INTRODUCTION:

The use of antioxidants therapy came into existence in the early 20^th century after the detail study on the cell aging at the molecular level with highly sophisticated equipment, procedures and skilled individuals. Antioxidants are compounds that help to inhibit the many oxidation reactions caused by free radicals thereby preventing or delaying damage to the cells and tissues of living organisms. Free radicals are highly reactive ions generated in the living body that damages the cell by “Absorbing” electrons from, nucleic acid, lipids, or any nearby molecule in a process called reduction. Antioxidants are important for food stability, human health and nutrition. They play major role in genesis of various diseases such as atherosclerosis, cancer, ageing, rheumatoid arthritis and inflammation. Plants provide a rich source of antioxidants which include tocopherols, vitamin C, Carotenoids and phenolic compounds. There are several other antioxidants namely olive oil phenolics, tocotrienols, oryzanol, squalene, sesame lignans, pycnogenol, flavonoids, isoflavones etc. which have been shown to possess health benefits¹. However, butylated hydroxylanisol (BHA) and butylated hydroxytoluene (BHT) are most commonly used synthetic antioxidants but both are suspected to cause liver damage^2,3. Therefore it is important to search safe and effective natural antioxidant by using various screening methods.

Bergenia ciliata is a perennial herb with stout rootstock, found in temperate Himalayas between altitudes 900-3000 meters. The drug possesses astringent, tonic, antisorbutic and laxative properties. Also, it is given in pulmonary affection, dysentery, ulcers, dysuria, spleen enlargement, cough and fever.

The acetone extract of rhizomes is cardio toxic in higher doses, and has depressant action on the central nervous system, with significant anti inflammatory activity, but the activity decreases on increasing dosage. In lower doses, the extract is mildly diuretic but in higher it exhibits anti diuretic action. The bruised rhizomes are applied in the eye diseases, boils, cuts and burns⁴. The phytochemistry and pharmacology about this leaf is not established. Literature survey of other species of Bergenia revealed that leaves contains flavonoids andrhizomes contains phenolic compounds chiefly – berginin^5,6. Rhizomes also reported to contain other phenolic compounds namely (+)- afzelechin, leucocyanidin , gallic acid , methyl gallate , catechin , catechin -7-O- β-D-glucopyranoside ,11- O- galloyl bergenin ; a lactone – Pashaanolactone and sterols viz. , sitoindoside I, β-sitosterol and β- sitosterol-D-glucoside^7,8.Literature survey revealed that no work has been done on the antioxidant activity of extractives/compounds against radical scavengers. Thus it was thought to carryout antioxidant activity of it various extracts against DPPH radical scavenging system.

MATERIALS AND METHODS:

Plant Material:

Bergenia ciliata leaves were collected in the month of June, from local forests of the Chota Shimla and were authenticated by Dr. Rakesh Kumar, Asst. Professor and head, Botany department, Dolphin Institute of Bio- Medical and Natural Sciences, Dehradun, Uttaranchal.

Preparation of Extractives:

Alcoholic Extraction:

Shade dried Bergenia ciliata leaves were powdered and extracted with 95% ethanol in a soxhlet extractor. The liquid extract was concentrated in a rotary flash evaporator. The residue was dried in a desiccator over sodium sulfite and the yield of extract was determined in table no. 01.

Table No.01: Percentage yield of various extracts of Bergenia ciliata leaves

Extract	Alcoholic	Successive Extraction
Extract	Alcoholic	Petroleum ether	Chloroform	Butanol	Ethyl acetate	Alcohol
Dry weight (% w/w)	22.58	2.74	2.06	10.98	0.6	4..5

Table No.02: Qualitative chemical analysis of various extracts of Bergenia ciliata leaves.

Tests	Alcoholic	Successive Extraction
Tests	Alcoholic	Petroleum ether	Chloroform	Butanol	Ethyl acetate	Alcohol
Alkaloids	-	-	-	-	-	-
Carbohydrates	+	-	-	+	+	+
Phenolic Compounds	+	-	-	+	+	+
Amino acids	+	-	-	+	+	+
Steroids	+	+	-	-	-	-
Triterpenoids	-	-	-	-	-	-
Saponins	-	-	-	-	-	-
Glycosides	+	-	-	+	+	+

+ Present, - Absent.

Table No.03: Antioxidant Activity of various extracts of various extracts of Bergenia ciliata leaves.

Samples	DPPH Radical Scavenging (%)	IC₅₀ values	Superoxide Scavenging (%)
Alcoholic Extract	55.93	51.85µg	67.34
Butanolic Fraction	62.80	40.50µg	72.78
Ethyl acetate Fraction	78.24	31.09µg	87.80
Standard	88.29	23.12µg	92.46

Successive extraction:

The plant material was air dried in shade, powdered was extracted subsequently with solvents of increasing polarity. The solvents were distilled under reduced pressure and yield of the extracts were determined in Table 01. All the extractives were subjected to phytochemical screening.

Phytochemical Screening of Different Extractives:

Various qualitative chemical tests for identifying phytoconstituents present were carried out on various extracts of Bergenia ciliata leaf. The presence of phytoconstituents was tabulated in the table no.02. Thin layer chromatographic (TLC) studies were carried out for various extracts to confirm their presence in the extract using silica gel G as a stationary phase and suitable mobile phase.

INVITRO ANTIOXIDANT ACTIVITY:

DPPH Radical Scavenging Method:

Alcoholic extract, butanolic fraction and ethyl acetate fraction were selected for the activity. Samples were prepared in different concentrations i.e. 10-70mgm/ml in AR grade methanol. The samples of above concentrations were mixed with 2ml of 90mM of DPPH prepared in AR grade methanol and make up the final volume up to 4ml with AR grade methanol. Methanol was used as negative control and BHT was used as positive control. The absorbance of the resulting solutions and the blank (with same chemicals except sample) were recorded after 60 minutes at room temperature, against BHT. The disappearance of DPPH was read spectrophotometrically at 517nm using a JASCO V 530 UV-Visible Spectrophotometer. Radical Scavenging Capacity (RSC) in percent was calculated by following equation.

RSC (%) =100 x A_{blank -} A_sample / A _blank

RSC = Radical Scavenging Capacity

A_{blank =} Absorbance of blank.

A_sample= Absorbance of sample.

From the obtained RSC values the IC₅₀were calculated, which represents the concentration of the scavenging compound that caused 50% neutralization^09,10.

Percentage scavenging capacity and IC₅₀of various extracts and standard were calculated by taking the mean of triplicate values and are mentioned in table no.03.

Xanthine/Xanthine Oxidase Superoxide Scavenging Method:

In this method the reaction mixture was prepared by dissolving sodium carbonate (0.53g), ETDA (0.004g) and xanthine (0.05g) in 100 ml distilled water and then 10ml NBT solution (0.025mM) was added. In 5mcL of each sample (50mg/ml), 995 mcL of reaction mixture and 0.1 mcL of xanthine oxidase solution were added. The solution was mixed and absorbance was measured at 560nm. The reaction mixture (1000mcL) and xanthine oxidase (0.1mcL) were used as negative control. Superoxide dismutase (5mcL), reaction mixture (995mcL) and xanthine oxidase were used as positive control.¹¹

RESULTS AND DISCUSSION:

In preliminary phytochemical screening, chemical examination of various extracts revealed the presence of phenolic compounds, steroids, amino acids and carbohydrates and their presence were substantiated by thin layer chromatographic studies. Three phenolic compounds (P-1, P-2, and P-3), a steroid and tannins were isolated.

Alcoholic extract, butanolic and ethyl acetate fraction were separately evaluated for antioxidant activity against DPPH radical scavenging and superoxide scavenging systems. The data (Table no. 03) indicate that ethyl acetate fraction exhibited highly significant antioxidant activity against free radical scavenging capacity of DPPH and superoxide scavenging with 72.24% and 87.80% inhibition respectively. Alcoholic extract and butanolic fraction also showed good antioxidant activity. However, the standard, BHT, was found to be potent in scavenging both DPPH and superoxide radical with 88.29% and 92.46% inhibition respectively. The data indicates that the ethyl acetate fraction is having promising antioxidant activity in both the systems and worth for further investigations.

REFRENCES:

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4. Anonymous. ‘The Wealth of India: A Dictionary of Indian Raw Materials and Industrial Products.’ Publication and Information Directorate, CSIR; New Delhi, 1952, 78-79.

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6. Kirtikar KR., Basu BD. Indian Medicinal Plants Text, International Distributors, Dehradun Vol. 2, 1995; 4th ed: pp. 278-279.

7. Anonymous. Indian Herbal Pharmacopoeia, Indian Drug Manufacturers Association, Mumbai, 2002; Revised New ed: 94-96.

8. Reddy UD, Chawla SA, Mundkinajeddu D, Singh D, Handa SS. High Pressure Liquid Chromatographic determination of bergenin and (+)- afzelechin from different parts of Paashaanbhed (Bergenia ligulata ). Phytochemical Analysis. 1998; 10(01): 44-47.

9. Dukic NM, Bozin B, Sokovic M, Mihajlovic B. Matavulj M. Antimicrobial and Antioxidant activities of three menthe species essential oils. Planta Medica. 2003; 69: 413-419.

10. Bios MS, Antioxidant determinations by the use of a stable free radical. Nature, 1958; 4617: 1199 -1200.

11. Chang WS, Lin CC, Chuang SC, Chiang HC. Superoxide anion scavenging effect of coumarins. Am J Chin Med. 1996; 24(01): 11-17.

Received on 06.12.2008 Modified on 23.02.2009

Research J. Pharm. and Tech. 3(2): April- June 2010; Page 399-401