INTRODUCTION:

The free radicals shows very important role in human health and are effective against several diseases. It is necessary to have a brief idea about free radicals. During a chemical reaction (oxidation), one reactant loses an electron and is called oxidant or free radical¹, while the other gains an electron. In living organisms oxygen in unstable form is the most common free radical. This is called Reactive Oxygen Species (ROS)² and they are produced continuously in cells, either as accidental byproducts of metabolism or deliberately. The most common cellular oxygen free radicals are superoxide radical (O₂‾), hydroxyl radical (OH) and nitric oxide (NO)³. Other molecules, such as hydrogen peroxide (H₂O₂) and peroxynitrate (ONOO) are not free radicals themselves but can lead to their generation through various chemical reactions. Cells normally employ a number of defense mechanisms against damage induced by free radicals ^3,4. Antioxidants have been reported to prevent oxidative stress by free radical and ROS, and may avoid the occurrence of disease, cancer and aging. It can interfere with the oxidation process by reacting with free radicals, chelating, catalytic metals, and also by acting as oxygen scavenger⁵.

Since time immemorial man has been using plant extracts to protect himself against several diseases and also to improve his health and life-style. With the development in techniques and recent researches, it has been proved that certain non-nutritive chemicals in plants such as terpenoids and flavonoids which were earlier thought to be of no importance to human diet possess antioxidant properties. The plants are susceptible to damage caused by active oxygen and thus develop numerous antioxidant defense system resulting in formation of numerous potent antioxidants⁶.

Many aromatic, medicinal and spice plants contain compounds that possess confirmed strong antioxidative components. There is also a considerable amount of evidence revealing an association between individuals who have a diet rich in fresh fruits and vegetables and the decreased risk of and certain forms of cancer^7,8 and cardiovascular diseases⁹.

Benincasa hispida Cogn (family: Cucurbitaceae) is a widely used vegetable in India and other tropical countries¹⁰. Plants belonging to the Benincasa species have been the subjects of many investigations for their biologically active components. Some species of Benincasa have been used as medicinal plants for the treatment of diabetes mellitus, urinary infection, epilepsy, peptic ulcer, and hemorrhages from internal organs¹¹. Therefore, the objective of the present study was to determine the antioxidant activity of fruit extract of Benincasa hispida cogn. using in vitro antioxidant models.

MATERIAL AND METHODS:

Collection of plant:

The fresh fruit of Benincasa. hispida was collected from the local vegetable market of Thane, Maharashtra. The authentification of the plant was done in the Department of Life Science, Ruia College, Matunga, Mumbai.

Extract preparation.

The fruit was peeled off and seeds were removed. Pulp was mashed using an electric juicer to afford a soft mass and later on macerated with ethanol (1: 4) for seven days at room temperature with occasional stirring daily. On eighth day, the pulp mass was filtered and the filtrate was heated (below 55^OC) and evaporated under reduced pressure till a strong brownish yellow liquid was obtained. It was then stored at 2-4^OC and protected from direct sunlight ¹².

Preliminary phytochemical screening:

The ethanolic extract of Benincasa Hispida (EEBH) was taken for various qualitative chemical tests to determine the presence of various phyto constituents like alkaloids, glycosides, carbohydrates, phenolics and tannins, phytosterols, fixed oils, protein and amino acids, flavanoids and saponins, using reported method¹³.

In vitro antioxidant methods :-

1) Hydrogen peroxide radical scavenging activity.

The ability of the extract to scavenge hydrogen peroxide was determined according to the method of Ruch, Cheng and Klaunig (1989)¹⁴. A solution of hydrogen peroxide was prepared in phosphate buffer (pH 7.4). Extracts (20–1000 μg /ml) were added to hydrogen peroxide solution (0.6 ml). Absorbance of hydrogen peroxide at 230 nm was determined after 10 min against a blank solution containing phosphate buffer without hydrogen peroxide. For each concentration, a separate blank sample was used for background subtraction.

2) Free radical scavenging activity (DPPH method):

The antioxidant activity of the plant extracts and standard were assessed on the basis of the radical Scavenging effect of the stable DPPH free radical by Blois M.S. (1958)¹⁵. About 20-1000 μl of each extract or standard was added to 2 ml of DPPH in methanol (0.33%) in a test tube. After incubation at 37^oC for 30 minutes the absorbance of each solution was determined at 517 nm using spectrophotometer.

3) Nitric oxide radical scavenging activity:

Nitric oxide was generated from nitroprusside and measured by the Griess reaction according to the Marcocci L.et al. (1994). Sodium nitroprusside in aqueous solution at physiological Ph spontaneously generates nitric oxide, which interacts with oxygen to produce nitric oxide which, interacts with oxygen to produce nitric ions that can be estimated by use of Griess reagent^{16, 17}. Scavengers of nitric oxide compete with oxygen leading to reduced production of nitric oxide. Sodium nitroprusside (5 mM) in phosphate–buffered saline (PBS) was mixed with 3.0 ml of different concentrations (20-1000 μg /ml) of the drugs dissolved in the suitable solvent systems and incubated at 25^oC for 150 min. The samples from the above were reacted with Griess reagent (1% sulphanilamide, 2% H₃PO₄ and 0.1% napthylethylenediamine dihydrochloride). The absorbance of the chromophore formed during the diazotization of nitrite with sulphanilamide and subsequent coupling with napthylethylenediamine was read at 546 nm using spectrophotometer.

4) Reducing power assay:

The reducing power of the EEBH was determined according to the method of Oyaizu (1986) ¹⁸. Different concentrations of the EEBH in 1.0 ml of deionised water were mixed with phosphate buffer (2.5ml, 0.2 M, pH 6.6) and potassium ferrocyanide (2.5 ml, 1%). The mixture was incubated at 50^oC for 20 min. A portion of trichloroacetic acid (2.5 ml, 10%) was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. The upper layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl₃(0.5 ml, 0.1%) and the absorbance was measured at 700 nm and compared with standards. Increased absorbance of the reaction mixture indicated increased reducing power.

RESULT AND DISCUSSION:

Preliminary phytochemical screening:

The phytochemical screening are shown at table-1

Table 1) Preliminary phytochemical screening.

Class	EEBH
Alkaloids	-
Carbohydrates	+
Terpenoids	+
Protein and Amino Acid	+
Flavanoids	+
Saponins	+
Phenolic compounds and Tannins	+

*(+) Indicate Present and (-) Indicate Absent

In vitro antioxidant assays –

1) Hydrogen peroxide radical scavenging activity.

Table 2 and Figure 1 shows that EEBH shows less scavenging activity (H₂O₂) than that of Ascorbic acid. The IC₅₀value for scavenging of H₂O₂ for EEBH was 397.85 μg/ml while IC₅₀value for ascorbic acid was 201.69 μg/ml. Hydrogen peroxide is a weak oxidizing agent and can inactivate a few enzymes directly, usually by oxidation of essential thiol (-SH) groups. Hydrogen peroxide can cross cell membranes rapidly, once inside the cell, H₂O₂ can probably react with Fe^2+,and possibly Cu²⁺ions to form hydroxyl radical and this may be the origin of many of its toxic effects ¹⁹.

Table 2) Percentage H₂O₂ radical scavenging activity of EEBH and Ascorbic acid.

Concentration (μg/ml)	% Inhibition by EEBH	% Inhibition by Ascorbic acid
20	10.99 ±0.0791	21.45 ±0.4114
40	19.78 ±0.0901	29.77 ±0.5319
80	25.10 ±0.2014	37.92±0.2701
100	32.078 ±0.1871	41.98 ±0.4105
200	38.85 ±0.1921	49.58 ±0.1927
400	50.27 ±0.3007	61.73 ±0.2309
800	62.81 ±0.0989	78.191 ±0.5014
1000	73.23 ±0.0819	89.74 ± 0.3115

Values are expressed as a mean ±SEM of triplicate tests

Fig. 1: H₂O₂ radical scavenging activity of Benincasa hispida Fruit.

2) Free radical scavenging activity (DPPH method).

Table 3) and Figure 2. Shows the dose-response curve of DPPH radical scavenging activity of the EEBH, compared with ascorbic acid, as standard. IC₅₀ value of the EEBH was found to be 379.21 μg/ml and for ascorbic acid was 182.32 μg/ml. In the DPPH radical scavenging assay, DPPH radical was used as a substrate to evaluate free radical scavenging activity of fruit extract. It involves reaction of specific antioxidant with a stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH)²⁰.

Table 3) Percentage DPPH radical scavenging activity of EEBH and Ascorbic acid

Concentration (μg/ml)	% Inhibition by EEBH	% Inhibition by Ascorbic acid
20	9.019 ±0.1019	14.666 ±0.3901
40	12.98 ±0.217	19.223 ±0.2021
80	20.39 ±0.1339	31.409 ±0.1908
100	29.27 ±0.0976	39.891 ±0.078
200	38.910 ±0.0891	54.847±0.1478
400	52.741 ±0.0927	64.927 ±0.0991
800	69.190 ±0.209	77.612 ±0.0072
1000	75.970 ±0.0712	82.871 ±0.409

Values are expressed as a mean ±SEM of triplicate tests

Fig. 2:DPPH radical scavenging activity of Benincasa hispida Fruit.

3) Nitric oxide radical scavenging activity:

Table 4) and Figure 3. shows that EEBH exhibited moderately good nitric oxide scavenging activity between 20 and 1000 μg/ml. IC₅₀ value for scavenging of nitric oxide for EEBH was 457.44 μg/ ml while IC₅₀ value for ascorbic acid was 340.62 μg/ ml. Although nitric oxide and superoxide radicals are involved in host defense, over production of these two radicals contributes to the some inflammatory diseases^21,22. Moreover in the pathological conditions, nitric oxide reacts with superoxide ion and form toxic molecule, peroxynitrite.

Table 4) Percentage Nitric Oxide radical scavenging activity of EEBH and Ascorbic acid.

Concentration (μg/ml)	% Inhibition by EEBH	% Inhibition by Ascorbic acid
20	7.983 ±0.0998	12.131 ±0.0695
40	13.801 ±0.1004	19.169 ±0.0834
80	21.291 ±0.0796	26.883 ±0.0251
100	28.743 ±0.0819	37.310 ±0.1072
200	33.771 ±0.110	43.39 ±0.0867
400	43.721 ±0.1019	58.716 ±0.2109
800	60.910 ±0.0713	73.99 ±0.0926
1000	79.667 ±0.854	95.351 ±0.3710

Values are expressed as a mean ±SEM of triplicate tests

Fig. 3: Nitric Oxide radical scavenging activity of Benincasa hispida Fruit.

4) Reducing power assay:

Table 5) and Figure 4) shows that EEBH exhibited good reducing power. The reducing power of EEBH was very potent and the power of extract increased with quality of sample. High absorbance indicates high reducing power. The reducing power of EEBH increases with increase in the concentration. The plant extract could reduce the most Fe³⁺ions, which had a lesser reductive activity than the standard of ascorbic acid ^23,
24.

Table 5) Percentage antioxidant activity of EEBH and Ascorbic acid in Reducing Power Assay.

Concentration (μg/ml)	Absorbance by EEBH	Absorbance by Ascorbic acid
20	0.092 ±0.001	0.119 ±0.003
40	0.109 ±0.002	0.151 ±0.003
80	0.208 ±0.002	0.232 ±0.002
100	0.217 ±0.001	0.246 ±0.001
200	0.348 ±0.002	0.419 ±0.003
400	0.414 ±0.003	0.527 ±0.002

Values are expressed as a mean ±SEM of triplicate tests.

Fig. 4: Reducing Power assay of Benincasa hispida Fruit.

CONCLUSION:

In summary, we observed that, the ethanolic extract of fruit of Benincasa hispida (EEBH) could dose- dependently and significantly exhibits high antioxidant and free radical scavenging activities. The results in the present report suggest that the plant extract we have examined exhibited potent antioxidant effect in vitro, can serve as good candidates for further evaluation of their bio-efficacies. Possible applications of the selected target plant extract as food supplement for human health care are also under evaluation and also might be helpful in preventing the progress of various oxidative stresses.

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Received on 03.03.2011 Modified on 07.04.2011

Research J. Pharm. and Tech. 4(7): July 2011; Page 1082-1085