Full Text

IJPRD, 2015; Vol 6(11):January-2015 (033 – 039)
International Standard Serial Number 0974 – 9446
-------------------------------------------------------------------------------------------------------------------------------------------------FREE RADICAL SCAVENGING POTENTIAL OF 'THULASI VETTILA', AN INDIGENOUS CULTIVAR OF BETEL VINE
(PIPER BETLE L.) IN KERALA
D K Sathish1*,
Vrinda1, Thomas Bennans2
1
Dept. of Pharmaceutical Chemistry, Padm. Dr. D. Y. Patil College of Pharmacy, Akurdi, Pune-411044, India
ABSTRACT
Betel Vine (Piper betle L.), commonly known as ‘paan’, is
Correspondence Author
a plant of antiquity with its global spread in terms of
distribution, its acceptance by diverse cultural groups and
known for the ethnomedicinal properties. The cultivar ‘thulasi’
is indigenous to Southern Kerala and is well known for its
traditional medicinal properties and rarely cultivated for
commercial purposes. The present study attempts to assess the
antioxidant potential of the cultivar in relation to its
phytochemical
composition
and
major
phenolic
compounds.Total carbohydrates, proteins, flavanoids, lipids,
D K Sathish
phenolic compounds, chlorophylls, vitamins etc were analyzed
Dept. of Pharmaceutical Chemistry,
and quantified from the aqueous and methanolic extracts of
Padm. Dr. D. Y. Patil College of
the plant material in order to assess the phytochemical
Pharmacy, Akurdi, Pune-411044,
composition. Antioxidant potential of a plant is measured in
India
terms of its ability to scavenge free radicals. So super oxide
anions and hydrogen peroxide were localized and quantified. It
Email:- drdksathish@gmail.com
was followed by the localization of the major indicator enzymes
associated with free radical scavenging such as catalase and
peroxidase. Lignin was also localized to verify the end impact of
free radical scavenging. The investigation showed that P.betle
cv ‘thulasi’ constitutes a significant level of major phenolics and
antioxidant phytoconstituents that account for the anti oxidant
potential of the plant. Both DPPH and FRAP assays revealed a
high degree of free radical scavenging ability of betel vine. The
investigations clearly suggest that the organic and aqueous
extract of P.betle could be further exploited in the future as a
source of useful phytochemical compounds for the
pharmaceutical industry owing to its strong anti oxidant
potential.
Keywords- Betel vine, Piper betle, ‘thulasi’, antioxidant
potential, free radicals, DPPH, FRAP.
33
International Journal of Pharmaceutical Research & Development
ISSN: 0974 – 9446
southern regions of Kerala and is not usually
INTRODUCTION
cultivated for commercial purposes as its
Human dependence on plants as a
chewing quality is poor because of its very
source of medicine dates back to prehistoric
stringent taste. However, thulasivettila has been
times. Piper betle L., commonly known as betel
traditionally used for religious and medicinal
vine or Paan, is one of the important plants in the
purposes and is grown as a household plant.
Asiatic region which ranks second to coffee and
Hence the present investigation attempts to
tea in terms of daily consumption. Use of Piper
assess the antioxidant potential
betle leaf was known for centuries for its curative
properties such as to reduce or prevent body
MATERIALS AND METHODS
odour and bad breath, throat and lung problems,
Plant Material
cough prevention and healing (1). In Peninsular
Betle (Piper betle) is a tropical shade-loving
Malaysia, the leaves of betel vine are used by the
perennial evergreen vine cultivated mainly for
rural population as an antimalarial remedy. The
leaves.
Development of the leaf beyond a
liquid extract of the plant has been used
particular level is of no commercial value because
traditionally in curing inflammation and infection
of the increased production of undesirable
of the respiratory tract, cough, indigestion,
phenolic compounds. These leaves were taken for
diphtheria, hysteria as well as general and sexual
the whole biochemical and antioxidant analysis.
debility. A concoction of indigenous Indian drugs
For experimentation, the plant species was grown
containing P. betle dry extract was found to be an
in two environmental conditions, namely the
effective long-lasting oral contraceptive. The
normal and drought stressed conditions.
parts of P.betle utilized are leaves, roots, stems,
Analytical Studies
stalks and fruits. The plant has got large number
The total protein was estimated as per Peterson
of
biomolecules
which
show
diverse
(3). The total Carbohydrates was estimated by
pharmacological activity. Medicinally P.betle leaf
the method of Rose et al., (4). Total polyphenol
is an aromatic, carminative, stimulant and
content was measured by the method of Mayer
astringent used as a preventive for worms and in
et al., 1995 (5). Total flavanoids was measured by
snake bite.
the method of Chang et.,al (6). The lipid content
The pharmacological profiling of betel vine
in the sample was estimated by following the
reveals its antimicrobial activity, protective and
method of Bligh and Dyer (1959). Superoxide
healing
activity,
antidiabetic
and
anion was quantified, following the method of
gastroprotective activity, and hepato-protective
Doke (7). H2O2 concentration of leaves was
activity etc, owing to the predominant presence
of essential oils, hydroxycatechol, chavicol,
estimated as per the procedure of Bellincampi et
Methyleugenol, caryophyllene, phenyl propane
al (8).
etc. Phytochemical studies have shown that Piper
Histochemical Studies
betle contains a wide variety of biologically active
Superoxide anions were localized by the
compounds whose concentration depends on the
procedure of Frahry and Schofer (9).
variety of the plant, season and climate (2).
Histochemical localization of hydrogen peroxide
Piper betle L., belongs to the family Piperaceae, is
was done by procedure of Ros Barcelo (10). POX
a dioecious, shade loving perennial Malay
activity was localized by the procedure of
creeper extensively cultivated in hot damp
Bestwick (1998). Cytochemical localization of
localities. Leaves usually opposite, cordate,
catalase was carried out by the method of Fang
petioled, all 4 to 7-ribbed and glabrous. There
et al. (11). Lignins can be localized by the
are about 125 to 150 cultivars of betel vine in
procedure of Faulkner and Kimmins (12).
India. In Kerala the most prevalent cultivars are
Biochemical Analysis
Venmani, Arikodi, Kalkodi, Karilanchi, Karpuram
The amount of vitamin –E was determined
etc. ‘Thulasivettila’ is a variety restricted to the
following the method of Rosenberg (13). DPPH
Available online on www.ijprd.com
34
International Journal of Pharmaceutical Research & Development
ISSN: 0974 – 9446
(1,
1-Diphenyl-2-Picrylhydrazyl)
radical
Lignin, being the end product of this biochemical
scavenging activity was estimated by the
sequence of free radical scavenging, was also
procedure of Saeed et al., (14). The FRAP
localized which showed very distinct deep red
procedure described by Benzie and Strain (15)
colour of lignin deposition in the stress induced
was followed for assessing the antioxidant
leaves (Figure 4). Normally lignin deposition is
activity. Antimicrobial activity and Antifungal
comparatively higher in ‘thulasi’ cultivar as evident
activity were analyzed following the procedure
from the rough texture of the leaves.
The increased production of super oxide anions
of NCCLS,1993 (16).
and hydrogen peroxide was further verified by
localizing the enzyme catalase which is responsible
RESULTS AND DISCUSSION
for the conversion of hydrogen peroxide back into
Phytochemical Composition
molecular oxygen and water. The figures 5 shows
Phytochemical composition is a significant factor in
the localization of the increased production of both
determining the antioxidant potential of a plant
the enzymes which clearly justify the enhanced
apart from the nutritional view point. Analytical
production free radicals as revealed by the
studies were conducted to estimate the
histochemical data.
phytochemical composition of the plant material
The histochemical data of super oxide anions and
and to relate their quantitative variation with the
hydrogen peroxide is well supported by the
antioxidant potential of the plant especially during
quantification data which also show a substantial
the stress conditions. Total carbohydrates, total
increase in the production of both the substances
proteins, phenols, chlorophylls, carotenoids,
during stress induction in P.betle.( Figure 6). The
flavanoids, vitamin C, vitamin E, total lipids etc
reducing activity of NBT by superoxide anions
were analyzed and estimated as per the standard
present in the extract was taken as a measure of
procedures. Pre-weighed fresh leaves were taken
quantification.
for the whole studies and either water or
DPPH Assay – The Radical Scavenging Activity
methanolic extracts were used for estimation
The relatively stable organic radical DPPH has been
purposes. Figure 1 shows the comparative
widely used to determine the antioxidant activity
distribution of the major phytoconstituents in Betel
of different plant extracts (18). Table 1 shows the
vine cultivar ‘thulasi’. Higher content of phenolic
free radical scavenging ability of both aqueous and
compounds and other major antioxidants such as
methanolic extracts of the plant material at
carotenoids, flavanoids and vitamin C and E justify
different concentrations. It is expressed as the
the increased antioxidant capacity of the cultivar
percentage inhibition of free radicals by the
and is comparable that of many medicinal plants.
antioxidants available in the extracts. The aqueous
Phytochemical screening of cultivars from Sri lanka
extracts showed a comparatively higher inhibition
also revealed the same kind of phytochemical
than that of both control and methanolic extract.
composition in methanolic extracts (17).
More over higher the concentration greater was
Histochemical and Quantification Data
the percentage inhibition may due to the higher
Figure 2 shows the localization of super oxide
quantity of antioxidants present in the extracts.
anions in the leaves of P.betle cv thulasi. The
FRAP Assay
induction of water stress in the leaves has resulted
The total antioxidant potential of the plant material
in an increased production of superoxide anions as
visible in the form of blue deposits. Since H2O2 is
was also determined using ferric reducing ability of
plasma FRAP assay based on the reducing power of
derived from the molecular oxygen by the action of
the antioxidant. Table 2 shows the free radical
the enzymes NADPH oxidase and super oxide
scavenging ability of both aqueous and methanolic
dismutase (SOD) and super oxide anions as
extracts of the plant material at different
intermediate products, hydrogen peroxide was
concentrations. Contrary to the DPPH assay,
further localized in order to verify its increased
methanolic extract showed a comparatively higher
production during stress conditions ( Figure 3).
Available online on www.ijprd.com
35
International Journal of Pharmaceutical Research & Development
ISSN: 0974 – 9446
free radical scavenging ability especially at the
domestic cultivar of P.betle,
P.betle which has not yet been
maximum concentration of the extract.
exploited as a commercially or pharmacologically
The antioxidant potential of P.betle as revealed by
significant cultivar of the plant. Since P.betle also
both the DPPH and FRAP assays
ssays showed results
offers an alternative drug delivery mechanism
similar to that of many medicinal plants with high
through buccal mucosa
sa bypassing the gastric route
antioxidant ability(14).
owing to its chewable quality, its potential as an
effective antioxidant agent can be utilized for
CONCLUSION
developing new medicines in the coming years.
The present investigation substantiates the free
radical scavenging potential of the ‘thulasivettila’,
‘thulasivettila’ a
Concentration(mg/g FW)
8
7
6
5
4
3
2
1
0
-1
-2
Biochemical Compounds
Figure 1:: Estimation of major phytoconstituents and antioxidants in Betel Vine ( Piper betle L.)
a
b
Figure 2 :Localization of Superoxide anion in the leaves of Betel vine ((Piper betle.L)
.L) cv Thulasi a) Normal leaf.
b) Stress induced leaf.
a
b
Figure 3:: Localization of Hydrogen Peroxide in the leaves of Betel vine (Piper
(Piper betle.L)
betle cv Thulasi a) Normal leaf.
b) Stress induced leaf.
Available online on www.ijprd.com
36
International Journal of Pharmaceutical Research & Development
a
ISSN: 0974 – 9446
b
Figure 4: Localization of Lignin in the leaves of Betel vine (Piper betle.L) cv Thulasi a) Normal leaf.
induced leaf.
a
b) Stress
b
Figure 5: Localization of Catalase and Peroxidase in the leaves of Betel vine (Piper betle.L) cv Thulasi a)
Catalase. b) Peroxidase
QUANTIFICATION OF H2O2 AND O2(Concentration µ/g.)
10
8
6
NORMAL
4
STRESS
2
0
-2
H2O2
O2-
Figure 6: Quantification of Superoxide anions and hydrogen peroxide in the leaves of P.betle
Sl.NO
1
2
3
Concentration
Aqueous
Methanol
L-Ascorbic acid*
(µg/ml)
(%of inhibition)
(%of inhibition)
(%of inhibition)
10
78.7±0.75
47.35±0.50
51.12 ± 0.35
50
90.5±0.45
66.37±0.75
67.92 ± 0.25
100
96.8±0.76
91.05±0.58
84.38 ± 0.69
Table 1: In vitro Free Radical Scavenging Activity of Different Extracts of Piper betle L.
by DPPH Assay. *Positive control (L-Ascorbic acid)
Available online on www.ijprd.com
37
International Journal of Pharmaceutical Research & Development
ISSN: 0974 – 9446
Sl.No
Concentration
Aqueous
Methanol
1
20
125.88±0.65
161.82±.72
2
60
181.04±0.34
285.52±.54
3
100
611.65±0.34
465.99±.42
BHT*
100
190±0.02
Table 2:In vitro Free Radical Scavenging Activity of Different Extracts of Piper betle L. by FRAP Assay. *Positive
control (BHT)
Physiology and Plant Pathology. Vol: 23, 1983
ACKNOWLEDGEMENTS
pp.359–367.
We express our sincere gratitude to Kerala State
8. Bellincampi D, Dipierro N, Salvi G, Cervone F
Council for Science, Technology and Environment
and De Lorenzo G. Extracellular H2O2 induced
for their financial support.
by oligogalacturonides is not involved in the
REFERENCES
inhibition of the auxin-regulated rol B gene
1. Nadkarni, A. K., and Nadkarni, K. M. Indian
expression in Tobacco leaf explants. Plant
Materia Medica, Eastern Book Corporation,
Physiology. Vol : 122, 2000 pp.1379–1385.
Mumbai, 2007.Vol.1.
9. Frahry G and Schopfer P. NADH-stimulated,
2. Satish, A. Bhalerao, Deepa R, Verma, Rohan V,
cyanide resistant superoxide production in
Gavankar, Nikhil C, Teli, Yatin Y Rane,
maize
coleoptiles
analyzed
with
a
VinodkumarS Didwana ,Ashwin Trikannad.
tetrazolium- based assay. Planta, Vol:212,
Phytochemistry and Pharmacological Profiles
2001 pp.175-183.
and Therapeutic Uses of Piper betle Linn. An
10. Ros Barcelo A. Hydrogen peroxide production is
Overview. Journal of Pharmacognosy and
a general property of the lignifying xylem from
Phytochemistry. Vol1(2), 2013. pp.177-186.
vascular plants. Annals of Botany 82 : 1998, 973. Peterson, G.L. Review of the Folin phenol
103.
protein quantification method of Lowry,
11. Fang T.K., Donaldson, R.P. and Vigil, E.L.
Rosebrough, Farr and Randall. Anal. Biochem.
Electron transport in purified glyoxysomal
Vol:100, 1979, pp.201–220.
membranes from castor bean endosperm.
4. Rose,
J.H.
Determination
of
food
Planta, Vol: 172, 1987 pp.1-13.
carbohydrates. D.A.T Sathgate , Uni. Cambr.,
12. Faulkner, G. and Kimmins, W. C. Staining
England. 1965. pp.258.
reactions of the tissue bordering virus lesions
5. Mayr VD, Trutter C, Dudga, HS and Senchitt,
in bean. Phytopathology, Vol:65 , 1975,
W. Developmental changes in the phenol
pp.1396-1400.
concentration of golden delicious apple fruit
13. Rosenberg, H.R. Chemistry and Physiology of
and leaves. Journal of Phyto. Chem.Vol:38(5)
Vitamins. Inter science Publishers Inc., New
1995, pp.1151-1155.
York, 1992, pp.452-453.
6. Chang CC, Yang MH, Wen HM and Chern JC.
14. Saeed, N., Khan, M.R,. and Shabbir, M.
Estimation of total flavonoid content in propolis
Antioxidant activity, total phenolic and total
by two complementary colorimetric methods,
flavonoid contents of whole plant extracts
Journal of Food and Drug Analysis, Vol:10, 2002
Torilis leptophylla L. BMC Complementary
pp.178-182.
and Alternative Medicine, Vol:12 , 2012, pp.
7. Doke N. Generation of superoxide anion by
1-12.
potato tuber protoplasts during hypersensitive
response to hyphal wall components of
Phytophtora infestans and specific inhibition of
the reaction with supressors of hypersensitivity.
Available online on www.ijprd.com
38
International Journal of Pharmaceutical Research & Development
ISSN: 0974 – 9446
15. Benzie, I.F.F., and Strain, J.J. Ferric
Edition: Approved Standard M2-A5. NCCLS,
reducing/antioxidant power assay: direct
1993, Villanova, P A.
measure of total antioxidant activity of
17. Lakshmi Arambewela, Menuka Arawwawala
biological fluids and modified version for
and Damisha Rajapaksa. International Journal
simultaneous measurement of total antioxidant
of Food Science and Technology, 41
power and ascorbic acid concentration.
(Supplement 1), 2006, 10-14
Methods in Enzymology. Vol: 299, 1999, pp.15–
18. Kanjwani, D.G., Marathe, T.P., Chiplunkar, S.V.,
27.
Sathaye, S.S. (): Evaluation of Immunodulatory
16. National Committee for Clinical Laboratory
Activity of Methanolic Extract of Piper betle
Standards. Performance Standards for
.Journal of Immunology.Vol:67(6), 2008,
Antimicrobial Disk Susceptibility Tests—Fifth
pp.589-593.
*****
Available online on www.ijprd.com
39