INTRODUCTION:

Oxidation is a basic part of aerobic life and our metabolism. During oxidation, many free radicals are produced which have an unpaired electron, nascent electron. Atoms of oxygen or nitrogen having central unpaired electron, are called reactive oxygen or nitrogen species ^1-4. Nitric oxide (NO) is an important chemical mediator generated by endothelial cells, macrophages, neurons and is involved in the regulation of various physiological processes. Excess concentration of nitric oxide is implicated in the cytotoxic effects observed in various disorders like AIDS, cancer, alzheimer’s and arthritis ⁵. This may be harmful to the body and may cause peroxidation of membrane lipids, aggression of tissue membranes and proteins or damage to DNA and enzyme ⁶. These can be related to some pathology, such as arthritis, haemorrhagic shock, coronary artery diseases, cataract, cancer, AIDS as well as age related degenerative brain diseases ⁷.

Overproduction of NO can mediate toxic effects, for eg. DNA fragmentation, cell damage and neuronal cell death⁸. NO does not interact with the bioorganic macromolecules such as the DNA or proteins directly.

However, in the aerobic conditions, the NO molecule is very unstable and reacts with the oxygen to produce intermediates such as NO₂, N₂O₄, N₃O₄ the stable products nitrate, nitrite^9,10 and peroxynitrite when reacted with superoxide¹¹.The production of these reactive species in healthy organism is approximately balanced by antioxidant defense systems. Currently, there’s a great interest in the study of antioxidant substances mainly due to the findings of the therapeutic effects of free radical scavengers on the organisms. The antioxidant activity of phenolic plays an important role in the adsorption or neutralization of free radicals¹². Antioxidant agents of natural origin have attracted special interest as they can protect human body from free radicals^{13, 14}. Antioxidants work in several ways like they may stop the free radical from forming in the first place, or interrupt an oxidizing chain reaction to minimize the damage caused by free radicals. Reactive oxygen species, especially OH radical, play a major role in oxidative damage of gastric mucosa in almost all forms of gastric ulcer^15,16. Increased lipid per oxidation, increased protein carbonyl content and decreased level of endogenous GSH are the characteristic features of OH radical –mediated oxidative damage of the gastric mucosa during ulceration^16-18. Inhibition of the release of free radicals is a potential strategy to control inflammation and is implicated in the mechanism of action of a number of anti-inflammatory drugs including the representative ones like dexamethasone¹⁹.

MATERIAL AND METHODS:

Phytochemical evaluation:

Plant material:

The leaves and stem of E. laevis were collected from Pune; Maharashtra, India during the month of July. The taxonomic identification is accomplished with the help of flora of Bombay Presidency ²⁰and Flora of Maharashtra ²¹ for identification. It was identified and authenticated at Botanical Survey of India, Pune, Maharashtra, India. Its voucher number is BSI / WC / Tech / 2006 /185.

Extraction Protocol using 80% Ethanol:

Air shade dried and pulverized material (0.100 g) of leaves and stem was used separately. Each material was triturated using ethanol (10 ml, 80%), centrifuged for 20 minutes and filtered. These extracts (A and B respectively) were evaporated to dryness in vaccum using a rotary evaporator and employed for the assessment of antioxidant capacities.

Extraction Protocol of Methanol Extract of Leaves:

Air shade dried and pulverized material (60.0 g) of leaves was extracted successfully with methanol (360 ml, 1:6 w/v) by keeping it for seventy two hours at room temperature and filtered. The solvent was evaporated to dryness in vacuum using a rotary evaporator to yield thick viscous mass (5.8 %). This methanol extract (C) was used for the assessment of antioxidant capacity.

Fractionation of methanol extract:

Broad fractionation of the crude extract (C, 15g) by column chromatography was carried out employing gradient polarity of solvents (Hexane to methanol) using silica gel (60- 120, 200g). Total five fractions were collected.

Hexane fraction (D) was further rechromatograhed. Fraction (D, 12g) was rechromatograhed using silica gel (60- 120, 160g). It was fractioned using gradient polarity of solvents (Hexane to ethanol). Total eight fractions were collected. Fraction (E, 5% ethyl acetate - hexane) was composed of other unidentified compounds which, on further purification gives pure compound, aromatic ester.

Nitric Oxide scavenging activity:

Nitric oxide scavenging activity was measured according to the standard method ²². The absorbance was measured at 546 nm. The free radical scavenging activity (% antiradical activity) was calculated using the equation:

% Antiradical Activity =

Control Absorbance - Sample Absorbance × 100

Control Absorbance

IC ₅₀value is used to measure the free radical scavenging activity. Each experiment was carried out in triplicates.

RESULTS AND DISCUSSION:

The comparison of antioxidant activity of stem and leaves, prepared in 80 % ethanol, reveals that activity of leaves (A) is higher than that of stem (B) Fig. 1. The IC ₅₀ values of methanol extract and its fractions by Nitric Oxide method Table 1, are found to be more potent in case of extract (C) and fraction (E) Fig. 2. Each value is mean ± SD of three measurements.

Table 1- IC ₅₀ Values of E. laevis

Extracts	Anti-oxidant Capacity ( IC₅₀ µg/ml ) Nitric Oxide Assay
Standard	17.80± 0.05
A	37.03± 0.08
B	90.7575± 1.02
C	9.453± 0,09
D	25.44± 1.00
E	10.43± 1.04

Fig 1 Comparative Study of IC 50 Values of Leaves and Stem of E. laevis

Fig 2 Comparative Study of IC 50 Values of Methanol Extract and Its Fractions

STATISTICAL ANALYSIS:

Results are expressed as the mean ± S.D. of three independent experiments. Student’s t-test was used for statistical analyses; P values > 0.05 were considered to be significant.

CONCLUSION:

This study indicates that the extracts obtained from the aerial parts of T. stans have significant free radical scavenging activity and high reactive hydroxyl radical. Since reactive oxygen species are important contributors to several serious ailments, aerial parts of T. stans might be useful for the development of novel and more potent natural antioxidant.

ACKNOWLEDGEMENT:

Authors are thankful to the Principal S. P. College Pune and the Head, Department of Chemistry, S. P. College, Pune, Maharashtra, India for providing the necessary laboratory facilities for the work.

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Received on 18.09.2011 Modified on 11.10.2011

Asian J. Research Chem. 4(12): Dec., 2011; Page 1864-1866