Pharmaceutical Biology
2005, Vol. 43, No. 9, pp. 766–772
Antinociceptive Activities of Aqueous and Ethanol Extracts
of Piper betle Leaves in Rats
L.S.R. Arambewela1, L.D.A.M. Arawwawala1, and W.D. Ratnasooriya2
1
Industrial Technology Institute, Bauddhaloka Mawatha, Colombo, Sri Lanka; 2Department of Zoology,
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University of Colombo, Colombo, Sri Lanka
Abstract
Leaves of Piper betle Linn (Piperaceae) possess a broad
spectrum of pharmacological and therapeutic properties.
However, its antinociceptive activity has not been inves-
tigated so far. The aim of this study therefore, was to
examine the antinociceptive activity of hot water extract
(HWE) and cold ethanol extract (CEE) of P. betle leaves
using rats and three models of nociception (tail flick, hot
For personal use only.
plate, and formalin tests). Different concentrations of
HWE (125, 200, 300, 500 mg=kg) and CEE (125, 200,
300, 500 mg=kg) were made and orally administrated to
rats, and the reaction times were determined. The results
showed that the extracts have marked antinociceptive
activity when evaluated in the hot plate and the formalin
tests but not in the tail-flick test. The overall antinocicep-
tive effect of CEE was higher than that of HWE.
The antinociceptive effect was mediated via opioid
mechanisms.
Keywords: Antinociception, opioid receptor, Piper betle
leaves.
Introduction
Piper betle Linn (Piperaceae) is a perennial dioecious,
semi-woody climber. Stems strongly swollen at the
nodes, papillose when young. Leaves alternate, simple,
and yellowish green to bright-green in color. Leaves of
fertile branches with a petiole 1–2 cm long, 1.2–1.8 mm
thick when dry, and glabrous at maturity. Flowers are
naked, unisexual, dioecious in dense cylindrical spikes,
male spikes not seen, female spikes 2.5–5 cm long, pendu-
lous. Bracts peltate, orbicular to obcordate, broadly
stipitate with a membranous margin (Jayaweera, 1982;
Address correspondence to: Prof. W.D. Ratnasooriya, Department of Zoology, University of Colombo, Colombo 03, Sri Lanka.
E-mail: wdr@zoology.cmb.ac.lk
DOI: 10.1080/13880200500406545 # 2005 Taylor & Francis Ltd.
Dassanayake & Fosberg, 1987). P. betle is cultivated in
Sri Lanka, India, Malay Peninsula, the Philippines, and
East Africa (Dassanayake & Fosberg, 1987). The chief
constituent of the leaves of this plant is a volatile oil
known as betel oil. The volatile oil is a bright-yellow to
dark-brown liquid possessing a clove-like flavor and con-
sists of terpenes and phenols (Anonymous, 1992).
In Asian countries, betel leaves are used for chewing
and are credited with many medicinal properties such
as digestive, stimulative, carminative, and aphrodisiac
(Anonymous, 1992). However, Sri Lankan betel inhibits
male sexual behavior in rats and possesses antiaphrodi-
siac activity (Ratnasooriya & Premakumara, 1996).
Further, betel juice is given to children for cough and
administered to the eye for night blindness in adults.
It is used to treat catarrh and diphtheria. The leaves
are given for gastric and lung disorders in children
and applied to purulent ulcers (Jayaweera, 1982). Exper-
imentally, leaves of P. betle are shown to possess
antimicrobial (Tewari & Nayak, 1991), gastroprotective
(Majumdar et al., 2003), wound healing (Santhanam &
Nagarajan, 2002), hepatoprotective (Saravanan et al.,
2002), antioxidant (Choudhary & Kale, 2002; Saravanan
et al., 2002; Santhakumari et al., 2003), antifertility on
male rats (Ratnasooriya & Premakumara, 1997), and
antimotility effects on washed human spermatozoa
(Ratnasooriya et al., 1990). According to available litera-
ture, antinociceptive activity of P. betle is not scientifi-
cally investigated yet. However, it is possible that
P. betle leaves may possess antinociceptive properties, as
P. longum (Vedhanayaki et al., 2003) a close relative of
the plant, was shown to have antinociceptive properties.
Therefore, this study was undertaken to examine whether
extracts of leaves of P. betle possess antinociceptive
 Piper betle and antinociceptive activity
activity. This was tested in rats using oral administration
of hot water and cold ethanol extracts.
Materials and Methods
Plant material
P. betle leaves were purchased from the main vegetable
markets in the western province of Sri Lanka in May
2002. The leaves were identified and authenticated by
the curator of National Herbarium, Royal Botanical
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Gardens, Peradeniya, Sri Lanka. A voucher specimen
(PS 01) was deposited in the Industrial Technology Insti-
tute, Colombo, Sri Lanka.
Animals
Healthy adult cross-bred male albino rats (weighing 200–
250 g) were used throughout the experiment. They were
housed under standard environmental conditions with
free access to pelleted food (Vet House Ltd., Colombo,
Sri Lanka) and tap water.
For personal use only.
Preparation of hot water extract (HWE)
P. betle leaves were air-dried for 3–5 days in the shade
and cut into small pieces. Five hundred grams were
boiled with 2.5 l of distilled water (DW) for 4 h. The
hot water extract was concentrated under vacuum (yield
26.2% w=w, dry weight basis) and stored at 4
C until use.
Preparation of cold ethanol extract (CEE)
P. betle leaves were air dried for 3–5 days in the shade
and cut into small pieces. Five hundred grams were
macerated with ethanol (80% v=v) and kept for 48 h at
room temperature (28–30
C). The extraction was filtered
and the filtrate was evaporated to dryness under reduced
pressure (yield 15.6% w=w, dry weight basis) and stored
at 4
C until use.
Administration of extracts
Doses of 125, 200, 300, and 500 mg=kg of HWE and CEE
were prepared in 1 ml of DW and given orally to separate
groups (n¼ 12 or 6 per group per extract) of rats. Doses
selected were comparable to what has been generally used
in investigating pharmacological activities of herbal
extracts (Saravanan et al., 2002; Majumdar et al., 2003).
Phytochemical screening of HWE and CEE
Extracts were subjected to qualitative testing for alka-
loids, polyphenols, steroids, saponins, and tannins as
described by Farnsworth (1996).
767
Evaluation of antinociceptive activity
Hot-plate and tail-flick tests
The reaction times of rats were measured 1 h prior to the
treatment (pretreatment) and then at hourly intervals
for 5 h after the treatment (post-treatment) either with
HWE, CEE, vehicle, or reference drug using hot-plate
and tail-flick techniques as described by Langerman
et al. (1995). In the hot-plate test, the rat was placed
on an enclosed hot plate (Model MK 35 A, Muromachi
Kikai Co. Ltd., Tokyo, Japan) maintained at 50
C and
the time taken (in seconds) either to lick the hind paw
or jump from the surface of the hot plate (the reaction
time) was determined. In the tail-flick test, the tail of
the rat was immersed (5–6 cm from the tip) in a water
bath at 55
C and the time taken (in seconds) to flick
the tail was determined by using a stop watch. Rats
showing a pretreatment reaction time greater than 15 s
in the hot-plate test and 5 s in the tail-flick test were
not used in the experiment. A cutoff time of 25 s was
set to avoid tissue damage. The reference drug, pethidine
(25 mg=kg), was injected intramuscularly to a separate
group (n ¼ 9 per group) of rats.
Formalin test
The method of Dubuission and Dennis (1977) was used
with slight modifications to evaluate the antinociceptive
activity. In brief, 1 h after the administration (either of
CEE, HWE, vehicle, or reference drug), each rat was
injected subcutaneously with 50 ml of 2.5% formalin sol-
ution into the subplantar surface of the left hind paw.
Rats were then observed for 60 min, and the time spent
licking the injected paw was recorded in two phases.
The first phase, 1–5 min post–formalin injection, is
known as the early phase and the period between
15–60 min as the late phase. The reference drug, aspirin
(100 mg=kg), was given orally to another group of rats
(n ¼ 9 per group).
Mechanism for antinociceptive activity
This was investigated using CEE because the antinoci-
ceptive activity was higher compared to HWE. Further,
200 mg=kg was selected because the maximal antinoci-
ceptieve activity was evident with this dose.
Investigation of involvement of opioid receptor
Eighteen rats were randomly divided into two equal
groups. Rats in group 1 were injected subcutaneously
with 5 mg=kg of naloxone hydrochloride in 1 ml of
normal saline. For group 2, 1 ml of normal saline was
injected subcutaneously. After 45 min, both groups of
rats were given 200 mg=kg of CEE orally, kept for 1 h,
and examined on the hot plate.
 768 L.S.R. Arambewela et al.
Investigation of involvement of dopamine receptor
Eighteen rats were randomly divided into two equal
groups. Rats in group 1 were orally administered
1.5 mg=kg of metoclopramide, a dopamine (D2) anta-
gonist, in 1 ml of 1% methyl cellulose. Rats in group 2
were orally treated with 1 ml of 1% methyl cellulose.
After 1 h, both groups of rats were orally treated with
200 mg=kg of CEE extract and the hot-plate test
performed.
Sedative effect of the extract
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Eighteen rats were randomly divided into two equal
groups. One group was treated with 1 ml of distilled
water (DW) and the other group was treated with
200 mg=kg of CEE in 1 ml of DW. After 1 h, each rat
was placed in the center of the rat hole-board and
observed for 7.5 min. The number of rears, number of
head dips, locomotor activity, and number of fecal
boluses were recorded as described by File and Wardill
(1975).
Effect of the extract on muscle relaxation
For personal use only.
and muscular coordination
Eighteen rats were randomly divided into two equal
groups. Group 1 was treated with 200 mg=kg of CEE
in 1 ml of DW and group 2 was treated with 1 ml of
DW. After 1 h, these rats were subjected to the bar-hold-
ing test (to evaluate the muscle relaxation) followed by
the bridge test (to evaluate the muscle coordination),
and the latency to fall and slide off (in seconds) were
determined, respectively as described by Plaznic et al.
(1993).
Figure 1. Effect of different doses of cold ethanol extract (CEE) of P. betle leaves on the reaction time of rats (n ¼ 9; hot-plate test,
means  SEM).  p < 0.05 as compared with control.
Statistical analysis
Data are given as means  SEM. Statistical comparisons
were made using one-way ANOVA followed by Tukey’s
family error test. A p value 0.05 was considered as sig-
nificant. ID50 values were determined graphically.
Results
Phytochemical screening of HWE and CEE
Phytochemical screening revealed the presence of alka-
loids, polyphenols, steroids, saponins, and tanins in both
extracts.
Hot-plate and tail-flick tests
In the hot-plate test, significant (p < 0.05) prolongation
of the reaction time was evident with all the tested doses
of CEE (Fig. 1) at 2 h post-treatment. Further pro-
longation of the reaction time lasted up to 5 h (until
the termination of the experiment) with 200 and
300 mg=kg doses. On the other hand, only 200 and
300 mg=kg doses of HWE significantly (p < 0.05) pro-
longed the reaction time up to 5 h and 3 h, respectively
(Fig. 2). The ID50 for the increase in the reaction time
at 1 h after the treatment of HWE and CEE was
145 mg=kg and 92 mg=kg, respectively. The best antinoci-
ceptive activity was evident with 200 mg=kg dose of both
CEE and HWE. Further antinociceptive effect of
200 mg=kg dose of CEE was comparable to the reference
drug pethidine at each point of time (CEE: 1 h, 105%;
2 h, 77%; 3 h, 76%; 4 h, 56%; 5 h, 35%; pethidine: 1 h,
118%; 2 h, 73%; 3 h, 61%; 4 h, 44%; 5 h, 41%). Likewise,
 Piper betle and antinociceptive activity 769
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Figure 2. Effect of different doses of hot water extract (HWE) of P. betle leaves on the reaction time of rats (n ¼ 9; hot-plate test,
means  SEM).  p < 0.05 as compared with control.
For personal use only.

the antinociceptive effect of 200 mg=kg dose of HWE
also was comparable to the pethidine at each point of
time apart from the 1 h (HWE: 1 h, 84%; 2 h, 61%; 3 h,
67%; 4 h, 45%; 5 h, 32%).
There was no significant increase in the reaction time
with any of the doses of HWE or CEE in the tail-flick
test (data not shown).
Formalin test
did not significantly (p > 0.05) change the reaction time
induced by the 200 mg=kg dose of CEE (Table 3).
Sedative effect of the extract
In the rat hole-board tests, none of the parameters inves-
tigated was significantly (p > 0.05) altered by the dose
200 mg=kg of CEE (control vs. treatment: number of
rears 28.9  1.0 vs. 29.8  0.9, number of head dips

All the tested doses of CEE significantly (p < 0.05)

reduced the licking time of the formalin-injected paw in Table 1. Effect of different doses of hot water extract (HWE)
the early phase (by 17–52%). However, other tested and cold ethanol extract (CEE) of P. betle leaves on the reac-
doses of CEE showed a significant (p < 0.05) reduction tion time of rats (formalin test, means  SEM).
of the licking time in the late phase (by 12–22%) apart Treatment n Early phase (s) Late phase (s)
from the 125 and 500 mg=kg doses. On the other hand,
only 200 and 300 mg=kg doses of HWE significantly Control 6 71.3  3.2 312.0  8.9
(p < 0.05) reduced the licking time in both the early (1 ml of DW)
(by 17–32%) and late (by 13–17%) phases. Compared 125 mg=kg
HWE 6 67.3  4.4 295.3  6.1
to the control, the reference drug, aspirin, significantly
CEE 6 59.1  3.9a 296.6  9.5
(p < 0.05) reduced the licking time in both early (by
200 mg=kg
65%) and late (by 75%) phases (Table 1). HWE 6 48.2  3.9a 258.0  4.3a
CEE 6 34.3  5.7a 245.0  5.9a
Investigation of involvement of opioid receptor 300 mg=kg
HWE 6 59.3  4.0a 270.3  4.5a
In the naloxone study, with the hot-plate technique, sub-
CEE 6 43.2  3.6a 274.7  5.1a
cutaneous administration of naloxone significantly 500 mg=kg
(p ¼ 0.0025) impaired the reaction time induced by HWE 6 64.0  2.4 286.6  8.5
200 mg=kg dose of CEE (Table 2). CEE 6 54.2  2.8a 289.7  6.6
Aspirin 9 25.1  1.9a 75.7  2.8a
Investigation of involvement of dopamine receptor (100 mg=kg)
In the metoclopramide experiment, with the hot-plate DW, distilled water.
a
technique, the oral administration of metoclopramide As compared with control: p < 0.05.
 770 L.S.R. Arambewela et al.
Table 2. Effect of naloxone on the reaction time of rats
induced by 200 mg=kg dose of cold ethanol extract (CEE) of
P. betle leaves (hot-plate test, means  SEM).
Treatment Reaction time (s)
200 mg=kg CEE þ saline 16.2  0.6
200 mg=kg CEE þ naloxone 8.3  0.7a
a
Significant at p < 0.05.
Table 3. Effect of metoclopramide on the reaction time of rats
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induced by 200 mg=kg dose of cold ethanol extract (CEE) of
P. betle leaves (hot-plate test, means  SEM).
Treatment Reaction time (s)
200 mg=kg CEE þ 1% methyl cellulose 13.3  1.1
200 mg=kg CEE þ metoclopramide 14.5  1.2
Not significant at p < 0.05 level.
13.7  0.5 vs. 14.2  0.9, number of crossings 17.1  0.6
vs. 15.1  0.8, and number of fecal boluses 0.77  0.34
For personal use only.
vs. 0.88  0.33).
Effect of the extract on muscle relaxation and muscular
coordination
When compared with control, 200 mg=kg dose of CEE
did not significantly (p > 0.05) change the latency to fall
in the bar-holding test (control vs. treatment 58.9  1.0
vs. 59.2  0.9 s) or the latency to slide off in the bridge
test (control vs. treatment 58.1  0.4 vs. 58.3  1.7 s).
Discussion
The results demonstrate that extracts of P. betle leaves
have antinociceptive activity as evaluated in the hot-plate
test and not in the tail-flick test. This indicates centrally
mediated antinociceptive activity of the plant extracts
against the acute pain: both hot-plate and tail-flick tests
measure centrally medicated transient pain. (Lopez–
Munoz et al., 1993). The hot-plate test predominately
measures supraspinally organized responses, wherein
the tail-flick test predominately measures spinal reflexes
(Wong et al., 1994). Because both extracts are effective
only against hot-plate test, the antinociceptive is likely
to mediated supraspinally. The lack of activity in
the tail-flick method could be due to coexistence of
compound(s) that interacts with other receptors such as
a2-adrenoceptors in the spinal cord (Ossipov et al.,
1990). The antinociceptive activity of extracts was genu-
ine as there was no change in the retain times of bar and
bridge tests as compared with controls. The highest
antinociceptive activity was evident with 200 mg=kg dose
of both HWE and CEE. Further, antinociceptive activity
of CEE was higher than that of HWE extract in terms of
ID50 values. The dose-response curves of the P. betle
extracts were bell-shaped. Such an action may result
from desensitization (Seuka & Mazrzymas, 1991) or
downregulation of receptors (Stewart & Badiani, 1993).
Bell-shaped dose-response curves have been reported with
other synthetic (Li et al., 1996) and herbal (Arambewela
et al., 2004) analgesics.
Both 200 and 300 mg=kg doses of P. betle extracts
markedly reduced the licking time in early and late
phases of the formalin test in a bell-shaped dose-response
curve. In the formalin test, the pain in the early phase is
caused due to the direct stimulation of the sensory nerve
fibers by formalin, whereas the pain in the late phase is
due to the inflammatory mediators, like histamine, pros-
taglandin, serotonin, and bradykinin (Murray et al.,
1988; Tjolsen et al., 1992). It is reported that NSAIDs
reduce both phases of the formalin test (Martindale
et al., 2001). Therefore, extracts induced interruptions
of both phases of this test, suggesting possible impair-
ments of sensory transmission and release of inflamma-
tory mediators.
Some sedatives possess antinociceptive activity (Rang
et al., 1995). However, the antinociceptive action of
CEE is unlikely to be mediated via sedation as none
of the parameters monitored in the rat hole-board tech-
nique was changed. Antinociception can be induced via
dopaminergic mechanisms (Jensen & Yaksh, 1986), but
such a mode of action is unlikely in this study as meto-
clopramide, a dopamine receptor antagonist, failed to
block antinociception induced by the extract. The
opioid receptor, antagonist naloxone, blocked the anti-
nociception induced by the CEE, suggesting that the
antinociception was mediated through opioid mechan-
isms. Further alkaloids were present in the CEE, and
alkaloids, which possess antinociceptive properties
usually, act via opioid mechanisms (Badio et al.,
1995). The overall antinociceptive effect of CEE was
higher than that of HWE. In CEE, most of chemical
compounds kept intact without major changes. In hot
water extract, there is a strong possibility that some
of the heat-labile constituents are destroyed. According
to Arambewela et al. (2003), both extracts were devoid
of unacceptable side effects even after chronic adminis-
tration at a dose of 1500 mg=kg. There were no overt
signs of toxicity, hepatotoxicity (in terms of serum
AST, ALT levels), or renotoxicity (as judged by serum
urea and creatine levels).
In conclusion, our results demonstrate, for the first
time, antinociceptive activity of P. betle leaves, and
further studies will be undertaken to correlate the antino-
ciceptive activity with the chemical constituents. It may
be possible to develop safe and potent antinociceptive
agents from P. betle leaves.
 Piper betle and antinociceptive activity
Acknowledgments
The authors express their gratitude to Mr. J.R.A.C.
Jayakody, University of Colombo, Department of
Zoology, for assisting in this study and National Science
Foundation for the research grant (SIDA (1L)
2000=BT=03).
References
Anonymous (1992): The Wealth of India. The Dictionary of
Pharmaceutical Biology Downloaded from informahealthcare.com by Chulalongkorn University on 12/30/14
Indian Raw Materials and Industrial Products. Raw
Material Vol. 5, Publication and Information director-
ate, CSIR, pp. 84–94.
Arambewela LSR, Arawwawala LDAM, Ratnasooriya WD
(2004): Antinociceptive activities of aqueous and etah-
nolic extracts of Alpinia calcarata rhizomes in rats. J
Ethnopharmacol 95: 311–316.
Arambewela LSR, Arawwawala LDAM, Ratnasooriya WD
(2003): Safety evaluation of Sri Lankan Piper betle leaf
extracts in rats. J Trop Med Plants 4: 195–198.
Badio B, Shi D, Garraffo HM, Daily JN (1995): Antinoci-
ceptive effects of the alkaloid epibatidine: Further stu-
For personal use only.
dies on involvement of nicotine receptors. Drug
Develop Res 36: 46–59.
Choudhary D, Kale RK (2002): Antioxidant and nontoxic
properties of Piper betle leaf extract: in vitro and in vivo
studies. Phytother Res 61: 461–466.
Dassanayake MD, Fosberg FR (1987): A Revised Hand
Book to the Flora of Ceylon. New Delhi, Amreind, pp.
287–288.
Dubuission D, Dennis SG (1977): The formalin test: A
quantitative study of the analgesia effects of morphine,
meperidine and brain stem stimulation in rats and cats.
Pain 4: 167–174.
Farnsworth NR (1996): Biological and phytochemical
screening of plants. J Pharm Sci 55: 225–276.
File SE, Wardill A (1975): Validity of head dipping as a
measure of exploration in modified hole-board. Psycho-
pharmacol 44: 53–57.
Jayaweera DMA (1982): Medicinal Plants Used in Ceylon,
Vol. 5. Colombo, National Science Council of Sri
Lanka, p. 201.
Jensen TS, Yaksh TL (1986): Effects of an intrathecal dopa-
mine agonists, apomorphine, on thermal and chemical
evoked noxious responses in rats. Brain Res 296: 285–
293.
Langerman L, Zakouski ML, Piskoun B, Grant GJ (1995):
Hot plate versus tail flick: Evaluation of acute tolerance
to continuous morphine infusion in the rat model.
J Pharmacol Toxicol Methods 34: 23–28.
Li BY, Nalwalk JW, Barker LA, Cumming P, Parsons MF,
Hougn LB (1996): Characterization of the antinocicep-
771
tive properties of cimetidine and a structural analog.
J Pharmacol Exp Ther 276: 500–508.
Lopez–Munoz FJ, Salazar LA, Castaneda–Hernandes G,
Villarreal JE (1993): A new model to assess analgesic
activity: Pain induced functional impairement in the
rat (PIFIR). Drug Develop Res 28: 169–175.
Majumdar B, Chaudhuri SGR, Ray A, Bandyopadhyay SK
(2003): Effect of ethanol extract of Piper betle Linn leaf
on healing of NSAID-induced experimental ulcer—A
novel role of free radical scavenging action. Indian J
Exp Biol 41: 311–315.
Martindale J, Bland–Ward PA, Chessell IP (2001): Inhi-
bition of C-fibre mediated sensory transmission in the
rat following intraplantar formalin. Neurosci Lett 316:
33–36.
Murray CW, Porreca F, Cowan A (1988): Methodological
refinements in the mouse paw formalin test an animal
model of tonic pain. J Pharmacol Methods 20: 175–186.
Ossipov MH, Harris S, Lloyd P, Messineo E, Lin BS,
Bagley J (1990): Antinociceptive interaction between
opioids and medetomidine: Systemic additivity and
spinal synergy. Anesthesiology 73: 1227–1235.
Plaznic A, Stefanski R, Palejko W, Kotawski W (1993): The
role of accumbense GABA-B receptors in the regu-
lation of rat behavior. Neurosci Res Common 12: 23–30.
Rang HP, Dale MM, Ritter JM (1995): Pharmacology.
London, Churchill Livingstone.
Ratnasooriya WD, Premakumara GAS (1996): Piper betle
leaves impairs masculine sexual behavior of rats. Med
Sci Res 24: 303–306.
Ratnasooriya WD, Premakumara GAS (1997): Piper betle
leaves reversibly inhibits fertility of male rats. Vidyo-
daya J Sci 7: 15–21.
Ratnasooriya WD, Jayawardena KGI, Premakumara GAS
(1990): Antimotility effects of Piper betle (L) leaf
extract on washed human spermatozoa. J Natl Sci Coun
Sri Lanka 18: 53–60.
Santhanam G, Nagarajan S (1990): Wound healing activity
of Curcuma aromatica and Piper betle. Fitoterapia 61:
458–459.
Saravanan R, Prakasam A, Ramesh B, Pugalendi KV
(2002): Influence of Piper betle on hepatic marker
enzymes and tissue antioxidant status in ethanol-treated
wistar rats. J Med Food 5: 197–204.
Santhakumari P, Prakasam A, Pugalendi KV (2003): Modu-
lation of oxidative stress parameters by treatment with
Piper betle leaf in streptozotocin induced diabetic rats.
Indian J Pharmacol 35: 373–378.
Scuka M, Mazrzymas JW (1991): Post synaptic potentiation
and desensitization at the vertebrate end-plate recep-
tors. Prog Neurobio 38: 19–33.
Stewart J, Badiani A (1993): Tolerance and sensitization to
the behavioural effects of drugs. Behavi Pharmacol 4:
289–312.
 772 L.S.R. Arambewela et al.

Tewari SN, Nayak M (1991): Activity of four plant leaf
extracts against three fungal pathogens of rice. Trop
Agric 68: 373–375.
Tjolsen A, Berge DG, Hunskaar S, Rosland JH, Hole K
(1992): The formalin test: An evaluation of the method.
Pain 51: 5–17.
Pharmaceutical Biology Downloaded from informahealthcare.com by Chulalongkorn University on 12/30/14
For personal use only.
Vedhanayaki G, Shastri GV, Kuruvilla A (2003): Analgesic
activity of Piper longum Linn. root. Indian J Exp Biol
41: 649–651.
Wong CH, Day P, Yarmush J, Wu W, Zbuzek UK (1994):
Nifedipine-induced analgesia after epidural injections in
rats. Anesthes Analges 79: 303–306.