Evaluation of Free Radical Scavenging, Anti-inflammatory and Analgesic Potential of Benincasa hispida Seed Extract

MATERIALS AND METHODS

Plant material: The seeds were purchased from the Khari Baoli (spice market) Delhi (India) in August 2009. The healthy looking seeds were selected for authentication and voucher specimen number 0389 has been deposited in the Botanical and Environmental Science Department, Guru Nanak Dev University, Amritsar (India). The seeds were cleaned, washed, dried at low temperature and powered.

Drugs and chemicals: Ascorbic acid and carrageenan were obtained Central Drug House Pvt. Ltd., Mumbai, India. Hydrogen peroxide was obtained from E-Merck Ltd., Mumbai. 1,1-Diphenyl-2-picrylhyderazyl (DPPH) was obtained from Sigma Chemical Co., USA. Physician sample of morphine and diclofenac sodium was procured from Government Medical College and Hospital, Patiala. All other chemical reagents used were of analytical grade which were procured from different companies (Loba Chem, Mumbai and Merck Limited, Mumbai).

Animals: The wistar albino rats (200-250 g) and swiss albino mice (25-30 g) of either sex were obtained from NIPER Mohali. They were kept at standard laboratory diet, environmental temperature and humidity. A 12 h light and dark cycle was maintained throughout the experimental protocol. The experimental protocol was duly approved by Institutional Animal Ethics Committee (IAEC) 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 powdered seeds were extracted for 72 h with methanol at room temperature. The solvent was filtered off and residue macerated again with the fresh solvent. Both solvents were combined and concentrated under reduced pressure on a rotary evaporator (Hedolph) at 40°C. The concentrated filtrate was suspended in distilled water and partitioned successively with hexane. The aqueous layer was separated and concentrated on water bath. The crude extracts were used for further investigation.

Phytochemical screening: The crude extract was studied for the presence of phytochemicals such as alkaloids, tannins, saponins, flavonoids, steroids, triterpenoids, carbohydrates, proteins and amino acids using standard procedures (Harborne, 1973).

The alkaloids were tested using various reagents such as Dragendroff’s reagent (potassium bismuth iodide solution), Hagers reagent (picric acid solution), Mayer’s reagent (potassium iodide solution) and Wagner’s reagent (solution of iodine in potassium iodide). The sterols were tested by Liebermann’s test (blue green color with acetic anhydride plus sulphuric acid). The triterpenoids were tested by Liebermann-Burchard’s test. In this test a few mg of the extract was dissolved in chloroform, a few drops of acetic anhydride were added followed by sulphuric acid. Red/pink/purple/violet color confirms the presence of triterpenoids. The ferric chloride test was used for tannins (blue green color with ferric chloride), Molish test for carbohydrates, Ninhydrin reagent test for proteins and amino acids. The flavonoids were tested by concentrated nitric acid (crimson or magenta color with concentrated nitric acid).

Free radical scavenging activity
DPPH radical scavenging activity: The free radical scavenging activity of MEBH was determined by using 1,1-diphenyl-2 picryl-hydrazyl (DPPH) method (Sreejavan and Rao, 1997). Briefly, a 0.05 mM solution of DPPH in methanol was prepared and 1.5 mL of this solution was added to 0.5 mL of extract solution in methanol at different concentrations (100-300 μg mL^-1). The mixture was shaken vigorously and allowed to stand at room temperature for 30 min. Then the absorbance was measured at 517 nm using a spectrophotometer (Shimadzu UV- 1700 Pharma spec). A blank without DPPH was used to remove the influence of the color of the samples. A methanolic solution of DPPH was used as negative control. Ascorbic acid was used as a reference drug. All measures were carried out in triplicate. Lower absorbance of the reaction mixture indicates higher free radical scavenging activity. The DPPH radical scavenging activity was calculated using the Eq. 1:

(1)

where, A_o is absorbance of the negative control A_s is the absorbance of the sample.

Hydrogen peroxide radical scavenging activity: Hydrogen peroxide radical scavenging activity was determined according to the method of Wettasinghe and Shahidi (2000). The extract solution (1.0 mL) in various concentrations (25-200 μg mL^-1) was mixed with 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.

After 10 min the absorbance values of the reaction mixtures were recorded against a blank solution containing phosphate buffer without H₂O₂ at 230 nm using a spectrophotometer (Shimadzu UV- 1700 Pharma spec). For each concentration, a separate blank sample was used for background subtraction. Ascorbic acid was used as a standard and mixture without sample was taken as a control. The percentage scavenging of H₂O₂ was calculated as:

(2)

where, A_o is the absorbance of the control and A₁ is the absorbance of the extract/standard. All tests were done in triplicate (Sood et al., 2009).

Anti-inflammatory activity
Carrageenan-induced rat paw edema: The carrageenan-induced rat paw edema assay was carried out according to Winter et al. (1962). Wistar rats were divided into 5 groups each consisting of 6 animals.

Edema was induced on the left hind paw of the rats by subplantar injection of 0.1 mL of a solution of 1% (w/v) carrageenin in a 0.9% NaCl (w/v). The paw volume was measured at intervals of 60, 120, 180 min by the mercury displacement method using a plethysmograph after administration of the extract/drug orally. The percentage inhibition of paw edema in drug treated group was compared with the carrageenan control group and calculated according to the following in Eq. 3.

(3)

where, V_c is the inflammatory increase in paw volume of control group of animals and V_t is the inflammatory increase in paw volume of drug-treated animals.

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

Tail immersion test: The procedure is based on the observation that morphine like drugs selectively prolongs the reaction time of the typical tail withdrawal reflex in mice (Toma et al., 2003). The tail of mice was immersed in warm water kept constant at 52.5"0.5°C. The reaction time of the tail-withdrawal response was determined at 0, 30, 60, 90 and 120 min after the administration of drugs. A cut off time of 15 sec was maintained to prevent tissue damage (Grotto and Sulman, 1967).

Tail flick test: 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 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. The temperature was maintained at 52"0.5°C (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-value<0.05 was considered to be statistically significant.

RESULTS AND DISCUSSION

Phytochemical screening: The results of the phytochemical screening of the methanolic extract are shown in Table 1; the results reveal the presence of chemical constituents such as proteins and amino acids, triterpenoids, carbohydrates, sterols and tannins.

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 MEBH showed concentration-dependent DPPH radical scavenging activity. The highest radical scavenging activity of MEBH was found to be 79.8% at concentration of 300 μg mL^-1; the results are shown in Table 2.

Table 1:	Phytochemical chemical screening of MEBH
+: Presence of chemical constituent, -: Absence of chemical constituent

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

Hydrogen peroxide radical scavenging activity: The free radical scavenging activity of the MEBH was evaluated by H₂O₂ scavenging method. The results are shown in Table 3, the MEBH showed concentration dependent activity. The H₂O₂ scavenging effect of MEBH was 63.7% at a concentration of 200 (μg mL^-1) which was comparable to the scavenging effect of ascorbic acid.

Anti-inflammatory activity: Table 4 shows the results of the anti-edematous effect of orally administered MEBH on carrageenin induced paw edema in rats; the MEBH showed dose dependent anti-inflammatory activity in carrageenan induced paw edema in rats. At dose of 100 mg kg^-1, MEBH caused a reduction in paw edema (19.4%) 3 h after the subplantar injection of carrageenin. Doses of 200 and 300 mg kg^-1 caused a significant reduction of paw edema (41.7 and 59.7%, respectively) from the first till the third hour compared with the disease control group.

Analgesic activity
Tail immersion test: Table 5 depicts the analgesic activity shown by MEBH by tail immersion method. MEBH showed dose dependent analgesic activity against conduction of heat-induced algesia in mice. Medium (200 mg kg^-1) and high (300 mg kg^-1) doses showed significant difference in the analgesic activity when compared with control group. Maximum analgesic effect was observed at 90 min interval.

Tail flick test: Table 6 depicts the analgesic activity shown by MEBH by tail flick method. The MEBH showed dose dependent analgesic activity against radiant heat-induced algesia. Medium (200 mg kg^-1) and high (300 mg kg^-1) doses showed significant difference in the analgesic activity when compared with control group. Maximum analgesic effect was observed at 90 min interval. Hence, it was observed that the MEBH significantly reduced the pain sensation in mice at medium and higher dose.

In the present study, the methanolic extract of Benincasa hispida seeds was evaluated for its free radical scavenging, anti-inflammatory and analgesic potential. Results have revealed that MEBH possess in vitro free radical scavenging activity. Hence, the extract was further evaluated for its in vivo anti-inflammatory and analgesic potential. Carrageenan induced rat paw edema test has frequently been used to assess the anti-edematous effect of natural products. Carrageenan brings about inflammation by the release of mediators of inflammation (prostaglandins, leukotrienes, histamine, bradykinin etc.) (Crunkhorn and Meacock, 1971); also, several free radicals are released during such inflammation (Koblyakov, 2001). The MEBH showed significant free radical scavenging activity, so this can be responsible for the reduction of inflammation in the carrageenan-induced paw edema in rats (Cuzzocrea et al., 2001). Tail immersion and tail methods were carried out to evaluate the analgesic potential of seed extract. Significant decrease in the tail withdrawal latency indicated that the methanolic extract of seeds possess analgesic property. This analgesic activity may be due to its free radical scavenging activity as these free radicals are involved during pain stimulation and antioxidants show reduction in such pain (Kim et al., 2004). 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., 2010). The seed extract of Momordica Charantia normalizes the impaired antioxidant status in streptozotocin induced-diabetes by scavenging of free radicals (Sathishsekar and Subramanian, 2005).

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).

Table 4:	Effect of MEBH on carrageenin induced paw edema in rats
The values are Mean±SEM of 6 animals. ap<0.05 compared with disease control group, bp<0.05 compared with diclofenac treated group

Table 5:	Analgesic effect of the MEBH 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 MEBH 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

Cucurbitacin glucosides are reported to possess antioxidant and free radical scavenging activities (Tannin-Spitz et al., 2007). Thus, the triterpenoids might be responsible for the free radical scavenging, analgesic and anti-inflammatory activity.

CONCLUSION

The MEBH was found to have free radical scavenging potential and this may be one of the probable reasons for the reduction in carregeenan-induced paw edema in rats along with decrease in algesia in mice models.

ACKNOWLEDGMENTS

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 help to carry out this project.