In vitro antioxident and free radical scavenging studies of Carissa Sp

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K. Sumathi et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-41 2016 108-111 108 IJAMSCR |Volume 4 | Issue 1 | Jan – Mar - 2016 Research article Pharmacological research In vitro antioxident and free radical scavenging studies of Carissa Spinarum L K.Sumathi Dr.N.Senthil Kumar J.K.K.M.M.R.F’s A.J.K.K. Sampooraniammal College of Pharmacy Komarapalayam Tamil Nadu 638183 Corresponding author: K. Sumathi Email: ABSTRACT Free radicals are implicated for many diseases including cancer diabetes mellitus arthritis ageing etc. In the treatment of these diseases antioxidant therapy has gained very importance. Various extract of Carissa spinarum L was studied for its in vitro antioxidant activity used different models viz. ABTS radical scavenging DPPH radical scavenging H2O2 Hydroxyl radical lipid peroxidation and Super oxide. The results were analyzed statistically by the regression methods. The scavenging effect of plant extracts and standard L.ascorbic acid on the various scavenging methods in the following order L-ascorbic acid EthanolEthyl acetateChloroformPet ether. Its antioxidant activity was estimated by IC50 value and the values a re 102.6 to 244.2 μg/ml ABTSradical scavenging 200.41 to354.42 μg/ml DPPH radical scavenging 152.31to 236.12 μg/ml H2O2 188.74 to 296.41 μg/ml Hydroxyl radical 142.12 to291.41 μg/ml Lipid peroxidation and 157.42 to 306.16 μg/ml Super oxide. The results clearly indicate that Carissa spinarum L has a significant potential to use as a natural antioxidant agent. Keywords: ABTS DPPH H2 O2 Hydroxyl radical Lipid peroxidation Super oxide Carissa spinarum.L INTRODUCTION According to WHO 1993 80 of the world’s population is dependent on the traditional medicines and a major part of the traditional therapies involves the use of plant extract or their bioactive compounds Mathur A 2011.Plant – based natural constituents can be derived from any part of plant Gordon M C and David 2001.Medicinal herbs constitute effective sources of antimicrobial anticancer and antioxidant natural products Calixto B.J. 2000. Medicinal herbs are an important source for the therapeutic remedies of various ailments Doss A. and S.P. Anand 2012.. Antioxidants are substances that prevent damage to cells caused by free radicals and search for free radicals lend them electron which stabilizes the molecule thus preventing damage to other cells. Antioxidants also turn free radicals into waste by products and they eventually get eliminated from the body. They also have the ability to repair previous damage to cells. These antioxidants are found naturally in fruits and vegetables. C spinarum L Apocynace is a shrub growing 2 to 3 meters high. Branches are numerous rigid and spreading with 2 straight simple or forked thorns ISSN:2347-6567 International Journal of Allied Medical Sciences and Clinical Research IJAMSCR

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K. Sumathi et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-41 2016 108-111 109 up to 5 centimeters in length on the axils and nodes. Leaves are smooth ovate or oblong-ovate 4 to 7.5 centimeters in length 2.5 to 4 centimeters in width rounded or notched at the base and blunt tipped. Flowers are fragrant white or pale rose- colored clustered in twos or threes Malik SK 2010. Calyx-segments are very slender pointed and hairy.Maheswari R 2012 Corolla tube is about 2 centimeters long smooth with a swollen throat and hairy lobes the lobes being lanceolate pointed spreading and about half as long as the tube. Fruit is a drupe broadly ovoid 1.5 to 2.5 centimeters long bluntly pointed and blackish or reddish-purple containing 2 to 4 small flat seeds. Pulp is reddish-purple and sour. The purpose of this study was to evaluate Carissa spinarum L as a potential source of natural antioxidants. MATERIAL AND METHODS All chemicals and solvents were of analytically grade and were of obtained from Ranbaxy Fine Chemicals Mumbai India. Plant material The fresh aerial parts of Carissa spinarum L Apocynaceae were collected from the local region of Bhavani Tamil nadu India in November 2011 and were authenticated by Prof.P. Jayaraman Ph.D Director Plant Anatomy Research CenterChennai- 5. Plant extract About 500 g of the crude powder was taken and extracted in a soxhelet extractor with various solvents such as petroleum ether chloroform ethyl acetate and ethanol 2 Lit.. The crude extract was concentrated to dryness in rotary flash evaporator under reduced pressure and controlled temperature 40-50 0 C. The extract was preserved in vacuum desiccators for subsequent use in study. ABTS radical scavenging assay To the reaction mixture containing 0.3 ml of ABTS radical 1.7 ml phosphate buffer and 0.5 ml extract was added at various concentrations from 2 to 500 μg/ml. Blank was carried out without drugs. Absorbance of recorded at 734 nm. Experiment was performed in triplicate Sreejayan 1996 John 1984. DPPH radical scavenging assay To the methanolic solution of DPPH 1 mM an equal volume of the extract dissolved in alcohol was added at various concentrations from 2 to 1000 μg/ml in a final volume of 1.0 ml. an equal amount of alcohol was added to the control. After 20 min absorbance was recorded at 517 nm. Experiment was performed in triplicate Sreejayan 1996 John 1984. The scavenging activity of extract towards Hydrogen peroxide radicals was determined by this method Subin Mary Zachariah et al 2012. 2ml of hydrogen peroxide 40mM was prepared in phosphate buffer pH 7.4 and 1.0 ml of methanolic sample 5-1000µg/ml of extract of plant /standard ascorbic acid was added to hydrogen peroxide solution. The absorbance of hydrogen peroxide at 230nm was determined after 10 minutes against a blank solution containing phosphate buffer without hydrogen peroxide. The experiment was repeated in triplicate. Hydroxyl radical scavenging activity. Various concentration of the extract / Standard of ascorbic acid 5-1000µg/ml or standards in 0.5 ml of distilled DMSO were added to a solution mixture containing 0.5 ml of ferric chloride 0.1 mM 0.5 ml of EDTA 0.1 mM 0.5 ml of ascorbic acid 0.1 mM 0.5 ml of hydrogen peroxide 2 mM and 0.5 ml of p-NDA 0.01 mM in phosphate buffer pH 7.4 20 mM to produce a final volume of 3 ml. Absorbance was measured spectrophoto metrically at 440 nm. All the measurements were carried out triplicate. Babu et al. 2001 Lipid peroxidation assay The mixture Egg phosphatidylcholine in 5 ml saline was sonicated to get a homogeneous suspension of liposome. Lipid peroxidation was initiated by adding 0.05 mM ascorbic acid to a mixture containing liposome 0.1 ml. The pink chromogen was extract with a constant volume of n-butanol and absorbance of the upper organic layer was measured at 532 nm. The experiment was performed in triplicate Sudheerkumar 2003. Superoxide scavenging Alkaline DMSO was used as a super oxide generating system.Shirwaiar2007 To 0.5 ml of different concentrations of the test compounds 1 ml of alkaline DMSO and 0.2 ml of NBT 20 mM in

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K. Sumathi et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-41 2016 108-111 110 phosphate buffer pH 7.4 was added. Experiment was performed in triplicate Govindarajan 2003. Statistical analysis All results are expressed as mean ± S.E.M. Linear regression analysis Origin 6.0 version was used to calculate the IC 50 values. RESULTS AND DISCUSSION Several concentrations ranging from 2 to 1000 μg/ml of the various extract of Carissa spinarum L were tested for their antioxidant activity in different in vitro models. It was observed that free radical was scavenged by the test compounds in a concentration dependent manner up to the given concentration in all the models. The percentage scavenging and IC 50 values were calculated for all models given in table 1. Table 1. In vitro antioxidant activity of Carissa spinarumextractsIC50 values ± SE µg/ml Extract ABTS DPPH H2O2 Hydroxyl radical Lipid peroxidation Super oxide Pet ether 244.2 ± 0 . 36 354.42 ± 2 . 02 236.12 ± 0 . 22 296.41 ± 0 . 28 291.41 ± 0 . 16 306.16 ± 0 . 12 Chloroform 178.4 ± 0 . 42 302.38 ± 3 . 12 212.38 ± 0 . 32 285.32 ± 0 . 41 188.42 ± 0 . 22 202.16 ± 0 . 38 Ethyl acetate 136.21 ± 0 . 16 212.36 ± 2 02 168.12 ± 0 . 24 216.42 ± 0 . 26 164.18 ± 0 . 42 169.38 ± 0 . 42 Ethanol 102.6 ± 0 . 22 200.41 ± 2 . 68 152.31 ± 0 . 46 188.74 ± 1 . 62 142.12 ± 0 . 12 157.42 ± 0 . 68 Ascorbic Acid 99.8 ± 0 . 16 198.32 ± 2 . 82 136.29 ± 0 . 52 168.92 ± 1 . 87 128.28 ± 0 . 06 128.92 ± 0 . 82 Oxidative stress has been implicated in the pathology of many diseases and conditions includes in diabetes cardiovascular diseases inflammatory conditions cancer and ageing Joyce 1987 Velioglu 1998. The reduction capacity of DPPH radicals was determined by the decrease in its absorbance at 517 nm Ganapathy 2007 The antioxidant activity of the extract by this assay implies that action may be by either inhibiting or scavenging the ABTS radicals since both inhibition and scavenging properties of antioxidants towards this radical have been reported in earlier studies Rice-Evans 1997 Initiation of the lipid peroxidation by ferrous sulphate takes place either through the ferryl- perferryl complex or through OH radical by Fenton’s reaction. Thus the decrease in the MDA level in egg phosphatidylcholine with the increase in the concentration of the extract indicated the role of the extract as an antioxidant Sudheerkumar 2003 Superoxide can cause oxidation or reduction of solutes depending on the reductions potential. Both aerobic and anaerobic organisms possess superoxide dismutase enzymes which catalysis the breakdown of superoxide radical Shirwaiar 2007. In our study alkaline DMSO used for superoxide generation indicates that C.spinarum L a potent superoxide scavenger CONCLUSION C.spinarumLan ever green deciduous shrub with immense medicinal value has been revived with the aim to provide a reference source for biology photochemistry ethnopharmacology and conservation strategy for further research on the plant. The results of the present study show that the extract of Carissa spinarum L contains the greatest antioxidant activity through the scavenging of free radicals which participate in various pathophysiology of diseases including ageing. Acknowledgement The authors sincerely thank J.K.K.M.M.R F’s Annai J.K.K. Sampooraniammal College of Pharmacy Komarapalayam Tamilnadu for providing the necessary facilities to carry out this research work.

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K. Sumathi et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-41 2016 108-111 111 REFERENCES 1. Calixto B.J. 2000. Efficacy safety quality control marketing and regulatory guidelines for herbal medicines phytotherapeutic agents. Brazilian Journal of Medical and Biological Research 332: 179- 189. 2. Doss A. and S.P. Anand 2012. Preliminary Phytochemical Screening of Asteracantha longifolia and Pergulariadaemia. World Applied Sciences Journal 182: 233-235. 3. N.Sreejayan M.N.A.Rao 1996 Free radical scavenging activity of curcuminoids Drug Res 46 pp169. 4. M.Sudeerkumar P.C.Jagadish R.B.Kiran M.K.Unnikrishnan 2003 Invitro evaluation of antioxidant properties of Cocusmicifera Linn. Nahrung/Food 47 pp126-131. 5. Govindarajan R.M.Vijaya Kumar A.K.S.Rawat S.Mehrotra2003Free radical scavenging potential of picrroroakurroa Royle ex Benth. IndianJ.Exptl.41pp875. 6. Rice-Evans C. N.J.Miller197Factors affecting the antioxidant activity determined by the ABTSradicalcationassayFreeRadic.Res.195pp26-27. 7. Mathur A et al .Antimicrobial potential of roots of Riccinus communis against pathogenic micro- organisms. International Journal of pharmacy and Biological Sciences 201121:545-548. 8. Gorden M C David J N Nayural product drug discovery in the next millennium. Journal of pharmaceutical Biology 2001 39:8-17. 9. Maheswarai Rsharma AVerma D.Phyto-therapeutic Significance of Karanda. Bull Environ Pharmacol Life Sci2012134-6. 10. Malik S KChaudhury R Dhariwaln OPBhandari DCGenetic Resources of Tropical Underutilized Fruits in India. New Delhi: NBPGR 2010 p.178. 11. S.Ganapathy V.M.Chandrashekhar H.R. Chitme N.M. Lakshmi 2007 Free radical scavenging activity of gossypin and nevadensin: An in vitro relation Indian Journal of Pharmacology39pp.281-283. 12. Babu B.H.Shylesh B. S.Padikkala.J.2001. Antioxidant and hepatoprotecctive effect of Alanthusicicifocus Fitoterapia 72272-277. 13. Barnes J. L.A.Anderson J.D.Phillipson 2002A guide for health- care professionals. In Herbal Medicines Edited by Barnes J Anderson I.A Phillipson JD. London Pharmaceutical Press pp 38. 14. Colondny L.R Montgomery M. Houston. 2001. The role of esterin processed alfalfa saponins in reducing cholesterol J Am Nitraceutical Assoc 3 pp. 1-10. 15. K.M Nadkarni A. K. Nadkarni 1976 Indian Materia Medica Popular Prakashan pvt Ltd. Bombay India pp.123-125. 16. A. Shirwaiar A .ShirwaikarI.S.R.Punitha2007Antioxidant studieson the methanol stem extract of Coscininum fenestratum Natural Product Science131pp40-45. 17. Houghton P.J.Hylands P.J.Mensah A.Y. Hensel A. Deters A. M. 2005. In vitro tests and ethno pharmacological investigations wound healing as an example. Journal of Ethnopharmacology 100 100- 107. How to cite this article: K.Sumathi N.Senthil Kumar In vitro antioxident and free radical scavenging studies of carissa Spinarum L. Int J of Allied Med Sci and Clin Res 2016 41: 108-111. Source of Support: Nil. Conflict of Interest: None declared.

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