DPPH (1,1-diphenyl-2-picrylhydrazyl) was obtained from Hi-media, carrageenan, ascorbic acid and diclofenac sodium from Jackson Laboratories, Amritsar. Punjab), Physician sample of morphine was procured from Government Medical College and Hospital, Patiala (Punjab) and the solvents like hexane, chloroform, ethyl acetate and methanol were of analytical grade and purchased from SD fine chemical.

Citrullus lanatus seeds were purchased from the local grain market, sector 26 in Chandigarh. The seeds were authenticated and the voucher specimen no: 0394 has been deposited in the Botanical and Environmental Science Department, Guru Nanak Dev University, Amritsar. For examination only healthy looking seeds were chosen (without mechanical damages and bacterial infection). The seeds were cleaned, washed, shade dried and carefully powdered in the grinder by maintaining the temperature below 20°C. All samples were kept in air tight light-protected containers.

Extraction was carried out using solvents of increasing polarity such as hexane, chloroform, ethyl acetate and methanol by simple maceration process for 24 hrs. The solvents were completely removed by rotary evaporator and crude extracts were obtained and stored in the refrigerator. These crude extracts were further used for their antioxidant anti-inflammatory and analgesic potential.

Wister albino rats (160-180 g) of either sex, Swiss albino mice (20- 30 g) were purchased from Sanjay Biologicals, Amritsar for experimental study. They were acclimated to standard animal house conditions such as temperature (24.0±1.0 ⁰C), relative humidity

(55-65%) and 12hrs light/12hrs dark cycle. They were fed with commercial pelleted rat feed and had free access to water. The experimental protocol was approved by the IAEC (Institutional Animal Ethical Committee) of CPCSEA, (Committee for the Purpose of Control and Supervision of Experiments on Animal) registration no. 874/ac/05/CPCSEA.

Qualitative evaluation of the DPPH scavenging activity

Quantitative evaluation of the DPPH scavenging activity

Quantitative evaluation of the hydrogen peroxide scavenging activity

The animals of either sex were divided into five groups each composed of six animals.

Group I (Control): Carrageenan (1%, p.o.)

Group II (Standard): Diclofenac sodium (12.5 mg/kg, p.o.)

Group III: Methanolic extract of Citrullus lanatus (MECL), (50 mg/kg, p.o.)

Group IV: MECL (100 mg/kg, p.o.)

Group V: MECL (200 mg/kg, p.o.)

Percentage inhibition of edema = (V_c - V_t / V_c) ×100

Where, V_c is the inflammatory increase in paw volume in control group of animals and V_t is the inflammatory increase in paw volume in drug-treated animals. Paw edema was induced by injecting 0.1 ml of 1% carrageenan in physiological saline into the sub plantar tissue of the left hind paw of each rat (17-18). The different doses of the extract were administered orally 30 min prior to carrageenan administration. The paw volume was measured at intervals of 60, 120, 180 and 240 min by the mercury displacement method using a plethysmometer (Labco,India). The percentage inhibition of paw volume in treatment group was compared with the carrageenan control group (Group- I). Diclofenac sodium (12.5 mg / kg p.o.) was used as reference drug.

The albino mice were divided into five groups of six animals each.

Group I (Control): Carboxy methyl cellulose suspension (0.3 ml, 1% w/v, p.o.)

Group II (Standard): Morphine (10 mg/kg, p.o.)

Group III: Methanolic extract of Citrullus lanatus (MECL), (50 mg/kg, p.o.)

Group IV: MECL (100 mg/kg, p.o.)

Group V: MECL (200 mg/kg, p.o.)

All analgesic tests were performed in different time intervals i.e. 0, 30, 60, 120 and 180 minutes.

In the present study analgesia was assessed according to the reported method (19). The terminal part of the tail (about 1cm) of the mice was placed on analgesiometer, at uniform distance from the nichrome wire. Temperature of heating element of the instrument was maintained at 52±0.5°C. Cut-off time 20 seconds was maintained.

In present study analgesia was assessed according to the reported method (20). 3-4 cm area of the tail was marked and immersed in the water bath thermo-statistically maintained at 51°C. The withdrawal time of the tail from hot water (in seconds) was noted as the reaction time or tail flick latency. The maximum cutoff time for immersion was 20 seconds to avoid the injury of the tissues of tail.

Descriptive statistics and comparisons of differences between each data set were calculated by the use of Sigma Stat 3.5 trial version software. The data were expressed as Mean ± SEM, and analyzed by one way ANOVA in each experiment. Statistical significance was accepted at the level of p<0.05. In the case of significant variation (p<0.05), the values were compared by Dunnet test.

The TLC of methanolic extract sprayed with 0.2% DPPH solution showed good antioxidant activity. The spots showed good yellow coloration over the violet coloration of the DPPH.

Scavenging of the DPPH radical: The reduction capability of DPPH radical was determined by the decrease in its absorbance at 517 nm induced by antioxidants. Due to rapid hydrogen donating ability of DPPH, it reacts with antioxidants and gets converted into 1, 1-diphenyl-2-picryl hydrazine and hence shows decrease in absorbance. All the extracts showed some concentration dependent antioxidant activity but the maximum activity was found in methanolic extract (300 µg/ml) (Fig.1).

Scavenging of Hydrogen Peroxide: The measurement of H₂O₂ scavenging activity is one of the useful methods of determining the ability of antioxidants to decrease the level of pro-oxidants such as H₂O₂ (15). _{Hydrogen peroxide itself is not very reactive, but sometimes it can be toxic to cells because of rise in the hydroxyl radicals in the cells (21). The hydrogen peroxide scavenging activity of various extracts is shown in Fig.2. The maximum activity was found in methanolic extract followed by ethyl acetate and chloroform. Concentration dependent antioxidant activity was observed for methanol extract.}

Figure 1

Figure 1. Percentage inhibition of DPPH free radical by seed extracts at different concentrations in vitro

Figure 2

Figure 2. Percentage inhibition of hydrogen peroxide free radical by seed extracts at different concentrations in vitro

Acute inflammation study in rats

Effect of MECL on acute inflammation in rats: The methanolic extract of Citrullus lanatus seeds significantly attenuated carrageenan induced paw volume in rats. However, the maximum reduction of paw volume was observed at higher dose (200mg/kg, p.o.) which was comparable to the effect of diclofenac sodium (12.5mg/kg, p.o.) as shown in Table 1.

Figure 3

Table 1. Anti inflammatory activity by carrageenan induced paw edema in rat

Table 1. Data were expressed as mean ± S.E.M., n = 6 rat in each group. a = p<0.05 vs Carrageenan control groups, b = p<0.05 vs diclofenac control group. Results were expressed as paw volume in ml.

Analgesic activity in mice

Effect of MECL on Tail flick test: The methanolic extract of Citrullus lanatus significantly attenuated the spinal pain sensation against conduction heat in mice. Moreover, this ameliorative effect of tail withdrawal response was observed in a dose dependent manner. The maximum analgesic effect was shown at higher dose (200mg/kg, p.o.) which was comparable to that of morphine (10mg/kg, p.o.) as shown in Table 2.

Figure 4

Table 2. Tail flick test

Table 2. Data were expressed as mean ± S.E.M., n=6 mice in each group. a = p<0.05 vs CMC control groups, b = p<0.05 vs morphine control group. Results were expressed as tail withdrawal latency in seconds.

Effect of MECL on Tail immersion test: The methanolic extract of Citrullus lanatus significantly attenuated the spinal pain sensation against hot water in mice. Moreover, this ameliorative effect of tail withdrawal response was observed in a dose dependent manner. The maximum analgesic effect was shown at higher dose (200mg/kg, p.o.) which was comparable to that of morphine (10mg/kg, p.o) as shown in Table 3.

Figure 5

Table 3. Tail immersion test

Table 3. Data were expressed as mean ± S.E.M., n=6 mice in each group. a = p<0.05 vs CMC control groups, b = p<0.05 vs morphine control group. Results were expressed as tail withdrawal latency in seconds.

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