IN-VITRO ANTIOXIDANT ACTIVITY OF ASPARAGUS RACEMOSUS ROOTS

Objective: The objective of present study is to evaluate the antioxidant activity of methanolic extract of Asparagus racemosus roots (liliaceae). Material and methods: The antioxidant activity of the methanolic extract of A. racemosus was determined by using a method based on the reduction of methanolic solution of coloured-free radical 1, 1 diphenyl-1-2 picrylhydrazyl (DPPH). The radical scavenging activity of tested sample was expressed as an inhibition percentage. Butylated hydroxyl toluene was used as reference standard. The absorbances of all the dilutions were taken after 30 minutes at λ max 517 nm using methanol as blank. Results and Discussion: The IC 50 value of A. racemosus was 4158.8 whereas butylated hydroxyl toluene used as a standard showed an IC 50 of 46.25µg. The absorbance of samples (Methanolic extract of A. racemosus and standard Butylated hydroxytoluene) were taken in triplicate. Conclusion: The present study showed that the methnolic extract of roots of A. racemosus have moderate free radical scavenging activity.


INTRODUCTION
Antioxidants or inhibitors of oxidation are the compounds which retard or prevent the oxidation in general and prolong the life of oxidizable matter 1 . Antioxidants can interfere with the oxidation process by reacting with free radicals, chelating catalytic metals and also by acting as reactive species scavenger. Polyphenolic compounds like flavonoids and phenolic acids, commonly found in plants, have been reported to have multiple biological effects, including antioxidant activity 2 . The antioxidant activities of the individual compounds may depend on structural factors, such as number of phenolic, hydroxyl or methoxyl groups and other structural features 3 . Among the antioxidative compounds vitamin A, C, E, selenium, carotenoids, ascorbic acid show very strong intensity of antioxidative activities 4 . A. racemosus is an indigenous medicinal plant of the family Liliaceae 5,6 , commonly it is known as Satavari. It is found in all over India, especially in Northern India. It is important for its sapogenin content 7 , the precursor of many pharmacologically active steroids. The roots of A. racemosus (Liliaceae) mentioned in Ayurveda, have been used to treat, Anticancer activity , antdysenteric activity, antifungal activity antibacterial activity, anti-inflammatory activities, antiulcer activity, antioxidant activity, anti-abortifacient activity (Shatvarin 1),

IJBR2[4][2011]228-235
www.ijbr.ssjournals.com Antioxytoxic (shatavarin 4), spasmodic to uterus Hypoglycaemic, hypotensiv activity, anticoagulant activity 8 . A free radical is a compound with one or more unpaired electrons in its outer orbital 9 . Such unpaired electrons make these species very unstable and therefore quite reactive with other molecules due to the presence of unpaired electrons and try to pair their electrons and generate a more stable compound 10 . The molecule of 1, 1-diphenyl-2-picrylhydrazyl (DPPH) is characterized as a stable free radical by virtue of the delocalization of the spare electron over the molecule as a whole, so that molecules do not dimerise, as would be the case with most other free radicals. The delocalisation also gives rise to the deep violet colour, characterised by an absorption band in ethanol solution centred at about 517 nm. When a solution of DPPH is mixed with that of a substance that can donate a hydrogen atom, then this gives rise to the reduced form with the loss of this violet colour (although there would be expected to be a residual pale yellow colour from the picryl group still present). Representing the DPPH radical by Z   and the donor molecule by AH, the primary reaction is Where, ZH is the reduced form and A   is free radical produced in this first step. This latter radical will then undergo further reactions which control the overall stoichiometry, that is, the number of molecules of DPPH reduced (decolorised) by one molecule of the reductant. The reaction [1] is therefore intended to provide the link with the reactions taking place in an oxidising system, such as the auto-oxidation of a lipid or other unsaturated substance; the DPPH molecule Z   is thus intended to represent the free radicals formed in the system whose activity is suppressed by the substance AH 11 .

Collection and Identification of Plant Material
The plant material was collected from the Khari Baoli market, Old Delhi and was identified as Asparagus racemosus (Root). Famaly (liliaceae) by the Dr. Anjula Pandey (Taxonomist), National Bureau of Plant Genetic Resources (NBPGR), Pusa Campus, New Delhi. A voucher specimen (Specimen No: NHCP/NBPGR 2010-5/6650) is preserved in herbarium section of taxonomic deptt. of NBPGR, New Delhi and also in the Pharmacognosy laboratory, department of pharmacy, Ram-Eesh Institute of Vocational and Technical Education, Greater Noida, Uttar Pradesh.

Preparation of plant material
The air dried roots were coarsely powdered and extraction was carried out in Soxhlet apparatus with help of methanol as a solvent. The methanolic extract was stored at 4 o C.

Preparation of reagents
The 500 µM solution of DPPH was prepared by dissolving 23 mg of DPPH in 100 ml of methanol. TRIS [2-amino-2 (hydroxy methyl) propane 1-3di-ol] buffer (pH 7.4) was prepared by adding 0.605g of TRIS buffer in 30 ml of water and adding 0.33 ml of concentrated hydrochloric acid, diluted to 100 ml with distilled water. TRIS buffer prevents the sudden pH change during the preparation of test dilutions 12 .

Measurement of in vitro antioxidant activity
The antioxidant activity of the methanolic extract of A. racemosus was determined by using a method based on the reduction of methanolic solution of coloured-free radical 1, 1 diphenyl-1-2 picrylhydrazyl (DPPH). The radical scavenging activity of tested sample was expressed as an inhibition percentage. Butylated hydroxyl toluene was used as reference standard. In 5 ml volumetric flasks added 1 ml of DPPH solution, 0.5 ml of TRIS Buffer and 0.5 ml of final dilutions of different concentrations range prepared from A. racemosus (methanolic extract) stock solution and made up the volume to 5 ml with methanol. In same way prepared the control dilutions of DPPH, replacing 0.5 ml of prepared dilutions (the drug solution under investigation) with methanol. The absorbances of all the dilutions were taken after 30 minutes at λ max 517 nm using methanol as blank.

Statistical analysis
The percentage inhibition was calculated using: Where, Ac is absorbance of control; As is the absorbance of sample. IC 50 value (a concentration at 50% inhibition) was determined from the curve between percentage inhibition and concentration. All determinations were done in triplicate and the IC 50 value was calculated by using the equation of line 13 .

RESULTS AND DISCUSSION
The methnolic extract of A. racemosus tested for in vitro using DPPH showed moderate free radical scavenging activity, as evidenced by low IC 50 values. Fig. 1 depicted that the IC 50 value of A. racemosus was 4158.8 whereas butylated hydroxytoluene used as a standard showed an IC 50 of 46.25µg. The absorbance of sample (Methanolic extract of A. racemosus and standard Butylated hydroxytoluene) were taken in triplicate. With the increase of concentration, the decrease of absorbance value and the calculated percentage inhibition has shown with the help of tables.