mt-259-kajal jain

Transcription

mt-259-kajal jain

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STANDARDIZATION OF AYURVEDIC
FORMULATION CATPUSPHADHYA CHURNA
A Dissertation
Submitted as a Partial Fulfillment for Degree of
Master of Pharmacy
In
The Faculty Of Pharmacy
(PHARMACOGNOSY DEPARTMENT)
To
GANPAT UNIVERSITY, KHERVA
April, 2010
Research Guide:
Submitted By:
Hardik. P. Patel
Kajal L. Jain
M.Pharm.
B.Pharm.
Department of Pharmacognosy
S. K. Patel College of Pharmaceutical Education and Research
Ganpat University, Kherva-382711, Dist-Mehsana(Gujarat), India.
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CERTIFICATE
I hereby certify that Miss. Kajal. L.Jain has completed her dissertation
work for Master of Pharmacy in Pharmacognosy on the topic
“ STANDARDIZATION
OF
AYURVEDIC FORMULATION -
CATPUSPHADHYA CHURNA ”. I further certify that the work was
carried out under my supervision and guidance at Department of
Pharmacognosy, S. K. Patel College of Pharmaceutical Education And
Research, Ganpat University, Kherva during the academic year 20092010. This work is up to my satisfaction.
Guide:
Mr. Hardik. P. Patel
M.Pharm.,
Assistant Professor,
Head of Department :
Dr. Rakesh . K. Patel
M.Pharm., Ph.D.,
H.O.D. & Professor,
Forwarded Through:
Dr. N. J. Patel
M.Pharm., Ph.D.,
Principal,
S. K. Patel College of Pharmaceutical Education and Research,
Ganpat University, Kherva-382711.
Date:
Place: Ganpat Vidyanagar
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Declaration
I, KAJAL. L. JAIN, hereby declare that the dissertation work entitled
“ STANDARDIZATION OF AYURVEDIC
FORMULATION
-
CATPUSPHADHYA CHURNA ” which is submitted to the Ganpat
University, Kherva in partial fulfillment for the degree of Master of
Pharmacy in Pharmacognosy is my own project work carried under
The guidance of Hardik. P. PATEL , Assistant Proffessor , Department
of Pharmacognosy , S. K. Patel College of Pharmaceutical Education
and Research, Ganpat University.
I further declare that the work presented in this dissertation has not
been submitted for the award of any degree in this university or in any
other university. The period of my dissertation work from January
2010 to June-2010.
Miss Kajal L Jain
B.Pharm
S.K.P.C.P.E.R
Ganpat University
Date :
Place : Ganpat University, Kherva
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Acknowledgement
First, I would like to express my salutation to GOD for giving
me the strength, confidence and moral boost for successful completion
of this project.
Words are always poor approximation of what one intends to
say. This is great opportunity on my part to express my gratitude and
sincere respect to my research guide Mr. Hardik P. Patel . I am very
very thankful and express my gratitude and sincere respect to
Dr. Rakesh K. Patel, Associate professor and Department Head,
Pharmacognosy of S. K. Patel College of Pharma. Edu. & research for
his immense guidance, help and hearty support for the timely
completion of this work. I shall forever be indebted to him for
providing me the necessary facilities, his affection, love, support,
encouragement and confidence in me. All in all, it’s my fortune and so
I am proud to have him as my guide because it would have never been
possible for me to take this project to completion without his ideas and
support.
My sincere thanks to Dr. N. J. Patel, Principal and Head
department of Pharmacology of S. K. Patel College of Pharmaceutical
Education & research for his support and facilities provided during
my project work. I am thankful to Kapil sir and Nikunjana ma’m for
their help and support.
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I sincerely thank to Mr. P. I. Patel (Librarian), Mahadevbhai and
Mukeshbhai. Ms. Chaula Patel (Computer lab), Mr. Dineshbhai Patel
(Store in charge). Rakesh Patel (lab assistant), Madhuben and
Geminbhai are sincerely acknowledged.
I am extremely thankful to Mr. Sushilbhai M. Patel, Lab assistant,
Department of Pharmaceutical Analysis, for their moral support
during my research work and provide me laboratory facility and
every time to be helpful in my practical work during my study.
Words fail me in expressing my gratitude to my parents and it
was their blessing which led me to succeed through the odd hours. This
work and ultimately myself is not possible without their care, love,
guidance, co-operation, understanding and trust on me. It was the
blessing of them that gave me courage to face the challenges and made
my path easier.
My
warmest
of
warm
and
the
most
acknowledgement, by far, is to my father Shree Lalit
important
P. Jain, my
mother Smt. Aruna L. Jain and my elder brother Kamal L. Jain
whose moral support and encouragement has been the driving force
behind my efforts are responsible for whatever I am today.
Kajal L. Jain
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Abbreviations
API
: Ayurvedic Pharmacopoeia of India
IP
: Indian Pharmacopoeia
IHP
: Indian Herbal Pharmacopoeia
ICH
: International Conference on Harmonization
ICMR
: Indian Council of Medicinal Research
conc.
: Concentration
gm
: Gram
GMP
: Good Manufacturing Practice
HPTLC
: High Performance Thin Layer Chromatography
LOD
: Limit of Detection
LOQ
: Limit of Quantification
AOAC
: Association of Analytical Chemist
M
: Meter
mcg
: Micro gram
ml
: Mililiter
Rf
: Retention Factor
RSD (% CV) : Relative standard deviation
S.D
: Standard deviation
SOP
: Standard Operating Procedure
TLC
: Thin Layer Chromatography
UV
: Ultra violate
μl
: Micro liter
WHO
: World Health Organization
% w/w
: Percentage weight by weight
% v/w
: Percentage volume by weight
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List of Tables
Table
Name of Title
No:
Pg. No:
7.1.1
Foreign matter analysis
69
7.1.3
Loss on drying
72
Determination of ash
72
7.1.5
Determination of extractive value
73
7.1.6
Phytochemical investigation
73
7.4
7.1.7
Determination of volatile oil of dill fruits from powder of
various mesh size
74
7.1.8
Estimation of carvone in dill oil
75
7.2.1
Amount of active constituent in samples from suppliers
76
7.2.2
Recovery data for piperine
80
7.2.3
Recovery data for embeline
80
7.2.4
Recovery data for carvone
80
7.2.5
Results for Intraday study
81
7.2.6
Results for Interday study
81
7.3.1
standardization parameters of churna
82
7.4.1
7.4.2
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In vitro Anti oxidant activity of Water and Methanol
extract of churna
Antiinflammatory activity by rat paw edema method
83
84
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List of Figures
Fig. No.
Name of Title
Pg. No.
4.1
Photograph showing normal and arthritic joint
1
4.2
Chemical structure of piperine
12
4.3
TLC fingerprinting of piperine
13
4.4
Chemical structure of embelin
16
4.5
TLC fingerprinting of embelin
17
4.6
Chemical structure of carvone
20
4.7
TLC fingerprinting of carvone
21
7.1
Microscopic character of Anethum sowa
70
7.2
Microscopic character of Piper nigrum
70
7.3
Microscopic character of Embelia ribes
71
7.4
Photograph of TLC plate showing separation of piperine,
embelin and carvone from standard and test solution
76
7.5
Calibration curve of Piperine by HPTLC
77
7.6
Calibration curve of Embelin by HPTLC
77
7.7
Calibration curve of carvone by HPTLC
77
7.8
Chromatogram of standard piperine, embelin and carvone
78
7.9
3-D chromatogram of standard piperine, embelin and
carvone
78
7.10
Chromatogram of churna extract
79
7.11
3-D chromatogram of churna extract
79
7.12
Graph of antioxidant activity by DPPH method
83
7.13
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Graph of anti-inflammatory activity by rat paw edema
method
84
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Chapter 1
Preamble
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Chapter 2
Working hypothesis
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Chapter 3
Aim of work
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Chapter 4
introduction
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Chapter 5
Review of
literature
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Chapter 6
Experimental work
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Chapter 7
Result & discussion
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Chapter 8
Summary of work
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Chapter 9
references
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Chapter 1
Preamble
Chapter 1
Preamble
Arthritis and rheumatism form a complex array of disorders. There are significant sources of
suffering and disability, and their prevalence and impact are increasing all around the globe as
people are living longer lives. Humoral medicines and its numerous cross cultural variant offers
an important prespective on the conceptualization and treatment of arthritis. The present work
provides a descriptive account of use of Catpusphadhya churna in rheumatic disorders. The
discussion explores the pharmacognostical , chemical quantification of active markers and
pharmacological evaluation of Catpusphadhya churna. This work provides a framework for its
use in arthritis.
The drugs used in Catpusphadhya churna are physiologically beneficial and non toxic medicines.
These drugs individually has been shown to have antioxidant, anti-inflammatory and antiarthritic
activity.
The ayurvedic properties of these drugs suggests the potencial therapeutic use of these in
inflammatory and autoimmune disease. So, Catpusphadhya churna will be examined for these
activities.
M.Pharm (Pharmacognosy)
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Kajal L. Jain
Chapter 2
Working Hypothesis
Chapter 2
Working Hypothesis
The Ayurveda suggests that the arthritis is caused primarily by an excess of ama and lack of
agni. This can be caused by poor digestion and weakened colon, resulting in the accumulation of
undigested food and the build up of the waste matter. Poor digestion allows the toxins to
accumulate in the body and problems with the colon allows the toxin to reach the joins.
Ayurvedic approach to treat arthritis includes :
 Strengthening the metabolic pathways that prevent the blockage of circulatory channels
to joints, by impurities.
 Strengthening of digestion to create nutritional plasma that is pure and easy to assimilate.
 Improving the lubrication of joints.
 Anything that aggravate or overly activaes vata, will increase the sensitivity and
experience of pain in body.
According to this line of treatment for arthritis ayurveda suggest the use of drugs which can
stimulate the digestive fire (agni), reduce the ama, stabilize vata and reduce the arthritic pain.
 Black pepper stabilizes vata and stimulates agni.
 Vidang increases digestive fire.
 Dill seeds stabilzes vata and kapha.
The hypothetical model shown above depicts that Catpusphadhya churna may work as an antiinflammatory and antiarthritic agent, by this way decrease the complications of rheumatoid
arthritis.
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Kajal L. Jain
Chapter 3
Aim of Present Work
Chapter 3
Aim Of Present Work
Standardization can create trust and confidence in the products and increase market relevance. It
is an essential requirement for the open exchange of information; without it, the network will
simply not work. The Practitioner as well as the Consumer now seek assurance from the
manufacturer about quality, safety and efficacy of a readymade Herbal Supplement or
Medication.An average person's diet, lifestyle and other social habits, all which play important
roles in disease and treatment, are completely different today. Hence the earlier
recommendations for herbs for specific disease states may not hold true today unless validated in
today's times.Phenotypic changes in plant species. Hence the original pharmacological claims of
these medicinal plant species need to be revalidated. Pharmaceutical research is aimed at
meeting the medical needs of the population for whom appropriate therapeutic remedies are not
available or at those that are available are unsafe for prophylactic use for various disorders.
While meeting medical needs, research also has to ensure that market needs for such
exist.Rheumatoid arthritis is an autoimmune disease for which no sure allopathic treatment is
available.
The present study is mainly aimed to
Standardize “Catpusphadhya Churna” in terms of identity, purity, content of assay.
Chemical quantifiation of active markers present in the formulation.
Perform pharmacological activity of catpusphadhya churna.
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Kajal L. Jain
Chapter 4
Introduction
Chapter 4
Introduction
4.1 Introduction to disease
Rheumatoid arthritis (RA) is a form of arthritis that causes pain, swelling, stiffness and loss of
function in your joints. It can affect any joint but is common in the wrist and fingers. More
women than men get rheumatoid arthritis. It often starts between ages 25 and 55. You might
have the disease for only a short time, or symptoms might come and go. The severe form can last
a life time.
Rheumatoid arthritis - the second most common form of arthritis - affects other parts of the body
besides the joints. Osteoarthritis is the most common type of arthritis. Rheumatoid arthritis is
different from osteoarthritis, the common arthritis that mostly affects cartilage and often comes
with older age. RA can affect body parts besides joints, such as your eyes, mouth and lungs. RA
is an autoimmune disease, which means the arthritis results from your immune system attacking
your body's own tissues.
Figure 4.1 normal and arthritic joint
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Chapter 4
Introduction
In some patients with rheumatoid arthritis, chronic inflammation leads to the destruction of the
cartilage, bone, and ligaments, causing deformity of the joints. Damage to the joints can occur
early in the disease and be progressive. Moreover, studies have shown that the progressive
damage to the joints does not necessarily correlate with the degree of pain, stiffness, or swelling
present in the joints.
Rheumatoid arthritis (RA) is an autoimmune disease that causes chronic inflammation of the
joints. Rheumatoid arthritis can also cause inflammation of the tissue around the joints, as well
as in other organs in the body. Autoimmune diseases are illnesses that occur when the body's
tissues are mistakenly attacked by their own immune system. The immune system is a complex
organization of cells and antibodies designed normally to "seek and destroy" invaders of the
body, particularly infections. Patients with autoimmune diseases have antibodies in their blood
that target their own body tissues, where they can be associated with inflammation. Because it
can affect multiple other organs of the body, rheumatoid arthritis is referred to as a systemic
illness and is sometimes called rheumatoid disease. However, rheumatoid arthritis is typically a
progressive illness that has the potential to cause joint destruction and functional disability.
No one knows what causes rheumatoid arthritis. Genes, environment and hormones might
contribute. Treatments include medicine, lifestyle changes and surgery. These can slow or stop
joint damage and reduce pain and swelling.
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Chapter 4
Introduction
Treatment of Rheumatoid Arthritis
There's no cure for RA, although progression of the disease can be slowed, the symptoms can be
treated, and a person can be helped to adjust to the condition. Treatments include:

Physiotherapy – heat, cold and exercises to relieve pain and stiffness, improve joint
movements and strengthen muscles.

Rest – when there is a worsening of the joint inflammation.

Occupational therapy – including training, advice, counselling and provision of splints,
and aids such as walking aids and specialised cooking utensils – these can help people do
daily activities more easily and with less pain.

Drugs. These (often taken in combinations) play an important part in dampening the
inflammatory and autoimmune process.
The earlier drug therapy begins, the better the outcome. Drug treatments include:

Mild painkillers like paracetamol and aspirin.

NSAIDs (non-steroidal anti-inflammatory drugs) . These dampen the inflammation,
but they can have side effects: chiefly gastric ulcer and bleeding from the stomach and
duodenum. They're effective in reducing the pain and the swelling though, and in milder
cases, may be enough to control the condition. They seem to work well in some people
but not so well in others. They include older NSAIDs such as indomethacin, sulindac,
ibuprofen, diclofenac and naproxen; and newer NSAIDs called the COX 2 inhibitors
such as rofecoxib and celecoxib. The COX 2 inhibitors are said to produce less gastric
bleeding, but have been shown to increase the risk of heart disease – in 2004, rofecoxib
(trade name Vioxx) was withdrawn from the market. Recent evidence suggests that there
may be an increased risk of heart disease with the NSAIDs as well.
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Kajal L. Jain
Chapter 4

Introduction
Corticosteroids may be used in more severe cases. They suppress the immune response,
and are used either continuously as tablets or capsules, or as injections directly in the
affected joint in a flare-up.

Disease-modifying anti-rheumatic drugs (DMARDs) . These don't cure the condition
either, but can slow its progress, though they can be associated with serious side effects –
so people on these drugs need to be carefully monitored. They include: antimalarial drugs
like hydroxychloroquine; penicillamine; immunosuppressant drugs like azathioprine and
methotrexate; sulphasalazine; leflunomide; and intramuscular gold injections. Another
class of drugs acts by blocking tumour necrosis factor (and are very effective but very
expensive). These include infliximab, etanercept and adalimumab. Another drug
sometimes used is anakinra, which blocks interleukin-1.
In some cases, surgery (for example a knee replacement) is an option, where a joint has been
badly damaged.
Because of the side effects associated with the allopathic treatment as mentioned above, now the
demand for herbal treatment for this disease is now increasing which ensures safety.
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Kajal L. Jain
Chapter 4
Introduction
4.2 Herbals and Ayurveda
The ayurvedic system of medicine is prevalent in India, since the vedic period and as earlt as the
dawn of human civilization.these medicine are of plant, animal or mineral origin or mixture of
them. There are well laid down procedures to make these preparation and as a result of which it
is claimed that their bioavailability is enhance.
Though ayurveda has undergone a number of changes in the course of its history, it still remains
the mainstay of medical relief to a large section of population of nation. The World Health
Organization (WHO) estimates that about 80 % of the population living in the developing
countries rely almost exclusively on traditional medicine for their primary health care
needs.there has been a tremendous increase in the usage of traditional and herbal medicines in
the past few years, not only among modern consumers of herbal products.
India has been a traditionally rich source of various types of medicinal plants. Traditional
medicines are and will be the indomitable area of pharmacognosy. Currently, plant based drugs
are researched, dispensed, formulated and manufactured in modern framework rather than in
form of galenical preparation or conventional dosage forms.hence pharmacognosy has become
an interface among various branches of pharmaceutical sciences. It is now emerging as
interdisciplinary science that incorporate inputs from chemistry, pharmacology, pharmaceutical
technology and biotechnology directed towards natural products based drug discovery.
Because of the various side effects associated with the current therapies for rheumatoid arthritis
and as the reason for its occurrence is not clear as per medical research, no drug therapy
effectively works on it. So there is need to switch to ayurvedic treatment for its cure.
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Chapter 4
Introduction
4.3 Churna
4.3.1 Introduction
Churnas are preparations comprising of fine powders of drugs and may be simple or compound.
Simple churna comprises of only one ingredient while a compound one consists of more than
one ingredient. The principle of using churna is due to the fact that therapeutic value of most of
the substances greatly increases when they are reduced to very fine state of subdivision.
4.3.2 Method of preparation
Drugs are cleaned and dried properly. Drugs are kept separately and powdered. They are sieved
and each one of them powdered and weighed separately, and then mixed together. It is then kept
in air tight containers in cool and dry place.
4.3.3 Characteristics and preservation
The powder is fine of atleast 80-mesh sieve. It should not adhere together or become moist. The
finer the powder, the beter irs therapeutic value. They retain potency for one year and should be
kept in airtight containers.
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Kajal L. Jain
Chapter 4
Introduction
4.4 Catpusphadhya churna1
4.4.1 Formulation
This formulation is mentioned in book of “The Ayurvedic System of Indian Medicine”, by
Kaviraj Nagendranath Sen Gupta for treatment of acute rheumatoid arthritis. It is a compound
powder comprising three drugs of plant origin and one drug of mineral origin, in equal
quantities. It consists of dried powders of fruits of following drugs:
Anethum sowa
Embelia ribes
Piper nigrum
Rock salt
4.4.2 Method of preparation
Drugs mentioned above were purchased from two local vendors and were studied for
preliminary pharmacognostical parameters like macroscopy, microscopy. Drugs are sieved and
then foreign matter removed manually using magnifying lens. Drugs are powdered in grinder
separately. It is then passed through 40-mesh sieve separately. Then all powders weighed in
equal quantities and mixed properly. Then this formulation kept in air tight container in cool and
dry place.
4.4.3 Dosage
One to two teaspoonful of powder with warm water.
4.4.4 Indication
It kills acute rheumatoid arthritis.
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Chapter 4
Introduction
4.5 Introduction to drugs used in formulation
4.5.1 Black pepper
Botanical Source 2:
It consist of fully matured dried fruits of Piper nigrum.
Family Piperaceae
Botanical description of Piper nigrum 3:
Much branched climbingshrub, rooting at the nodes. Leaves simple, alternate, cordate, broadly
ovate, 5- 9 nerved, dark green. Flowers minute,in spikes of variable length. Fruits ovoid or
globose, one seeded bright red when ripe. Seeds globose, testa thin, perisperm hard and white.
Seed morphology 2:
Fruits are grayish black to black, hard, wrinkled, 0.4-0.5 cm in diameter, odour, aromatic; taste,
pungent.
Vernecular names 2,3:
English
: black pepper
Hindi
: kalimirch
Gujarati
: kalimori
Sanskrit
: usana
Bengali
: morichmenaru
Kannad
: galmirch
Urdu
: kalimirch
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Chapter 4
Introduction
Ayurvedic properties 2,3:
Rasa
: katu, tikta
Guna
: laghu, ruksa, tiksna
Virya
: usna
Vipaka : katu
Karma : slesmahara, pittakara, chedana, dipana, rucya, antunashana, medohara,
chedi, hrdroga, vataroga.
Ayurvedic formulations 2:
Marikadi Gutika
Marikadi Taila
Trikatu Churna
Powder study 2:
Blakish grey, shows with a debris characteristic, in groups, more or less isodiametric or slightly
elongated stone cells, interspersed with thin walled polygonal hypodermal cells, beaker shaped
stone cells from endocarp and abundant polyhedral, elongated cells from perisperm, packed
tightly with masses of minute compound and single, oval to round, starch grains having 2-3
components and a few aleurone grains and oil globules.
Chemical constituents 2,3 :
It contains an alkaloid Piperine about 5-8 % , piperidine, caffeic acid,volatile oil 1.5-2.5% which
includes 15-25 % of sabinene, β alanine, vitamins and minerals.
Figure : 4.2 1-[5-(1,3-benzodioxol- 5-yl)-1-oxo-2,4-pentadienyl]piperidine
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Chapter 4
Introduction
TLC profile 2:
Mobile phase
: Toluene : Ethylacetate ( 7 : 3)
Standard solution : Dilute 5 mg of piperine in 5 ml of methanol
Test solution
: Extract 5 gm of pepper powder with 25 ml methanol for 30 min
Filter, then concentrate and then apply on tlc plate along with
standard compound
Detection
: U.V 254, 366 nm
Rf value
: 0.5
Sample id
: S – std piperine,
P – pepper powder extract
C – churna extract
D – dill powder extract
E - vidang extract
S
P
C
U.V 254 nm
S
P
C
U.V 366 nm
S
P
D E
C
U.V 254 nm
Figure 4.3: TLC fingerprinting of piperine
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Chapter 4
Introduction
Pharmacopeial standards 2:
Foreign matter
: NMT 2%
Total ash
: NMT 5%
Acid insoluble ash
: NMT 0.5 %
Alcohol soluble extractive : NLT 6%
Water soluble extractive
: NLT 6%
Pharmacology 3:
The drug is found to be a powerful antioxidant. It is also used in as anticonvulsant, sedative,
analgesic. It has a property of inhibiting the growth of some pests , so can be used as
insecticidal, pesticidal against various pests. It has also be found to possess activities like cns
depressant,
muscle
relaxant,
antipyretic,
anti-inflammatory,
antifungal,
taenicidal,
hepatoprotective, antimicrobial, antiulcer, antibacterial, lipolytic, cyclo oxigenase inhibitory
activity and this may be attributed to its active principle piperine.
Therapeutic indication 2:
It is used as antimicrobial. It improves digestion and also is bioavailability enhancer. It is found
that it has melanocyte proliferating activity, thermogenic activity, anticancer, anti-inflammatory,
antioxidant, antiarthritic , anticonvulsant.
Dosage 2:
250 mg – 1 gm of drug in powder form.
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Kajal L. Jain
Chapter 4
Introduction
4.5.2 Vidang
Botanical Source 4:
It is the dried mature fruits of Embelia ribes.
FAMILY :- Myrsinaceae.
Botanical description 5:
A large ,scandant shrub with long slender , flexible,terete branches, bark studded with lenticles.
Leaves simple, alternate,elliptic_ lanteolate,gland dotted,short and obtusely
acuminate,
entire,shiny above. Flowers small white or greenish , in both terminal and axillary panicles.
Fruits globose, wrinkled or wavy, dull red to nearly black, a short padicle often present, usually
one seeded; seeds globose.
Seed morphology 4:
Fruit brownish black, globular, 2-4 mm in diameter, warty surface with a beak like projection at
apex, often short, thin pedicle and persistent calyx with usually 3 or 5 sepals present; pericarp
brittle enclosing a single seed covered by a thin memberane; entire seed, reddish and covered
wih yellowish spots; odour, slightly aromatic, taste, astringent.
Vernecular names 4,5,6:
English
: Embelia
Hindi
: Vayavidanga
Gujrati
: Vavading
Marathi
: Vavding
Punjabi
: Babrung
Telgu
: Vayu – vilamgam
Oriya
: Bidanga
Urdu
: Baobrang
Bengali
: Biranga
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Kajal L. Jain
Chapter 4
Introduction
Ayurvedic properties 4,5,6:
Rasa
: tikta, katu
Guna
: lagu,ruksha,teekshana
Veerya
: ushna
Vipaka
: katu
Prabhava
: krimighna
Ayurvedic formulation 4:
Vidangarista
Vidanga lauha
Vidangadi lauha
Powder study 6:
Powder shows abundant pitted, lignified stone cells and sclerids of various size and thickness,
isolated or in groups, groups of abundant fragments of dark brown colored palisade like cells of
endocarp, fragments of endosperm filled with aleurone grains and fixed oils, thick walled cells
of epicarp in surface view with striated cuticle.
Chemical constituents 5,6:
Embelin,quercitol,tannin,coloring matter, an alkaloid called as christembine, embolic acid, fatty
ingredients,resinoid,volatile oil .a colorless crystalline constituent vilangin from berries on
oxidation gives orange flakes.it gives a dark.colored aliphatic oil. A crystalline compounds of
embolic acid with soda,potash, and ammonia are obtained.
Figure 4.4: 2,5-dihydroxy -3- undecyl-2,5-cyclohexadiene-1,4-benzoquinon
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Chapter 4
Introduction
TLC profile 6:
Mobile phase
: toluene : ethylacetate : acetic acid ( 5 : 4 :1 )
Standard soluttion : dissolve 1 mg of embeline in 1 ml of chloroform
Test solution
: dissolve 5 gm of powdered vidang in 25 ml of chloroform for
20 min. Filter, then concentrate and then apply on tlc plate along
with standard compound .
Detection
: UV 254 nm, 366 nm
Rf value
: 0.71
Sample id
: S – std embeline
E – embelia powde extract
C – Churna extract
S
E C
U.V 254 nm
S
E C
U.V 366 nm
S
E C
Normal light
Figure 4.5: TLC fingerprinting of embelin
Page
17
Kajal L. Jain
Chapter 4
Introduction
Pharmacopeial standards 4,5,6:
Foreign matter
: NMT 2%
Total ash
: NMT 6%
Acid insoluble ash
: NMT 1.5 %
Water soluble extractive : NLT 9 %
Alcohol soluble extractive : NLT 10 %
Pharmacology 5,6:
This drug has many important pharmacological activities like nematicidal, hypoglycemic,
anthelmentic, antibiotic, antitubercular, anti
implantation, anti-inflammatory, estrogenic,
antifertility, antispermatogenic, antiandrogenic, anticancer, immunostimulant.
Therapeutic indication 5:
Dried berries are useful as powder especially to expel tape worms. The worm is expelled dead.
Berries prevent flatulence and are useful in dyspepsia. A paste of seed is applied locally in ring
worm and other skin infections. It is given for piles. Powder made from dried bark of the root is
a reputed remedy for toothache. A paste of the bark is a valuable application to chest in lung
disease like pneumonia. The drug is also used in scorpion sting and snake bite.
Dosage 4:
5-10 gram of drug in powder form.
Page
18
Kajal L. Jain
Chapter 4
Introduction
4.5.3 Dill seeds
Botanical Source 10:
It consist of the dried ripe fruits of Anethum graveolens.
Family :- Umbelliferrae
Botanical description 9:
A herb 30 cm in height. Flowers – yellow, fruits – blackish, leaves 5-6 cm long. Wing shaped
and flowering, fruiting season is winter.
Seed macroscopy 10:
Fruits,dark brown, often stalk attached, broadly oval and compressed dorsally, mericarps usually
separate and free, 4 mm long, 2-3 mm broad and 1 mm thick, glabrous, traversed from the base
to apex by 5 lighter colored primary ridges of which 3 dorsal, slightly raised, brown, filiform and
inconspicuous, 2 lateral prolonged into thin, yellowish membranous wing. odour, faintly
aromatic resembling that of caraway, and a war, slightly sharp taste, akin to caraway.
Vernecular names 8,9,10:
Sanskrit
: mishroya, sthatapushpi
English
: dill
Hindi
: sowasoya
Gujarati : surva-nu-bi
Punjabi
: soya
Bengali
: soolpha
Marathi
: shepu
Telgu
: shatakupivittulu
Arab
: shubhit
Page
19
Kajal L. Jain
Chapter 4
Introduction
Ayurvedic properties 8,9,10:
Rasa
: katu, tikta
Guna
: laghu, ruksha, trikshna
Veerya
: ushna
Vipaka : katu
Dosa
: vatakaphanashak
Ayurvedic formulation10:
Brhatphala Ghrta
Gorocanadi vati
Narayana churna
Sadbindu Taila
Powder study 9:
Powder shows fragments of yellow colored vittae in surface view, exhibiting thin
walled
cells containing volatile oil globules, fragments of epicarp in surface view with striated cuticle
and infrequent stomata, fragments of thin walled polygonal cells of the endosperm filled with oil
globules, aleurone grains embedded with microrossete crystals of Ca oxalate, endocarp and
polygonal cells of mesocarp, sclerids with wide lumen.
Chemical constituents 7,8:
Dried ripe dill fruit contains a volatile oil 3-4 % and fixed oil.the volatile essential oil is
composed of anethine phellandrine and d- limonine, and apiol termed dill apiol; also
carvol(carvone)
and other hydrocarbon. It also contains monogalactolactosyl digalactosyl
diglycerides the chief constituents of glycolipids, phosphatidylcholine, linolenic acid,palmitic
acid, 4’methoxy kaempferol, 3’ 4’quercetin, coumarins, carvones and anethofuran
Page
20
Kajal L. Jain
Chapter 4
Introduction
Figure 4.8: (RS)-5-isopropenyl-2-methylcyclohex-2-en-1-one
TLC profile 9:
Mobile phase
: toluene : ethylacetate : acetic acid ( 8.8 : 1.0 : 0.2 )
Standard solution : dissolve 5 mg of carvone in 10 ml of methanol.
Test solution
: dissolve 5 gm of powdered sowa in 25 ml of methanol for 20 min.
Filter, then concentrate and then apply on tlc plate along with
standard compound.
Detection
: UV 254 nm
Rf value
: 0.6
Sample id
: S – std carvone
D – dill powder extract
C – churna extract
S
D
C
U.V 254 nm
Figure 4.6 : TLC fingerprinting of c arvone
Page
21
Kajal L. Jain
Chapter 4
Introduction
Pharmacopeial standards 9,10:
Foreign matter
: NMT 5%
Total ash
: NMT 14%
Acid insoluble ash
: NMT 1.5%
Alcohol soluble extractive : NLT 4%
Water soluble extractive
: NLT 15%
Volatile oil
: NLT 3%
Pharmacology 7,8:
Dill seeds is mainly used as carminative, stomachic, aromatic, stimulant, diuretic, resolvent,
emmenagouge and galactogouge. Dill water prepared from the fruit is regarded as stimulant,
carminative and aromatic, and like anise, popularly supposed to promote the secretion of milk .
Therapeutic indication 7,8,9,10:
Essential oil of the fruit and the distilled water of the fruit are much used inflatulence, hiccup,
colic and abdominal pain in children and in adults. It is used to diminish the gripping of
purgatives, and the tormina of dyssentry.it is also given as drink to women after
confinement.seeds bruished and boiled in water and mixed with the roots are applied externally
in rheumatic and other swellings of the joints. It increases the urine flow, increases sodium and
chlorine excretion, long lasting insect repellant activity.
Dosage 10:
0.5 – 1 gm of drug in powder form.
Page
22
Kajal L. Jain
Chapter 4
Introduction
4.5.4 Rock salt
Halite is the mineral form of sodium chloride , NaCl, commonly known as rock salt. Mineral solid homogeneous inorganic substances occurring in nature having a definite chemical
composition. Rock salt - naturally occurring crystalline sodium chloride (halite). Na, atomic
number 11, sodium - a silvery soft waxy metallic element of the alkali metal group; occurs
abundantly in natural compounds (especially in salt water); burns with a yellow flame and reacts
violently in water; occurs in sea water and in the mineral halite (rock salt).
Halite forms isomeric crystals. The mineral is typically colorless or white, but may also be light
blue, dark blue, purple, pink, red, orange, yellow or gray depending on the amount and type of
impurities. It commonly occurs with other evaporate deposit minerals such as several of the
sulphates, halides and borates. The primary difference between rock salt and ordinary table salt
is the size. Rock salt forms in very large, chunky crystals, as opposed to the small crystals seen
in table salt. Like table salt, rock salt also has an assortment of trace minerals which can have an
impact on how it behaves chemically. Because of the large crystal size, rock salt is not usually
used directly in cooking, since it takes a long time to dissolve.
Rock salt, purified by nature, is considered superior to table salt, due to its richness in natural
minerals and lack of chemicals and additive. Many micro organisms cannot live in an overly
salty environment: water is drawn out of their cells by osmosis. For this reason salt is used to
preserve some foods, such as smoked bacon or fish. It can also be used to detach leeches that
have attached themselves to feed. It is also used to disinfect wounds. It Improve Digestion.
Rock Salt as the name says is obtained from Salt mines in its pure natural form. It is very low in
sodium content as compared to the common salt. In Ayurveda, Rock Salt is recommended in
place of Sea Salt or common salt which is termed as a non-vegetarian salt as it has many
impurities like animal and human excreta's, dead animal flesh etc. Rock Salt is also an effective
Page
23
Kajal L. Jain
Chapter 4
Introduction
medicine for various problems related to the digestive system and those due to worms.
Benefit from the purity of rock salt.
One of the benefits of rock salt is what it lacks, which is chemicals and additives. Regular table
salt goes through a harsh refining process in which the salt from the mine is crushed, refined and
packaged. This removes the natural minerals found in rock salt, including iodine, which has to
be added back in the manufacturing process of table salt.
From ancient times to today, it is believed that salt that comes naturally from mines benefit the
digestive system in many ways. When taken with food, salt derived from pure rock improves
appetite, improves digestion, decreases gas, and promotes proper bowel function. It is used as a
spice and an ingredient in ice cream. It Alleviate respiratory symptoms. It is said that the salt
cleans mucus from the airways and decreases the severity of inflammation in the sinuses. After
the discovery of the first natural rock salt cave in Britain, scores of people attest to cures of
many respiratory symptoms ranging from the common cold to asthma and allergies. It Increase
your intake of dietary minerals. It is common knowledge that our diets should be rich in
minerals, which aid in
body metabolism, water balance, and bone health. Rock salt is rich in
minerals. In addition to sodium chloride (salt), such as calcium, magnesium, copper and iron. In
addition, over 50 trace minerals are found in rock salt.
Moreover, there are no side effects of consuming Rock Salt. It is also an effective remedy for
teeth and Gum problems as an external food constituent. Scientists do not advise use of common
salt because of the logical reason that Sea water which contains so much of harmful wastes
cannot be purified however one tries to purify it. Therefore, according to scientists, common salt
may be harmful to us as compared to the controlled use of Rock Salt. The chemical composition
also of both the salts are different for (e.g.), the Rock Salt has a low sodium content, is a
Naturally refined salt, and has a natural anti-caking property whereas, common salt has a
comparatively high sodium content, is a chemically refined salt and toxic anti-caking chemicals
like potassium/sodium ferro-cyanide are added for common salt to achieve the anti-caking
property. Rock salt in one's diet is beneficial specially for people having heart ailments and
therefore advised to reduce the salt intake.
Page
24
Kajal L. Jain
Chapter 4
Introduction
Composition of rock ( saindhav) salt
Nutrient values / 100 g
Reference
Nutrient factor
Content
Unit Method
Acquisition
Macro-components
energy, calculated
0 (0)
carbohydrate, available 0
imputed/estimated,
kJ (kcal)
generic
g
value
created
within host-system
food
table
composition
other method type
composition
composition
fat, total
0
g
other method type
food
table
protein, total
0
g
other method type
food
table
alcohol
0
g
imputed/estimated,
generic
150
150
150
value
created
within host-system
Carbohydrate Components
food composition
table
150
starch, total
0
G
other method type
sugars, total
0
G
value
created
summation
from
within host-system
constituent components
sucrose
0
G
value
created
estimated according to
within host-system
logical deduction
lactose
0
g
other method type
g
value
created
within host-system
logical deduction
fructose
0
Page
25
food composition
table
150
Kajal L. Jain
Chapter 4
Introduction
fibre, total
0
g
other method type
food composition
table
150
fibre, water-insoluble
0
g
other method type
value
created
within host-system
polysaccharides,
non0
cellulosic, water-soluble
G
value
created
within host-system
logical deduction
glucose
G
value
created
within host-system
logical deduction
G
value
created
within host-system
logical deduction
0
maltose
0
Fat
fatty
acids,
calculated as
equivalents
total,
TAG 0
g
value created within
summation from constituent
host-system
components
0
g
summation from constituent
host-system
components
0
g
imputed/estimated
related food
fatty
acids,
total
0
monounsaturated cis
G
estimated according to logical
host-system
deduction
fatty
acids,
polyunsaturated
G
host-system
deduction
G
host-system
deduction
Mg
host-system
deduction
fatty acid 18:3 n-3
0
(alpha-linolenic acid)
Mg
host-system
deduction
fatty acid
(EPA)
Mg
host-system
deduction
fatty acids, total
fatty
acids,
saturated
total
total
fatty acids, total trans
0
0
fatty acid 18:2 cis,cis n0
6 (linoleic acid)
20:5
n-3
0
Page
26
from
host-system
Kajal L. Jain
Chapter 4
fatty acid
(DHA)
Introduction
22:6
n-3
cholesterol (GC)
sterols, total
0
mg
host-system
deduction
0
mg
imputed/estimated, generic
mg
imputed/estimated
related food
0
host-system
from
host-system
Minerals
sodium
38700.0
mg
other method type
food
table
composition
191
salt
98607.6
mg
summation
from
host-system
37.0
mg
other method type
food
table
composition
potassium
food
table
composition
food
table
composition
magnesium
140.0
mg
other method type
calcium
230.0
mg
other method type
phosphorus
70.0
mg
imputed/estimated,
generic
iron, total
0.3
mg
other method type
zinc
< 0.1
mg
imputed/estimated,
generic
394
191
191
host-system
food
table
composition
191
host-system
iodide (iodine)
5.0
µg
other method type
food
table
composition
selenium, total
0
µg
other method type
food
table
composition
mg
imputed/estimated,
generic
host-system
191
394
Nitrogen Components
tryptophan
0
Page
27
Kajal L. Jain
Chapter 4
Introduction
Vitamins
vitamin A retinol activity
0
equivalents
µg
summation
from value created within
constituent components host-system
vitamin D
µg
other method type
0
mg
summation
from
host-system
0
µg
imputed/estimated,
generic
host-system
vitamin C (ascorbic acid) 0
mg
imputed/estimated,
generic
host-system
folate (HPLC)
0
µg
other method type
food
table
niacin equivalents, total
0
mg
imputed/estimated,
generic
host-system
niacin,
preformed
(nicotinic
acid
+0
nicotinamide)
mg
imputed/estimated
from related food
host-system
riboflavine
0
mg
imputed/estimated,
generic
host-system
thiamin (vitamin B1)
0
mg
imputed/estimated,
generic
host-system
vitamin B-12 (cobalamin) 0
µg
other method type
food
table
vitamers
pyridoxine
0
(hydrochloride)
mg
imputed/estimated,
generic
host-system
carotenoids, total
µg
summation
from value created within
constituent components host-system
vitamin
alphatocopherol
vitamin K, total
0
E
0
Page
28
food
table
composition
composition
composition
150
150
150
Kajal L. Jain
Chapter
Review of Literature
Chapter 5
Review Of Literature
For piperine :
1. Indian Herbal Pharmacopoeia1 described the analytical method for the determination of
Piperine by HPTLC.
2. In official methods of Analysis of AOAC International2 stated that piperine in solution
isomerizes when exposed to light.
3. Indian Council of Medical Research of India3 described the HPTLC method for analysis
of piperine.
4. M. K . Santosh et.al.4 discussed that Piper longum Linn. and Piper nigrum Linn. are used as
spices and medicines. Quantitative determination of piperine was undertaken to provide an
easy and simple analytical method, which can be used as a routine quality control method.
RP-HPLC was performed using methanol and water as mobile phase. The detection and
quantification was performed at a wavelength of 345 nm. Linearity of detector response for
piperine was between the concentrations 0.005% to 0.1%. The correlation coefficient
obtained for the linearity was 0.998. The assay value of piperine for fruit and root of P.
longum was found to be 0.879% and 0.31%. The assay value of piperine for fruit of P.
nigrum was 4.5%. The recovery value of standard piperine was 99.4%. Low value of
standard deviation and coefficient of variation are indicative of high precision of the method.
5. Kedar Kumar Rout et.al.5 developed a simple, sensitive, and rapid high-performance thin
layer chromatographic (HPTLC) method has been established for estimation of piperine in
commercial Ayurvedic formulations and in the fruits of Piper nigrum Linn, and Piper
longum Linn. Chromatography was performed on aluminum foil HPTLC plates coated with
0.2 mm layers of silica gel F 254 , with hexane-acetone 6.5: 3.5 ( v/v ) as mobile phase. The
Page 29
Kajal L. Jain
Chapter
development distance was 76 mm, the temperature 25 ± 5°C, and the chamber was saturated
for 5 min. Piperine was quantified at 340 nm, its wavelength of maximum absorbance. Under
the conditions used the R F of piperine was 0.33 and the limit of detection (LOD) was 4 ng
per zone. The calibration plot was linear in the range of 10 to 60 ng per zone with a
correlation coefficient of 0.9996. Recovery was in the range 98.76 to 100.70%. This HPTLC
method was found to be reproducible, accurate, and precise and could be used to detect
piperine at nanogram levels. The method is a very simple and cost-effective means of
quantitative estimation of piperine in Ayurvedic formulations.
6. S.K Chauhane et.al.6 developed HPLC method to determine pipeine in different Piper
species. This method can be used for detection, monitoring and quantification of piperine.
7. A Kumar et. al.7 studied the antiinflammatory activity of the Piper longum dried fruit's oil
in rats using the carrageenan-induced right hind paw edema method. The activity was
compared with that of standard drug ibuprofen. The dried fruit's oil inhibited carrageenaninduced rat paw edema. The results indicated that the dried fruit's oil produced significant
(p< 0.001) antiinflammatory activity when compared with the standard and untreated
control.
8. S.C. Jagdale et.al.8 worked on Myocardial ischemia, a serious cardiac problem which
should be diagnosed and treated effectively to prevent its complications. Many plant based
medicine have been utilized for the effective treatment in cardiac problems. Myocardial
ischemia was induced in rats by administration of isoproterenol. Petroleum ether extract of
root and piperine from roots of Piper longum Linn. were subjected for evaluation of their
anti-oxidant activity by DPPH scavenging method. Lipid peroxide and Glutathione values in
myocardial ischemic rats have also been estimated by inducing myocardial ischemia by
using isoproterenol. It has been found that at 50 mg mL-1 concentration pet ether extract and
piperine exerts 74.12 and 72.13% of inhibition. Pet ether extract and piperine pretreatment
decreases lipid peroxide level and maintain glutathione content to near normal in treated rats.
The present study shows that the extract of the root of the plant and piperine exert antioxidant activity and are protective in the myocardial ischemic condition.
Page 30
Kajal L. Jain
Chapter
9. Dipak Kulkarni et. al.9 provided easy and simple analytical HPTLC method for
determination of piperine from Piper nigrum.
10. Samudram P et.al.10 studied the combined hepatoprotective effect of Bi- herbal ethanolic
extract (BHEE)
against carbon tetra chloride (CCl4) induced hepatic damage in rats.
Ethanolic extract from the leaves of Eclipta alba and seeds of Piper longum at a dose level
of 50 mg/kg body weight was administered orally daily once for 14 days. The substantially
elevated serum marker enzymes such as SGOT, SGPT, ALP, LDH, ACP,
GT and 5’
Nucleotidase, due to CCl4 treatment were restored towards normalization. The biochemical
parameters like total protein, total bilirubin, total cholesterol, triglycerides, and urea were
also restored towards normal levels. In addition, BHEE significantly decreased the liver
weight of CCl4 intoxicated rats. Silymarin at a dose level of 50 mg/kg was used as a standard
reference also exhibited significant hepatoprotective activity against CCl4 induced
hepatotoxicity. The results of this study strongly indicate that BHEE has got a potent
hepatoprotective action against CCl4 induced hepatic damage in rats.
11. A. B. Wood et.al.11 developed a reversed-phase high-performance liquid chromatographic
(HPLC) method for the selective determination of piperine, in pepper or its oleoresins, is
described and evaluated. It employs a fully-capped, bonded C18 stationary phase (ODS-2)
and an acetonitrile - aqueous acetic acid mobile phase, with ultraviolet (UV) detection.
Piperine is extracted from ground pepper by refluxing 3h with ethanol (96% v/v), whilst
homogenized oleoresins are simply dissolved in the same solvent. At all stages, light must as
far as possible be excluded from the solutions, so as to minimize the photoisomerization of
piperine. Quantitation, based on peak areas, is achieved by reference to purified piperine as
external standard. Results obtained for a range of samples at four different detection
wavelengths showed virtual wavelength-independence, indicating a good degree of peak
purity. All samples were also analysed by the ISO spectrophotometric method (ISO 5564,
1982), which invariably yielded higher results because of the contributions from other
pepper alkaloids (e.g. piperyline and piperettine) to the UV absorption. Since some of these
other alkaloids are believed to contribute to the pungency of pepper, the HPLC method is
proposed as complementary to the spectrophotometric method, rather than as a replacement
for it. The HPLC method gave results with relative standard deviations less than ±3% for
Page 31
Kajal L. Jain
Chapter
black or white pepper, and piperine values between 75.8% and 90.9% of those yielded by
spectrophotometry.
12. Suthar A.C et. al.12 developed HPTLC method for identification of Piper nigrum and Piper
longum and mixture thereof by their fingerprinting pattern.
13. Koul Surrinder et. al.13 studied that piperine inhibits both constitutive and inducible
cytochrome P450(CYP) dependent drug metabolizing enzyme.
14. Koul I.B et. al.14 evaluated antihepatotoxic potencial of piperine in order to validate its use
in traditional therapeutic formulations. This plant principle exerted a significant protection
against tert butyl hydroperoxide and CCl4 hepatotoxicity by reducing both invitro and invivo
lipid peroxidation, enzymatic leakage of GPT and AP and by preventing the depletion of
GSH and total thiols in the intoxicated mice. Silymarin, a known hepatoprotective drug was
tested simultaneously for comparision. Piperine showed a lower hepatoprotective potencial
than silymarin.
15. Sharma G et. al.15 reported piperine as therapeutic agent and bioavailability enhancer.
16. Mujumdar A.M et. al.16 used different acute and chronic experimental models and reported
that piperine acted significantly on early acute changes in inflammatory processes and
chronic granulative changes. I also acted partially through stimulation of pituitary adrenal
axis. Exudaive changes in both acute and chronic models, however were insignificant.
17. Gupta S.K et. al.17 Stated that piperine is an inhibitor of various hepatic and other enzymes
involved in the biotransformation of drugs. Preliminary pharmacokinetic suggested the
increased bioavailability of nimesulide co administered with piperine. Results for the present
study suggest a better therapeutic index for the nimesulide-piperine combination indicating
that this combination would further reduce the frequency of adverse effects associated with
nimesulide alone.
18. Rauscher Frederick M et. al.18 studiedsubacute treatment (
10 mg/kg/day, i.p for 14
days) with piperine protect against diabetes induced oxidative stress in 30 days streptozocin
Page 32
Kajal L. Jain
Chapter
induced diabetic Sprague – Dawley rats and concluded that subacute treatment with piperine
for 4 days is only partially effective as an antioxidant therapy in diabetes.
19. İlhami Gülçin19 investigated water and ethanol crude extracts from black pepper (Piper
nigrum) for their antioxidant and radical scavenging activities in six different assay, namely,
total antioxidant activity, reducing power, 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) free
radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, and
metal chelating activities. Both water extract (WEBP) and ethanol extract (EEBP) of black
pepper exhibited strong total antioxidant activity. The 75 µg/ml concentration of WEBP and
EEBP showed 95.5% and 93.3% inhibition on peroxidation of linoleic acid emulsion,
respectively. On the other hand, at the same concentration, standard antioxidants such as
butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and α-tocopherol
exhibited 92.1%, 95.0%, and 70.4% inhibition on peroxidation of linoleic acid emulsion,
respectively. Also, total phenolic content in both WEBP and EEBP were determined as gallic
acid equivalents. The total phenolics content of water and ethanol extracts were determined
by the Folin-Ciocalteu procedure and 54.3 and 42.8 µg gallic acid equivalent of phenols was
detected in 1 mg WEBP and EEBP.
20. D Hooge R et. al.20 showed that piperine significantly block convulsions induced by
intracerebroventricular injection of threshold doses of kainite, but to have no or slight effects
on convulsions induced by L- glutamate, N-methyl D aspartate or guanidine succinate
receptor.
21. Ramnik Singh et.al.21 obtained various fractions R1, R2 and R3 from pet ether extract of
Piper nigrum Linn. (PEPN) and were investigated for invitro antioxidant activity.1,1Diphenyl-2-picryl-hydrazyl (DPPH) radical, superoxide anion radical, nitric oxide radical,
and hydroxyl radical scavenging assays were performed. The free radical scavenging activity
of the different fractions PEPN increased in a concentration dependent manner. R3 and R2
fraction of PEPN in 500Pg/ml inhibited the peroxidation of a linoleic acid emulsion by
60.48±3.33% and 58.89±2.51%, respectively. In DPPH free radical scavenging assay, the
activity of R3 and R2 was found almost similar. R3 (100Pg/ml) fraction of PEPN inhibited
Page 33
Kajal L. Jain
Chapter
55.68±4.48% nitric oxide radicals generated from sodium nitroprusside whereas curcumin in
the same amount inhibited 84.27±4.12%. Moreover, PEPN scavenged the superoxide radical
generated by the Xanthine/Xanthine oxidase system. The fraction R2 and R3 in the dose of
1000Pg/ml also inhibited 61.04±5.11% and 63.56±4.17% respectively, the hydroxyl radical
generated by Fenton's reaction. The amounts of total phenolic compounds were also
determined and 56.98Pg pyrocatechol phenol equivalents were detected in one mg of R3.
22. Leung A. Y et. al.22 showed pepper has been thought to cure many illness such as cancer,
malaria and cholera, however today it is most commonly used as food additive.
23. Karekar V.,R et. al.23 assessed genotoxic potencial of piperine using, four different test
systems, namely Ames test using Salmonella typhimurium, micronucleus test, sperm shape
abnormality test and dominant lethal test using albino mice which showed piperine to be
non-genotoxic chemical.
Page 34
Kajal L. Jain
Chapter
For embeline :
1. Paranjape A.S, Gokhale G.S24, “arch. Int. P’codyn Ther” 1932,42,212 studied that vidang
showed anthelmentiic activity and found that the drug was ineffective in man against
hookworms and tapeworms but found good response against ascariasis.
2. Sarin J.P.S. and Ray G.k.25 “ The Indian Journal of Pharmacy”,1961,23(12),330-31 studied
a simple colorimetric method for the estimation of embeline in Embelia ibes. The method is
based on color reaction between embeline and aniline, the color intensity of which is directly
proportional to the concentration of embeline.
3. Bheemasankara
Rao
and
Venkateswarlu
V26
“
The
Indian
Journal
of
Pharmacy”,1962,24(10),252 studied a new method for gravimetric determination of embeline
and vilangin in Embelia ribes.
4. Zutshi U, Johri R.K, Atal C.K27, ”Indian Journal of Experimental Biol.”,1989,27(7),656
showed that potassium embelate was established as a centrally acting analgesic in mice and
rats. Mixed µ and k receptor binding sites in the brain may be involved in the analgesic
activity. Analgesic activity was compared wih morphine, although it was not antagonized by
naloxone, indicating a central site of action differed from opiates.
5. Choudhary R.P, Ibrahim A, Bharati H.N28, “Electronic Journal of Food and Plt Chem”
2007, 2(1),20 showed that emeline in fruit of myrisana Africana was quantified using HPLC
and HPTLC. U.V detection was used in both analytical methods to examine the presence of
quantity of embeline. The assay provided a good accuracy and reproducibility for the
quantitative analysis of embeline.
6. Chauhan S.K, Singh B.P, Agrawal S29, “Indian Drugs”1999,36(1),41 showed a simple and
reproducible method for determination of embeline in Embelia ribes. A TLC method with
densitometric U.V detection has been developed for the quantification of embeline in
Lysimachia punctata.
Page 35
Kajal L. Jain
Chapter
7. Venketa Rao D, Ramana Rao V.V, Pulla Rao J, Subramanium B 30, “ Indian Journal of
Pharmaceutical Sciences”,1986,2,37 found that only iodoembeline showed enhanced
anthelmentic
activity
compared
to
embeline.
All
halo
compounds
possessed
increasedantibacterial activity against all organisms tested except S.typhi. the acetates of halo
embelines was found to be more active than either haloembeline or embeline itself. P- amino
benzoate derivative of embeline was also synthesized and studied for biological activity.
8. Patel R.K, Pundarikakshadu K., Patel M.M31, “Indian Journal of Pharmaceutical
Sciences”, 2006, 227 developed a tablet formulation of embeline employing the wet
granulation and direct compression techniques. This study was also carried out to design a
suitable dissolution medium for embeline. Effects of different diluents like lactose,
microcrystalline cellulose, and co-crystallized lactose- microcrystalline cellulose were studied
for improving the flow and compressibility. Binders such as starch paste and alcoholic
polyvinyl pyrrolidine were used to optimize the crushing strength of the formulation.
Solubility study of embeline in different media revealed that embeline has optimum solubility
in phosphate buffer pH 8 and in 2% aq. SLS solution. Incorporation of 10% v/v ethanol in
phosphate buffer of ph 7.4, significantly increased the solubility of embeline. These solutions
were also found to be the most suitable media for dissolution of embeline in dissolution
studies.
9. Chitra M, Sukumar E, suja V, Devi C.S32, “ Journal of Chemoherapy”, 1994,109-13
studied embeline, plant based benzoquinone derivative and this found to exhibit significant
antitumor activity in methyl cholanthrene induced fibrosarcoma in albino rats besides
enhancing their survival time. The drug also has an appreciable action on pain and
inflammation. The changes in DNA, RNA and protein levels in various organs in the tumors
bearing control and the drug treated animals were also studied.
10. Guru L.V, Misra D.N33, “Journal Res. Indian Med”,1996,1,47 studied anthelmentic
activity for aqueous and alcoholic extract of vidang on children infested with ascarides and
found that alcoholic extract cured 80% of cases, whilst the aqueous extract cured 55%,
expelling the worms and rendering the stool free from ova.
Page 36
Kajal L. Jain
Chapter
11. Chander H, Ahemed S.D34, “J. of Stored Prod. Res.’,1989,25(2),87 studied embeline as
insecticidal. The efficiency as insecticidal against Tribolium castaneum and other insect was
established in wheat sample by embeline at 0.18 % brought about mortality even after 8
months of storage. The reproduction of insect was also reduced significantly.
12. M.Chitra,
C.S
Shyamala
Devi,
E.Sukumar35,
“Journal
of
Natural
Remedies”,2004,4(1),77-80 studied the effect of embeline on carbohydrate moieties of
glycoprotein in tumor bearing rats.
13. M.Chiitra et . al.36, Indian Journal of Pharmacology 2003; 35: 241-244 investigated the
effect of a naturally occurring antitumour compound embelin on its lipid lowering activity
in experimental fibrosarcoma. Methylcholantherene induced fibrosarcoma was transplanted
in rats. After 30 days, embelin (50 and 100 mg/kg, p.o.) was administered as suspension for
a period of 20 days. The blood sample was collected on 21st day and the liver and the
kidney were also removed for studying the lipid profile in serum and the tissues. The levels
of total cholesterol, phospholipids, triglycerides and free fatty acids were markedly elevated
in the serum of tumour bearing rats. Significant alterations were also observed in the lipid
profile of liver and kidney. These changes were significantly reversed in embelin (100
mg/kg) treated animals. The reversal of altered lipid to normal values by embelin in rats
with experimentally induced tumour further supports the antitumour potential of embelin.
14. R. Paul Choudhury et.al.37 ,quantified Embelin in fruits of Myrsine africana L.
(Myrsinaceae)
using high performance liquid chromatography (HPLC) and high
performance thin layer chromatography (HPTLC) at 288 nm and 333 nm respectively. UV
detection was used in both analytical methods to examine the presence and quantity of
embelin. The assays provided a good accuracy and reproducibility for the quantitative
analysis of embelin. The results obtained in both analytical methods did not show any
significant overall difference (P = 0.66 ) although a trend to slightly lower range could be
found for the HPTLC method. Embelin content in Myrsine africana was similar to that
found in Embelia ribes Burm. f. and therefore has potential use as a substitute and an
alternative source of embelin
Page 37
Kajal L. Jain
Chapter
For carvone :
1.
Rakesh.K.Patel, M.M.Patel and K.Pundarikakshadu38 , Planta indica”, 2006, vol 2(3),
35-36 discussed the standardization of herbs in present scenario and the factors standing in
the way of acceptance of herbal drugs and checking quality by modern metods.
2.
J.Minija, J.E.Thoppil39 , “Journal of Natural Remedies”,2001, vol 1(2),147-150 found the
volatile oil constitution and microbial activities of essential oils of Corriandrum sativum.
3.
B.T Umesh et.al.40 , “Journal of Natural Remedies”, 2003, vol 1(2),199-201 showed the
superiority of microbial potency of essential oil of Curcuma haritha over commercial
bacterioside gentamicin.
4.
S.H Ansari et.al.41 ,“Indian Journal of Natural Products”,1991, vol 7(2),15 performed
chromatographic analysis of oil from Pistacia intigerima galls of family Anacardiaceae .
5.
E.Porchezhian et.al.42 , “Indian Journal of Natural Products”,2000, vol16(1),24 showed
analgesic and anti-inflammatory activity of volatile oil from Daucus carota.
6.
Syed Imam Rabbans43 , “Indian Journal of Natural Products”,2003,vol 19(2), 13 showed
invivo clastogenic effects of citral in mice.
7.
Gurinder Jeet Kaur and Daljit Singh Arora44 , “Journal of Medicinal Plants
Research”vol. 4(2), 87-94 showed that plants belonging to the family Umbelliferae are used
in traditional
medicine to alleviate gastrointestinal disorders and various biological
activities have been accredited to these plants. Antimicrobial potential shown by these
plants further warrant their exploration for the development of novel effective
chemotherapeutic agents.
8.
Amr Amin, Mohamed Mousa45 ,“Cancer Therapy”,2007, vol 5, 55-66 concluded that
Individual isoprenoids suppress the proliferation of B16 and HL-60 promyelocytic leukemia
cells with varying degrees of potency. Cell cycle arrest at the G0-G1 phase and apoptosis
account, at least in part, for this suppression. These findings suggest that the cancer-
Page 38
Kajal L. Jain
Chapter
protective property of products is partly conferred by the cumulative impact of volatile
isoprenoid constituents. Carvone, a monoterpene, has been shown to reduce fore-stomach
tumor formation by about 60% and pulmonary adenoma formation by nearly 35%, due to its
capacity to inhibit Nnitrosodiethylamine- induced carcinogenesis. Dietary monoterpenes
were found to be helpful, both in prevention and therapy of cancer (Crowell, 1999). After
being extracted and purified, carvone has been utilized throughout the centuries. Ancient
Arabs were the first to use this herb for hysterical complaints and pale complexions.
9.
Kawther F. Abed46 , “Saudi Journal of Biological Sciences”,2007, vol 14 (1) 53-60 showed
antimicrobial activity of Dill and Fennel extracts by simple solvent extraction method
against some Mycobacterium species essential oils from both fennel and dill have shown
considerable antifungal activity against commonly encountered Candida species
10. V. V. Semenov
et.al.47,“Russian
Chemical
Bulletin”,2007,
vol
56(12),
isolated
polyalkoxybenzenes from CO2 extracts of Umbelliferae plant seeds. Here the conditions of
CO2 extraction for obtaining concentrates with minimum contents of the distillation residues
were selected. Using high performance fractional distillation, polyalkoxyallylbenzenes with
98–99% purity were isolated from the concentrates on a pilot unit. By isomerization of some
allylbenzenes followed by ozonolysis under specially selected conditions, apiol-and
dillapiolaldehydes were obtained in 75–80% yields.
11. Malihezaman Monsefi et.al.48 , “DARU”, 2006, volume 14,(3),131 showed the effect of
Anethum graveolens on female reproductive system of rats. Anethum graveolens extract
increased duration of diestrus phases and total time of estrous cycle in high dose groups but
had no significant effect in duration of estrus and proestrus phases.
12. Kevin Cusack49 ,”Abbot Role of Stereochemistry in Drug Development”, 2006, vol 3(22)
showed stereochemistry and distinct receptor binding profiles synthesis of carvone via
pinene and oxidative routes via carveol
13. Carol A. Clausen Vina W. Yang50 , “American Wood Protection Association” suggest that
dill weed, lemongrass, tea tree, rosemary, or geranium (Egyptian) essential oils could be
Page 39
Kajal L. Jain
Chapter
used alone or as a co-termiticide fumigant for wood product applications to protect
structures from termite attack.
14. Hossein Hosseinzadeh, Gholam_Reza Karimi, and Maryam Ameri51 , “pubmed”showed
the Effects of Anethum graveolens L. seed extracts on experimental gastric irritation models
in mice and indicated that the extracts of A. graveolens seed have effective antisecretory
and anti-ulcer activity against HCl- and ethanol-induced stomach lesions.
15. K.R.Kirtikar and Basu52 ,Indian Medicinal Plants, suggests its use as carminative,
anthelmentic, jaundice, skin disease, expelling tape worms, bronchitis.
Page 40
Kajal L. Jain
Chapter
References :
1. Anonymous “ Indian Herbal Pharmacopeia”, Indian drug manufacturer’s Association,
Mumbai, 2062, 312-314.
2. Anonymous “ Official methods of Analysis of AOAC International”, vol II, AOAC
International,16th edition, 1995, CH- 43, 5.
3. Anonymous “ Indian Council of medical Research of India”,vol I,2003, New Delhi, 171.
4. M. K. Santosh, D. Shaila, I. Rajyalakshmi and I. Sanjeeva Rao, RP - HPLC Method for
Determination of Piperine from Piper longum Linn. and Piper nigrum Linn “ Electronic
Journal of Chemistry”, 2005, 2(2), pp 131- 135.
5. Kedar Kumar Rout, Om Prakash Rout, Sagar Kumar Mishra, Estimation of piperine in
commercial Ayurvedic formulations Journal of Planar Chromatography, 2007,2(6), pp
447-450.
6. S.K.Chauhan, G.P kimothi, B.P Singh and S.Agrawal, “Indian drugs”, july
1998,35(7),408-411.
7. A Kumar, S Panghal, SS Mallapur, M Kumar, Veerma Ram, BK Singh
Antiinflammatory activity of Piper longum fruit oil.
8. Jagdale, S.C., B.S. Kuchekar, A.R. Chabukswar, P.D. Lokhande and C.G. Raut, 2009.
Anti-oxidant activity of Piper longum Linn. Int. J. Biol. Chem., 3: 119-125.
9. Dipak Kulkarni, Sukhdar P.Apte, Francis Mary and Sane R.T, “Indian Drugs”, june
2001,38(6), 323-326.
Page 41
Kajal L. Jain
Chapter
10. Samudram P., Rajeshwari Hari, Vasuki R., Geetha A. and Sathiya moorthi P.
Hepatoprotective activity of Bi - herbal ethanolic extract on CCl4 induced hepatic
damage in rats“African Journal of Biochemistry Research”, 2008,.2 (2), pp. 061-065.
11. A. B. Wood, Maureen L. Barrow, D. J. James, Piperine determination in pepper (Piper
nigrum
L.)
and
its
oleoresins - a
reversed-phase
high-performance
liquid
chromatographic method “ Flavour and Fragrance Journal”, 3(2), pp 55 – 64.
12. Suthar A.C, Sohoni D.P, Banvalikar M.M and Biyani M.K ”Indian Drugs”, December
2003,40(12), 692-694.
13. Koul Surrinder, Koul Jawahir L., Taniya SubashC.,Dhar Kanaya L etal, SAR of piperine
and its syn thetic analogues for their inhibitory potencials of rat hepatic microsomal
constitutive and inducible cytochrome P 450 activities “ Bioorganic and Medical
Chemistry” 8(1),2000,251-268.
14. Koul I.B, Kapil A., Evaluation of the liver protective potencial of piperine, an active
principle of black and long peppers, “Planta Medica”, 59(5),1993,413-17.
15. Sharma G. And Mishra B., “Journal of Pharmaceutical research”, vol-6(3),july 2007,
129-133.
16. Mujumdar A.M, Dhuley J.N, Deshmukh V.K, Raman P.H, Naik S.R, Antiinflammatory
activity of piperine,” Japaneese Journal of Medicinal Science Biol.”, 43(3),1990, 95-100.
17. Gupta S.K, Bansal P, Bharadwaj R.K, Velpendian T, comparative antinociceptive, antiinflammatory and toxicity profile of nimesulide v/s nimesulide and piperine combination,
Pharmacological Research, 41(6),2000,657-662.
18. Rauscher Fedrick M., Sanders
Ruth A., Watkins John B., Effects of piperine on
antioxidant pathways in tissues from normal and streptozocin induced diabetic rats, Inc,
“Journal of Biochem Mol Toxicol” 14 2000,329-334.
Page 42
Kajal L. Jain
Chapter
19. İlhami Gülçin, The antioxidant and radical scavenging activities of black pepper (Piper
nigrum) seeds International Journal of Food Sciences and Nutrition 2005, 56( 7), pp
491-499 ,
20. D’ Hooge R., Pei Y.CS, Raes A, Lebrun P, van Bogarrt P.P, de Deyn P.P,
Anticonvulsant activity of piperine on seizures induced by excitatory aminoacid receptor
agonist, Arzneimittelforschung, 4696): 1996, 557-60.
21. Ramnik Singh and H.S. Rao invitro antioxidant activity of Piper nigrum Linn.
22. Leung A.Y, Ed Encyclopedia of Common Natural Ingredients used in Food, Drugs and
Cosmetics, John Wiley and Sons: New York 1980.
23. Karekar V.R, Mujumdar A.m, Joshi S S, Dhuley J, Shindey SL, Ghaskadhi S.,
Assessment of genotoxic effect of piperine using salmonella typhimurium and somatic
and and germ cells of swiss albino mice, Arzneimittelforschung 46(10), 1996, 972-5.
24. Paranjape A.S, Gokhale G.S, “arch. Int. P’codyn Ther” 1932,42,212.
25. Sarin J.P.S. and Ray G.k. “ The Indian Journal of Pharmacy”,1961,23(12),330-31.
26. Bheemasankara
Rao
and
Venkateswarlu
V
“
The
Indian
Journal
of
Pharmacy”,1962,24(10),252.
27. Zutshi U, Johri R.K, Atal C.K, ”Indian Journal of Experimental Biol.”,1989,27(7),656.
28. Choudhary R.P, Ibrahim A, Bharati H.N, “Electronic Journal of Food and Plt Chem”
2007, 2(1),20.
29. Chauhan S.K, Singh B.P, Agrawal S, “Indian Drugs”1999,36(1),41.
30. Venketa Rao D, Ramana Rao V.V, Pulla Rao J, Subramanium B, “ Indian Journal of
Pharmaceutical Sciences”,1986,2,37.
Page 43
Kajal L. Jain
Chapter
31. Patel R.K, Pundarikakshadu K., Patel M.M, “Indian Journal of Pharmaceutical
Sciences”, 2006, 227 .
32. Chitra M, Sukumar E, suja V, Devi C.S, “ Journal of Chemoherapy”, 1994,109-13.
33. Guru L.V, Misra D.N, “Journal Res. Indian Med”,1996,1,47.
34. Chander H, Ahemed S.D, “J. of Stored Prod. Res.’,1989,25(2),87.
35. M.Chitra, C.S Shyamala Devi, E.Sukumar “Journal of Natural Remedies”,2004,4(1),7780.
36. M.Chitra et . al Indian Journal of Pharmacology 2003; 35: 241-244.
37. R. Paul Choudhury, A. Md. Ibrahim, H.N. Bharathi, and Padma Venkatasubramanian
Quantitative Analysis of Embelin in Myrsine africana L. (Myrsinacee) using HPLC and
HPTLC Electronic Journal of Food and Plants Chemistry 2 (1) 2007 20-24.
38. Rakesh.K.Patel, M.M.Patel and K.Pundarikakshadu”Planta indica”, 2006, vol 2(3), 3536.
39. J.Minija, J.E.Thoppil “Journal of Natural Remedies”,2001, vol 1(2),147-150.
40. B.T Umesh et.al “Journal of Natural Remedies”, 2003, vol 1(2),199-201.
41. S.H Ansari et.al “Indian Journal of Natural Products”,1991, vol 7(2),15.
42. E.Porchezhian et.al “Indian Journal of Natural Products”,2000, vol16(1),24.
43. Syed Imam Rabbans “Indian Journal of Natural Products”,2003,vol 19(2).
Page 44
Kajal L. Jain
Chapter
44. Gurinder Jeet Kaur and Daljit Singh Arora “Journal of Medicinal Plants Research”vol.
4(2), 87-94.
45. Amr Amin, Mohamed Mousa “Cancer Therapy”,2007, vol 5, 55-66.
46. Kawther F. Abed “Saudi Journal of Biological Sciences”,2007, vol 14 (1) 53-60.
47. V. V. Semenov et.al “Russian Chemical Bulletin”,2007, vol 56(12).
48. Malihezaman Monsefi et.al “DARU”, 2006, volume 14,(3),131.
49. Kevin Cusack”Abbot Role of Stereochemistry in Drug Development”, 2006, vol 3(22).
50. Carol A. Clausen Vina W. Yang “American Wood Proteetion Association”.
51. Hossein Hosseinzadeh, Gholam_Reza Karimi, and Maryam Ameri, “pubmed”.
52. K.R.Kirtikar and Basu, Indian Medicinal Plants.
Page 45
Kajal L. Jain
Chapter 6
Experimental Work
Chapter 6
Experimental Work
6.1 Pharmacognostic And Physicochemical Investigation Of Raw Materials
6.1.1 Introduction
Plant based medicines have been used by mankind since time immemorial. According to the
report of Whorld Health Organization(WHO), over 80 % of world population relies on
traditional system of medicine, largely plant based, to meet their primary health care. The
effectiveness of these system, in turn, mainly depends upon the proper use and sustained
availability of genuine raw material. Global resurgence of the interest in herbal drugs has led to
the need of their mass production which perhaps was not even contemplated by the traditional
medicine practitioners who prepared and dispensed the medicine on a personal and individual
bias.
Large scale production of medicinal plant products nesseciate the availability of standards to
ensure their quality, efficacy and safety. These are mainly concerned with the quality of raw
material and methodology adopted during the procurement, handling and processing them. The
prime importance is given to standardization of raw materials which are used in such
formulation to ascertain the efficacy and safety. Many guidelines are published especially by
WHO, IHP, API, Ayurvedic formulary, ICMR, AOAC and many others for the standardization
and evaluation of these materials.
Hence in the present investigation the attempt was made to standardize the materials under the
study for the pharmacognostic, physicochemical and phytochemical parameters according to the
guidelines.
Page 46
Kajal L. Jain
Chapter 6
Experimental Work
6.1.2 Experimental
6.1.2.1 Materials
Different samples of crude drugs i.e black pepper, dill seeds, vidang fruits and rock salt were
procured from two different ayurvedic drug supplier viz. Ms. L.V.Gandhi & Sons, Ahmedabad
and Ms. Sanjivani Aushadhalaya, Bhavnagar which are shown in the following table.
Name
of Name of sample
supplier
LVG
Pepper 1
Dill 1
Vidand 1
Saindhav 1
S.A
Pepper 2
Dill 2
Vidand 2
Saindhav 2
Procured samples were powdered and used for standardization. Powder passed through 40 mesh
sieve. Standardization of these powdered samples was carried out for the usual pharmacognostic,
physicochemical and chromatographic parameters as described below.
6.1.2.2 Methodology
6.1.2.2.1 Foreign matter analysis
Here drug sample are sieved and then with the help of magnifying lens checked for the presence
of impurities. Then impurities and fines are considered as foreign matter and weighed and %
foreign matter calculated.
6.1.2.2.2 Microscopic examination method
The coarse seed powder was boiled with chloral hydrate for 5 minutes, then stained with
phloroglucinol and HCL (1:1) and observed for the microscopic features under low power (10x)
and high power (40 x).
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Kajal L. Jain
Chapter 6
Experimental Work
6.1.2.2.3 Loss on drying
To estimate loss on drying, 5 gm of air dried powdered plant material were accurately weighed
in a dried and tarred flat weighing bottle. The sample was dried to constant mass in a hot air
oven at temperature 105 ºC. The loss on drying was determined with respect to air dried plant
material.
6.1.2.2.4 Determination of ash value
a.) Total ash : 3g of accurately weighed powdered drug was taken in a tarred silica crucible and
incinerated at a temperature not exceeding 4500C until free from carbon and constant weight,
cooled and weighed .
b.) Acid-insoluble ash : Total ash obtained was boiled for five minutes with 25 ml of
dilute
Hydrochloric acid. The insoluble matter was collected on an ash less filter paper, washed with
hot water and ignited, cooled and weighed. The percentage of acid insoluble ash was calculated
with reference to powder.
c.) Water-soluble ash : Total ash obtained was boiled for five minutes with 25ml of distilled
water, cooled and collect the insoluble matter on an ash-less filter paper, washed with hot water
and ignited for 15 minutes at temperature not exceeding 4500C. The percentage of water-soluble
ash was calculated with reference to powder.
Page 48
Kajal L. Jain
Chapter 6
Experimental Work
6.1.2.2.5 Determination of extractive value
The extractive value of drug helps to determine the amount of soluble constituents
in a
given amount of medicinal plant material, when extracted with solvents. The extraction of any
crude drug with a particular solvent yields a solution containing different phytoconstituents. The
composition of these phytoconstituents in a solvent depends upon the nature of drug and solvent
used.
a.) Alcohol soluble extractive value: 5g seed powder of A, subulatum was macerated
with
100ml of ethanol in a closed flask, shaking frequently during the first 6hrs and allowed to stand
for 18hrs. Thereafter it was filtered rapidly taking precaution against loss of ethanol. Evaporate
25ml of filtrate to dryness in a tared flat bottom shallow dish dried at 1050C and weighed.
Percentage ethanol soluble extractive was calculated with reference to the dried powder.
b.) Water soluble extractive value: Above procedure was repeated using water in place of
alcohol.
6.1.2.2.6 Phytochemical investigation:
All drugs were rxtracted in methanol and extract was concentrated. These extracts
were then subjected to various qualitative tests for identification of various plant constituents
like alkaloid, glycoside, volatile oil, flavaoids , tannins etc.
Qualitative chemical tests for identifying various phytoconstituents present were carried out on
various extracts of A. subulatum Roxb fruit constituents as follows.
 Tests for Glycosides :
Test I: Extract 200 mg of powder drug by warming in a test tube with 5 ml of dilute (10%)
sulphuric acid on a water bath at 100oC for two minutes, centrifuge or filter, pipette out
supernatant or filtrate. Neutralize the acid extract with 5% solution of Sodium hydroxide
(note the volume of NaOH added). Add 0.1 ml of Fehling’s solution A and B until
Page 49
Kajal L. Jain
Chapter 6
Experimental Work
alkaline (test with pH paper) and heat on a water bath for 2 minutes. Note the quantity of
red precipitate formed and compare with that formed in Test II.
Test II: Extract 200 mg of the drug using 5 ml of and boil on water bath. After boiling add equal
volume of water to the volume of NaoH used in the above test. Add 0.1 ml of Fehling’s
A and B until alkaline (red litmus changes to blue) and heat on water bath for two
minutes. Note the quantity of the red precipitate formed.
Compare the precipitates of Test II with Test I. If the precipitate in Test-II is greater than
in Test-I, then Glycoside may be present. Since Test I represent the amount of free
reducing sugar already present in the crude drug, whereas Test-II represents the
Glycoside after acid hydrolysis.
 Tests for Alkaloids :
a. Mayer’s test (Potassium mercuric iodide solution): To extract/sample solution, add few
drops of Mayer’s reagent, creamy white precipitate is produced
b. Dragendroff’s test (Potassium bismuth iodide solution): To extract/sample solution, add
few drops of Dragendroff’s reagent, reddish brown precipitate is produced.
c.
Wagner’s test (Solution of Iodine in Potassium Iodide): To extract/sample solution,
add few drops of Wagner’s reagent, reddish brown precipitate is produced.
d.
Hager’s Test (Saturated solution of Picric acid): To extract/sample solution, add few
drops of Hager’s reagent, yellow precipitate is produced.
e.
Tannic acid test: To extract/sample solution, add few drops of Tannic acid solution,
buff precipitate is produced.
 Tests for Phenolic Compounds :
Page 50
Kajal L. Jain
Chapter 6
Experimental Work
a. Ferric chloride test: Extract solution gives blue-green color with few drops of Fecl3.
b. Shinoda Test (Magnesium Hydrochloride reduction test): To the extract solution, add
few fragments of magnesium ribbon and concentrated Hydrochloric acid drop wise,
yellowish, yellow- orange occasionally orange color appears after few minutes.
c. Zinc-Hydrochloride reduction test: To the extract solution, add a mixture of Zinc dust
and conc. Hydrochloric acid. It gives yellowish, yellow- orange occasionally orange
color appears after few minutes.
 Tests for Flavonoids :
a. Shinoda Test (Magnesium Hydrochloride reduction test): To the extract solution add
few fragments of magnesium ribbon and concentrated Hydrochloric acid drop wise, pink
scarlet, crimson red or occasionally green to blue color appears after few minutes.
b. Zinc-Hydrochloride reduction test: To the extract solution, add a mixture of Zinc dust
and conc. Hydrochloric acid. It gives red color after few minutes.
c. Alkaline reagent test: To the extract solution, add few drops of Sodium hydroxide
solution; formation of an intense yellow color that turns to colorless on addition of few
drops of dilute acetic acid indicates the presence of flavonoids.
 Tests for Tannins :
a.
Gelatin test: Extract solution with 1% gelatin solution containing 10% sodium chloride
gives white precipitate.
b.
Ferric chloride test: Extract solution gives blue-green color precipitate with Fecl3.
c.
Vanillin Hydrochloride test: Extract solution when treated with few drops of Vanillin
Hydrochloride reagent gives purple red color.
d.
Alkaline reagent test: Extract solution with sodium hydroxide solution gives yellow
to red precipitate within short time.
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Kajal L. Jain
Chapter 6
e.
Experimental Work
Metchell’s test: With iron and ammonium citrate or iron and sodium tartarate, tannins
give a water-soluble iron-tannin complex, which is insoluble in solution of ammonium
acetate.Tannins get precipitated in the solution when treated with heavy metals.
f.
Tannins yield bulky precipitate with phenazone especially in the presence of sodium
and phosphate.
 Test for Steroidal Glycosides :
Kedde’s test: Extract the leaf powder with chloroform, evaporate to dryness, and add one drop
of 90% of alcohol and 2 drops of 2% 3,5-dinitro benzoic acid (3,5, dinitrobenzene carboxylic
acid - Kedde’s reagent) in 90% alcohol. Make alkaline with 20% sodium hydroxide solution. A
purple color is produced. The color reaction with dinitrobenzoic acid depends upon the presence
of an ,  unsaturated –  lactone in the aglycone.
6.1.2.2.7 Determination of volatile oil content from dill fruit powder of various
mesh size
Volatile oil is isolated or determined by using clavenger apparatus. In order to
determine the
volume of oil, the powder material is distilled with water and the distillate is collected in a
graduated tube. The aqueous portion separates automatically and is returned to the distillation
flask. Since the volatile oil possesses the mass density lighter than water, it floats on the top of
the aqueous phase.
Apparatus/Method:
Clavanger apparatus was used to isolate volatile oil. Here drug powder ws mixed with water and
heated at constant temperature.Volatile oil was lighter than water, so it started floting on water
layer. After few hours when oil ceased to isolate, the process was stoped and after measuring
volume oil was removed and stored carefully.
6.1.2.2.8 Estimation of carvone in volatile oil by titrimetry
Weigh accurately 1.5 gram of dill oil (specific gravity- 0.916 gm/ml) in conical flask. Add 10 ml
of hydroxyl amine hydrochloride reagent prepared in 90 % ethanol. Place the tightly closed flask
in water bath for heatingt at 80 °C neutralize the liberated acid with 1N potassium hydroxide
Page 52
Kajal L. Jain
Chapter 6
Experimental Work
prepare in ethanol until the red color changes to yellow. Continue heating and titrate again until
yellow color remains persistent. The reaction will acquire 30-40 miniutes for completion.The
indicator for notice of change in color is already added in preparation of hydroxylamine
hydrochloride reagent. Here after addition of reagent, the red color produced is titrated with
KOH to yellow color and then heated. This is repeated till persistent yellow color is obtained.
Calculate content of carvone by following factor:
Each ml of 1 N KOH ~ 0.1514 g of carvone.
6.2 Simultaneous estimation of piperine in Piper nigrum, embeline in Embelia
ribes, carvone in Anethum sowa.
6.2.1 Introduction
Herbal medicine has been enjoying renaissance among the customers throughoutthe world.
However one of the impediments in the acceptance of the Ayurvedicformulation is the lack of
standard quality control profiles (Bagul and Rajani, 2005). The quality of herbal medicine, that
is, the profile of the constituents in the final products has implication in efficacy and safety. Due
to the complex nature and inherent variability of the chemical constituents of the plant based
drugs, it is difficult to establish quality control parameters and modern analytical techniques.are
expected to help in circumventing this problem.
Standardization and analysis of chemical markers of ayurvedic and other polyherbal formulation
is always difficult. Quantitative estimation of chemical markers of each ingredient in the
polyherbal formulation requires ideal separation technique by which these markers are separated
with highest purity and with least interference from each other. For botanicals and herbal
preparation, there is a requirement of scientific proof. HPTLC is the most commonly applied for
the analysis of multiple constituents present in the medicinal plants and herbal preparation.
In the present study we report the development of a HPTLC method for the
simultaneous
estimation of piperine, carvone and embeline in black pepper, dill seeds and vidang respectively.
Page 53
Kajal L. Jain
Chapter 6
Experimental Work
Three chemical markers, one from each medicinal plant were selected, piperine from black
pepper, carvone from dill seeds, embeline from vidang, for the quantification as these are
responsible for physiological action.
6.2.2 Materials and chemicals :
Standard Piperine
Standard carvone
Standard embeline
Pepper powder
Vidang powder
Dill seed powder
AR grade Methanol
AR grade Toluene
AR grade Ethylacetate
AR grade n- hexane
AR grade gl. Acetic acid
AR grade formic acid
6.2.3 Instrumentation
Analysis was performed on 10cm x 10cm plates cut from 20cm x 20cm aluminium backed silica
gel 60 F254 plates. Samples were applied to the plates by means of a Linomat-V automatic
spotter with the aid of Hamilton 100 μl syringe. TLC plates were developed in flat bottom twin
trough chamber. Densitometry was performed with a TLCscanner-3 with Win CATS 4 software
resident in a Pentium IV computer.
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Kajal L. Jain
Chapter 6
Experimental Work
6.2.4 Chromatographic condition :
Stationary phase
: 15 x 10 cm aluminium backed silica gel 60 F254
Plates (E.Merck)
Mobile phase
: toluene : ethyl acetate : n hexane : gl acetic acid : formic acid
Chamber saturation
: 15 min
Band width
: 4 mm
Distance between tracks
: 5 mm
Rate of spotting
: 5 µl/sec
Distance run
: 85 mm
Visualization
: observe plate under U.V 254 nm and 366 nm
Scanning wavelength
: 254 nm
Scanning speed
: 5mm/sec
Slit dimension
: 5 x 0.45 mm
Temperature
: 25 °C
6.2.5 Preparation of standard solution :
6.2.5.1 Preparation of standard solution of Piperine :
Standard solution of piperine was prepared by weighing and diluting 10 mg of
standard
piperine upto 10 ml methanol.
6.2.5.2 Preparation of standard solution of carvone :
Standard solution of carvone was prepared by diluting 10 µl of standard carvone upto 10 ml
methanol.
6.2.5.3 Preparation of sample solution of embeline :
Standard solution of embeline was prepared by weighing and diluting 1 mg of standard embeline
upto 10 ml methanol.
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Kajal L. Jain
Chapter 6
Experimental Work
6.2.5.4 Preparation of test solution :
500 mg of vidang powder,500 mg of pepper powder and 1gm of dill seeds powder were weighed
separately and refluxed for 1 hr in a seperate flask. It is then filterd and diluted upto 50 ml.
6.2.6 Preparation of standard curve :
Analysis was performed on 10 x 10 cm precoated silica gel 60 F254 TLC plate (E.Merck) of
uniform thickness. For preparation of standard curve 1-9 µl volume of each of the standard
solution were spotted bt linomat v spotter. Plate was kept in tlc chamber and allowed to run upto
85 mm. the plate was then removed and dried in air and densitometric scan was performed at
254 nm with scanner -3 with the conditions as mentioned above. A caliberation equation relating
to the standard concentration to scan area was determined.
6.2.7 Estimation of piperine, carvone and embeline :
5 µl of the test solutions of samples from two suppliers was spotted along with the standard spots
on precoated silicagel plate. Amount of the active constituent was calculated from the
caliberation equation.
6.2.8 Validation of the HPTLC method :
The method was validated as per ICH guidelines for Linearity, Precision, Limit of Detection,
Limit of Quantification, Accuracy and Specificity
Linearity
Linearity of the method was performed by analyzing standard solution of Piperine, embeline and
carvone by the proposed method in concentration range 0.5 to 10, 0.1 to 0.5 mcg/spot and 1 to 9
nl/spot respectively.
Accuracy
Accuracy of the proposed method was determined by recovery study. Recovery study was
carried out by adding three different quantities of Piperine, Embeline and Carvone (2.5, 5 and
7.5 mcg/spot) to preanalyzed solution of respectively sample of churna. All the procedure was
repeated three times. From the linear regression percentage recovery of Piperine, Embeline and
Carvone were determined.
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Kajal L. Jain
Chapter 6
Experimental Work
Precision
Precision was determined by repeatability, intra day and inter day (Table 3) reproducibility
experiment of the proposed method. Repeatability was evaluated for degree of repeatability of
spotting by preparing and analyzing the standard solution of the drug three times. The intra day
reproducibility was determined by analyzing freshly prepared solution in triplicate at three
different concentration whereas inter day reproducibility was checked by analyzing the standard
solutions at three different days under same operative condition.
Limit of Detection and Limit of Quantification
Limit of Detection of Piperine, Embeline and Carvone were found o be 0.2 mcg,0.05 mcg and
0.2 nl and Limit of Quantification were found to be 0.5mcg, 0.1mcg and 0.5 nl respectively.
Specificity
Specificity of an analytical method is its ability to measure the analyte accurately and spefically
in the presence of component that may be expected to be present in the sample matrix. Test and
standard of Piperine, embeline and carvone were spotted on the TLC plate, developed and
scanned as described above and it was observed that other constituents present in the formulation
did not interfere with the peak of the markers. Therefore the method was specific. The test
chromatogram (Figure 14) was compared with the standard (Figure 12) and were found to be
similar.
6.3 Standardization Of Catpusphadhya Churna
6.3.1 Introduction
According to the report of World Health Organization (WHO), over 80 per cent of the world
population relies on the traditional systems of medicine, largely plant based, to meet their
primary health care. Efficacy and safety of the herbal drugs and formulations including
ayurvedic and other traditional formulations are always under a big question mark. Furthermore,
limited availability of medicinal plants of consistent quality has been perhaps one of the toughest
impediments for reliable biological, pharmacological, chemical and clinical evaluation besides
their use in health care. Lack or inadequacy of quality standards has been a discouraging factor
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Kajal L. Jain
Chapter 6
Experimental Work
for many potential herbal drug manufacturers and also a handicap in regulating the herbal drug
market.
Hence, in the present investigation the attempt was made to standardization of Catpusphadhya
churna under the study for the pharmacognostic, physicochemical and
Phytochemical and parameters according to these guidelines.
6.3.2 Experimental
6.3.2.1 Materials
Catpusphadhya churna consists of three herbs, Black pepper (Pepper nigrum), Vidang (Embelia
ribes), Dill (Anethum suwa) and Rock salt. Formulation was prepared according to the ayurvedic
system of Indian medicine from the powder of raw materials and other ingredients of well
known ayurvedic pharmacy, L. V. Gandhi & Sons, Ahmedabad and Sanjivani aushadhalaya,
Bhavnagar in the laboratory.
6.3.2.2 Method of preparation :
Following drugs were used in equal quantities :
Pepper nigrum
1 part
Embelia ribes
1 part
Anethum suwa
1 part
Rock salt
1 part
These drugs were evaluated for pharmacognostic and physicochemical evaluation. These drugs
were powdered , then weighed and mixed in equal quantities. It is then kept in air tight
container in cool and dry place.
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Kajal L. Jain
Chapter 6
Experimental Work
6.3.2.3 Standardization of catpusphadhya churna
6.3.2.3.1 Loss on drying
To estimate the loss on drying, 5 gm of the air dried Churna is accurately weighed in a dried and
tared flat weighing bottle. The sample was dried to constant mass in a hot air oven at
temperature 105o C. The loss on drying was determined with respect to air-dried plant material.
6.3.2.3.2 Determination of ash value
a.) Total ash
4 g of the air-dried Vasavaleha was taken and accurately weighed, in a previously ignite and
tared crucible (silica). The material was placed in an even layer and ignited by gradually
increasing the heat to 500-600o C until it appeared completely white, indicating the absence of
carbon. The material called ash was cooled in desiccators for 30 min and then weighed. The
content of total ash was determined with respect to air-dried material.
b.) Acid insoluble ash water soluble ash
To the crucible containing the total ash, 25 ml of HCL (~70g/l) was added, covered with a watch
glass and boiled gently for 5 minutes. The watch glass was rinsed with 5 ml of hot water and
washing was added to the crucible. Insoluble matter was collected on an ash less filter paper and
washing of this filter paper was carried out with hot water until the filtrate was remaining
neutral. The filter paper containing the insoluble matter was transferred to the original crucible,
which is then dried on a hot plate and ignited to constant weight. Allowed the residue to cool in a
suitable desiccator for 30 minutes, and then weighed without delay. The content of acidinsoluble ash was calculated with respect to the weight of air dried material.
c.) Water-Soluble Ash Value
To the crucible containing total ash, 25 ml of water was added and boiled for 5 minutes.
Insoluble matter was collected on an ash less filter paper. The residue was washed with hot
water and ignited in a crucible for 15 minutes at a temperature not exceeding 450o C. The
weight of the residue was subtracted from the weight of total ash. The content of water soluble
ash was determined with respect to the weight of the air dried material.
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Kajal L. Jain
Chapter 6
Experimental Work
6.3.2.3.3 Determination of extractive value
a.) Water soluble extractive
Accurately weighed 10 gm of Vasavaleha was taken in glass-stopper conical flask. The
content was macerated with 100 ml of distilled water for 6 hours with frequent shaking
and then allowed to stand for 18 hours. The content was filtered taking care not to lose
any solvent. 25 ml of filtrate was transferred to a tarred flat-bottomed dish and evaporated to
dryness on a water-bath and dried at 105o C for 6 hours, cooled in desiccators for 30 min and
weighed without delay. Percent water soluble extractive value was calculated with respect to the
original weight of the air-dried material.
b.) Ethanol soluble extractive
Accurately weighed 10 gm of Vasavaleha was taken in glass-stopper conical flask. The content
was macerated with 100 ml of methanol for 6 hours with frequent shaking and then allowed to
stand for 18 hours. The content was filtered taking care not to lose any solvent. 25 ml of filtrate
was transferred to a tarred flat-bottomed dish and evaporated to dryness on a water-bath and
dried at 105o C for 6 hours, cooled in desiccators for 30 min and weighed without delay. Percent
alcohol soluble extractive value was calculated with
respect to the original weight of the air-dried material.
6.3.2.3.4 Powder properties
Here powder properties like bulk volume, tapped volume, bulk density tapped density are
calculate to find carr, s index, hausssner,s ratio and angle of repose.
6.3.2.3.5 Determination of pH
Page 60
Kajal L. Jain
Chapter 6
Experimental Work
5 gm churna sample was taken in conical flask. A 10 % w/v suspension of churna in water was
made by adding 50 ml of water and thoroughly mixed with frequent shaking for 30 min. The
suspension was centrifuged and the supernatant liquid was used for the determination of pH
using digital pH meter.
6.3.2.3.6 Estimation of piperine, carvone and embeline in churna by HPTLC
We have developed chromatographic mobile phase and other specification for Catpusphadhya
churna which was successfully performed for maximum separation of present phytoconstitutes.
Materials and chemicals :
Standard piperine
Standard carvone
Standard embeline
Pepper powder
Vidang powder
Dill seed powder
AR grade Methanol
AR grade Toluene
AR grade Ethylacetate
AR grade n- hexane
AR grade gl. Acetic acid
AR grade formic acid
Instrumentation
Page 61
Kajal L. Jain
Chapter 6
Experimental Work
Chromatographic condition
Stationary phase
: 10 x 10 cm aluminium backed silica gel 60 F254
Plates (E.Merck)
Mobile phase
: toluene : ethyl acetate : n hexane : gl acetic acid
: formic acid
Chamber saturation
: 15 min
Band width
: 4 mm
Distance between tracks : 5 mm
Rate of spotting
:
Distance run
: 85 mm
Visualization
: observe plate under U.V 254 nm and 366 nm
Scanning wavelength
: 254 nm
Scanning speed
: 5mm/sec
Slit dimension
: 5 x 0.45 mm
Temperature
: 25 °C
5 µl/sec
Preparation of sample solution :
500 mg of vidang powder, 500 mg of pepper powder and 1gm of dill seeds powder alonf with
equal quantity of salt were weighed separately and refluxed for 1 hr in a seperate flask. It is then
filterd. Mark is again refluxed with the solvent and then filtered. Both filtrate were combined
and solvent evaporated. This concentrated mass was diluted upto 50 ml. then amount of three
active constituent were calculated accordingly.
Estimation of 3 components:
From the different sample solutions, 5.0 μl was applied on the precoated silica gel plate and
process was repeated to develop and scan the plate as mentioned above. The amount of Piperine,
embeline and carvone in the samples was calculated from the calibration equation generated in
previous chapter by using the average area of triplicate sample aliquots. The results were
recorded.
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Kajal L. Jain
Chapter 6
Experimental Work
Validation of the HPTLC method for Catpusphadhya churna :
This method was validated for the accuracy by performing recovery study. Recovery study was
carried out by adding three different quantities of Piperine, embeline and carvone
to the
preanalyzed laboratory sample of churna. Extraction, separation and analysis were carried out as
discussed in the estimation procedure. Percent recovery of Piperine, embeline and carvone was
determined.
6.4 Pharmacological activity of Catpusphadhya churna
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Kajal L. Jain
Chapter 6
Experimental Work
6.4.1 In vitro Anti oxidant activity Methanol extract of churna along with
individual ingredients
6.4.1.1 Preparation of extract :
1g per batch of powder of each fruit individually was extracted with Methanol using soxhlet
apparatus. The extracts were concentrated and air dried.
6.4.1.2 Instruments :
UV Visible double beam Spectrophotometer (Shimadzu UV 1601), Centrifuge Machine (Eltek
research Centrifuge-TC-4100D)
6.4.1.3 1,1-Diphenyl-2-picryl hydrazyl (DPPH) radicals scavenging activity
Chemicals & reagents : α- α diphenyl β picryl hydrazyl DPPH. A solution of 500 μgM in
methanol was made & protected from light by covering the test tubes with aluminum foil.
Preparation of sample extracts : Both the extract above mentioned was taken in range of 10100 μg/ml in methanol.
Preparation of standard solution: Ascorbic acid was used as standard. Aliquots of 10-100
μg/ml in methanol were prepared.
Procedure: 1ml of DPPH solution was added to 2ml methanol & absorbance was taken after 30
minute at 517nm for control reading. 1 ml of different concentrations of extracts and sample
were taken,to it 1ml of methanol and 1 ml of DPPH was added. The mixture was kept in dark
for 30 minutes and absorbance was measured at 517nm after 30 minutes. The absorbance of
control reduce dose dependently.
The % reduction was calculated as follow.
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Kajal L. Jain
Chapter 6
Experimental Work
% Reduction = (AB – AA / AB) x 100
AA is the absorbance of the tested sample after 30 minutes.
AB is the absorbance of blank sample.
IC50 is the concentration required to reduce % reduction by 50 %.
6.4.2 Antiinflammatory activity by rat paw edema method
6.4.2.1 Grouping of animals :
Page 65
Kajal L. Jain
Chapter 6
Experimental Work
Here four group of animals containing six in each group were taken as follows
Gp I: Cotrol animals (1% cmc in d.w 2 ml/kg orally)
Gp II: Methanolic extract (10 mg/kg)
Gp III: Water extract (10 mg/kg)
Gp IV: Standard control (indomethacin 10 mg/kg)
6.4.2.2 Method
Animals were fasted overnight. Doses of drug as per their groups were given orally. After 1 hr
freshly prepared 1% carageenan in normal saline is given by subplantar injection in left hind
paw. Paw thickness measured by plethysmometer as degree of edema is proportional to paw
thickness . Paw volume were measured for three hours and then % inhibition was calculated.
PC = increase in paw thickness of control group
PT = increase in paw thickness for test drug
= Pt-Po i.e thickness at time t and 0 hr.
% Inhibition = (Pc-Pt/Pc)x 100
Page 66
Kajal L. Jain
Chapter 1
Preamble
Chapter 7
Result and Discussion
7.1 For Pharmacognostic And Physicochemical Investigation
Of Raw Materials
7.1.1 Foreign matter :
PHARMACOPEIAL
DRUGS
LVG (%)
S.A (%)
Dill fruits
0.06
2.01
NMT 5
Pepper fruits
0.729
1.74
NMT 2
Embelia ruits
1.575
1.9
NMT 2
Rock salt
--
--
M.Pharm Dissertation
LIMITS (%)
Kajal L
Page 69 Jain
Chapter 1
Preamble
7.1.2 Microscopic charaters of three drugs :
For dill seeds
innermost layer of mesocarp
with thick walls
Fragment of vittae in
surface view
Sclerids
Endosperm cell with microrossete crystals of ca- oxalate and oil globules
Figure 7.1 : Microscopic character of Anethum sowa
For black pepper
Perisperm cells filled
with oleoresin
Stone cells of
black pepper
Non glandular trichomes of
black pepper
Kajal L
Page 70 Jain
Chapter 1
Preamble
Tracheids of black pepper
Figure 7.2 : Microscopic character of Piper nigrum
For embelia ribes
Stone cell
Brown colored Palisade
like cells of mesocarp
Sclerids
sclerified layer of
mesocarp
Perisperm cells
Figure 7.1 : Microscopic character of Embelia ribes
Kajal L
Page 71 Jain
Chapter 1
Preamble
7.1.3 Loss on drying :
DRUGS
LVG (%)
S.A (%)
Dill fruits
7.4 ± 0.02
8.4 ± 0.032
Pepper fruits
10 ± 0.013
10.6 ± 0.016
Embelia ruits
9.8 ± .051
9 ± 0.04
Rock salt
1.2 ± 0.002
1 ± 0.004
7.4 Determination of ash :
DRUGS
TOTAL ASH (%)
AYURVEDIC
LVG
S.A
Dill fruits
7.67±0.62
4.83±0.24
NMT 14
Pepper fruits
4.33±0.47
6±0.2
NMT 5
Embelia fruits
3.67±0.24
3.83±0.23
NMT 6
Rock salt
98.75±0.36
98.92±0.47
--
DRUGS
ACID INSOLUBLE ASH (%)
LVG
Dill fruits
0.67±0.24
S.A
PHR. LIMITS
PHR.
LIMITS
WATER SOLUBLE
ASH (%)
LVG
1.17±0.24 NMT 1.5 4.42±0.51
S.A
1.35±0.25
Pepper fruits 0.42±0.062 0.62±0.103 NMT 0.5
3±0.2
1.33±0.24
Embelia fruits 1.17±0.21 0.82±0.193 NMT 1.5
2.6±0.51
0.83±0.243
94.25±1.2
92.1±0.89
Rock salt
9±0.33
10.1±0.12
--
Kajal L
Page 72 Jain
Chapter 1
Preamble
7.1.5 Determination of extractive value :
DRUGS
WATER EXTRACTIVE
PHARMACOPEIAL
VALUE (%)
LIMITS (%)
LVG
S.A
Dill fruits
20.67±1.25
17±2.1
NLT 15
Pepper fruits
9.17±0.85
8.3±1.3
NLT 6
Embelia fruits
13.58±2.5
14.96±1.8
NLT 9
7.1.6 Phytochemical investigation :
Pepper
Dill
Embelia
extract
extract
extract
Steroids and Triterpenoids
-
-
-
Alkaloids
+++
-
+
Flavanoid glycoside
-
++
-
Cyanogenetic glycoside
-
-
-
Tannins
-
-
+
Cardiac glycoside
-
-
-
Anthraquinone glycoside
-
-
-
Saponin glycoside
-
-
-
Coumarin glycoside
-
++
-
Quinones
-
-
+++
Volatile oil
+
+++
-
Test for
Kajal L
Page 73 Jain
Chapter 1
Preamble
For rock salt
Ca
Mg
Na
K
Fe
SO4
PO4
Cl
CO3
NO3
++
++
++
-
-
+++
-
+
++
-
“ +, ++, +++ ” Present, “ – ” Absent
7.1.7 Determination of volatile oil of dill fruits from powder of
various mesh size :
Powder of
Amount of
mesh size
Volatile oil (%)
10
0.75
20
2.73
30
3.02
40
3.62
60
2.88
V.Oil of 40 mesh
LVG
2.82
S.A
3.62
Kajal L
Page 74 Jain
Chapter 7
7.1.8 Estimation of carvone in dill oil :
Sample from
suppliers
Carvone content (%)
LVG
49.59 ± 0.53
S.A
69.42 ± 0.48
M.Pharm (
Page 75
Chapter 7
7.2 For HPTLC Method Development For Simultaneous Estimation Of
Piperine, Embeline, Carvone .
1
2
3 4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
Figure 7.4 TLC plate showing separation of Piperine, Embeline and Carvone from the
standard (Tracks 1–5) and test solutions (Tracks 6-8).
Table 7.2.1 Amount of active constituent in samples from suppliers
Piperine
Embeline
Carvone
LVG
2.35
4.86
0.93
S.A
1.86
3.37
1.48
M.Pharm (
Page 76
Chapter 7
std curve of piperine
25000
Series1
20000
Linear (Series1)
15000
Auc
y = 1337.9x + 5906.9
R2 = 0.9954
10000
5000
0
0
2
4
6
8
10
12
conc. in mcg
Figure 7.5 : standard curve of piperine
std curve of embeline
3000
2500
Series1
Linear (Series1)
Auc
2000
y = 4964.2x - 65.164
R2 = 0.998
1500
1000
500
0
0
0.1
0.2
0.3
0.4
0.5
0.6
conc. in mcg
Figure 7.6 : standard curve of embeline
std curve of carvone
30000
25000
Series1
Linear (Series1)
Auc
20000
y = 1316.8x + 13609
R2 = 0.9936
15000
10000
5000
0
0
2
4
6
8
10
conc. of solution in mcl
Figure 7.7 : standard curve of carvone
M.Pharm (
Page 77
Chapter 7
Figure 7.8 : Chromatogram of standard Piperine, Embeline and Carvone
peak: Piperine (Rf :0.53), Embeline (0.63), carvone (0.74)
Figure 7.9: 3-D chromatogram of standard piperine, embelin and carvone
M.Pharm (
Page 78
Chapter 7
Figure 7.10 : Chromatogram of churna extract peak: Piperine (Rf :0.54),
Embeline (Rf :0.63) and Carvone (Rf :0.74)
Figure 7.11 : 3 D Chromatogram of churna extract
M.Pharm (
Page 79
Chapter 7
Table 7.2.2 : Recovery data for piperine
Practical amount
of piperine
Amount of
Amount of
piperine added piperine found
% recovery ±
S.D
(mcg/spot)
(mcg/spot)
(mcg/spot)
10
2.5
12.66
101.28 ± 0.995
10
5
14.85
99 ± 1.017
10
7.5
17.27
98.69 ± 0.34
Average %
99.65±0.786
recovery
%CV
0.786
Table 7.2.3: Recovery data for embeline
Practical
amount of
embeline
(mcg/spot)
Amount of
embeline added
(mcg/spot)
Amount of
embeline
% recovery ±
found
S.D
(mcg/spot)
1.2
0.25
1.48
102.66 ± 0.81
1.2
0.5
1.76
100.57 ± 0.46
1.2
0.75
1.947
99.83 ± 0.713
Average %
101.02±0.654
recovery
M.Pharm (
%CV
Page 80
0.654
Chapter 7
Table 7.2.4: Recovery data for carvone
Practical
Amount of
Amount of
carvone added
carvone found
(mcg/spot)
(mcg/spot)
11
2.5
13.32
98.67 ± 1.51
11
5
16.08
100.52 ±0.184
11
7.5
18.7
101.09 ±0.183
amount of
carvone
(mcg/spot)
Average %
S.D
100.09±0.625
recovery
Table 7.2.5 Results for Intraday (n=3) study
Piperine
mean ± S.D
% C.V
0.5
6650.6± 38.46
0.61
1
73679.6± 3
0.55
2
8564.5± 38.6
0.36
Embeline
mean ± S.D
% C.V
0.1
355.23± 11.08
0.56
0.2
949.67± 9.81
0.2
0.4
1959.7v5.5
0.95
Carvone
mean ± S.D
% C.V
3
17205.2± 11.11
0.21
4
18660.3± 10.05
0.34
5
20621.2± 17.43
0.12
M.Pharm (
% recovery ±
Page 81
%CV
0.625
Chapter 7
Table 7.2.6 Results for Interday (n=6) study
Piperine
mean ± S.D
% C.V
0.5
6624.6± 40.4
0.58
1
7406.3± 40.42
0.13
2
8562.9± 30.8
0.1
Embeline
mean ± S.D
% C.V
0.5
339.1± 18.62
0.31
1
933.03± 18.88
1.03
2
1941.8± 18.51
0.28
Carvone
mean ± S.D
% C.V
0.5
17171.5± 35.23
0.084
1
18628.7± 62.71
0.065
2
20647.7± 23.82
0.057
Limit of Detection and Limit of Quantification
Limit of Detection of Piperine, Embeline and Carvone were found o be 0.2 mcg,0.05 mcg and
0.2 nl and Limit of Quantification were found to be 0.5mcg, 0.1mcg and 0.5 nl respectively.
7.3 For Standardization of Churna
7.3.1 Loss on drying
Loss on drying in all samples are mentioned in Table 7.3.1
7.3.2 Ash value
Results of the ash values of all the samples of vasavaleha are given in Table 7.3.1
7.3.3 Extractive value
The results of water, ethanol soluble extractives are mentioned in Table 7.3.1 High values of
ethanol soluble extractive indicate the presence of good amount of ethanol soluble chemical
constituents.
M.Pharm (
Page 82
Chapter 7
7.3.4 Determination of pH
The results of pH of churna are mentioned in Table 7.3.1.
Table 7.3.1 : standardization parameters of churna
Parameters (%)
Obtained values
Total ash value
31.5±0.82
Acid insoluble ash value
1.16±0.23
Water soluble ash value
29.83±0.24
Water extractive value
37.67±0.94
Ethanol extractive value
10.33±2.35
Loss on drying
7.8
Cars index
18.1
Hausnerrs ratio
1.12
Angle of repose
37.34
pH
6.2
7.4 For Pharmacological activity of Catpusphadhya churna
7.4.1 In vitro Anti oxidant activity of Water and Methanol extract of churna
along with individual ingredients by DPPH method.
DRUG
IC 50 VALUE
Ascorbic acid
18.73
Pepper ext
39.168
Dill ext
26.99
Embelia ext
29.8
M.Pharm (
Page 83
Chapter 7
Churna water ext
32.5
Churna met ext
25.51
Figure 7.12 : Graph of antioxidant activity by DPPH method
7.4.2 Antiinflammatory activity by rat paw edema method.
Drug (10 mg/kg)
% inhibition after 3 hr
indomethacin
65.2
Water extract of churna
47.67
Methanolic extract of churna
60.47
M.Pharm (
Page 84
Chapter 7
antiinflammatory activity
70
65.2
60.47
60
% inhibition
50
47.67
40
30
indo
20
water
meth
10
0
0.5
1
1.5
2
2.5
3
time (hr)
Figure 7.13 : Graph of anti-inflammatory activity by rat paw edema method
M.Pharm (
Page 85
Chapter 8
Summary of Work
Chapter 7
Summary Of Work
Standardization of formulation is required to have assurance about biological activity and
clinical consistency. Efforts are made here to standardize Catpusphadhya churna in terms of
various pharmacognostic, physicochemical and phytochemical parameters to establish reliable
values for respective and correlate with further work on instrumental analytical methods like
HPTLC.
Following conclusion have been drawn from the detailed study carried out under the aim of
“Standardization of atpusphadhya churna”
The raw materials for the formulation were procured from different location. It is concluded
from the pharmacognostical, physicochemical and phytochemical studies that all the raw
materials are genuine.
The proposed HPTLC methods for simultaneous estimation of piperine, embeline and carvone
seems to be accurate, precise, reproducible and repeatable. It is for the first time, when different
samples of this formulation and its raw materials are estimated and compared for the respective
active constituents.
The pharmacognostic, physicochemical and phytochemical parameters can now be laid down for
this formulation
This formulation showed very good antioxidant and anti-inflammatory activity, so an attempt
must be made to extend the investigations for the pharmacological study like
immunomodulatory and antiarthritic study.
Page 85
Kajal L Jain
Chapter 8
Summary of Work
References:
1.
Kaviraj Nagendranath Sen Gupta, “ The Ayurvedic System of Indian Medicine” , volume I,
year, page.
2.
Anonymous, “The Ayurvedic Pharmacopoeia of India”, Part-I, Vol.-III, 1st ed., Govt. of
India, Ministry of Health and Family Welfare, 1986, 115-117.
3.
“Database on Medicinal Plants used in Ayurveda”, Vol-V, Central Council for Research
in Ayurveda and Siddha, 2005, 187-190.
4.
Anonymous, “The Ayurvedic Pharmacopoeia of India”, Part-I, Vol.-I, 1st ed., Govt. of
India, Ministry of Health and Family Welfare, 1986, 123-124.
5.
“Database on Medicinal Plants used in Ayurveda”, Vol-V, Central Council for Research
in Ayurveda and Siddha, 2005, 478-482.
6.
“Quality Standards of Indian Medicinal Plants”, Vol-IV, Published by Indian Council of
Medicinal Research, New Delhi, 2003, 130-136.
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Medicinal Research, New Delhi, 2003, 18-26.
10. Anonymous, “The Ayurvedic Formulary of India”, Part-II, Govt. of India,
11. Ministry of Health and Family Welfare, Dept. of Indian Systems of Medicine and
Homeopathy, New Delhi.
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Ministry of Health and Family Welfare.
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Ministry of Health and Family Welfare.
14. Mukherjee P. K., “Quality Control of Herbal Drugs”, Business Horizons, New Delhi,
India, 2005.
15. Khanderwal K. R., “Practical Pharmacognosy: Techniques and Experiments”, Niral
Prakashan, 157.
16. Anonymous, “Indian Herbal Pharmacopoeia”, Indian Drug Manufacturer’s Association,
Mumbai, 2002.
Page 86
Kajal L Jain
Chapter 8
Summary of Work
17. ICH Guidelines “Validation of analytical methodology”.
18. Rajpal V., “Standardization of Botanicals”, vol-I, 2005.
19. Rajpal V., “Standardization of Botanicals”, vol-II, 2005.
20. Anonymous, “Official Methods of Analysis of AOAC International”, vol-II, AOAC
International, 16th edition, 1995, CH-43, 5.2006 dopne
21. Shah, C.S. and Qadry, J.S., “A Text Book of Pharmacognosy”, 7th edition,
22. B.S.Shah Prakashan, Ahmedabad-380001, 1990.
23. WHO guidelines.
24. “The Merk index”, An Encyclopedia of chemicals, drugs and botanicals, thirteenth edition,
1889, 3587, 7553.
25. http://www.thefreedictionary.com/rock+salt
26. http://answers.yahoo.com/question/index?qid=20081001101318AAOajig
27. http://www.fineli.fi/food.php?foodid=79&lang=en
28. S.K.Chauhan, G.P kimothi, B.P Singh and S.Agrawal, “Indian drugs”, july 1998,35(7),408411.
29. A Kumar, S Panghal, SS Mallapur, M Kumar, Veerma Ram, BK Singh Antiinflammatory
activity of Piper longum fruit oil.
30. Jagdale, S.C., B.S. Kuchekar, A.R. Chabukswar, P.D. Lokhande and C.G. Raut, 2009. Antioxidant activity of Piper longum Linn. Int. J. Biol. Chem., 3: 119-125.
31. Mujumdar A.M, Dhuley J.N, Deshmukh V.K, Raman P.H, Naik S.R, Antiinflammatory
activity of piperine,” Japaneese Journal of Medicinal Science Biol.”, 43(3),1990, 95-100.
32. Gupta S.K, Bansal P, Bharadwaj R.K, Velpendian T, comparative antinociceptive, antiinflammatory and toxicity profile of nimesulide v/s nimesulide and piperine combination,
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33. İlhami Gülçin, The antioxidant and radical scavenging activities of black pepper (Piper
nigrum) seeds International Journal of Food Sciences and Nutrition 2005, 56( 7), pp 491499 ,
34. Venketa Rao D, Ramana Rao V.V, Pulla Rao J, Subramanium B, “ Indian Journal of
Pharmaceutical Sciences”,1986,2,37.
35. Patel R.K, Pundarikakshadu K., Patel M.M, “Indian Journal of Pharmaceutical Sciences”,
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36. M.Chitra, C.S Shyamala Devi, E.Sukumar “Journal of Natural Remedies”,2004,4(1),77-80.
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Kajal L Jain
Chapter 8
Summary of Work
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Kajal L Jain

mt-259-kajal jain

Transcription

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