INTRODUCTION:

There has been growing interest in the investigation of natural products from plants, for the discovery of new antioxidant agents as an alternative route for the substitution of synthetic chemicals, side effects of which are always in question. For this many plants extracts have been screened for antioxidant activities¹. In living system free radicals are generated as part of the body’s normal metabolic process and oxidation is a basic part of the metabolic process. During oxidation many free radicals are produced which have an unpaired, nascent electron. Atoms of oxygen (or) nitrogen having central unpaired electron are called reactive oxygen (or) nitrogen species. 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 enzyme². Currently there is a great interest in the study of antioxidant substances mainly due to therapeutic effects of the free radical scavengers on the living organs. The antioxidant activity of phenolics plays an important role in the adsorption or neutralization of free radicals, due to its redox potential, which allow them to act as reducing agents, hydrogen donors and single let oxygen quenchers³.

Rumex vesicarius L. is an annual herb, belongs to family polygonaceae. Rumex vesicarius L. occupies a special significance due to its several beneficial uses. It is commonly known as “Bladder dock”, grows up to 15-30cm height, monoecious, glabrous, pale green herb, leaves: elliptic, ovate (or) oblong, 2.5-7.5cm long, base cuneate, hastate (or) cordate. Flowers: pedicelled, terminal and leaf- opposed racemes. The plant is widely cultivated as green leafy vegetable in many parts of India. The leaves are used as asperient, diuretic, and considered as antitode for snake venom. In unani system of medicine the herb is used as analgesic, astringent, antiulcer, hepatoprotective agent and is useful in scabies, leucoderma, toothache, asthma, heart troubles, tumours, and scurvy^4-8.

In the present study we have evaluated the antioxidant potential of hexane, chloroform, ethylacetate, ethanol and aqueous extracts of Rumex vesicarius L. The evaluation of antioxidant activity was performed in vitro by Nitric oxide radical scavenging method and total antioxidant capacity.

MATERIALS AND METHODS:

Plant material:

Rumex vesicarius L. was collected from plains of Tiruvannamalai, Tiruvannamalai District, Tamilnadu, India and Taxonomically identified by Dr.G.V.S.Murthy, Scientist “F”, BSI, South regional centre, Coimbatore, India. The voucher specimen has been retained in Botany Department, Government Arts College (Autonomous), Kumbakonam, Tamilnadu, India. The collected material was shade dried and powered with mechanical griender, and well preserved for further use. The dried powder material was subjected to extraction with various solvents of increasing polarities such as hexane, chloroform, ethylacetate and ethanol by soxhlet apparatus and the aqueous extract was prepared by maceration method⁹. The extracts were filtered using Whatmann number 1 filter paper and then concentrated in vacuum and dried.

Chemicals:

The sodium nitroprusside (SNP), griess reagent, and Quercitin were purchased from Sisco research laborites Pvt. Ltd., India. All other chemicals and solvents used were of analytical grade.

Preliminary phytochemical tests:

The preliminary phytochemical screening¹⁰ of all the extracts were carried out to know the different constituents present in Rumex vesicarius L. as per the standard procedure.

Nitric oxide generation and assay of Nitric oxide scavenging method:

Nitric oxide (NO) was generated from sodium nitroprusside (SNP) and was measured by the Griess reagent. SNP in aqueous solution at physiological pH spontaneously generates NO¹¹, which interacts with oxygen to produce nitrite ions that can be estimated by the use of Griess reagent. Scavengers of NO compete with oxygen leading to reduce production of NO. SNP (3ml) in phosphate buffer saline (PBS) was mixed with different concentrations of extracts (1.5-1000µg/ml) of the drug dissolved in respective solvents and incubated at 25⁰C for 150minutes. The samples from the above were reacted with Griess reagent (1% Sulphanilamide, 0.1% napthylethyienediamine dichloride and 3% phosphoric acid) the absorbance of the chromophores formed during the diazotization of nitrite with sulphanilamide and subsequent coupling with napthylethylenediamine dichloride was read at 546nm and referred to the absorbance of quercitin used as positive control treated in the same way with Griess reagent. The experiments were repeated in triplicates. The percentage scavenging of nitric oxide radical activity was calculated by using the below formula

Nitric oxide Inhibition % = (A control – A test) x 100

A control

Where A control = Absorbance of control reaction, A test = Absorbance in the presence of the sample of extracts.

The Ic ₅₀ values were determined as the concentration of the test mixture that gave 50% reduction in the absorbance from control blank. The experiments were repeated in triplicates. Quercitin was used as standard positive antioxidant.

Determination of total antioxidant capacity:

The total antioxidant activity of Rumex vesicarius L. extracts was estimated by the phosphomolybdenum method according to the procedure of Prieto¹². The assay is based on the reduction of Mo (VI) to Mo (V) by the extract and subsequent formation of a green phosphate (or) Mo (V) complex at acidic pH. An aliquot 3ml of sample or Vitamin E (equivalent to 500µg) was combined with 3ml of reagent solution (0.6m sulfuric acid, 28mM sodium phosphate and 4mM ammonium molybdate). In case of blank, methanol was used in place of sample. The tubes containing the reaction solutions were capped and incubated in a boiling water bath at 95⁰C for 60-90min. samples were cooled to room temperature the absorbance of the aqueous solution of each was measured at 695nm against the blank in a Perkin Elmer-UV-Visible Spectrophotometer. Vitamin E was used as standard antioxidant; the total antioxidant activity is expressed as the number of equivalents of vitamin E.

RESULTS AND DISCUSSION:

Nitric oxide (NO) is a potent pleotropic inhibitor of physiological process such as smooth muscle relaxation, neuronal signaling, inhibition of platelet aggregation and regulation of cell mediated toxicity. It is a diffusible free radical that plays many roles as an effectors molecule in diverse biological systems including neuronal messenger, vasodilatation, antimicrobial and antitumor activities¹³.

The preliminary phytochemical screening of Rumex vesicarius L. extracts revealed the presence of various bioactive components of phenols and flavonoids were most predominant and the results of phytochemical test has been summarized in the Table 1. The phenolic compounds and flavonoids are associated with antioxidative action in biological systems, acting as scavengers of singlet oxygen and free radicals¹⁴.

Table 1: Preliminary Phytochemical screening of Rumex vesicarius L.

Phytoconstituents	n-Hexane	Ethy lacetate	Chlor oform	Eth anol	Wa ter
Phenols	+++	+++	+++	+++	+++
Tannins	+	+	+	+	+
Flavonoids	+	++	+	+	+
Saponins	-	-	-	+	+++
Triterpenoids	-	-	-	++	-
Anthraquinones	-	-	-	+	-
Quinones	-	-	-	+	+

The nitric oxide scavenging activity suppressed the release of NO may be partially attributed to the direct NO scavenging, as the extracts of Rumex vesicarius L. decreased the amount of nitrate generated from the decomposition of SNP in vitro. The scavenging of NO by the extracts was increased in dose dependent manner. Figure1, Illustrates a significant decrease in the NO radical due to the scavenging ability of extracts and positive standard Quercitin. Of all the five extracts analyzed and compared the ethanol extract showed the maximum inhibition activity of 76.86% followed by ethylacetate extract with 75.52%, aqueous extract with 62.94%, chloroform extract with 61.93% while the hexane extract showed a minimum of 31.92% inhibition respectively at 1000µg/ml concentration. The standard querictin exhibited 81.67% of inhibition activity at same concentration. The IC₅₀values of the extracts were 153.72 for ethanol, 151.04 for ethylacetate, 125.88 for aqueous, 123.86 for chloroform and hexane with 117.9, the IC₅₀ of quericitin is 192.28 respectively. Of all the five extracts the ethanol extract showed a significant Nitric oxide activity on compared to other extracts.

The total antioxidant capacity of the extracts is given in figure 2. Total antioxidant capacity of Rumex vesicarius L. is expressed as the number of equvivalents of vitamin E. The phosphomolybdenum method was based on the reduction of Mo (VI) to Mo (V) by the antioxidant compound and the formation of green phosphate or Mo (V) complex with a maximal absorption at 695nm. The assay is successfully used to quantify total antioxidants in the plant extracts. The assay is a quantitative one since the activity is expressed as numbers of equivalents of vitamin E. On comparing the total antioxidant activity to standard antioxidant vitamin “E”, the study reveals that the antioxidant activity of the extracts exhibited increasing trend with the increasing concentration of the plant extract. While the standard antioxidant showed a reversal trend as the concentration of the standard antioxidant increased from 1.5µg/ml to 1000µg/ml the activity decreased from 90.14% to 4.64% as the concentration increased the activity decreased.

Figure 1: Nitric oxide free radical scavenging activity of Rumex vesicarius L. and Quercitin.

Figure 2: Total antioxidant capacity of Rumex vesicarius L. and Vitamin “E”.

Statistical Analysis:

All the treatments were performed in triplicates and each data point in the results is the mean of three replicates. All experiments were repeated at least once. The statistical significance of a treatment effect were expressed as mean + SEM.

CONCLUSION:

The ethanol and ethyl acetate extracts of Rumex vesicarius L. exhibited significant inhibition activity on compare to standard quercitin. The activity may be related to the phenols and flavonoid compounds present in the plant extract. Since reactive oxygen species are important contributors to several serious ailments. In the present study the observed NO scavenging activity of the extracts of Rumex vesicarius L. might be useful for the development of newer and more potent natural antioxidants.

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Received on 12.05.2011 Modified on 20.05.2011

Asian J. Research Chem. 4(9): Sept, 2011; Page 1482-1484