MATERIALS AND METHODS

Plant material: The seeds were purchased from the local market of Ropar (India) in August 2010. The healthy looking seeds were selected for authentication and voucher specimen number 0388 has been deposited in the Botanical and Environmental Science Department, Guru Nanak Dev University, Amritsar (India). The seeds were cleaned, washed, dried at room temperature and powered at low temperature.

Drugs and chemicals: All chemical reagents used were of analytical grade which were procured from different companies (Loba Chem, Mumbai and Merck Limited, Mumbai). Sample of morphine and diclofenac sodium was procured from Government Medical College and Hospital, Patiala. Ascorbic acid, Hydrogen peroxide were obtained from Sigma Chemical Co., USA.

Animals: The wistar albino rats and swiss albino mice of either sex were obtained from Sanjay biological, Amritsar. Institutional Animal Ethics Committee duly approved the experimental protocol and care of the animals was carried out as per the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Government of India (Reg No. 874/ac/05/CPCSEA).

Extraction: The seeds of Cucumis melo (500 g) were cleaned, shade dried, coarsely powdered and extracted with various solvents (methanol, ethyl acetate, chloroform etc.) for 72 h at room temperature. The extract was evaporated and concentrated under reduced pressure in rotary evaporated. The concentrated filtrate was suspended in distilled water and partitioned successively with hexane. The aqueous layer was separated and concentrated on the water bath. These crude extracts were further used for various investigations such as antioxidant, anti-inflammatory, analgesic activity (Uchikoba et al., 1998; Banerjee et al., 2008).

Phytochemical screening: The crude extracts were analyzed for alkaloids, tannins, saponins, flavonoids, steroids, terpenoids and phenolic acids using standard procedures of analysis (Harborne, 1973).

Free radical scavenging activity
Quantitative evaluation of the DPPH free radical scavenging activity: DPPH scavenging activity of cantaloupe extract was determined according to the method described with slight modifications. In brief, 1 mL of methanolic extract of Cucumis melo seeds (MECM) at various concentrations were respectively added to 1 mL 0.05 mM DPPH in methanol and was made up to 5 mL with methanol. Then, the mixtures were vortexed vigorously and allowed to stand in the dark for 60 min. Finally, the absorbance of these mixtures was measured by using a spectrophotometer (Pharmaspec UV-1700, Shimadzu, Kyoto, Japan) at 517 nm using methanol as blank. Ascorbic acid was used as standard. Control was prepared by diluting 1 mL of 0.05 mM DPPH with 4 mL of methanol. The capability to scavenge the DPPH radical was calculated using the following equation:

where, A_c = Absorbance of the control reaction, A_t = Absorbance in presence of the sample of the extracts (Singh et al., 2002).

Hydrogen peroxide radical scavenging activity: In this 1 mL of extract (25-200 μg mL^-1) was added to 2.4 mL of 0.1 M phosphate buffer (pH 7.4), and then 0.6 mL of a 43 mM solution of H₂O₂ in the same buffer were added. The absorbance of hydrogen peroxide at 230 nm was determined after 10 min against a blank solution containing phosphate buffer without hydrogen peroxide. Ascorbic acid was taken as control. The percentage of hydrogen peroxide scavenging by the extracts and standard compounds was calculated as follows:

Anti-inflammatory activity
Carrageenan-induced rat paw edema: Wistar rats were divided into 5 groups each consisting of 6 animals.

Five groups each consisting of six wister rats were used. Edema was induced by subcutaneous injection (0.1 mL) of 1% freshly prepared suspension of carrageenin in normal saline, into the plantar side of the right hind paw of each rat. Methanolic extract of Cucumis melo (100, 200, 300 mg kg^-1 p.o) and Diclofenac sodium (12.5 mg kg^-1 p.o) as a standard drug were given 30 min before the carrageenin injection. Control animals received the vehicle only. The volume of the paw was measured at intervals of 60, 120, 180 min by the mercury displacement method using a plethysmograph. The percentage of inflammation decrease was calculated in relation to carrageenan control group and calculated according to the following formula:

Analgesic activity: Swiss albino mice of either sex were divided into 5 groups each consisting of 6 animals.

Tail immersion method: Male Swiss albino mice weighing 20-25 g were divided into six groups each containing five animals. The tail of the mouse was immersed to a constant level (3 cm) in a water bath maintained at 55±0.5°C. The control group was fed with the vehicle (1% CMC) orally. The Standard group was treated with morphine (10 mg kg^-1 p.o.). The methanolic extract was administered to other groups in doses 100, 200 and 300 mg kg^-1 orally. The time to flick the tail from water (reaction time) was recorded. A maximum immersion time of 15 sec was maintained to prevent thermal injury to the animals. The reaction time was the time taken by the mice to deflect their tails. The reaction time of the tail-flick response was determined at 0, 30, 60, 90 and 120 min after the administration of drugs. A significant increase in reaction time compared with control animals was considered a positive analgesic response (Patwardhan and Hooper, 1992).

Tail flick method: Swiss albino mice of either sex were divided into six groups each consisting of five animals. The tail of mice was placed on the radiant heat source (1 cm distance from the nichrome wire) of an analgesiometer and time taken by the animals to withdraw its tail from the radiant heat source was taken as the reaction time. The temperature was maintained at 55° to 56°C. Reaction time was recorded at 0, 30, 60, 90 and 120 min after the drug administration. A cut off time of 15 sec was maintained to prevent tissue damage (Davies et al., 1946).

Statistical analysis: All the results were expressed as Mean±standard error of means (SEM). The data was statistically analyzed by one way analysis of variance (ANOVA) followed by Tukey’s multiple range tests by using Sigmastat Version-2.0 Software. The p<0.05 was considered to be statistically significant.

RESULTS

Phytochemical screening: The results in Table 1 reveals that methanolic extract contain maximum amount of tannins, alkaloids, steroids, triterpenoids, carbohydrates, proteins and amino acids using standard procedures. Further studies were carried out by using the methanolic extract.

DPPH radical scavenging activity: DPPH reacts with antioxidants and gets converted into 1,1-diphenyl-2- picrylhydrazine by accepting a hydrogen atom and hence shows decrease in absorbance. The highest antioxidant activity of MECM was found to be 74.9% at concentration of 300 μg mL^-1 as shown in Table 2.

Hydrogen peroxide radical scavenging activity: The free radical scavenging activity of the MECM was evaluated by H₂O₂ scavenging method. The H₂O₂ scavenging effect of MECM was 58.91% at a concentration of 200 μg mL^-1 which was comparable to the scavenging effect of ascorbic acid (Table 3).

Table 1:	Phytochemical screening of MECM
+: Presence of chemical constituent, -: Absence of chemical constituent, ++: Maximum presence of chemical constituents

Table 2:	Percentage scavenging of DPPH radical
Values are the average of triplicate experiments and represented as Mean±SEM

Table 3:	Percentage scavenging of H2O2
Values are the average of triplicate experiments and represented as Mean±SEM

Table 4:	Effect of MECM on carrageenin induced paw edema in rats
The values are Mean±SEM of 6 animals

Table 5:	Analgesic effect of the MECM by tail immersion test
Values are Mean±SEM of 6 animals in each group. ap<0.05 compared with control group, bp<0.05 compared with morphine treated group

Table 6:	Analgesic effect of the MECM by tail flick test
Values are Mean±SEM of 6 animals in each group. ap<0.05 compared with control group, bp<0.05 compared with morphine treated group

Anti-inflammatory activity: Table 4 shows the results of the anti-edematous effect of orally administered MECM on carrageenin induced paw edema in rats. At dose of 100 mg kg^-1, MECM caused a reduction in paw edema (18.3%) 3 h after the subplantar injection of carrageenin. Doses of 200 and 300 mg kg^-1 caused a significant reduction of paw edema (43.4 and 56.6%, respectively).

Analgesic activity: Table 5 and 6 depicts the analgesic activity shown by MECM by tail immersion and tail flick method. Maximum analgesic effect was observed at 90 min interval.

The results of the current study demonstrate that Cucumis melo seeds displays potent anti-inflammatory, antioxidant, analgesic activity. Results have revealed that MECM seeds possess in vitro free radical scavenging activity. Hence, the extract was further evaluated for its in vivo anti-inflammatory and analgesic potential (Peters et al., 1997).

DISCUSSION

Tail flick and tail immersion methods were carried out evaluate the analgesic potential of Cucumis melo seeds. Free radicals are involved during pain stimulation and antioxidants show reduction in such pain. Thus methanolic extract of seeds possess analgesic property by inhibiting the free radical formation. Anti-inflammatory effect of natural products has frequently been studied by Carrageenan induced rat paw edema. Carrageenan-induced accumulation of leukocytes in the pleural cavity, as well as the enhancement of LTB₄ levels in pleural exudate occur after inflammatory stimulus. Migration of neutrophils to the affected area constitutes an important pro-inflammatory factor, as they liberate toxic oxygen radicals in the extracellular medium. Cucumis melo inhibited the leukocyte influx and raised LTB₄ levels. It also possesses significant free radical scavenging activity (Borgeat et al., 1976; Peters et al., 1999). The literature also reveals the antioxidant capacity of the pumpkin seed extract against DPPH free radical formation (Xanthopoulou et al., 2009). The seeds of Citrullus lanatus are found to possess antioxidant, anti-inflammatory and analgesic activities (Gill et al., 2009). The seed extract of Momordica Charantia normalizes the impaired antioxidant status in streptozotocin induced-diabetes by scavenging of free radicals (Sathishsekar and Subramanian, 2005).

Thus the seeds of Cucumis melo can be used as source of antioxidants in yhe treatment of pain and inflammation. The triterpenoids isolated from various species of cucurbita family have been reported to possess anti-inflammatory activities (Miro, 1995). The cucurbitacins from Cucurbita andreana were evaluated for their anti inflammatory and inhibitory effects on the growth of cancer cell lines (Jayaprakasam et al., 2003). Thus the triterpenoids might be responsible for the free radical scavenging, analgesic and anti-inflammatory activity.

CONCLUSION

Free radical scavenging activity of traditionally consumed Cucumis melo seeds were proved as it causes reduction in carregeenan-induced rat paw edema along with decrease in algesia in mice models.

ACKNOWLEDGEMENT

Thanks to Professor A. C. Rana and all faculty members of Rayat Institute of Pharmacy for their encouragement and support. We are also grateful to Rayat and Bahra Educational and Research Trust for their unconditional helps to carry out this project.