal Ethics
Committee (IEC), Faculty of Pharmacy, University
of Sargodha (Approval No. 40-B21 IEC UOS).
Experiments comply with the declarations of
National Research Council (8).
EXPERIMENTAL
Acute toxicity test
The objective of these experiments was to
determine the LD50 of the crude extract. For this purpose both male and female albino mice weighing 3040 g were randomly divided into five groups of two
animals each. Group I served as control and received
normal saline (10 mL/kg) while other groups (Group
2, Group 3, Group 4 and Group 5) were given different doses (intraperitoneally) of crude extract of RML
in an ascending order i.e., 100, 500, 1000, 1500
mg/kg, respectively. The mortality rate was observed
for 24 h. Since no mortality occurred in any group
treated with RML extract, so another five groups of
mice were taken. They were again treated with the
various doses of crude extract in an increasing order
i.e., 2000, 2500, 3000, 3500, 4000 mg/kg, respectively.The highest dose, which did not kill any animals, and the lowest dose, which killed only one
mouse, was noted. LD50 was calculated from the geographic mean of these two doses (9).
Preliminary phytochemical test
The methanolic extract of RML was analyzed
for the presence of different phytochemical constituents such as flavonoids, reducing sugars, tannins, phenolic compounds, saponins, alkaloids and
cardiac glycosides by using standard methods (10).
Effects of crude extracts on various cardiac
parameters by Langendorff method
The experiments were performed according to
the method prescribed by Langendorff, 1895. A rabbit (n = 6) was injected with 1000 IU of heparin
intravenously through the marginal ear vein, 30 min
before dissection. Five minutes later, the rabbit was
sacrificed; its heart was dissected out with about 1
cm of aorta attached, and transferred as quickly as
possible into a Petri dish containing Krebs-Henseleit
solution. The heart was then cleaned of any excessive tissue and mounted on the Langendorffís apparatus containing Krebs-Henseleit solution maintained at 37OC. The aorta was tied to the glass cannula with a pressure transducer. A clip was attached
to the apex of heart to measure the FC (g) by forcedisplacement transducer. Both the transducers were
attached to the Power Lab data acquisition system
and the recordings were measured using Chart 5.0
Pro software. The preparation was then allowed to
equilibrate for 30 min before starting the experiment. After stabilization, different doses (1 ng, 10
ng, 100 ng, 1 µg, 10 µg, 100 µg, 1 mg and 10
mg/mL) of the extract were applied to assess various
cardiac parameters (HR, FC and PP) with each heart
serving as its own control. The drug in a fixed volume of 5 mL was injected with 5 mL syringe
through a three way port. Each dose was first filtered
with micro filter before injection. In order to elucidate the possible mechanism of action, the effect of
selected dose of the extract was assessed both in the
Phytochemical analysis and cardiotonic activity of methanolic extract of...
951
absence and presence of propranolol (10-5 M) and
verapamil (10-6 M) (7, 11).
produced significant changes in behavior, breathing,
sensory and nervous system responses in mice.
The results were expressed as the mean ± standard error of the mean (SEM). Studentís t-test was
applied; with p < 0.05 and p < 0.001 considered as
significant and highly significant.
Preliminary phytochemical test
Alkaloids, indole alkaloids, tannins, saponins,
reducing sugars, cardiac glycosides, steroids and terpeniods were present in the plat as shown in Table 1.
RESULTS
Acute toxicity
LD50 of the RML was calculated from the data
and was found to be 4000 mg/kg. The methanolic
extract from 100 to 3500 mg/kg body weight did not
Effect of crude extract on various cardiac parameters
The crude extract of RML in the doses from 1
ng to 10 mg exhibited a significant increase in PP
and FC. The maximum (p < 0.001) increase in PP
and inotropic effect was observed at 1 µg and 1 mg
(83.5 ± 8.14 and 27.8 ± 0.08), respectively. The
Figure 1. Effect of different doses of crude extract of RML on PP, FC and HR of isolated heart, where a = (p < 0.001), b = (p < 0.01), c =
(p < 0.1), NS = Non-significant as compared to control
Tracing 1. Effect of crude extract of Ranunculus muricatus Linn. on FC and PP
952
ALAMGEER et al.
crude extract at a dose from 1 ng to 1 µg produced a
significant increase in chronotropic effect. The maximum increase (p < 0.001) in HR was observed at a
dose of 10 ng (56.8 ± 6.18). It was interesting to note
that at higher doses from 10 µg to 10 mg, a non-significant effect was produced. The remarkable effects
in all the three parameters were observed at 1 ng
(Fig. 1) (Tracing 1).
Effect of crude extract of RML on isolated heart
in the presence of verapamil
The crude extract of RML at a dose of 1 ng, in
the presence of verapamil 10-6 M exhibited a significant reduction in PP (-8.12 ± 2.59) of the isolated
heart. However, the increase in FC and HR was not
reduced in the presence of verapamil (Fig. 3).
DISCUSSION
Effect of crude extract of RML on isolated heart
in the presence of propranolol
The crude extract of RML at a dose of 1 ng, in
the presence of propranolol 10-5 M exhibited a significant decrease in FC (-20.1 ± 0.23) and HR (19.0% ± 6.96). However, there was not any reduction in PP in the presence of propranolol (Fig. 2).
RML are important medicinal plants commonly found in Pakistan. They are of great utility and
have been reported to be effective in the treatment of
cough, fever, stomach ache, and various cardiac
complications (6, 12).
Table 1. Phytochemical analysis of Ranunculus muricatus Linn.
TEST
OBSERVATIONS
INFERENCES
Alkaloids
Colored precipitate
+
Indole alkaloids
Change in coloration
+
Tannins
Dark blue or greenish
+
Saponins
Persistent froth
-
Reducing sugars
Red precipitate formed
-
Cardiac glycosides
Reddish brown
+
Steroids
Greenish/blue rings
+
Terpeniods
Reddish brown
+
Key: + = present, - = absent
Figure 2. The effects of extract of Ranunculus muricatus Linn. on PP, FC and HR before and after the use of propranolol, where a = (p <
0.001) increased from the control before propranolol and A = (p < 0.001) increased as compared to the control after propranolol, b = (p <
0.05) increased from the control before propranolol, B = (p < 0.05) and C = (p < 0.001) decreased as compared to the control after propranolol
Phytochemical analysis and cardiotonic activity of methanolic extract of...
953
Figure 3. The effects of crude extract of RML on three parameters of isolated rabbit heart before and after the use of verapamil, where a =
(p < 0.001) increased from the control before verapamil, b = (p < 0.05) increased from the control after verapamil, c = (p < 0.05) decreased
from the control after verapamil
The methanolic extract of RML was evaluated
for its cardiovascular activities on isolated rabbit
heart. The extract produced a significant cardiotonic
effect. The crude extract of RML produced a maximum (p < 0.001) increase in FC, HR and PP at a
dose of 1 µg and 1 ng, respectively. This cardiotonic activity of the crude extract might be due to the
involvement of β1 receptors or Ca2+ channels. In
order to investigate the possible role of β1 receptors,
both the extracts were tested in the presence of propranolol. The results revealed that the positive
inotropic and chronotropic effects were significantly reversed to a decrease indicating an adrenaline
like activity. These effects were in accordance with
the prior studies (13).
The rise in PP in the presence of propranolol
suggested that the constriction of coronary blood
vessels might be due to the involvement of Ca2+
channels or α-receptors. In order to determine the
possible role of Ca2+ channels, verapamil was used to
block these channels and then the crude extract was
tested in the presence of verapamil. The extracts
produced a significant reduction in PP, which was in
agreement with previous studies (14). This effect
might be due to the Ca2+ entry through the voltagedependent Ca2+ channels. However, the role of αreceptors should also be studied in order to determine the exact mechanism of action.
The preliminary phytochemical analysis has
shown that the extract contains certain important
chemical constituents that might be involved in an
increase of all the three parameters. The methanolic
extract of RML contains compounds such as alka-
loids, flavonoids, cardiac glycosides, tannins,
steroids and indole alkaloids. Previous studies have
indicated that cardiac glycosides and steroids appear
to be involved in a positive inotropic activity (15). It
has been reported that certain alkaloids might be
involved in the cardiotonic effect. Moreover, glycoalkaloids have also been known to exert their cardiotonic activities in isolated frog heart (16, 17).
Cardiac glycosides, on the other hand, are known to
work by inhibiting the Na+/K+ pump. This inhibition
increases the level of Ca2+ ions available for contraction of the heart muscle, which improves cardiac
output and reduces distention of heart (18).
Similarly, plant steroids are known to be effective
for their cardiotonic activities (19). It has also been
documented that tannins also appear to exert a significant cardiotonic activity in frog heart. They form
insoluble calcium salts and the stimulant action
depends on the presence of Ca2+ in the perfusion
fluid. It could be inferred from these studies that
methanolic extract of RML possesses certain active
phytochemical constituents that might be responsible for their cardiotonic activity in isolated rabbit
heart. Moreover, tannins present in the extract might
be involved in a rise in perfusion pressure as reported in previous studies (20).
CONCLUSION
It is concluded from the study that various biologically active compounds in the methanolic
extracts of RML has produced positive inotropic and
chronotropic effects on Langendorffís isolated rab-
954
ALAMGEER et al.
bit heart preparation. The results suggested that
active principle(s) responsible for positive inotropic
and chronotropic effects of RML extract act by β1
receptors and Ca2+ channels. However, further studies are required to isolate these pharmacologically
active phytochemical constituents and elucidate
their exact mechanism of action.
REFERENCES
1. Trivedi P. C., Nehra S.: Herbal drugs and
biotechnology, Plant which cures heart disease.
p. 3, Pointer Publishers, Jaipur 2004.
2. Ali S.I., Qaiser M.A.: Proc. R. Soc. Edinburg
89, 89 (1986).
3. Shinwari Z.K., Gilani S.S.: J. Ethnopharmacol.
84, 289 (2003).
4. Mashour N.H., Lin G.I., Frishman W.H.: Arch.
Intern. Med. 158, 2225 (1998).
5. Mabberley D.I. The Plant Book. Cambridge
University Press, Cambridge, New York 1987.
6. Khuroo A.A., Akhtar H., Malik D., Dar G.H.,
Khan Z.S.: Asian J. Plant Sci. 6, 148 (2007).
7. Farid A., Shahid R., Mansoor A.K., Usman G.:
J. Islamic Acad. Sci. 5, 67 (1992).
8. NRC. Guide for the care and use of laboratory
animals. National Academy Press, Washington
DC 1996.
9. Akhila J.S., Deepa S., Alwar M.C.: Curr. Sci.
India 93, 917 (2007).
10. Khandelwal K.R.: Practical Pharmacognosy,
pp. 149-153, Nirali Prakashan Publishers, Pune
2006.
11. Al-Hashem F.H., Dallak M.A., Nwoye L.O.,
Bin-Jaliah I.M., AL-Amri H.S. et al.: Saudi J.
Biol. Sci.19, 93 (2012).
12. Hamayun M., Khan A., Afzal S., Khan M.A.:
Indian J. Tradit. Know. 5, 407 (2006).
13. Bain W.A.: Exp. Physiol. 19, 297 (1929).
14. Hearse D.: Cellular damage during myocardial
ischemia. John Wiley and Sons Ltd., New York
1979.
15. Tanz R.D., Kerby C.F.: J. Pharmacol. Exp.
Ther. 131, 56 (1961).
16. Daly M.N.E., Gusovsky F.F.: J. Med. Chem. 31
477 (1988).
17. Nishie K., Fitzpatrick T.J., Swain A.P., Keyl
A.C.: Res. Commun. Chem. Pathol. Pharmacol.
15, 601 (1976).
18. Schneider G., Wolfling J.: Curr. Org. Chem. 8,
1381 (2004).
19. Callow R.K., Young, F.G.: Proc. Royal Soc.
London Series A 157, 194 (1936).
20. Broadbent J.L.: Br. J. Pharmacol. 18, 167
(1962).
Received: 21. 02. 2015
ISSN 0001-6837
APPLICATION OF DRY HAWTHORN (CRATAEGUS OXYACANTHA L.)
EXTRACT IN NATURAL TOPICAL FORMULATIONS
ADA STELMAKIENE1*, KRISTINA RAMANAUSKIENE1, VILMA PETRIKAITE2,
VALDAS JAKSTAS3 and VITALIS BRIEDIS1
Department of Clinical Pharmacy, 2Department of Drug Chemistry, 3Department of Pharmacognosy,
Lithuanian University of Health Sciences, Mickeviciaus 9, Kaunas, Lithuania
1
Abstract: There is a great potential for a semi-solid preparation for topical application to the skin that would
use materials of natural origin not only as an active substance but also as its base. The aim of this research was
to model semisolid preparations containing hawthorn extract and to determine the effect of their bases (carriers) on the release of active components from experimental dosage forms, based on the results of the in vitro
studies of the bioactivity of hawthorn active components and ex vivo skin penetration studies. The active compounds of hawthorn were indentified and quantified by validated HPLC method. The antimicrobial and antiradical activity of dry hawthorn extract were evaluated by methods in vitro. The penetration of active substances
into the full undamaged human skin was evaluated by method ex vivo. Natural topical composition was chosen
according to the results of release of active compounds. Release experiments were performed with modified
Franz type diffusion cells. B.cereus was the most sensitive bacteria for the hawthorn extract. Extract showed
antiradical activity, however the penetration was limited. Only traces of hyperoside and isoquercitrin were
founded in epidermis. Protective topical preparation with shea butter released 41.4-42.4% of active substances.
Four major compounds of dry hawthorn extract were identified. The research showed that extract had antimicrobial and antiradical activity, however compounds of hawthorn stay on the surface of the undamaged human
skin. Topical preparation containing beeswax did not release active compounds. Beeswax was identified as suspending agent. Topical preparations released active compounds when shea butter was used instead of beeswax.
Keywords: hawthorn, natural, release, skin penetration, topical
tions modeling. Crataegus spp. is a member of the
rose family which has been used for its medical
properties since ancient times. It is well known that
bioactivity of hawthorn is associated with the treatment of cardiovascular diseases (4-7), however
recently, a broader spectrum of the effect of
hawthorn bioactivity has been found. Hawthorn
extracts were incorporated into semisolid drug
forms (8). Crataegus spp. is indeed one of the
species that is highly recommended in folk medicine, being regarded as particularly important in the
management and prevention of age-related diseases
(for instance, cardiovascular disease, atherosclerosis, arthritis, and hypertension), nervous system disorders (such as migraines, confusion, irritability and
memory loss) and treatment of upper respiratory
infections, cellulite, obesity and menopause disturbances (9, 10). The juice of its fruits is a topical
preparation for skin application that relieves pain
and stiffness (10). C. monogyna extracts have been
verified to be highly effective against Candida albi-
The base of semisolid preparations is an active
factor that may affect the release of the drug substances from a pharmaceutical form and their absorption through biological membranes, which in turn
may have an effect on the pharmacokinetic parameters of a pharmaceutical preparation (1, 2). The base
composition of a semi-solid preparation affects its
stability and drug release kinetics. As products from
natural materials are becoming increasingly popular,
scientists and manufacturers face a challenging task
of creating stable products that meet modern requirements by using materials of natural origin (2). There
is a great potential for a semi-solid preparation for
topical application to the skin that would use materials of natural origin not only as an active substance
but also as its base. When modeling an ointment
basis, the following components were selected:
beeswax, shea butter, cholesterol, cocoa butter,
honey, avocado and olive oils (3).
Dry hawthorn (Crataegus oxyacantha L.)
extract was used as an active substance for formula-
* Corresponding author: e-mail ada.stelmakiene@yahoo.com
955
956
ADA STELMAKIENE et al.
cans and Herpes simplex virus (11). However, no
effect of hawthorn extracts on C. albicans was
observed by other authors (4). The O-glycosidic
flavonoids and the oligomeric proanthocyanidins
from Crataegus sinaica exhibited significant
inhibitory activity against Herpes simplex virus type
1 (HSV-1), which was shown to be due to an extracellular mechanism for procyanidin C-1 (12).
Extracts of Crataegus spp. have anti-inflammatory,
gastro-protective and antimicrobial properties (4,
13-15). Antioxidant, radical-scavenging activity of
different parts of hawthorn have been evaluated by a
number of authors (16-19).
Research has shown that C. monogyna extract
can be used as a photoprotective agent, which provides stronger protection against UVA than UVB
radiation (20). Crataegus monogyna has been shown
to be cytoprotective by scavenging free radicals.
Jalali et al. study has shown that Crataegus monogyna fruits aqueous extract with antioxidant properties could serve as a protective agent against reproductive toxicity during cyclophosphamide treatment
in a rat model (21). Furthermore, Crataegus pinnatifida extract significantly inhibited the generation of
reactive oxygen species and the phenomena of
inflammation induced by TPA. Also, extract inhibited benzopyrene/TPA-induced skin tumor formation
and decreased the incidence of tumor (22). The
extracts obtained from C. monogyna parts revealed
antiproliferative activity associated with cancer pro-
gression and development, such as cell proliferation,
apoptosis, cell differentiation and neovascularization, and did not show toxicity for non-tumor cells
(23). Other authors also found antitumor activity of
Crataegus spp. demonstrated on various cell lines
(24).
There are little data published in the scientific
literature about the use of hawthorn extract in the
production of topical preparations. The literature
search did not yield any data pertaining to the skin
penetration of the active components of hawthorn
extract or the effect of carriers on the release of
active components from topical preparations. The
physical and chemical properties of the drug substance in the topically applied pharmaceutical forms
determines the permeation of a given drug substances through the skin from applied carrier matrix
(25). Topical bioavailability of a drug depends on
the amount of drug diffusing from the dosage form
and reaching the surface of the skin where it can be
absorbed. Thus, it is relevant not only to produce
stable semisolid preparations but also to study the
skin penetration of hawthorn active components and
to select suitable carriers that would ensure a proper
release of active compounds.
The aim of this research was to model semisolid preparations containing hawthorn extract and to
determine the effect of their bases (carriers) on the
release of active components from experimental
dosage forms, based on the results of the in vitro
Figure 1. Chemical structure of active compounds of hawthorn flowers and leaves extract. A - chlorogenic acid; B - vitexin-2íí-O-rhamnoside; C - hyperoside; D - isoquercitrin
Application of dry hawthorn (Crataegus oxycantha L.) extract...
957
Figure 2. Typical chromatogram of active compounds of hawthorn extract: 1 - chlorogenic acid; 2 - vitexin-2íí-O-rhamnoside; 3 - hyperoside; 4 - isoquercitrin
studies of the bioactivity of hawthorn active components and ex vivo skin penetration studies.
Materials
Vitexin-2?-O-rhamnoside, HPLC grade acetonitrile and acetic acid were purchased from Sigma
Aldrich Chemie GmbH (Steinheim, Germany).
Hyperoside, isoquercitrin and chlorogenic acid were
purchased from Carl Roth. Standardized dry
hawthorn extract was purchased from Naturex
(France).
Methods
Preparation of hawthorn extract for analysis
The hawthorn leaf and flower dry extract was
dissolved in purified water at the ratio 1 : 20 (w/v)
by mixing it with a magnetic stirrer for one hour at
room temperature.
HPLC
Amounts of hawthorn active components were
evaluated by HPLC method with Agilent 1260
Infinity
Capillary
LC
System
(Agilent
Technologies, Santa Clara, USA) using the Agilent
diode array detector (DAD) and applying validated
HPLC method for quantification. The separation
was performed in C18 ACE (5 µm) 150 ◊ 0.5 mm
i.d. column. The mobile phase consisted of the solvent (A) 0.5% aqueous acetic acid (v/v) and (B) acetonitrile, using gradient elution of 10% (B) at 0-3
min, 14% (B) at 3.1-25 min, and 70% (B) at 26-29
min and then returned to the initial conditions with
10 min re-equilibration, with total 40 min run time.
The analysis was carried out at a flow rate of 10
µL/min with the detection wavelength set at 340 nm.
Total phenolic content
Total phenolic content was determined according to the method of Ramanauskiene et al. (26). The
quantity (n = 3) of phenolic compounds was determined using Agilent 8453 UV-Vis spectrophotometer (Agilent Technologies, Inc., Santa Clara, USA)
according to p-coumaric acid equivalents after reaction with Folin-Ciocalteu phenol reagent.
Antiradical scavenging activity
A 0.1 mmol/L DPPH solution was prepared in
96% ethanol. The solution was kept in a cool, dark
place for 24 h. The reaction (n = 3) was initiated in
a cuvette to which 2.9 mL of DPPH solution and 0.1
mL of the analyzed solution were added. The
absorbance of the reaction mixture was measured at
the wavelength of 518 nm 30 min after the start of
the reaction. The results were expressed in percentage of bound DPPH according to Molyneux (27).
Antimicrobial activity
The microbiological examination (n = 5) was
performed in aseptic conditions. During the microbiological study, the antimicrobial activity of the
studied preparation was determined using solid
growth media and the well technique. Resistance to
preparations from natural material was examined in
Mueller-Hinton agar (Mueller-Hinton Agar II, BBL,
Cockeysville, USA) with standard cultures of
Pseudomonas aeruginosa ATCC 27853, Proteus
mirabilis ATCC 12459, Staphylococcus aureus
ATCC 25923, Staphylococcus epidermidis ATCC
12228, Escherichia coli ATCC 25922, Bacillus
cereus ATCC 8035 and Candida albicans ATCC
60193. Agar was poured into sterile Petri dishes 85
mm in diameter (20 mL in each dish). Agar wells
were 7 mm in diameter and 8 mm deep. The density
of microorganism suspensions applied in the test
was 0.5 Mac Farland standard scale (5.107 n 1.108
CFU/mL). The density of the bacterial suspension in
the Mueller-Hinton agar was 106 CFU/mL. After
the agar solidified, wells were formed in the medium and were filled with the studied preparation. The
cultures were incubated for 24 h in a thermostat at
958
37OC, and then the microorganism growth in the
whole agar volume was evaluated.
Rheological flow behavior test
The test (n = 5) was performed using the Carrimed CSL2 500 rheometer (TA Instruments, USA),
by applying the cone-and-plate geometry system
(cone diameter ñ 60 mm, angle ñ 2O, sample thickness ñ 150 µm. The shear rate was increased for 2
min from 0 to 500 s-1. Rheological characteristics
were calculated according to the Ostwald de
Waeleís mathematical model.
In vitro release study
In vitro release experiments (n = 3) were performed using the modified Franz type diffusion
cells. The semisolid sample (1.00 ± 0.02 g) was
placed into these cells with a dialysis membrane.
The CuprophanÆ dialysis membrane (Medicell
International Ltd., London, UK) was made of natural cellulose. Area of the diffusion was 1.77 cm2.
Purified water was used as an acceptor medium. The
temperature of acceptor medium was kept on 37 ±
0.2OC. The medium was stirred using magnetic stirrer. The samples from the acceptor solution were
taken at 1, 2, 4, 6 h and immediately replaced with
the same volume of fresh acceptor solution.
Penetration experiment through the full-thickness
undamaged human skin
Abdominal skin of Caucasian women (age
range: 25-40 years) was obtained from the
Department of Plastic and Reconstructive Surgery
(the Hospital of the Lithuanian University of Health
Sciences, Lithuania) after cosmetic surgery. It was
stored at -20OC for not longer than 6 months before
use. Kaunas Regional Biomedical Research Ethics
Committee has approved the use of human skin for
transdermal penetration studies. A Bronaugh-type
flow-through diffusion cell with full-thickness
human skin was used for ex vivo skin penetration
experiments (n = 3). The experiment was performed
according to the methods proposed by Kezutyte et
al. and Zilius et al. (25, 28, 29).
Statistical data evaluation was performed using
the SPSS software with one-way ANOVA. Tukeyís
post hoc test was performed for multiple comparisons. Level of significance was determined as p <
0.05.
The experimental research found phenolic
compounds of different classes in the standardized
extract of dried hawthorn leaves and flowers: phe-
S. epidemidis
B. cereus
E. coli
S. aureus
Figure 3. Inhibition zones (mm) of bacterial growth including well diameter (7 mm)
Table 1. Amounts of active components found in dry hawthorn extract.
Analyzed
sample
Total phenolic content
based on p-coumaric
acid equivalent, mg/g
Chlorogenic
acid, mg/g
Vitexin-2î-Orhamnoside, mg/g
Hyperoside,
mg/g
Isoquercitrin,
mg/g
Dry hawthorn
extract
92.00 ± 0.53
7.91 ± 0.52
23.00 ± 1.38
3.65 ± 0.43
1.95 ± 0.89
959
Table 2. Penetration (n = 3) of standard mix of hawthorn active compounds into human skin ex vivo.
Epidermis, µg/mL
Sample
Chlorogenic acid
Vitexin-2"-O-rhamnoside
Hyperoside
Isoquercitrin
Standard mix and
taped skin
-
-
tr
-
Standard mix
-
-
tr
tr
tr = traces
Figure 4. Antiradical activity (n = 3) of the total phenolic content of hawthorn based on the binding of DPPH radical
nolic acid, flavone-C-glycoside, flavonol-O-glycoside. The prevailing compounds, the chemical structure of which is provided in Figure 1, were quantitatively assessed in the hawthorn extract.
A typical chromatogram of active components
of hawthorn dried leaves and flowers extract was
obtained (seen in Fig. 2), based on the adapted and
validated HPLC method. The quality of the dry
hawthorn extract was assessed by measuring the
amounts of prevailing active components as well as
the total phenolic content (Table 1). It has been determined that the hawthorn leaf and flower extract contained the highest levels of vitexin-2íí-O-rhamnoside.
While there are research data indicating that
hawthorn extracts show antimicrobial activity, the
activity against specific microorganisms varies
between different authors (4, 14, 15). The aforementioned differences are probably determined by different hawthorn species analyzed, different
hawthorn plant materials as well as different solvents. The antimicrobial research carried out by the
authors of this study analyzed an aqueous solution
of Crataegus oxycanta dried flowers and leaves
extract. The results of the antimicrobial activity in
vitro studies showed that the active components of
the hawthorn extract possess antimicrobial activity.
Seven microorganisms were analyzed during the
antimicrobial study. The extract of hawthorn flowers and leaves inhibited the growth of S. epidermidis, B. cereus, E. coli and S. aureus, whereas P.
mirabilis, P. aeruginosa and C. albicans remained
resistant. B. cereus showed the highest degree of
sensitivity (the widest zone of inhibition) to the
hawthorn extract (Fig. 3).
Different methods can be found in scientific
literature to determine antiradical and antioxidant
activity of hawthorn extract (14, 16-19). Differences
in values of DPPH antiradical activity occur as a
result of methodological differences. The study
results of antiradical activity showed that the
hawthorn extract possesses antiradical activity (Fig.
4). The binding of the free radical DPPHï depends
on the concentration of compounds: the greater
amount of phenolic compounds leads to more powerful radical binding effect.
The results of the bioactivity tests have confirmed the data reported in scientific literature (4,
16) that hawthorn extract is a suitable component for
topical preparations due its antimicrobial and
antioxidant properties.
960
2). This can be associated with the fact that the solubility of hyperoside and isoquercitrin is higher in a
lipophilic environment than in a hydrophilic one
(logP is 0.4), as compared to the predicted
hydrophilic properties of chlorogenic acid and vitexin-2ì-O-rhamnoside because their logP is -0.4 and 1.3, respectively. The permeation test results
showed that the hawthorn active compounds do not
permeate across the undamaged human skin and
their activity is limited to the surface of the skin
index,
Ex vivo penetration experiment was conducted
using full-thickness undamaged human skin to
determine the capacity of hawthorn active compounds for skin penetration. The study results
(Table 2) showed that the epidermis acts as a permeability barrier; no analyzed compounds were
found in the dermis. After 24 h of testing, it was
determined that the more lipophilic hyperoside and
isoquercitrin accumulate in the epidermis but their
amounts are below the limit of quantification (Table
Figure 5. Rheological characteristics of group I samples at the temperature of 30OC
Table 3. Compositions of modelled natural topical formulations (g).
Group
I
II
III
Sample
no.
Beeswax
Honey
Butter
cacao
Cholesterol
Avocado/Olive
oil
Shea
butter
Dry hawthorn
extract
N1
5.0
10.0
10.0
3.0
72.0
-
-
N2
7.0
10.0
10.0
3.0
70.0
-
-
N3
9.0
10.0
10.0
3.0
68.0
-
-
N4
11.0
10.0
10.0
3.0
66.0
-
-
N5
13.0
10.0
10.0
3.0
64.0
-
-
N6
15.0
10.0
10.0
3.0
62.0
-
-
N7
-
-
-
-
98.0
-
2.0
N8
0.5
-
-
-
97.5
-
2.0
N9
1.0
-
-
-
97.0
-
2.0
N10
1.5
-
-
-
96.5
-
2.0
N11
2.0
-
-
-
96.0
-
2.0
N12
3.0
-
-
-
95.0
-
2.0
N13
5.0
-
-
-
93.0
-
2.0
N14
-
10.0
10.0
3.0
60.0
-
2.0
N15
-
10.0
10.0
3.0
60.0
15.0
2.0
N16
0.5
10.0
10.0
3.0
60.0
14.5
2.0
N17
1.0
10.0
10.0
3.0
60.0
14.0
2.0
961
Figure 6. The kinetics of hawthorn active compounds release (n = 3) from group II formulations
(Table 2). This is determined by their lipophilicity
and skin layer properties. In order to determine
whether the stratum corneum limits the penetration
of hawthorn active compounds into the human skin,
this skin layer was removed. Ex vivo penetration
results showed that the permeability of active compounds across the human skin with no stratum
corneum did not increase over 24 h of the test. No
active compounds were found in the dermis layer of
the skin. Meanwhile, hyperoside and isoquercitrin
(Table 2) were identified in the epidermis with no
stratum corneum. The study results showed that the
stratum corneum does not limit the permeability of
the studied compounds. Based on the results
obtained, it can be assumed that the permeability of
active compounds is affected by the viable epidermis which is more lipophilic, the closer it is to the
stratum corneum (30). The study results have supported the data reported in scientific literature that
the physical and chemical properties of the drug
substance in the topical dosage forms determines the
permeation of the drug substance across the skin
(31, 32). Based on the results of these hawthorn skin
penetration tests, it is appropriate to select carriers
that act in the epidermis and on its surface and allow
modeling a stable protective semisolid preparation
for topical application (33). The ointment base for
incorporating active substance was selected by combining components of natural origin (Table 3).
Beeswax and olive oil were the main components of semisolid bases of groups I and II. Also, a
N7 suspension and a N14 formulation without
beeswax were prepared to determine the effect of this
material on the release of active substances. Shea
butter and olive oil were used as the main components for the base of group III semisolids. An emulsifier ñ cholesterol for enhanced stability and cocoa
butter for better spreadability were incorporated into
the base of I and III group semisolids (34).
Furthermore, honey was incorporated for its antibacterial, anti-inflammatory and wound-healing properties (35, 36). According to the data reported in scientific literature, honey, olive oil and beeswax mixture
is useful on patients with atopic dermatitis, psoriasis
vulgaris and eczema (37, 38). Shea butter has moisturizing, anti-inflammatory, UV protection, anti-age-
962
ing properties (39, 40). Two percent of active substance, extract of dried hawthorn leaves with flowers,
was incorporated into the bases (Table 3).
A comparative analysis of rheological characteristics (Fig. 5) between samples of group I revealed
a high variety of consistency index from 1.33 to
129.40 (Pa s)n. It was noticed 2-fold increase of the
consistency index when increasing the amount of
beeswax by 2 grams (the amount of beeswax range
was between 5 to 15 grams) compared with a sample
containing less than 2 grams of beeswax. An exception was the range of beeswax between 11 to 13
grams that had a slight effect on rheological characteristics. The highest change (decrease) of flow
behavior index was seen when increasing the amount
of beeswax from 5 to 7 grams, and later, a consistent
reduction of the flow behavior index to 0.1 was
observed. All tested samples had pseudoplastic characteristics. Statistical evaluation of rheological characteristics revealed that samples can be divided into
four homogeneous subsets (p > 0.05) (N1-N2, N2N3, N4-N5 and N6) according to the consistency
index. On the other hand, there was five homogeneous subsets (N1, N2, N3, N4-N5 and N6) according to the flow behavior index.
The 2% of dry hawthorn extract were added to
the semisolid formulations, however the results of
the in vitro release testing of the active compounds
of group I formulations with 2% of dry hawthorn
extract showed that these bases are not suitable carriers because the analyzed hawthorn compounds
were not released after 6 h of testing. The hypothesis that beeswax may suspend active compounds has
been suggested. Therefore, samples of group II
(Table 3) were modeled. The experimental tests
determined the effect of beeswax on the release of
hawthorn active compounds by analyzing the samples of group II (Fig. 6). The results demonstrated
that an increase in beeswax content had a statistically significant effect on the release of active substances. The samples (N10-N13) with 1.5-5% of
beeswax did not release active substances. The data
presented in Figure 5 show that formulations N7 and
N8 released significantly higher amounts of active
Figure 7. The kinetics of hawthorn active compounds release from group III formulations
963
Figure 8. Rheological characteristics of group III samples at the temperature of 25OC
substances compared to the formulations N9 and
N14. The results demonstrated that 0.5% of beeswax
did not affect the release of active compounds,
whereas 1% of this material caused a statistically
significant reduction in the amounts of active compounds released. Other components contained in the
semisolid formulations (Table 3) decelerate the
release of active compounds but their cumulative
effect is not significant as compared to the effect of
beeswax. The mathematical analysis were performed for cumulative amounts of active substances
released per square cm from modeled formulations.
It revealed that R2 values of Higuchi model were
0.829, 0.952, 0.974, 0.830 of formulations N7, N8,
N9 and N14, respectively. The study results showed
that the release of a drug substance is determined by
the components of the selected carrier matrix (41).
After analyzing the results obtained from the tests,
shea butter was chosen as a thickening agent for the
semisolid formulations of group III (Table 3) as a full
or partial substitution for beeswax. An in vitro release
test of hawthorn active compounds from formulated
semisolid formulations was conducted (Fig. 7).
The results of the release test (Fig. 7) showed
that the formulation N15 without beeswax released
the highest amounts (41.4-42.4%) of active substances, and the formulation N17 with 1% of beeswax
released the lowest amounts (5.37-6.63%).
Formulation N16 released moderate amounts (32.035.4%) of active compounds. Moreover, the amount
of hyperoside (N17) and amounts of isoquercitrin
released from all the formulations were lower than the
limit of quantification. According to the release kinet-
ic profile given in Figure 7, the most rapid processes
occurred during the first and the second hour. The
deceleration of release was observed after 4 h of the
test and stabilization was found after 6 h. The results
of statistical analysis showed that amounts of released
substances differed statistically significantly between
all the tested formulations. The mathematical analysis
of kinetic profile of active substances released per
square cm from modeled formulations revealed that
R2 values of Higuchi model were 0.864, 0.981, 0.892
of formulations N15, N16 and N17, respectively. The
analysis of rheological characteristics (Fig. 8) of
group III formulations confirmed literature data that
higher amounts of active compounds were released
from formulations with lower consistency indices and
higher flow behavior indices (42). The rheological
characteristics of group III formulations showed that
even small amounts of beeswax (up to 1%) statistically significant influenced rheological characteristics of formulations (Fig. 8).
The study results confirmed the hypothesis that
the amount of beeswax had an effect of modeled formulation on active substance release. A statistically
significant difference was found between group III
samples and the sample N14. The amounts of active
compounds released from the formulation N14 were
lower than those released from the formulations N15
and N16, and higher than those released from the
formulation N17. The test results demonstrated that
shea butter does not limit the release of hawthorn
active components from semisolid forms.
The ex vivo skin penetration tests of semisolid
samples containing shea butter (Table 3, group III)
964
revealed that hawthorn active substances do not penetrate into the skin. The test results demonstrated
that the formulated semisolid preparation can only
affect the surface of the skin. The test results supported the data found in the scientific literature that
the physical and chemical properties of a drug substance in topical dosage forms determines the permeability of that substance across the skin from the
applied carrier matrix (31, 32).
CONCLUSIONS
The study analyzed the extract of dried hawthorn
(Crataegus oxyacantha L.) leaves with flowers in
which the following were identified as the main phenolic compounds: chlorogenic acid, vitexin-2î-Orhamnoside, hyperoside and isoquercitrin; the extract
possesses antibacterial activity against B. cereus, S.
aureus, S. epidermidis, and E. coli, and antiradical
activity based on the DPPH radical binding capacity,
and can be used as an active ingredient in the topical
formulations. The ex vivo penetration test using
undamaged human skin demonstrated that the permeability of hawthorn active substances is limited: only
the traces of hyperoside and isoquercitrin were found
in the epidermis. The in vitro release tests revealed that
beeswax in semisolid formulations has a limiting
effect on the release of hawthorn active components.
Declaration of interest
The authors report no declarations of interest.
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Received: 30. 06. 2015
ISSN 0001-6837
EVALUATION OF ANTI-DIABETIC EFFECTS OF POLY-HERBAL PRODUCT
ìDIABETIC BALî IN ALLOXAN-INDUCED DIABETIC RABBITS
ALAMGEER1*, MUHAMMAD NUMAN1, SAYED ATIF RAZA2, MUHAMMAD NAVEED
MUSHTAQ1, SAYED ATIF RAZA2, ZAHID KHAN3, TASEER AHMAD4, HASEEB AHSAN1, HIRA
ASIF1, NABEELA NOOR1, AMBREEN MALIK UTTRA1 and LAIBA ARSHAD1
1
Faculty of Pharmacy, University of Sargodha, Sargodha, Pakistan
University College of Pharmacy, University of Punjab, Lahore, Pakistan
3
Faculty of Pharmacy, Federal Urdu University, Karachi, Pakistan
4
Shifa Pharmaceutical Sciences, STMU, Islamabad, Pakistan
2
Abstract: The current study was conducted to evaluate the anti-diabetic effect of polyherbal product ìdiabetic
balî in normal and alloxan induced diabetic rabbits. Glibenclamide was used as standard drug. Diabetes was
induced by single i.v. injection of 150 mg/kg b.w. of alloxan monohydrate in rabbits. ìDiabetic balî (250 and
500 mg/kg) significantly decreased the blood glucose level both in normal and diabetic rabbits in dose dependent manner. In oral glucose tolerance test, ìDiabetic balî demonstrated a significant inhibitory effect on rise of
blood glucose level compared to control. ìDiabetic balî showed synergistic anti-hyperglycemic effect with different units of insulin in diabetic rabbits. The ìdiabetic balî decreased the glucose level and prevented the
weight loss of diabetic rabbits as compared to control for an extended period of one month. It caused a significant increase (p < 0.001) in the insulin level of treated diabetic rabbits in 30 days study. In addition AST, ALT,
ALP, cholesterol, LDLs, VLDLs and triglyceride level were significantly reduced whereas HDLs level was significantly elevated in diabetic rabbits with 500 mg/kg dose. The herbal product did not cause any significant
change in CBC as compared to normal control in diabetic rabbits for one month. It is conceivable; therefore,
that ìdiabetic balî is effective in diabetes and its associated complications which support its use in folklore.
Keywords: alloxan, blood glucose, diabetic bal, lipid profile, LFTs
their beneficial effects in the control of hyperglycemia, these agents have not been able to establish adequate control of DM, being unable to suppress the associated chronic and acute complications
(5). In addition to specific adverse effects for each
one of these medicines, administration and dosage
problems have also been reported. Moreover, these
drugs are of high cost. Therefore, the interest in
herbal medicine is increasing among the health professionals for the search of more safer and effective
treatments (5). Medicinal plants continue to contribute significantly to modern prescription drugs by
providing lead compounds upon which new drugs
can be synthesized. Pakistan has been blessed with
abundant medicinal plants that are easily available at
economic price. A large number of these herbs are
used in different ways by the local community for
the cure of diabetes. Some of these plants are
Adhatoda vasica, Aloe vera Mill., Fagonia indica
L., Tylophora hirsuta L., Ficus bengalensis L.,
Psidium guajava L., Momordica charantia L.,
Diabetes mellitus (DM) is characterized by elevated glucose concentrations resulting from insufficient insulin, insulin resistance and/or both, leading
to metabolic abnormalities in carbohydrates, lipids
and proteins (1). DM is a global epidemic affecting
approximately 285 million people worldwide, that is
expected to increase to 439 million by 2030 (2).
Diabetes is the fourth leading cause of death in most
developed countries. Pakistan is currently ranking at
7th position in the list of countries with major burden
of DM and it is expected to move to 4th position if
present situation continues (3). Diabetes is associated with acute and chronic complications, such as
ketoacidosis, hyperosmolar coma, macro- and microangiopathy, nephropathy, neuropathy and recurrent
infections. These complications are the principal
causes of illness and mortality in diabetics (4).
The most commonly used medicines for management of DM are sulfonylureas, biguanides, thiazolidinedione derivatives and insulin. Although
these drugs have been extensively used because of
* Corresponding author: e-mail: alam_yuchi@yahoo.com; phone: +923437547785
967
968
ALAMGEER et al.
Cajanus cajan, Vigna mungo (Burm. f.) Walp, Allium
cepa L. and Zizyphus jujuba (6).
Diabetic bal ìthe poly-herbal productî manufactured by Amhro Herbal Research Laboratories
(AHRL), Gilgit-Baltistan, has been used by the local
inhabitants for the prevention and treatment of diabetes.
So, it was thought worthwhile to design this study to
authenticate its empirical use through scientific work.
Drugs and reagents
Alloxan monohydrate and methanol were purchased from Sigma Chemicals Co. Glibenclamide
was obtained from Biorax Pharmaceuticals,
Islamabad, Pakistan.
Herbal product ìDiabetic balî
Crude powder of herbal product ìdiabetic balî
was provided by Amhro Herbal Research
Laboratory, Gilgit-Baltistan, Pakistan. The powdered material was stored in well closed cellophane
bags at 4OC in the refrigerator.
Experimental animals
Healthy adult rabbits of a local strain
(Oryctolagus cuniculus) weighing 1-1.8 kg of either
sex were used in the study. The animals were kept at
animal house of Faculty of Pharmacy, University of
Sargodha .The animals were housed in stainless cages
under standard laboratory conditions (light period:
8:00 a.m. to 8:00 p.m., 21 ± 2OC, relative humidity
55%). Animals were provided with a balanced rabbitís
diet consisting of green fodder and water ad libitum.
The study protocol was approved by the Institutional
Animal Ethics Committee, Faculty of Pharmacy,
University of Sargodha. Experiments comply with the
declarations of National Research Council (7).
Induction of experimental diabetes
After overnight fasting, rabbits were made diabetic by intravenous injection of fresh solution of
150 mg per kg/body weight of alloxan monohydrate
in jugular vein. This dose destroys the β cells of pancreas and produces diabetes mellitus (8). Three days
(72 h) after injecting the alloxan-monohydrate blood
glucose level of surviving rabbits was measured
with the help of glucometer and rabbits with blood
glucose level between 250-300 mg/dL were considered diabetic and were used for further study (5).
Collection of blood samples
Blood was collected from one of the ear marginal vein of rabbits for the determination of blood
glucose level. However, for the estimation of total
cholesterol, TG, HDL-cholesterol and serum insulin
blood samples were collected from the jugular vein
of rabbits in clot activator gel tubes. Blood serum
was separated by allowing 5 mL of blood into each
tube containing clot-activating gel to stand for 10
min, then blood was centrifuged at 2000 rpm for 10
min for the separation of serum that was preserved
in Eppendorf tubes (serum cups) and stored in
refrigerator.
For the determination of CBC, the collected
blood samples were transferred to tubes containing
EDTA because these tests were performed on fresh
and un-clotted blood.
Biochemical analyses
Blood glucose level was measured by Optium
Xceed Glucometer using glucose oxidized optium
kits (Abbott Laboratories, USA). Serum insulin
level was measured by ELISA reader (Stat Fax
2100, Awareness Technology, Inc. USA). CBC of
rabbits was measured with the help of photo analyzer. Total serum cholesterol, TG, HDL were estimated by enzymatic test kit (Randox) using Chemistry
analyzer biolyzer 100. The LDL-cholesterol level
was calculated by using formula (9):
LDL = total cholesterol ñ HDL-cholesterol ñ
(triglyceride/5) and
VLDL = Triglyceride/5
Hypoglycemic effect of crude powder of ìdiabetic balî in normoglycemic rabbits
Rabbits were divided into four groups of six
animals each. Group 1 served as untreated normal
control and was administered orally 20 mL of 2%
aqueous gum acacia solution. Groups 2 and 3 were
given orally 250 and 500 mg/kg body weight of
ìdiabetic balî suspended in 2% gum tragacanth
aqueous solution, respectively. Group 4 was treated
orally with 3 mg/kg body weight of glibenclamide.
Blood glucose levels were checked at 0, 2, 4, and 6
h intervals after administration of drugs (6).
Oral glucose tolerance test (OGTT)
Oral glucose tolerance test was run in healthy
normal rabbits. The overnight fasted rabbits were
divided into three groups of six animals each. Group
1 served as untreated normal control and was administered orally 20 mL of 2% aqueous gum acacia
solution. Groups 2 and 3 were administered orally
crude powder of ìdiabetic balî 500 mg/kg of powder and glibenclamide 3 mg/kg body weight, respectively. After half hour, glucose was administered
orally (1 g/kg) to all the three groups. Then, blood
969
Evaluation of anti-diabetic effects of poly-herbal product...
glucose levels were estimated at 0, 0.5, 1, 2, 3, 4 and
6 h interval after administration of drugs (10).
exogenous insulin only. Blood glucose levels were
estimated at 0, 0.5 1, 2, 3, 4 and 6 h interval after the
administration of insulin and ìdiabetic balî (6).
Hypoglycemic effect of crude powder of ìdiabetic balî in alloxan-induced diabetic rabbits
Rabbits were fasted overnight and divided into
five groups of six animals each. Group 1 and 2
served as untreated normal and diabetic control and
were administered orally 20 mL of 2% gum tragacanth aqueous solution in water only. Groups 3 and
4 were administered 250 mg/kg and 500 mg/kg body
weight of ìdiabetic balî while Group 5 received 3
mg/kg body weight of glibenclamide. The blood
glucose level of all the groups was estimated at 0, 2,
4 and 6 h intervals after the administration of drugs
(5).
Hypoglycemic effect of ìdiabetic balî on blood
glucose and weight of diabetic rabbits for 30 days
animals each. Rabbits in Groups 1 and 2 were served
as untreated normal and alloxan-induced diabetic
control and were administered orally 20 mL of 2%
tragacanth aqueous solution once daily for 30 days.
Group 3 was given ìdiabetic balî (500 mg/kg) and
group 4 was treated with glibenclamide (600 µg/kg)
for 30 days. Blood glucose level and weight of rabbits in all groups were determined at 0, 7th, 14th, 21th,
and 30th day (11, 12).
Hypoglycemic effect of ìdiabetic balî with and
without different doses of insulin in diabetic rabbits
This experiment was conducted to determine the
synergistic effect of insulin and ìdiabetic balî on blood
glucose level of alloxan-induced diabetic rabbits.
Rabbits were fasted overnight and divided into five
groups of six rabbits each. Groups 1, 2 and 3 were
administered 1, 2 and 3 units of exogenous insulin and
500 mg/kg of ìdiabetic balî, respectively. Group 4 was
administered 500 mg/kg of ìdiabetic balî only, whereas Group 6 was administered 6 units/kg body weight of
Effect of ìdiabetic balî on liver enzymes and
lipid profile of diabetic rabbits
animals each. Groups 1 and 2 were treated as normal
control and diabetic control and were administered
with 20 mL of 2% gum tragacanth solution daily for
30 days. Rabbits in Groups 3 and 4 were treated with
500 mg and 600 µg /kg body weight of ìdiabetic
balî and glibenclamide, respectively, for 30 days.
Blood sample were collected on 0 and 30th day from
the jugular vein and estimated for liver enzymes and
lipid profile (11).
Table 1. Hypoglycemic effect of crude powder of ìdiabetic balî in normoglycemic rabbits.
Groups
0h
1h
2h
ns
4h
ns
6h
ns
Normal
87.16 ± 3.73
86.33 ± 3.43
86.16 ± 3.41
87.66 ± 3.79
87.16 ± 3.78ns
Diabetic bal
(250 mg/kg)
80.5 ± 3.73
91.66 ± 3.43ns
86.16 ± 14.12ns
82.16 ± 12.54ns
72.33 ± 8.9c
Diabetic bal
(500 mg/kg)
105.66 ± 2.21
99.83 ± 7.92ns
92.5 ± 4.93b
86.33 ± 5.54c
77.33 ± 5.2c
Glibenclamide
97.33 ± 2.64
79.83 ± 1.40c
64.66 ± 1.66a
54.66 ± 1.64c
45.33 ± 1.49c
Results (blood glucose levels = mg/dL) are expressed as the mean ± SEM (n = 6) where, ns = non significant change, a = (p < 0.05), b = (p
< 0.01) and c = (p < 0.001) significant as compared to 0 hour.
Table 2. Hypoglycemic effect of crude powder of ìdiabetic balî in diabetic rabbits.
Groups
0h
1h
2h
4h
6h
Normal
89.33 ± 4.15
89.33 ± 4.32ns
88.83 ± 4.16ns
89.5 ± 3.94 ns
90.16 ±4.37 ns
Diabetic
273.66 ± 5.19
273.83 ± 4.9ns
273 ± 5.54 ns
274 ± 5.20 ns
272.16 ± 5.3 ns
250 mg/kg
278.16 ± 5.53
268.33 ± 5.7b
252.83 ± 4.76 c
237.16 ± 4.2 c
c
c
c
500 mg/kg
281.5 ± 4.80
257.66 ± 4.42
235.66 ± 3.17
Glibenclamide
271.5 ± 5.35
239.5 ± 6.41c
212.66 ± 8.08 c
221.66 ± 2.9
223 ± 1.84 c
209.66 ± 2.98 c
189.5 ± 8.10 c
ns
163.83 ± 8.2 c
b
Results (blood glucose levels = mg/dL) are expressed as the mean ± SEM (n = 6) where, = non significant change, = (p < 0.01) and c =
(p < 0.001) significant as compared to 0 hour.
970
ALAMGEER et al.
The results were expressed as the mean ±
SEM. The statistical analysis was carried out using
paired t-test and one way analysis (ANOVA); p
value < 0.05 was considered significant.
Effect of ìdiabetic balî on serum insulin level of
diabetic rabbits
The aim of this experiment was to study the
effect of ìdiabetic balî on the serum insulin level
of diabetic rabbits. The rabbits were divided into
four groups of six animals each. Groups 1 and 2
served as normal and diabetic control and
received orally 20 mL of 2% aqueous gum tragacanth solution receptively for 30 days. Group 3
was administered orally 500 mg/kg of ìdiabetic
balî for 30 days. Blood samples were collected
from each group after 30 days and estimated for
serum insulin level (13).
RESULTS
Hypoglycemic effect of crude powder of ìdiabetic balî in normoglycemic rabbits
The crude powder of herbal product ìdiabetic balî in a dose of 500 mg/kg decreased the blood
glucose level significantly (p < 0.001) after 2 h to
6 h of drug administration, however, at a dose of
250 mg/kg, there was significant reduction of
blood glucose level after 6 h of drug administration. Glibenclamide also significantly (p < 0.001)
reduced the blood glucose level. There was no
significant change in blood glucose levels of control group receiving 2% gum tragacanth solution
(Table 1).
Effect of ìdiabetic balî on complete blood cell
count (CBC) of diabetic rabbits
This study was conducted to determine the
effect of ìdiabetic balî on complete blood count of
diabetic rabbits. The rabbits were divided into three
groups of six rabbits each. Groups 1 and 2 were
administered 20 mL of 2% gum tragacanth solution
and served as normal control and diabetic control.
Group 3 served as treated group and received 500
mg/kg of ìdiabetic balî orally for 30 days. After 12
h after last dosing, blood samples of all the rabbits
in three groups were taken for the estimation of
CBC (14).
Hypoglycemic effect of crude powder of ìdiabetic balî in diabetic rabbits
The crude powder of ìdiabectic balî significantly (p < 0.001) decreased the blood glucose
Table 3. Blood glucose levels (mg/dL) of normal rabbits after oral glucose load.
Groups
Normal control
0h
83 ± 2.74
0.5 h
1h
C
154.16 ± 3.13
2h
C
127.33 ± 3.72
c
a
Glibenclamide
92.8 ± 3.78
135.66 ± 4.15
87.83 ± 2.57
Diabetic bal
84 ± 4.80
140.16 ± 2.41c
111.16 ± 3.44c
4h
ns
101.33 ± 4.22
61.5 ± 1.7
c
77 ± 2.55 b
6h
C
108.5 ± 2.24C
c
48.16 ± 2.18
42.16 ± 1.27c
63.33 ± 3.59c
58.66 ± 6.34c
84.5 ± 2.17
Results are expressed as the mean ± SEM (n = 6) where, ns = non significant change, a = (p < 0.05), b = (p < 0.01) and c = (p < 0.001) significant decrease, C = (p < 0.001) significant increase as compared to 0 hour.
Table 4. Hypoglycemic effect of ìdiabetic balî with and without different units of insulin (ins.) in diabetic rabbits.
Groups
0h
Diabetic bal +
1 unit ins.
280 ± 7.03
Diabetic bal +
2 units ins.
2h
3h
4h
6h
234.6 ± 92.0c 192 ± 10.5c
152.1 ± 8.7c
127.1 ± 7.4c
101.3 ± 5.3c
89 ± 1.8c
284 ± 6.0
229.8 ± 4.7c
181.8 ± 5.7c
128.1 ± 9.9c
87.1 ± 5.1c
84.2 ± 3.4c
80.2 ± 3.4c
Diabetic bal +
3 units ins.
279.3 ± 5
185.6 ± 12.2c 129.1 ± 11.5c
79.16 ± 4.8c
76.16 ± 4.8c
72.16 ± 45c 66.16 ± 3.7c
Diabetic bal
500 mg
283.1 ± 3.1
248.8 ± 3.09c 231.3 ± 3.7c
206.3 ± 3.3c
189.6 ± 2.3c
176.5 ± 2.1c 176.5 ± 2.7c
c
c
73.1 ± 4.20c 68.1 ± 5.20c
6 units insulin
274.5 ± 5.3
0.5 h
1h
c
188.6 ± 11.1
c
124 ± 7.4
82.1 ± 6.20
79.1 ± 5.10
Results (blood glucose levels = mg/dL) are expressed as the mean ± SEM (n = 6) where, ns = non significant change, c = (p < 0.001) significant as compared to 0 hour.
971
Table 5. Hypoglycemic effect of ìdiabetic balî on blood glucose levels (mg/dL) in diabetic rabbits for 30 days.
Groups
0 day
Normal
7 days
14 days
ns
86.8 ± 4.66
ns
300.33 ± 6.71
300.5 ± 3.92
Glibenclamide
274 ± 4.41
218.33 ± 7.69c
86.8 ± 1.16ns
84.2 ± 3.72
ns
294.33 ± 10.88
295.83 ± 10.02
306.33 ± 3.52ns
179.5 ± 4.05c
139.33 ± 2.38c
110.83 ± 3.38c
c
259.33 ± 19.4
30 days
ns
86 ± 2.23
84.4 ± 4.72
Diabetic control
Diabetic bal
21 days
ns
c
232.16 ± 12.87
ns
c
180 ± 5.80
83.66 ± 7.99c
122.83 ± 8.84
Results are expressed as the mean ± SEM (n = 6) where, ns = non significant change, c = (p < 0.001) significant as compared to 0 hour.
Table 6. Effect of ìdiabetic balî on the weight (kg) of diabetic rabbits treated for 30 days.
Groups
0 day
7 days
14 days
ns
21 days
ns
30 days
ns
Normal control
1.337 ± 0.03
1.338 ± 0.03
1.340 ± 0.03
1.342 ± 0.03
1.345 ± 0.03ns
Diabetic control
1.323 ± 0.05
1.320 ± 0.05ns
1.318 ± 0.05ns
1.315 ± 0.05ns
1.312 ± 0.05ns
Glibenclamide
1.368 ± 0.03
ns
1.370 ± 0.03
ns
1.373 ± 0.03
ns
1.375 ± 0.03
1.378 ± 0.03ns
Diabetic bal
1.427 ± 0.02
1.432 ± 0.02c
1.437 ± 0.02c
1.440 ± 0.02c
1.444 ± 0.02c
Results are expressed as the mean ± SEM (n = 6) where, ns = non significant as compared to 0 days, c = significant increase as compared to
0 day.
Table 7. The effect of ìdiabetic balî on ALT, AST and ALP in diabetic rabbits.
Normal
LFTs
0 day
Diabetic
30 days
0 day
Glibenclamide
30 days
0 day
Diabetic bal
30 days
ALT
49.66 ± 3.7 46.83 ± 3.06ns 77.5 ± 6.5 86.16 ± 5.25A 68.16 ± 10.64 48.5 ± 3.77c
AST
41.5 ± 1.9
ALP
41.83 ± 1.8ns 59.5 ± 3.4 73.83 ± 2.46C
ns
41.16 ± 3.5 41.33 ± 2.8
61.1 ± 3.2
72 ± 2.47
0 day
30 days
47 ± 4.15
29 ± 4.9c
56.33 ± 7.59a 58.66 ± 4.2 28.5 ± 4.8c
C
71.5 ± 2.78 109.16 ± 8.08 71.33 ± 5.96c 64.33 ± 2.3 36.83 ± 5.7c
LFTs (U/L) = liver function tests, ALT = alanine aminotransferase, AST = aspartate aminotransferase, ALP = alkaline phosphatase.
Results are expressed as the mean ± SEM (n = 6). ns = non significant change, a = (p < 0.05), and c = (p < 0.001) significant decrease compared to zero day. A = (p < 0.05), and C = (p < 0.001) significant increase compared to zero day.
Table 8. The effect of ìdiabetic balî on the lipid profile in alloxan induced diabetic rabbits.
Lipid profile
Normal
0 day
Triglyceride
65.5 ± 2.2
Diabetic
30 days
0 day
ns
65 ± 2.8
ns
Cholesterol
39.8 ± 1.1
40.8 ± 0.6
HDL
24.5 ± 2.1
23 ± 2.1ns
ns
Glibenclaimide
30 days
0 day
C
164.6 ± 4.8 216.8 ± 7.5
0 day
30 days
c
142.1 ± 3.7 64.8 ± 1.95 134.3 ± 14.1 101.3 ± 12.8b
39.3 ± 1.5c
57.6 ± 2.8
50.1 ± 3.6a
13.5 ± 1.8 10.33 ± 1.4a 11.83 ± 0.87 20.66 ± 0.8c
15.5 ± 0.4
20.8 ± 2.7A
57 ± 3.05
C
Diabetic bal
30 days
67.6 ± 2.7
c
51.5 ± 1.2
c
LDL
5.23 ± 2.1
4.96 ± 1.2
8.30 ± 2.1
15.4 ± 2.6
15.63 ± 0.39 8.26 ± 1.3
15.6 ± 13.1
8.2 ± 8.0c
VLDL
13.1 ± 0.4
13.1 ± 0.5ns
32.9 ± 0.9
43.3 ± 1.5c
28.4 ± 0.74 12.9 ± 0.9c
26.8 ± 2.8
21.26 ± 1.9b
HDL = high density lipoprotein, LDL = low density lipoprotein, VLDL = very low density lipoprotein. Results are expressed as the mean
± SEM (n = 6) ns = non significant change, a = (p < 0.05), b = (p < 0.01) and c = (p < 0.001) significant decrease compared to zero day. A =
(p < 0.05), and C = (p < 0.001) significant increase compared to zero day.
972
ALAMGEER et al.
level of diabetic rabbits treated with 250 and 500
mg/kg doses in dose dependent manner. The standard drug glibenclaimide also significantly
reduced (p < 0.001) blood glucose levels of diabetic rabbits. There was no significant change in the
blood glucose level of normal and diabetic control
(Table 2).
Oral glucose tolerance test (OGTT)
ìDiabetic balî significantly (p < 0.001) inhibited the increase in the blood glucose level after oral
glucose load in normal rabbits. The results were
comparable with glibenclamide. However, blood
glucose level of normal control group of rabbits
receiving oral glucose was significantly increased
(Table 3).
Table 9. Effect of ìdiabetic balî on serum insulin level of diabetic rabbits.
Grouping of rabbits
Serum insulin level (IU/L)
Normal control
4.37 ± 0.306
Diabetic control
0.54 ± 0.051
Diabetic bal treated
2.41 ± 0.208C
Results are expressed as the mean ± SEM (n = 6), C = significant
increase as compared to diabetic control (p < 0.001).
Hypoglycemic effect of ìdiabetic balî with and
without different units of insulin in diabetic rabbits
The crude powder of ìdiabetic balî produced
significant synergistic effect (p < 0.001) with all
doses of insulin. However, maximum decrease in
the blood glucose level was observed when the
crude powder of herbal product ìdiabetic balî was
administered with 3 units/kg of insulin which was
comparable to 6 units/kg of insulin alone (Table 4).
Hypoglycemic effect of ìdiabetic balî on blood
glucose and weight of diabetic rabbits for 30 days
ìDiabetic balî significantly (p < 0.001)
reduced the blood glucose level of diabetic rabbits
treated for 30 days. The results were comparable
with glibenclamide which also significantly
decreased the blood glucose level. However, no significant change was observed in the blood glucose
level of diabetic control. ìDiabectic balî and glibenclamide prevented weight loss in the diabetic rabbits
as compared to diabetic control group during one
month treatment. However, weight of diabetic rabbits were significantly (p < 0.05) decreased during
30 days study (Tables 5, 6).
Effect of ìdiabetic balî on liver enzymes and
lipid profile of diabetic rabbits
The crude powder of herbal product ìdiabetic
balî significantly (p < 0.001) reduced all the three
Table 10. Complete blood cell count (CBC) of normal control, diabetic control and ìdiabetic balî treated rabbits. (RBCs = red blood corpuscles, PVC = packed cell volume, MCH = , MCHC = mean corpuscular hemoglobin concentration, ESR = erythrocyte sedimentation rate.
CBC
Normal control
Diabetic control
Diabetic bal
Hb (mg/dL)
RBCs (1012 L-1)
12.3 ± 0.32
5.6 ± 0.27
8.05 ± 0.30
4.38 ± 0.27c
12.95 ± 0.32ns
5.73 ± 0.21ns
PVC (%)
44.36 ± 0.43
40.98 ± 0.12c
45.8 ± 0.32ns
MCH (pg)
29.8 ± 0.42
c
c
30.00 ± 0.83ns
c
158 ± 0.38
MCHC (%)
TLC (L-1)
33.51 ± 0.99
6.73 ± 0.29
181 ± 0.10
6.88 ± 0.38ns
33.50 ± 0.68ns
6.91 ± 0.42ns
Platelets (109 L-1)
270 ± 0.07
831 ± 0.319c
272.83 ± 0.56ns
ESR (mm/ 1st h)
6.8 3 ± 0.60
15 ± 0.60C
6.33 ± 0.49ns
c
Neutrophils(%)
56.5 ± 0.88
405 ± 0.99
55.33 ± 0.77ns
Lymphocytes(%)
35.5 ± 0.43
196 ± 0.43c
36.10 ± 0.27ns
ns
Monocytes(%)
6.83 ± 0.60
5 ± 0.16
6.67 ± 0.47ns
Eosinophils(%)
3.5 ± 0.42
3.4 ± 0.42ns
3.33 ± 0.49ns
MCH = mean corpuscular hemoglobin, MCHC = mean corpuscular hemoglobin concentration, TLC = total
leukocyte count. Results are expressed as the mean ± SEM (n = 6) ns = non significant change, c = (p < 0.001)
significant decrease compared to zero day. C = (p < 0.001) significant increase compared to zero day.
liver enzymes (ALT, AST and ALP) level from the
start to the end of month as compared to diabetic
control. ìDiabetic balî also significantly (p < 0.01)
reduced the levels of cholesterol, triglycerides,
LDLs and VLDLs of diabetic rabbits whereas the
HDL levels of diabetic treated rabbits was found to
be increased as compared to diabetic control (Tables
7, 8).
Effect of ìdiabetic balî on serum insulin level of
diabetic rabbits
The effect of ìdiabetic balî on serum insulin
level of diabetic rabbits clearly showed that there
was a significant increase in insulin level in diabetic
treated rabbits as compared to diabetic control, however, the product did not cause the increased level of
insulin near normal value as observed in normal
control rabbits (Table 9).
The effect of ìdiabetic balî on complete blood
count (CBC) in diabetic rabbits
There was no significant difference in complete blood count between the normal control and
ìdiabetic balî treated rabbits. However, red blood
corpuscles (RBCs), hemoglobin (Hb) level, erythrocyte sedimentation rate (ESR), packed cell volume
(PVC), platelets, neutrophils, lymphocytes, mean
corpuscular volume (MCV), mean corpuscular
hemoglobin concentration (MCHC) were significantly decreased in diabetic control group of rabbits
(Table 10).
DISCUSSION AND CONCLUSION
A significant number of hypoglycemic plants
and herbs are known through folklore.
Ethnomedicinal information has indicated that more
than 800 plants have been traditionally used for the
treatment of diabetes (15). However, their introduction into modern therapy requires pharmacological
testing by modern scientific methods. Diabetic bal is
a poly-herbal formulation which has been used as
empirical therapy for the management of diabetes
and its associated complications. The current study
was designed to confirm its use for the diabetes
treatment. The herbal product ìdiabetic balî exhibited hypoglycemic effect in normal and alloxan
induced diabetic rabbits after an oral dose of 500
mg/kg body weight. A number of possible mechanisms which includes the stimulation of β-cells and
subsequent release of insulin and activation of the
insulin receptors may be involved. The effect may
also be due to the potentiation of pancreatic secretion of insulin, which was clearly supported by the
973
increased level of insulin in ìdiabetic balî treated
rabbits.
In the light of the present study, it is clear that
the herbal product ìdiabetic balî exhibited effective
glycemic control by reversing high plasma glucose
level to basal glycemia following oral glucose load.
Similar explanation has been put forward on a number of plants believed to have anti-hyperglycemic and
insulin stimulatory effects (16).
Diabetic bal produced synergistic effect when
administered with different units of insulin which
clearly showed that there was/were some biological
active principle(s) in ìdiabetic balî that exhibited
insulin like action or some active compounds that
protect insulin biotransformation. The results were
also in agreement with previous study.
Diabetes is associated with hyperlipidemia
(17). It is well documented that there is elevation of
serum lipid concentration in diabetics. It is reported
that in uncontrolled type-II diabetes mellitus, there
is increased levels of TC, TG, LDLs, VLDLs and
decreased level of HDL cholesterol, all of which
contribute to the coronary artery diseases in diabetic patients (18). The herbal product ìdiabetic balî
significantly decreased the TC, TG, VLDL and
LDL-cholesterol levels and significantly increased
the HDL cholesterol level in the treated animals.
The results were in accordance with the previous
study which clearly demonstrated the presence of
hypolipidemic agent(s) in the ìdiabetic balî (19).
The ability of the ìdiabetic balî to manage dyslipidemia is a potential beneficial effect on cardiovascular risk factors which is a major cause of death in
diabetes mellitus (20). Increased activities of liver
enzymes such as AST, ALT and ALP are associated
with hepatocellular injury accompanied with insulin
resistance, metabolic syndrome, and type II diabetes
(21-23). A significant decrease in ALT, AST and
ALP levels occurred from the start to the end of the
month in the diabetic rabbits treated with herbal
product ìdiabetic balî. The decrease in the liver
enzymes may be due to the presence of some active
constituent like flavonoids and terpenoids in the
ìdiabetic balî which have hepatoprotective effect
against hepatotoxins (24). ìDiabectic balî was
observed to produce some positive effect on the
hemopoietic system of the treated animal. This was
manifested by keeping the hemoglobin, red blood
cell, PCV, total white blood cell and platelet counts
near normal in the ìdiabetic balî treated animals
whereas the CBC was changed in the diabetic control group. This gives an indication that herbal product ìdiabetic balî may have some phytochemicals
that can stimulate the formation or secretion of ery-
974
ALAMGEER et al.
thropoietin in the stem cells of the animals, which in
turn stimulates stem cells in the bone marrow to produce the red blood cells. The treatment with ìdiabetic balî caused the increase in weight of the alloxan treated rabbits. The anti-diabetic drugs like sulfonylureas are also associated with an increase in
weight of the diabetic patients. After one month
treatment of the experimental animals with the
herbal product ìdiabetic balî, a considerable
increase occurred in the weight of both rabbits to
diabetic control group. This result agrees with the
other investigators who observed the increase in
body weight upon improvement of diabetes status
(25). However, the mechanism of action is unknown
but suggests a protective effect of ìdiabetic balî in
controlling muscle wasting and maintenance of
metabolism as reported previously (26).
From this study it is concluded that ìdiabetic
balî may contain some active constituents which are
responsible for maintenance of blood glucose level
of treated animals within normal range and exert
beneficial effects on diabetic complications. So, it
needs further attention and work to find out the constituents that are responsible for hypoglycemic
activity and their exact mechanism of action.
Acknowledgment
The authors would like to express gratitude to
Amhro Herbal Research Laboratory, GilgitBaltistan, Pakistan for providing crude powder of
herbal product ìdiabetic balî and also to Faculty of
Pharmacy, University of Sargodha for granting
required chemicals and relevant equipment for conducting research work.
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Received: 12. 07. 2015
ISSN 0001-6837
ACTIVITY OF THYME OIL (OLEUM THYMI) AGAINST
MULTIDRUG-RESISTANT ACINETOBACTER BAUMANNII
AND PSEUDOMONAS AERUGINOSA
DANUTA TROJANOWSKA, PAULINA PALUCHOWSKA*, £UKASZ SOJA and ALICJA BUDAK
Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Jagiellonian University Medical
College, 9 Medyczna St., 30-688 KrakÛw, Poland
Abstract: Almost as soon as antibiotics were introduced to treat infectious diseases, it could be observed that
bacteria were able to develop resistance against them. Currently, multidrug-resistant strains are being isolated
mainly in the hospital environment. These are primarily non-fermenting Gram-negative bacilli, which exhibit
both natural and acquired resistance to multiple antibiotics and disinfectants rendering them difficult to eradicate. The development of new, effective and safe substances that prevent troublesome infections is greatly needed to provide alternative therapeutic options for patients. There is increasing interest in drugs of natural origin,
including essential oils. It is of particular interest that, although active against many bacterial strains, they do
not contribute to antibacterial resistance against their components. The aim of our study was to evaluate the in
vitro antibacterial activity of thyme oil against multidrug-resistant strains of A. baumannii and P. aeruginosa
using the disc diffusion and macrodilution methods. The strains were isolated from patients hospitalized in the
years 2013-2014. The in vitro antibacterial activity of thyme oil was assessed by the disc diffusion method and
the inhibition zones for the oil at different concentrations, produced against A. baumannii, ranged from 7 to 44
mm. Low level of activity of thyme oil was observed against P. aeruginosa strains. The results of serial dilution tests confirmed the high activity of thyme oil against A. baumannii isolates, expressed as MIC values ranging from 0.25 to 2 µL/mL. These results suggest the need for further studies of antibacterial activity of essential oils, especially against multidrug-resistant bacterial isolates.
Keywords: thyme oil, activity, non-fermenting bacilli, multidrug-resistant strains
rich composition of essential oils determines their
extremely wide range of biological activity, including activity against bacteria, viruses and fungi which
depends mainly on the nature of the leading chemical component. Essential oils containing phenols
(thymol and carvacrol), eugenol and cinnamaldehyde exhibit the strongest antibacterial activity. Oils
have lipophilic properties, which allow them to rapidly penetrate the cell wall and cytoplasmic membrane causing damage to the cell. Essential oils
interfere with the transport proteins, which leads to
leakage of bacterial cell components, inhibits the
hydrogen pump and ATP formation, and affects the
cytoplasmic enzymes, which ultimately causes cell
lysis (2-4). The above-mentioned activity of essential oils and their major constituents shows significant therapeutic potential in the treatment of infections. Antioxidant, immunostimulatory and antiinflammatory properties of the oils are of equal
importance (5).
The healing powers of essential oils, the secondary metabolism products of oil plants, have been
known for centuries. Despite the long history of
their use, chemical composition of essential oils was
not analyzed until the nineteenth century, when the
development of chromatographic techniques
allowed researchers to study the plant products in far
greater detail, but only chromatography combined
with mass spectrometry could be used in more
advanced analysis of the compounds (1). In chemical terms, oils are mixtures of carbohydrates, alcohols, aldehydes, ketones as well as esters and ether
derivatives of both terpenes (mainly mono- and
sesquiterpenes) and propylbenzene. The specific
chemical composition of an essential oil depends on
the plant species but the relative percentages of the
different components will vary according to the climate, growing conditions, stage of vegetation as
well as the part of the plant used (roots, rhizomes,
herbs, leaves, flowers, fruits, seeds or bark). The
* Corresponding author: e-mail: paulina.mrowiec@uj.edu.pl; phone: +48 12 620 57 53; fax: +48 12 620 57 58
975
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DANUTA TROJANOWSKA et al.
An outstanding achievement of the 20th century
was the discovery of antibiotics which revolutionized the treatment of infectious diseases but unfortunately, shortly after their introduction to clinical
practice, the emergence of bacterial resistance to
antimicrobial agents began. Recently, a progressive
increase in drug resistance among pathogenic
microorganisms has been confirmed, which
adversely affects the outcomes of infected patients.
Among the bacterial strains, the following
groups of multidrug-resistant microorganisms can
be currently distinguished: MDR (multidrug-resistant), XDR (extensively drug-resistant) and PDR
(pandrug-resistant), referred to as alert pathogens.
Multidrug-resistant microorganisms are most commonly found in the hospital environment, where
they can cause serious nosocomial infections, occurring particularly in the following hospital units:
intensive care, burn therapy, surgical, hematological
and oncological (6). These are primarily non-fermenting Gram-negative bacilli belonging to the
species Acinetobacter (A.) baumannii or
Pseudomonas (P.) aeruginosa. Microorganisms,
which can survive in nutrient-poor conditions, colonize the hospital environment including sanitation
facilities, ventilation and the medical equipment,
mainly respirators, inhalators and venous or urinary
catheters (7, 8). They are responsible for surgical
site infections and infections of burns in patients
hospitalized in surgical wards and burn units,
whereas in the intensive care units (ICU) they are
the most important species that cause infections of
the respiratory system and urinary tract. Their ability to form biofilm contributes to their survival and
colonization of respirators and urinary catheters (9,
10). Resistance to many antibiotics and disinfectants, primarily acquired but also natural, exhibited
by many harmful pathogens makes them difficult to
eradicate, therefore the search for new, effective and
safe substances, that would prevent troublesome
infections, is becoming increasingly more urgent. It
should be noted that recently drugs of natural origin,
including essential oils, have been the subject of
growing interest. Essential oils, although active
against many bacterial strains, do not contribute to
antibacterial resistance against their components. It
cannot be ruled out that using essential oils in the
treatment of infections caused by multidrug-resistant microorganisms could be a powerful alternative
to synthetic antibiotics whose effectiveness has
declined over the last decade. In the search for compounds or substances that prevent infections or the
spread of infectious agents in hospital environment,
we studied the effect of thyme oil on some selected
multidrug-resistant clinical strains of non-fermenting bacilli.
The aim of the study was to evaluate the in
vitro antibacterial activity of thyme oil (Oleum
Thymi) against the selected multidrug-resistant
strains of A. baumannii and P. aeruginosa, using the
disc diffusion and macrodilution methods.
EXPERIMENTAL
Material
We studied the natural thyme oil (Bakalland,
Warszawa, Poland) certified according to the
National Institute of Hygiene standards (PZH certificate No. HØ/15994/96).
The original, 100% oil was serially diluted in
dimethyl sulfoxide (DMSO; Merck, Warszawa,
Poland) to achieve the final concentrations of 50, 25,
12.5, 6.25 and 3.125%. We examined the following
strains of A. baumannii: 59 clinical isolates and the
reference strain ATCC 19606 and P. aeruginosa
strains: 29 clinical isolates and the reference strain
ATCC 27853. The strains were collected from subjects hospitalized between 2013 and 2014 at the L.
Rydygier Memorial Specialized Hospital in
KrakÛw, in the following wards: anesthesia and
intensive care, burn therapy, multiple trauma care,
orthopedics and neuro-orthopedics, neurology and
brain injuries, plastic surgery, nephrology, cardiology and internal medicine as well as toxicology.
Acinetobacter baumannii isolates were cultured from endotracheal aspirates (22 strains), blood
samples (17), wound swabs (12), urine (4), and
catheters (4), whereas P. aeruginosa isolates were
cultured from the wound swabs (11), endotracheal
aspirates (9), urine (4), swabs of ear (4) or conjunctival sac (1).
All the examined A. baumannii and P. aeruginosa strains were multidrug-resistant. Susceptibility
testing of A. baumannii isolates (59) was performed
against imipenem (IPM), meropenem (MEM),
amikacin (AMK), gentamicin (GEN), tobramycin
(TOB), ciprofloxacin (CIP), colistin (CST), and
trimethoprim/sulfamethoxazole (SXT). The following antibiotics were used to determine the susceptibility of P. aeruginosa strains (29): piperacillin
(PIP), piperacillin/tazobactam (TZP), ceftazidime
(CAZ), cefepime (FEP), imipenem, meropenem,
aztreonam (ATM), amikacin, gentamicin,
tobramycin, ciprofloxacin, and colistin.
Methods
The antimicrobial susceptibility of A. baumannii and P. aeruginosa strains was evaluated by disc
Activity of thyme oil (Oleum Thymi) against multidrug-resistant...
diffusion technique (Oxoid Ltd., Hampshire, United
Kingdom) and, to selected antimicrobials, with the
use of the automated system Vitek 2 Compact
(bioMÈrieux, Marcy lí…toile, France) and the minimum inhibitory concentrations (MICs) were determined. The obtained results suggest that these isolates should be classified as multidrug-resistant
(Figs. 1 and 2).
Antibacterial activity of thyme oil was determined by disc diffusion and tube macrodilution
methods in order to find the minimum inhibitory
concentration capable of inhibiting the growth of
bacteria.
The isolates were inoculated onto solid medium, Trypticasein Soy Agar (bioMÈrieux, Marcy lí…toile, France) and incubated at 35OC for 20 h. Next,
977
the colonies were suspended in 0.85% saline solution in order to obtain an equivalent of 0.5
McFarland units.
The disc diffusion tests were performed on
Mueller-Hinton agar (bioMÈrieux, Marcy lí…toile,
France) in 90 mm-diameter Petri dishes. Bacterial
suspensions were inoculated onto the culture plates
and then 6 mm sterile filter paper discs, containing
decreasing concentrations of thyme oil (15 µL),
were placed on the surface of the plates. After 20 h
of incubation at 35OC, the diameters of the growth
inhibition zones were measured in mm.
Minimum inhibitory concentrations were
determined by a broth macrodilution method in
Trypticasein Soy Broth (BioCorp, Warszawa,
Poland). The original thyme oil was diluted in
Figure 1. Susceptibility of Acinetobacter baumannii strains to selected antimicrobial agents
Figure 2. Susceptibility of Pseudomonas aeruginosa clinical strains to selected antimicrobial agents.
978
dimethyl sulfoxide (DMSO). The volumes were
matched in a way that the solvent volume was the
smallest possible. First, a stock solution was prepared by dissolving the original thyme oil in DMSO
to the concentration of 16 µL/mL, and then serial
dilutions ranging from 8 µL/mL do 0.125 µL/mL
were made in test-tubes containing 1 mL of broth.
Next, 100 µL of bacterial suspension, equivalent to
0.5 McFarland units, was added to each tube and
incubated at 35OC for 20 h. After this time, the minTable 1. Range of zone diameters of inhibition of Acinetobacter
baumannii (clinical and reference strains) growth in relation to the
tested thyme oil concentrations determined using the disc diffusion technique.
imum inhibitory concentration (MIC µL/mL) was
determined as the lowest thyme oil concentration at
which no bacterial growth could be observed.
Controls of the broth, thyme oil and bacterial growth
were run in parallel with all tests.
RESULTS
In the present study we investigated in vitro the
antimicrobial activity of thyme oil against bacterial
reference strains. Disc diffusion tests were performed and the diameters of zones of inhibition
Table 2. Range of zone diameters of inhibition of clinical
Pseudomonas aeruginosa strains growth in relation to the tested
thyme oil concentrations determined using the disc diffusion technique.
Tiyme oil
concentration [%]
Range of inhibition
zones [mm]
100
26 ñ 44
50
19 ñ 30
Tiyme oil
concentration [%]
Range of inhibition
zones [mm]
25
11 ñ 22
100
0 ñ 12
12.5
8 ñ 17
50
0ñ7
6.25
7 ñ 11
25
0
Figure 3. Growth inhibition zones against an Acinetobacter baumannii clinical strain, thyme oil at the concentration of: A. 100%; B. 50%;
C. (25%, 12.5%, 6.25%). Comparison of the susceptibility to thyme oil with the resistance to the selected antibacterial agents: IPM, GEN,
TOB, CIP, SXT (D).
979
Figure 4. Thyme oil MIC values (µL/mL) determined against clinical strains of Acinetobacter baumannii
around the discs impregnated with thyme oil were
measured. The zone diameters for the reference
strain ATCC 19606 of A. baumannii ranged from 9
to 38 mm at the oil concentrations of 100, 50, 25,
12.5 and 6.25%, whereas at the oil concentrations of
100, 50 and 25%, the reference strain ATCC 27853
of P. aeruginosa produced inhibition zones ranging
from 7 to 18 mm. The same concentrations were
used to evaluate the antibacterial activity of thyme
oil against pathogens isolated from inpatients. Our
results confirmed that the antibacterial activity of
thyme oil against the studied strains of A. baumannii was high. The size of the inhibition zones ranged
from 26 to 44 mm for thyme oil at the concentration
of 100%, from 19 to 30 mm for 50%, from 11 to 22
mm for 25% and from 8 to 17 mm for 12.5%. The
smallest inhibition zones, ranging from 7 to 11 mm,
were produced at the oil concentration of 6.25%
(Table 1). The results of bacterial susceptibility testing of thyme oil against a selected clinical isolate of
A. baumannii are shown in Figure 3.
Our study revealed very low level of activity of
thyme oil against the tested clinical strains of P.
aeruginosa. Thyme oil produced bacterial growth
inhibition zones ranging from 7 to 12 mm for 11 P.
aeruginosa strains, but it proved to be totally inactive
against eighteen isolates of this pathogen (Table 2).
The next step of the study was to determine
MIC values of thyme oil at different concentrations
against A. baumannii strains using the serial dilution
method. The obtained results confirmed that the
antibacterial activity of thyme oil, expressed as MIC
values, ranging from 0.25 to 2 µL/mL, was high.
The MIC50 and MIC90 of these isolates were found to
be 0.5 and 1 µL/mL, respectively. The lowest MIC
of 0.25 µL/mL was observed against ten A. baumannii strains while the values of 0.5 µL/mL and 1
µL/mL were found against 31 and 17 isolates,
respectively. Only one strain exhibited MIC of 2
µL/mL (Fig. 4).
DISCUSSION AND CONCLUSION
In the twenties of the last century, a French
chemist RenÈ GattefossÈ became interested in the
healing properties of essential oils when he applied
lavender oil to a burn on his hand. In 1937,
GattefossÈ coined the word ìaromatherapyî to
describe the use of essential oils in therapy (2).
Recently, there has been a growing interest in the
use of natural products in medicine. Despite bringing new antimicrobial drugs, mainly antibacterial, to
market they are still inefficient. There is an urgent
need to search for new agents, including the natural
products showing high antimicrobial activity and no
adverse effects. Research has proven that essential
oils have a remarkable antimicrobial potential and
are highly effective against both Gram-positive and
Gram-negative bacteria. Several studies have documented that essential oils can increase the bacterial
susceptibility to drugs, even of the most resistant
strains (3, 4, 11), therefore therapeutic strategies of
essential oils in combination with antibiotics not
only could improve treatment outcomes but could
also slow down the increase in antibiotic-resistant
bacteria. The strongest antibacterial activity is
exhibited by phenolic-rich essential oils; one of
them is thyme oil which is derived from thyme herb
980
(Thymus vulgaris) (12). Thymol, the main component, accounts for 30-50% of thyme oil and carvacrol up to 5%. Thyme essential oil also contains a
range of additional compounds, such as terpenes,
tannins, flavonoids, saponins and minerals (2).
Thyme acts as an expectorant so it is used in respiratory tract diseases. Due to its antimicrobial and
antiseptic activity, thyme is used in skin disinfection, treatment of seborrheic dermatitis and bacterial eczema as well as to accelerate wound healing.
Thyme has also many uses in aromatherapy and skin
care: massage, baths, inhalation and facial cleansing
(2, 13, 14).
The results of our studies confirmed the high
activity of thyme oil against multidrug-resistant
clinical strains of A. baumannii isolated from
patients hospitalized in various wards. We obtained
similar results using the disc diffusion technique
(zones of bacterial growth inhibition ranged from 7
to 44 mm) and the serial dilution method (MICs
from 0.25 to 2 µL/mL). Our results are consistent
with that of £ysakowska et al., who studied multidrug-resistant bacilli of the genus Acinetobacter
isolated from the hospital environment and hospitalized patients. They determined the MIC values in
the range from 0.25 to 1 µL/mL (15).
Hersch-MartÌnez et al. studied susceptibility of
various species of bacteria to thyme oil using disc
diffusion method and they obtained an average
diameter of the zones of inhibited bacterial growth
ranging from 6.4 to 35.7 mm. For P. aeruginosa
strains, this value was 6.4 mm (16). In our study, P.
aeruginosa zone of inhibition did not exceed the
diameter of 12 mm, which was regarded as very low
antibacterial effect.
Hammer et al. determined the antimicrobial
activity of 53 essential oils, including oil of thyme,
against various bacterial species with the use of serial dilution technique, both agar and broth. The minimum inhibitory concentrations with thyme oil to
the reference strain NTCT 7844 of A. baumannii
was 0.12 µL/mL and P. aeruginosa NTCT 10662 over 2 µL/mL (17).
KÍdzia et al. examined the antimicrobial activity of thyme oil against 31 bacterial strains of various species, including three A. baumannii strains
and one strain from each of the species: P. aeruginosa and P. stutzerii. They used serial dilutions of
thyme oil in Mueller-Hinton agar. The MIC values
for A. baumannii strains were found to range from
0.5 to 2 mg/mL, for P. aeruginosa over 4 mg/mL and
for P. stutzerii 0.5 mg/mL (18).
Sienkiewicz and Wasiela tested 30 P. aeruginosa strains using thyme oil prediluted in ethanol
and then serially diluted in agar. The MIC values
were found to range from 1 to 2.5 µL/mL (19).
In agreement with these findings, we also
demonstrated high antimicrobial activity of thyme
oil against A. baumannii strains.
Although these findings are encouraging, it is
necessary to continue research on antibacterial
activity of essential oils, especially with regard to
multidrug-resistant strains of clinically important
bacterial species. It is worth mentioning that many
products containing essential oils and recommended
in treatment of various conditions, are protected by
patents. Synergistic antimicrobial effect of essential
oils and antibacterial agents not only could help to
eradicate the etiological agent of infection but could
also slow down the increase in antibiotic-resistant
bacteria (20). Current treatments of serious bacterial
infections caused by resistant strains include combined therapies of broad-spectrum antibiotics (21).
Particular attention should be given to colistin,
a polymyxin antibiotic, which has been reintroduced
in clinical practice for treatment of infections due to
multidrug-resistant bacterial strains including A.
baumannii and P. aeruginosa (MDR, XDR, PDR).
Colistin, even though effective, exhibits adverse
effects and may interact with other medicines. This
is particularly of concern in patients with serious
comorbidities. Unfortunately, among the colistinonly-susceptible hospital isolates, colistin-resistant
strains have emerged recently (22). Prolonged
antimicrobial therapy in hospitalized patients perturbs the normal flora, promoting worsening of the
serious opportunistic infections.
In the recent years, multidrug-resistant strains
have been increasingly isolated but also a small
number of new antimicrobial drugs have been introduced for clinical use. Therefore, it is necessary to
continue research on new products, including essential oils, because their use in combination therapy
with antibiotics could be an alternative therapy for
selected infections.
It is necessary to continue research on antibacterial activity of essential oils as bacterial drugresistance to currently administered treatments is
ever increasing. Our results show that the in vitro
activity of thyme oil against multidrug-resistant
strains of A. baumannii is high and this suggests that
this oil can be used in the treatment of infectious diseases and to eradicate alert pathogens from the hospital environment.
Conflict of interest
The authors have declared no conflict of interest.
REFERENCES
1. Kubeczka K.-H.: in Handbook of essential oils:
science, technology, and applications, Baser
K.H.C., Buchbauer G. Eds., pp. 3-5, CRC Press,
Boca Raton 2010.
2. Pisulewska E., Janeczko Z.: Polish oil plants:
occurrence, cultivation chemical composition
and uses, pp. 7-11, Know-How, KrakÛw 2008.
3. Yap P.S., Yiap B.C., Ping H.C., Lim S.H.: Open
Microbiol. J. 8, 6 (2014).
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Coppola R., De Feo V.: Pharmaceuticals
(Basel) 6, 1451 (2013).
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Zakaøenia 2, 30 (2006).
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159 (2013).
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Antimicrob. Agents 35, 219 (2010).
9. Mulcahy L.R., Isabella V.M., Lewis K.:
Microb. Ecol. 68, 1 (2014).
10. Longo F., Vuotto C., Donelli G.: New
Microbiol. 37, 119 (2014).
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Dosw. (Online) 67, 1000 (2013).
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12. Sienkiewicz M., Denys P., Kowalczyk E.: Int.
Rev. Allergol. Clin. Immunol. 17, 36 (2011).
13. Nowak G., Nawrot J.: Herba Polonica 55, 178
(2009).
14. Angelucci F.L., Silva V.V., Dal Pizzol C., Spir
L.G., Praes C.E., Maibach H.: Int. J. Cosmet.
Sci. 36, 117 (2014).
15. £ysakowska M., Denys A., Sienkiewicz M.:
Cent. Eur. J. Biol. 6, 405 (2011).
16. Hersch-MartÌnez P., Leaños-Miranda B.E.,
SolÛrzano-Santos F.: Fitoterapia 76, 453
(2005).
17. Hammer K.A., Carson C.F., Riley T.V.: J.
Appl. Microbiol. 86, 985 (1999).
18. KÍdzia A., Dera-Tomaszewska B., ZiÛ≥kowskaKlinkosz M., KÍdzia A.W., KochaÒska B.,
GÍbska A.: PostÍpy fitoterapii 2, 67 (2012).
19. Sienkiewicz M., Wasiela M.: PostÍpy fitoterapii
3, 139 (2012).
20. Wolska K.I., Grzeú K., Kurek A.: Pol. J.
Microbiol. 61, 95 (2012).
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Received: 16. 07. 2015
ISSN 0001-6837
ANALGESIC, ANTIPYRETIC AND ANTI-INFLAMMATORY ACTIVITIES
OF GREWIA ASIATICA FRUIT EXTRACTS IN ALBINO MICE
BUSHRA AKHTAR1, MUHAMMAD ASHRAF2, AQEEL JAVEED2, ALI SHARIF2,
MUHAMMAD FURQAN AKHTAR2, AMMARA SALEEM3, IRFAN HAMID4, SADIA ALVI2
and GHULAM MURTAZA5*
1
Institute of Pharmacy, Physiology and Pharmacology, University of Agriculture, Faisalabad, Pakistan
2
Department of Pharmacology and Toxicology, UVAS, Lahore, Pakistan
3
Department of Pharmacy, Government College University, Faisalabad, Pakistan
4
Akson college of Health Sciences, Mirpur University of Science and Technology, AJK, Pakistan
5
Department of Pharmacy, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan
Abstract: The present study was aimed to assess biological (analgesic, antipyretic and anti-inflammatory)
activities of methanolic and aqueous fruit extracts of Grewia asiatica. The study was performed on albino mice.
Analgesic effect of the extracts was determined by acetic acid induced writhing. Antipyretic potential of the
tested fruit extracts was assessed by brewerís yeast induced pyrexia. Carrageenan induced paw edema method
was used to evaluate the anti-inflammatory activity. Both the extracts showed biological effects in a dose
dependent fashion at doses 125 mg/kg, 250 mg/kg and 500 mg/kg orally. Analysis of variance (ANOVA) was
used for data analysis and the values having p-value smaller than 0.05 were considered significant. Both the
extracts had significant analgesic, antipyretic and anti-inflammatory activities.
Keywords: Grewia asiatica fruit, carrageenan induced paw edema, analgesic, antipyretic
(dolor), redness (rubor), swelling (edema) and
loss of function (4). Flurbiprofen is a nonsteroidal anti-inflammatory drug and belongs to
propionic acid derivatives (5). Inflammation is of
two types, i.e., acute and chronic inflammation.
Acute inflammation is characterized by five classical cardinal signs. These signs are triggered by
tissue infiltration by leucocytes and serum.
Progressive transfer in cell-type, present at injury
site, leads to chronic inflammation. It is characterized by concurrent healing and destruction of
injured tissues (6).
Pain is a sensorial modality and sometimes it
is the only feature for diagnosis of various diseases.
It has a protective function sometimes. Man has
used different forms of therapy for curing this condition, among them medicinal herbs are widely
used (7). In response to tissue injury, visceral distensions and some other factors, peripheral nociceptors are stimulated so the pain develops.
Perception of pain is a normal physiological
response and it is mediated by the healthy nervous
system (8).
Plants are very important and integral part of
universe. After various experimentations, several
plants have been found to possess medicinal activities. From the ancient times, medicinal plants have
been used for curing various illnesses (1). Drugs
which are used nowadays for managing inflammation, pain and fever are categorized into opioids and
non-opioids. All of these drugs have well known
side effects. Numerous medicinal compounds, since
long time, have been used with no adverse effects.
So, there is necessary to develop new drugs of plant
origin having low cost (2).
Inflammation is chief physiological mean by
which body is defended against allergens, infections, lethal chemicals and numerous other stimuli.
If inflammation remains uncontrolled, then it may
become a leading cause for many chronic diseases.
Even though inflammation is a protection mechanism, many multifarious mediators and events of
inflammation can initiate, maintain and worsen
many diseases (3).
Five fundamental signs are characteristics of
inflammation. These are warmth (calor), pain
* Corresponding author: e-mail: gmdogar356@gmail.com; phone: 0092-314-2082826
983
984
BUSHRA AKHTAR et al.
Pyrexia or fever occurs as bodyís natural
defense mechanism in response to any infection or
disease. This is the bodyís natural function by which
an environment is created in body in which the
infectious pathogens and damaged tissues are unable
to survive (9). Normal body temperature is maintained and regulated by hypothalamus that maintains
a balance between heat loss and production. When
any infectious agent enters the body through shatter
in its protective barriers, will interact with immune
cells and endorse the endogenous mediators release
like prostaglandins, cytokines and endothelins. In
pre-optic area of anterior hypothalamus, PGE2 plays
a vital role in fever induction (10).
The plant which is evaluated here for its analgesic activity is Grewia asiatica. It is also called
phalsa. It is cultivated in Punjab province of Pakistan
and can be used for medicinal purposes and as food.
Its fruit is laxative, mordant, stomachic and an
aphrodisiac. Unripe fruit can be used for treatment of
fever, blood, respiratory and cardiac problems, diarrhea and is also helpful for relieving inflammation
(11). It contains anthocyanin, antioxidants like vitamin A and C, carotenes and folate (12).
The present study was aimed to assess biological (analgesic, antipyretic and anti-inflammatory)
activities of methanolic and aqueous fruit extracts of
Grewia asiatica.
Extract preparation
Fruit of Grewia asiatica plant was carefully
washed and shade dried. Then, it was grinded into a
coarse powder, passed through sieve no. 20 and
stored in containers. Two extracts, aqueous and
methanolic, were prepared. Triple maceration process
was used to prepare the aqueous and methanolic
extracts of Grewia asiatica fruit. In amber colored
glass bottle, 500 g of powdered drug was soaked in
500 mL of distilled water. Solvent was removed next
day by using muslin cloth. Water (500 mL) was again
added to the remaining powdered drug and the whole
process was repeated thrice. Methanolic extract was
also prepared in the same manner. Extracts were dried
to a semi-solid mass by using rotary vacuum evaporator. Finally, the extracts were dried in oven and
freezed in plastic bags for storage (2).
Animals
Albino male mice were collected from
Department of Theriogenology UVAS, Lahore. Each
animal was collected from the same breeding colony
and batch. The weight of albino mice varied from 20 to
25 g. Animals were given fresh water ad libitium.
Temperature was maintained at 22-25OC. All the experiments were conducted in compliance with Institutional
Ethical Committee (reference no. DAS 3091) of experimental animals and also considering international standards for laboratory animal use and care (10).
Plant collection
Fruit of Grewia asiatica plant was collected in
summer season, since this plant grows well in warm
atmosphere. In this season, fruit has maximum
medicinal activity. Identification of fruit was done
in Botany Department of Government College
University, Lahore and voucher number ìGC Herb.
Bot. 2210î was obtained in this context.
Phytochemical analysis
Various tests were performed for detection of
various phytochemical constituents (13).
Pharmacological evaluation
For pharmacological evaluation, animals were
placed into 5 groups. Each group had five animals.
Treatment strategy for these groups was as shown in
Table 1.
Table 1. Experimental design.
Groups
Drug
Dose
Route
No. of
animals
Group 1
(Negative control)
Normal
saline
1 mL/kg
Oral
5
Group 2
(Positive control)
Indomethacin/
Paracetamol
10 mg/kg/
150 mg/kg
Oral
5
Group 3
Plant extract
125 mg/kg
Oral
5
Group 4
Plant extract
250 mg/kg
Oral
5
Group 5
Plant extract
500 mg/kg
Oral
5
985
Analgesic, antipyretic and anti-inflammatory activities of...
Table 2. Tests for phytochemical constituents of Grewia asiatica fruit extract.
Phytochemical
constituent
Test procedure
Color
Result
Anthraquinones
5 g of crude extract + 10 mL of
1% HCl, boiled, filtered. Filterate
+ 5 mL of benzene and shacked.
Benzene layer was removed from
the solution and then added 10%
NH4OH into remaining solution.
Red color in
alkaline phase
Present
5 g of crude extract + 5 mL distilled
water + few drops of neutral 5%
ferric chloride solution
Bluish green
Present
Aqueous extract solution +
ammonium hydroxide solution
Yellow
fluorescence
Present
Tannins
Flavonoids
Carrageenan, 1% solution was used to induce
the inflammation in mice. It was injected to right
hind paw of mice at a dose of 0.05 mL. In control
group, only vehicle was given to mice. Test drug
was given 1 h before the administration of carrageenan. A mark was made at the paw of each mice
up to the ankle joint. Paw voume was measured up
to ankle joint in drug treated and untreated groups
before the drug administration and also at times of 1,
2 and 3 h (14). For evaluation of anti-inflammatory
activity, paw edema was determined at 1, 2 and 3 h,
respectively, by using digital vernier caliper. Results
were expressed in terms of mean values of each
group ± SEM. Reduction in paw edema was calculated by measuring percentage inhibition of positive
control and extract treated groups with respect to the
negative control. The following formula was used:
Inhibition (%) = Control ñ Treated / Control ◊ 100
Brewerís yeast induced pyrexia
Fever was induced in mice by subcutaneous
administration of brewerís yeast solution below the
nape of the neck. Initial rectal temperature was
recorded by using digital clinical thermometer.
After 18 h of brewerís yeast administration, animals
which showed a mean rise of 0.3-0.5OC in temperature were selected. Animals were treated with normal saline, standard drug and drug extracts were
administered according to design of experiment.
Animals temperature were recorded at 0, 1, 2 and 3
h post dosing (15).
Acetic acid induced analgesic activity
Analgesic activity was conducted in mice by
acetic acid induced writhing method. Drugs were
given orally one hour before acetic acid injection.
Acetic acid (300 mg/kg) injection was administered
to induce writhing in mice. Each animal was kept in
glass container and number of writhings were counted for 20 min after acetic acid injection. Results
were shown in terms of standard error mean.
Percentage inhibition of extract groups versus control group was determined. Percent inhibition of
writhing for both the extracts was calculated by
using the following formula:
Inhibition (%) = Nc ñ Nt/Nc ◊ 100
where, Nc = mean number of writhing in control
group, and Nt = mean number of writhing in treated
group.
Analysis of variance (ANOVA) was applied to
the data while data were expressed in terms of mean
± SEM. By using the software, Statistical package of
Social Sciences (SPSS), ANOVA was applied to all
the data sets.
Various biochemical constituents were detected. Results are shown in Table 2.
Paw edema values of mice of all groups were
recorded in millimeters and are summarized in Tables
3-5 for methanolic and aqueous extracts, respectively.
Percentage inhibition of paw edema was determined
then at different time intervals. Both the extracts
showed dose dependent anti-inflammatory activity
and 500 mg/kg showed the maximum activity in case
of both extracts. Percentage inhibition of aqueous and
methanolic extracts is shown in Tables 4 and 5.
Methanolic extract showed better anti-inflammatory
activity as compared to aqueous extract.
986
Analgesic activity
Analgesic activity was evaluated by acetic acid
induced writhing method. Results are presented in
Tables 7 and 8 for both the aqueous and methanolic
extract, respectively. Both the extracts showed significant analgesic activity in a dose dependent fashion. Extract dose of 500 mg/kg, in case of both
extracts, showed maximum analgesic activity
(Tables 7, 8).
Various pharmacological activities are known
to be present in different parts of the plant Grewia
asiatica. Leaves have antimicrobial, antiplatelet,
antiemetic and anticancer activities. Fruit has
antioxidant, anticancer, antihyperglycemic and
radioprotective properties. Stem bark is known to
possess anti-inflammatory and analgesic activities
(16).
Both the extracts showed dose dependent pharmacological activities. Methanolic and aqueous
extracts showed a dose dependent anti-inflammatory activity as the average percentage inhibition
shown by the three doses are 28.29, 32.16, 36.12%
and 8.86, 22.13 and 32.44%, respectively.
Methanolic extract has been proved to contain more
anti-inflammatory activity as compared to the aqueous extract (3).
Antipyretic activity
Temperature values expressed in (OF) ± SEM
of all positive and negative control groups, aqueous
and methanolic extract groups were recorded. It was
observed that there is a dose dependent decrease in
temperature with increasing effect from 125 mg/kg
to 500 mg/kg. (Tables 9, 10).
Table 3. Anti-inflammatory activity of Grewia asiatica (methanolic extract).
Groups
Mean values of paw edema (mm) ± SEM
1h
2h
3h
Negative control
4.63 ± 0.06
4.85 ± 0.04
3.91 ± 0.03
Indomethacin
Extract 125 mg/kg
2.99 ± 0.21
3.52 ± 0.02
2.71 ± 0.06
2.85 ± 0.04
2.68 ± 0.30
3.20 ± 0.16
Extract 250 mg/kg
Extract 500 mg/kg
3.40 ± 0.01
3.11 ± 0.03
2.71 ± 0.07
2.50 ± 0.02
2.90 ± 0.43
2.85 ± 0.03
Table 4. Percentage inhibition of carrageenan induced paw edema (methanolic extract).
Groups
Percentage inhibition of carrageenan
induced paw edema
Average %
inhibition
Indomethacin
1h
35.42%
2h
44.12%
3h
31.45%
36.39%
Extract 125 mg/kg
23.97%
41.23%
19.69%
28.29%
Extract 250 mg/kg
26.56%
44.12%
25.8%
32.16%
Extract 500 mg/kg
32.82%
48.45%
27.10%
36.12%
Table 5. Anti-inflammatory activity of Grewia asiatica (aqueous extract).
Groups
Mean values of paw edema (mm) ± SEM
1h
2h
3h
Negative control
4.63 ± 0.06
4.85 ± 0.04
3.91 ± 0.03
Indomethacin
2.99 ± 0.21
2.71 ± 0.06
2.68 ± 0.30
Extract 125 mg/kg
3.10 ± 0.11
2.99 ± 0.12
2.76 ± 0.34
Extract 250 mg/kg
3.12 ± 0.03
2.78 ± 0.16
2.99 ± 0.02
Extract 500 mg/kg
3.21 ± 0.08
2.66 ± 0.028
3.16 ± 0.21
987
Table 6. Percentage inhibition of carrageenan induced paw edema (aqueous extract).
Groups
Indomethacin
Percentage inhibition of carrageenan
induced paw edema
1h
2h
3h
35.42%
44.12%
31.45%
Average percentage
inhibition
36.39%
Extract 125 mg/kg
7%
13.08%
6.5%
8.86%
Extract 250 mg/kg
8.48%
32.71%
25.20%
22.13%
Extract 500 mg/kg
13.93%
44.39%
39.02%
32.44%
Table 7. Acetic acid induced analgesic activity of Grewia asiatica (methanolic extract).
Dose
Number of writhing
(mean) ± SEM
Percent inhibition
of writhing
Groups
Drug
Group 1
Normal saline
1 mL/kg
61.8 ± 0.44
-
Group 2
Indomethacin
10 mg/kg
15.4 ± 0.89
75.1%
Group 3
Test extract
125 mg/kg
30.6 ± 0.89
50.49%
Group 4
Test extract
250 mg/kg
28.0 ± 0.44
54.70%
Group 5
Test extract
500 mg/kg
23.6 ± 0.54
61.81%
Table 8. Acetic acid induced analgesic activity of Grewia asiatica (aqueous extract).
Groups
Drug
Dose
Number of writhing
(mean) ± SEM
Percent inhibition
of writhing
Group 1
Normal saline
1 mL/kg
61.8 ± 0.44
-
Group 2
Indomethacin
10 mg/kg
15.4 ± 0.89
75.1%
Group 3
Test extract
125 mg/kg
40.6 ± 0.54
34.30%
Group 4
Test extract
250 mg/kg
31.2 ± 0.44
49.51%
Group 5
Test extract
500 mg/kg
27.6 ± 0.54
55.34%
Peripheral analgesic activity is evaluated by
acetic acid induced writhing method (17). In this
method, abdominal constrictions associated with
irritation of peritoneal cavity leads to pain sensation.
Elevated levels of prostaglandins biosynthesis via
cyclooxygenase and lipoxygenase are produced due
to prolonged irritation by acetic acid in peritoneal
fluids. This leads to enhanced secretion of free
arachidonic acid from tissue phospholipid.
Inflammatory pain is enhanced in peritoneal cavity
of abdomen due to these increased levels of lipoxygenase and prostaglandins (18, 19). Analgesic drugs
exert their effect by inhibiting the synthesis of
prostaglandins and lipoxygenase products and hence
writhing number is decreased (20).
Aqueous and methanolic extract of Grewia asiatica fruit showed significant analgesic activity by
reducing the writhing number at doses of 125, 250
and 500 mg/kg. Analgesic activity of both extracts
was compared with reference indomethacin.
Analgesic activity of methanolic extract was found
to be slightly greater than aqueous extract.
Analgesic activity of aqueous and methanolic fruit
extract of Grewia asiatica indicates that the peripherally active analgesic principles might be present.
The study revealed strong antipyretic activity
of both the extracts of Grewia asiatica. Fever is
caused in animal models by numerous exogenous
substances like microbial infections and bacterial
endotoxins. Exogenous pyrogen stimulate the production of pro-inflammatory cytokines like IL-6,
interferon-α (INF-α), tumor necrosis factor (TNFα) and interleukin-1β. They trigger the release of
local prostaglandins (PGs) after entering into the
988
Table 9. Brewer's yeast induced antipyretic activity (methanolic extract).
Mean values of temperature (OF) ± SEM
Groups
Before drug administration
After drug administration
Normal temp.
18 h
1h
2h
3h
Normal saline
1 mL/kg
96.99 ± 0.04
102.5 ± 0.03
102.14 ± 0.02
101.91 ± 0.04
101.80 ± 0.01
Paracetamol
150 mg/kg
97.55 ± 0.09
102.31 ± 0.03
100.51 ± 0.04
100.11 ± 0.09
99.97 ± 0.07
Extract 125 mg/kg
96.71 ± 0.02
101.90 ± 0.08
101.72 ± 0.05
100.61 ± 0.05
100.50 ± 0.08
Extract 250 mg/kg
97.93 ± 0.03
102.54 ± 0.02
101.63 ± 0.01
101.5 ± 0.01
101.55 ± 0.02
Extract 500 mg/kg
97.51 ± 0.10
101.71 ± 0.02
100.91 ± 0.03
100.8 ± 0.03
100.81 ± 0.19
Table 10. Brewer's yeast induced antipyretic activity (aqueous extract).
Mean values of temperature (OF) ± SEM
Groups
Before drug administration
After drug administration
Normal temp.
18 h
1h
2h
3h
Normal saline
1 mL/kg
96.71 ± 0.07
102.9 ± 0.07
102.30 ± 0.07
102.21 ± 0.08
102.0 ± 0.01
Paracetamol
150 mg/kg
97.25 ± 0.04
102.14 ± 0.03
100.71 ± 0.05
100.0 ± 0.03
98.64 ± 0.04
Extract 125 mg/kg
96.62 ± 0.02
102.2 ± 0.01
101.20 ± 0.05
100.60 ± 0.05
100.0 ± 0.08
Extract 250 mg/kg
96.98 ± 0.03
102.12 ± 0.02
101.34 ± 0.01
101.84 ± 0.01
100.40 ± 0.02
Extract 500 mg/kg
97.25 ± 0.03
101.74 ± 0.02
101.10 ± 0.03
100.90 ± 0.03
100.50 ± 0.12
hypothalamic circulation. As a result, thermal set
point of hypothalamus is reset (21).
Most of the antipyretics exert their effect by
blocking prostaglandin synthetase, which leads to
the decreased levels of PGE2 in hypothalamic region
(22). Nonsteroidal anti-inflammatory drugs
(NSAIDs) are widely used for treating pyrexia. The
NSAIDs act by inhibition of prostaglandins via
cyclooxygenase pathway (22). The oral administration of aqueous and methanolic extract of Grewia
asiatica fruit showed significant decrease in temperature of mice. Since NSAIDs act by blocking of
cyclooxygenase pathway so these both extracts are
believed to exert antipyretic activity in the same pattern of NSAIDís. It is attributed that components
obtained after the metabolism of primary constituents were responsible for the antipyretic actions
of aqueous and methanolic extracts. The results
were compared with the standard drug paracetamol,
which showed that the extracts have good antipyretic activity. Both extracts showed antipyretic activity
in a dose dependent manner and 500 mg/kg dose
showed the highest activity.
CONCLUSION
Aqueous and methanolic extracts of Grewia
asiatica fruit have significant analgesic, antipyretic,
and anti-inflammatory activities. It can be used for
toxicity testing after subjecting to purification
processes and then clinical trials for use in humans
as alternative to NSAIDs.
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Received: 28. 07. 2015
ISSN 0001-6837
PROTECTIVE EFFECT OF POMEGRANATE SEED OIL AGAINST MERCURIC
CHLORIDE-INDUCED HEPATOTOXICITY IN RAT
MOHAMMAD TAHER BOROUSHAKI1,2, AZAR HOSSEINI1, KARIM DOLATI2,
HAMID MOLLAZADEH2 and AREZOO RAJABIAN2*
1
Pharmacological Research Center of Medicinal Plants, 2Department of Pharmacology, Faculty of Medicine,
Mashhad University of Medical Sciences, Mashhad, Iran
Abstract: Mercuric chloride (HgCl2) is an environmental and industrial toxicant that affects many tissues.
Considering oxidative stress is an important component of mercury induced hepatotoxicity, antioxidants are
expected to play a protective role against it. The present study was designed to investigate the probable effects
of pomegranate seed oil (PSO) on hepatotoxicity induced by HgCl2 administration in rats. Rats were divided
into five groups. Group 1 and 2 received corn oil (1 mL/kg, i.p.) and PSO (0.8 mL/kg, i.p.), respectively. Group
3 was treated with HgCl2 (5 mg/kg, i.p.) for 3 days. In groups 4 and 5 PSO (0.4 and 0.8 mL/kg i.p., respectively)
was given 1 h before HgCl2 administration. Twenty four hours after last injection of HgCl2, blood samples and
specimens of liver were taken. HgCl2 administration led to significant increase in liver malondialdehyde level,
significant reduction in total sulfhydryl content and significant changes in serum alanine aminotransferase
(ALT) and aspartate aminotransferase activity (AST), compared to control group. The histopathological
changes such as necrosis, inflammatory cell infiltration and hepatocellular vacuolization were observed. PSO
administration (0.8 mL/kg i.p.) improved the liver function in HgCl2 intoxicated animals as indicated by the
significant decline in increased levels of AST, ALT in serum, MDA level and significant elevation in decreased
total sulfhydryl content. Histological studies revealed milder hepatic lesions in PSO treated samples. The results
indicated that oxidative stress may be an important mechanism of HgCl2 induced hepatic injury and dysfunction and PSO may be a useful agent for the prevention of HgCl2 induced oxidative damage in rat liver.
Keywords: HgCl2, liver, malondialdehyde, oxidative stress, pomegranate seed oil
the liver (3). Mercuric chloride toxicity causes overproduction of reactive oxygen species (ROS) in
liver, leading to oxidative damage and cellular dysfunction (1). Regarding the non-negligible oxidative
stress in mercury induced hepatotoxicity, antioxidants are expected to have a protective role against
it (7). Recently, there is overwhelming attention to
herbal products and natural antioxidants for treatment and prevention of many diseases compared to
synthetic antioxidants (2, 8). Pomegranate (Punnica
granatum L.) from Punicaceae family is native to the
region from northern India to Iran. It is also widely
cultivated in parts of Southwest America,
California, Arizona and Africa (9). Pomegranate has
extensively been used in folk medicine for various
purposes. Recently, studies have shown that pomegranate has several pharmacological activities such
as antimicrobial (10, 11), antioxidant, anti-inflammatory and anticarcinogenic effects (12). Pome-
Mercury (Hg) is naturally distributed throughout the environment. Anthropogenic activities and
industrialization have increased the release of Hg to
the environment (1). Since mercury is widely used
in agriculture, medicine, industrial manufacture, it is
impossible to avoid being exposed to some forms of
mercury (2). Mercury is found in three forms (elemental, inorganic and organic) and all of them have
toxic effects (3). Mercury intoxication can occur
during the environmental, occupational or accidental exposure. The toxicity of mercury depends on the
dose, route and duration of exposure (4). The liver
plays an important role in mercury biotransformation and metabolism. Mercury localizes in the liver
tissue after exposure to its compounds (5, 6).
Mechanism of Hg-induced hepatotoxicity is not
completely understood, but in many studies has
been found that mercury induced oxidative stress is
one of the major molecular mechanisms, damaging
* Corresponding author: e-mail: rajabianar@gmail.com; phone & fax: +98 511 8828567
991
992
MOHAMMAD TAHER BOROUSHAKI et al.
granate derived products have shown beneficial
effects on the treatment and prevention of various
diseases such as cancer, cardiovascular disease, neurological disorders, diabetes etc. (13, 14). PSO comprising 12-20% of total seed weight consists of
approximately 80% conjugated octadecatrienoic
fatty acids mainly punicic acid (9cis, 11trans, 13cis
acid). Also, catalpic acid (C18:3-9trans, 11trans,
13cis) and α-eleostearic acid (C18:3-9cis, 11trans,
13trans) are isomers of conjugated linolenic acids
(CLnAs) found in PSO. The oil is characterized by
a high content of fatty acids, of which 99% are
triglycerides. The fatty acid composition of PSO
obtained from specific varieties of pomegranate has
been reported (punicic acid, linoleic acid, oleic acid,
stearic acid, palmitic acid, others) (15-18). Sterols,
steroids, cerebroside, lignins, hydroxycinnamic
acids and the potent antioxidant lignin derivatives
are considered as minor components of the oil. The
oil consists of high contents of phytosterols such as
β-sitosterol, campesterol, stigmasterol and tocopherols (α and γ-tocopherol) (12, 19). Although
some published reports demonstrated the protective
effects of pomegranate seed oil (PSO) against atherosclerosis and nephrotoxicity (20-22) but there is no
evidence concerning the hepatoprotective effects of
PSO against HgCl2-induced acute toxicity. Therefore, the present study was designed to evaluate the
protective effects of PSO against liver damage
induced by i.p. injection of HgCl2 in rat.
Chemicals
Mercuric chloride (HgCl2) was obtained from
May & Baker company (England), TMP (tetramethoxypropane), Trizma base (Tris (hydroxymethyl)
aminomethane), TBA (2-thiobarbituric acid), nbutanol , DTNB (2,2í-dinitro-5,5í-dithiodibenzoic
acid), NaOH (sodium hydroxide), phosphoric acid,
HCl (hydrochloric acid), Na2EDTA (ethylenediaminetetraacetic acid disodium salt), KCl (potassium
chloride) and diethyl ether were purchased from
Merck Co. (Darmstadt, Germany). Pomegranate seed
oil (d = 0.81 g/mL at 25OC) was a kind gift from Urom
Narin Co. (Uromeya, I. R. Iran).
Animals
The study was performed on 24 adult male
W/A rats with 170ñ190 g body weight (Animal
House, School of Medicine, Mashhad, Iran). The
animals were housed in a humidity and temperature
controlled environment and maintained on a lightdark cycle of 12 h. They were allowed free access to
standard laboratory diet and water ad libitum. All
experimental procedures were conducted in accordance with the university ethics committee and were
conducted under national laws and the National
Institutes of Health guidelines for the use and care of
laboratory animals.
Experimental design
After acclimatization, animals were randomly
divided into five groups (six each), Group 1 (control group) and 2 received corn oil (1 mL/kg) and
PSO (0.8 mL/kg) for 3 days, respectively. Group 3
was injected a single dose of HgCl2 (with the concentration of 5 mg/kg body weight in 0.9% NaCl).
In group 4 and 5, PSO were administered 0.4, 0.8
mL/kg, one hour before HgCl2 (5 mg/kg) injection
for 3 consecutive days. All injections were performed intraperitoneally. Twenty four hours after
last HgCl2 injection, all rats were anesthetized with
ether. Blood was directly collected from heart, centrifuged at 1000 ◊ g for 15 min to separate the
serum for assessment of some biochemical parameters. Ratís liver were then removed, the piece of
liver was homogenized in cold KCl solution (1.5%,
pH = 7) to give a 10% homogenate suspension for
biochemical assays. The other piece was fixed in
10% formalin and sectioned for histopathological
studies.
Biochemical study
Serum ALT and serum AST
ALT and AST level measurement was according to the International Federation of Clinical
Chemistry (IFCC) method and was expressed as
units per liter (23).
Lipid peroxidation (LPO)
MDA (malondialdehyde) level is identified as
a main products of lipid peroxidation that reacts
with TBA to give a red color species (thiobarbituric
acid reactive substance (TBARS)), which have peak
absorbance at 532 nm). Half milliter of homogenate
in centrifuge tube was mixed with 3 mL phosphoric
acid (1%) and 1 mL TBA (0.6%). Then, all tubes
were placed in a boiling water bath for 45 min. The
tubes were then cooled and 4 mL of n-butanol was
added to the reaction mixture, vortexed for 1 min,
and centrifuged at 60,000 ◊ g for 20 min. The
absorbance of supernatant was determined at 532
nm. MDA content was expressed as nanomoles per
gram of tissue.
MDA concentration of the liver samples were
calculated using the standard curve of MDA (concentration range of 0ñ40 µM) (24).
Protective effect of pomegranate seed oil against...
Total sulfhydryl (SH) groups assay
The sulfhydryl group content in liver was
measured spectrophotometrically using DTNB
(2,2¥- dinitro-5,5¥-dithiodibenzoic acid) as a coloring reagent. This reagent produces a measurable yellow-colored product when it reacts with sulfhydryl
group.
Fifty µL of homogenate was mixed with 1 mL
Tris-EDTA buffer (pH = 8.6) in test tube and its
absorbance was read at 412 nm (A1). Twenty µL of
10 mM DTNB was added to the mixture. After 15
min at laboratory temperature, the absorption was
measured again (A2). Blank (B) was the absorbance
of DTNB reagent. Total SH groups are calculated
using an equation (25):
Total thiol concentration (mM) =
(A2 - A1 - B) ◊ 1.07/0.05 ◊ 13.6
Histological study
Liver tissue samples were fixed in 10% formalin for at least 24 h. The fixed specimens were
processed, using paraffin-embedding technique and
hematoxylin and eosin (H&E) staining was performed for histopathological examinations through
the light microscope (7).
993
The values are expressed as the mean ± SEM.
All statistical analyses were performed using Prism
5 software. Data were analyzed using one-way
analysis of variance followed by Tukey-Kramer
post-hoc test for comparison between groups. The
minimum level of significance was set at p < 0.05.
RESULTS
Biochemical studies
Serum ALT, AST enzyme activity
The activities of serum ALT, AST after mercuric chloride exposure and PSO pretreatment (0.4,
0.8 mL/kg) are shown in Figures 1 and 2. HgCl2
intoxicated rats displayed a significant elevation in
serum ALT enzyme activity, compared to the control
group (p < 0.01). Pretreatment of PSO (0.8 mL/kg)
caused a significant decline in ALT activity as compared to HgCl2 intoxicated animals. Also HgCl2
injection caused significant elevation in AST level as
compared with the control group (p < 0.05). Rats,
which were pretreated with PSO (0.8 mL/kg), exhibited a significant reduction in AST serum level with
respect to HgCl2 group (p < 0.05) (Figs. 1, 2).
Figure 1. Effect of the pomegranate seed oil pre-treatment against HgCl2 intoxication on serum ALT activity. Values are expressed as the
mean ± SEM (n = 6). * p < 0.05, ** p < 0.01 as compared with HgCl2-treated group
Figure 2. Effect of the pomegranate seed oil pre-treatment against HgCl2 intoxication on serum AST activity. Values are expressed as the
mean ± SEM (n = 6).* p < 0.05 as compared with HgCl2-treated group; # p < 0.05 as compared with control group
994
However, AST levels in rats pretreated with PSO
(0.8 mL/kg) showed no significant difference when
compared with the control group, but there was a significant difference in AST levels between the control
group and the PSO (0.4 mL/kg) pretreated rats (p <
0.05). Rats treated with PSO (0.8 mL/kg) alone did
not show significant differences in AST and ALT
levels when compared with the control group.
Lipid peroxidation
The lipid peroxidation (LPO) level in liver is
expressed as malondialdehyde levels (MDA). As
shown in Figure 3, mercury chloride administration
significantly increased tissue level of malondialdehyde compared to control group (p < 0.001).
Compared with the HgCl2 group, MDA levels
decreased significantly in the PSO (0.8 mL/kg) pretreatment group (p < 0.01). However, the rats pretreated with PSO (0.4, 0.8 mL/kg) showed significant differences in MDA levels when compared
with the control group (p < 0.001, p < 0.05).
Total thiol content
Figure 4 shows that HgCl2 treatment decreased
the total thiol content significantly in the rat liver
homogenates (p < 0.001). PSO pretreatment (0.8
mL/kg) exhibited a significant elevation in total
thiol concentrations, as compared with HgCl2 group
(p < 0.01). However, significant differences were
observed in the total thiol content between the PSO
(0.4, 0.8 mL/kg) pretreated rats and the control
group (p < 0.001, p < 0.01).
Rats treated with PSO (0.8 mL/kg) alone did
not show significant differences in lipid peroxidation and the total thiol content, as compared with the
control group (Figs. 3, 4).
Histopathological observations
Histopathological analyses showed focal
necrosis of hepatocellular, inflammatory cell infiltration and cytoplasmic vacuolization in the HgCl2treated rat liver sections (Fig. 5). Liver section of rat,
pre-treated with PSO, showed partial recovery and
very small hepatocellular necrosis (Fig. 5).
DISCUSSION
Mercury as a toxic transition metal, induces
oxidative stress through overproduction of ROS
(25). ROS cause damage to cellular proteins, nucle-
Figure 3. Effect of pomegranate seed oil pretreatment against HgCl2 Intoxication on MDA content in rat liver. Values are expressed as the
mean ± SEM (n = 6). * p < 0.05 as compared with HgCl2-treated group; # p < 0.05, ### p < 0.001 as compared with control group
Figure 4. Effect of pomegranate seed oil pretreatment against HgCl2 intoxication on total thiol concentration in rat liver. Values are
expressed as the mean ± SEM (n = 6). ** p < 0.01,*** p < 0.001 as compared with HgCl2-treated group; ## p < 0.01, ### p < 0.001 as
compared with control group
995
Figure 5. Photomicrographs showing the pathological changes of liver tissues after HgCl2 exposure and pomegranate seed oil pretreatment.
A) Control: normal liver tissue. B) Group treated with 5 mg/kg HgCl2 showing focal necrosis, inflammatory cell infiltration. C) Group
treated with 5 mg/kg HgCl2 showing cytoplasmic vacuolization (400◊). D) Pomegranate seed oil (0.4, 0.8 mL/kg) + HgCl2: liver sections
show partial recovery with less cytoplasmic vacuolization and occasional hepatocytic vacuolations
ic acid, and lipids, leading to cell membrane damage
and cellular dysfunction (3). The liver is a major site
involved in mercury metabolism, hence, resulting in
mercury accumulation in the liver (1). Results of the
present study showed a significant increase in activity of serum transaminases (AST and ALT) after
HgCl2 treatment. There are many reports indicating
that activity of these enzymes were significantly elevated in rat, given HgCl2 (5, 7). This may be due to
hepatocellular necrosis, which causes the release of
these enzymes into the blood circulation after cellular damage and rupture of the plasma membrane (2).
The biochemical findings were also confirmed by
histopathological changes after HgCl2 injection.
These changes were mostly in hepatocellular necrosis, vacuolations and inflammatory cell infiltration.
Similarly, Sharma et al. (2) and Kumar et al. (26)
reported that mercuric chloride induces centrilobular
necrosis and cytoplasmic vacuolization in liver. Oda
and El-Ashmawy (7) showed that HgCl2 caused
hepatic injury characterized by periportal hepatocytic vacuolations and hepatic necrosis in rats. Also
Deng et al. (1) observed several histopathological
changes such as necrosis after HgCl2 exposure.
Lipid peroxidation (LPO) plays a crucial role in
HgCl2-induced hepatotoxicity (26). LPO can destroy
biological membranes and alter cell membrane per-
meability (27, 28). MDA is the main product of LPO
and considered as a marker of oxidative damage
(29). The present study revealed that administration
of HgCl2 caused significant enhancement in LPO
level as expressed by increased tissue levels of
MDA. Similar results were reported by Oda and ElAshmawy (7) and Deng et al. (1). The increased
LPO level could be due to the overproduction of free
radicals and decreased antioxidant enzyme activities
which cause oxidative tissue damage (6).
Sulfhydryl groups are highly-reactive constituents of non-protein and protein molecules such
as catalytic or binding domains of enzymes, and
they play important roles in several metabolic and
biochemical processes such as detoxification mechanisms, maintenance of protein systems and activation of enzymes including antioxidant enzymes
(superoxide dismutase, catalase, etc.). They can
scavenge oxygen-derived free radicals (5, 22, 30).
There are several reports suggesting that mercury
exerts toxic effects mainly through the formation of
complexes with sulfhydryl groups and depletion of
intracellular thiol pool, which may induce oxidative
stress (2, 5, 31). The results revealed significant
decrease of total sulfhydryl groups in HgCl2-treated
rats. It was observed that pretreatment with PSO significantly increased total sulfhydryl group levels.
996
Deng et al. (1) found that overproduction of ROS in
the liver was caused by HgCl2 exposure and oxidative stress is probable mechanism for HgCl2-induced
hepatic damage.
Studying the section of hepatic tissue following administration of PSO revealed a milder lesion
in liver samples of PSO treated rat compared to rats
treated with HgCl2 alone. On the other hand, treatment with PSO was found to suppress (p < 0.05) the
elevation of serum AST and ALT activities induced
by HgCl2 treatment in rats. This finding implies that
PSO play a protective role in liver tissue against
HgCl2 injury. It may be due to the prevention of the
leakage of intracellular enzymes by its membrane
stabilizing activity. In several studies, PSO administration led to significant reduction of MDA concentration in tissue homogenate samples (5, 21, 24).
PSO indicated antioxidative properties by reduction
in the MDA levels and the enhancement of
decreased glutathione (20). PSO administration may
decrease HgCl2 induced hepatotoxicity by quenching these toxic metabolites. PSO did not exert any
significant alteration when administered alone but
significantly reduced or normalized the alterations
caused by HgCl2 injection. Pomegranate seed oil,
consists of high content of conjugated fatty acids
among which punicic acid is the most common.
Other components of pomegranate seed include γtocopherol, ursolic acid, sterols (daucosterol,
campesterol, stigmasterol, β-sitosterol), hydroxybenzoic acids (gallic and ellagic), coniferyl 9-O-[βD-apiofuranosyl(1ñ6)]-O-β-D-glucopyranoside,
sinapyl 9-O-[β-D-apiofuranosyl(1-6)]-O-β-D-glucopyranoside (12, 32) which showed antioxidant
activity (32). Moreover, it contains polyphenolic
compounds with antioxidant and anti-inflammatory
activity (22, 33). Several studies reported anti-cancer and anti-inflammatory effects for these components: e.g., hydroxybenzoic acids cause inhibition of
growth and induce apoptosis in human DU-145
prostate cancer cells (34) and sterols inhibit proinflammatory cytokine production in mice (35).
PSO contains ellagic acid, an antioxidant compound
that removes peroxy radicals and prevents lipid peroxidation induced by Cu2+ (36). According to the
presence of a variety of biologically active compounds in PSO, its antioxidant and free free-radicals
scavenging properties may be partially responsible
for its hepatoprotective activity.
CONCLUSION
In the present study, HgCl2 administration
induced hepatic damage which was alleviated by
PSO pretreatment. Oxidative stress may be one of
the most important mechanisms of HgCl2 induced
hepatic injury and dysfunction. Our data suggest that
PSO has protective and preventive effects against
oxidative damage in the liver of HgCl2-treated rats.
Acknowledgments
The authors thank Dr. Mehdi Farzadnia for
histopathological studies. This investigation was
financially supported by the Pharmacological
Research Center of Medicinal Plants, Faculty of
Medicine, Mashhad University of Medical Sciences,
Mashhad, Iran.
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Toxicol. 49, 1426 (2011).
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Vosough S.: J. Food Biosci. Technol. 2, 35
(2012).
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J.: J. Hum. Nutr. Food Sci. 2, 1024 (2014)
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Food Sci. 7, 85 (2012).
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Asadpour E., Hosseini A.: Iran J. Pharm. Res.
12, 821 (2013).
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M., Yavari S.: J. Ren. Fail. 32, 612 (2010).
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H.R., Dolati K.: J. Food Sci. Technol. 51, 3510
(2012).
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26. Kumar M., Sharma M.K., Kumar A.: J. Health
Sci. 51, 424 (2005).
997
27. Ghosh A., Sil P.C.: Biol. Pharm. Bull. 31, 1651
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Danaee A.: J. Pharm. Pharm. Sci. 8, 387 (2005).
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Received: 2. 08. 2015
ISSN 0001-6837
PHARMACEUTICAL TECHNOLOGY
COMPARATIVE BIOAVAILABILITY ANALYSIS OF ORAL ALENDRONATE
SODIUM FORMULATIONS IN PAKISTAN
HUMAYUN RIAZ1, BRIAN GOODMAN2, SAJID BASHIR1, SHAHZAD HUSSAIN3,
SIDRA MAHMOOD4, FARNAZ MALIK3, DURDANA WASEEM5, SYED ATIF RAZA6,
WAJAHAT MAHMOOD7 and ALAMGEER1*
1
Faculty of Pharmacy, Sargodha University, Sargodha, Pakistan
Department of Laboratory Medicine, Division of Clinical Pharmacology, Karolinska Institute, Karolinska
University Hospital Huddinge, SE-141 86, Stockholm, Sweden
3
Drugs Control and Traditional Medicines Division, National Institute of Health, Islamabad, Pakistan
4
International Islamic University, Islamabad, Pakistan
5
Department of Pharmacy, Quaid-i-Azam University, Islamabad, Pakistan
6
University College of Pharmacy, University of Punjab Lahore, Pakistan
7
Department of Pharmacy, COMSATS, Abbottabad, Pakistan
2
Abstract: Alendronate sodium, a bisphosphonate drug, it is used to treat osteoporosis and other bone diseases.
The present study was designed to conduct comparative bioavailability analysis of oral formulations of alendronate sodium through an open-label, randomized, 2-sequence, 2-period crossover study. Healthy adult male
Pakistani volunteers received a single 70 mg dose of the test or reference formulation of alendronate sodium followed by a 7 day washout period. Plasma drug concentrations were determined using a validated HPLC post
column fluorescence derivatization method. AUC0-t, AUC0-8, Cmax and Tmax were determined by non-compartmental analysis and were found within the permitted range of 80% to 125% set by the US Food and Drug
Administration (FDA). Results show that both in vitro and in vivo assays of all test brands were within the specification of the US Pharmacopoeial limits and were statistically bioequivalent. No adverse events were reported in this study.
Keywords: alendronate, bioavailability, bioequivalence, pharmacokinetic parameters, HPLC
Abbreviations: PK - pharmacokinetic parameter, Cmax - maximum plasma concentration, Tmax - time to reach
maximum plasma concentration, AUC - area under curve, t1/2 - half life, Ke - elimination rate constant
The importance of generic drugs in health care has
made it crucial to consistently monitor their pharmaceutical quality and in vivo performance since
these are alternative to innovator products in the
market (8). Alendronate sodium [sodium (4-amino1-hydroxybutylidene) bisphosphonate] is prescribed
for the management of bone diseases such as osteoporosis, hypercalcemia, and Pagetís disease (9).
Alendronic acid is released immediately in solution
and irritate mucosal lining during deglutition. As a
consequence, it can cause esophagitis. Thus, it is
administered orally as tablets in its monosodium salt
form to prevent esophagitis (10, 11). Moreover, it
should be taken in morning with a glass of water at
least 30 min before food and the patient should
avoid lying down during that time. The fast release
In recent years, the provision of quality health
care is receiving global interest and medicines have
become a core component of health care system to
cater health care needs of patients (1-3). In some
countries, up to 60% of total health care expenditure
is allocated to the cost of medicines (4) which can be
controlled with rational and judicious use of medicines. An essential cost reduction measure is to
encourage the prescription of low cost generics. The
generic drugs have captured more than 65% of the
global pharmaceutical market (5, 6). Average prescription spending in the United States topped 286
billion in 2007, prompting calls for greater generic
drug use to reduce costs without sacrificing quality.
Generic drugs account for 66% of prescriptions
filled in the US but less than 13% of the cost (7).
* Corresponding author: e-mail: alampharmacist@gmail.com
999
1000
HUMAYUN RIAZ et al.
of alendronic acid exhibits a potential health risk,
which should be kept as low as possible (12).
Alendronate sodium is selectively accumulated
in the bone, and its oral absorption is < 1% of the
administered dose in the fasted state (13). It is
reported that about 40% of an oral dose is excreted
within 8-12 h, while the remaining is gradually
released from the bone, depending on the rate of
bone turnover, and is eventually eliminated in the
urine (14). It is an effective inhibitor of osteoclast
and bone resorption. The plasma half-life of alendronate is 1.7-1.8 h in healthy individuals (13-15).
An increasing number of generic alendronate formulations have become available. Although expected to have the same tolerability and efficacy, headto head comparison of generic and brand alendronate was never performed. A study compared the
tolerability and efficacy of generic and brand alendronate and found no significant differences in overall tolerance between treatment groups (16). It was
also found that the level of bone turnover markers
were significantly decreased over 12 weeks of follow-up for generic and branded alendronate.
Generic caused significantly higher abdominal pain
scores. Therefore, generic alendronate may not have
the same tolerability and efficacy as branded alendronate in the first weeks after starting treatment in
patients with a recent fracture. Patients who were
previously stable on doses of brand alendronate
experienced an increase in adverse events causing
discontinuation after introduction of automatic substitution to generic alendronate (17).
In Pakistan, currently multiple pharmaceutical
formulations containing alendronic acid are manufactured and marketed by many local pharmaceutical companies with varied prices and may differ in
their quality towards the innovator product (18). In
line of this, the objectives of the present study were
to conduct in vitro and in vivo studies of oral formulations of alendronate sodium 70 mg. The research
was conducted according to standard specifications
of United States Pharmacopoeia (USP) and an openlabel, randomized, two-period crossover comparison in Pakistani male adult volunteers was done to
compare bioavailability and pharmacokinetic
parameters (PK) of leading brands with generic
product. This study will help the policy makers in
guiding various Government and private institutions
to buy good quality and cheap generic alendronate
sodium in order to cater the demands of patients suffering from osteoporosis and will also save a lot of
foreign exchange incurred on medicines. It will also
help boosting the confidence of the medical professionals to prescribe locally manufactured drugs and
also aid the local industry to export to neighboring
countries to earn foreign exchange as well.
EXPRIMENTAL
In vitro analysis
A total of ten leading brands of alendronate
sodium 70 mg tablets available in the market were
purchased, i.e., brand leader and generic; and were
tested according to standard USP methods. The
samples were tested for their in vitro characteristic,
i.e., physical and chemical parameters, disintegration time and dissolution percentage (19). In vitro
tests were performed according to USP30 guidelines. The dissolution test was performed by paddle
method (dissolution apparatus ERWEKA DT6,
GmbH, Germany) with following parameters: water
as medium; speed 50 rpm; dissolution medium volume 900 mL, time 15 min, temperature 37.0 ±
0.5OC. Disintegration test (ERWEKA ZT3-A,
GmbH, Germany) was also performed according to
the specifications of USP30.
Assay for quantication of alendronate was conducted based on USP monograph for alendronic acid
tablets. Alendronic acid tablets contain an amount of
alendronate sodium equivalent to 90-110% of the
labeled amount. Briefly, after mixing standard/test
solution with sodium citrate dihydrate diluent and
borate buffer for 3 min, 0.05% 9-fluorenylmethyl chloroformate solution was agitated with previous mixture
for 30 s. The resultant mixture was allowed to stand for
25 min at room temperature and then centrifuged after
adding methylene chloride for 5 min. Upper layer was
analyzed by high performance liquid chromatographic
(HPLC) and amount of alendronate in test/standard
sample was estimated by comparing the chromatogram
peaks of respective samples with calibration standards.
The same method was used to determine percentage of
active compound in dissolution test (19).
In vivo analysis
Study design
In vivo studies were materialized through
open-label, randomized, 2-sequence, 2-period
crossover study in healthy male adult population to
compare their bioavailability and PK parameters of
the brand leader with generic product. The studies
were carried out at Drugs Control and Traditional
Medicines Division (DCTM), National Institute of
Health (NIH), Islamabad, Pakistan.
Subjects
Healthy Pakistani adult male subjects were eligible to be enrolled in the study. Inclusion criteria
Comparative bioavailability analysis of oral alendronate sodium...
consisted of unremarkable results on medical history, physical examination, and clinical laboratory
tests (vital signs, serum chemistry and hematology,
and urinalysis). Subjects with HIV or hepatitis B
were excluded from the study. Concurrent medications and the consumption of alcohol were not
allowed from 2 weeks before administration of the
first dose until the end of the study period. Subjects
were informed about the aims and risks of the study,
and written informed consent was obtained from all
volunteers before screening. The study protocol was
approved by the board of studies of Sargodha
University, Sargodha, Pakistan and DCTM, NIH,
Islamabad, Pakistan. All protocols were in accordance with the revised Declaration of Helsinki (20)
and the Good Clinical Practice guidelines (21). All
the drugs used for bioequivalence studies were procured from market and reference standard of alendronate sodium for the study was provided by M/S
MSD (OBS), Pakistan. All chemicals used in the
study were from Sigma/Merck (analytical or HPLC
grade). Distilled water was used wherever required
in the study.
Blood sampling and sample processing
Twenty four healthy adult male volunteers
were randomly assigned to receive a single 70 mg
dose of test or reference formulation of alendronate
sodium, administered with 240 mL of water, followed by a 7 day washout period and subsequent
administration of alternate formulation. Drugs were
administered after 12 h overnight fasting. Serial
blood samples were collected and adverse events, if
any, were monitored by a clinical investigator via
observation, personal interview, and measurement
of vital signs (blood pressure, heart rate and body
1001
temperature) over a 7 h period (at 0.25, 0.5, 0.75, 1,
1.5, 2, 3, 4, 5, 6 and 7 h) after drug administration.
After collection, the blood sample was immediately
centrifuged (Cat. No. 9527-16, Abbott Diagnostics
Centrifuge machine) at 4000 rpm for 15 min and
separated plasma was frozen (UNI-TRAP UT-50L
refrigeration system) at -20OC in Eppendorf tubes
until analysis.
Plasma alendronate sodium concentrations
were determined using a validated HPLC post column fluorescence derivatization method, with visible
detection in the range of 2 to 100 ng/mL and lower
limit of quantification set at 2 ng/mL (22, 23). PK
properties including AUC0-t, AUC0-8, Cmax, Tmax, t1/2,
and the elimination constant (Ke) were determined by
non-compartmental analysis. The formulations were
considered bioequivalent if 90% CI ratios for Cmax
and AUC were within predetermined interval of 80%
to 125%, that is the regulatory definition set by US
Food and Drug Administration (24).
Standard curve
Stock solution of alendronate sodium in a concentration of 1 mg/mL was prepared in mobile phase
(buffer solution: acetonitrile : methanol, 75 : 20 : 5,
v/v/v). The stock solution was then diluted to 100
µg/mL with methanol to make a working stock solution. From this working stock solution, calibration
standards were prepared in plasma in concentrations
of 1, 2, 3, 4, 5, 6, 7, 9, 10, 12 and 15 µg/mL (Fig. 1).
Extraction and assay of alendronate from plasma
Stored frozen plasma was thawed and 200 µg
of plasma was vortexed (Model MA-1, Torika) with
1 mL of methanol in Eppendorf tube for 2 min. The
sample was then centrifuged (Cat. No. 9527-16,
Figure 1. AUC standard curve for alendronate sodium
1002
HUMAYUN RIAZ et al.
Abbott Diagnostics Centrifuge machine) at 10000
rpm for 10 min and 800 µL of the supernatant was
separated in another tube. The sample was dried in a
vacuum oven (VOS-300, EYELA) and the residue
left after drying was reconstituted with 200 µL of
mobile phase that was composed of buffer solution
(14.7 g sodium citrate dihydrate and 7.05 g anhydrous disodium hydrogen phosphate, pH 8) acetonitrile, and methanol in ratio 75 : 20 : 5 (v/v/v). The
sample (100 µL) was injected into the HPLC system
(LC-9A; Column: L-21, 4.1 ◊ 250 mm, 5 µm particle size; temperature 35OC) for analysis (19). Mobile
phase was set to flow through sample at the rate of
1 mL/min. Analyte detection was done with a fluorescence detector (LC 9A, Shimadzu, Japan). The
fluorometric detector was operated at 260 nm (excitation) and 310 nm (emission). Amount of alendronate in plasma was estimated from calibration
curve by recording the chromatogram and measuring the responses for the major peaks.
RESULTS
Pharmacokinetic and statistical analysis
A non-compartmental pharmacokinetic model
was used to determine the pharmacokinetic (PK)
parameters of alendronate sodium. The PK parameters i.e., AUC0→t, AUC0→∞, Cmax, Tmax, t1/2 were determined using MinitabÆ 16 software (Minitab, Inc.,
USA) for each of the volunteers for both the drugs.
Both the AUC and AUMC were calculated using the
trapezoidal rule, more specifically, the AUMC was
calculated by constructing the area under the concentration (C) times time (T) versus time (T) curve
(C.T vs. T). The mean residence time (MRT) for the
drugs was calculated as MRT = AUMC/AUC.
Statistical comparisons between pharmacokinetic
parameters of the two products were analyzed by
two-way ANOVA with p < 0.05 for statistical significance. Data were presented as the mean ± SD of
multiple values.
The first-order terminal elimination rate constant (Ke) was determined by linear regression using
points describing the elimination phase on a log-linear plot. The Cmax and Tmax parameters were obtained
directly from the curves. The areas under the curve
for alendronate plasma concentration versus time for
0→t (AUC0→t) were calculated by applying the linear trapezoidal method.
The 90% confidence intervals of the test/reference ratio of Cmax, AUC0→t and AUC0→∞ were determined using log transformed data. The bioequivalence between the two formulations was accepted if
90% CI of the log transformed Cmax, AUC0→t and
AUC0→∞ of test was within 80-125% of the original
product (19).
Alendronate sodium quantification in plasma
The analytical method for alendronate sodium
quantification in plasma samples had good specificity, sensitivity, linearity, precision, and accuracy
over the entire range of clinically significant and
therapeutically achievable plasma concentrations,
thereby enabling its use in bioequivalence trials. The
linearity was observed within the range of 1 to 100
ng/mL (alendronate sodium; y = 45579x + 21172, r2
= 0.988; x = plasma concentration, y = peak area
ratio) (Fig. 1). The intra-day precision ranged from
0.29 to 1.78%, whereas intra-day accuracy ranged
from 97.9% to 100.9%. Inter-day precision ranged
from 0.82% to 1.56%, whereas inter-day accuracy
ranged from 98.30 to 102.6%. The amount extracted
of alendronate sodium was determined with 5 repetitions. The mean absolute recovery was 92.82%,
whereas the relative recovery ranged from 94.9 to
101.2%.
Pharmacokinetic parameters were determined
from the plasma level-time curve drawn for either of
the drugs in each volunteer (annex 1). The results of
ANOVA revealed that Cmax, ln Cmax, Tmax, AUC, ln
AUC0→t, AUC0→∞, and ln AUC0→∞ were statistically
insignificant for effect subject, period and treatment.
Statistical analysis of the pharmacokinetic parameters of the test and reference drug are presented in
Table 2. The geometric mean ratio (90% CI) of these
parameters along with the results of bioequivalence
test are presented in Table 3. Since 90% CI for all
parameters was within the predefined bioequivalence acceptance limits (80-125% of the originator);
therefore, the test and reference formulations were
In vitro parameters
The chemical assay, disintegration time and
dissolution rate of all test brands were found within
the specifications of USP (Table 1). The brand
leader FOSAMAXÆ from MSD, Pakistan and the
one having best dissolution rate were selected for
bioequivalence studies.
In vivo parameters
Among 24 volunteers enrolled in the study, no
serious adverse event was found throughout the
study period. There was insignificant difference in
all analyzed pharmacokinetic parameters among all
brands of alendronate sodium. The demographic
parameters of subjects were as follows: mean age
23.5 ± 3.5 years (range 21ñ29 years); mean height
175.4 ± 3.8 cm (range 152.0ñ173.0 cm); and mean
weight 68 ± 4.8 kg (range 64ñ76 kg).
BONAFIDE
BONATE
BONGARD
FOSAMAX
ORTHONATE
OSTEOPOR
REVENTA-70
ALENDRATE
OSTIM
DRATE
1
2
3
4
5
6
7
8
9
10
SJG Fazal Ellahee
Genome
Global
Getz
Werrick
Schazoo
OBS Pharma
PharmEvo
Wilshire
Medisure
Manufacturer
2251T
003
051
24
1414
051T
Ont013
NM22110
9N033
007
004
Batch
No.
08/12
05/12
01/12
12/12
12/09
01/12
10/09
12/09
09/11
12/12
Mfg.
Date
08/12
05/14
01/14
12/14
12/11
01/14
10/12
12/11
08/13
12/14
Expiry
date
0.566 ± 0.12
0.445 ± 0.18
0.335 ± 0.06
0.201 ± 0.18
0.553 ± 0.24
0.442 ± 0.09
0.746 ± 0.42
0.205 ± 0.26
0.216 ± 0.14
0.330 ± 0.21
Avg.
weight
99.7%
100.1
101.7
101.5
101.7
100.3
100.2
101.7
96.6
95.6
Assay
%
69.8
70.07
71.19
71.05
71.2
70.21
70.1
71.2
67.6
66.92
Amount of
active (mg)
9
7
2
8
10
8
12
11
13
11
D-Time
(min)
82.6
84.8
80.6
88.6
94.4
82.4
90.2
86.6
80.1
81.4
Dissolution
%
49.5
1.00*
Cmax (ng/mL)
Tmax (h)
SD
6.86
0.61
0.55
0.11
17.65
8.26
1.43
0.13
0.11
0.02
3.68
1.72
SE
Test drug
Min
0.75
41.47
0.81
1.90
0.10
73.00
99.00
1.5
67.7
3.08
3.50
0.57
150.00
131.00
Max
1.00*
58.22
1.85
2.66
0.37
119.04
116.39
Mean
SD
8.98
0.45
0.82
0.16
9.05
6.97
1.13
0.09
0.17
0.03
1.89
1.45
SE
Reference drug
Min
0.75
43.87
1.19
0.97
0.11
105.00
105.00
Max
1.5
79.99
2.83
3.78
0.75
137.00
131.00
AUC = area under curve; Ke = elimination rate constant; MRT = mean residence time; t1/2_e = absorption half life; Cmax = maximum plasma concentration; Tmax = time to reach maximum plasma concentration; *median values were calculated.
2.78
1.92
0.25
Ke (L/h)
t1/2_e (h)
117.09
AUC0→∞ (ng◊h/mL)
MRT (h)
Mean
112.17
profile
AUC0→t (ng◊h/mL)
Pharmacokinetic
Table 2. Statistical analysis of pharmacokinetic parameters for test and reference drug in 1st and 2nd periods.
D-Time = disintegration time; average weight of tables is expressed as the mean ± SD. % active is determined with assay limit of 90-110%. Dissolution assay limit is 80%. All tests are according to USP.
Brand
(70 mg)
No.
Table 1. In vitro analysis of various brands of alendronate sodium (70 mg).
1003
1004
HUMAYUN RIAZ et al.
unless an oral dosage form (pill or tablet) disintegrates into small aggregates, it can not be efficiently
absorbed by the body (28). However, a switch from
disintegration testing to dissolution testing is due to
the fact that only tablet disintegration does not
ensure its availability in solution form for absorption. Today, disintegration is typically conducted as
an in-process test during tablet manufacture. Thus,
in the present study, dissolution and disintegration
tests of all brands of alendronic acid tablets analyzed
were rapid, complete and conformed to the established USP30 specifications (Table 1) ensuring adequate drug absorption from the generic products.
However, the generic products of alendronic acid
tablets are approved based on the results of singledose bioavailability studies in healthy subjects,
which is not an adequate test to establish similar disintegration characteristics (29). In United States, if a
drug substance and a drug product monograph exist
in USP, the generic version is expected to conform
to these established quality specifications. The
generic version is also required to use the same salt
form of the active ingredient, as the salt form can
affect the inherent solubility and subsequent absorption of the active ingredient. Thus, alendronic acid
must also be formulated as sodium salt. There is cur-
considered bioequivalent. Hence, OsteoporÆ could
be concluded as having comparable pharmacokinetic profiles with FosamaxÆ.
Tolerability
Both formulations of alendronate sodium 70
mg were well tolerated by all volunteers. No clinical
undesirable events were observed throughout the
study and for up to two weeks after the study.
DISCUSSION
The appearance of off-patent generic drugs in
the worldís pharmaceutical market is a highly interesting fact from the socio-economic point of view
and can bring about an increased efficiency in health
systems and increase the percentage of population
benefitting from a medical care plan (25). The
World Health Organization has reported various
copies of substandard quality of original pharmaceutical products (26). The substandard drugs contain low quality ingredients or lack the specified
ingredient altogether. Moreover, even when the definite ingredient is present, in some cases, it does not
dissolve satisfactorily or the amount is improper
(27). It was recognized some 50 years ago that
Figure 2. Plasma concentration-time plots of alendronate sodium. The plot is obtained after a single oral dose (70 mg) of reference and test
formulation in healthy volunteers (n = 24). No statistical significant difference was recorded. Data are presented as the mean values
Table 3. Pharmacokinetic parameters of test and reference drugs.
Parameter
Test
Standard
Reference
Lower
Upper
Observed
Lower
Upper
Within Equivalent Limit
Lower
Upper
ln AUC0→t (h◊ng/mL)
4.72 ± 0.07
4.75 ± 0.10
80%
125%
0.967
1.04
Yes
Yes
ln AUC0→∞ (h◊ng/mL)
4.72 ± 0.04
4.78 ± 0.09
80%
125%
0.969
1.04
Yes
Yes
ln Cmax (ng/mL)
3.90 ± 0.07
4.06 ± 0.08
80%
125%
0.962
1.06
Yes
Yes
rently a USP monograph for alendronate sodium
(active substance) and for alendronic acid tablets
(19) and unless stated otherwise the generic form
should conform to these standards. In our study, all
generics and brands tested conformed to USP standards (Table 1). Recently, a study has shown that for
all drug categories investigated, the patients who
experienced a generic switch did not have more concerns about their index medicine than patients who
did not switch and patients having high confidence
in the health care system showed less concern (30).
Generic versions of alendronate have been
reported to be bioequivalent to branded alendronate
(22, 23). The development of a brand name formulation requires the demonstration of its pharmacokinetics, efficacy and tolerability in both healthy subjects and in the target patient population. However,
the development of the generic equivalent requires
only the demonstration of its bioequivalence with
the brand name product in healthy subjects (31).
WHO guidelines state that 18 to 24 healthy male and
female volunteers aged 18 to 55 years of normal
body weight should be used in a crossover study
design to determine whether bioequivalence is
achieved between two formulations (21). It is
assumed that bioequivalence demonstrated in
crossover studies performed in this typically
younger and healthier population would be equivalent to that observed in the patient population. It is
further supposed that this bioequivalence would
translate into comparable clinical efficacy and tolerance; however, evidence to support the existence of
a well defined relationship between these parameters is lacking (32). These differences have fostered
concern as to whether bioequivalence, as ascertained in crossover studies of healthy adults, should
be used to make claims of comparable clinical effectiveness and tolerability in patients who are older and
often have numerous underlying disorders or diseases.
Further, older individuals tend to have greater difficulty in swallowing pills (orientation of pill in mouth)
and have reduced GI motility as compared to younger
individuals. These differences may increase the exposure time of the alendronate tablet to the upper GI
tract, which could then increase the probability of
alendronate exposure to the esophagus in older adults.
Much dissimilarity exists between brand and
generic alendronate including: disintegration time,
bio-adhesion to the esophagus, patient compliance
to treatment, adverse event prevalence, and conservation of bone mineral density. Generic forms of
alendronate warrant closer clinical study before their
credibility for clinical usefulness and acceptability
of brand. Differences in the excipient composition
1005
between the brand and generic formulations of alendronate may alter the bioavailability of the generic
alendronate to bone if this substitution changes the
behavior of the tablet such that the alendronate
tablet becomes more easily bound to food or drink
and unavailable for absorption in the gut (33).
Since, the bioavailability of alendronate is low
and dosing requirements are strict; therefore, any
characteristic of the tablet that changes the speed of
delivery of alendronate, such as time required for
disintegration or dissolution, can have important
implications on drug effectiveness or tolerance. For
regulatory approval, explicit dissolution parameters
are required to allow for the generic substitution of
brand alendronate; however, there are no specifications for the required disintegration characteristics
of the generic forms.
It has been reported (29) that the dissolution and
disintegration rates of a number of generic formulations of alendronate from Canada, Netherlands,
Germany, and United Kingdom were compared to
United States manufactured brand risedronate and
alendronate. All of the generics tested had an acceptable dissolution rate as compared to brand alendronate. Commercially available oral tablets designed
to dissolve in mouth without water prior to swallowing were purposefully included to act as disintegration comparators. Six of the 26 generic versions of
alendronate tested had disintegration times that were
comparable to oral tablets disintegrating in mouth.
Furthermore, substantial evidence indicates
that many generic formulations of alendronate are
more poorly tolerated than the proprietary preparations resulting in ominously poorer adherence and
efficacy (34). Reduced effectiveness may result
from faster disintegration times of many generics
that increase the likelihood of adherence of particulate matter to the esophageal mucosa. Unfortunately,
market authorization, based on the bioequivalence
of generics with a proprietary formulation, does not
take into account the potential concerns about safety. Poor adherence of many generic products has
implications for guideline development, cost effectiveness and impact of treatment on the burden of
disease. The impact of generic bisphosphonates
requires formal testing to re-evaluate their role in the
management of osteoporosis.
The majority of generic versions of alendronate disintegrate faster or slower than brand alendronate, whereas dissolution times are largely similar between brand and generic alendronate.
Moreover, a qualitative study had shown two general themes: first, complications in recognizing the
substituted medicine and second, lack of confidence
1006
HUMAYUN RIAZ et al.
in the identical effect of the substitutable medicines
(35). Previous interview and questionnaire based
analysis have shown that some patients felt anxious
and insecure about generic substitution and furthermore expressed uncertainty with regard to inferior
quality of the generic drugs compared with the original products. Moreover, side effects were experienced by some users of generic drugs (35-37).
The Drug Regulatory Authority of Pakistan
(DRAP) has not made it mandatory requirement for
marketing a generic in Pakistan with bioequivalence
studies, nevertheless, bioequivalence studies have
been carried out in recent years either for the purpose of quality enhancement or as a marketing tool
(38, 39). We are aware of the fact that there are strict
guidelines in Europe and USA for marketing authorization of oral (small molecule) generics to ensure
good quality and enhance their utilization. This is
recognized as the first step to enhance the use of
generics versus originators and patented products in
a class. Without such legislation and subsequently
its implementation in its true letter and spirit, there
will always be concerns with the quality of generics
especially the one that are locally produced. It will
also be difficult to promote low priced generics in
Pakistan. There is a dire need to revisit the drug policy of DRAP to promote and encourage the prescribing of low cost generics with highest quality,
especially with generics priced at 2% to 10% of prepatent loss prices as established in some (5, 6, 40).
Our study is a step towards this goal.
CONCLUSIONS
It is concluded that in the present study in
healthy male Pakistani volunteers, no statistically
significant differences in AUC0→t, AUC0→∞, and Cmax
were found between the test and reference formulations of alendronate sodium. The single 70 mg dose
of these formulations met the regulatory criteria for
bioequivalence and all test brands were within the
acceptable quality limits.
The study was carried out for educational purposes and the authors have declared no conflict of
interest.
Acknowledgments
The authors wish to thank the laboratory staff
at the National Institute of Health, Islamabad,
Pakistan for their technical support.
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Received: 26. 01. 2015
ISSN 0001-6837
DEVELOPMENT AND CHARACTERIZATION OF SMART DRUG
DELIVERY SYSTEM
UME RUQIA TULAIN, MAHMOOD AHMAD*, AYESHA RASHID
and FURQAN MUHAMMAD IQBAL
Faculty of Pharmacy and Alternative Medicine, Khawaja Fareed Campus,
The Islamia University of Bahawalpur, Pakistan
Abstract: Present work concerned with development and evaluation of an innovative drug carrier, as smart
drug delivery system for highly acid labile drug. Free radical polymerization technique was employed to develop pH sensitive drug delivery system by using carboxymethyl cellulose (polymer), methacrylic acid
(monomer), potassium persulfate (initiator) and N,N methylene bisacrylamide (crosslinker). Prepared
crosslinked polymer was characterized by swelling analysis at acidic and basic pH to evaluate pH responsive
swelling, instrumental analysis (SEM, FTIR and thermal analysis) and pH responsive release of model drug
rabeprazole sodium. Characterization of smart drug delivery concluded that pH responsive swelling and drug
release parameters were directly related with methacrylic acid concentration in the crosslinked polymer. It was
observed that by raising methacrylic acid contents swelling at basic pH enhanced and crosslinker contents increment reduce swelling. Among nine formulations with varying formulation contents, CMA2 exhibited more pH
sensitive swelling and cumulative drug release at alkaline pH. Results of investigation recommended that
CMA2 can be a best crosslinked polymer as smart drug carrier.
Keywords: crosslinked polymer, pulsatile behavior, hydrogel, gel fraction
The distinctive features of hydrogels have promoted meticulous attention in their use in drug
delivery applications. Highly porous structure can
be regulated by crosslinking density of hydrogels.
Porosity imparts affinity with aqueous medium to
swell, thus allows drug loading and subsequent
release through gel matrix (1).
Intelligent hydrogels can recognize and
respond to small alteration in external circumstances
such as pH, temperature, light, and ionic strength.
The smart drug delivery systems hold some vital
characteristic such as predetermined rate, self-controlled, targeted, predefined time and monitor the
delivery (2). The prime qualification of the system is
the presence of ionizable weak acidic or basic moieties attached to a backbone. Transition from collapsed state to expanded state also modified by electrostatic repulsion in response to alterations in environmental states like pH, temperature, ionic contents
etc. Proper selection between polyacid or polybase
is essential for desired application. Polyacidic polymers will be unswollen at low pH, since the acidic
groups will be protonated and unionized. When
increasing the pH, a negatively charged polymer
will swell. The opposite behavior is exhibited in
polybasic polymers, since the ionization of basic
groups will increase when decreasing the pH (3).
In modern era, copolymers have originated
new and extremely progressing horizons in front of
the precincts of conventional drug delivery system
to develop into one of the focal compounds as drug
delivery vehicles. Copolymers are heteropolymers
of miscellaneous types of monomers and with varying length of structural repeating units. The amalgamation of different chemical units in copolymer
structures consequences in new materials with
numerous novel features (4).
Researchers have struggled to engineer the
controlled drug delivery system to regulate the
bioavailability of drugs. The most striking way to
improve bioavailability of acid sensitive drugs is pH
responsive release. Hydrogels having such desired
characteristics, make them an ideal vehicle for intelligent drug delivery system (5).
Chemically, crosslinked hydrogels (as compared to physical hydrogels) are mechanically stable
* Corresponding author:e-mail: ma786_786@yahoo.com; phone: 0092-062-9255556; mobile: 0092300-9682258; fax: 0092-062-9255565
1009
1010
UME RUQIA TULAIN et al.
due to the covalent bond. These hydrogels can be
prepared by radical polymerization of low molecular weight monomers/polymers in the presence of a
crosslinking agent (6)
Rabeprazole sodium is an inhibitor of gastric
proton pump. It restrain gastric acid secretion by
particularly blocking the H+/K+-ATPase enzyme
system at the secretory surface of gastric parietal
cell considerably plummeting gastric acid levels and
allowing acid-related disorders to cure, as well as
mitigate symptoms of chronic conditions, like gastric and duodenal ulceration and also in Zollinger
Ellison syndrome and reflux esophagitis (7).
This significant and substantial piece of
research work includes development, characterization and evaluation of polymeric formulations by
using anionic polymer (carboxymethyl cellulose)
and monomer (methacrylic acid) for stable delivery
of acid labile drug. Rabeprazole sodium have very
short half-life (1-1.5 h), and is highly acid-labile so
low bioavailability (52%), presents many formulation challenges.
EXPERIMENTAL
Materials
Rabeprazole sodium (Getz Pharma Islamabad
Pakistan), carboxymethyl cellulose CMC (Sigma
Aldrich, Finland), potassium persulfate (AnalaR,
BDH, England), N,N-methylene bisacrylamide
(Fluka, Switzerland), tris(hydroxymethyl)aminomethane (Fluka, Switzerland), methanol, potassium
dihydrogen phosphate, ethanol absolute, methacrylic acid (all from Merck, Germany), sodium
hydroxide (Sigma Aldrich Lab, Riedel-de Haen
GmbH).
Methods
Weighed amount given in Table 1 of carboxymethyl cellulose was dissolved in degassed distilled water to obtain a sticky transparent solution at
70OC. Then potassium persulfate (initiator) solution
in water was added to carboxymethyl cellulose solution and stirred for 10 min at 70OC to generate radicals. Following this, reaction mixture was cooled
down to room temperature. At room temperature,
solution containing monomer and cross linker was
added under magnetic stirring. The final weight of
solution was made by adding distilled water. Air
above the solution in the tube or any dissolved oxygen was removed by bubbling nitrogen for 15-20
min which acts as free radical scavenger. For polymerization, solution was heated in water bath with
gradual increase in temperature from 45 to 65OC by
10 degree per hour. Temperature of reaction was
maintained at 65OC for further 8 hours to complete
reaction (8). Hydrogels obtained were cut into discs.
0.1 M sodium hydroxide and ethanol were used to
remove unreacted monomer and catalyst. These
discs were thoroughly washed until the pH of washing water resembles distilled water. After washing,
the discs were dried first at room temperature and
then in oven at 50OC till constant weight of hydrogels obtained. These discs were kept in desiccator
and further used for characterization and drug
release study (9).
Swelling analysis
The equilibrium swelling of CMC-g-MAA
graft copolymer were determined by swelling the
dried hydrogels disks in acidic and basic pH buffer
solution at 37OC. Dried hydrogels of 0.45 g were
immersed in 100 mL solution of 0.1 M HCl (pH 1.2)
Table 1. Composition of 100 g CMC-g-MAA hydrogel preparation with varying monomer polymer and crosslinker concentration.
No.
Formulation code
CMC
MAA
Crosslinker %
mole ratio
of monomer
1
CMA1
0.75
20
0.1
2
CMA2
0.75
30
0.1
3
CMA3
0.75
35
0.1
4
CMA4
1
25
0.1
5
CMA5
1.5
25
0.1
6
CMA6
2
25
0.1
7
CMA7
0.75
25
0.2
8
CMA8
0.75
25
0.3
9
CMA9
0.75
25
0.4
Development and characterization of smart drug...
and USP phosphate buffer of pH 7.4 at 37OC. The
swollen samples were weighed at preschedule intervals. The studies were performed in triplicate and
average values were taken for data analysis. The
swelling of various samples were continued until
they attained constant weight (11). The dynamic
swelling ratio (q) was calculated using following
equation (12).
q = Ws/Wd
(1)
where q is dynamic swelling ratio, Ws is the weight
of swollen gel at time t and Wd is the initial weight
of dry hydrogel.
pH responsive/pulsatile behavior
The transition of the swelling/deswelling
behavior was proved by recurrently cycling the
CMC-g-MAA hydrogel between buffers at pH 1.2
and pH 7.4. For controlled delivery of drug from
graft copolymer, the swelling process must be
reversible to ensure that the release of drug could be
initiated and stopped promptly upon change in pH.
To investigate reversibility of swelling/deswelling
process of polymer networks with respect to environmental pH change, selected hydrogel sample was
swollen in a buffer solution of pH 7.4 until equilibrium swelling, placed them in a buffer solution of
pH 1.2, returned them to a buffer solution of pH 7.4,
and finally collapsed them in a buffer solution of pH
1.2 (10).
Drug loading
Selected hydrogels were soaked in 0.1 M sodium hydroxide solution containing 1% rabeprazole
sodium for time period until swelling equilibrium
achieved. Loaded hydrogels were washed after
swelling with water to remove surface adhered drug
on disc. For drug loading, 0.1 M sodium hydroxide
solution was selected due to maximum swelling
ratio of hydrogels and drug stability in that solution.
The drug loaded hydrogels were freeze dried
because drug was unstable at oven temperature (13).
Determination of drug loading
Two methods were used for determination of
drug loading in hydrogels. The first method used to
calculate the amount of drug loaded in hydrogel was
determined by following equation:
Amount of drug = WL - Wo
(2)
Wo and WL are weight of dried hydrogels before and
after immersion in drug solution, respectively.
In the second method, amount of drug
entrapped in hydrogels was premeditated by extraction method. Weighed quantity of powdered loaded
gels was extracted by using 0.1 M sodium hydrox-
1011
ide solution. Each time, fresh 0.1 M sodium hydroxide solution was replaced after specific interval until
there was no drug in the solution. Drug concentration was determined spectrophotometrically at λmax
284 nm. Amount of drug present in all portions was
considered as total amount of drug loaded into
hydrogel (14).
Determination of the equilibrium water content
and gel fraction of hydrogel.
Water absorbed by CMC-g-MAA copolymeric
hydrogel was quantitatively signified by the equilibrium water content (EWC). Dried hydrogel samples
were soaked in buffer of pH 1.2 and pH 7.4 at 37OC
to measure water uptake of hydrogel in a thermostatically controlled chamber to the equilibrium state.
Fully swollen samples were removed and weighed
after removal of excess of solvent with absorbent
paper. The equilibrium water content in swollen
samples (Weq) was calculated as follows (15):
Ws ñ Wd
Weq% = ñññññññññ
× 100
(3)
Ws
where Ws is the weight of swollen sample at equilibrium state and Wd is weight of the dry sample.
By extraction of prepared hydrogels insoluble
part (gelled part) was collected and washed with
water to remove unreacted contents. Hydrogels were
cut into disc of 4-5 mm diameter and oven dried.
Then, few dried hydrogels were extracted with
water at room temperature in order to extract the
insoluble parts of hydrogel until the weight became
constant. The gel fraction was calculated by following equation (16):
Wo ñ W1
Sol fraction % = ñññññññññ × 100
(4)
Wo
Gel fraction (%) = 100 - Sol fraction
(5)
where Wo is weight of hydrogel before extraction
and W1is weight of hydrogel after extraction.
Instrumental analysis
SEM analysis
SEM images deliver evidence about pore
size and geometry and homogeneity/heterogeneity of graft copolymeric network. Surface morphology of crosslinked hydrogel was evaluated by
Quanta 250 SEM (FEI), working at 10 kV with
secondary electrons, in low vacuum mode. For a
better observation of the pores, swollen hydrogels
were previously freeze-dried in freeze dryer
(Christ Alpha 1-4 Germany), for 24 h at -65OC.
The sample was prepared by grinding the dry
hydrogels into powder, in order to expose the
internal structures (17).
1012
FTIR analysis
The chemical structures for new CMC-g-MAA
hydrogels and their individual components were
confirmed by FTIR analysis. Grafting was examined
by FTIR spectroscopy. IR spectra for components
and prepared hydrogel were recorded in a Fourier
transform infrared (FTIR) spectrophotometer
(Bruker, Tensor-27, Germany). For FTIR analysis
hydrogel samples were ground to powder. A small
quantity of sample was placed in crystal area and
pressure arm was locked to push the sample over
zinc selenide crystal. Bands were observed in the
region from 4000 to 400 cm-1 (17).
Thermal analysis
Thermal stability of the copolymer (CMA) and
individual constituents CMC and methacrylic acid
were studied by TGA analyzer in the temperature
range from 0 to 600OC under inert nitrogen atmosphere. Thermal behavior of the prepared graft
copolymers were studied by Thermogravimetric
Analyzer model SDT Q 600 series Thermal
Analysis System (TA Instruments, New Castle DE,
UK). All the measurements were made in triplicate.
Thermograms were recorded by software (18).
In vitro release studies and release kinetics
The selected in vitro dissolution conditions
were in accordance with US Food and Drug
Administration, CDER (Center for Drug Evaluation
and Research). Drug release studies were carried out
using a USP type II dissolution test apparatus (PTCF
II Pharma Test, Germany) at 100 rpm for 24 h in 0.1
M HCL (900 mL) maintained at 37 ± 0.5OC. Five
mL of sample was collected at 0, 0.5, 1, 1.5, 2, 4, 6,
8, 10, 12 and 24 h with an automated sample collector (PT-DT7Pharma Test, Germany) after filtering
through sinter filters (10 µm). The collected samples
were diluted up to 50 mL and analyzed at 284 nm
using a UV-spectrophotometer (UV-1600 Shimadzu. Germany). The same studies were conducted
with 0.6 M Tris buffer, pH 8.0 (900 mL) and tested
for drug release for 24 h at the same temperature and
rotation speed. Samples were taken out and volume
of fresh Tris buffer pH 8.0 was added to kept volume of dissolution medium constant and samples
were analyzed using UV spectrophotometer at 284
nm. The in vitro cumulative drug release study was
conducted in triplicate (19).
Dissolution profile can be described by different mathematical functions. To obtain a more quan-
Figure 1. Comparative swelling ratios of CMC-g-MAA hydrogels using different concentrations of MAA
Figure 2. Comparative swelling ratios of CMC-g-MAA hydrogels using different concentrations of CMC
1013
Figure 3. Comparative swelling ratios of CMC-g-MAA hydrogels using different concentrations of MBA (crosslinker)
Figure 4. On-off switching behavior as reversible pulsatile swelling (pH 7.4) and deswelling (pH 1.2) of CMC-g-MAA hydrogel
titative understanding of the transport kinetics in
hydrogel, the drug release data were analyzed as a
function of time. The release models with major
application and best describing drug release phenomena are, in general, the Higuchi model, zero
order model, first order model and Korsmeyer
Peppas model (20).
Pulsatile behavior of hydrogel
The transition of the swelling/deswelling
behavior was proved by recurrently cycling the
CMC-g-MAA hydrogel between buffers at pH 1.2
and pH 7.4. As shown in Figure 4, the hydrogel
quickly constricted when placed in a pH 1.2 buffer
but slowly resumed to nearly the original swollen
size at pH 7.4.
RESULTS
Swelling studies at pH 1.2 and pH 7.4
Methacrylic acid contents effect on swelling
pattern have been given in Figure 1 and CMC weight
ratio effects on swelling behavior was described in
Figure 2 and crosslinker effect in Figure 3.
Equilibrium water contents and gel fraction
Water absorbed by CMC-g-MAA copolymeric hydrogels was quantitatively signified by the
equilibrium water content (EWC) The EWC values
of the hydrogels were calculated and tabulated in
Table 2. By extraction of prepared hydrogels insol-
1014
uble part (gelled part) was collected and washed
with water to remove unreacted contents.
Calculated gel fraction of hydrogel was summarized in Figure 5.
SEM analysis
SEM images deliver evidence about pore size
and geometry and homogeneity/heterogeneity of
graft copolymeric network. SEM images of CMC-gMAA hydrogels are presented in Figure 6.
FTIR analysis
The chemical structures for new CMC-g-MAA
hydrogels and their individual components were
confirmed by FTIR analysis. FTIR spectra of CMC
(carboxymethyle cellulose), MAA (methacrylic
Figure 5. Gel fraction of CMC-g-MAA hydrogel with different concentrations of MAA, CMC and crosslinker
Figure 6. SEM images of lyophilized hydrogels (CMC-g-MAA) at magnification of 100◊ and 200◊ and 10 µm, 30 µm, 300 µm, and 500
µm scale bar, respectively
1015
Table 2. Equilibrium water contents and gel fraction of CMC-g-MAA hydrogels using different concentrations
of MAA, CMC and crosslinker.
Formulation code
Contents w/w %
EWC %
Gel fraction
CMA1
MAA 20
96
67.41
CMA 2
MAA 30
93
88.45
CMA 3
MAA 35
90
92.33
CMA 4
CMC 1
95
89.54
CMA 5
CMC 1.5
96
92.65
CMA 6
CMC 2
97
74.14
CMA 7
MBA 0.45
89
84.64
CMA 8
MBA 0.65
86
86.38
CMA 9
MBA 0.85
66
93.75
Table 3. DTG data of methacrylic acid (MAA), CMC and CMC-g-MAA hydrogel (CMA).
Sample
CMA
MAA
CMC
Step
Tdi
(OC)
Tdm
(OC)
Tdf
(OC)
Weight loss %
at Tdf
1
67
91
130
8.8
II
447
460
531
21.79
I
48
130
144
97.02
I
51
67
89
4.76
II
255
290
314
36.08
acid), CMA (formulation) shown in Figure 7 confirmed modifications of CMC with methacrylic acid
by grafting.
sion exponent ëní to find out the release pattern of drug from
hydrogel given in Table 5.
DISCUSSION
Thermal analysis
Thermal stability of the copolymer (CMA) and
individual constituents CMC and methacrylic acid
were studied by TGA analyzer in the temperature
range from 0 to 600OC under inert nitrogen atmosphere. DTG data are summarized in Table 3. TGA
curves of CMC (carboxymethyl cellulose), MAA
(methacrylic acid) and CMA (CMC-g-MAA)
hydrogel formulation are depicted in Figure 8. DSC
curves of CMC, MAA and CMA hydrogel formulation are presented in Figure 9.
Drug loading and in vitro release kinetics of
rabeprazole sodium from CMC-g-MAA hydrogel
Release of rabeprazole sodium from CMC-g-MAA
was carried out at acidic and basic pH to evaluate the pH
sensitive release. Effect of methacrylic acid on cumulative
release of rabeprazole sodium was described in Figure 10.
Effect of CMC and crosslinker (MBA) on drug release was
demonstrated in Figure 11 and 12, respectively. Release
kinetics of drug can be determined by fitting in vitro release
data into mathematical release models and calculate diffu-
Effect of variation of pH, monomer, polymer and
cross-linker on swelling behavior of CMC-gMAA hydrogel
The best exigent task in the development of
drug delivery systems is to deal with instabilities of
drugs in the cruel environment of the stomach.
Swelling capacity in varying pH buffer solutions is
of prime significance practical applications for
smart drug delivery system. Equilibrium swelling
capacity of hydrogels depends on hydrogel structure, crosslinking density, ionic contents and
hydrophilicity of hydrogel (21).
In the present study, dynamic swelling studies of CMC-g-MAA were executed to scrutinize
the swelling behavior of hydrogels prepared using
a different molar ratio of carboxymethyl cellulose,
methacrylic acid and N,N MBA (crosslinker).
Figure 1 shows swelling profile at different pHs
for hydrogels prepared with varying methacrylic
acid (monomer) contents. It was observed that
hydrogels reveal pH sensitive behaviour. At pH
1016
Figure 7. FTIR spectra of CMC, MAA, and prepared hydrogel (CMA)
1.2 hydrogels remain in collapsed state, show less
swelling. At alkaline pH swelling ratio increased
owing to dissociation of pendant acididc group
(carboxylate group) of hydrogel. It has been proposed that as the methacrylic acid contents (20, 30
and 35%) increased, pH sensitivity of hydrogels
augmented. At pH 7.4 exhibits chain relaxation
process due to repulsion among ñCOO- groups
sideways the macromolecular chains formed from
the ionization of carboxylic groups. The electrostatic repulsion causes the network to expand and
solvent enters causing swelling at high pH. The
Figure 8. TGA curves of methacrylic acid (MAA), CMC and CMC-g-MAA
Figure 9. DSC curves of methacrylic acid (MAA), CMC and CMC-g-MAA
1017
1018
in the free space for lodging of water molecules.
Additionally, the increased firmness of the network
also limits the relaxation of macromolecular chains
in the matrix, thus directing to minor degree of
swelling. Swelling profile of CMC-g-MAA hydrogels at alkaline pH revealed pH sensitivity, owing
to fact that neg (-COOCH3) in CMC increase the
electrostatic repulsions between the polymer
chains and permit entry of fluid into hydrogel network thus enhance swelling ratio. Swelling at
acidic pH was inhibited by collapsing the polymeric chains (protonation) and hindering the solvation
of the hydrogel. Previous studies described similar
CMC behavior in hydrogel swelling (23).
In order to evaluate the effect of crosslinker
contents on swelling, three samples with varying
concentration (0.2, 0.3 and 0.4%) of N, N MBA has
been prepared and observed their swelling profile.
equilibrium water absorption of hydrogel manifests its swelling capacity and is a function of the
network configuration, the crosslinking ratio,
hydrophilicity and the degree of dissociation of
the functional groups. Similar swelling behavior
has been depicted by HEMA-co-MAA hydrogel.
HEMA-co-MAA revealed highly pH sensitive
behavior but less equilibrium water uptake
because of methyl groups, which promote
hydrophobicity to polymeric network thus
obstruct expansion (22).
The effect of carboxymethyl cellulose concentration on swelling capacity and pH sensitivity
have been depicted in Figure 2. It was concluded
that increased contents of CMC (1, 1.5 and 2%
w/w), decreased swelling. This fact may be related
with an increase in gel fraction a number of
crosslinks per unit volume, thus causing a decrease
Table 4. Drug loading in different hydrogel formulations (CMC-g-MAA).
Amount of rabeprazole sodium loaded
(mg per 0.45 g of dry disk)
Formulation
code
By extraction
By weight
CMA1
48
49.3
CMA2
45
45.9
CMA3
41
41.7
CMA4
52
52.8
CMA5
59
60.2
CMA6
66
67.3
CMA7
39
39.4
CMA8
37
38
CMA9
34
35
Table 5. Release kinetic parameters of rabeprazole sodium from hydrogel CMA.
Formulation
Higuchi
First order
Zero order
code
R
R
R
R2
n
CMA1
0.997
0.567
0.971
0.995
0.604
CMA 2
0.992
0.639
0.982
0.993
0.783
CMA 3
0.994
0.541
0.936
0.996
0.439
CMA 4
0.996
0.572
0.954
0.988
0.598
CMA 5
0.993
0.613
0.980
0.994
0.718
CMA 6
0.990
0.618
0.987
0.997
0.774
CMA 7
0.996
0.540
0.971
0.994
0.711
CMA 8
0.997
0.560
0.962
0.992
0.654
CMA 9
0.998
0.575
0.974
0.992
0.631
2
2
2
Korsmeyer-Peppas
1019
Figure 10. Effect of methacrylic acid concentration on cumulative drug release of rabeprazole sodium
Figure 11. Effect of CMC concentration on cumulative drug release of rabeprazole sodium
Figure 12. Effect of crosslinker concentration on cumulative drug release of rabeprazole sodium
Figure 2 shows that increased crosslinker contents
reduced swelling due to compact and dense polymeric network. Similar findings have been
described by other researchers that high degree of
crosslinking of hydrogels produce less porous network which has a low swelling ratio (24).
Pulsatile behavior of hydrogels
To evaluate the pH responsive behavior of prepared hydrogels, reversible oscillatory swelling
experiment has been conducted. Pulsatile behavior of
CMC-g-MAA has been shown in Figure 4, demonstrating that hydrogels undergo volume phase transi-
1020
tion at acidic and basic pH due to protonation or
deprotonation of pendant groups attached with
copolymer chain leading to conformational changes.
The pH-responsive swelling and collapsing style of
CMC-g-MAA hydrogel is required for controlled
release of acid sensitive model drug (rabeprazole
sodium) in our study. Literature has been supported
our findings that ionic hydrogels represent reversible
swelling and deswelling behavior in response to pH
transition due to electrostatic interactions of hydrogels and swelling medium. The most common pHsensitive hydrogels are poly(acrylic acid) (PAA),
poly(methacrylic acid) (PMAA), poly(diethylaminoethyl methacrylate) (PDEAEMA), and poly(dimethylaminoethyl methacrylate) (PDMAEMA),
and their copolymers (25).
Equilibrium water contents and gel fraction
It has been reported that constituent that impart
hydrophilic character to hydrogel improve water
contents of hydrogels. Table 2 showed the variation
of EWC of CMC-g-MAA hydrogels in varying
quantities of CMC, MAA and croslinker. The values
of EWC increase with increase of CMC content in
the hydrogels. It increases from 95 to 97% for the
concentration of CMC in the range 1 to 2% in the
hydrogels. CMC has high attraction to water due to
existence of carboxyl group in it, as a result EWC of
CMC-g-MAA hydrogels increases. Such characteristics of carboxymethyl cellulose (CMC) has also
been reported that it improves EWC properties of
poly (vinyl alcohol)/sago blend hydrogel due to the
presence of the carboxylic group in the CMC molecules. Results also described that increasing
methacrylic acid amount and crosslinker contents
decreased EWC values of hydrogels because of
hydrophobic nature (26).
Figure 5 showed the effect of different concentration of carboxymethyl cellulose, methacrylic acid
and crosslinker (N,N MBA). It has been evaluated
from the results that increasing concentration of
individual constituents increased gel fraction and
reduced sol fraction. Optimized reaction conditions
and increased contents provide sufficient grafting
site, monomers and crosslinking density favor high
gel fraction. It was found that at high the concentration of crosslinker in the hydrogels formulation,
crosslinking density will be higher, reducing sol
fraction (27).
Instrumental analysis
SEM analysis
Porous structure of hydrogels is prerequisite
for their application in controlled drug delivery.
Because porosity enhances the swelling capacity so,
reduce drug transport resistance. CMC-g-MAA photomicrographs shown in Figure 6 were exhibiting
that hydrogel was compact, smooth and with dense
surface at low magnification and at high magnification exhibited heterogeneous pore distribution in the
structure. These morphological modifications related to grafting of methacrylic acid onto CMC accelerate penetration of water, promote swelling. The
porosity plays multiple roles for drug loading and
release from the hydrogels. Similar findings have
been reported for pH-sensitive poly(ethylene oxide)
grafted methacrylic acid and acrylic acid hydrogels
(17).
FTIR spectrum analysis
In Figure 7 FTIR spectrum of pure sample, evidently exposes the major peaks allied with NaCMC.
Previous studies have described that absorption
bands seen at wave numbers of 1500-1700 cm-1, due
to carboxyl groups and their salts are respectively
(28). The band at 1030 cm-1 is due to carboxymethyl
ether group (CH O CH2-) stretching. Strong absorption band at 1589 cm-1 shown in Figure 7 confirmed
the presence of C=O group, designated CMC. The
band at 2924 cm-1 is due to CñH stretching of the
ñCH2 and CH3 groups.The band around 1322 cm-1 is
assigned to OH bending vibration. General absorption band observed at 3200 ñ 3600 cm-1, were due to
the stretching frequency of the ñOH group (29).
From FTIR spectrum associated with CMC-gMAA (CMA) hydrogel, it can be perceived that
important peaks at 1691 cm-1, 1641 cm-1 and 1443
cm-1 authenticating the development of graft copolymer product. These peaks are ascribed to carbonyl
stretching of the carboxylic acid groups and symmetric and asymmetric stretching styles of carboxylate anions, respectively.This fact practically proves
grafting of vinyl monomer onto carboxymethyl cellulose polymer backbone. Previous study revealed
that FTIR spectra of the grafted (starch grafted with
methacrylic acid) sample specify the advent of identical absorption bands which were not observed in
the spectrum of polymer backbone (30).
Thermal analysis
Thermogram given in Figure 8 CMC exhibited
two distinct decomposition phases in its thermogravimetric curve. The first one is in temperature
range of 51-89OC allied with loss of moisture (4.76
wt %), and the second one is in the range 255-314OC
with maximum weight loss (36.08%), related with
decomposition of carboxymethyl group. The maximum decomposition of the CMC-g-MAA hydrogels
occurred in a temperature range of 447ñ531OC, with
approximately 21.79% weight loss. Significantly,
the remaining weight of the hydrogels at Tdf was far
higher than the parent constituents. Higher remaining mass in the thermal profile of hydrogels designated higher thermal stability of the hydrogels than
of the individual constituents. Preceding researches
have also been designated that the graft copolymerization of natural polymers with vinyl monomers
could augment their thermal stability (31). DSC is
the thermal analysis method practiced to evaluate
the temperatures and heat flows linked with shifts as
a function of time and temperature. Transition related with absorption or emission of heat creates alteration in heat flow. Difference in energy is recorded
as peak. Area under the peak is directly related with
enthalpy changes and direction of peak designates
the thermal episode as endothermic or exothermic.
The endothermic peak of CMC below 100OC is
evidently punier than that of CMC-g-MAA, and the
new endothermic peak at 495OC also seemed in the
DSC curve of CMC-g-MAA. This signposts that the
thermal decomposition progression was proved by
grafting. In previous work has been described that
shift to higher decomposition temperature could be
accounted to formation of covalent bonds in the
graft copolymers, and improved thermal stability
(32).
In vitro release kinetics of rabeprazole sodium
from CMC-g-MAA hydrogel
Rabeprazole sodium release studies were conducted to a maximum period of 24 h in buffer of pH
1.2 and Tris buffer of pH 8 in accordance with the
US Food and Drug Administration. Results displayed in Figure 10 show rabeprazole sodium
released from a gel containing 20, 30 and 35%w/w
MAA at constant CMC and cross-linker contents. It
was viewed that maximum 11.69, 7.80 and 5.59%,
respectively, of the total loaded drug was released
after 24 h at pH 1.2 with increasing contents of
methacrylic acid. However, 63.11 to 71.85% of the
total drug loaded was released at pH 8 in 24 h period of time. These results are correlated with pH
responsive swelling of hydrogels. Analogous findings have been depicted in previous studies, pH sensitive poly(methacrylic acid-g karaya gum) synthesized graft copolymer exhibited pH responsive
swelling and drug release pattern (33).
Effect of carboxymethyl cellulose contents on
percent cumulative drug reease have been studied.
Results displayed in Figure 11 revealed that percent
cumulative drug release at acidic pH (1.2) and at
alkaline pH (8) were in accordance with swelling
1021
fashion of CMC-g-MAA hydrogels. High CMC
contents increase hydrophilicity and dominent
anionic properties to hydrogels, augment pH sensitivity and drug release accordingly.
Effect of concentration of crosslinker has also
been studied. Figure 12 revealed that by modulation
in crosslinking density of hydrogels, decreased percent cumulative drug release due to compact and
highly dense network restricted permeation of
release medium ultimately declined swelling. Our
findings are correlated with previous work that high
crosslinker contents reduced free spaces for drug
transport from meshwork of polymer (34).
To evaluate the release mechanism from
hydrogels drug release data were analyzed by various release kinetics models, zero order kinetics, first
order kinetics, Higuchi model, and KorsmeyerPeppas equation. Most appropriate mechanism was
explained on the basis of best fitness of release
model. The release model can be anticipated by
deliberating the regression value nearby 1. The
kinetics of drug release governed by comparative
drive of the erosion and swelling/diffusion fronts.
To comprehend the rabeprazole sodium release
from loaded graft copolymeric network, in vitro
release studies data were fitted into release models,
and release profile at basic pH is best explained by
Higuchi model, as plots expressed high linearity
with regression value of between 0.992-0.998 of
series of hydrogels with varying composition weight
ratios. Drawback of Higuchi model is that it is
unable to explain effect of swelling on matrix upon
hydration. Therefore, the in vitro release data were
also fitted to exponential Korsmeyer-Peppas equation and value of release exponent (n) explains the
exact release mechanism. The detected ëní values
for release profiles of hydrogels were in between
0.50 and 1 indicating anomalous release behavior.
Similar release kinetics have been exhibited by poly
(vinyl caprolactam) grafted on to sodium alginate,
the values of ëní were in the range of 0.616-0.918
and were accredited to the anomalous type of diffusive transport of drug (35).
CONCLUSION
Among various graft copolymer formulations
prepared with varying contents of carboxymethyl
cellulose, methacrylic acid and N,N MBA CMA2
present superior properties in regards with swelling,
pulsatile behavior, mechanical strength, sustained,
and pH responsive drug release. CMC-g-MAA
hydrogels high methacrylic acid concentration may
lead to more efficient network formation (a lower
1022
sol fraction) due to the higher concentration of reactive vinyl groups in the polymer mixture. The concept of formulating graft copolymer CMC-g-MAA
containing rabeprazole sodium offers an appropriate, sensible approach to accomplish a lingering
therapeutic outcome by continuously releasing the
drug over extended period of time.
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Received: 30. 03. 2015
ISSN 0001-6837
RELATIVE BIOAVAILABILITY STUDY OF SUCCINIC ACID COCRYSTAL
TABLET AND MARKETED CONVENTIONAL IMMEDIATE RELEASE
TABLET FORMULATION OF CARBAMAZEPINE 200 MG IN RABBITS
MAJEED ULLAH, GHULAM MURTAZA*, IZHAR HUSSAIN
Department of Pharmacy, COMSATS Institute of Information and Technology, Abbottabad, Pakistan
Abstract: A single-dose study was performed to observe the bioequivalence of the newly formulated carbamazepine-succinic cocrystal (CBZ-SUC) immediate release tablet (F1) with marketed immediate release formulation EpitolÆ 200 mg tablet (F0). In this study on albino rabbits, the plasma levels resulting from 250 mg
cocrystal equivalent to 200 mg of carbamazepine and conventional tablets 200 mg immediate release tablets
were compared. An open-label, randomized 2 ◊ 2 crossover study design, with a 1-week washout period, was
used. Carbamazapine (CBZ) plasma concentrations were determined by a high-performance liquid chromatography validated method using ultraviolet detection. CBZ plasma levels were measured at predose and various
postdose time points up to 72 h and the following pharmacokinetic parameters were used for evaluation: area
under the curve (AUC), maximum plasma drug concentration (Cmax), time to achieve Cmax (tmax), and elimination rate constant (Ke). By applying paired t-test to AUC0-72 (calculated by linear trapezoidal rule), the experimental formulation F1 was found to have statistically significant (***p < 0.05) improvement in bioavailability
of CBZ. However, these statistical differences do not have practical implications and the two formulations (F0
and F1) were found to be bioequivalent as the relative bioavailability of both formulations (106.9%) falls within the acceptable FDA set range of two bioequivalent products 80-125%.
Keywords: carbamazapine, cocrystal, bioequivalence
Carbamazepine (CBZ) (5H-dibenz[b,f]azepine-5-carboxamide) has been routinely used clinically in the treatment of trigeminal neuralgia and
epilepsy since 1965. When CBZ is given in solution
in ethanol or propylene glycol or in aqueous suspension, its absorption is rapid from GI tract with peak
plasma levels of 1-7 h (1-3). However, absorption of
drug from commercially available tablets formulations seems to be sluggish, as the peak plasma level
varies from 6 to 24 h (4, 5). This is probably due to
poor aqueous solubility of CBZ, which may be
reflected in differences in the rates of dissolution
and absorption of the drug from different tablet formulations. Not surprisingly, its oral bioavailability
depends on dissolution (6), which is affected by the
crystalline form used in the dosage form (7). The
extremely low solubility is also responsible for the
incomplete, slow and erratic gastrointestinal absorption (8). Owing to its narrow therapeutic index as
well as relatively high variation in drug plasma concentration (9), a uniform distribution of CBZ in the
solid dosage form and reproducible dissolution rate
are essential for achieving the desired therapeutic
effect without high risk of toxicity.
In recent years, pharmaceutical cocrystals have
emerged as a potential strategy to boost the solubility concerns of weakly soluble drugs (10). CBZ is a
BCS class II drug, thus shows dissolution limited
bioavailability, and thus cocrystals with a number of
soluble coformers have been reported and extensively studied in order to improve the CBZ solubility. About 40 different coformers have been accounted in the literature that formed cocrystals with CBZ
(11). Hickey and co-workers studied the in vivo performance of carbamazepine-saccharine (CBZ-SAC)
cocrystal in comparison to brand product TegretolÆ.
The cocrystal in powder form was found to be bioequivalent as it gave similar oral bioavailability in
four dogs to that of marketed immediate release (IR)
product (11). Jung et al. also observed similar results
with indomethacin-saccharine (IND-SAC) cocrystal, outcomes of the study revealed that the bioavailability of cocrystal was above pure indomethacine
powder but was found to be equivalent to that of the
* Corresponding author: e-mail: gmdogar356@gmail.com; phone: 00923142082826; fax: 0092992383441
1023
1024
MAJEED ULLAH et al.
IND immediate release commercial formulation IndomeeÆ (12). Similarly, in our recent study on
bioavailability of CBZ powder, carbamazepine-succinic (CBZ-SUC) cocrystal powder filled in ë0í
sized capsule shells, the bioavailability of CBZSUC cocrystal powder was almost double to that of
CBZ powder and was equivalent to marketed product EpitolÆ tablets 200 mg in four healthy rabbits n
= 4 (12).
The widespread approach for in vivo evaluation of cocrystals is centred on deliberately excluding additional formulations so as to compare the
ìneatî aqueous cocrystal suspension or filling
ëëneatíí cocrystal into capsule shells of suitable
sizes. But, studies on cocrystals conducted with the
intention of using this solid in a proper drug product
are odd. This work aims to use this solid form in
tablet dosage form and compare its performance
with marketed formulation of CBZ in order to facilitate and ease the use of this solid form as a podium
in dosage form design. Tablet formulation development is usually time and resource intensive. We
were encouraged by recent study on mefloquine
(MFL) where significant improvement in the dissolution rate was observed in cocrystals tablets over
pure MFL tablets (13). In this work, we used intrinsic dissolution rate (IDR) as a material-sparing tool
to guide appropriate polymer for an efficient tablet
formulation development of an inherently unstable
cocrystal, and compared its in vivo performance to
the marketed immediate release tablet formulation
of carbamazepine i.e., EpitolÆ tablets 200 mg.
EXPERIMENTAL
Materials
Kollidon VAÆ 64 (Lot No. 46581856Po) was
purchased from BASF SE, Germany. Carbamazepine (Lot No. SLBB3655V) was received from
Sigma Aldrich, USA. Croscarmellose sodium (Lot
TN08819630) and Avicel PH-102Æ (Lot No.
XN06817380) were obtained from FMC Biopolymer, USA. Lactose monohydrate, spray dried (Lot
No. 8508091061) was purchased from Foremost
Farms, USA. Magnesium stearate (Lot No. J03970)
was procured form Mallinkrodt, USA.
Formulations
Slurry crystallization and liquid assisted grinding methods were used for cocrystal synthesis,
phase purity of cocrystal were confirmed by solid
state characterization techniques powder x-ray crystallography (PXRD), Raman spectroscopy, FTIR,
and thermogravimetric analysis (TGA). Final tablet
formulation contained 5 : 1 cocrystal to polymer
(KollidonÆ VA/64) ratio and was finalized based on
IDR and in vitro studies of prototype formulations in
comparison to EpitolÆ tablets 200 mg in physiologically relevant 900 mL of modified simulated intestinal fluid (SIF containing 0.2% sodium lauryl sulfate) (12).
In vivo evaluation
Ethical approval
For in vivo studies, the protocols used were
approved by Research Ethical Committee (Ref. No.
PHM-0023/E.C/M-4), Department of Pharmacy,
COMSATS Institute of Information and Technology, Abbottabad. These studies were conducted in
agreement with Helsinki declaration and Animal
Scientific Procedure Act (1986 UK).
Design of the study
This study was an open-label, single-dose, randomized, 2-period, 2-sequence, crossover design
under fasting conditions.
Subjects and treatments
Albino rabbits (n = 4 for each treatment) aged
between 1.5 to 2.5 years, having body mass indices
1.8-2 kg, were isolated for two weeks before initiation of the experiment. During the experimental
period, the animals were maintained on fresh green
fodder thrice a day and water was provided ad libitum. All animals were kept under the same experimental conditions with natural day and night cycle.
After an overnight fasting period, these subjects
were given single oral doses of the 2 treatments: F1
and F0 (eq. to 200 mg of CBZ). The treatments were
taken apart by one-week washout periods.
Blood samples were collected in heparinized
test tubes by jugular vein puncture prior to and after
administration of the drug at predetermined time
point i.e., 0, 1, 2, 3, 4, 6, 8, 10, 12, 24, 48 and 72 h.
The collected samples were centrifuged at 3500 rpm
for 5 min, 200 µL of serum was collected in
Eppendorf tube and stored at -20OC for further studies. The extraction of drug was carried out with acetonitrile (1 : 1), vortexed for 90 s and centrifuged at
12000 rpm for 10 min and were analyzed by HPLC
(14).
Analysis of drug in plasma samples
For chromatographic separation, Phenomenex
LunaÆ 5 µm (particle size) C18 having pore size 100
Å, LC column (250 ◊ 4.6 mm) was used.
Separations of drug in plasma samples were carried
out by slight adjustment of already reported method
Relative bioavailability study of succinic acid cocrystal tablet and...
(11). Mobile phase used was methanol : water (50 :
50, v/v). The temperature of the column oven was
maintained at 50OC and flow rate was kept at 1
mL/min. For HPLC analysis, 25 µL of aliquot was
injected into the column. Drug concentrations were
detected at 285 nm. Under the prescribed chromatographic conditions CBZ eluted at 12.5 min (15).
1025
Statistical analyses
Non-compartment model was utilized for
analysis of serum drug concentration versus time
data. KineticaÆ was used for the calculation of various pharmacokinetic parameters, i.e., AUC, Cmax, tmax
and Ke. Normal log of AUC and Cmax was taken for
determination of relative bioavailability. Moreover,
Figure 1. In vivo performance of marketed product F0 and experimental formulation F1
Figure 2. AUC0-72 of commercial product F0 and experimental formulation F1 in rabbits, n = 4. Bioavailability of F0 and F1 are significantly different at equal doses of 200 mg/animal. All values are expressed as the mean ± SEM, applying paired ëtí test (two tailed) ***p ≤ 0.05
1026
MAJEED ULLAH et al.
significance of difference was demonstrated by
using SPSS version 19.0. Paired ëtí test with p <
0.05 was applied.
All solid state characterization techniques
(PXRD) and thermal technique (TGA) authenticated
the formation of carbamazepine-succinic acid CBZSUC cocrystal in accordance with all reported reference data. The solubility of this soluble cocrystal
has been reported to be 4.5 times higher than carbamazepine dihydrate (CBZDH) at 25OC at pH 3 (1518). Recently, the solubility behavior of CBZ-SUC
cocrystal in four different buffers at 37OC i.e., simulated intestinal fluid (SIF), simulated gastric fluid
(SGF), phosphate buffer pH 1.2 and pH 6.8 phosphate buffer for 24 h have been examined, and CBZSUC cocrystal demonstrated higher aqueous solubility in all the four buffers studied than the stable
CBZ dihydrate form (13). Solid state analysis of
solid residue confirmed CBZ dihydrate formation in
three buffers while the cocrystal maintained its
integrity in SGF only and gave maximum concentration of CBZ than all other buffers. Similarly, in
our recent study on solution stability of CBZ-SUC
cocrystal with and without added polymers, the
cocrystal converted immediately to the stable carbamazepine dihydrate form immediately as confirmed
by in situ Raman spectroscopy and off line PXRD
and FTIR techniques (12). Childs et al., have also
reported similar results (1). Regardless of aforesaid
solubility advantages presented by CBZ-SUC
cocrystal, no study has been reported in literature
that describes concrete formulation approach, that
thermodynamically stabilizes the cocrystal to translate in vitro higher aqueous solubility of this cocrystal system, when used in a suitable medical application. Therefore, we opted for this soluble cocrystal
system, and devised suitable tablet formulation with
proper crystallization inhibition/solubility enhancing polymer KollidonÆ VA/64 (5 : 1 cocrystal : polymer ratio with added excipients) and studied its
bioequivalence study to carbamazepine tablets in
rabbits.
Plasma concentration-time curves of EpitolÆ
tablets and experimental formulation F1 after oral
administration at equal doses of 200 mg of CBZ/rabbit (250 mg of CBZ-SUC is equivalent to 200 mg of
CBZ) are demonstrated in Figure 1, and pharmacokinetic (PK) parameters are shown in Table 1.
Comparison of PK parameters of F1 with F0 tablets
demonstrated that values of AUC0-72 and Cmax of F1
were higher than marketed formulation F0. AUC0-72
of F0 and F1 were found to be 58 ± 2 µg.h/mL and
64.18 ± 1.9 µg.h/mL, while Cmax values were in the
order of 4.9 ± 0.35 µg/mL and 5.3 ± 0.3 µg/mL,
respectively. Similarly, Tmax of F1 reduced from 5.3 ±
0.31 of F0 tablets to 3.9 ± 0.43. By applying paired
ëtí test (two tailed), the means of the four pairs of
bioavailability of F1 and F2 were found to be significantly different statistically (***p < 0.05) as shown
in Figure 2. This increase in AUC0-72 of F1 formulation is due to the stability of cocrystal caused by
polymer in the formulation due to hydrogen bonding
between the polymer and cocrystal (data not shown
here), and as cocrystal is more soluble than the
CBZDH, thus, resulted in statistically significant
improvement in bioavailability. We attribute the
improved drug release from the experimental tablets
formulation F1 to the higher dissolution rate, which
leads to improved in vivo performance of CBZ than
F0. These findings were promising since cocrystal
given as pure powder filled in capsule shells did not
yield the required results and formulation with crystallization inhibitor polymer improved in vivo performance of cocrystal. More studies are needed to
explore the functions of excipients on phase transition of cocrystals in order to maximize the benefits
offered by cocrystals. The relative bioavailability
calculated for F1 with the marketed product F0 was
found to be 106.9%. Thus, the main end point was
bioequivalence, as bioequivalence is believed to be
established if 90% confidence intervals (CIs) for
AUC and Cmax (ln-transformed ratios) fall within the
80-125% range (17-20).
Table 1 Pharmacokinetic parameters of F0 and F1 in albino rabbits, obtained after single oral administration of
single dose (200 mg of CBZ/animal, n = 4).
Formulation
AUC0-72 (µg.h/mL)
Cmax (µg/mL)
Tmax (h)
Ke (/h)
F0 (tablet)
58 ± 2.00
4.9 ± 0.35
5.3 ± 0.31
0.04
F1 (tablet)
64.18 ± 1.9
5.3 ± 0.3
3.9 ± 0.43
0.05
AUC0ñ72 = area under the plasma concentration vs. time curve from time 0 extrapolated to 72 h; Cmax = maximum plasma concentration; Ke = elimination rate constant; Tmax = time to reach Cmax
Relative bioavailability study of succinic acid cocrystal tablet and...
CONCLUSION
There is a dire need for using cocrystals as an
alternative solid form in pre-formulation studies;
however, the selection of a cocrystal strategy at the
pre-clinical formulation stage over other available
formulation techniques for improving bioavailability has been relatively rare. Cocrystal tablet formulation F1 was bioequivalent to the marketed immediate
formulation F0 under fasting conditions. Formulation of soluble cocrystal with crystallization
inhibitor polymer improved in vivo performance of
cocrystal. These findings are encouraging, to use
more soluble cocrystal of otherwise unstable cocrystal system in tablet formulations without phase transition to stable polymorph or parent drug by judicious selection of appropriate polymers.
Acknowledgments
M.U. thanks Higher Education Commission,
Pakistan, for Indigenous PhD scholarship as well as
for supporting the visit to University of Minnesota
under International Research Support Initiative
Programme for six months. Dr. Changquan Calvin
Sun is thanked for accepting M.U. in his lab and
useful discussion throughout this project.
REFERENCES
1. Childs S.L., RodrÌguez-Hornedo N., Reddy
L.S., Jayasankar A., Maheshwari C.:
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Bull. 31, 875 (2006).
1027
6. Jung M.S., Kim J.S., Kim M.S., Alhalaweh A.,
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(2006).
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Clarkeís isolation and identification of drugs.
Vol. 2 pp. 936-938, The Pharmaceutical Press,
London 1986.
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Riccio A.: Epilepsia 16, 759 (1975).
12. Mowafy H.A., Alanazi F.K., El Maghraby
G.M.: Saudi Pharm. J. 20, 29 (2012).
13. Qiao N.: Investigation of CarbamazepineNicotinamide cocrystal solubility and dissolution by a UV imaging system. Ph.D. thesis,
URL: http://hdl.handle.net/2086/10201 (2014).
14. Rawlins M., Collste P., Bertilsson L., Palmer
L.: Eur. J. Clin. Pharmacol. 8, 91 (1975).
15. Riad L.E., Chan K.K., Wagner W.E., Sawchuk
R.J.: J. Pharm. Sci. 75, 897 (1986).
16. RodrÌguez-Hornedo N., Murphy D.: J. Pharm.
Sci. 93, 449 (2004).
17. Shete A., Yadav A., Murthy M.: Drug Dev. Ind.
Pharm. 39, 716 (2013).
18. US Department of Health and Human Services.
Guidance for industry: statistical approaches to
establishing bioequivalence. Available at:
http://www.fda.gov/downloads /Drugs/Guidance-ComplianceRegulatoryInformation/ Guidances/ucm070244.pdf. Published: January 2001.
Accessed: April 28 (2010).
19. Department of Health and Human Services.
Guidance for industry: bioavailability and bioequivalence studies for orally administered drug
products-general considerations. Available at:
http://www.fda.gov/downloads/Drugs/Guidanc
e/ucm070124 .pdf. Published: March 2003.
Accessed: April 28 (2010).
20. Xu C., Zou M., Liu Y., Ren J., Tian Y.: Arch.
Pharm. Res. 34, 1973 (2011).
Received: 20. 06. 2015
ISSN 0001-6837
EFFECT OF SIMULTANEOUSLY SILICIFIED MICROCRYSTALLINE
CELLULOSE AND PREGELATINIZED STARCH ON THE THEOPHYLLINE
TABLETS STABILITY
EDYTA MAZUREK-W•DO£KOWSKA1, KATARZYNA WINNICKA1,
URSZULA CZYØEWSKA2 and WOJCIECH MILTYK2
1
Department of Pharmaceutical Technology, Medical University of Bia≥ystok,
Mickiewicza 2c, 15-222 Bia≥ystok, Poland
2
Department of Pharmaceutical Analysis, Medical University of Bia≥ystok,
Mickiewicza 2d, 15-522 Bia≥ystok, Poland
Abstract: High profitability and simplicity of direct compression, encourages pharmaceutical industry to create universal excipients to improve technology process. ProsolvÆ SMCC - silicified microcrystalline cellulose
and Starch 1500Æ - pregelatinized starch, are the example of multifunctional excipients. The aim of the present
study was to evaluate the stability of theophylline (API) in the mixtures with excipients with various physicochemical properties (ProsolvÆ SMCC 90, ProsolvÆ SMCC HD 90, ProsolvÆ SMCC 50Æ, Starch 1500Æ and magnesium stearate). The study presents results of thermal analysis of the mixtures with theophylline before and
after 6 months storage of the tablets at various temperatures and relative humidity conditions (25 ± 2oC /40 ±
5% RH, 40 ± 2oC /75 ± 5% RH). It was shown that high concentration of Starch 1500Æ (49%) affects the stability of the theophylline tablets with ProsolvÆ SMCC. ProsolvÆ SMCC had no effect on API stability as confirmed by the differential scanning calorimetry (DSC). Changes in peak placements were observed just after
tabletting process, which might indicate that compression accelerated the incompatibilities between theophylline and Starch 1500Æ. TGA analysis showed loss in tablets mass equal to water content in starch. GC-MS
study established no chemical decomposition of theophylline. We demonstrated that high content of Starch
1500Æ (49%) in the tablet mass, affects stability on tablets containing theophylline and ProsolvÆ SMCC.
Keywords: theophylline, ProsolvÆ SMCC, Starch 1500Æ, DSC, drug-excipient compatibility studies, tablets
Direct compression is the preferred method for
the preparation of tablets because it is more economic and ease of manufacture process.
Unfortunately, only less than 20% of active components can be compressed directly into tablets (1).
Most API requires the addition of suitable excipients, which improve physico-chemical properties
and enable compression of the tablet mass (2).
Directly compressible adjuvants are the speciality
products prepared by chemical or physical modification, spray drying, fluid bed drying or co-crystallization but one of the most widely explored and
commercially used method is co-processing (3-5).
This technique combines two or more excipients
without altering the chemical structure of the final
product. Although the co-processed excipients
maintain their independent chemical characteristics,
synergistically they obtain superior properties compared to the simple mixture of components (6-8).
ProsolvÆ SMCC - silicified microcrystalline
cellulose (SMCC) - is multifunctional, co-processed
excipient consisting of 98% microcrystalline cellulose (MCC) and 2% colloidal silicone dioxide
(CSD), combined in a patented co-processed intimate mixture (9). ProsolvÆ SMCC shows a five-fold
bigger surface than microcrystalline cellulose,
which provides better compressibility and ensures
better flow properties than regular MCC or than traditional physical mixture of MCC with colloidal silicone dioxide. Moreover, in direct compression,
SMCC is 10-40% more compactable than regular
MCC (10-13). Starch 1500Æ (partially pregelatinized
maize starch) is a multifunctional excipient that is
used in oral solid dosage forms to improve disintegrant properties, enhance flow and lubricity (14).
In the solid dosage form API is in direct contact with the other excipients used. It might affect
the potential physical and chemical interactions, so
* Corresponding author: e-mail: edyta.wadolkowska@umb.edu.pl
1029
1030
EDYTA MAZUREK-W•DO£KOWSKA et al.
an important part of preformulation is assessment of
possible incompatibilities between the drug and
excipients used. One of the employed techniques in
drug-excipient compatibility screening is differential scanning calorimetry (DSC) a thermoanalytical
method used to determine the differences in the heat
flow generated or absorbed by the sample (15).
The aim of this study was to examine stability
of theophylline (API) and other simultaneously used
excipients with various physico-chemical properties
(silicified microcrystalline cellulose: ProsolvÆ
SMCC 90, ProsolvÆ SMCC HD 90, ProsolvÆ SMCC
50Æ; pregelatinized starch ñ Starch 1500Æ and magnesium stearate). Theophylline is a challenge to formulators because it could inconvert between crystalline anhydrate and monohydrate forms as a function of relative humidity (RH) (16-19).
The study presents results of thermal analysis of
mixtures of these substances with theophylline,
before and after 6 months storage of tablets at various
temperatures and humidity conditions (25 ± 2OC /60 ±
5% RH, 40 ± 2OC /75 ± 5% RH). For the identification of possible changes of API chemical structure,
gas chromatograph mass spectrometry (GC-MS) with
electron impact ionization (EI) was employed to
determine the fragmentation pattern of API.
Thermogravimetric analysis (TGA) was used to characterize moisture content of the materials. Tablets
were also evaluated for thickness, crushing strength,
drug content uniformity and dissolution profile.
EXPERIMENTAL
Materials
Theophylline, pyridine and the silylation
reagent N,O-bis(trimethylsilyl)trifluoroacetamide
(BSTFA) with 1% trimethylchlorosilane (TMSC)
were obtained from Sigma-Aldrich (Steinheim,
Germany). ProsolvÆ SMCC 50, ProsolvÆ SMCC 90,
ProsolvÆ SMCC HD 90 were a gift from JRS Pharma
(Rosenberg, Germany), Starch 1500Æ was received
from Colorcon (Indianapolis, IN, USA). Magnesium
stearate, methanol and chloroform (GC grade) were
purchased from POCH (Gliwice, Poland). All chemicals and reagents were of analytical grade.
Methods
Preparation of tablets
Tablets were prepared by direct compression
method according to the formulae given in Table 1.
Nine formulations of tablets with theophylline (100
mg), containing various types and various percent
ratio (10, 50 and 59% by weight of the tablet) of silicified microcrystalline cellulose (ProsolvÆ SMCC
90, ProsolvÆ SMCC HD 90, ProsolvÆ SMCC 50),
pregelatinized starch (Starch 1500Æ) and the same
ratio of magnesium stearate were prepared. All
ingredients except magnesium stearate were blended in a plastic bag by hand for 10 min. Magnesium
stearate was added and blended for an additional 2
min. The mixed powder was compressed into tablets
using an 8-mm diameter punch in the single punch
tabletting press (Erweka EP1, Heusenstamm,
Germany).
Evaluation of tablets
Physical properties of tablets
Physical characteristics of the tablets were
evaluated according to European Pharmacopoeia 8.0
(EP) (20). All tablet formulations were tested for
weight variation (n = 20), hardness (n = 10) and friability (n = 10). Hardness was applying using the
Schleuniger tablet hardness tester (Dr. Schleuniger
Pharmatron Model 5Y, Thun, Switzerland). The friability test was determined by using Electrolab friabilator (EFñ1W Electrolab, Mumbai, India).
Drug content determination
Content of theophylline in tablets was carried
out by measurement of the absorbance of the sample at 272 nm using a spectrophotometer (Hitachi
U-1800, Tokyo, Japan) The amount of theo-
Table 1. Composition of manufactured tablet formulations.
Ingredient
Formulation
(mg/tablet)
F1
F2
F3
F4
F5
F6
F7
F8
F9
Theophylline
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
ProsolvÆ SMCC 90
147.5
125.0
25.0
147.5
125.0
25.0
147.5
125.0
25.0
22.5
122.5
22.5
122.5
2.5
2.5
2.5
2.5
ProsolvÆ SMCC HD 90
Æ
Prosolv SMCC 50
Starch 1500Æ
Magnesium stearate
2.5
22.5
122.5
2.5
2.5
2.5
2.5
Effect of simultaneously silicified microcrystalline cellulose and...
phylline was calculated with a calibration curve
with an analytically validated method (R2 = 0.9989,
repeatability coefficient of variation (CV) =
1.112%).
In vitro dissolution studies
To evaluate release profile of theophylline
from prepared tablets, the test was performed using
USP type II dissolution apparatus (Erweka DT600,
Heusenstamm, Germany) under the following conditions: 900 mL of distilled water at 37 ± 0.5OC and
50 rpm. The absorbance of the solutions was measured at 272 nm (USP) (21).
Stability studies
Tablets were placed into Petri dishes and
exposed inside humidity chambers (Binder,
Tuttlingen, UK) at various temperatures and relative
humidity (RH) (25 ± 2OC /60 ± 5% RH, 40 ± 2OC /75
± 5% RH) for a period of 6 months. After this time
physical properties, in vitro dissolution and compatibility studies of tablets were tested.
Compatibility studies
Differential scanning calorimetry (DSC)
DSC measurements were performed by using
an automatic thermal analyzer system (TA Q 2000,
New Castle, DE, USA). All precisely weighed samples (approximately 4 mg) were placed in sealed
aluminium crucibles. Temperature calibrations were
performed using indium and zinc as standards. An
empty sealed pan was used as a reference. The entire
samples were run at a scanning rate of 10OC/min.
from 50 to 300OC in nitrogen atmosphere (20
mL/min.). The temperatures range 200-300OC is
presented.
Sample preparation for GC-MS analysis
The powdered samples of each formulation
were dissolved in the mixture of chloroform,
methanol (1 : 1, v/v) and sonificated for 5 min.
Then, the resultant solution was filtered using 0.45
µm PTFE membrane. Aliquots (100 µL) of filtrate
were placed into the vials and the solvents were
evaporated to dryness under argon. The derivatization of samples was performed using N,Obis(trimethylsilyl)trifluoroacetamide (BSTFA) with
1% trimethylchlorosilane (TMSC), (99 : 1, v/v)
reagents. The dry residue was dissolved in 50 µL
pyridine and 50 µL of BSTFA was added into the
vial. The reaction mixture was sealed and heated
during 45 min. at 80OC to obtain TMS derivatives
(22). All the experiments were performed in triplicates.
1031
Gas chromatography - mass spectrometry (GCMS)
GC-MS analyses were carried out on an
Agilent Technologies 7890A gas chromatograph
coupled to an Agilent 5970C VL quadrupole mass
spectrometer equipped with an autosampler 7693
(Agilent Technologies, Wilmington, DE, USA). The
mass spectrometer was operated using electron
impact ionization mode (70 eV). Samples were separated on a fused silica capillary column HP-5MS
(30 m ◊ 0.25 mm i.d., 0.25 µm film thickness) from
J&W (Agilent Technologies, Wilmington, DE,
USA). Aliquots of 1 µL were injected in the split (50
: 1) mode. The injector was kept at 300OC, MS
source and quadrupole temperatures were 230 and
150OC, respectively. The following oven temperature program was used with helium as the carrier gas
at a constant flow rate of 1 mL/min.: 2 min. at 70OC,
then increased to 250OC at rate 10OC/min. held for 5
min., next increased to 280OC at rate 10OC/ min.
Oven temperature of 280OC was held for 10 min.
Thermogravimetric analysis (TGA)
In thermogravimetric studies, the thermogravimetric analyser TGA Q50 (TA Instruments, New
Castle, DE, USA) was used. Samples were heated in
an open platinum pan from room temperature to
260OC, under nitrogen purge, at a rate of 10OC/ min.
Quantity variables were expressed as the mean
and standard deviation. All studies were performed
in triplicate. Statistical analysis was performed
using analysis of variance and Tukeyís test conducted by using STATISTICA 10.0 software.
Differences between groups were considered to be
significant at p < 0.05.
Theophylline is a methylxanthine derivative
commonly used to treat asthma. It exists as a crystalline monohydrate and four anhydrous polymorphs
(I, II, III and IV) (23-25). Theophylline anhydrate
(TA) can transform into theophylline monohydrate
(TM) at high relative humidity (16, 26). In low relative humidity, TM form has been shown to lose
water to produce form II, which is the most prevalent
form and has been considered as the only stable form
at room temperature (27, 28). Form I is produced by
heating form II and is reported to be stable at higher
temperatures (24, 29). Form III is a highly metastable
and converts easily to form II during storage (25, 30).
Form IV has been identified recently, it occurs as a
1032
result of slow, solvent-mediated conversion from
form II, and is now claimed as the most thermodynamically stable anhydrous polymorph of theophylline (31). Debnath and Suryanarayanan found
that wet-granulation process induced polymorphic
transformation of theophylline, which can result in
many difficulties during the compaction (32-34).
Therefore, in this study, tablets with theophylline
were obtained by direct compression method. All the
manufactured formulations showed very low weight
Figure 1. DSC thermograms of pure theophylline and theophylline in tablets containing 10, 50, 59% of ProsolvÆ 90, ProsolvÆ HD 90 and
ProsolvÆ 50 and respectively, 49, 9, 0% of Starch 1500Æ just after compression
Figure 2. DSC thermograms of pure theophylline and theophylline in tablets containing 10, 50, 59% of ProsolvÆ 90 (A), ProsolvÆ HD 90
(B) and ProsolvÆ 50 (C) and respectively, 49, 9, 0% of Starch 1500Æ after 6 months storage at 40 ± 2OC/75 ± 5% RH
variation, satisfactory drug content uniformity,
mechanical strength and friability, indicating that
direct compression is appropriate method to prepare
proper quality tablets with theophylline.
The physicochemical stability of tablets is
important for the quality of pharmaceutical products.
Some factors such as heat and moisture accelerate
most drug-excipient reactions and might increase the
API degradation (35-37). In the present study, an
assessment of theophylline melting point was conducted in multicomponent individually performed
mixtures with various percentage ratio of different
types of ProsolvÆ (of various particle size and bulk
density) and Starch 1500Æ with magnesium stearate.
1033
The thermogram of pure theophylline is characterized by the sharp peak at 272OC due to its melting
(270-274OC, EP). It was found that DSC thermograms of tablet mixtures with 59% and 50% content
of ProsolvÆ SMCC 90 (F1, F2), ProsolvÆ SMCC HD
90 (F4, F5) and ProsolvÆ SMCC 50 (F7, F8) after
tabletting process did not show significant changes in
peak placement in comparison to the peak obtained
from pure theophylline - suggesting compatibility of
the compounds. However, changes were observed in
all tablets, with 10% content of ProsolvÆ (F3, F6, F9)
and 49% of Starch 1500Æ (Fig. 1). Similar differences
in peak placement and shape were observed in DSC
thermograms achieved from analysis of the tablets
Figure 3. DSC thermograms of pure theophylline and theophylline in binary mixtures with Starch 1500Æ, ProsolvÆ 90, ProsolvÆ HD 90 and
ProsolvÆ 50 just after tabletting
Figure 4. Thermogravimetric analysis of moisture content in tablets containing 10% of ProsolvÆ SMCC and 49% of Starch 1500Æ, after 6
months storage at 40 ± 2OC/75 ± 5% RH
1034
after 6 month storage at the room temperature (25 ±
2OC /60 ± 5% RH ñ data not shown) and in tablets
after accelerated storage conditions (40 ± 2OC /75 ±
5% RH) (Fig. 2). Tablets prepared with ProsolvÆ
SMCC at concentrations 50% and 59%, resulted in a
proper long term storage conditions.
Pharmaceutical dosage forms are exposed to
water present in an atmosphere (during production or
storage) or excipients possess a high water content
which is able to equilibrate between various components (38). DSC changes in theophylline peak were
observed in all cases of tablets containing 10% con-
tent of ProsolvÆ SMCC - that is, with a high content
of Starch 1500Æ. It is known that excipient may possess a high water content (the equilibrium moisture
content of starch is about 8-10%), which can lead to
a physicochemical change of the tablets and may
affect drug stability (39, 40). Therefore, to investigate whether the variation is related to Prosolv or
Starch content, mixtures of two component (theophylline and ProsolvÆ SMCC 90, theophylline and
ProsolvÆ SMCC HD 90, theophylline and ProsolvÆ
SMCC 50, theophylline and Starch 1500Æ) were prepared. The results of DSC study showed that change
Figure 5. The dissolution profile of theophylline tablets prepared with 10% of ProsolvÆ 90 (A), ProsolvÆ HD 90 (B) and ProsolvÆ 50 (C)
and 49% of Starch 1500Æ before ( ï ) and after 6 month storage ( ■ ) at high temperature and humidity conditions (40 ± 2OC/75 ± 5% RH)
Figure 6. EI-mass spectra of theophyllin
in theophylline peak placement was observed only in
the mixture of the drug and Starch 1500Æ (Fig. 3).
The significant shift of melting temperatures and
broaden peak of theophylline were also related to
changes of the tablets physical and mechanical properties. Formulations containing 10% of all types of
ProsolvÆ and 49% of Starch 1500Æ (F3, F6, F9) after
6 month storage at high temperature and humidity
conditions presented lower hardness (the range from
28 to 36 N) and higher friability (> 1.4%). However,
no significant changes in the tablets weight after storage were shown. It suggests that the tablets do not
absorb moisture from the atmosphere and probably
moisture content in the Starch 1500Æ is able to equilibrate between the tablets component and lead to
drop of physical properties of theophylline (in the
case of F3, F6, F9). Additionally, evaluated by TGA
water amount in tablets was about 5%, which is related to the water content in the starch (based on the
weight of the tablet) (Fig. 4). This phenomenom has
been observed by Otsuka et al. - theophylline anhydrate changed into theophylline monohydrate at
more than 75% RH (27). Sandler et al. demonstrated
that only storage at 99% RH lead to theophylline
transition from TA to TH (38). The results of the
study indicate that water present in Starch 1500Æ content could induce incompatibilities in theophylline
tablets.
Figure 5 showed the dissolution profiles of the
theophylline tablets with 10% of ProsolvÆ SMCC. It
was demonstrated that after storage all tablets
showed the accelerated release of theophylline compared to tablets just after compression but significant changes in the in vitro release profile was
observed in tablets containing 10% ProsolvÆ (F3,
F6, F9). The formulations release the active substances more than two times faster than just after
compression. Because dissolution rate of TA
depends on the degree of hydration and TH is less
soluble than TA, probably theophylline anhydrate
does not pass in monohydrate (16, 26).
In order to exclude the effect of chemical
decomposition on the change of theophylline DSC
peak, prepared tablets were analyzed by GC-MS
method. The electron-impact mass spectra, the fragmentation patterns and molecular ion m/z 295 of
theophylline are shown in Figure 6. The gas chromatogram of silylated extracts from tablets did not
reveal other peaks and linear temperature programmed retention index of theophylline was calculated. GC-MS study revealed that there were no
changes of theophylline chemical structure, what
might indicate that changes observed in the DSC
thermograms were the result of physical reactions.
1035
Changes in peak placements were observed
just after tabletting, which may suggest that compression technology accelerate the incompatibilities
between theophylline and Starch 1500Æ in the
tablets. The type of interaction will be investaigated
in future experiments.
CONCLUSION
In the present study, we demonstrated that high
concentration of Starch 1500Æ (49%) in the tablet
mass affects stability of the tablets containing theophylline and ProsolvÆ SMCC. The determining factor is probably water from starch content.
The results confirmed that differential scanning calorimetry, could be used as a quick screening
test to evaluate the compatibility between theophylline and ProsolvÆ SMCC as well as Starch
1500Æ.
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Received: 3. 08. 2015
ISSN 0001-6837
FORMULATION, EVALUATION AND IN VITRO DISSOLUTION
PERFORMANCE OF ENALAPRIL MALEATE SUSTAINED RELEASE
MATRICES: EFFECT OF POLYMER COMPOSITION AND VISCOSITY GRADE
AAMNA SHAH1, GUL M. KHAN2, HANIF ULLAH3, KAMRAN AHMAD KHAN1,
KALEEM ULLAH3 and SHUJAAT A. KHAN3*
1
Department of Pharmaceutics, Faculty of Pharmacy, Gomal University, Dera Ismail Khan, Pakistan
2
Department of Pharmacy, Quaid-i-Azam University, Islamabad, Pakistan
3
Department of Pharmacy COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan
Abstract: The present study aimed at developing the sustained release matrix tablets of enalapril maleate and
evaluating the effect of polymer concentration and viscosity grade on drug release. The sustained release
enalapril maleate tablets were successfully formulated by direct compression method using nonionic cellulose
ethers HPMC K15, HPMC K100 and HPC polymers either alone or in combination. In-vitro drug release study
was carried out in phosphate buffer (pH 6.8) for a period of 24 h following USP dissolution apparatus II i.e.,
paddle apparatus. Model dependent approaches like zero-order, first order, Higuchiís model and KorsmeyerPeppas model were used to assess drug release from various formulations. All the three polymers alone or in
combination sustained the drug release. The drug release characteristics from HPMC and HPC polymer followed zero order release kinetics except for 45% concentration of all polymers alone or in combination where
by the drug release followed Higuchiís model. In all cases, the drug release mechanism was both diffusion as
well as erosion.
Keywords: enalapril maleate, HPMC, HPC, sustained release, viscosity grades
Different types of sustained release (SR) formulations have been formulated for improving clinical efficacy of active pharmaceutical ingredients
(APIs) and patient compliance. The SR oral dosage
forms have been documented for improvement in
therapeutic efficacy and maintenance of steady state
plasma concentration of drug (1). Nonionic cellulose ethers and hydroxypropylmethyl cellulose
(HPMC) have been extensively considered for the
purpose of formulating oral SR formulations. Such
hydrophilic and water soluble polymers are wellliked due to their broad regulatory acceptance, cost
effectiveness and flexibility in order to get a desired
drug release profile (2). HPMC always finds preference in formulation of hydrophilic matrices due to
cost effectiveness, choice of viscosity grades, nonionic nature, robust mechanism and utilization of
existing conventional equipment and methods (3). It
has been most extensively employed as a gel forming agent in the formulations of controlled release,
semisolid, liquid and solid dosage forms. Variation
in viscosity grades, polymer concentration and the
addition of various excipients to the matrices can
alter the release of drug (4).
HPC is used as film coating agent or tablet
coating agent at a 5% (w/w) concentration.
Similarly, HPC in a concentration of 15-35% (w/w)
is used for the preparation of SR, controlled release
(CR) and extended release (ER) dosage forms.
Additionally HPC in a concentration of 2-6% is used
as tablet binder in both dry and wet granulation
processes used for tablet manufacturing (5).
Enalapril is angiotensin-converting enzyme
(ACE) inhibitor that is administered orally. It undergoes in vivo hydrolysis to form its bioactive metabolite. Biotransformation most likely takes place in the
liver. Biotransformation beyond bio-activation is
not observed in human (6). Enalapril maleate (EM)
is considered as effective candidate for SR dosage
forms due to its gastric absorption, high water solubility and shorter half-life (7). Foods do not affect its
bioavailability. It is primarily excreted through renal
* Corresponding author: e-mail: drshujatalikhan@ciit.net.pk
1037
1038
AAMNA SHAH et al.
route. It reduces blood pressure (BP) by lessening
systemic vascular resistance. The reduction in BP is
not associated with reflex tachycardia. Moreover,
there is slight increase in cardiac output without any
impairment in cardiovascular reflexes (8).
Direct compression (DC) is the most inexpensive, fast, and simple method for the preparation of
SR tablet formulations (9). Moreover, it does not
change the physical nature of the drug. It is most
commonly used in the formulation of crystalline
powders due to good cohesive properties (10). DC
can be effectively used for drugs sensitive to moisture and humidity as this technique do not require
water and any other solvent (9).
Materials
Disodium hydrogen orthophosphate and potassium dihydrogen phosphate were procured from
Guideís Corporation (Pvt.), Ltd., Islamabad.
Sodium hydroxide and hydrochloric acid (37%) was
purchased from Merck, Germany, EM was supplied
as a gift by Warrick Pharmaceuticals, Islamabad.
Lactose and magnesium stearate were procured
from BDH Chemical Ltd., England. PVP K-30,
Talc, AvicelÆ (PH-102 and PH-200) were procured
from Guideís Corporation (Pvt.) Ltd., Islamabad.
Moreover, Methocel K100M Premium, Methocel K
15 and HPC (Dow Chemicals Co. Midland USA)
were supplied as a gift by Allied Pharmaceuticals,
Islamabad. All the chemicals were used as such
without any further purification.
Compositions and preparation of enalapril
maleate matrix tablets
SR matrix tablets of EM were formulated using
Methocel K15, Methocel K100 and HPC in different
drug to polymer ratio i.e., D : P ranging from 4 : 3 to
4 : 10. Moreover, they were also used concomitantly in some formulations and their combined effect
on drug release profile was observed. Lactose MH
and Lactose SD, Avicel 102 and 200, magnesium
stearate, talcum powder and PVP K-30 were used as
excipients. Formulations pattern using HPMC K15,
HPMC K100 and HPC alone or in combination are
shown in Table 1.
Direct compression technique was used for the
preparation of tablets. This method is used for drugs
or substances with crystalline form and having good
cohesive and compressibility properties. For tablet
preparation Methocel K-15, Methocel K-100M premium and HPC were used as polymers. In this
method drug, polymer and all the excipients except
magnesium stearate were thoroughly mixed with
one another and geometrically blended in pestle and
mortar for 5-10 min. The mixture was passed
through sieve number 24 followed by addition of
magnesium stearate and mixed for 2 min. Again, the
mixture was passed through sieve number 24. The
powder mixture was compressed using rotary tableting machine (ZP-19, Lahore, Pakistan). Eight mm
flat punches were used for tablet preparation. The
batch size selected for each formulation was 300
tablets while the compression weight was 200 mg
per tablet. The compositions of all formulations are
shown in Table 1.
Table 1. Compositions of various formulations (F1-F12).
Composition (mg) per tablet (200 mg)
Code
HPC
Methocel
K15
Methocel
K100
Drug :
Polymer
Lactose
Drug
Talc
Magnesium
stearate
PVPK30
F1
30
--
--
4:3
92.68
52.32
10
5
10
F2
60
--
--
4:6
62.68
52.32
10
5
10
F3
90
--
--
4:9
32.68
52.32
10
5
10
F4
--
30
--
4:3
92.68
52.32
10
5
10
F5
--
60
--
4:6
62.68
52.32
10
5
10
F6
--
90
--
4:9
32.68
52.32
10
5
10
F7
--
--
30
4:3
92.68
52.32
10
5
10
F8
--
--
60
4:6
62.68
52.32
10
5
10
F9
--
--
90
4:9
32.68
52.32
10
5
10
F10
10
10
10
4:3
92.68
52.32
10
5
10
F11
20
20
20
4:6
62.68
52.32
10
5
10
F12
30
30
30
4:9
32.68
52.32
10
5
10
1039
Table 2. Mathematical models applied to formulations.
Kinetic model
Zero-order kinetics
W = k1 t
Higuchi kinetics
W = k2 t1/2
First order kinetics
ln (100 - W) = ln 100 ñ k3 t
Korsmeyer-Peppas equation
Mt / M8 = k4 tn
Table 3. Physical properties of formulations.
Weight (mg)
Formulation
Hardness (kg/cm2)
Thickness (mm)
Friability (%)
Average
SD (±)
Average
SD (±)
Average
SD (±)
F1
203.6
2.836
3.0
0.07
0.19
8.1
0.152
F2
203.2
3.553
3.1
0.05
0.17
7.5
0.217
F3
202.3
2.584
3.3
0.07
0.15
6.8
0.295
F4
202.8
2.44
3.3
0.05
0.25
8.9
0.109
F5
203.4
3.627
3.5
0.07
0.19
7.7
0.336
F6
203.2
3.293
3.6
0.07
0.13
6.4
0.179
F7
203.7
3.860
3.4
0.09
0.11
7.1
0.123
F8
202.8
2.440
3.6
0.04
0.08
6.6
0.152
F9
202.8
3.293
3.7
0.07
0.04
5.4
0.144
F10
202.3
2.214
3.5
0.08
0.13
7.6
0.219
F11
204.6
3.718
3.6
0.07
0.10
6.4
0.192
F12
202.8
3.327
3.7
0.08
0.08
5.6
0.261
Physical evaluation of prepared formulations
After tablet preparation, tablets were evaluated
by applying various official tests according to USP.
Dimensional test which was applied on tablets
include thickness, while the QC test include weight
variation, friability and hardness test. For weight
variation test, 20 tablets were taken and weighed
individually using the electronic weighing balance
(AX-120, Shimadzu, Japan). Then, average weight
of all the 20 tablets was calculated. According to
USP, the individual weight of not more than 2
tablets should vary from average weight by not more
than 5%. For hardness test, 10 tablets were selected
randomly and their hardness/average breaking
strength was tested using the hardness tester (PTB3112, Germany). USP acceptable limit of hardness
is 5-10 kg/cm2. For friability test, 10 pre-weighed
tablets were taken and placed in friabilator (FB0606, Curio, Lahore, Pakistan). The friability apparatus was turned on for 100 revolutions at 25 rpm
and the tablets were weighed again. The % friability
was then calculated using the equation:
W1 ñ W2
Friability (%) = ñññññññññ
× 100
(1)
W1
USP limit of friability is less than 0.8% w/w.
For dimensional test, 10 tablets were selected randomly for checking tablet thickness using the digital
Vernier caliper (China). The USP acceptable range
for thickness is 2-4 mm for the tablets having diameter of 4-13 mm.
In vitro drug release studies
In vitro drug release test of controlled release
matrix tablets of EM was performed on prepared
tablets in accordance with the ìDissolution
Procedureî described by USP applying Apparatus 2
and involving use of Paddle Dissolution System.
Pharma test dissolution apparatus (DT/7-13372,
Germany) was used for the study. Phosphate buffer
solution of pH 6.8 was used as dissolution medium.
Volume of the medium was 900 mL, temperature
was maintained at 37 ± 1OC while rotating speed of
paddles was 50 rpm. The test was performed on six
tablets from each batch. The drug release pattern
was evaluated by taking 5 mL samples at specific
time interval of 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 18
and 24 h. The samples collected were filtered
through Whatman filter paper or through filter mem-
1040
AAMNA SHAH et al.
brane having pore size of 0.45 µm and then analyzed
with UV-visible spectrophotometer (7200, Cecil,
England) at λmax of 206 nm. The analysis of dissolution data was performed using various kinetic models i.e., zero order release kinetics, first order release
kinetics, Higuchi and Korsmeyer-Peppas models.
Drug release kinetics
The data obtained from the dissolution studies
were fitted into the mathematical models shown in
Table 2.
In these equations, ëWí represents the percent
drug released at time ëtí compared with total amount
of drug present in tablets. k1, k2 and k3 are rate constants depending upon the models. Mt/M∞ designates
fractional drug release form the matrix tablet into
the dissolution medium. Diffusion exponent ëní
characterizes the release mechanism of drug. The
mechanism of drug release was elucidated by calculating the value of ëní from the dissolution data
obtained from the initial 60% release. If the ëní
value is greater than 0.43 and less than 0.85 then,
release of drug is anomalous or non-Fickian, while
if value of ëní is greater than 0.85 then, in that case
release kinetics is super case-II transport.
RESULTS
Physical evaluation of prepared formulations
The weight variation test was performed for all
formulations (F1-F12) and results were found to be
in the range of 202.3 ± 2.584 mg to 204.6 ± 3.718
mg (Table 3). No tablet deviate from the official
limit, which is 5% for 200-250 mg tablet. Thus, all
formulations were within limits and passed the
weight variation test. The friability tests performed
for the formulations (F1-F12), were in the range of
0.04 to 0.25%, which fall within the limit of standard (less than 0.8%). The thickness was carried out
according to the procedure. The thickness of the
tablets ranges from 3.0 ± 0.07 to 3.8 ± 0.08 mm. The
hardness for all formulations ranged from 5.6 ±
0.261 to 8.1 ± 0.154 kg/cm2, which showed that
hardness was within the limits.
The release profile from SR tablet matrices of
EM using different polymers i.e., HPMC K100,
HPMC K15 and HPC in varying drug to polymer
ratio are shown in Figure 2. As shown in Figure 2,
the release profile for formulation (F1) with 15% of
HPC was extended to about 99% of drug in 12 h.
The drug release was found to be about 91% for
Figure 1. Physical properties of formulations F1-F12: A. Weight (mean ± SD), B. Thickness (mean ± SD), C. Friability (%), D. Hardness
(mean ± SD)
1041
Table 4. Drug release kinetics.
Determination coefficient (R2)
Zero-order
First-order
Higuchi
Korsmeyer-Peppas
Value of n for
Korsmeyer-Peppas
F1
0.905
0.803
0.778
0.966
0.638
F2
0.959
0.702
0.877
0.94
0.838
F3
0.942
0.664
0.982
0.99
0.903
F4
0.951
0.749
0.846
0.988
0.741
F5
0.985
0.541
0.947
0.987
0.833
F6
0.917
0.681
0.982
0.994
0.95
F7
0.959
0.68
0.862
0.966
0.765
F8
0.982
0.724
0.978
0.973
0.862
Formulation
F9
0.91
0.772
0.978
0.973
0.994
F10
0.981
0.566
0.926
0.99
0.843
F11
0.984
0.789
0.984
0.984
0.933
F12
0.96
0.821
0.995
0.941
0.855
Figure 2. Drug release profile of all formulations (F1-F12)
30% (F2) and 72% for 45% of HPC (F3) in 12 h,
respectively.
In case of HPMC K15, the formulation with
15% of HPMC K15 (F4) extended drug release
and about 99% of drug was released in about 12 h.
Therefore, for obtaining further extended release
profile, the concentration of polymer was
increased and proportional decrease in percent
drug release was observed. The formulation with
30% (F5) and 45% of HPMC K15 (F6) released
about 88% of drug and 62% of drug in 12 h,
respectively.
Similarly, the formulation with 15% of HPMC
K100 (F7), the release profile was extended, however, the release extent was higher than the desired
results and about 96% of drug release was observed
in about 12 h. And the formulation with 30% of
HPMC K100 (F8) released about 77% of drug in 12
1042
AAMNA SHAH et al.
h. The formulation with 45% of HPMC K100 (F9)
released 55% of drug in 24 h.
When assessed combination of all the three
polymers in equal ratios, the drug release in 12 h,
was 92, 72 and 52% for 15, 30 and 45% of polymers
blend, respectively. Moreover, the polymer blend
retarded the drug release more efficiently than that
of polymer used alone in the same corresponding
ratios (15, 30 and 45%).
The release rate of drug was reduced significantly when HPMC K100, HPMCK 15 and HPC
were used in combination.
Table 3 represents the results of in vitro drug
release kinetic models applied to the sustained
release matrix tablets of EM using HPMC K15,
HPMC K100, HPC and all these three polymers in
combination. Drug release from HPC, HPMC K15
and HPMC alone as well as in combination followed
zero order at concentration of 15% and 30%, whereas at concentration of 45% all three polymers alone
or in combination followed Higuchiís model. The
value of ëní in Korsmeyer-Peppas equation was
observed to be greater than 0.45 for all the prepared
formulation which indicates that all the formulations
were exhibiting non-Fickian or anomalous drug diffusion. The drug release mechanisms followed by all
formulations was found to be the combination of two
mechanisms i.e., diffusion and erosion (Table 4).
DISCUSSION
In order to mimic the physiological conditions
of lower GIT, all the formulations were subjected to
drug release studies in phosphate buffer (pH = 6.8)
for 24 h. HPMC K100 showed more efficient drug
release retardant property than HPMC K15 due to
relatively higher viscosity, good gelling characteristics and strong linking capacity of HPMC K100.
Furthermore, the slow release profile observed with
combination of polymers may be due to the high
concentration of polymers, high viscosity, strong
cross linking and high molecular weight achieved.
The swelling of matrix tablets were detected
during the dissolution process. HPMC is well
known for swelling controlled release mechanism
and hydrophilic drug are released from this polymer
by diffusion process (2). The hydration of polymer
is the reason for swelling of tablets. Glass transition
temperature of the polymer is reduced as compared
to the temperature of the dissolution medium due to
swelling of the tablets. The dissolution solvent
exerts stress on the polymeric chains due to which
there occurs relaxation effect within polymeric
chains. This relaxation effect, in turn, increases distance among the polymeric chains. In hydrated
polymer the molecular volume of polymer is
increased that reduces the free volume due to the
presence of microspores, which is observed clearly
as shift in the drug release mechanism. Other
researchers also observed similar type of results in
their studies (11).
In all cases, an increase in polymer concentration retarded the drug release from the matrices.
This might be due to the reasons that an increase in
the concentration of polymer and drug to polymer
ratio lags the release of drug while reducing the
amount of polymer and D : P ratio enhance the drug
release from the polymeric matrices (11).
Moreover, an increase in concentration and/or
viscosity grade of polymer may lead to an increase
in gel viscosity upon absorption of water, which acts
as a barrier to diffusion that, in turn, may lead to a
decrease of the diffusion coefficient. Thus, the drug
release may be retarded. The gel barrier also provides hindrance to the penetration of water thus preventing the wetting of tablet core. Consequently, the
tablet disintegration is hindered, which further sustains the drug release (12).
CONCLUSION
The SR tablet matrices of EM were successfully formulated using HPC and different viscosity
grades of HPMC. As evident from in vitro drug
release studies, the drug release was retarded by
increasing viscosity grade or quantity of polymer as
well as changing the type of polymer. The findings
of this study obviously documented that matrix
tablets of HPC and HPMC are a promising and effective drug delivery tool for once daily administration
of enalapril maleate. The analysis of the release profiles in the light of distinct kinetic models led to the
conclusion that the drug release characteristics from
HPMC polymer matrices follows zero order kinetics
except for 45% of all polymers alone or in combination where the drug release was following Higuchiís
model. In all cases, the mechanism of drug release
was both diffusion as well as erosion.
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Received: 18. 08. 2015
ISSN 0001-6837
PREPARATION AND EVALUATION OF pH RESPONSIVE
POLY(2-HYDROXYETHYL METHACRYLATE-CO-ITACONIC ACID)
MICROGELS FOR CONTROLLED DRUG DELIVERY
1
1
ZERMINA RASHID *, NAZAR MUHAMMAD RANJHA ,
1
1
HINA RAZA , RABIA RAZZAQ and ASIF MEHMOOD2
1
Faculty of Pharmacy, Bahauddin Zakariya University, Multan, Pakistan
2
Faculty of Pharmacy & Alternative Medicine, Bahawalpur, Pakistan
Abstract: In this study, a series of pH sensitive microgels (MGs) were prepared by modified free radical suspension polymerization of 2-hydroxyethyl methacrylate (HEMA) and itaconic acid (IA), using ethylene glycol
dimethacrylate (EGDMA) as crosslinker. Equilibrium swelling technique was employed for esomeprazole magnesium trihydrate (EMT) loading. Prepared microgels were characterized through Fourier transforms infrared
spectroscopy (FTIR), thermogravimetric analysis (TGA), dynamic light scattering technique (DLS), scanning
electron microscopy (SEM), equilibrium swelling and in vitro drug release kinetics. FTIR and TGA confirmed
the formation of copolymeric p(HEMA-co-IA) network. SEM and DLS revealed smooth, round and uniformly
distributed microspheres with particle size up to 10 µm. Developed microgels found to be pH responsive in
nature. All the formulations (HID1 ñ HID5) followed Higuchi model with non-Fickian diffusion mechanism of
drug release. It was concluded that p(HEMA-co-IA) microgels have potential to be used as drug carriers for site
specific and controlled drug delivery.
Keywords: esomeprazole magnesium trihydrate, microgels, itaconic acid, pH responsive
An ideal drug delivery system (DDS) delivers
drug to the site of action, at a controlled rate according to the needs of the body and over the treatment
period. To make it, in practice various controlled
and targeted drug delivery systems are introduced
(1, 2). Intelligent biomaterials have been widely
employed in pharmaceutical applications for the
design of such ideal drug carriers. Among available
biomaterials, hydrogels have proved their value
because of their good mechanical properties, threedimensional physical structure and tuneable chemical structure. In addition, high water content and low
surface tension contribute to their biocompatibility
to natural tissues (3). In recent years, with the
advancement of technology, an interest in micro and
nano sized hydrogels has been increased (4, 5).
Microgels (micro-sized hydrogels), as drug
delivery carriers are often regarded as good alternatives to other DDSs like liposomes, polymer drug
conjugates and micelles because these DDSs have
limitations in their composition materials and lack
controlled load ability of drug (6). Porous network
of MGs serves as an ideal reservoir for drug loading
and protects the drug from degradation and environmental hazards. In addition, MGs exhibit exceptional properties like colloidal stability, large surface
area, facile synthesis, good control overparticle size
and ease in incorporation of stimuli responsive
behavior (7, 8).
Stimuli-responsive MGs are smart drug delivery carriers, have capability to incorporate and
release their host molecules in response to stimuli
(pH, ionic strength and temperature), for targeted
drug delivery (9). MGs belong to group of swelling
controlled DDS and the swelling depends on pH of
respective medium (10). Due to the large variations
in physiological pH values, as well as the pH variations in pathological conditions, pH-responsive
polymeric networks have been extensively studied
(11). Proposed polymer with ionizable ñCOOH
group shows a pH sensitive swelling nature in basic
pH (12).
HEMA is biocompatible, inert to biological
processes, has water content similar to living tissue
and is resistant to degradation and absorption by
body. PHEMA has been studied in tissue engineer-
* Corresponding author: e-mail: zermina_malik@yahoo.com
1045
1046
ZERMINA RASHID et al.
ing (13) and is a backbone for stimuli responsive
hydrogels (14, 15). It is practically insensitive to pH
(16). Itaconic acid (IA), on the other hand, provides
polymeric chains with carboxylic side groups and
can be copolymerized easily. It has two carboxylic
acid (ñCOOH) groups with pKa1 = 3.85 and pKa2 =
5.45, thus renders good pH sensitivity and
hydrophilicity. Furthermore, IA is obtained from
natural source and therefore has good biocompatibility (17, 18).
Esomeprazole magnesium trihydrate (EMT) is
an acid-labile proton pump inhibitor used in the
treatment of peptic ulcer. It has a bioavailability of
48% when administered orally (19, 20). In the literature, delayed release tablets, sustained release
matrix tablets and enteric coated multiparticulate
DDS have been discussed (21, 22).
In the present study, an attempt was made to
synthesize and evaluate pH responsive copolymeric
microgels, by chemically crosslinking hydrophobic
monomer, HEMA and hydrophilic monomer, IA in
the presence of EGDMA by free-radical suspension
polymerization technique. EMT was successfully
loaded as model drug to investigate the drug release
kinetics of p(HEMA-co-IA) microgels. Previously,
(HEMA-co-IA) hydrogels have been prepared and
evaluated (14), to the best of authors knowledge no
synthesis and investigation of micro-sized
p(HEMA-co-IA) hydrogels have been reported in
the literature.
Materials
EMT was received as generous gift from
Unison Chemicals Works, Lahore, Pakistan. 2hydroxyethyl methacrylate (HEMA) (Aldrich) and
itaconic acid (IA) (Aldrich) were used as monomers,
benzoyl peroxide (BPO) (Merck) was used as an ini-
tiator, and ethylene glycol dimethacrylate
(EGDMA) (Aldrich) was used as crosslinking agent.
Polyvinyl alcohol (PVA) (Aldrich) was used as suspending agent. All the chemicals and reagents were
of analytical grade.
Synthesis of crosslinked p(HEMA-co-IA) microgel
p(HEMA-co-IA) MGs were produced by suspension polymerization method as reported earlier
(23), with slight modifications. Double deionized
water was used as dispersion medium. Mixture of
different combinations of HEMA, IA, EGDMA and
BPO dispersed in toluene was added into 500 mL
conical flask containing 0.5% PVA solution. This
reaction mixture was purged with nitrogen gas,
stirred well at 700 rpm, heated to 70OC for 2 h and
then at 90OC for 1 h. At the end of the reaction
crosslinked copolymeric particles were separated
and washed with water and ethanol. Obtained microgels were vaccum dried and stored in airtight glass
vials for further studies. Figure 1 shows the possible
structure of synthesized p(HEMA-co-IA) microgel.
A list of different formulations is given in Table 1.
Percent yield
Yield of the copolymeric microgels was determined gravimetrically. Washed microgels were
dried at 40OC till constant weight was achieved.
Percent yield was calculated as:
W
Percent yield = (ññññññ) × 100
(1)
M
where, W is weight of the obtained dry copolymeric
microgels and M is the weight of the monomeric
load.
Preparation of buffer solution
USP phosphate buffer solutions of pH 1.2, 4,
5.5 and 7.5 were prepared with potassium dihydro-
Table 1. Results of percent yield, mean size and percent drug loading of different formulations MGs.
Formulation
code
HEMA : IA
(mol %)
EGDMA
(%)
Percent yield
± SD (%)
Percent loading
± SD (%)
Mean size
(µm) ± SD
HID1
90 : 10
5
68 ± 1.69
48.34 ± 2.45
6±2
HID2
80 : 20
5
72 ± 1.24
60.93 ± 2.12
3±4
HID3
70 : 30
5
78 ± 2.44
65.42 ± 1.33
5±3
HID4
60 : 40
5
65 ± 3.26
56.07 ± 3.65
2±3
HID5
50 : 50
5
62 ± 2.44
55.63 ± 1.73
4±1
HID6
70 : 30
8
70 ± 1.24
50.98 ± 2.175
8±4
HID7
70 : 30
10
66 ± 2.49
54.31 ± 3.37
10 ± 5
1047
Preparation and evaluation of pH responsive...
Figure 1. Possible structure of synthesized p(HEMA-co-IA) microgels
Table 2. Effect of IA and pH on drug release kinetics.
Formulation
HID1
HID2
HID3
HID4
HID5
pH
Zero-order kinetics
-1
First-order kinetics
-1
Higuchi equation
R
K0 (h )
r
K1 (h )
r
K2 (h-1)
1.2
0.5488
0.8451
0.0058
0.851
0.026
0.965
6.5
4.057
0.759
0.074
0.841
0.203
0.927
7.5
4.425
0.716
0.101
0.834
0.225
0.901
1.2
0.6416
0.905
0.006
0.913
0.030
0.988
6.5
5.783
0.851
0.080
0.799
0.208
0.897
7.5
6.357
0.819
0.111
0.810
0.229
0.877
1.2
0.863
0.977
0.0092
0.979
0.034
0.982
6.5
6.417
0.851
0.123
0.927
0.251
0.946
7.5
6.479
0.815
0.171
0.944
0.256
0.925
1.2
0.715
0.915
0.0077
0.923
0.033
0.99
6.5
6.257
0.825
0.102
0.805
0.226
0.880
7.5
6.29
0.785
0.150
0.834
0.228
0.854
1.2
0.8099
0.935
0.008
0.943
0.037
0.988
6.5
6.054
0.811
0.106
0.807
0.219
0.874
7.5
6.231
0.777
0.176
0.863
0.226
0.849
1048
gen phosphate and 0.2 M HCl or NaOH solutions
were used to adjust the pH of these solutions. NaCl
was used to keep the ionic strength of all buffer
solutions constant.
Equilibrium swelling behavior
Swelling of microgels was studied by gravimetric method. The weighed amount of the dry
microspheres (W0) were immersed in USP phosphate buffer solutions of various pH (1.2, 4, 5.5
and 7.4) and mixed at 50 rpm. Swelling process of
the microgels was monitored continuously till
equilibrium swelling condition were achieved,
i.e., no weight gain of swollen microgels was
recorded. Then, weight of the swollen microspheres (We) was recorded and the equilibrium
degree of swelling (qe) was calculated by following equation (24):
We ñ Wo
qe = ññññññññññññ
(2)
Wo
Figure 2. SEM of HID3 formulation (a) stirring speed 700 rpm and (b) stirring speed 900 rpm
1049
Figure 3. FTIR analysis of HEMA, IA, EGDMA and p(HEMA-co-IA) microgels
Drug loading and percent loading
Model drug (EMT) was loaded into MG networks by using simple equilibrium swelling technique (25). Dried microgels (100 mg) were
immersed in drug solution prepared in ethanol-0.05
M phosphate buffer pH 7.4 mixture (50 : 50% v/v)
for 24 h at 37OC. Drug loaded MGs were immediately dipped in ethanol to remove unloaded drug and
dried at room temperature. For the determination of
percent drug loading (Dl), EMT loaded microgels
(10 mg) were crushed and incubated in 15 mL of the
same solvent used for drug loading at room temperature for 24 h. The suspension was sonicated and filtered through Whatman filter paper no. 41. After
appropriate dilutions the solution was assayed for
EMT at 301nm (Perkin Elmer, Japan). Results of %
loading (Dl) were calculated as follows:
Wd
% Dl = (óó)
◊ 100
(3)
W
where, Wd = weight of drug in microgel and W =
weight of microgel.
Fourier transform infrared spectroscopy (FTIR)
FTIR spectra of HEMA, IA and empty microgels were recorded by attenuated total reflectance
ATR-FTIR (Bruker IR Affinity 1 Model, Japan). All
the spectra were recorded over the wavelength range
4000 to 500 cm-1.
Thermogravimetric analysis (TGA)
Thermal analysis was performed by TA instrument, USA model Q600 series. The samples were
ground and passed through mesh 40. For TGA, the
measurements were carried out under dry nitrogen at
1050
the rate of 20OC/min, in temperature range of 20500OC.
Scanning electron microscopy (SEM)
Surface morphology of empty micogel and
drug loaded microgel was investigated by scanning
electron microscopy (SEM-JEOL Instruments,
JSM-6360, Japan). Microgels were fixed on support
with carbon-glue, and coated with gold using a SPI
sputter module in a high-vacuum evaporator for
SEM images at 5 kV.
Dynamic light scattering (DLS)
Particle size and distribution measurement
were carried out through dynamic light scattering
technique using zeta analyzer (Zetasizer Nano-series
ZEN3600, Malvern Instruments, USA).
Dissolution studies
Dissolution studies were performed in USP II
dissolution apparatus (Pharma Test, Germany) using
0.05 M USP phosphate buffer at various physiological pH values (1.2, 6.5 and 7.4). Accurately
weighed microgels (equivalent to 100 mg of drug)
were loaded in a small dialysis bag (dialysis membrane 800 Da), fixed with paddle and immersed in
dissolution flask containing 500 mL of dissolution
medium at constant speed of 100 rpm. The temperature was maintained at 37 ± 0.5OC. At regular intervals of time, sample aliquots were withdrawn and
analyzed at 301 nm using UV spectrophotometer
(Perkin Elmer, Japan). Amount of drug was calculated by preparing standard curve. Each withdrawn
sample was replaced with same volume of fresh
medium. All the measurements were carried out in
triplicate.
Drug release data were fitted to various kinetic
models including zero-order (26), first order (27),
Higuchi (28) and Korsmeyer-Peppas models (29).
From the Korsmeyer-Peppas equation the diffusion
coefficient ëní was calculated. Value of ëní determines the mechanism of drug release. For the spherical matrices, if n is less than 0.43 a Fickian (case-I),
if n is between 0.43 and 0.85 a non-Fickian, and if n
is greater than 0.85 a case-II (zero order) drug
release mechanism dominates.
Figure 4. TGA thermograms of HEMA, IA and p(HEMA-co-IA) MG
1051
Figure 5. Swelling behavior (a) Effect of IA and pH, (b) Effect of EGDMA
Preparation and characterization of p(HEMA-coIA) microgels
In the present work, p(HEMA-co-IA) microgels of different composition were successfully prepared. The method of preparation was simple using
water as dispersion medium. The unreacted
monomers and crosslinker were easily removed by
immersing MGs in water and ethanol. Initially,
experiments were performed with varying stirring
speed and time of stirring to optimize the experimental conditions. Microgels were found to be deshaped at speed above 700 rpm while very large
spheres with irregular shape were obtained at stirring speed below 700 rpm. SEM images of microgels prepared at 700 rpm and 900 rpm are presented
in Figure 2. It can be observed that the microgels
were spherical in shape and uniformly distributed
over the grid with smooth outer surfaces (Fig. 2a) at
700 rpm stirring speed, while large irregular spheres
appeared when stirring speed was 900 rpm (Fig. 2b),
therefore 700 rpm speed was selected for the preparation of microgels. On DLS analysis the drug
loaded microgels showed mean particle size in the
range of 2 to 10 µm (Table 1). Our results showed
that amount of monomers and crosslinking agent
used are affecting the size of MGs.
FTIR and TGA studies
Results of FTIR analysis are presented in
Figure 3. All characteristic bands present in FTIR
spectra of components (HEMA, IA and EGDMA),
appeared in the FTIR spectrum of the prepared
p(HEMA-co-IA) microgel. The spectral characteristics of p(HEMA-co-IA), revealed the characteristic
1052
Figure 6. Cumulative percent drug release of formulations HID1-HID4 at pH 1.2, 6.5 and 7.4
Table 3. Effect of IA and pH on drug release mechanism.
Formulation
pH
Release
exponent (n)
r
Mechanism of
release
HID1
1.2
0.731
0.9869
Non-Fickian
6.5
0.693
0.940
Non-Fickian
7.4
0.607
0.927
Non-Fickian
1.2
0.670
0.993
Non-Fickian
6.5
0.674
0.952
Non-Fickian
HID2
HID3
HID4
HID5
7.4
0.592
0.908
Non-Fickian
1.2
0.646
0.994
Non-Fickian
6.5
0.699
0.936
Non-Fickian
7.4
0.552
0.909
Non-Fickian
1.2
0.622
0.988
Non-Fickian
6.5
0.628
0.950
Non-Fickian
7.4
0.462
0.913
Non-Fickian
1.2
0.606
0.986
Non-Fickian
6.5
0.559
0.942
Non-Fickian
7.4
0.430
0.908
Non-Fickian
band around 3450 cm-1 indicating -OH group of
HEMA, an absorption bands around 2956 cm-1, correspond to the aliphatic ñCH2, CñH and ñCH3
groups, the C=O stretching vibration of α, β unsaturated ester group also appeared around 1715 cm-1
and the increased peak intensity of the C=O group at
1720 cm-1 associated with the presence the additional C=O groups from IA. In addition, several bands
between 1600 and 1000 cm-1 appeared for ethylene
glycol units, originating from EGDMA component
(30). These results confirmed the formation of
crosslinked p(HEMA-co-IA) microgels.
TGA was performed to study thermal behavior
of the copolymeric network. TGA thermograms of
pure HEMA, IA and cross linked poly(HEMA-coIA) microgels are presented in Figure 4.
Temperatures of the 10% mass degradation were
112, 206 and 225OC for HEMA, IA and p(HEMAco-IA), respectively. Results showed that p(HEMAco-IA) microgel showed much better thermal stability than the individual components, which could
probably be due to copolymerization and crosslinking of HEMA and IA (14).
Equilibrium swelling studies of microgels
Swelling behavior of microgels against external stimuli like temperature or pH is a measure of
their usefulness as biomaterials in the field of pharmaceuticals. The swelling behavior of microgels
significantly effects the diffusion of drug from the
microgel matrix (30). In the present study, the effect
of pH, IA and EGDMA on swelling behavior have
been studied.
Mostly, ionic microgels are sensitive to pH and
swell significantly at a pH that favors their swelling.
Anionic microgels swell at basic pH and collapse at
acidic pH. The carboxylic groups present in anionic
polymeric networks ionized at pH above its pKa,
repel each other and cause swelling of hydrogel (29,
31, 32). Figure 4a represents the swelling behavior
of the synthesized microgels with increasing IA concentration (code HID1 to HID5 with fixed
EGDMA), as a function of pH (1.2, 4, 5.5 and 7.4).
Our results revealed that there was little or no
swelling the MGs at pH 1.2. At pH 4, significant
jump in equilibrium degree of swelling (qe) has been
observed might be due to ionization of first carboxylic group (pKa1 3.85) of IA. At and above pH
5.5, further increase in qe was observed due to dissociation of second -COOH group (pKa2 5.45) of
IA. Similar swelling behavior has been observed by
other authors (16, 35, 36). In all the formulations,
maximum swelling was reached at pH 7.4. The reason might be in complete dissociation of -COOH
1053
groups of IA. Such behavior of these copolymeric
MGs with IA recommends them for application as
DDS in biological fluids with pH 4 and above (23).
Hydrophilicity of the microgels also seems to
be improved due introduction of IA (Fig. 4a). The
reason might be with the increase in IA more COOions would be formed, thus the electrostatic repulsion became stronger and the microgel network
expands (35). Similar kind of expansion of the polymeric chains was observed when there was an
increase of ionic groups in the formulations (36).
Swelling was also seemed to be dependent on
concentration of crosslinking agent (EGDMA).
With increasing concentration of EGDMA from 5 to
10% (HID3, HID6 and HID7), the equilibrium
swelling ratio decreased (Fig. 4b). It might be due to
decreased mesh size and decreased polymer chain
relaxation with increasing crosslinker concentration.
Literature supports the present results (37, 38).
Drug loading and in vitro dissolution studies
Percent loading of drug found up to 65.24 ±
0.85% w/w as shown in Table 1. It was found that
microgels having greater swelling accommodated
higher amounts of drug (33, 39). Release of drug
molecules from hydrogels depends on various characteristics such as the chemical composition of the
polymer, the network structure, the release conditions, etc. To assess release pattern of EMT from the
MGs, in vitro kinetics studies were performed.
Major aim of the in vitro drug-release studies was to
illustrate the pH-sensitive drug-release properties of
the p(HEMA-co-IA) microgel and effect of IA content on drug release. Figure 6 represents the effect of
IA content and pH on drug release kinetics. Results
clearly indicated that the p(HEMA-co-IA) microgels
showed pH-sensitive drug-release behavior. All the
formulations showed higher drug release at pH 7.4
than at pH 6.5 and at pH 1.2. The reason might be
higher degree of swelling of microgel at pH 6.5 and
7.4. Our result showed direct relationship between
drug release measured and the swelling studies, as
reported earlier (31, 40). On the other hand, %
cumulative drug release was found higher in formulation with higher concentration of IA, as presented
in Figure 6. Reason might be connected with the
increase in IA concentration as hydrophilic nature of
the polymer matrix increased as reported earlier
(35). Our results showed lower concentration of
drug release at acidic pH, while at basic pH sustained release pattern over time was observed, therefore p(HEMA-co-IA) MGs would be recommended
for the formulation of site specific controlled DDS
to release drug at pH ≥ 4.
1054
Drug release data obtained from dissolution
studies were fitted to various kinetics models (zeroorder, first-order, Higuchi and Korsmeyer-Peppas
models) to evaluate the drug release mechanism.
The best fitted model was selected on the basis of
regression coefficient (r). The r values obtained for
p(HEMA-co-IA) MGs at varying contents of
HEMA and IA are given in Table 2. It has been
observed that the r values obtained for Higuchi
model (0.849-0.99) were higher than those of first
order kinetics (0.799-0.979) and zero-order kinetics
(0.716-0.977), suggesting that drug release followed
Higuchi model. The Korsmeyer-Peppas equation,
Mt/M• = ktn was used to study the mechanism of
drug release, where Mt/M• is the fraction of drug
release at time t, k is the kinetic rate constant, and n
is the diffusion exponent describing the release
mechanism. The n values calculated by the above
equation were above 0.43 and below 0.85 indicating
non-Fickian diffusion release (Table 3).
CONCLUSION
The study concludes that p(HEMA-co-IA)
MGs are an efficient DDS with a diverse pH-responsive mechanism. FTIR and TGA confirmed the formation of crosslinked copolymeric network. MGs
were of uniform size, spherical in shape and have
potential to be used as platform for the controlled
site specific release of drugs.
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Received: 2. 01. 2016
ISSN 0001-6837
PHARMACOLOGY
QUINIDINE AND DOMPERIDONE INTERACTIONS IN THE RAT
EXPERIMENTAL MODEL OF REPEATED ADMINISTRATION
MAGDALENA BAMBUROWICZ-KLIMKOWSKA, TADEUSZ SZOST, ANNA MA£KOWSKA
and MIROS£AW SZUTOWSKI
Department of Toxicology, Faculty of Pharmacy, Medical University of Warsaw,
Banacha 1 St., 02-097 Warszawa, Poland
Abstract: This study has investigated domperidone (DOM) and quinidine (QD) interaction in the Wistar rat
experimental model of repeated administration. We used nonconventional administration model consistent with
occasional administration method. Difference in administration was related to sequence of domperidone alone
or with quinidine dosage. Expected domperidone-quinidine interactions could have its origin both in the ability of quinidine to inhibit P-glycoprotein (P-gp) activity as well as cytochrome P450-mediated metabolism of
both compounds. There also were examined kinetics of acetaminophen (PAM) administered (30 mg/kg) with
domperidone as an indicator of gastric emptying, showing domperidone prokinetic activity, as well as quinidine anticholinergic activity. Domperidone (30 mg/kg) with PAM and with/without quinidine (25 mg/kg) was
administered orally according to the disposition regiment different for six examined rat groups. DOM and PAM
concentrations in plasma were assayed by HPLC method. Following changes were observed: domperidone did
not modify the duration of the uptake phase of acetaminophen; quinidine prolongs gastric emptying time (as a
result of anticholinergic action); quinidine given as the fourth or fifth dose with domperidone promotes growth
of its concentration in plasma; analysis of changes in the value of AUC0-2 at the initial three weeks of experiment suggests intensity of domperidone absorption processes, the following week increase in the value AUC4-6
suggests inhibition of domperidone hepatic biotransformation and the mechanism of induction of absorption
during domperidone administration is different from the absorption - inducing effects of quinidine. Both effects
are superimposed and produce large, 2, 3-fold change in domperidoneís AUC0-6.
Keywords: domperidone, P-glycoprotein, quinidine, multi-dosage administration model
Systemic preclinical studies of therapeutic substances can not answer the question how they
behave when irregularly or occasionally administered. This kind of application is becoming more
common due to increasing pace of life and growing
number of drugs available without a prescription, a
situation which favors self-treatment (1, 2). More
drugs are permitted for use in an increasing number
of ailments. Hence, the possibility of interactions
between active drug compounds. These may be
related to pharmacokinetic interactions connected
with intestinal wall transporters and cytochrome
P450 in liver and intestine. Complex influences
between drugs or medicines together with the structures of the body - enzymes, transporters, cause
great difficulty in predicting occurrence of possible
interactions.
A variety of drug transporters present in the
body control the fate of drugs by affecting absorp-
tion, distribution, and elimination processes. In clinical pharmacotherapy, ATP-dependent efflux transporters expressed on the apical membrane of the
intestinal epithelial cells determine oral bioavailability, intestinal efflux clearance, and the site of drugdrug interaction of certain substances (3). P-glycoprotein (P-gp) belongs to the ABC transporters super
family (4). P-gp is expressed on the luminal surface
of the intestinal epithelia, the renal proximal tubule,
the bile canalicular membrane of the hepatocytes, the
placenta and the blood-brain barrier (5). This
anatomical localization together with its broad substrate profile contributes to the significant role of Pgp in drug absorption and disposition. P-gp distribution within many organs and tissues results in the
absorption or excretion of xenobiotics and drug-drug
interactions with this protein may change the
bioavailability of simultaneously administered active
drugs. It must be considered that administration of
* Corresponding author: e-mail: emjbamburowicz@wum.edu.pl
1057
1058
MAGDALENA BAMBUROWICZ-KLIMKOWSKA et al.
one drug can influence metabolism of another drug
via modulation of P-gp activity (6) but also via modulation of the cytochrome P450 enzymes (7).
Quinidine (QD), an antiarrhythmic drug is a
well-known cause of drug-drug interactions (8). It is
P-gp (9) and P450 (10) as well as CYP2D6 substrate
and inhibitor (11, 12). QD is listed on sixth place as
substance involved in CYP-mediated drug-drug
interaction by Kato et al. (10)
Domperidone (DOM) is a dopamine D2-receptor antagonist. Due to its high molecular weight and
low lipid solubility DOM does not enter the CNS
(13). It is rapidly absorbed after oral administration
but with low bioavailability caused by intestinal Pgp activity (14). Both domperidone and quinidine
interact with P450 enzymes (especially CYP2D6)
and with P-gp in each phase of disposition of these
pharmaceutical compounds within an organism (15,
16). QD has been shown in our previous paper to
increase rat plasma DOM concentration during the
first two hours after their single-dose administration
(17). This study has investigated interaction
between DOM and QD in multi-dosage (once a
week repeated single disposition) administration to
Wistar rat model. There also were examined kinetics
of acetaminophen (PAM) administered (30 mg/kg)
with domperidone as an indicator of gastric emptying, showing domperidone prokinetic activity, as
well as quinidine anticholinergic activity (18, 19).
The aim of the study was to examine the
changes of domperidone kinetics in rat serum
assuming multiple administration at weekly intervals (during 7 weeks), while using different dosing
regimens of quinidine. Among the different variants
of medication, in addition to conventionally used
doses of single and multiple, daily, administered at
different times, the periodic, and/or occasional manner of medication, is virtually no examined. Raises
the question whether dose used from time to time,
separated by a longer period of absence of drug in
the body, may interact as to lead to specific pharmacokinetic and pharmacological effects? In this
model domperidone as occasionally used drug and
quinidine as well-known cause of drug-drug interactions were used. We also have examined the course
of the kinetics of paracetamol co-administered with
domperidone as an indicator of gastric emptying,
showing the prokinetic activity of domperidone.
Domperidone (C22H24ClN5O2; ≥ 98%, Pub
Chem CID: 44349952), quinidine hydrochloride
monohydrate (C20H24N2O2 HCl H2O; ≥ 5.5% dihydroquinidine, Pub Chem CID: 16219921), acetaminophen (C8H9NO2; ≥ 99%, Pub Chem CID:
1983), propranolol hydrochloride (C16H21NO2 HCl;
≥ 99%), 8-chlorotheophylline (C7H7ClN4O2; 98%),
β-glucuronidase from Helix pomatia (type H-1, partially purified powder, ≥ 300,000 units/g solid) and
carboxymethyl cellulose sodium salt (high viscosity,
1500-3000 cP, 1% in H2O, in 25OC) were obtained
from Sigma-Aldrich (Germany). Sodium phosphate
monobasic (NaH2PO4 ≥ 99%), phosphoric acid
(H3PO4 ≥ 85%), potassium phosphate monobasic
monohydrate (KH2PO4 H2O ≥ 99.5%) and sodium
chloride (NaCl ≥ 99.5%) were supplied by POCh
Gliwice (Poland). HPLC grade dichloromethane and
methanol were supplied by Labscan (Ireland). Water
was obtained from Mili-Q purification system.
Table 1. Experimental design.
Number of administration
1
2
3
4
PAM
PAM
PAM
PAM
PAM
DOM
DOM
DOM
DOM
group I
5
6
7
QD
PAM
group II
PAM
PAM
PAM
PAM
PAM
PAM
DOM
DOM
DOM
DOM
DOM
DOM
PAM
PAM
PAM
PAM
PAM
PAM
DOM
DOM
DOM
DOM
DOM
DOM
QD
PAM
group III
QD
PAM - acetaminophen, DOM - domperidone, QD - quinidine.
QD
QD
Quinidine and domperidone interactions in the rat experimental model of...
Male Wistar Cmd: (WI) WU rats weighing 191
± 11 g at the beginning and 321 ± 25 g at the end of
the experiment (Medical Research Center, Polish
Academy of Sciences) were used. Rats were treated
according to the Guiding Principles for the Care and
Use of Laboratory Animals approved by the Local
Animal Research Ethic Committee in an independent room with controlled temperature at 23OC, 12/12
light/dark cycle and 66% humidity. Animals had
free access to water and food supplied as pellets.
The general condition of the animals was checked
daily. The animals were acclimated for 10 days
before the beginning of the experiment. Thereafter,
twelve rats were randomly divided into three
groups, which were treated as shown in Table 1.
Food was withdrawn for 12 h before drug administration, free access to water was allowed during the
experiment.
DOM powder was suspended to concentration
of 14 mg/mL in 1% water solution of carboxymethyl
cellulose sodium salt. DOM was administered at the
dose of 30 mg/kg b.w. The dose of DOM was chosen on the basis of doses used by Michiels and
Heykants in their animal pharmacokinetic study (20,
21).
QD was dissolved in water to a final concentration of 8 mg/mL and given per os at the dose of
25 mg/kg (dose calculated as a free base). We
decided on this dose due to the fact that in both
studies the influence of quinidine on the metabolism of various compounds such as quinidine
impact on P-glycoprotein in rats doses of 10 to 50
mg/kg were used (22, 23).
PAM was administered at the dose of 30 mg/kg
b.w. (24), as a water suspension with 1% carboxymethyl cellulose sodium salt at a final concentration 24 mg/mL.
All compounds were given via gastric gavage,
concomitant or alone with vehiculum (1% water
suspension of carboxymethyl cellulose sodium salt
or water) as a one shut.
Blood samples (250 µL) were collected without anticoagulants from tail vein at 30, 120, 240,
360, and 480 min after DOM treatment. The samples were centrifuged at 8000 ◊ g for 5 min. to
obtain the plasma which was stored at ñ20OC until
quantitative determination of DOM and PAM.
For DOM extraction, to 0.1 mL of each sample, 80 ng of propranolol hydrochloride as internal
standard, 0.1 mL 0.1 M NaOH, and 3 mL of
dichloromethane were added. The mixture was
shaken for 10 min and centrifuged at 1200 ◊ g for 15
min. Next, organic phase was transferred to another
glass tube and evaporated to dryness under gentle
1059
nitrogen stream. Dried residue was dissolved in 200
µL of mobile phase, and 20 µL of the aliquot was
injected into the HPLC column.
A recovery study was performed by spiking 0.1
mL of plasma with 20 µL DOM standard solutions
and the samples underwent extraction procedure
described above. For three tested concentration an
average of 92.7% and 86.7% recovery was obtained,
respectively, for DOM and propranolol hydrochloride.
PAM was isolated from plasma by solid phase
extraction after enzymatic hydrolysis. In the
Eppendorf tubes were placed 20 µL of serum, 20 µL
β-glucuronidase solution in 0.2 M potassium phosphate monobasic solution and after thorough mixing
the tubes were subjected to incubation at 37OC for 18
h. Afterwards, for the incubation of tubes, 20 mL
methanol solution of 8-chloroteophylline at concentration of 15 µg/mL as an internal standard and 440
µL 0.9% NaCl were added. Prepared samples were
mixed and placed on the SPE columns (BAKERBOND C18, 3 mL) for the extraction process. After
final elution, methanol was evaporated to dryness
under gentle nitrogen stream. Dried residue was dissolved in 200 µL of mobile phase, and 20 µL of the
aliquot was injected into the HPLC column.
Efficiency of the extraction process was 89.0% for
acetaminophen and 104.9% for the 8-chlorotheophylline (average for three concentrations).
Concentration of DOM in plasma samples
was measured by reversed phase HPLC method
(Shimadzu, Germany) with fluorescent detection
(RF-10Axl, Shimadzu, Germany) by use of the
reported method with modifications (25).
Chromatographic separation was performed using
a Discovery HS PEG stainless steel column 150 ◊
4.6 mm I.D., 5 µm (Supelco, Bellefonte, PA,
USA), preceded by a 20 ◊ 4.6 mm I.D., Discovery
HS PEG guard column. The column was kept at
40OC. Injection volume was 20 µL. The mobile
phase consisted of 0.02 M water solution of natrium dihydrogen orthophosphate (adjusted to pH 3.5
with orthophosphoric acid) ñ methanol (82 : 18,
v/v) and was delivered at a flow rate of 1.0
mL/min. The run time was 10 min. The mobile
phase was ultrasonically degassed prior to use. Six
point calibration curve was linear ranging between
10 and 1000 ng/mL with correlation coefficient (r2)
0.999. The precision was calculated as percent
coefficient of variation (CV) and for each analyzed
concentration did not exceed 6.5%. Accuracy of
measurement for 6 different concentrations (bias)
was 5%. The limit of detection for the assay was
7.97 ng/mL of plasma.
1060
PAM plasma concentration was quantified by
HPLC (Shimadzu, Germany). The method was based
on the procedure developed and used in the
Toxicology Department (26). Discovery C18 150 ◊
4.6 mm I.D., 5 µm column (Supelco, Bellefonte, PA,
USA), preceded by a 20 ◊ 4.6 mm I.D., Discovery
C18 guard column were used for resolution. The column was kept at 40OC. The mobile phase consisted of
0.025 M water solution of potassium phosphate
monobasic monohydrate ñ acetonitrile - methanol
(85 : 10 : 5, v/v/v) and was delivered at a flow rate of
1.5 mL/min. Injection volume was 20 µL. PAM was
detected by UV-detection (SPD Shimadzu,
Germany) at a wavelength of 230 nm. The run time
was 10 min. The mobile phase was ultrasonically
degassed prior to use. The six point calibration curve
was linear ranging between 2.5 and 100 µg/mL with
correlation coefficient (r2) 0.9921. Precision was calculated as percent CV and it was 2.5% (average for
5, 50 and 100 µg/mL of plasma concentration).
Accuracy of measurement for 6 different concentration (bias) was 4.3%. The limit of detection for the
assay was 2.35 µg/mL of plasma.
The peak serum concentration (Cmax) and the
time to peak concentration (Tmax) were obtained
from visual observation. Due to the limited number
of measurement points, pharmacokinetic parameters
were determined in real time and pharmacokinetic
parameters of DOM and PAM were analyzed by
noncompartmental method of the program Kinetica
(version 5.0, Thermo Scientific, USA). The concentration-time profiles for domperidone and acetaminophen were analyzed for partial area under the con-
centrationñtime curve from zero to 6 h (AUC0-6).
The area under the serum concentration-time curve
was calculated using linear trapezoidal rule.
All statistical analyses were conducted with
Statistica (version 9.0, StatSoft, Poland). All results
were tested for parametric assumptions (ShapiroWilk and Brown-Forsythe tests). For each parameter
for which assumptions were satisfied the Repeated
Measures ANOVA tests were used. When assumptions of the analysis of variance had been violated,
the Friedmanís ANOVA test to determine significant differences within groups was used. The limit
for statistical significance was set at p < 0.05.
RESULTS
Analysis of the results should be conducted
based on experimental design scheme (Table1).
After conducting analysis in groups it should
be noted that in group I (where quinidine was
administered in 3rd week) the Cmax in this kinetics
although less markedly significant (p < 0.06) was
higher than in the 2nd and 4th week kinetics, where
domperidone was given without quinidine. In 7th
week kinetics, 5-fold increase in the values of the
pharmacokinetic parameters for domperidone in
relation to the initial data was observed. Mean values of parameters in the latter kinetics are up to 5
times higher than the initial ones (for example
AUC0-6). There also was statistically significant 2fold increase in the partial AUC values in relation to
the 4th week kinetics. A general upward trend in values of these parameters over time was shown (Fig.
Figure 1 Values of domperidone area under the time-concentration curve and Cmax in group I. Doses of drugs: DOM 30 mg/kg, PAM 30
mg/kg and QD 25 mg/kg. Data represent the mean ± SD for n = 4. Administrations were compared by the Friedmanís ANOVA test, * p
< 0.05 vs. 2nd administration; # p < 0.06 vs. 2nd administration
1061
Figure 2. Values of domperidone area under the time-concentration curve and Cmax in group II. Doses of drugs: DOM 30 mg/kg, PAM
30 mg/kg and QD 25 mg/kg. Data represent the mean ± SD for n = 4. Administrations were compared by the Repeated Measures ANOVA
test (for AUC values), and by the Friedmanís ANOVA test (for Cmax), * p < 0.05 vs. foregoing administrations; # p < 0.05 vs. 2nd, 3rd, 4th
administration; ^ p < 0.05 vs. 2nd administration
Figure 3. Values of domperidone area under the time-concentration curve in group III. Doses of drugs: DOM 30 mg/kg, PAM 30 mg/kg
and QD 25 mg/kg. Data represent the mean ± SD for n = 4. Administrations were compared by the Repeated Measures ANOVA test, * is
p < 0.05, vs. remaining administrations; # is p < 0.05, vs. 2nd, 3rd and 4th administration
1). Moreover, a rise in the value of domperidone
AUC0-2/AUC4-6 ratio in 3rd week kinetics, when
quinidine was administered, was observed.
In group II, during 6th administration there was
a sharp increase in the AUC areas after first time
quinidine co-administration with domperidone
(especially for AUC0-6, 2193 ng/mL ◊ h compared to
783 ng/mL ◊ h in 5th week kinetics). The remaining
AUC: AUC0-0.5, AUC0-1 and AUC0-2 after administration of quinidine increased by approximately 100%,
as shown in Figure 2. In these kinetics, statistically
significant increase in both parameters characterizing the absorption process - AUC0-Tmax, and other
phases of the fate of the drug in the body - from the
AUC0-0.5 to AUC0-6 and AUC4-6, was revealed.
Group III, 6th week kinetics is characterized by
a significant increase in partial AUC from 245% for
AUC0-4 to 467% for AUC0-1 one week after QD disposition. Reduction of domperidone AUC0-2/AUC4-6
ratio in group III where quinidine was administrated
every other week was observed in the 3rd, 5th and 7th
administrations (Fig. 3)
In group I, quinidine administration in 3rd week
kinetics resulted in a decrease in all AUC values but
recorded decreases were not statistically significant.
However a week after the administration of quinidine (4th administration) AUC areas were significantly higher and this effect persisted in the following 7th week kinetics, after three weeks (Fig. 4).
Slight decrease in PAM AUC0-2/AUC4-6 ratio after
1062
QD dosage was observed due to statistically significant rises of AUC4-6 values in 4th and 5th dispositions.
Significant increases in PAM pharmacokinetic
parameters in 7th week kinetics in both the group I
(PAM administration with DOM after a two week
break) and in group II - one week after administration of quinidine, are shown in Figures 4 and 5. In
Group II - one week after quinidine administration,
the highest values of the AUC in 7th week kinetics
were observed.
Alternate administration of PAM with domperidone and PAM with domperidone and quinidine
cause an increase in average AUC values in the
week after the administration of quinidine, which is
observed in 4th and 6th week kinetics and showed in
Figure 6.
DISCUSSION
Among the different variants of drug administrations, apart from conventionally used single and
multiple doses daily, administered at different periods of time, there is virtually unexplored way of
irregular, occasional intake. The question is whether
Figure 4. Values of acetaminophen area under the time-concentration curve and Cmax in group I. Doses of drugs: DOM 30 mg/kg, PAM
test, * p < 0.05, vs. 1st, 2nd and 3rd administration
Figure 5. Values of acetaminophen area under the time-concentration curve and Cmax in group II. Doses of drugs: DOM 30 mg/kg, PAM
test, * p < 0.05, vs. 2nd, 3rd and 5th administration; # p < 0.05, vs. remaining administrations; ^ p < 0.05, vs. 2nd, 3rd, 4th and 5th administration
1063
Figure 6. Values of acetaminophen area under the time-concentration curve and Cmax in group III. Doses of drugs: DOM 30 mg/kg, PAM
30 mg/kg and QD 2 5mg/kg. Data represent the mean ± SD for n = 4. Administrations were compared by Repeated Measures ANOVA test
for AUC0-0.5 and Cmax, Friedmanís ANOVA test for AUC0-6, * p < 0.05, vs. remaining administrations; # p < 0.05, vs. 1st administration;
^ p < 0.05, vs. 1st, 2nd, 3rd and 5th administration; ^# p < 0.05, vs. 3rd and 5th administration
medicine applied from time to time, separated by a
longer period of absence of drug in the body may
interact so, as leading to the specific pharmacokinetic and pharmacological effects. According to the
traditional approach, it is believed that the majority
of pharmacokinetic interactions occurs due to drug
biotransformation in the liver with the participation
of cytochrome P450 enzymes (12, 27). P-glycoprotein may be perceived as a barrier due to the nature
of its activities - regulated efflux of toxic substances
and xenobiotics from the light outside the cell. Due
to its characteristics, P-gp plays an important role in
the processes of absorption and distribution of medicines (4, 28). Knowledge about the distribution and
mechanisms of action of this membrane protein is
crucial to understanding its impact on the kinetics of
drugs, especially in the face of polytherapy and
increasingly frequent phenomenon of self-medication. Therefore, an animal model was built by us.
We examined rat plasma domperidone kinetics
changes employing multiple dosing at weekly intervals, while using different dosing regimens of quinidine. Expected interactions between domperidone
and quinidine may have its origin both in the ability
of quinidine to inhibit P-gp activity as well as
cytochrome P450-mediated metabolism of the two
compounds. The kinetics of acetaminophen were
also investigated when administered concomitantly
with domperidone as an indicator of gastric emptying, showing domperidone prokinetic activity, as
well as quinidine anticholinergic activity.
In our investigation, we used DOM, the drug
administered in many disease entities but not with
repeated dosage regiment and with occasionally
repeated dosage as in dyspepsia, nausea and vomiting treatment or travel sickness. Domperidone was
administered in various systems with acetaminophen
and quinidine at weekly intervals for 5-7 weeks.
Animal study shows that domperidone undergoes rapid and extensive hepatic metabolism by
hydroxylation and N-dealkylation (20). In vitro
metabolism in human liver microsomes with
inhibitors revealed that CYP3A4 is a major form of
cytochrome P-450 involved in the N-dealkylation of
domperidone, whereas CYP3A4, CYP1A2 and
CYP2B6, CYP2C8 and CYP2D6 are involved in
domperidone aromatic hydroxylation (16).
Domperidone is a very good substrate for MDR1
and mdr1a P-gp (4, 5). Quinidine is metabolized by
CYP3A superfamily of cytochrome P450 enzymes
to hydroxylated derivatives excreted with urine. QD
is known as a good P-gp inhibitor (9, 29).
There is no data tied to pharmacokinetics after
occasionally repeated dosage. However, the importance of CYP3A and P-glycoprotein in limiting oral
delivery is suggested by: their joint presence in
small intestinal enterocytes, the significant overlap
in their substrate specificities, and by the poor oral
bioavailability of drugs that are substrates for both
CYP3A and P-gp, e.g., cyclosporine (30) and examined in this trials QD (10). Additionally, enzymes
and drug transporter proteins may be induced or
1064
inhibited by the same compounds - QD is found to
be an inhibitor and substrate for CYP2D6 (8, 11, 12)
or CYP3A4 (10).
The increase in value of DOM AUC0-2/AUC4-6
ratio after first quinidine and domperidone coadministration at 3rd week of experiment was the
result of QD-DOM interactions at the level of
absorption (17). This effect is visible even in spite
of overall upward trend in concentrations and
AUC levels in subsequent administrations (Fig. 1).
This augmentation is consistent with quinidine
inhibitory action on intestinal P-gp. However, in
group II, when first QD and DOM co-administration was preceded by four domperidone dispositions much greater growth of DOM pharmacokinetic parameters was observed. DOM AUC0-6 was
3-fold higher and others were 100% higher (Fig.
2). In this case, the action of QD as an inhibitor of
P-glycoprotein is evident by a statistically significant increase in parameters characterizing the
absorption - AUC0- Tmax, AUC0-0.5 and other phases
of ADME - AUC0-6 and AUC4-6 (data not shown),
which may indicate an inhibition of domperidone
hepatic biotransformation by cytochrome P450.
Note that where DOM and QD co-administration
was done after one DOM premedication only, an
increase in Cmax took place, while the other
examined parameters did not show statistically
significant changes.
When considering acetaminophen, it should
be noted that in contrast to group I PAM, DOM and
QD co-administration (in the 6th kinetics, Fig. 4 and
Fig. 5) caused statistically insignificant increase in
PAM AUC values. A reduction of domperidone
AUC0-2/AUC4-6 ratio when quinidine was administered every other week, at 3, 5 and 7 weeks (in
group III) demonstrated the inhibitory action of
quinidine on domperidone metabolism presumably
by inhibiting the activity of hepatic CYP3A4.
Moreover, in this group at 6th week, statistically significant increase in absorption of domperidone was
noted. The fact that the largest increases in domperidone AUC values in this drug delivery system
(Fig. 3) took place in the week after the quinidine
administration is also worth mentioning. The
largest increases in PAM pharmacokinetic parameters one week after quinidine administration in
group I (4th disposition, Fig. 4) and II (7th disposition, Fig. 5) was observed as well. Analysis of
changes in the value of AUC0-2 at the initial three
weeks of experiment suggests intensity of domperidone absorption processes. The following week
increase in the value AUC4-6 suggests inhibition of
domperidone hepatic biotransformation.
Many studies have demonstrated that in
response to increased intestinal metabolism caused
by the interaction of P-gp and metabolizing
enzyme substrate concentration is stabilized at a
lower level, thus affecting the AUC and Cmax (4,
31). This has occurred in our study - domperidone
as a substrate for CYP3A, CYP2D and P-gp undergoes extensive metabolism associated with the
induction of intestinal cytochrome P450 system,
which is a response to the previously given quinidine. This results in low levels of pharmacokinetic
parameters, lower than implied by the simultaneous administration of quinidine with domperidone,
especially with regard to high levels of pharmacokinetic parameters in the week after the QD administration. Induction of cytochrome may depend on
many factors, such as the accumulation of an
inducer or the enzyme capacity synthesis de novo.
According to Shapiro et al., induction of CYP3A
with a model inducer - rifampicin can take several
weeks (12).
Wandel et al. describes the ability of quinidine
to inhibit CYP3A4 activity, thereby inhibiting the
metabolism of substrates of cytochrome P450 isoforms. They studied the activity of CYP3A in the
metabolism of nifedipine to dehydronifedipine in
human microsomes in the presence of 14 different
compounds, which are inhibitors of P-glycoprotein
(32). All the increases in pharmacokinetic parameters in conducted experiment may be partially due to
inhibitory action of quinidine on domperidone
metabolizing enzymes.
P-glycoprotein is a widely recognized cause of
reduced bioavailability of orally administered drugs
due to its active role in limiting absorption from the
intestinal lumen. A classic example of it is the interaction of quinidine-digoxin. Digoxin as a model Pglycoprotein substrate (as also is domperidone studied in this work), undergoes no further metabolism by
cytochrome P450 and has greatly limited oral
bioavailability. Quinidine given orally to patients
with digoxin causes total increase of examined compound absorption by 15%, while increasing the AUC
by 77% and the maximum concentration by 81%.
These data suggest that inhibition of P-gp activity by
quinidine in the intestine at the stage of absorption,
which significantly affects the growth of the pharmacokinetic parameters of digoxin, takes place in our
study in the group I, II and III with different intensities after quinidine co-medication (33). Weekly domperidone administration causes increases in its AUC
maximum at 4 and 5 week of study. These growths
are visible in all AUC bands. They are the most
marked in the range of 0-0.5 h - 6-fold, while for
AUC0-6 is a 4.7-fold increase observed. Because significant increases in the parameters values describing
all phases of drug present in the system took place,
there is difficulty in classifying the reasons for the
observed effect. Given that DOM bioavailability is
approximately 20% and after several domperidone
administration absorption system as a result of compensating effects can operate more efficiently or start
a new factor responsible for its absorption, DOM concentrations have the right to grow. Moreover, vehiculum, in which the test substances were administered is
sodium carboxymethyl cellulose. Sodium carboxymethyl cellulose salt, as shown by Clausen et al.,
has the ability to increase the permeability of fluorescein, bacitracin and insulin in guinea pig small intestine (34). One percent addition of carboxymethyl cellulose significantly increased absorbtion within 180
min of fluorescein dose of 4.93 to 6.42%. This feature
was additionally confirmed by measurements of
TEER (Transendothelial Electrical Resistance).
Sodium carboxymethyl cellulose salt causes 10%
decrease in transepithelial electrical resistance compared to baseline.
CONCLUSION
Based on statistical analysis of the results, it
can not be unambiguously determined what mechanisms are responsible for the observed effects.
Clarification of the mechanisms of this phenomenon
requires additional study despite the fact that there
are not documents which take into account repeated
administration during occasional irregular administrations. Especially it is worth to remember that
irregular intake of drugs with concomitant treatment
of chronic disease can lead to unpredictable effects
of interactions.
Acknowledgment
Authors would like to thanks Ms. Sylwia
Sztermel for help in acetaminophen isolation and
determination.
All procedures performed in studies involving
animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted which are consistent with
European Communities Council Directive of 24
November 1986 (86/609/EEC). The research was
financed by: the Statutory Funds of the Medical
University of Warsaw (FW13/N/14) and received no
special grant from any founding agency in the public, commercial or not-for-profit sectors.
1065
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Wang Y.F. et al.: J. Pharmacol. Exp. Ther. 304,
1161 (2003).
1066
23. Okura T., Morita Y., Ito Y., Kagawa Y.,
Yamada S.: J. Pharm. Pharmacol. 61, 593
(2009).
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Morikawa K., Kato H. et al.: Jpn J. Pharmacol.
56, 261 (1991).
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Chromatogr. B 720, 251 (1998).
26. BrzeziÒski J., Bamburowicz-Klimkowska M.,
Jab≥oÒska J., Koteras M., £ukasik M., OsickaKoprowska A., Szost T., Szutowski M., Wysocka-Paruszewska B.: Toxicological Analysis
(Analiza toksykologiczna), Medical University
of Warsaw, Warszawa 2007 (in Polish).
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Beijnen J.H., Schellens J.H.M.: Toxicol. Appl.
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1788 (2003).
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Received: 23. 06. 2015
ISSN 0001-6837
EXCRETION OF ETHYL GLUCURONIDE IN THE URINE OF WARSAW
HIGH PREFERRING RATS DEPENDS ON THE CONCENTRATION
OF INGESTED ETHANOL
ANNA MA£KOWSKA1*, MIROS£AW SZUTOWSKI1, WANDA DYR2
and MAGDALENA BAMBUROWICZ-KLIMKOWSKA1
1
Department of Toxicology, Medical University of Warsaw, Banacha 1, 02-097 Warszawa, Poland
2
Department of Pharmacology and Physiology of the Nervous System, Institute of Psychiatry and
Neurology, Al. Sobieskiego 1/9, 02-957 Warszawa, Poland
Abstract: Ethyl glucuronide (EtG) is a direct ethanol metabolite. The presence of EtG in urine can be used as
a laboratory test to detect recent alcohol consumption. Several earlier studies in humans and in rats revealed
that the same amount of ethanol ingested at different concentrations results in different blood ethanol concentrations. The effect of different concentrations of ingested ethanol on the resulting EtG levels in urine was tested in WHP rats. The EtG concentration was also measured in rat hair. A significant (p < 0.05) decrease in the
total amount of urine EtG after administration of the higher concentration (50%) ethanol solution as compared
to 30% ethanol at the same dose of ethanol (3 g/kg) was observed. Median EtG concentration in rat hair of 1.5
ng/mg (range: 0.7ñ2.3 ng/mg) was observed. Our results demonstrate that EtG production and excretion in
WHP rats is dependent on alcohol concentration administered orally. EtG levels in hair closely reflect the fate
of EtG in the rat.
Keywords: ethanol concentration, ethyl glucuronide excretion, urine, WHP rats
Abbreviations: AUC - area under the curve, EI - electron impact, EtG - ethyl glucuronide, MG - methyl glucuronide, WHP - Warsaw high-preferring (line of rats)
Harmful effects of alcohol consumption have
caused a recent interest in the search for biomarkers
of alcohol abuse. These biomarkers play an important role in identifying the nature of alcohol consumption from an occasional alcohol intake to abuse
or dependency status (1-3). Ethyl glucuronide (EtG)
is a direct ethanol metabolite formed by ethanol conjugation with glucuronic acid mediated by UDP-glucuronyltransferase (4). EtG is non volatile, water
soluble and stable upon storage; it can be detected in
urine for extended time periods of up to several days
after complete elimination of alcohol from the body
(5). It has been estimated that only about 0.02% of
total ethanol dose was recovered as EtG in the urine
in humans and about 0.13% in rats (6, 7). As a direct
ethanol metabolite, EtG can be regarded as a highly
specific marker of alcohol intake, which may be useful in monitoring drinking during withdrawal treatments or in situations where it is important to
exclude alcohol use for example, in workplace, dur-
ing pregnancy and in transplantation patients (8).
The presence of EtG in urine provides a strong indication of recent drinking, even if ethanol is no
longer detectable. Thus, urine EtG levels can be
used as a laboratory test to detect recent alcohol consumption.
Determination of EtG in urine samples helps to
fill the gap in evaluations between short-term biomarkers (ethanol) and long-term ones [carbohydrate
deficient transferrin (CDT), γ glutamyltransferase
(GGT), mean corpuscular volume (MCV)], used as
traditional markers of alcohol consumption (9).
In recent years, EtG has been receiving more
and more attention as a biomarker of acute alcohol
intake and has been recommended for use in clinical and forensic investigation of alcohol intake (10,
11). The usefulness of its detection has been repeatedly demonstrated in situations where alcohol consumption was tentatively assumed, but not confirmed.
* Corresponding author: e-mail: amalkowska@wum.edu.pl
1067
1068
ANNA MA£KOWSKA et al.
One study conducted in alcohol-dependent
patients during an alcohol detoxification period after
admission to the hospital demonstrated high interindividual variability in EtG detectability, ranging
from 40 to 130 h (5).
Several earlier studies in humans revealed that
the same amount of ethanol ingested at different
concentrations results in different blood ethanol
concentrations (blood alcohol content, BAC)
depending on the prandial state (12-14).
These differences in BAC were also observed
in rats. The studies in rats have shown that first-pass
metabolism of ethanol is also dependent on the concentration of administered ethanol (13).
The aim of this study was to establish whether
the amount of ethyl glucuronide formed and excreted with the urine of WHP rats could be affected by
the concentration of alcohol given orally. In addition, the EtG concentration in rat hair was measured
and evaluated.
Animal experiments
This study was conducted using adult male
Warsaw high preferring (WHP) rats, weighting
320ñ390 g at the beginning of the experiment. The
WHP rat line is selectively bred from Wistar rats for
voluntary alcohol consumption. WHP rats were
obtained from Department of Pharmacology and
Physiology of the Nervous System, Institute of
Psychiatry and Neurology, Warszawa, Poland. Rats
were kept in individual cages in a temperature-controlled room with a 12-h light/12-h dark cycle.
Animals were quarantined for 1 week before ethanol
dosing. Rats received the ethanol solution orally
according to their body weight on 5 consecutive
days per week for five weeks. During weeks 1, 2 and
3, the rats were administered 1.5 g/kg of ethanol at
concentrations 15, 30 and 50%, respectively. During
weeks 4 and 5, the same rats were administered 3
g/kg of ethanol at concentrations 30 and 50%,
respectively.
Urine was collected in metabolic cages at the
following time periods: 0ñ3, 3ñ6, 6ñ9, 9ñ12, 12ñ15,
15ñ21, 21ñ27, 27ñ33 h after ethanol administration.
The rats received continuously water ad libitum and
food was withdrawn two hours before ethanol
intake.
The study protocol was approved by the II
Local Ethics Committee at the Medical University
of Warsaw, Poland (37/2009).
Urine samples were collected weekly from
each animal by housing each animal separately in
metabolic cages, with water ad libitum. Samples
were stored frozen (-20OC) in Eppendorf vials until
their analysis. Hair samples were collected as close
as possible to the skin from the back right side (the
same place on each animal) using an electric shaver
before ethanol administration and on the seventh
day after the last ethanol intake.
Materials
The following chemical reagents were used in
this study: ethyl glucuronide (EtG) and methyl glucuronide (MG) standard from Medichem
(Germany)], GC/MS-grade ethyl acetate (Sigma),
acetone (POCH S.A.), methanol (LAB-SCAN) and
N-methyl-N-(trimethylsilyl)trifluoroacetamide
(MSTFA; Sigma-Aldrich).
Stock standard solutions of EtG and MG were
prepared by dissolving of each compound in
methanol to obtain a concentration of 1 mg/mL.
Analytical procedures
EtG levels were determined using a gas chromatograph / mass spectrometer (GC/MS-QP2010
Plus, Shimadzu), equipped with a ZB-5MSi column
(length 30 m; inner diameter 0.25 mm; film thickness
0.25 µm; Zebron, Phenomenex). The injector and
GC/MS interface temperatures were 270OC, and the
ion source temperature was 250OC. The oven temperature was maintained at 100OC for 10 min, then
increased to 270OC at a rate of 10OC/min, then maintained at 270OC for 5 min. Helium was used as a carrier gas with a flow rate of 1 mL/min. The mass
selective detector was operated in the electron impact
(EI) mode with 70 eV ionization energy. Under these
conditions the retention time was 10.82 min for silylated EtG and 10.44 min for silylated MG. The following ions were monitored: 261, 292 and 405 for
EtG and 261, 292 and 391 for MG, the underlined
ones being used for quantification. With 261 and 391
ions, biological background interference was negligible. Methyl glucuronide was chosen as the internal
standard according to Skopp et al. (15).
Sample preparation and EtG extraction procedure
Two hundred microliters (10 µg/mL) of internal standard (IS) was added to the 50-µL urine sample and the solution was made up to 1 mL in volume
with methanol.
After 10 min shaking and centrifugation at
10,600 ◊ g, 50 µL of the supernatant was transferred
to vials and evaporated to dryness under a stream of
nitrogen at 70OC using a heated metal block.
Anhydrous ethanol (100 µL) was added and evapo-
Effect of dietary flavonoid naringenin on bones in rats with...
rated to dryness. The residue was derivatized with
50 µL MSTFA and 50 µL ethyl acetate (60 min,
80OC) and the volume of 1 µL of the processed samples was injected into the GC/MS system.
The calibration curve covered a concentration
range of 10ñ2,000 µL/mL for the analyte in urine.
The limit of detection (LOD) and limit of quantitation (LOQ) were calculated to be 3.3 µL/mL and 10
µL/mL, respectively.
The rat hair EtG concentrations were measured
according to a previously published validated
method (16). The limit of detection was 0.03 ng/mg,
and the limit of quantification was 0.1 ng/mg.
The Shapiro-Wilk test was used to test for normal distribution of data and the Leveneís test - for
1069
homogeneity of variance. Analysis of variance
(ANOVA) was used to test for significance between
the different ethanol concentrations and total EtG
excreted with urine.
The nonparametric test of Wilcoxon was used
when the data did not meet the requirements for a
parametric test (such as normal distribution). Values
of p < 0.05 were considered statistically significant.
The analyses were performed using Statistica
10 software (StatSoft, Poland).
RESULTS
Urine EtG cumulative profiles after ethanol
administration at 1.5 g/kg and 3 g/kg in different
concentrations are presented in Figure 1. All urine
samples collected immediately before alcohol
Figure 1. The cumulative profiles of urine EtG excretion after ethanol administration by oral gavage at 1.5 g/kg (at concentrations 15, 30
and 50%) and 3 g/kg (at concentrations 30 and 50%) (n = 7). a - 1.5 g/kg - 15%; R2 = 0.344, b - 1.5 g/kg - 30%; R2 = 0.162, c - 1.5 g/kg
- 50%; R2 = 0.123, d - 3 g/kg -15%; R2 = 0.565, e - 3 g/kg - 50%; R2 = 0.585
Figure 2. Individual profiles of urine EtG excretion after ethanol administration by oral gavage at 1.5 g/kg (at concentrations 15, 30 and
50%) and 3 g/kg (at concentrations 30 and 50%)
1070
administration were negative for EtG. The synthesis
of EtG was dependent on the amount of ethanol
ingested and increased with the dilution of ethanol.
EtG was detectable for up to 33 h but the EtG concentration in the last collected urine samples
(between 27ñ33 h after ethanol administration) was
6 times higher after the dose of 3 g of ethanol/kg
(93.4 ± 12.41 µg/mL) than after the smaller one
(15.5 ± 10.46 µg/mL).
This indicates that higher doses of ethanol
resulted in a greater urine EtG concentration.
Individual profiles of urinary excretion of EtG
revealed varied EtG formation between rats. We
observed a tendency for reduced total EtG excreted
with urine depending on increasing the ethanol concentration
Total amounts of EtG excreted with urine in
individual rats are depicted in Figure 2.
This study showed an ethanol dose- and concentration-dependent correlation with EtG excretion
in rat urine. The higher ethanol dose of 3 g/kg b.w.
resulted in higher EtG levels and the total amount
excreted with urine were significantly higher (p <
0.01) (Table 1).
When the administered ethanol was more concentrated, the total excretion of EtG was decreased.
The difference was statistically significant at the
higher dose of 3 g/kg (p < 0.01). Furthermore, we
observed a tendency toward a reduction in total
amount of EtG excreted with urine depending on the
ethanol concentration after the lower ethanol dose of
1.5 g/kg as well, however, the differences between
the three concentrations 15, 30 and 50% were not
statistically significant (p > 0.05) (Table 1).
It was interesting to find out if the inter-individual differences in rats alter the EtG deposition in
rat hair. Table 2 presents the EtG content in rat hair
after a completed set of experiments. The rats
received the same amounts of ethanol at the same
time and at the same concentration. A mean of 1.5
ng EtG/mg hair was found. This result reflects a
43% coefficient of variance that is comparable to the
mean urine AUC coefficient of variance (49%) or
the coefficient of variance (44%) of the total amount
of EtG excreted.
DISCUSSION
Biotransformation of ethanol to EtG was
observed in various rat strains and lines (17, 18). We
chose to study EtG production in WHP rats because
this line has been used in experimental models in
alcohol studies (19-21).
Ethyl glucuronide (EtG) is formed by the net
addition of activated glucuronic acid to ethanol.
This reaction is catalyzed by the UDP-glucuronosyltransferase (UGT) enzymes, which are characterized
by a high genetic polymorphism. Foti and Fisher
(22) observed the highest rate of EtG formation in
humans by isoform UGT 1A1, whereas Schwab and
Skopp found high activity of UGT 1A9 and UGT
2B7 (23). The increase of UGT 1A1 expression was
also reported in the liver of ethanol consuming
Wistar rats (24). While comparing to humans, more
efficient glucuronidation of ethanol has been
observed in tree shrews and Sprague-Dawley rats
(7). Ethanol biotransformation to EtG after a single
dose has been described in Sprague-Dawley (SD)
Table 1. Maximum concentrations (Cmax), time to-maximum concentrations (Tmax) and area under the curve (AUC) for EtGand the total
amount of EtG excreted in rat urine after alcohol administered in two doses at different concentrations.
Doses of ethanol
1.5 g/kg b.w.
3 g/kg b.w.
Ethanol
concentrations
(v/v)
15%
30%
50%
30%
50%
Cmax (µL/mL)
1687
1010
1212
2407
1919
3-6
3-6
3-6
6-9
Tmax (h)
AUC µg/mL∑h
RSD%
Total urine EtG
RSD%
EtG in hair ng/mg
a
6036 ± 3262
a
12713 ± 5244
9051 ± 5574
41.2
61.6
54.0
4600 ± 2110
3746 ± 1955
3162 ± 2219
45.9
52.2
70.2
1.49 ± 0.64
26548 ± 9709
36.6
Mean RSD%
6-9
a
18524 ± 9401 a
50.8
48.8
12462 ± 3124* 8428 ± 2068*
25.1
24.5
43.6
43.0
*Denotes a statistically significant difference between 30 and 50% ethanol at dose 3 g/kg b.w. (p < 0.01) (Wilcoxon test). aThe value differed significantly for ethanol administered in two doses of 1.5 g/kg b.w. and 3 g/kg b.w. (p < 0.01) (two-way ANOVA). RSD% - relative
standard deviation.
Effect of dietary flavonoid naringenin on bones in rats with...
Table 2. EtG concentration in WHP rat hair.
Rat number
EtG ng/mg
1
1.17
2
2.30
3
1.45
4
2.06
5
0.79
6
1.98
7
0.70
Mean
1.49
S.D.
0.64
RSD%
42.95
S.D. - standard deviation, RSD% - relative standard deviation.
rats and its similarity to that in humans was indicated (18).
The variations in urine EtG with different concentrations of the same ethanol dose observed in our
study correspond to findings reported by other
authors, who studied the effect of ingested ethanol
concentration on blood alcohol levels (13, 25). As was
demonstrated, the consumption of more concentrated
ethanol resulted in lower blood alcohol levels than
consumption of a dilute solution did. When rats were
tested in a fed state, they exhibited decreasing mean
AUC with increasing ethanol concentrations (4, 16
and 40%) after intragastrically administered ethanol
(1.0 g/kg). In fasted rats, only the highest ethanol concentration (40%) produced a lower mean AUC (13).
Likewise in humans, when ethanol was ingested postprandially, the mean AUC and the mean peak
blood alcohol concentrations were significantly
lower with a concentrated (40% w/v) than with a
dilute (4%) solution (13). Differences were also
observed after ingestion of beer and whiskey and
showed higher peak blood alcohol levels with beer
than with whiskey in the postprandial condition,
with the opposite findings in the preprandial state
(25). Our assumption is that the changes in blood
ethanol concentration in the postprandial state are
followed by similar changes in the excretion of EtG
with urine. In this study, rats were given ethanol two
hours after their feed had been withdrawn in the
morning. Therefore, it can be assumed that the rats
were in postprandial state, considering the fact that
rats feed mainly at night.
The Tmax of EtG in urine after 1.5 g/kg of alcohol was shorter than after 3 g/kg of alcohol (Table
1). Similar trends have been observed in humans. In
a study conducted by Lostia et al. (26), the authors
1071
found a statistically significant increase in urine EtG
Tmax after higher doses of alcohol. An increase in
EtG Tmax after three doses of alcohol was also
observed in rat serum. In rats receiving ethanol at 1,
2 and 3 g/kg body weight, the Tmax for blood EtG
were 1, 2 and 4 h, respectively (17).
EtG concentrations in blood and hair of LongEvans rats were compared after ingestion of different
ethanol doses. Higher blood EtG AUC values were
observed after higher ethanol doses and a correlation
with hair EtG concentrations was found. Median hair
EtG concentrations. were 21, 104 and 189 pg/mg in
groups receiving ethanol in doses of 1, 2 and 3 g/kg,
respectively, on four consecutive days per week for
three weeks (17). All WHP rats having received
ethanol in our study showed a median EtG concentration in hair of 1490 pg/mg (range: 700ñ2300).
This concentration was higher than that observed by
Kharbouche et al. (17) but we administered ethanol
for longer time, i.e. on 5 days a week for 5 weeks.
It is worth emphasizing that EtG that accumulated in hair was a result of five different ethanol dosing procedures, which is closer to the pattern of alcohol consumption by humans (in different forms and
in different amounts). The rats in the experimental
study group also varied in ethanol glucuronidation
levels. Despite these differences, relative standard
deviation (RSD) values for total urine EtG excretion
(44%), urine EtG AUC values (49%) and EtG content in hair (43%) were very similar, which indicates
a close relationship between EtG contents in urine
and in hair, as both parameters are derived from the
blood EtG concentration. Thus, the increased ethanol
concentration in consumed beverages would
decrease ethanol blood concentration and, in turn,
decrease the EtG synthesis that results in lower urine
EtG levels and likely EtG hair content.
In summary, the present study showed that EtG
production in WHP rats was depended on the concentration of orally administered alcohol, with differences more pronounced at higher ethanol doses.
These findings may have implications for individuals who consume alcoholic beverages with food.
Drinking less concentrated ethanol is very likely to
result in greater EtG production than ingesting the
same amount of ethanol in a more concentrated
form, particularly in the postprandial state. EtG levels in hair closely reflect the fate of EtG in the rat.
There are no conflicts of interest relevant to the
presented study. The authors alone are responsible
for the content and writing of the paper.
1072
Acknowledgments
Authors would like to thank Ms. Katarzyna
KosiÒska for help in EtG determination. This work
was supported by the Statutory Funds of the Medical
University of Warsaw (FW13/N/12) and received no
special grant from any founding agency in the public, commercial or not-for-profit sectors.
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Received: 23. 06. 2015
ISSN 0001-6837
EFFECT OF DIETARY FLAVONOID NARINGENIN ON BONES IN RATS
WITH OVARIECTOMY-INDUCED OSTEOPOROSIS
ILONA KACZMARCZYK-SEDLAK*, WERONIKA WOJNAR, MARIA ZYCH,
EWA OZIMINA-KAMI—SKA AND ANNA BO—KA
Department of Pharmacognosy and Phytochemistry, School of Pharmacy with the Division of Laboratory
Medicine in Sosnowiec, Medical University of Silesia, JagielloÒska 4, Sosnowiec, Poland
Abstract: Naringenin is a dietary flavanone which can be found in many products such as citrus fruits. This
substance reveals multiple pharmacological activities such as antiinflammatory and antioxidative. During the
menopause, the estrogen deficiency occurs, thus naringenin, which is also considered as a phytoestrogen, may
be useful in the treatment of menopause-associated osteoporosis. The aim of the presented study was to examine the effect of naringenin on the mechanical properties, chemical composition and the histomorphological
parameters of bones in the rats with ovariectomy-induced osteoporosis. The female Wistar rats were divided
into 4 groups: sham-operated, ovariectomized, ovariectomized treated with estradiol (0.2 mg/kg p.o.) and
ovariectomized treated with naringenin (50 mg/kg p.o.), and the tested substances were administered for 4
weeks. The obtained results show that ovariectomy caused the characteristic changes in the skeletal system of
rats ñ there was deterioration in mechanics, chemistry and histomorphometry. The estradiol administered to the
rats served as positive control for the experiment. Administration of naringenin to the ovariectomized rats
affected neither the bone chemical content nor the mechanical properties, however, there was a slight improvement in the bone microarchitecture in the tissue affected by osteoporosis. It can be concluded that the intake of
naringenin in dietary products is not harmful and may even bring beneficial effect on the bones histomorphometry during postmenopausal osteoporosis.
Keywords: ovariectomized rats, naringenin, bones, postmenopausal osteoporosis, histomorphometry, chemical
composition, mechanical properties
Naringenin is a flavanone which occurs in
many dietary products. As an aglycone and in glycoside forms, naringenin can be found in pistachios,
almonds, potatoes and some edible sprouts such as
soy, broccoli or lentil sprouts (1-6). It was also
reported to occur in propolis and herbal tea named
Honeybush tea, obtained from Cyclopia intermedia
(7, 8). This substance can be also detected in trace
amounts in tomatoes, however, during processing
tomatoes into ketchup its concentration is increasing
due to transformation from naringenin chalcone (9).
The greatest source of naringenin and its glycosides
are citrus fruits such as mandarins, oranges, pummelos while the richest in naringenin are bitter
oranges and grapefruits. The highest concentration
of these compounds is in solid tissues, but they are
also present in the citrus fruit-derived juice (9, 10).
Naringenin as a polyphenolic compound
reveals many pharmacological activities such as
antioxidant, neuroprotective and antiinflammatory
ones (11-13). This substance also acts as a protective
agent in cadmium-induced nephrotoxicity and hepatotoxicity (14, 15). There are reports indicating that
this flavanone shows vasorelaxant, antiulcer and
antiallergic activities, as well as antibacterial and
antiviral effects. Moreover, it can inhibit capillary
permeability, shows protective properties in hypertension and Alzheimer disease and it also protects
against UV and X radiation (16-19). The majority of
the above activities are connected with the antioxidative properties of naringenin, which can be
observed only in the hydrophilic environment. It
was proved that naringenin shows prooxidative
effect in lipophilic solvents (20). The hypoglycemic,
hypocholesterolemic and cytochrome P450 isoforms modulating properties of naringenin have
been also reported (10, 21, 22). Due to its structural
similarity to 17β-estradiol, naringenin is also classified among phytoestrogens (23). The estrogenic
activity of this substance is several times lower than
* Corresponding author: e-mail: farmafit@sum.edu.pl; phone +48-32-364-15-20
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ILONA KACZMARCZYK-SEDLAK et al.
estradiol, and its affinity towards estrogen receptors
β (ERβ) is higher than to estrogen receptors α
(ERα) (24-26). There are also evidences that naringenin may show antagonistic activity toward ERα,
but only in some specific pathways, thus it cannot be
considered as selective estrogen modulator (SERM).
This antiestrogenic properties of naringenin can
mostly be observed when it is co-administered with
estradiol or in ER-dependent cancers (26-31).
Osteoporosis is a disorder characterized by the
increased bone loss leading to bone fractures. The
pathogenesis of osteoporosis is connected with
many factors, including oxidative stress or estrogen
deficiency e.g., during menopause (32, 33). As
naringenin may act both as an antioxidant or prooxidant and as estrogen agonist or antagonist, the aim
of the presented study was to analyze whether the
intake of this dietary flavanone is safe, harmful or
beneficial on skeletal system during osteoporosis.
Since studies on mechanical properties and chemical
composition of bones are impossible to conduct in
human, this research was carried out on rats with
ovariectomy-induced osteoporosis.
The experiment was carried out on the 3months-old female Wistar rats provided by the
Centre of Experimental Medicine at the Medical
University of Silesia. The animals were fed a standard laboratory chow and since the day preceding
the experiment, the standard chow was replaced
with chow containing no soybean. The research was
conducted with the approval of the Local Ethics
Commission in Katowice.
Rats were divided into four groups (n =7 ):
(I) the control sham-operated, vehicle-treated rats
(SHAM),
(II) the control ovariectomized, vehicle-treated rats
(OVX),
(III) the ovariectomized rats receiving estradiol at a
dose of 0.2 mg/kg (OVX + ES),
(IV) the ovariectomized rats receiving naringenin at
a dose of 50 mg/kg (OVX + NRG).
The tested substances were administered orally
(per os ñ p.o.) for four weeks, starting one week
after the performance of sham surgery and bilateral
ovariectomy in general anesthesia induced by the
mixture of ketamine and xylazine. The body mass
gain was controlled during the whole experiment.
The body weight of the rats was recorded both on
the first day of the research and after four weeks following the administration of the substances. After
four weeks of drugs administration, all rats were
sacrificed with the use of general anesthesia induced
by ketamine and xylazine. The uterus, the thymus
and following bones: the right and left tibia and the
right and left femur were excised from each rat, and
the L-4 vertebra was also collected. After the cessation the mass of organs was recorded.
Analysis of the mechanical properties of the
bones
The assessment of the mechanical properties of
analyzed bones was performed as previously
described (34, 35). For femoral diaphysis the bending test and for tibial proximal metaphysis the
method of Str¸mer et al. (35, 36) were applied in
order to determine the mechanical properties. For
these bones the following parameters were measured: the maximal load, the fracture load, displacement for maximal load, and displacement for fracture load. Youngís modulus was also evaluated. The
mechanical properties of the femoral neck were analyzed by performing a compression test with the
evaluation of the maximal load affecting the femoral
neck. The analysis of the bone mechanical properties of the left femoral diaphysis, left tibial proximal
metaphysis, and right femoral neck was performed
using the Instron apparatus, model 3342 500 N.
Results were evaluated by using the software
Bluehill 2, version 2.14.
Assessment of the chemical content of the bones
The analyzed bones were examined in regard
to the content of water, organic substances, and mineral substances as well as the content of calcium and
phosphorus. These analyses were performed on L-4
vertebra and left femur and left tibia (following the
bone mechanical properties analysis) as described
before (34, 37). Briefly, in order to determine the
water content, all the bones were lyophilized in the
lyophilizer Labconco FreeZone 6 for five days (temperature: -53OC, pressure: 0.03 mBa) and then
weighed. The difference between the bone mass
obtained directly after the isolation and the bone
mass determined after lyophilization corresponds to
the water content. Subsequently, the lyophilized
bones were mineralized for 48 h in the muffle furnace (type LG/11/C6, Nabertherm) and weighed
again. The difference between bone mass determined after lyophilization and bone mass after mineralization represents the organic substances content. The bone ash remained after the mineralization
process comprised the mass of the mineral substances in bones. The obtained results were presented as the ratio of water, organic, and mineral substances mass per 100 mg of the bone mass after iso-
1075
Effect of dietary flavonoid naringenin on bones in rats...
lation. The bones after mineralization were dissolved in 6 M HCI, then diluted in distilled water in
order to determine the calcium and phosphorus content by the colorimetrical method, using the kit
Pointe Scientific, Inc. The content of calcium and
phosphorus in the analyzed bones was presented as
the ratio of the quantity of calcium and phosphorus
per 100 mg of mineral substances.
Analysis of histomorphometric parameters of the
bones
The histological specimens were prepared and
measured as previously described (38. 39). Briefly,
from the right femoral diaphysis the transverse
cross-sections and the longitudinal sections of the
right femoral distal epiphysis were made. The
obtained sections were ground on the tarnished
glass. These histological specimens were tested for
the following parameters: the width of the trabeculae in the distal femoral epiphysis and metaphysis,
the width of the femoral epiphyseal cartilage, the
area of the transverse cross-section of the femoral
diaphysis, the area of the transverse cross-section of
the cortical part of the femur, the area of the transverse cross-section of the marrow cavity of the
femur and the periosteal transverse growth of the
femoral diaphysis. The ratio of the transverse crosssection area of the marrow cavity per area of whole
diaphysis was evaluated.
The measurements of histomorphometric
parameters were conducted with the use of an
Optiphot 2 microscope connected with an RGB
camera with final magnifications of 200 and 500
times. The results were analyzed by Lucia G
4.51software (Laboratory Imaging). The lanameter
with the magnification of 50 times was used to
assess the areas of the transverse cross-section of the
cortical part and the marrow cavity in the femur.
The results obtained during experiment were
evaluated by one-way ANOVA, followed by the
Duncanís post hoc test. The non-parametric tests:
Kruskal-Wallis and Mann-Whitney U test were performed when necessary (lack of normality of variance). All the results are presented as arithmetic
means ± SEM.
The differences were considered to be statistically significant if in ANOVA test p < 0.05.
The estrogen deficiency had an effect on the
body weight gain after 4 weeks ñ this parameter was
higher by 127.5% in the OVX group in statistically
significant manner than in the SHAM group. After
administration of estradiol, the tendency to lower
body mass gain was noted by 21.9% in comparison
with the OVX group. Treatment with naringenin did
not affect the body mass gain in statistically significant manner, however slight increase of this parameter was observed. After ovariectomy, the uterus
weight was statistically significantly lower in the
OVX rats by 83.9% than in the SHAM rats, and the
administration of estradiol caused, in statistically
significant way, the higher uterus weight in the
OVX + ES rats compared with the OVX rats by
139.6%. The tendency to the decrease of the uterus
weight after administration of naringenin was noted
in comparison with the OVX rats. Pang et al.
observed in their study on ovariectomized mice that
glycoside of naringenin ñ naringin did not cause any
significant changes in the uterus weight in comparison to the untreated animals (40). The thymus
weight was affected by ovariectomy by 62.4% (statistically significant increase) in comparison to the
SHAM rats. The treatment with estradiol caused no
Table 1. Effects of estradiol and naringenin on the body weight gain and weight of organs in ovariectomized rats.
Parameter
SHAM
OVX
OVX + ES
OVX + NRG
Body weight at
the start of the
experiment [g]
222.3 ± 4.5
221.4 ± 6.0
217.7 ± 5.3
217.1 ± 5.7
Body weight gain
after 4 weeks [g]
13.5 ± 0.8
30.7 ± 2.3AA
24.0 ± 1.7
34.9 ± 4.0
Uterus weigh [g]
0.643 ± 0.096
0.104 ± 0.004AA
0.248 ± 0.009BB
0.093 ± 0.004
0.313 ± 0.021
AA
0.503 ± 0.028
0.556 ± 0.030
Thymus weight [g]
0.508 ± 0.051
Results are presented as the means ± SEM (n = 7). AA ñ p < 0.01 ñ statistically significant differences between the OVX and the SHAM
groups; BB ñ p < 0.01 ñ statistically significant differences in comparison with the OVX group.
1076
significant changes in this parameter in ovariectomized rats and administration of naringenin to the
OVX rats caused insignificant increase of this
parameter (Table 1). The analogous results for the
effect of ovariectomy and estradiol administration
had been observed before in other studies (34, 37,
39).
Both the ovariectomy surgery and the administration of estradiol and naringenin did not affect the
weight, length and diameter of the analyzed bones,
only the tendency towards a decrease of the tibia
weight after ovariectomy by 6.8% was noted. The
weight of the L-4 vertebra was altered neither by
ovariectomy nor by administration of analyzed substances (Table 2). The lack of the effect of ovariectomy and estradiol administration on macrometric
parameters of bones was also noted in another study
(37).
The results obtained in the mechanical properties analysis showed that ovariectomy caused the
worsening of several parameters in the tibial bone.
In the OVX rats a statistically significant decrease of
maximal load by 44.1% and fracture load by 44.1%
was observed when compared with the SHAM rats.
The femoral bone was not affected by the ovariectomy, only the tendency in the Youngís modulus (a
decrease by 19.7%) was recorded. The analysis of
the results obtained for estradiol and naringenin
indicated that these substances caused neither
improvement nor the deterioration of the mechanical
properties of analyzed bones, except for the
Youngís modulus in the femoral bone and the displacement for fracture load in the tibia which
changed after estradiol treatment (increase by 21.7%
and decrease by 17.6%, respectively; tendency)
(Table 3). In other studies, ovariectomy also led to
the worsening of mechanical properties of the bones
(34, 37, 39, 40). Pang et al. demonstrated that glycoside of naringenin ñ naringin, shows beneficial
effect on the biomechanical parameters of the tibia
in ovariectomized mice (40). However, there are
reports that naringenin is less bioavailable than its
glycosides (41), thus its effects may be weaker than
those reported for naringin. On the other hand,
Habauzit et al. did not noted any changes in mechanical properties of femur after administration of
naringenin to the old male rats either (42).
In the analyzed bones there was a statistically
significant decrease of the mineral substances content observed in ovariectomized rats when compared
to the SHAM rats by 5.4% in femur and 3.9% in
tibia. Moreover, an increase of the organic compounds by 5.6% in the tibia and an increase of water
content by 11.9% in the L-4 vertebra in the OVX
rats in comparison with the SHAM rats was reported. In addition, in the L-4 vertebra calcium content
was statistically significantly lower by 5.5% in the
OVX rats as compared with the SHAM. The administration of estradiol brought the statistically significant effects only in the L-4 vertebra, where it caused
a decrease of water and an increase of the mineral
content by 6.6% and 6.0%, respectively. The tendency towards an increase of calcium content by
7.2% in the tibia after treatment with estradiol was
also observed, when compared to the OVX. Other
parameters in all the analyzed bones remained
unchanged in the OVX + ES rats. Naringenin
administered at a dose of 50 mg/kg affected the
organic compounds content in the tibia. This parameter was significantly lower by 5.6% than in the
Table 2. Effects of estradiol and naringenin on bone macrometric parameters in ovariectomized rats.
Parameter
SHAM
OVX
OVX + ES
OVX + NRG
FEMUR
Weight [g]
0.668 ± 0.013
0.643 ± 0.016
0.647 ± 0.020
0.652 ± 0.022
Length [mm]
33.37 ± 0.31
33.97 ± 0.51
33.14 ± 0.11
33.58 ± 0.25
Diameter [mm]
3.25 ± 0.02
3.25 ± 0.04
3.23 ± 0.05
3.22 ± 0.0
TIBIA
Weight [g]
0.521 ± 0.008
0.485 ± 0.015
0.496 ± 0.017
0.507 ± 0.020
Length [mm]
37.31 ± 0.29
37.94 ± 0.49
36.95 ± 0.10
37.57 ± 0.35
Diameter [mm]
2.63 ± 0.06
2.58 ± 0.04
2.63 ± 0.06
2.60 ± 0.08
0.164 ± 0.007
0.163 ± 0.010
L-4 VERTEBRA
Weight [g]
0.172 ± 0.005
Results are presented as the means ± SEM (n = 7).
0.162 ± 0.007
1077
Table 3. Effects of estradiol and naringenin on bone mechanical properties in ovariectomized rats.
Parameter
SHAM
OVX
OVX + ES
OVX + NRG
Maximal load [N]
100.8 ± 5.7
102.6 ± 4.1
100.8 ± 5.7
101.6 ± 6.9
Displacement for
maximal load [mm]
0.490 ± 0.023
0.556 ± 0.037
0.473 ± 0.034
0.551 ± 0.040
Fracture load [N]
100.4 ± 5.6
102.6 ± 4.1
100.8 ± 5.7
101.6 ± 6.9
Displacement for
fracture load [mm]
0.493 ± 0.025
0.555 ± 0.037
0.473 ± 0.034
0.551 ± 0.040
Youngís modulus [MPa]
6265 ± 303
5029 ± 289
6120 ± 218
5428 ± 505
82.7 ± 8.3
73.6 ± 6.1
FEMORAL DIAPHYSIS
FEMORAL NECK
Maximal load [N]
83.5 ± 11.1
78.3 ± 6.1
TIBIAL METAPHYSIS
Maximal load [N]
123.8 ± 4.8
69.2 ± 4.3AA
87.4 ± 12.0
75.3 ± 8.7
Displacement for
maximal load [mm]
0.885 ± 0.069
0.953 ± 0.037
0.834 ± 0.077
1.021 ± 0.040
Fracture load [N]
121.1 ± 4.9
67.3 ± 4.5AA
86.6 ± 12.3
72.5 ± 7.8
Displacement for
fracture load [mm]
0.979 ± 0.074
1.104 ± 0.069
0.909 ± 0.065
1.133 ± 0.038
Youngís modulus [MPa]
3171 ± 705
2847 ± 963
5470 ± 1308
3664 ± 850
Results are presented as the means ± SEM (n = 7).
groups.
AA
ñ p < 0.01 ñ statistically significant differences between the OVX and the SHAM
OVX rats and its value was close to that reported in
the SHAM group. Moreover, the tendencies towards
the statistically significant differences after naringenin administration were also observed in the
organic content in the femur (decrease by 3.9%) and
in the L-4 vertebra (decrease by 5.0%). The tendency to increase of calcium and phosphorus content in
the tibia by 9.6% and by 12.0%, respectively, was
also noted (Table 4). Svarnkar et al. demonstrated
that naringenin and its glycoside naringenin-6-Cglucoside reveal beneficial effect on the bone mineralization in vitro. In our study, the administration
of naringenin did not cause significant changes in
the bone mineralization (41). Similar observations
were recorded by åliwiÒski et al, where genistein,
another phytoestrogen, added to osteoblast in vitro
culture enhanced the mineralization of the bones,
but, on the contrary, the administration of this substance to the ovariectomized rats revealed no effect
on this parameter (43).
The periosteal transverse growth of the femoral
diaphysis was statistically significantly higher by
34.9% in the OVX rats than in the SHAM group.
The administration of estradiol to the ovariectomized rats caused a decrease in this parameter by
17.0% and the treatment with naringenin resulted in
a decrease by 12.2% in comparison to the OVX rats.
The changes in these groups were statistically significant. Similar results after ovariectomy and estradiol administration were obtained in the study conducted by Folwarczna et al. (39). The transverse
cross-section area of the cortical bone in the diaphysis in all groups of rats remained unchanged, however, there were tendencies to statistically significant differences in the transverse cross-section area
of the marrow cavity ñ in the OVX group in comparison with the SHAM, there was an increase by
15.4% and a decrease in the OVX + NRG group by
13.9% in comparison with the OVX rats. As a result
of these statistically significant and tendentious
changes, the ratio of the transverse cross-section
area of the marrow cavity per transverse cross-section area of the cortical bone was statistically significantly higher in the OVX rats by 14.5% than in the
SHAM, and statistically significantly lower by 9.0%
in the OVX + NRG than in the OVX group. In
another study, conducted by Klasik-Ciszewska et
al., the ovariectomy also induced an increase of the
ratio of transverse cross-section area of the marrow
cavity per transverse cross-section area of the cortical bone, what indicates the harmful effect of estrogen deficiency on the skeletal system in rats. Also,
in this study, the administration of isoflavone genistein to ovariectomized rats resulted in a decrease in
1078
this parameter, what overlaps with our result after
the treatment with naringenin (44).
Ovariectomy caused a statistically significant
decrease of the width of trabeculae in epiphysis and
metaphysis of femur by 15.2 and 6.0%, respectively, in comparison to the SHAM rats. The administration of estradiol caused a statistically significant
increase of the width of the trabeculae in epiphysis
and metaphysis by 5.2 and 4.0%, respectively, and
the treatment with naringenin by 6.9 and 5.4%,
respectively (Table 5). The worsening of these
parameters after ovariectomy was reported by
Folwarczna et al. as well. The authors also noted an
increase of the trabeculae width after the administration of estradiol to the ovariectomized rats. In
some reports it is concluded, that changes in bone
microarchitecture observed after ovariectomy are
connected with the intensification of bone resorption
and/or with inhibition of bone formation process
(39, 44). Habauzit et al. tested the effect of the citrus
flavonoids naringenin and hesperidin included in the
diet on the skeletal parameters in old male rats. In
this study animals were treated with 0.5% naringenin in the chow. The results obtained in their
study indicated that the administration of naringenin
improves the selected parameters in histomorpho-
Table 4. Effects of estradiol and naringenin on the content of H2O, organic and mineral substances, and calcium and phosphorus content
in bones in ovariectomized rats.
Parameter
SHAM
OVX
OVX + ES
OVX + NRG
FEMUR
H2O
[mg/100 mg bone weight]
27.56 ± 0.79
29.75 ± 0.99
29.11 ± 0.60
30.39 ± 0.52
Organic substances
24.33 ± 0.23
24.77 ± 0.32
24.10 ± 0.18
23.81 ± 0.24
Mineral substances
48.11 ± 0.69
45.49 ± 0.74A
46.79 ± 0.60
45.81 ± 0.46
Calcium [mg/100 mg
mineral substances]
40.01 ± 0.50
39.03 ± 0.83
40.55 ± 0.42
40.13 ± 0.53
Phosphorus [mg/100 mg
mineral substances]
15.47 ± 0.30
15.18 ± 0.33
15.54 ± 0.31
15.84 ± 0.31
TIBIA
H2O
26.11 ± 0.63
26.55 ± 1.02
25.90 ± 0.40
28.22 ± 0.49
Organic substances
25.66 ± 0.42
27.10 ± 0.40A
26.47 ± 0.28
25.56 ± 0.20B
Mineral substances
48.24 ± 0.39
46.35 ± 0.79A
47.63 ± 0.35
46.21 ± 0.34
Calcium [mg/100 mg
mineral substances]
41.43 ± 6.36
38.48 ± 1.05
41.26 ± 0.42
42.18 ± 0.82
mineral substances]
14.79 ± 1.34
14.57 ± 0.76
15.32 ± 0.24
16.31 ± 0.15
L-4 VERTEBRA
H2O
26.51 ± 0.25
29.68 ± 0.58AA
27.72 ± 0.81B
29.33 ± 0.57
Organic substances
27.98 ± 2.02
27.29 ± 0.74
26.67 ± 0.40
25.93 ± 0.18
Mineral substances
45.51 ± 1.97
43.03 ± 0.88
45.61 ± 0.70B
44.75 ± 0.48
Calcium [mg/100 mg
mineral substances]
44.65 ± 0.78
42.21 ± 0.57A
42.28 ± 0.79
41.13 ± 0.68
mineral substances]
16.71 ± 0.55
16.11 ± 0.37
15.46 ± 0.58
16.68 ± 0.47
Results are presented as the means ± SEM (n = 7). A ñ p < 0.05, AA ñ p < 0.01 ñ statistically significant differences between the OVX and
the SHAM groups; B ñ p < 0.05 ñ statistically significant differences in comparison with the OVX group.
1079
Table 5. Effects of estradiol and naringenin on histomorphometric parameters of the femur in ovariectomized rats.
Parameter
SHAM
OVX
OVX + ES
OVX + NRG
Periosteal transverse
growth of the
diaphysis [mm]
33.08 ± 3.87
44.61 ± 1.99A
37.03 ± 1.41B
39.17 ± 0.38B
Transverse crosssection area of the
cortical bone in the
diaphysis [mm2]
6.514 ± 0.127
6.185 ± 0.172
6.240 ± 0.173
6.158 ± 0.302
Transverse crosssection area of the
marrow cavity [mm2]
2.821 ± 0.191
3.256 ± 0.128
3.029 ± 0.151
2.805 ± 0.122O
Transverse crosssection area
of the marrow cavity/
diaphysis ratio
0.301 ± 0.015
0.345 ± 0.007A
0.326 ± 0.013
0.313 ± 0.007B
Width of trabeculae
in epiphysis [mm]
63.36 ± 1.20
53.70 ± 0.77AA
56.50 ± 0.51B
57.39 ± 0.65BB
Width of trabeculae
in metaphysis [mm]
41.78 ± 0.76
39.26 ± 0.34A
40.82 ± 0.25BB
41.39 ± 0.63B
Width of cartilage [mm]
55.21 ± 2.76
53.82 ± 2.59
47.91 ± 1.76
50.84 ± 2.29
A
AA
Results are presented as the means ± SEM (n = 7). ñ p < 0.05, ñ p < 0.01 ñ statistically significant differences between the OVX and
the SHAM groups; B ñ p < 0.05, BB ñ p < 0.01 ñ statistically significant differences in comparison with the OVX group.
logical analysis of the bones; an increase of the bone
mineral density in the femur metaphyseal zone was
recorded (42). Swarnkar et al. demonstrated that
ovariectomy caused the deterioration of all the analyzed histomorphological parameters in mice while
the administration of naringenin showed the preventive effect on these bones. They also pointed that
glycoside of naringenin administered at the same
dose as the aglycone (5 mg/kg) to the ovariectomized mice induced a better trabecular response
than naringenin itself, due to the fact that glycoside
was 1.5-fold more bioavailable (41).
The results obtained in our study indicate that
the most evident effects of naringenin can be
observed in the histomorphometric parameters in
trabecular bone. The increase of the width of trabeculae may be connected with resorption inhibition by
this flavanone. The scientific report presented by La
et al. indicates that naringenin shows the potency to
inhibit the osteoclastogenesis and osteoclastic bone
resorption by decreasing the secretion of the interleukins related with osteoclastogenesis (45).
Moreover, the profitable effect of naringenin on
bone tissue may be associated with its higher affinity towards ERβ than ERα. Roelens et al. examined
affinity of naringenin to ERs and they reported that
this flavanone shows 10 ◊ 103 times lower affinity
towards ERα and 6 ◊ 103 times lower towards ERβ
than estradiol. Estrogen receptors β play crucial role
in the maintenance of the correct bone mass during
menopause (26, 46), thus naringenin binding to this
type of receptors may prevent the further deterioration of the bones in postmenopausal osteoporosis.
As mentioned above, Swarnkar et al. observed
that naringenin proved to have a preventive effect in
some parameters in femoral epiphysis microarchitecture, but there were no signs of the bone formation stimulation ñ there were no changes in the bone formation
rate or the mineral apposition rate after naringenin treatment in comparison with the OVX mice (41).
There are also some reports indicating that
naringenin shows antiestrogenic effects. This flavanone exhibits a weak antagonistic activity towards
ERα ñ receptors that mostly occur in endometrium,
ovaries and mammary gland. Blocking the ERα may
result in the intensified body weight gain and the
decrease of the uterus weight, what was proved in
estrogen receptor-α knockout mice (47, 48). The
estrogen deficiency also leads to an increase of the
thymus weight (49). In our study, as mentioned
above, some changes which may be associated with
antiestrogenic activity of naringenin were observed
in comparison with the OVX group ñ enhanced
body weight gain, decreased weight of the uterus
and increased thymus weight, yet all these alterations were statistically insignificant (Table 1).
1080
The lack of beneficial effect of naringenin on
the mechanical or chemical parameters of bones in
ovariectomized rats may also be a result of aromatase (estrogen synthetase) inhibition by this
flavonoid. Naringenin can bind to the aromatase due
to its structural similarity to the androgens (A and C
rings of naringenin resemble D and C ring of
steroidal aromatase substrates) what may lead to the
suppressing of estrogen biosynthesis and the bone
formation processes (50, 51). The decrease of
periosteal transverse growth of the femoral diaphysis observed after naringenin administration may
be associated with this inhibited bone turnover
(Table 5).
CONCLUSION
Due to its both agonistic and antagonistic
activity towards ERs, naringenin shows bidirectional effect on the ovariectomized rats. This flavonoid
does not induce improvement in the chemical composition or mechanical properties in bones and may
suppress the bone formation and resorption processes, but it does not lead to further osteoporotical
changes during menopause. On the other hand, the
preventive effect on bones is observed in histomorphological parameters in the femoral bone. Thus, it
can be assumed that naringenin does not reveal any
harmful effect on osteoporotically altered bone tissue and it even shows the beneficial effect on the
bone microarchitecture in postmenopausal osteoporosis in ovariectomized rats.
Acknowledgment
The authors are grateful to the staff of
Department of Pharmacology (School of Pharmacy
with The Division of Laboratory Medicine, Medical
University of Silesia) for making this study possible.
This study was supported by grant no. KNW-1021/N/4/0.
The authors state that they have no conflict of
interests.
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Received: 13. 09. 2015
ISSN 0001-6837
GENERAL
TELEVISION ADVERTISING OF SELECTED MEDICINAL PRODUCTS
IN POLAND AND IN THE UNITED STATES ñ A COMPARATIVE ANALYSIS
OF SELECTED TELEVISION COMMERCIALS
EWA WIåNIEWSKA1, ALEKSANDRA CZERW1,
MARTA MAKOWSKA2 and ADAM FRONCZAK1
1
Public Health Department, Medical University of Warsaw,
Banacha 1a, 02-097 Warszawa, Poland
2
Department of Social Sciences, University of Life Sciences,
ul. Nowoursynowska 166, no. 4, 02-787 Warszawa, Poland
Abstract: The aim of the analysis was to establish the differences between television commercials of OTC
drugs broadcast in Poland and in the U.S. The study covered 100 commercials of medicinal products of various producers applied to treat a variety of symptoms and diseases. The analysis demonstrated that there are both
similarities and differences. The differences concerned e.g., spot length, the time of placement of a brand name
and the diversity of advertising slogans. The most significant similarities concerned applied manipulation techniques, locations featured in commercials and the choice of actors.
Keywords: television advertising, OTC drugs, content analysis, USA, Poland, comparative analysis
As an element of marketing television advertisement facilitates the communication between a
company and market, and stimulates product sales.
The most popular form of television advertising are
the so-called TV commercials broadcast during commercial breaks between TV programs or during the
programs. The length of an advertising transmission
per one hour of a program in Poland is 12 min (1).
Until recently, commercials had an average length of
60 s. Today, 30-s commercials prevail. In a report
that comprised an analysis of the market in 2013 the
National Broadcasting Council of Poland stated that
average Pole spends 4 h and 7 min daily in front of a
TV (2), and thus comes before Americans who spend
2.5 h per day watching television (3). The report of
Polish Internet Research (Polskie Badanie Internetu)
demonstrates that television advertising is still an
essential element of promotion. Sixty eight % of the
society in Poland learns about new products or new
offer via this form of advertising (4). The greatest
advertisers in 2014 in Poland were pharmaceutical
companies selling OTC drugs (5).
Medicinal product advertising in Poland is regulated by a few legal acts. These include:
Pharmaceutical Law Act of 6 September 2001, as
amended; the Act on Reimbursement of Costs of
Drugs, Foodstuffs for Particular Nutritional Uses
and Medicinal Products of 12 May 2011; the
Regulation of the Minister of Health of 21
November 2008 on advertising of medicinal products (6). The legislative actions in this respect are
guided by European Union directives, i.e. in particular, the Directive of the European Parliament and
Council no. 2001/83/EC of 6 November 2001 on the
Community code relating to medicinal products for
human use.
In the United States, the issue of advertising of
medicinal products is regulated by the United States
Federal Food, Drug and Cosmetic Act (abbreviations: FFDCA, FDCA, FD & C), the regulations of
the Food and Drug Administration (FDA) and the
Federal Trade Commission Act (FTC Act). Unlike
Poland, the United States allows for advertising of
prescription drugs (DTC ñ Direct-to-consumer)
alongside OTC drugs to a wide circle of consumers.
Both Polish and U.S. laws provide for a range of
penalties for breach of regulations regarding advertising of medicinal products (7).
* Corresponding author: e-mail: aleksandra.czerw@wum.edu.pl
1083
1084
EWA WIåNIEWSKA et al.
Table 1. Types of drugs covered by the study.
Drug type
Number of
products
Painkillers
17
Cough medicines
10
Allergy medications
10
Cold medicines
8
Sore throat medicines
8
Heartburn drugs
7
Constipation medications
4
Medications for symptoms associated
with venous and lymph circulation
3
Runny nose medications
3
Sinus symptoms medications
2
Antidiarrhoeal medicines
2
Drugs for symptoms of liver diseases
1
Motion sickness medications
1
Drugs for eye irritation/fatigue
1
Herpes simplex medications
1
Drugs for symptoms of prostatic
hyperplasia
1
Antifungal drugs
1
Excessive flatulence gas medications
1
Various effects
19
EXPERIMENTAL
Method
The aim of the analysis was to establish the differences in television commercials of OTC medicinal products broadcast in Poland and in the United
States. A hundred purposefully sampled commercials were covered by the study ñ 50 for each country. The sample covered commercials of medicinal
products of various producers intended to treat various symptoms. For Poland the analysis covered e.g.,
nine commercials of USP Zdrowie products, three
commercials of Polpharma S.A. products, three
commercials of Bayer Group products, and two
commercials of GlaxoSmithKline, PPF Hasco-Lek
S.A., Berlin-Chemie, Novartis, Sandoz, Sanofi,
Teva, Omega Pharma Poland Sp. z o.o., Aflofarm
and Polfa Warszawa S.A. (the company is currently
a member of Polpharma S.A. group ñ mentioned
earlier ñ but the commercial was broadcast at a time
when the companies were not linked) products. For
the United States the analysis covered e.g., eleven
commercials of Bayer Group products, six of
McNeil products, five of Reckitt Benckiser, Pfizer
Inc. and Novartis products, four of Sanofi products
and two for Prestige Brands, Procter & Gamble and
Boehringer Ingelheim products. In all, the analysis
covered 37 producers. The study examined commercials of medicinal products for 19 registered
indications for use (Table 1). In Poland these included: 8 pain medications, 7 cough medicines, 6 sore
throat medicines, 4 cold medicines, 3 runny nose
medications, 3 medications for symptoms associated
with venous and lymph circulation, 2 heartburn
drugs, 2 constipation medications, 2 antidiarrhoeal
medicines and 1 sinus symptoms medication, 1
medication for symptoms of liver disease, 1 allergy
medication, 1 herpes simplex medication, 1 medication for symptoms of prostatic hyperplasia, 1 antifungal drug. Furthermore, in 7 cases the producers
listed more than one indication for use for one drug:
2 medications for pain, cold, fever and runny nose;
1 for pain and cold; 1 for pain and fever; 1 for constipation and indigestion; 1 for pain, cough, fever
and sinus symptoms, 1 for pain, cough, cold, fever
and runny nose. The drugs advertised in the United
States included: 9 pain medications, 9 allergy medications, 5 heartburn drugs, 4 cold medicines, 3
cough medicines, 2 constipation medications, 2 sore
throat medicines, and 1 sinus symptoms medication,
1 motion sickness medication, 1 drug for eye irritation / fatigue, 1 excessive flatulence gas medication.
Furthermore ñ similarly to the situation in Poland ñ
the producers sometimes listed several indications
for one drug. This was found in 12 cases: 2 medications for cough and cold; 1 for pain and fever; 1 for
cold and runny nose; 1 for pain and good sleep; 1 for
cold and pain; 1 for cough and sore throat; 1 for
sinus and allergy symptoms; 1 for sinus symptoms
and runny nose; 1 for pain, cold and fever; 1 for
sinus symptoms, runny nose and allergy symptoms;
1 for pain, cold, fever and runny nose. The differences also applied to the active ingredients of drugs
(Table 2).
For the purposes of the analysis of commercials broadcast in Poland commercials uploaded on
YouTube between March 2007 and December 2014
were used (www.youtube.com). Despite significant
differences ñ including phonetic and grammatical ñ
between various English dialects spoken in the
English speaking countries, the use of videos
uploaded on the above website for the purposes of
the analysis of commercials broadcast in the United
States was unfeasible due to high risk of confusion
of videos from the U.S. with videos from the other
English speaking countries. This is why videos
uploaded on the American website iSpot.tv
(www.ispot.tv) were used for the purposes of the
analysis. In a Microsoft Excel file the following data
were gathered: country, drug name, brand name,
commercial length, date of publication on
Television advertising of selected medicinal products in Poland and...
YouTube/last broadcast according to iSpot.tv, the
time of placement of drug name, the time of placement of brand name, type of drug, distinctive words,
music, actors, actorsí age, atmosphere, setting,
manipulation, dominant color, advertising slogan
and recognizable character.
RESULTS
Thirty-second long commercials prevailed
among those examined. This applied to both Poland
(84%) and the United States (50%). A relatively
higher percentage of 15-s commercials was recorded for the U.S. (48%). In Poland, shorter commercials made up only 10%. In the case of Poland there
were also some commercials of 20 and 45 s (4% and
2%), and in the United States commercials of 10 s
1085
(2%) (Chart 1).
The trade name of drugs read aloud or displayed in a textual form in the commercials broadcast in Poland usually, i.e., in 48% of cases,
appeared at the beginning of the commercial, i.e., up
to 1/3 of its length (for 30-s long commercials up to
the 10th s) and in the middle, i.e., for 30-s long commercials between the 10th and 20th s. Occasionally
(4%), the name was displayed at the top or at the
bottom of the screen throughout the entire commercial. In none of the analyzed commercials did the
advertisers choose to place the trade name of the
medicinal product at the end of the commercial. In
the United States, the names of drugs that appeared
at the beginning of the commercial made up 58%. In
38% of the commercials the trade name was placed
in the middle of the commercial. Placement during
Chart 1. Commercial length
Chart 2. Time of trade name placement
1086
Table 2. Active ingredients of drugs covered by the study.
Poland
Active ingredient
Ibuprofen
United States
No. of drugs
3
Active ingredient
Ibuprofen
No. of drugs
4
Diosmin
3
Loratadine
3
Xylometazoline hydrochloride
2
Acetaminophen
2
Loperamide hydrochloride
2
Acetaminophen, aspirin, caffeine
2
Omeprazole
2
Calcium carbonate
2
Paracetamol, pseudoephedrine
hydrochloride, dextromethorphan
hydrobromide
2
Cetirizine hydrochloride
2
Acetylcysteine
1
Acetaminophen, caffeine
1
Aciclovir, hydrocortisone
1
Acetaminophen, chlorpheniramine maleate
1
2,4-Dichlorobenzyl alcohol,
amylmetacresol
1
Acetaminophen, dextromethorphan
hydrobromide, doxylamine succinate
1
2,4-Dichlorobenzyl alcohol,
amylmetacresol, levomenthol
phenylephrine hydrochloride
1
hydrobromide, doxylamine succinate,
Benzocaine, cetylpyridinium chloride
1
hydrobromide, guaifenesin,
1
Bisacodyl
1
hydrobromide, phenylephrine
hydrochloride
1
Dextromethorphan hydrobromide,
tilia flower extract
1
Acetaminophen, phenylephrine
hydrochloride
Benzoxonium chloride, lidocaine
hydrochloride
1
Anhydrous citric ccid, sodium
bicarbonate
1
Ambroxol hydrochloride
1
Aspirin
1
Benzydamine hydrochloride
1
Aspirin , caffeine
1
Bromhexine hydrochloride
1
Aspirin, chlorpheniramine maleate,
phenylephrine bitartrate
1
Drotaverine hydrochloride
1
Benzocaine
1
1
Fenspiride hydrochloride
1
Bisacodyl USP
1
Oxymetazoline hydrochloride
1
Dextromethorphan
1
Pseudoephedrine hydrochloride,
triprolidine hydrochloride
1
doxylamine succinate
1
Terbinafine hydrochloride
1
1
Butamirate citrate
1
Diphenhydramine hydrochloride
1
Diclofenac diethylamine
1
Diphenhydramine hydrochloride,
naproxen sodium
1
Soybean phospholipids with
(3-sn-phosphatydyl)choline
1
Diphenhydramine hydrochloride,
1
Ibuprofen, pseudoephedrine
hydrochloride
1
Docusate sodium, benzocaine
1
Ketoprofen
1
Dyclonine hydrochloride
1
Acetylsalicylic acid
1
Esomeprazole magnesium
1
Acetylsalicylic acid, phenylephrine
hydrochloride, chlorphenamine
maleate
1
Fexofenadine hydrochloride
1
1087
Table 2. Cont.
Poland
United States
Active ingredient
No. of drugs
Active ingredient
No. of drugs
Acetylsalicylic acid, ascorbic acid
1
Fexofenadine hydrochloride,
pseudoephedrine hydrochloride
1
Dehydrocholic acid
1
Fluticasone propionate (glucocorticoid)
1
Chlorpheniramine maleate, pseudoephedrine
hydrochloride, paracetamol
1
Glycerin, hypromellose, polyethylene
glycol
1
Meloxicam
1
Guaifenesin, dextromethorphan
hydrobromide
1
Metamizole sodium
1
Loratadine, pseudoephedrine sulfate
1
Paracetamol, guaifenesin, phenylephrine
hydrochloride
1
Meclizine hydrochloride
1
Paracetamol, caffeine
1
Phenol
1
Paracetamol, ascorbic acid,
pheniramine maleate
1
Pseudoephedrine hydrochloride
1
Rutin, ascorbic acid
1
Ranitidine hydrochloride
1
Naproxen sodium salt
1
Simethicone
1
Dried senna fruit extract standardized
to hydroxyanthracene glycosides
content
1
Triamcinolone acetonide
1
Coltsfoot leaf extract, thyme extract
1
Trolamine salicylate
1
Saw palmetto extract
1
Table 3. Distinctive words used in adverts.
Percentage
Words
Poland
United States
fast/instant
20%
32%
relieve/alleviate
8%
52%
combat/fight off
26%
0%
powerful/the most
powerful
12%
26%
disappear
22%
0%
remove/eliminate/get
rid of/free from
18%
4%
effectively
16%
10%
make easier
6%
2%
protection
2%
6%
safe
2%
2%
restore
2%
2%
improve
4%
0%
anaesthetize
4%
0%
support/help
4%
0%
well-tried
4%
0%
comprehensive
4%
0%
serious
0%
4%
No distinctive words
20%
6%
the whole length of a commercial (2%) or at the end
of it was rare (Chart 2).
In the commercials broadcast in Poland the
brand name was usually read aloud or displayed on
the screen at the end of the commercial (52%) or in
the middle of it (26%). In 10% of cases the information about the producer was placed at the start, and
in 6% of commercials the name was displayed on
the screen the whole time. In 2% of the analyzed
commercials the information about the producer of
the medicinal product did not appear at all, which is
inconsistent with the applicable laws. In the case of
American commercials the situation was different.
As many as 76% of commercials failed to give the
name of the producer. In 2% of cases the brand
name was displayed on the screen the whole time or
appeared in the middle of the commercial. In none
of the analyzed commercials did the brand name
appear at the start or at the end of the commercial
(Chart 3).
The commercials were also analyzed in terms
of the use of the so-called distinctive words. In this
respect it was observed that the most frequently used
distinctive words in Polish commercials are ìcombat/fight offî and ìpowerful/the most powerfulî
(26%), while in the United States ìrelieve/alleviateî
prevailed (52%). In both countries the use of
1088
Chart 3. Time of brand name placement
Chart 4. Music in television adverts of OTC drugs
Chart 5. Actors in television adverts of OTC drugs
Chart 6. The age of actors in television adverts of OTC drugs
Chart 7. Emotions in television adverts of OTC drugs
Chart 8. The setting of television adverts of OTC drugs
1089
1090
Table 4. Advertising slogans that appeared in the analysed commercials (arbitrary sequence).
Advertising slogans
Poland
Delicious raspberry Ibum, and the fever and
pain are gone
United States
Fast acting/relief doesn't get any better than this
Heartburn under control
Fast acting
Aspiryn for cold. Aspirin for pain
Break the grip of pain
We have a well-tried remedy for dry cough
Targeted relief with the power of Bayer
Naxii for a whole day without pain
Tough on pain, gentler to your stomach
Xenna Extra for constipation - works the
way Nature wanted
Headache gone
I know nothing will stop me - even pain
No pill relieves heartburn faster
It improves your quality of life!
Get moving again
Apap Extra - Discover its Extra power!
Oh what a relief it is
Aspirin. Stronger than cold
Live Claritin Clear
And you're back in action
Six is greater than one. This changes everything
Gripex - fights off all symptoms of flu
Nexium Level Protection
Relief in discomfort
Aleve pm for a better am
FlavamedÖ and the cough is gone
For what matters most
Diarrhoea attacks? Laremid acts!
Same relief as dramamine, less drowsy
Ketonal Lek - power and trust
Wow! That was fast!
Tabcin. Don't waste your time on flu
Up to 10 hrs comfort
Fervex. Pass it on/ Fervex. A healthy family
Start the relief. Ditch the misery. Let's end this.
No-Spa. Enjoy the moment, don't waste time on pain
Silence is relief
Strepsils. First aid in throat issues
Enjoy the relief
Number 1 during cold and flu season
Relief with a Smile
There isn't any faster painkiller
Stop Suffering. Start Living.
After Flegamina the cough will be gone
Nasacort stops more of what makes you miserable
100% with you
Satisfaction guaranteed or your money back
One medication, two benefits for your health
Live Claritin Clear. Every Day.
Acatar. My choice for a runny nose
Live Claritin Clear
SUDAFED XyloSpray HA. A break from allergy
Muddle No More
Pressure and pain are gone
The nighttime, sniffling, sneezing, coughing,
aching, fever, best sleep with a cold, medicine
Stops viruses. Treats herpes simplex. Fast.
Powerful sinus & allergy medicine from
the makers of Vicks NyQuil and DayQuil
Helps to eliminate sore throat EVEN in men
Serious Power
First for sick throat
Enjoy the Relief
Well-tried medication for your child
Oh what a relief it is
A wise Pole before a sore throat
Start the Relief. Ditch the Misery. Let's End This
UNDOFEN MAX KREM Brings mycosis to its knees
Mucinex In. Mucus out
The only gel effective for up to 12 h
Don't Suffer the Coughequences
Excellent for dry cough!
Serious medicine. Seriously great taste
Moms don't take sick leave, moms take Vicks /
Breathe in life to the fullest
Real relief. Real fast
Runny nose will go away with the gel
Be Ready
Modern remedy for heartburn
We care for kids
1091
Table 4. Cont.
Advertising slogans
Poland
Fast acting and mild
United States
The secret to feel better is simple - Simply Saline
Relief from constipation every morning
It's all in the tin
PYRALGINA No. 1 for great pain
Open Up
No pain during the day and at night
Trust in Triaminic
SOLID SUPPORT for your legs
With the decongestant pharmacist trust most
and the formula our communities trust, too
AFLAVIC - It's time for healthy legs
DocuSol, That's All!
Positive effect
The overnight relief you're looking for
Fast remedy for cough
The Gas Xperts
Tantum Verde - it really works!
Fast acting
Comprehensive medicine for cold and flu
Man's thing. Man's medicine
ìfast/instantî was equally frequent ñ in Poland they
were present in every fifth commercial (20%), and
in the United States in every third commercial
(32%). In 20% of commercials of medicinal products aired in Poland there were no distinctive words.
In all, there were 17 distinctive words used in drug
commercials in Poland and in the U.S. (Table 3).
For the purposes of comparative analysis of
commercials the authors also employed the music
criterion. In both countries the music used in drug
commercials usually had a fast tempo (from 93
BPM). This was observed for 38% of commercials
in Poland and 44% of commercials broadcast in the
U.S. In Poland, the use of a shifting tempo was
equally common (34%), while there were few commercials with such a tempo in the United States
(10%). In over 20% of analyzed commercials from
both countries music with a slow tempo (up to 92
BPM) was employed. In Poland there were few
commercials with no music (4%) while in the
United States every fourth commercial does not feature any music (Chart 4).
The actors starring in commercials of OTC
drugs broadcast in Poland and the United States
were usually adults. In Polish commercials women
prevailed over men (84%). Men were present in
66% of the analyzed commercials. In the United
States women were present in 76% of commercials
and men in 64% of commercials. In Poland there
were children in 26% of analyzed commercials, and
occasionally animated characters were present (4%).
In 4% of cases the actors were not present. In the
U.S. every third commercial featured children. In
14% of commercials there were animated characters
and animals. 8% did not feature any actors (Chart 5).
When it comes to the age of the actors starring in the
commercials it was found that in both countries the
majority of actors were adults ñ 94% for Poland and
88% for the United States (Chart 6). In 34% of commercials broadcast in Poland there were children.
Eight % of analyzed commercials did not feature
any actors. In the United States, children were present in 36% of commercials. In 12% of cases in the
U.S. there were no actors in the commercials.
However, the commercials featured animated characters/animals (Chart 6).
In 66% of commercials in Poland and in 76%
in the United States there was more than one actor in
one commercial. They were assigned separately to
the appropriate group (women and men), which is
why the total number in both analyzed cases exceeds
100%.
The atmosphere in the commercials was also
subject to analysis. It was found that in nearly half
of the commercials broadcast in both countries the
feelings were mixed. At first, the situation was presented in a negative light only to become positive
after the use of the drug. In Poland this was found in
44% of the analyzed commercials and in the United
States in 46% of commercials. In Poland, the opposite was observed in 28% of commercials. At first,
the atmosphere in the commercial was positive then
it evolved to a negative one, and finally, after drug
use, the situation was restored to an atmosphere of
satisfaction. In the United States, 26% of the commercials evoked positive associations. Other emotions present in commercials in both countries
included: mystery and tension, happiness, insecuri-
1092
Chart 9. Manipulation
Chart 10. Dominant colors in television adverts of OTC drugs
1093
Chart 11. The presence of a recognizable character in television adverts of OTC drugs
ty. Producers in Poland rarely referred to negative
associations (2%), and the American producers did
not refer to them at all. Particular data on feelings
are presented on Chart 7. The feelings are presented
according to the incidence ñ from the most frequent
to those that appeared rarely.
The analyzed commercials were subject to an
analysis also in terms of the setting. The setting usually employed in the commercials in both countries
was home (34% of Polish commercials and 40% of
American commercials). In 34% of commercials in
Poland and 26% in the United States other locations
were used (restaurant, airport, hotel, school, cinema,
ice hockey rink etc.). In both countries 26% of commercials were set outdoors (garden, meadow etc.).
In both countries the setting of around 20% of commercials was hard to determine. Six % of Polish
commercials and 10% of American commercials
were set in many locations i.e., the action at home,
on a busy street or in a shop etc. was combined. The
least popular location was the office ñ 4% in Poland
and 2% in the United States (Chart 8).
If any commercial was set in two places, the
locations were assigned individually to appropriate
groups. If there were three or more locations used in
one commercial they were assigned to ìVaried (3
and more)î group.
The manipulation mechanisms and techniques
present in the commercials were also subjected to
analysis. It was found that the most common manipulation employed in the analyzed commercials was
the suggestion that medication users would not need
to give up on pleasant activities/plans/work. This
pertained to half the commercials broadcast in
Poland (50%) and every third commercial broadcast
in the United States (34%). In Poland, 18% of com-
mercials appealed to parental feelings (ìbe a good
parent and give the medicine to your childî) and in
the same number of commercials the drug was recommended by one person to another person. In the
case of the United States, in 20% of commercials it
was suggested that the drug use will help in regaining the sense of joy in life, and 16% of commercials
used humor. In 4% (Poland) and 8% (the United
States) the employed manipulation was not classified. All types of manipulation techniques found in
the analyzed commercials of medicinal products are
listed on Chart 9. The data are presented according
to the incidence ñ from the most frequent to the
rarest.
When it comes to the analysis of the commercials in terms of the used colors it turned out that
46% of commercials broadcast in Poland and 68%
of commercials broadcast in the United States did
not have any dominant color. In 20% of commercials in Poland and in 16% of commercials in the
United States the dominant color was blue. In
Poland, 14% of commercials were dominated by
red, and 12% were dominated by white. In 6% of
commercials in the U.S. white and green dominated.
In 16% of cases in Poland and similarly in the
United States there was other dominant color (violet, yellow, grey, brown etc.) (Chart 10).
Sixteen percent (Poland) and 10% (the United
States) of commercials had two dominant colors.
Each was assigned individually to one of the groups
presented on Chart 10. If there were more than 2
dominant colors it was assumed that none of the colors predominates.
The analysis also covered the used advertising
slogans. It was observed that the slogans used in
commercials in both countries are quite diverse.
1094
Only one of the commercials broadcast in the United
States did not feature any advertising slogan. All
advertising slogans that appeared in the analyzed
commercials are presented in Table 4. They are presented in an arbitrary sequence.
In 44% of commercials in Poland and 22% of
commercials in the United States the advertising slogan featured the name of the drug, e.g., ìAfter
Flegamina the cough will be goneî (ìKaszel minie
po Flegaminieî) or ìTrust in Triaminicî.
The analysis of the commercials also concerned the presence of recognizable characters who
draw attention and make the commercial more
memorable, e.g., Mrs. Goüdzikowa who appeared in
Polpharmaís EtopirynaÆ commercials, or an animated droplet of mucus in the American commercial
of MucinexÆ by Reckitt Benckiser (Chart 11).
DISCUSSION
The pharmaceutical market in the U.S. was
estimated at $395.2 billion in 2014 (8). It made up
40% of the total value of the global pharmaceutical
market (9). Americans spent about $40 billion on
OTC medications in 2014 (10). The Polish pharmaceutical market seems small in comparison. In 2014,
the value of the market was PLN 28.5 billion ($7.4
billion1). Poles spent about PLN 828,000 ($215,000)
on OTC drugs in 2014 (11). Currently, the Polish
market is the sixth largest in Europe and the largest
in Central and Eastern Europe (12). In both countries the value of the pharmaceutical market
increased every year between 2002 and 2012. Year
2012 changed this trend in the U.S. and in Poland,
but the next year the market started to grow again in
both countries (13). The prognoses for both markets
are optimistic. Their value will rise for a number of
reasons: aging population, development of medicine, increased health awareness in the society,
increased incidence of diseases of civilization,
mutating diseases and their resistance to certain
drugs. In Poland it is also: improvement of the financial situation of society. In the U.S.: health reform
and insurance coverage for more people and an
increasing number of citizens (13). We cannot forget about one more crucial factor ñ advertising. In
both countries pharmaceutical companies spend a
lot of money for this purpose and are considered to
be those who conduct aggressive promotion.
In 2012, pharmaceutical companies in the U.S.
spent more than $27.5 billion dollars on promotion and
marketing, out of which $24 billion on promotion
among doctors and $3.5 billion on consumer promo-
1
Dollarís exchange rate ñ $1 to PLN 3.85 (02.11.2015)
tion. For DTC companies spend 12.5% of their marketing budget (14). The United States is the only country in the world, except New Zealand, where advertising of prescription drugs may be directed to the public.
In Poland, only OTC drugs can be advertised to
the public. In 2013, the pharmaceutical sectorís
spending to this end in the media increased by 20%
and amounted to PLN 3.7 billion ($0.96 billion).
The following companies invest the most in OTC
drug promotion: Aflofarm Farmacja Polska (PLN
860 million), USP Zdrowie (PLN 366 million),
Olimp Laboratories (PLN 171 million), Reckitt
Benckiser (PLN 162 million) GlaxoSmithKline
Consumer Healthcare (PLN 151 million). Their
largest expenses are associated with television
advertisements (15).
Advertising plays a crucial role in providing the
basic information about the pharmaceuticals and influencing patientsí decisions about the purchase. U.S.
research suggests that the general perception of OTC
advertisement is rather unfavorable (16, 17). U.S. customers watching television may see twice as many
OTC advertisements as DTC ads (16, 18). Polish
research shows that slightly more than a half of the
respondents have positive opinion about drug ads (19).
The results of a research study by Diehl et al. show
that people may not rely on pharmaceutical advertising, but they tend to believe it. Additionally, they are
less doubtful about their informational content compared to advertisements of other products (17).
A review of the literature revealed that comparisons between the U.S. and Poland concerning
OTC television advertisement had not been made
before. We have identified four studies in the U.S.
which analyzed the content of OTC television commercials (18, 20-23,) and three in which the content
of printed OTC advertisement was analyzed (22, 24,
25). They all used different methods of analysis,
which is the reason why they are described separately below.
In 1997, Tsao analyzed informational and symbolic content of 150 television OTC advertisements.
His study indicated that topicals, respiratory, central
nervous system, gastrointestinal, nutritional medications and antiperspirants were the most frequently
promoted products. He found that there were on
average 3 information cues per OTC advertisement.
The information in ads often highlighted benefits
instead of the reasons to use the drug. Only 18% of
OTC advertisements provided complete information
(name and functions) about the promoted drug. Tsao
was concerned about the readability and preciseness
of disclosure of side effects and drug performance in
commercials. He concluded that product awareness
is the primary communication goal of OTC advertisement, and commercials do not provide customers
with valuable information (20).
In their study Byrd-Bredbenner and Grasso
(1999) were brought to a similar conclusion. OTC
commercials make people believe that taking a drug
is an easy, rapid and risk free way of getting rid of
health problems. They analyzed the health content
of advertisements broadcast during prime-time network programs for children. Twenty nine (out of
298) commercials with health content concerned
drugs (25 were OTC drug commercials). All of them
advised to use the drug according to directions, few
named side effects, drug interactions or advised to
read the instructions on the label (21).
Brownfield et al. (23) analyzed quantity, frequency and placement of RX and OTC advertisements on television. They analyzed 504 hours of
network TV programs broadcast in the summer of
2001. A total of 18,906 commercials were analyzed. The OTC advisements accounted for 4.8%
and DTC for 2.4%. The OTC ads were more common, but the DTC were significantly longer. The
length of an average OTC commercial was 21.7 s
and DTC commercials lasted on average 42.1 s. The
length of all commercials of high cost drugs was 60
s. The OTC commercials were usually aired in the
midafternoon (2-4 p.m.) and the early evening (6-8
p.m.). The main target audience of those commercials were older adults and females. Brownfield et
al. estimated that Americans are exposed to more
pharmaceutical information each year compared to
other forms of health information ñ for example,
comparing time of doctorís visits and pharmaceutical advertising they discovered that for every
minute spent with a physician Americans watch
100 min of ads (23).
Faerber and Kreling (18) analyzed the content
of TV commercials of drugs that switched from prescription to OTC ñ there were 98 ads of three products (before and after switching) broadcast in the
period between 1996 and 2009. The OTC ads contained the same proportion of information as prescription ads, but they used more appeals (9.1 vs.
6.0). The most popular appeals were: control, convenience and long-lasting effect. That shows different marketing attitudes towards the same drug after
changing the way customers can buy it (18).
Similarly to other research studies (20, 21), they
found out that television advertisements of OTC
drugs often do not contain good informative content
about drug use and the medical condition which this
medicament treats.
While looking for a comparison of drug
advertisements aired in the two countries we
1095
found the Kansal (24) research that compared the
U.S and India. The study does not concern television commercials but OTC advertisements in
magazines. However, such comparisons seem to
be unique, so it is worth a mention. The results of
this study showed that the U.S. advertisements
were better balanced in comparison to Indian ones
in terms of the use of informative and attractiveness indications. The Indian ads had on average
fewer information cues and additionally they had
inferior quality. The results highlighted that in
India advertisements try to attract customers by
showing models or painting a nice picture by
using emotional, youth, attractive, excitement
appeals. The author claims that Indian OTC marketers should become more ethical and socially
responsible (24).
Sansgiry et al. (25) made a content analysis of
advertisements from U.S. consumer periodicals. The
OTC advertisements accuracy based on federal
guidelines was judged independently by five clinical
pharmacists. Reviewers found out that around 50%
of advertisements did not contain accurate statements. Only one did mention the side effects. This
study showed that the OTC advertisements did not
provide customers with adequate information that
would allow them to make responsible purchases.
The lack of proper information can lead to harmful
events (25).
Main, Argo, and Huhmann (22) made a content
analysis of 195 advertisements of DTC drugs, 137
advertisements of OTC remedies and 33 ads of
dietary supplements from 30 national circulation
magazines in the United States. They did not find
any difference in the use of rational appeals between
the three groups of products, although they discovered that DTC used more often positive and more
negative emotional appeals than advertisements of
OTC drugs and dietary supplements. DTC advertisements also contained more sex appeals than the
two other groups. Additionally, 73% of OTC models were female, 20% were men and 5% were intermediate. Only 6% of models were under 18 years
old, 51% had Caucasian ethnicity and 49% other
(22).
Our study will add some new information to
those earlier research studies as it was also conducted based on a different method. Some data seems to
confirm what has already been written (e.g., length
of TV commercials (23); the fact that the majority of
models are female (22); frequent use of the influence of emotions (20, 24)), but it also adds new
information (e.g., ways of changing emotional
appeals in commercials, room in which commercials
take place, dominant color, techniques of manipulation).
1096
CONCLUSION
Advertising of OTC drugs, in the rhetoric of
pharmaceutical companies, serves to provide information that enables the patients to self-use specific
medications for minor health problems without a
prior consultation with a physician. The OTC advertising research included in this article indicates that
for manufacturers it is very important that the
patient memorize the name of their drug (it is much
more important than remembering the name of a
producer ñ in the U.S. as many as 76% of commercials do not refer to it at all). To this end, producers
use ìcatchyî and ìsimpleî marketing slogans (only
one ad from the sample does not contain such a slogan). The name of the drug has to be associated
unambiguously with its indication for use. Most
commercials linked the drug with only one indication, however, sometimes there were two indications, and in some cases there were more of them.
The study presented in this article shows ways
of manipulation used in commercials. It analyzes
advertisements in terms of starring actors, used
music, colors, distinctive words and emotional
atmosphere. These elements are present in every
commercial and appropriate selection thereof can
facilitate memorizing the drug name, and evoke positive associations with the drug. Every day marketing specialists from pharmaceutical companies consider how the commercial will be received by viewers. Information included in this article can prove
valuable for them.
A proper composition of an advertisement is
very important. It should be properly thought out
and balanced. This is important in view of the criticism of larger drug manufacturers as they run
aggressive marketing campaigns that can lead to
undesirable health effects for the society (26).
Critics say that there are too many commercials and
they include a lot of false statements. Usually commercials show an ìenchantingî reality, promising
immediate health effects shortly after consumption
of the ìmagic pillî. This may lead to medicalization
and pharmaceuticalization of the society.
However, according to public data every year
the drug industry is giving more attention to the
advertising of OTC drugs in order to comply with
the requirements of Polish law. In 2007 the Main
Pharmaceutical Inspectorate withheld commercials
115 times, in 2010 ñ 47 times, and in 2014 ñ 7 times
(27). In the U.S., the Federal Trade Commission
(FTC) deals with the compliance of OTC drugs
advertising. Together with the Food and Drug
Administration it sends to Pharma warning letters
when they violate the law. In recent years there were
only a few of such warnings. In addition to these
state institutions there are also ethics committees
that take care of ethical promotion of drugs. In
Poland we have the Committee of Advertising
Ethics and in the United States, there are organizations such as: the National Advertising Division
(NAD), Council of Better Business Bureaus and the
National Advertising Review Board (NARB).
Future research should focus on a larger sample of advertisements in both countries and contain
more information about the analysis of informative
content of advertisements, for example the presence
of: ways of treatment, health outcomes, side effects.
It should also feature an analysis in terms of emotional, youth, attractive, excitement appeals. This
would give us a greater opportunity to compare the
study with research carried out earlier.
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Received: 4. 12. 2015
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1. Gadzikowska M., Grynkiewicz G.: Acta Pol. Pharm. Drug Res.
59, 149 (2002).
2. Gilbert A.M., Stack G.P., Nilakantan R., Kodah J., Tran M. et
al.: Bioorg. Med. Chem. Lett. 14, 515 (2004).
3. Roberts S.M.: Molecular Recognition: Chemical and
Biochemical Problems, Royal Society of Chemistry, Cambridge
1989.
4. Salem I.I.: Clarithromycin, in Analytical Profiles of Drug
Substances And Excipients. Brittain H.G. Ed., pp. 45-85,
Academic Press, San Diego 1996.
5. Homan R.W., Rosenberg H.C.: The Treatment of Epilepsy,
Principles and Practices. p. 932 , Lea & Febiger, Philadelphia
1993.
6. Balderssarini R.J.: in The Pharmacological Basis of
Therapeutics, 8th edn., Goodman L., Gilman A., Rall T.W., Nies
A.S., Taylor P. Eds., Vol 1, p. 383, Pergamon Press, Maxwell
Macmillan Publishing Corporation, New York 1985.
7. International Conference on Harmonization Guidelines,
Validation of analytical procedures, Proceeding of the
International Conference on Harmonisation (ICH), Commission
of the European Communities, Geneva 1996.
8. http://www.nhlbi.nih.gov/health/health-topics/topics/ms/
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