INTRODUCTION:

The role of oxygen derived free radicals in pathogenesis of number of degenerative diseases is well known. Many plants contain phytoconstituents like carotenoid. flavonoids, phenolics which can be used for scavenging free radicals in the human body. ¹Role of free radicals and reactive oxygen species in the pathogenesis of human diseases like cancer, aging, liver diseases, respiratory diseases has been widely recognized.² Such plants can be the potential candidates for the use in stress related diseases.³

Ficus glomerata belonging to family Urticaceae is known to possess astringent activity. It is used in dental preparations. ^4,
5 It is also useful in treatment of asthma, piles and skin diseases. The

tree is found to be stomachic, carminative and useful in the treatment of menorrhagia. The bark is useful in treating diabetes.⁶

MATERIALS AND METHODS:

The plant was collected from the local area of Pune in month of October and identified by Dr. Dinesh Shirodkar, Botanical survey of India, Pune.

Preparation of extracts:

Course powder (500 g) of the bark was macerated for overnight in distilled water (1.0 L). The extract so obtained was concentrated in a rotary vacuum evaporator and then placed in a desiccator. The Yield was of aqueous 7.5%w/w.

Preliminary phytochemical screening:

The powdered crude drug (100 gm) was subjected to successive solvent extraction using soxhelt apparatus.

The different successive extracts so obtained were then subjected to preliminary phytochemical screening by applying different qualitative testes for phytoconstituents.⁷ The different extracts showed presence of steroids, carbohydrates, phenolics, flavonoids, alkaloids, amino acids and volatile oil. The presence of these phytoconstituents was confirmed by TLC.

Determination of antioxidant activity:

The scavenging activity against hydroxyl radicals and superoxide anions was measured using two different assays such as deoxyribose and NBT assay. Initially deoxyribose assay to determine the rate constant for the reaction between either antioxidant and hydroxyl radicals (non site specific scavenging assay) or antioxidant and iron (site specific scavenging assay) was conducted as described by Haliwell.⁸

For nonspecific assay, varied concentrations of extracts were mixed with 1 ml of reaction buffer (100µM Ferric chloride, 10 µM EDTA, 1.5 mM hydrogen peroxide, 2.5 mM deoxyribose and 100µM L ascorbic acid, pH 7.4) and incubated for 1hour at 37ºC. 1 ml of 0.5% 2 thiobarbituric acid in 0.025 M sodium hydroxide and 1 ml of 2.8% trichloro acetic acid was added to the mixture and heated for 30 minutes at 80ºC. Finally the mixture was cooled and absorbance was measured at 532 nm using spectrophotometer.

Site specific scavenging activity which represents the ability of extracts to chelate iron ions and interfere with hydroxyl radical generation, was measured using the same reaction buffer without EDTA. The ability of the extracts to reduce Fe⁺⁺⁺- EDTA to Fe⁺⁺- EDTA complex acting as pro-oxidant was tested using non site specific scavenging assay without addition of ascorbic acid.

Scavenging activity on superoxide radical was accessed by the method described by Gotoh and Niki.⁹ Different concentrations of extracts were added to the reaction solution containing 100µl of 30mm EDTA, 10µl of 30mm of hypoxanthine in 50 mM sodium hydroxide and 200µl of 1.42 mM nitroblue tetrazolium. After preincubation at RT for 30 min, 100µl of 0.5u/ml of xanthine oxidase was added to the mixture and volume was made up to 3 ml with 50 mM phosphate buffer. After incubation for 20 min, absorbance was measured at 560 nm.

The inhibitory effect of all the assays was calculated as follows-

% inhibition = (OD _control- OD_sample) / OD _controlX100

Table 1: Pro-oxidant activity of aqueous extract of bark of Ficus glomerata on non site specific hydroxyl radical generation

Conc.(µg/ml)	Absorbance (532nm)	% stimulation
Control	0.151	-
10	0.098	0
20	0.107	0
50	0.112	0
75	0.116	0
100	0.136	0
200	0.152	0.6

Fig: 1 Inhibition effect of aqueous extract of bark of Ficus glomerata on NBT reduction.

RESULTS:

It had been observed that the extract showed excellent antioxidant activity. Concentration dependant inhibition of hydroxyl radical induced deoxyribose degradation was observed in both site specific and non site specific assay. Pro oxidant effect was not observed in case of the extract.(Table 1) Table 2 represents potential of the extract to scavenge hydroxyl radicals. The antioxidant effect of extract was tested on superoxide anions by reduction of NBT induced superoxide anions by xanthine mediated degradation of hypoxanthine. (Fig 1) The extract showed dose dependant inhibition of free radicals.

DISCUSSION:

Free radicals generated in the body have ability to attack healthy cell and are capable of producing several diseases like liver disorder, diabetes, cancer etc.¹⁰The ability of extract to reduce Fe⁺³ – EDTA complex which represents its ability to stimulate hydroxyl radical generation was not observed. It indicates that the extract was found to have no pro oxidant activity.¹¹ Furthermore, since superoxide anions produce other kinds of cell damaging free radicals and oxidizing agents.¹² The extract has a potential of scavenging the superoxide anions. This suggests that the extract is efficient natural antioxidant.

Table 2: Antioxidant activity of aqueous extract of bark of Ficus glomerata on non site specific hydroxyl radical generation

Conc.(µg/ml)	Absorbance (532nm)	% inhibition
Control	0.098	-
10	0.107	9.1
20	0.112	14.2
50	0.152	55
75	0.159	62.2
100	0.180	83.6
200	0.196	96.9

CONCLUSION:

The antioxidant activity of aqueous extract of bark of Ficus glomerata is may be due to phenolics and flavonoids present in the extract. This suggests the use of the plant in treatment of various human ailments.

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Received on 20.10.2008 Modified on 30.10.2008

Research J. Pharm. and Tech. 1(4): Oct.-Dec. 2008; Page 537-538