INTRODUCTION

Bitter melon (Momordica charantia L.) is an annual tendril herbage plant belonging to family Cucurbitaceae. Presence of ascorbic acid and iron in high concentation make this plant an important and valuable vegetable (Behera et al., 2008). Bitter melon is in use as a traditional medicine for diabetes in Central America, China and India and it also has antimicrobial properties (Grover et al., 2002; Yeh et al., 2003; Saeed and Tariq, 2005). M. charantia contains chemical constituents like charantin, linoleic acid, linolenic acid, momordicins, oleic acid, oxalic, trypsin inhibitors, v-insulin, ascorbigen, b-sitosterol-d-glucoside, lycopene, pipecolic acid as well as the fruit pulp has soluble pectin but no free pectic acid (Dhalla et al., 1961). Researchers found that, Bitter gourd fruit powder, in the form of an ointment showed a statically significant response in terms of wound contracting ability, wound closure time, tensile strength of the wound and regeneration of tissues at wound site when compared with a reference drug povidone iodine ointment in an excision, incision and dead space wound model in rats (Jayshri and Jolly, 1993). The phenolic compounds of bitter gourd have been reported to exhibit antioxidant activity (Horax et al., 2005; Budrat and Shotipruk, 2009). Grover and Yadav (2004) have reported anti-diabetes, anti-inflammatory, anti-bacterial and anti-cancer effects of Momordica charantia . Most of the synthetic drugs used at present for analgesic and anti-inflammatory effect that cause many side and toxic effects. Plants still represent a large untapped source of structurally novel compounds that might serve as lead for the development of novel drugs. Plant medicine are in use since a long time for analgesic and anti-inflammatory activity because of the reason that they are devoid of side effects (Ahmad et al., 1992). According to WHO there is about 82% population that depends upon herbal drugs and these are gaining popularity because of less side effects and low-priced (Kumara, 2001). The present study was therefore undertaken to investigate the secondary metabolite contents, acute toxicity, analgesic and anti-inflammatory potential of the ethanolic extract of M. charantia Linn. in experimental animal models.

MATERIALS AND METHODS

Collection of plant materials: The fruits of Momordica charantia (variety : charantia) were collected during the month of July, 2009 from the Rangamati District, Chittagong Hill tracts, Bangladesh and were taxonomically identified by standard procedure in the Department of Botany; University of Chittagong, Bangladesh.

Preparation of the plant extract: Fresh fruits were cleaned and dried under shade in clean dust free environment , grinded and stored in air- tight container. About 750 g powders were soaked in 4.5 L pure ethanol for about 7 days at room temperature with occasional starring. After 7 days, ethanol extract was filtered using muslin cloth and Whatman’s filter paper No.1. The solvent was concentrated by evaporating ethanol using a rotary evaporator. The ethanol extract thus obtained transferred to a petri dish and kept in an oven at 30°C until the solvent is completely evaporated. Finally, a brownish colored residue was obtained (yield 3.23% w/w) which was kept in refrigerator at 4°C for experimental purposes.

Phytochemical screening
Phytochemical analysis: Freshly prepared Momordica charantia ethanol extract was subjected to preliminary qualitative phytochemical investigation using the procedure as reported (Khandelwal, 2007).

Experimental animals: Swiss Albino mice of both sexes weighting between 25 to 30 g and Wister Albino rats of the either sex weighting between 150-200 g obtained from animal house of BCSIR laboratories, Chittagong, Bangladesh were used for the present study. All the animals were kept in standard polypropylene cages under standard conditions in animal room with alternating light-dark cycle of 12 h. The animal were acclimatized to the laboratory conditions for at least five days prior to the experiments. During the entire period of the study the animals were supplied with standard pellet diet and water ad libitum. In this study, all the animal experiments was carried out according to the guidelines of Institutional Animal Ethics Committee (IAEC).

Acute toxicity study: Acute toxicity study was carried out according to the Organization of Economic Corporation Development (OECD) guidelines No. 425. Ethanol extract of Momordica charantia was administered orally in doses of 100, 200, 400, 800, 1000 and 2000 mg kg^-1 to the group of mice (n = 3) and the percentage mortality was recorded for a period of 24 h. During the first 1 h after the drug administration, the mice were observed for any gross behavioral change and the parameters observed were hyperactivity, grooming, convulsions, sedation and loss of righting reflex, respiration, salivation, urination and defecation (Vogel, 2002). Based on the above toxicity study, direct limit test was done. Initially at a particular dose, on the basis of the above study was administered to single female rat and the rat was observed for 48 h with close surveillance up to initial 4 h (same as in case of first rat) and after 48 h (of the second administration), same dose was administered in two more female rats and the observation was done similarly as for the previous rat. The rats were observed for 14 days and no adverse observation was found morphologically. The weight of the animals was recorded on 7th and 14th day. Animals were divided into five groups of six animals each. The first group (Group I) served as a control group. And the second (Group II) was used as the reference standard. Three groups (Group III, IV and V ) received ethanol extract at three different doses (100, 250 and 500 mg kg^-1 p.o.). The doses were selected on the basis of our preliminary screening. The research was conducted in accordance with the ethical rules on animal experimentation, approved by Institutional Animal Ethics Committee (IAEC).

Drugs and chemicals: Indomethacin (Opsonin Pharma limited, Bangladesh) and Tramadol Hydrochloride (SQUARE Pharmaceuticals Limited, Bangladesh)were used in the study. Distilled water was used as vehicle. All the chemicals and solvents were of analytical grade. Carrageenan was purchased from Sigma-Aldrich, Germany, acetic acid was obtained from Merck, Germany.

Analgesic and anti inflammatory activity
Acetic acid induced writhing model in mice: Acetic acid induced writhing test model as described by Koster et al. (1959). This was performed to evaluate the analgesic activity of ethanol extract. In this model, the animals were pretreated with drugs 30 min before induction of writhing. The Group I animals received vehicle and Group II animals received the reference standard drug indomethacin (10 mg kg^-1 p.o.). Analgesic activity of ethanol extract at the doses of 100, 250 and 500 mg kg^-1 p.o. (Group-III, IV and V) was assessed by counting the number of writhing induced using 1% acetic acid. The number of writhing per animal was counted for 20 min. Percent reduction in writhing syndrome was calculated and compared with the standard drug. Percent reduction indicates the percentage protection against abdominal constriction which was taken as an index of analgesia. It was calculated by using the formula:

Where:

Tail immersion method: In this method (Turner, 1971) the animals were pretreated with drugs for 60 min before tail immersion. The animals received vehicle (Group I) and the standard drug Tramadol Hydrochloride (50 mg kg^-1, i.p.) (Group II) which served as reference standard. Analgesic activity of ethanol extract of Momordica charantia at doses100, 250 and 500 mg kg^-1 p.o. (Group III, IV and V), respectively was assessed by observing the reaction time in the treated groups .The distal 2-3 cm portion of mouse-tail was immersed in hot water maintained at 55±1°C. The time taken by the mouse to withdraw the tail from hot water was noted as reaction time. The cutoff time was considered about 10-12 sec.

Carrageenan-induced hind paw edema in rats: This test was conducted as per the method described by Winter et al. (1962). The paw thickness(0 h) was measured, in millimetres, using digital Plethysmometer (UGO Basile, Italy). The test substances and standard drug were administered one hour prior to the injection of a phlogistic agent via the oral route. The phlogistic agent carrageenan was prepared as 1% suspension, in sterile normal saline, a day before the study, to get a proper suspension. Carrageenan 0.1 mL^-1 was injected subcutaneously into the right hind paw of each mouse. The paw volume was measured at 0 and 3 h after injection of carrageenan by using a Plethysmometer (Arunachalam et al., 2002) after carrageenan injection. The edema thickness (mm) was calculated by the following formula Olumayokun et al. (1999):

where, Ct is the right hind paw thickness volume (in mm³) at time t, Co is the right hind paw thickness volume (in mm³) before carrageenan injection. Ct-Co is paw edema, (Ct –Co) control is edema or paw size after carrageenan injection to control rats at time t. (Ct –Co) treated is edema or paw size after carrageenan injection to treated (reference or sample drug) rats at time t.

Statistical analysis: The statistical analysis of data was done using the SPSS software (version 11.5). p<0.001 was considered as highly significant.

RESULTS

Phytochemical analysis: The crude bitter gourd extract was found to be positive for the presence of alkaloids, saponin, glycosides, steroids and sterol and phenolic compound as shown in Table 1.

Acute toxicity study: It was found that oral administration of Momordica charantia extract up to 2 g kg^-1 did not produce any toxic effects in the normal behavior of the mice. Further no mortality was observed and from this observation it might be suggested that the plant extent in safe at this given dose.

Table 1:	Phytochemical screening of ethanol extract of Momordica charantia
+: Presence of the constituents

Table 2:	Analgesic activity of Momordica charantia extract in acetic acid induced writhing method
All value are expressed as Mean±SEM (n = 6 in each group). All values are significant at *p<0.001 compared to control group (Student t-test). Percentage inhibition of inflammation is given within parentheses

Table 3:	Analgesic activity of Momordica charantia extract on tail-immersion tests in mice
All value are expressed as Mean±SEM (n = 6 in each group). All values are significant at **p<0.001 compared to control group (Student t-test)

Table 4:	Anti-inflammatory activity of Momordica charantia extract in Carrageenan induced paw edema model
All value are expressed as Mean±SEM (n = 6 in each group). All values are significant at *p<0.001 compared to control group (Student t-test)

Effect on acetic acid induced writhing model in mice: The extract produced significant (p<0.001) reduction in the number of writhing in mice in dose dependent manner. The ethanolic extracts (250 and 500 mg kg^-1, p.o.) significantly reduced the acetic acid-induced writhing by 42.62 and 50.14%, respectively but the standard drug indomethacin (10 mg kg^-1 p.o.) showed a reduction of 75.36% (Table 2).

Effect on the tail immersion test: From Table 3, It was observed that the ethanolic extracts (500 mg kg^-1) being the more active compared to 100 and 250 mg kg^-1.

Effect on carrageenan-induced edema in rats: The edema swellings induced by carageenan in rats was significantly (p<0.001) inhibited by the fruit extract (100, 250 and 500 mg kg^-1, p.o.) as well as by Indomethacin (10 mg kg^-1, p.o). As shown in Table 4 the extract 500 mg kg^-1 reduced the edema swellings by 42.10% as compared to 57.89% reduction produced by indomethacin (10 mg kg^-1, p.o.) (Table 4).