Introduction

The role of free radicals and reactive oxygen species (ROS) in the pathogenesis of human diseases such as cancer, aging, inflammatory response syndrome, respiratory diseases and liver diseases is widely recognized¹. Electron acceptors such as molecular oxygen readily react with free radicals to become free radicals themselves. Prominent manifestation of free radical activity in biological systems is lipid peroxidation and it is involved in the development of different diseases².Research is focused on the use of anti-oxidants with particular emphasis on naturally derived anti-oxidants which inhibit ROS production and display protective effects. Plant phenolics, for example, Phenolic acids3,4, Tannins5,6 and flavonoids7 are known to be potent anti-oxidants and occur in vegetables, fruits, seeds and barks. It is suggested there is an inverse relationship between dietary intake of anti-oxidant rich food and the incidence of human disease. Therefore the research for the determination of natural antioxidant source is important8-14.

Dpph radical scavenging activity

DPPH (1,1-diphenyl-2picryl-hydrazyl) free radical scavenging activity of the test compounds was determined by the method of Lamaison et. al15., which depends on scavenging of coloured free radical (DPPH) in methanol solution by the test drugs. The reaction mixture contains DPPD and test drug in a final volume of 3 mL. Gallic acid was taken as reference standard. The concentrations of test extract and the reference employed are shown in Table and Fig.1.Absorption of DPPH at it’s adsorption maximum 516nm is inversely proportional to the concentration of the scavenger (Test drug). The activity was expressed as inhibitory concentration 50  (IC50) i.e. the concentration of the test solution required to give 50% reduction in absorbance of the test solution as compared to that of blank solution. The results are expressed in Table and Fig. 1.

Figure 1: DPPH radical scavenging activity.    Click here to View figure

Material and Methods

Plant material extraction

Gymnema sylvestre plant root was collected Tanjavur (India). The root was dried under shade and powdered in a mechanical grinder.
The root powder was extracted with 70% ethanol by cold maceration. The extraction was done for 72 hours. After extraction the extract was separated from marc by filtration through filter paper. The marc was pressed with muslin cloth to remove the solvent which is left in the marc after filtration.
The filtrate was preserved in a well closed container. The marc left after extraction was further extracted by cold maceration with the same solvent. And the process was repeated one more time.
That is the drug was extracted with a gap of 3 days each.

a, a–diphenyl-b-picryl hydrazyl, galic acid were obtained from Sigma Chemical Co. (St.Louis, MO, U.S.A.) All other  the chemicals used are of analytical grade and the solvents used for extraction are from Ranbaxy fine chemicals, New Delhi.

The UV Visible spectrophotometric values
were recorded employing Varian-Cary-50
UV Spectrophotometer.

Superoxide radical scavenging activity

Method: Nbt / riboflavin photoreduction (mccord and fridovich)

The superoxide free radical scavenging activity of the test extracts is determined by the method of Mccord and Fridovich which depends on light induced super oxide generation by riboflavin and the corresponding reaction of vitro blue tetrazolium (NBT).

The assay mixture contained different concentrations of test extracts and EDTA (6 mm containing 3 mg of NaCN), NBT (50 mm), riboflafin (2 mm) and phosphate buffer (58 mm, pH 7.8) to give a total volume of 3 ml. The tubes are uniform by illuminated for 15 min, and there after the absorbances are measured at 560 nm.

The percentages inhibition by the test drug of super oxide production was evaluated by comparing the absorbance values of standard and experimental tubes. Gallic acid in distilled water at different concentrations is used as standard for comparison. Table and Fig. 2 (Given below).

Figure 2: Superoxide radical scavenging activity.   Click here to View figure

Results and Discussion

The extracts showed promising free radical scavenging activity and also super oxide radical scavenging activity. In between Andrographis serpyllifolia root extract and Gymnema sylvestre root extract, Gymnema sylvestre showed much higher activity with Andrographis serpyllifolia root extract showing little scavenging activity nature.  A maximum percentage inhibition of 81.29 was observed with Gymnema sylvestre root extract at dose of 250 µg/ml.  The IC50 µg/ml for Gymnema sylvestre root extract is found to be 136.6, where as for Andrographis serpyllifolia root extract even at a concentration of more than 100 µg/ml. the percentage inhibition is found to be 14.59. only. In case of Gymnema sylvestre root extract not only a good inhibition but also there is found to be a linear response with increasing dose (Table and Fig. 1). The dose vs. response relationship could be expressed in the form of equation y=02834 n +11.272. A good correlation coefficient (R2= 0.9901) indicated a good linear relationship.

References

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