Abstract
Leaves of Piper betle Linn (Piperaceae) possess a broad Dassanayake & Fosberg, 1987). P. betle is cultivated in
spectrum of pharmacological and therapeutic properties. Sri Lanka, India, Malay Peninsula, the Philippines, and
However, its antinociceptive activity has not been inves- East Africa (Dassanayake & Fosberg, 1987). The chief
tigated so far. The aim of this study therefore, was to constituent of the leaves of this plant is a volatile oil
examine the antinociceptive activity of hot water extract known as betel oil. The volatile oil is a bright-yellow to
(HWE) and cold ethanol extract (CEE) of P. betle leaves dark-brown liquid possessing a clove-like flavor and con-
using rats and three models of nociception (tail flick, hot sists of terpenes and phenols (Anonymous, 1992).
For personal use only.
plate, and formalin tests). Different concentrations of In Asian countries, betel leaves are used for chewing
HWE (125, 200, 300, 500 mg=kg) and CEE (125, 200, and are credited with many medicinal properties such
300, 500 mg=kg) were made and orally administrated to as digestive, stimulative, carminative, and aphrodisiac
rats, and the reaction times were determined. The results (Anonymous, 1992). However, Sri Lankan betel inhibits
showed that the extracts have marked antinociceptive male sexual behavior in rats and possesses antiaphrodi-
activity when evaluated in the hot plate and the formalin siac activity (Ratnasooriya & Premakumara, 1996).
tests but not in the tail-flick test. The overall antinocicep- Further, betel juice is given to children for cough and
tive effect of CEE was higher than that of HWE. administered to the eye for night blindness in adults.
The antinociceptive effect was mediated via opioid It is used to treat catarrh and diphtheria. The leaves
mechanisms. 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
Keywords: Antinociception, opioid receptor, Piper betle
antimicrobial (Tewari & Nayak, 1991), gastroprotective
leaves.
(Majumdar et al., 2003), wound healing (Santhanam &
Nagarajan, 2002), hepatoprotective (Saravanan et al.,
2002), antioxidant (Choudhary & Kale, 2002; Saravanan
Introduction et al., 2002; Santhakumari et al., 2003), antifertility on
Piper betle Linn (Piperaceae) is a perennial dioecious, male rats (Ratnasooriya & Premakumara, 1997), and
semi-woody climber. Stems strongly swollen at the antimotility effects on washed human spermatozoa
nodes, papillose when young. Leaves alternate, simple, (Ratnasooriya et al., 1990). According to available litera-
and yellowish green to bright-green in color. Leaves of ture, antinociceptive activity of P. betle is not scientifi-
fertile branches with a petiole 1–2 cm long, 1.2–1.8 mm cally investigated yet. However, it is possible that
thick when dry, and glabrous at maturity. Flowers are P. betle leaves may possess antinociceptive properties, as
naked, unisexual, dioecious in dense cylindrical spikes, P. longum (Vedhanayaki et al., 2003) a close relative of
male spikes not seen, female spikes 2.5–5 cm long, pendu- the plant, was shown to have antinociceptive properties.
lous. Bracts peltate, orbicular to obcordate, broadly Therefore, this study was undertaken to examine whether
stipitate with a membranous margin (Jayaweera, 1982; extracts of leaves of P. betle possess antinociceptive
Address correspondence to: Prof. W.D. Ratnasooriya, Department of Zoology, University of Colombo, Colombo 03, Sri Lanka.
E-mail: wdr@zoology.cmb.ac.lk
activity. This was tested in rats using oral administration Evaluation of antinociceptive activity
of hot water and cold ethanol extracts.
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
Materials and Methods for 5 h after the treatment (post-treatment) either with
Plant material HWE, CEE, vehicle, or reference drug using hot-plate
and tail-flick techniques as described by Langerman
P. betle leaves were purchased from the main vegetable et al. (1995). In the hot-plate test, the rat was placed
markets in the western province of Sri Lanka in May on an enclosed hot plate (Model MK 35 A, Muromachi
2002. The leaves were identified and authenticated by Kikai Co. Ltd., Tokyo, Japan) maintained at 50C and
the curator of National Herbarium, Royal Botanical the time taken (in seconds) either to lick the hind paw
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Gardens, Peradeniya, Sri Lanka. A voucher specimen or jump from the surface of the hot plate (the reaction
(PS 01) was deposited in the Industrial Technology Insti- time) was determined. In the tail-flick test, the tail of
tute, Colombo, Sri Lanka. the rat was immersed (5–6 cm from the tip) in a water
bath at 55C and the time taken (in seconds) to flick
Animals the tail was determined by using a stop watch. Rats
showing a pretreatment reaction time greater than 15 s
Healthy adult cross-bred male albino rats (weighing 200– in the hot-plate test and 5 s in the tail-flick test were
250 g) were used throughout the experiment. They were not used in the experiment. A cutoff time of 25 s was
housed under standard environmental conditions with set to avoid tissue damage. The reference drug, pethidine
free access to pelleted food (Vet House Ltd., Colombo, (25 mg=kg), was injected intramuscularly to a separate
Sri Lanka) and tap water. group (n ¼ 9 per group) of rats.
For personal use only.
Administration of extracts This was investigated using CEE because the antinoci-
ceptive activity was higher compared to HWE. Further,
Doses of 125, 200, 300, and 500 mg=kg of HWE and CEE 200 mg=kg was selected because the maximal antinoci-
were prepared in 1 ml of DW and given orally to separate ceptieve activity was evident with this dose.
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 Investigation of involvement of opioid receptor
extracts (Saravanan et al., 2002; Majumdar et al., 2003). 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
Phytochemical screening of HWE and CEE
normal saline. For group 2, 1 ml of normal saline was
Extracts were subjected to qualitative testing for alka- injected subcutaneously. After 45 min, both groups of
loids, polyphenols, steroids, saponins, and tannins as rats were given 200 mg=kg of CEE orally, kept for 1 h,
described by Farnsworth (1996). and examined on the hot plate.
768 L.S.R. Arambewela et al.
Eighteen rats were randomly divided into two equal loids, polyphenols, steroids, saponins, and tanins in both
groups. One group was treated with 1 ml of distilled extracts.
water (DW) and the other group was treated with
200 mg=kg of CEE in 1 ml of DW. After 1 h, each rat
Hot-plate and tail-flick tests
was placed in the center of the rat hole-board and
observed for 7.5 min. The number of rears, number of In the hot-plate test, significant (p < 0.05) prolongation
head dips, locomotor activity, and number of fecal of the reaction time was evident with all the tested doses
boluses were recorded as described by File and Wardill of CEE (Fig. 1) at 2 h post-treatment. Further pro-
(1975). 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
Effect of the extract on muscle relaxation
For personal use only.
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.
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 did not significantly (p > 0.05) change the reaction time
also was comparable to the pethidine at each point of induced by the 200 mg=kg dose of CEE (Table 3).
time apart from the 1 h (HWE: 1 h, 84%; 2 h, 61%; 3 h,
67%; 4 h, 45%; 5 h, 32%). Sedative effect of the extract
There was no significant increase in the reaction time
with any of the doses of HWE or CEE in the tail-flick In the rat hole-board tests, none of the parameters inves-
test (data not shown). tigated was significantly (p > 0.05) altered by the dose
200 mg=kg of CEE (control vs. treatment: number of
Formalin test rears 28.9 1.0 vs. 29.8 0.9, number of head dips
Table 2. Effect of naloxone on the reaction time of rats antinociceptive activity was evident with 200 mg=kg dose
induced by 200 mg=kg dose of cold ethanol extract (CEE) of of both HWE and CEE. Further, antinociceptive activity
P. betle leaves (hot-plate test, means SEM). of CEE was higher than that of HWE extract in terms of
Treatment Reaction time (s) ID50 values. The dose-response curves of the P. betle
extracts were bell-shaped. Such an action may result
200 mg=kg CEE þ saline 16.2 0.6 from desensitization (Seuka & Mazrzymas, 1991) or
200 mg=kg CEE þ naloxone 8.3 0.7a downregulation of receptors (Stewart & Badiani, 1993).
a
Significant at p < 0.05.
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
Table 3. Effect of metoclopramide on the reaction time of rats markedly reduced the licking time in early and late
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induced by 200 mg=kg dose of cold ethanol extract (CEE) of phases of the formalin test in a bell-shaped dose-response
P. betle leaves (hot-plate test, means SEM). curve. In the formalin test, the pain in the early phase is
caused due to the direct stimulation of the sensory nerve
Treatment Reaction time (s)
fibers by formalin, whereas the pain in the late phase is
200 mg=kg CEE þ 1% methyl cellulose 13.3 1.1 due to the inflammatory mediators, like histamine, pros-
200 mg=kg CEE þ metoclopramide 14.5 1.2 taglandin, serotonin, and bradykinin (Murray et al.,
1988; Tjolsen et al., 1992). It is reported that NSAIDs
Not significant at p < 0.05 level.
reduce both phases of the formalin test (Martindale
et al., 2001). Therefore, extracts induced interruptions
of both phases of this test, suggesting possible impair-
13.7 0.5 vs. 14.2 0.9, number of crossings 17.1 0.6 ments of sensory transmission and release of inflamma-
vs. 15.1 0.8, and number of fecal boluses 0.77 0.34 tory mediators.
For personal use only.
Indian Raw Materials and Industrial Products. Raw bition of C-fibre mediated sensory transmission in the
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