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In Vitro Evaluation of Free Radical Scavenging Activity of Codariocalyx Motorius Root Extract

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In Vitro Evaluation of Free Radical Scavenging Activity of Codariocalyx Motorius Root Extract Asian Pacific Journal of Tropical Medicine (2013)188-194 188 Contents lists available at ScienceDirect Asian Pacific Journal of Tropical Medicine journal homepage:www.elsevier.com/locate/apjtm Document heading doi: In vitro evaluation of free radical scavenging activity of Codariocalyx motorius root extract Uma Chidambaram1, Vanitha Pachamuthu1, Suganya Natarajan1, Bhakkiyalakshmi Elango1, Suriyanarayanan2, Kunga Mohan Ramkumar1* 1 Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur- 603 203, Tamilnadu, India 2 Department of Water and Health, JSS University, Mysore - 570015, Karnataka, India ARTICLE INFO ABSTRACT Article history: Objective: Codariocalyx motorius To determine the phenolic content in root extract and to Received 15 September 2012 in vitro Methods: evaluate its antioxidant properties using various assay systems. The antioxidant Received in revised form 27 December 2012 Accepted 28 January 2013 activity was evaluated based on scavenging of 1,1-diphenyl-2-picrylhydrazyl, hydroxyl Available online 20 March 2013 radicals, superoxide anions, nitric oxide, hydrogen peroxide, peroxynitrite, reducing power and by inhibitionResults: of lipid peroxidation which wasCodariocalyx estimated inmotorius terms of(C. thiobarbituric motorius) acid reactive substances. The root extract of the exhibited potent Keywords: Codariocalyx motorius total antioxidant activity that increased with increasing amount of extract concentration, which was compared with standard drug such as quercetin, butylated hydroxytoluene, tocopherol at Antioxidant activity different concentrations. The different concentrations of the extracts showed inhibition on lipid Medicinal plants peroxidation. In addition, the extracts had effective reducing power, free radical scavenging, Polyphenols super oxide anion scavenging, nitric oxide scavenging, lipid peroxidation, and total phenolic content depending on concentration. High correlation between total phenolic contents and r2 scavenging potential of different reactive oxygen species ( =0.831-0.978) indicated the Conclusions C. motorius polyphenols as the main antioxidants. : Codariocalyx motorius ( ) root possess the highly active antioxidant substance which can be used for the treatment of oxidative stress-related diseases. 1. Introduction defence system within the body can easily scavenge free radicals that are produced. In human diseases an increased free radical generation occurs and resulted in lipid Free radicals are produced either from by normal cellular peroxidation of the membrane lipids, causing damage to processes or from external sources such as exposure to phospholipids of cell membrane, lipoprotein by propagating radiation, cigarette smoking, air pollutants and industrial a chain reaction cycle[2]. Therefore, antioxidants with free • chemicals. The most common reactive -oxygen species radical scavenging activities may have great significance in (ROS) • O2 include: the superoxide1 anion ( ), the hydroxyl the protection and therapeutics of diseases involving free radical (OH ), singlet oxygen ( O2), and hydrogen peroxide radicals[3,4]. (H2O2). The oxidation induced by ROS may result in cell Recently, there has been a considerable interest in finding membrane disintegration, membrane protein damage and natural antioxidants from plant materials, though they are DNA mutations which play an important role in aging accepted to be safe[5,6]. All higher plants have characteristic and can further initiate or propagate the development of compounds, such as vitamins, carotenoids, and phenolics, many diseases, such as arteriosclerosis, cancer, diabetes have exhibited antioxidant property through scavenging of mellitus, liver injury, inflammation, coronary heart diseases free radicals. Numerous studies have demonstrated that the and arthritis[1]. Under normal conditions, the antioxidant consumption of plants and vegetables with high phenolic content can reduce the risk of cardio- and cerebrovascular *Corresponding author: Dr. K.M. Ramkumar, Department of Biotechnology, School diseases and cancer mortality[7,8]. Phytochemicals having of Bioengineering, SRM University, Kattankulathur - 603203, Tamilnadu. Tel: +91-9940737854 antioxidant properties are associated with a lower risk of Fax: +91-44-2745 2343 mortality from many of the diseases[9]. E-mail: [email protected] Uma Chidambaram et al./Asian Pacific Journal of Tropical Medicine (2013)188-194 189 Codariocalyx motorius C. motorius ( ) belongs to the 2.4. ABTS cation radical scavenging activity Fabaceae family and is also known as Telegraph plant[10]. It is widely distributed throughout Bangladesh, Bhutan, Codarioalyx motorius Cambodia, China, India and Indonesia[11]. In Siddha The total antioxidant activity of medicine, it is used as antidote, cardiac-tonic and wound root extract (CMRt) was measured by the ABTS [15] healing ointment[12]. It has also been used in curing snake radical cation-decolorization assay . ABTS was prepared by reacting 5 mL of 7 mM ABTS and 80 毺L bite poisons. In‘Codariocalyx China, it has motorius been widely ohashi used’ in Ayurvedic medicine as and the main of 140 mM potassium persulphate and the mixture was 12-16 constituent of herbal tea[13]. It also been reported to contain allowed to stand in the dark at room temperature for h ABTS few alkaloids including hypaphorine, phenethylamines before use. (1 mL) was added to glass test tubes 50 CMR 1-1 000 and 5-Methoxy-N,N-dimethyltryptamine[12]. To the best containing 毺L of t ( 毺g/mL) and mixed by vortex mixer for 30s. Absorbance was measured at 734 nm of our knowledge no other study on biological properties, after 2 min. BHT was used as a standard. The percentage of including antioxidant properties was carried out so far on radical scavenging activity was calculated by comparing the this plant. The main objectives of the present study were to absorbance values of control (not treated with the extract). determine the phenolic contentC. motorius and screening of free radical All determinations were performed six times. scavenging activities of root extract. 2.5. Scavenging of superoxide radical 2. Materials and methods • The scavenging activity towards the superoxide radical-. 2.1. Plant material was measured in terms of inhibition of generation of O2 The assay was performed using alkaline DMSO method[16]. C. motorius ’ Potassium superoxide and DMSO were allowed to stand in Fresh root of were collected in November 2011 contact for 24 h and the solution was filtered immediately from the Kolli hills/ Kollimalai, Namakkal district of Tamil before use. Filtrate (200 毺L) was added to 2.8 mL of an Nadu and authenticated in National institute of Herbal aqueous solution containing NBT (500 毺M), EDTA (10 Science, Chennai, India (Accession No: PARC/2012/1085). 毺M) and potassium phosphate buffer (10 mM). Test The plant material was deposited at the Department of compounds (1 mL) at various concentrations (1-1 000 毺g/mL) Biotechnology, SRM University. were added and the absorbance was recorded at 560 nm against a control in which pure DMSO was added instead 2.2. Polyphenol extraction from roots of alkaline DMSO. The results were compared with the reference antioxidant quercetin. The collected roots were washed and dried in shade for a week 2.6. Nitric oxide radical scavenging assay and powdered. Every dried and powdered root sample (5 g) was extracted using soxhlet apparatus, at 30 曟 for 6 h and The nitric oxide generation capacity was quantified by the acidified with 0.01 mol/L H2SO4 as previously described[14]. Griess Illosvoy reaction using sodium nitroprusside (SNP)[17]. The aqueous solution obtained was extracted three times The reaction mixture contained 10 mM SNP, phosphate with butanol (200 mL), using a separatory funnel. The buffer (pH 7.4) and different concentration of the extract three alcoholic phases which contain all the polyphenol (1-1 000 毺g/mL) and incubated for 2 hours at room temp. compounds were combined and the butanol extract was Then 1 mL of 0.33% sulfanilamide (in 20% glacial acetic 35 then brought to dryness using a rotating evaporator at 曟. acid) was added and allowed to stand for 5 min. Then 1 mL The dry matter obtained was dissolved with deionized water of 0.1% napthylethylenediamine dihydrochloride was added (100 mL) and extracted with ethyl acetate (3伊10 mL). The and the mixture was further incubated for 30 min. Quercetin organic layer containing the phenolics was finally dried. was used as the reference compound. The color developed 2.3. 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was measured spectrophotometrically at 540 nm against a scavenging assay blank sample. 2.7. Peroxynitrite scavenging assay To determine the DPPH scavenging activity of the extract, 3 mL of 60 毺M DPPH in ethanol was added to different An Evans Blue bleaching assay was used to measure concentration of CMRt (1-1 000 毺g/mL) and then incubated peroxynitrite scavenging activity[18]. The reaction mixture at room temperature (dark condition) for 15 min. Absorbance contained 50 mM phosphate buffer (pH 7.4), 0.1 mM DTPA, was read at 517 nm using a Genesys-5-spectrophotometer 90 mM NaCl, 5 mM KCl, 12.5 毺M Evans Blue, different (Spectronic Instruments, Rochester, NY). butylated concentration of plant extract (1-1 000 毺g/mL) and 1 mM hydroxytolune (BHT) was used as a standard. The percentage peroxynitrite in a final volume of 1 mL. After incubation 30 611 of DPPH radical scavenging activity was calculated by for min, the absorbance was measured- at nm. comparing
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