Antinociceptive Activities of Aqueous and Ethanol Extracts of Piper betle. Leaves in Rats

Abstract

Leaves of Piper betle. Linn (Piperaceae) possess a broad spectrum of pharmacological and therapeutic properties. However, its antinociceptive activity has not been investigated 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 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 antinociceptive 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, pendulous. Bracts peltate, orbicular to obcordate, broadly stipitate with a membranous margin (Jayaweera, 1982; 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 consists 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 antiaphrodisiac 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). Experimentally, 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 literature, antinociceptive activity of P. betle. is not scientifically 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 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 Gardens, Peradeniya, Sri Lanka. A voucher specimen (PS 01) was deposited in the Industrial Technology Institute, 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.

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 (n = 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 alkaloids, polyphenols, steroids, saponins, and tannins as described by Farnsworth (1996).

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 = 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 µl of 2.5% formalin solution 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 = 9 per group).

Mechanism for antinociceptive activity

This was investigated using CEE because the antinociceptive activity was higher compared to HWE. Further, 200 mg/kg was selected because the maximal antinociceptieve 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.

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) antagonist, 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

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 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-holding 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).

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 significant. ID₅₀ values were determined graphically.

Results

Phytochemical screening of HWE and CEE

Phytochemical screening revealed the presence of alkaloids, 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 prolongation 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) prolonged 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 antinociceptive 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, 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%).

Figure 1 Effect of different doses of cold ethanol extract (CEE) of P. betle. leaves on the reaction time of rats (n = 9; hot-plate test, means ± SEM). *p < 0.05 as compared with control.

Figure 2 Effect of different doses of hot water extract (HWE) of P. betle. leaves on the reaction time of rats (n = 9; hot-plate test, means ± SEM). *p < 0.05 as compared with control.

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

All the tested doses of CEE significantly (p < 0.05) reduced the licking time of the formalin-injected paw in the early phase (by 17–52%). However, other tested doses of CEE showed a significant (p < 0.05) reduction of the licking time in the late phase (by 12–22%) apart from the 125 and 500 mg/kg doses. On the other hand, only 200 and 300 mg/kg doses of HWE significantly (p < 0.05) reduced the licking time in both the early (by 17–32%) and late (by 13–17%) phases. Compared to the control, the reference drug, aspirin, significantly (p < 0.05) reduced the licking time in both early (by 65%) and late (by 75%) phases (Table 1).

Table 1. Effect of different doses of hot water extract (HWE) and cold ethanol extract (CEE) of P. betle. leaves on the reaction time of rats (formalin test, means ± SEM).

Treatment	n	Early phase (s)	Late phase (s)
Control (1 ml of DW)	6	71.3 ± 3.2	312.0 ± 8.9
125 mg/kg
HWE	6	67.3 ± 4.4	295.3 ± 6.1
CEE	6	59.1 ± 3.9^a.	296.6 ± 9.5
200 mg/kg
HWE	6	48.2 ± 3.9^a.	258.0 ± 4.3^a.
CEE	6	34.3 ± 5.7^a.	245.0 ± 5.9^a.
300 mg/kg
HWE	6	59.3 ± 4.0^a.	270.3 ± 4.5^a.
CEE	6	43.2 ± 3.6^a.	274.7 ± 5.1^a.
500 mg/kg
HWE	6	64.0 ± 2.4	286.6 ± 8.5
CEE	6	54.2 ± 2.8^a.	289.7 ± 6.6
Aspirin (100 mg/kg)	9	25.1 ± 1.9^a.	75.7 ± 2.8^a.

DW, distilled water.

^a.As compared with control: p < 0.05.

Investigation of involvement of opioid receptor

In the naloxone study, with the hot-plate technique, subcutaneous administration of naloxone significantly (p = 0.0025) impaired the reaction time induced by 200 mg/kg dose of CEE (Table 2).

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 + saline	16.2 ± 0.6
200 mg/kg CEE + naloxone	8.3 ± 0.7^a.

^a.Significant at p < 0.05.

Investigation of involvement of dopamine receptor

In the metoclopramide experiment, with the hot-plate technique, the oral administration of metoclopramide did not significantly (p > 0.05) change the reaction time induced by the 200 mg/kg dose of CEE (Table 3).

Table 3.. Effect of metoclopramide 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 + 1% methyl cellulose	13.3 ± 1.1
200 mg/kg CEE + metoclopramide	14.5 ± 1.2

Not significant at p < 0.05 level.

Sedative effect of the extract

In the rat hole-board tests, none of the parameters investigated 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 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 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 α.₂-adrenoceptors in the spinal cord (Ossipov et al., 1990). The antinociceptive activity of extracts was genuine 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, prostaglandin, 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 impairments of sensory transmission and release of inflammatory 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 technique was changed. Antinociception can be induced via dopaminergic mechanisms (Jensen & Yaksh, 1986), but such a mode of action is unlikely in this study as metoclopramide, a dopamine receptor antagonist, failed to block antinociception induced by the extract. The opioid receptor, antagonist naloxone, blocked the antinociception induced by the CEE, suggesting that the antinociception was mediated through opioid mechanisms. 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 administration 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 antinociceptive activity with the chemical constituents. It may be possible to develop safe and potent antinociceptive agents from P. betle. leaves.

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).