Free radical scavenging capacity of ethanolic extract of Pleiospermium Alatum leaves. School of pharmaceutical sciences,vels University Chennai-117.

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1 INTERNATIONAL JOURNAL OF FRONTIERS IN SCIENCE AND TECHNOLOGY Research Article ISSN Received On : ,Revised And Accepted On : Free radical scavenging capacity of ethanolic extract of Pleiospermium Alatum leaves ABSTRACT R Elumalai* and V.Ravichandiran School of pharmaceutical sciences,vels University Chennai-117. The ethanolic extract of the leaves of Pleiospermium Alatum was subjected to investigate its free radical scavenging capacity. The phytochemical screening demonstrated the presence of different types of compound like, tannins, proteins, alkaloids and phenolic compounds. The ethanolic extract of the plant was subjected to high pressure liquid chromatography, which confirms the presence of phenolic compounds Rutin and gallic acid. Phenolic compounds containing free hydrogen are largely responsible for antioxidant activity. The ethanolic extract of the plant was tested for antioxidant activity using scavenging activity of DPPH(1,1-diphenyl-2- picrylhydrazil) radical method and Nitric oxide method. The extract exhibited high free radical scavenging activity. IC50 was found to be significant to standard antioxidant Ascorbic acid. Keywords: Pleiospermium Alatum, EEPA, HPLC, DPPH, NO,Anti-oxidants assay. Corresponding Author:R Elumalai .id: sasimalai1965@gmail.com Introduction: Traditional herbal medicines are those which are naturally occurring, they have minimal or no industrial or synthetic manufacture that have been used to prevent, diagnose and treat illness within local or regional healing practices. Traditional herbal medicines are being brought forth to global vision by various global health debates. Herbal medicines are also known as botanical medicines or phytomedicines; these refer to the use of plant s seeds, berries, roots, leaves, bark or flowers for medicinal purposes. There July Sep 2013 Volume Issue 3 Page 114

2 are a variety of archeological evidences to support the fact that primitive man used plants and herbs for medicinal purposes. Man has been using herbals from the time immemorial for the cure and prevention of various diseases. It is evolved from recorded in books of folklore which require evidence of clinical efficacy and from books of medicinal practitioners of ancient times. Though the modern era has abundance and multiple scores of advanced synthetic drugs, a significant proportion of the population of developing and developed countries are still depending on herbal medicines for health care, prevention and cure of various diseases. At present various governments are collecting information on the collection, cultivation and use of these traditional medicines in a predetermined methodological approach and are officially publishing them as herbal pharmacopoeias. A survey taken by the world health organization states that a total of 4 billion people, almost 80% of the world population utilize herbal drugs for health care either directly or indirectly. The herbal drugs are being utilized by various systems of medicines viz., homeopathy, ayurveda, siddha, unani, naturopathy, traditional oriental, native American Indian medicines, Chinese system of medicines, etc., Most pharmaceutical companies are currently conducting extensive research on plant materials gathered from the forest and cultivated varieties of herbal medicines for their potential medicinal values (1). Pleiospermium alatum (Wight and Arn.) Swingle (Syn. Limonia alata) belonging to Rutaceae is a small tree native of India and Sri Lanka. Rutaceae family which includes citrus fruits is a known source of essential oils. In India it is distributed in the dry deciduous forests of July sep 2013 Volume Issue 3 Page 115

3 Karnataka, Tamil Nadu and Kerala regions of Western Ghats (2,3). Flowering and fruiting usually occurs in October. The leaves and bark are used for the fomentation of rheumatic pain; the dried fruit is useful in malignant and pestilent fevers and is used as an antidote for poisons (4), inflammation and pain management (5).The folklore claim suggests that the leaf is showing wound healing property. The aim of this present study to investigate the free radical scavenging capacity of ethanolic extracts of pleiosperrmium alatum leaves. 2. Materials and methods: 2.1 Collection of Plant Materials The plant materials (leaves) of Pleiospermium alatum (Wight & Arn.)Swingle were collected from the Jamnamaradhur forest vicinity, of Tiruvannamalai district. The collected plant materials were botanically identified and confirmed by the Botanist Dr. A. C. Tangavelou, Director, Bio-Science Research Foundation, Pondicherry. The herbarium specimen was prepared and deposited at Bio-Science Research Foundation, Pondicherry, for future reference. 2.2 Materials and reagents required: DPPH, Acetonitrile of HPLC grade was from Merck (Darmstadt, Germany); distilled water and HPLC grade methanol were used in the analysis. Other reagents were of analytical grade. Gallic acid (Lot: ), corilagin (Lot: ) and rutin (Lot: ) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products. Methyl brevifolincarboxylate and ellagic acid (purity > 98%) were isolated in our laboratory, and their purity and structures were confirmed by HPLC and by comparison of spectral data to those published in the literature. July sep 2013 Volume Issue 3 Page 116

4 2.3 Preparation of the Extracts The collected materials were shade-dried, and coarsely powdered using a pulverizor. The coarse powders were extracted with 80% ethanol by Soxhlet method hydro-alcoholic extraction by Soxhlet method (6). The extracts were collected and distilled off on a water bath at atmospheric pressure and the last trace of the solvents was removed in vacuo. The resulted extracts were used for preliminary phytochemical screening and free radical scavenging capacity studies. 2.4 HPLC Instrumentation and Chromatographic Conditions All analysis was performed using an Agilent 1200 series HPLC system equipped with a quaternary pump, a vacuum degasser, and data analysis was carried out by spinchrom software. The separation was carried out on a Diamonsil C18 (2) column (150 mm 4.6 mm i.d., 5 μm) under the following chromatographic conditions: Sample injection volume : 20 μl Column temperature : 25 C Flow rate Mobile phase : 1.0 ml/min : Acetonitrile and 0.1% aqueous phosphoric acid (v/v). A gradient program was used according to the following profile: 0 20 min, 2 10% acetonitrile; min, 10 14% acetonitrile; min, 14 17% acetonitrile; min, 17 30%; min, 30 50% acetonitrile. The wavelength of UV detection was set at 274 nm. (7) July sep 2013 Volume Issue 3 Page 117

5 Sample Preparation The ethanolic extract of Pleiospermium Alatum was filtered through a 0.45 μm Millipore membrane prior to HPLC analysis. 2.5 DPPH radical scavenging activity About 0.1 mm solution of DPPH in methanol was prepared and 1 ml of this solution was added to 3 ml of the different concentration ( µg/ml) of ethanol extract and control (without the test compound, but with an equivalent amount of methanol) in different test tubes. The mixture was shaken and allowed to stand at room temperature for 30 min and the absorbance was measured at 517 nm using a spectrophotometer.(8) % Inhibition= (Abs Control Abs Sample ) x 100/ Abs Control Where Abs Control is absorbance of control at time = 0 and Abs Sample is absorbance of test sample. The IC 50 Value for extracts was also calculated. The results are mentioned in table. 2.6 Nitric oxide scavenging assay Nitric oxide scavenging assay was measured by spectrophotometric,method described was mixed with different concentration of extract and fraction dissolved in methanol and incubated at 250c for 30 min. After 30 min 1.5 ml of the incubation solution were removed and diluted with 1.5 ml of modified Griess reagent (sulphanilic acid with naphylethylene diamine dichloride in acetic acid). The absorbance was measured at 546 nm.(9) % Inhibition = [Abs Control Abs Sample ] /Abs control x100 July sep 2013 Volume Issue 3 Page 118

6 3. Results: 3.1 Phytochemical analysis: The various extract of Pleiospermium alatum leaf is subjected to the phytochemical investigation [15].The extract indicates the presence of Alkaloids, Flavonoids, Glycosides, Tannin, Carbohydrates, terpenoids and proteins. 3.2 High performance liquid chromatography: HPLC chromatograms from the leaf of pleiospermium alatum showed 4 components. The retension time of the peaks was found to be 1.887,2.44, 5.89,7.6 respectively. In the present investigation, 2 flavonoids were quantified at 254 nm using peak area by comparison to a calibration curve derived from the standard rutin (0.25mg/ml), gallic acid (0.25mg/ml) and the retension time of the ethanolic extract of pleiospermium alatum given good agreement with standard Rutin and gallic acid. Figure: 1 HPLC chromatogram of ethanolic extract of Pleiospermium Alatum leaves July sep 2013 Volume Issue 3 Page 119

7 Figure: 2 HPLC of ethanolic extract of Pleiospermium Alatum leaves Compared with Standard Rutin and Gallic acid 3.3 Effect of Pleiospermium alatum leaf in DPPH radical scavenging assay: Figure 4 depicts the free radical scavenging capacity of EEPA using DPPH generated radical in in-vitro. It was observed that increase in % inhibition of free radicals has observed in increasing concentration of EEPA. The IC 50 values of EEPA were found to be 510µg/ml. All the fractions was compared with the standard ascorbic acid IC µg/ml. Effect of Pleiospermium alatum leaf in DPPH radical scavenging assay: July sep 2013 Volume Issue 3 Page 120

8 3.4 Effect of Pleiospermium alatum leaf in Nitric oxide scavenging assay: Figure depicts the ability of EEPA to quench NO radicals was tested in-vitro. The results indicate that EEPA were exhibited IC 50 values of 16.5 µg/ml and the extract was compared with standard vit C value of 19.5µg/ml. Effect of Pleiospermium alatum leaf in Nitric oxide scavenging assay: 4. Discussion and conclusion: Antioxidant is a chemical compound that works to protect the body from cell damage by inhibiting oxidation, rendering free radicals harmless. Antioxidants slow down, prevent and treat degenerative diseases and aging by scavenging free radicals. Antioxidant includes enzymatic antioxidants and non-enzymatic antioxidants. Plants and their products have been used for many years for human health. There are still many plants which have various medicinal values but still not explored and used. Plants contain many novel compounds with medicinal values which need scientific exploration. The free radicals are produced in aerobic cells due to consumption of oxygen in cell growth [10,11]. Free radicals cause decrease in membrane fluidity, loss of enzyme receptor activity and damage to membrane protein leading to death [12]. These free radicals are involved in different disorders like ageing, cancer, cardiovascular disease, diabetes, rheumatoid July sep 2013 Volume Issue 3 Page 121

9 arthritis, epilepsy & degradation of essential fatty acids [13] Antioxidant helps in treatment of above disorders. As the ethanolic extract of Pleiospermium alatum leaf extract showed the presence of dose dependent antioxidant activity comparable with standard antioxidant Ascorbic acid. Generally the phenolic compounds are responsible for free radical scavenging activity, the pleiospermium alatum also shows the presence of phenolic compounds like Rutin and gallic acid. In conclusion, the ethanolic extract of Pleiospermium alatum leaves shows the presence of flavonoids, alkaloids,tannins,proteins in the preliminary phytochemical analysis. Further the presence of flavonoids was confirmed by using high pressure liquid chromatography, compared with standard phenolic compounds Rutin and gallic acids. The ethanolic extract of Pleiospermium alatum shows the significant free radical scavenging capacity. The further studies are warranted to do with In-vivo models. Reference: 1. Salim Bastaki, Diabetes mellitus and its treatment, Int J Diabetes & Metabolism (2005) 13: M.T. Fauvel and B.R. Ramesh, Rutaceae of Peninsular India: Vegetative key, distribution and uses. J. Econ. Tax. Bot., 13, (1989). 3. K.M. Mathew,The Flora of Tamil Nadu, Carnatic, Part 1. p. 213, The Rapinat Herbarium, St. Joseph s College, Tiruchirapalli, India (1983). 4. Anonymous, Indian Pharmacopoeia. Government of India, Ministry of Health &Family Welfare, Published by The Controller of Publications, New Delhi, Vol. II,3rd Ed. Appendix IV , The Wealth of India, Raw Materials, Council of Scientific and Industrial Research, New Delhi, Vol. VI, Kokate CK, Purohit AP and Gokhale SB: Textbook of Pharmacognosy, Nirali Prakashan, 21st Ed July sep 2013 Volume Issue 3 Page 122

10 7. Rajalakshmi PV, Senthil KK. Direct HPLC analysis of quercetin in exudates of Abutilon indicum (Linn). Malvaceae. J Pharm Sci Technol 2009; 1(2): Sankaradoss Nirmala, KF Haja Nazeer ahamed, Velayudem Ravichandiran Comparative in vitro study on the free radical scavenging capacity of tannin and biflavone fraction from Ficus racemosa Linn and Araucaria bidwilli Hook International Journal of Chem Tech Research Vol. 3, No.3, pp Md. Sarfaraj Hussain, K. F. H Nazeer Ahamed, V. Ravichandiran, Md. Zaheen Hassan Ansari, Evaluation of in vitro free radical scavenging potential of different fractions of Hygrophila auriculata (K. Schum) Heine., Asian Journal of Traditional Medicines, 2009, 4 (5) 10. Barros L, Ferreira MJ, Queiros B, Ferreira ICFR & Baptista P, Total phenols, ascorbic acid, b-carotene and lycopene in Portuguese wild edible mushrooms and their antioxidant activities. Food Chemistry, 103( 2007) Singh R, Singh S, Kumar S & Arora S, Studies on antioxidant potential of methanol extract/fractions of Acacia auriculiformis, A. Cunn. Food Chemistry, 103 (2007) Li X M, Li X L & Zhou AG, Evaluation of antioxidant activity of the polysaccharides extracted from Lycium barbarum fruits in vitro. European Polymer Journal, 43 (2007) Bondet V, Brand-Williams W & Berset C, Kinetics and Mechanisms of antioxidant activity using the DPPH free radical method. Lebensm.-Wiss. u.-technol., 30, (1997) 609. July sep 2013 Volume Issue 3 Page 123

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