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Experimental studies on antioxidant properties of calotropis gigantea
Experimental studies on antioxidant properties of calotropis gigantea
Experimental studies on antioxidant properties of calotropis gigantea
Experimental studies on antioxidant properties of calotropis gigantea
Experimental studies on antioxidant properties of calotropis gigantea
Experimental studies on antioxidant properties of calotropis gigantea
Experimental studies on antioxidant properties of calotropis gigantea
Experimental studies on antioxidant properties of calotropis gigantea
Experimental studies on antioxidant properties of calotropis gigantea
Experimental studies on antioxidant properties of calotropis gigantea
Experimental studies on antioxidant properties of calotropis gigantea
Experimental studies on antioxidant properties of calotropis gigantea
Experimental studies on antioxidant properties of calotropis gigantea
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Experimental studies on antioxidant properties of calotropis gigantea

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  • 1. International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME168EXPERIMENTAL STUDIES ON ANTIOXIDANT PROPERTIES OFCALOTROPIS GIGANTEA SOLVENT EXTRACTSDavid Mandepudi1,2, Bharath Kumar Ravuru3, Bhavani Mandepudi21Faculty of Biotechnology, Jawaharlal Nehru Technological University-Hyderabad,Hyderabad-500085, Andhra Pradesh, India2Department of Biotechnology, School of Engineering, Sir Padampat Singhania University,Udaipur-313601, Rajasthan, India3School of Biotechnology, Vignan University, Guntur – 522213, Andhra Pradesh, IndiaABSTRACTThe active metabolites from plant sources had been explored and extracted to servethe societies as part of the medicine either internally or externally for the treatment ofailments. In the similar lines, the Calotropis gigantea, has been investigated for antioxidantproperties using ethanol, methanol, chloroform and aqueous solvent extracts of leaves, budsand flowers with the help of popular antioxidant testing methods of DPPH and hydrogenperoxide free radical scavenging activities and an additional reducing power test. In theresults, many of the solvent extracts were reaching to variable saturation levels beyond therespective extract concentrations of 5µg/ml. Further, comparable free radical scavengingactivities for some of the solvent extracts like, ethanol and methanol leaves, buds & flowersextracts in addition to methanol, chloroform and aqueous extracts of leaves, buds andflowers were observed with respect to free radical scavenging activities of the standardantioxidants, ascorbic acid and quercetin in the tests. However the reducing power of thesesolvent extracts of Calotropis gigantea leaves, buds and flowers were very low in comparisonto that of the standard antioxidants, ascorbic acid and BHT. So, the observed free radicalscavenging activities of the solvent extracts may be due to the presence of the detected activemetabolites like, alkaloids, tannins, polyphenols etc. Though these results favor the use ofCalotropisgigantea as part of the traditional medicine due to their auxiliary antioxidantproperties, similar studies may be carried out for the better utilization and management of theCalotropis species.INTERNATIONAL JOURNAL OF ADVANCED RESEARCH INENGINEERING AND TECHNOLOGY (IJARET)ISSN 0976 - 6480 (Print)ISSN 0976 - 6499 (Online)Volume 4, Issue 4, May – June 2013, pp. 168-180© IAEME: www.iaeme.com/ijaret.aspJournal Impact Factor (2013): 5.8376 (Calculated by GISI)www.jifactor.comIJARET© I A E M E
  • 2. International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME169Keywords: Antioxidant, Ayurveda, drugs of abuse, Prodrug, Polyphenols, Calotropisgigantean1. INTRODUCTIONThe molecular agents that prevent the oxidation of other molecules either by stoppingthe transfer of electron or hydrogen are known as the antioxidants. But then, these antioxidantmolecules get themselves oxidized often acting as reducing agents [1]. While the oxidationand reduction reactions are very important set of reactions in biological systems, the soldierlike antioxidants play a vital role in the sustenance of the life [2-3] on the planet, Earth. Onthe other hand, the reactive species and free radicals were known to cause severe damage tothe cellular components of the life systems especially of human and may lead to variousailments like early ageing, oxidative stress related with Alzheimers disease,Parkinsonsdisease,diabetes, rheumatoid arthritis, and neuro degeneration in motor neuron diseases[2,3,4-13]etc. Since, these oxidation and reduction reactions were found to have the greatersignificance in the realm of biological systems with antioxidant mechanism, plants andanimals established complex systems of antioxidants like glutathione, vitamins – C, D, A, K& E; enzymes like catalase, superoxide dismutase, peroxidases, β-carotene, lutein, lycopene,vitamin A, vitamin C, vitamin E, flavonoids, polyphenols, ascorbic acid [14-17] etc. toregulate and check the unnecessary burst of any reactive species [11] like hydrogen peroxide(H2O2), hypochlorous acid (HClO) and free radicals like hydroxyl radical (.OH) andsuperoxide anion (O2-). Owing to these facts, synthetic antioxidants likebutylatedhydroxytoluene (BHT) and buytlatedhydroxyanisole (BHA)were also developed orderived in the form of health supplements or drug molecules to serve the purpose by variouspharmaceutical and health care companies [7,18]. However the natural antioxidants are thebest means for the human consumption and in the lines of acceptability for the environmentwithout the creation of any xenobiotic compounds [19] by the synthetic processes. So, thesefacts elucidated the need to search for the novel and bioactive compounds [20] likeantioxidants directing the scientists to investigate the plant based solvent extracts to isolateand identify the antioxidants that may be used as dietary supplements [21-23] or the drugsthat can save the life. In such scenario, many plants like, Azadiractaindica, Meliaazedarach,Digitalis purpurea, Digitalis lanata, Daturastramonium, Atropa belladonna,Papaversomniferum, Catharanthusroseus, Colchicum autumnale, erythroxylon coca etc.were investigated for potential active metabolites likeazadiractin, cardenolides,parasympatholytic agents, analgesics, anticancer agents, antigout agent, local anesthetics etc.for the use of medicine and food supplements [20,24]. In the similar lines, the Calotropis sp.known for its poisonous nature for centuries and for medicine in the traditional medicalpractices [25-27] of several ethnic groups has been investigated for the possible activemetabolites using the residual solvent extracts of Calotropis gigantea leaves, buds andflowers.2. MATERIALS AND METHODS2.1 Plant material & solvent extractionThe Calotropis gigantia leaves, flowers and buds were collected [28] and processedto obtain the respective dry powders and these dry powders were added to the solvents ofethanol, methanol and chloroform and extracted the active metabolites by solvent extraction
  • 3. International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME170procedure and named them as the solvent extracts of ethanol leaves (EL), ethanol flowers(EF), ethanol buds (EB); methanol leaves (ML), methanol flowers (MF), methanol buds(MB); Chloroform leaves (CL), chloroform flowers (CF), chloroform buds (CB). Theaqueous extracts were also prepared by boiling the plant material (powder) individually andprocessed [28] to obtain the final extracts of Aqueous leaves (AL), Aqueous flowers (AF)and Aqueous buds (AB). These extracts were stored in the refrigerator until use in sterilebottles.2.2 Antioxidant Activity of solvent extractsThe extracted solvent extracts of leaves, buds and flowers of Calotropis giganteawere tested for the detection and estimation of antioxidant activity in terms of free radicalscavenging activity using DPPH [29-32] and Hydrogen peroxide [33-34] scavenging assaymethods.2.3 The DPPH free radical scavenging activityThe DPPH solution (3ml) of 0.02mg/ml in methanol was taken in three sets of clean& dry test tubes and the solvent extracts of 1 ml were added respectively with an increasingconcentration in the range of (0-25µg/ml) in multiples of 5µg/ml. Similarly a standardantioxidant, ascorbic acid of 1 ml was also added separately to the 3 ml DPPH solution inthree sets of test tubes and used as a reference for the assay. All those test tubes with thesolutions were vortexes and incubated at room temperature for 30 minutes under darkconditions. The absorbance of these resulting solutions were recorded at 517nm using UV-Visible double beam spectrophotometer. So the assay was performed in triplicates and theresults were expressed as mean values and the standard derivations were also calculated.The DPPH free radical scavenging activity of the standard, ascorbic acid and the solventextracts were calculated using the equation (1).(% ) DPPH Free Radical Scavenging Activity (DFRSA) =ሺ ୅ೞି୅೐ሻ୅ೞX100 (1)Where As is the absorbance of the DPPH in the absence of test solution (solventextract/ascorbic acid) and Ae is the absorbance of the test solution.2.4 Hydrogen peroxide free radical scavenging activityEstimation of antioxidant activity of enzyme, catalase [35] was adopted to detect andevaluate the hydrogen peroxide scavenging activity of solvent extracts of Calotropis gigantialeaves, buds and flowers. The solvent extracts were taken as test solutions of 0.4ml withvarying concentrations in multiples of 5µg/ml in the concentration range of (5-25) µg/mlseparately in three sets of clean and dry test tubes and then added 0.6ml of 40mM hydrogenperoxide solution (in 0.1M phosphate buffer of pH 7.4) to each test sample and incubatedthem for 15 min under dark conditions. Similarly, a standard antioxidant solution of quercetinwas prepared and used for the assay following the above standard procedure with varyingconcentration in multiples of 5µg/ml in the concentration range of (5-25)µg/ml. After theincubation period, 1ml of dichromate solution in acetic acid (5% potassium dichromateaqueous solution mixed with glacial acetic acid in the ratio of 1:3) was added to each solventextract test solution and standard quercetin solution separately and mixed them well. Thesetest solutions were further incubated in boiling water bath for 10min, where the characteristicgreen color appeared in the test tubes. 2ml of deionized water (MilliQ water) was added toeach test solution and the resultant solution was used for the measurement of absorbance at
  • 4. International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME171620nm using UV-Visible double beam spectrophotometer. The control sample was preparedwithout any extract or standard antioxidant, containing only deionized water and thehydrogen peroxide as well as dichromate solutions in parallel with test sample& standardsamples of quercetin. Hence the test was performed in triplicates and the measuredabsorbance at 620nm for each sample was taken as mean value and used this data for furtheranalysis.The (%) hydrogen peroxide free radical scavenging activity of solvent extracts & thestandard, quercetin were calculated as per the equation (2).(% ) Hydrogen peroxide Free Radical Scavenging Activity (HFRSA) =ሺ ୅ೃି୅೐ሻ୅ೃX 100(2)Where AR is the absorbance of control sample, and Ae is the absorbance of extract orstandard at 620nm.2.5 Reducing power assayThe solvent extracts of Calotropis gigantea leaves, buds and flowers were evaluated[36], where 1ml of the variable concentration of residual solvent extracts (0-1 mg/ml) weremixed with 2.5ml of 0.2M phosphate buffer (pH 6.6) and 2.5ml of 1% potassium ferricyanide(K3Fe CN6) solution respectively and then incubated the resulting solutions at 50oC for 20minutes. Added 2.5ml of 10% trichloroacitic acid to each reaction mixture in the test tubesand then centrifuged at 3000g for 10 min where the supernatant of 1ml was withdrawncarefully and mixed with 2.5ml of distilled water and further added 0.5ml of 0.1% ferricchloride solution to get the final solution. The absorbance of the resulting solution for eachtest sample was measured using UV-Visible double beam spectrophotometer at 700nm. Theascorbic acid and BHT were taken as the standard antioxidants for positive controls followingthe above procedure and the experiments were performed in triplicates. The mean absorbancefor each test sample was calculated and used the data for further analysis.3. RESULTSThe free radical scavenging activity of Calotropis gigantea leaves, buds and flowerswere calculated using DPPH and hydrogen peroxide methods as the (%) reduction in theabsorbance and used them to plot the free radical scavenging activity profiles of Calotropisgigantea leaves extracts, buds extracts and flowers extracts against their respectiveconcentrations as represented in the Figures 1-6. These profiles in the figures also include thestandard or reference antioxidant (Ascorbic acid for DPPH method and quercetin for H2O2Method) profile, providing a means for the comparison of the solvent extract antioxidantprofiles of Calotropis gigantea leaves, buds and flowers. The saturation free radicalscavenging activities and the corresponding Effective Dosage for the 50% free radicalscavenging activities (ED50) were noted for all the solvent extracts and represented in theFigures 7 & 8 with respect to DPPH and hydrogen peroxide methods. Similarly the reducingpower of the residual solvent extracts of Calotropis gigantea leaves, buds and flowers wererepresented as the absorbance profiles against their respective variable concentrations asshown in the Figures 9-11 along with the absorbance profiles of standards, ascorbic acid andBHT. The reducing power of the standards were so high that the ascorbic acid test resultswere diluted to 5% and the BHT results were diluted to 12% for the convenience. Howeverthe data represented in the profiles and the figures were taken as the average of the triplicates.
  • 5. International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, MayFig.1 DPPH free radical scavenging activity pFig.2 DPPH free radical scavenging activity pFig.3 DPPH free radical scavenging activity pInternational Journal of Advanced Research in Engineering and Technology (IJARET), ISSN6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME172DPPH free radical scavenging activity profiles of Calotropis gigantea leaves extractsDPPH free radical scavenging activity profiles of Calotropis gigantea buds extractsDPPH free radical scavenging activity profiles of Calotropis gigantea flowers extractsInternational Journal of Advanced Research in Engineering and Technology (IJARET), ISSNJune (2013), © IAEMEleaves extractsbuds extractsflowers extracts
  • 6. International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, MayFig.4 Hydrogen peroxide free radical scavenging activity ofFig.5Hydrogen peroxide free radical scavenging activity ofFig.6 Hydrogen peroxide free radical scavenging activity ofInternational Journal of Advanced Research in Engineering and Technology (IJARET), ISSN6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME173Hydrogen peroxide free radical scavenging activity of Calotropis giganteaextractsHydrogen peroxide free radical scavenging activity of Calotropis giganteaHydrogen peroxide free radical scavenging activity of Calotropis giganteaextractsInternational Journal of Advanced Research in Engineering and Technology (IJARET), ISSNJune (2013), © IAEMECalotropis gigantea leavesbuds extractsCalotropis gigantea flowers
  • 7. International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, MayFig.7 Variations in the saturation of DFRSA ofradicals and the corresponding ED50 concentrationsFig.8 Variations in the saturation of HFRSA ofperoxide radicals and the corresponding ED50 concentrationsFig. 9 Comparative reducing power ofstandard antioxidants, ascorbic acid and BHTInternational Journal of Advanced Research in Engineering and Technology (IJARET), ISSN6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME174Variations in the saturation of DFRSA of Calotropis gigantea extracts on DPPHradicals and the corresponding ED50 concentrationsVariations in the saturation of HFRSA of Calotropis gigantea extracts on hydrogenperoxide radicals and the corresponding ED50 concentrationsComparative reducing power of Calotropis gigantea leaves extracts along with thestandard antioxidants, ascorbic acid and BHTInternational Journal of Advanced Research in Engineering and Technology (IJARET), ISSNJune (2013), © IAEMEextracts on DPPHextracts on hydrogenleaves extracts along with the
  • 8. International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, MayFig. 10 Comparative reducing power ofstandard antioxidants, ascorbic acidFig. 11 Comparative reducing power ofstandard antioxidants, ascorbic acid and BHT4. DISCUSSIONThe free radical scavenging activity of many plant based extracts and the standardantioxidants like ascorbic acid and quercetin were dependent on their concentration [37however such free radical scavenging antioxidant properties ofextracts on free radicals like that of DPPH were found to be saturating (for concentrationsbeyond 5µg/ml) as shown in the Figures 1follows.4.1 Antioxidant properties of Calotropis giganteaThe leaves extracts of Calotropis giganteavalues of free radical scavenging activity by 5µg/ml concentration whereas the standardantioxidant, ascorbic acid followed a gradually increasing free radicalthe saturation level at around 15µg/ml concentration. Conversely, the quercetin, the otherstandard antioxidant was found (Figure 4) to reach the saturation levels of free radicalInternational Journal of Advanced Research in Engineering and Technology (IJARET), ISSN6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME175Comparative reducing power of Calotropis gigantea buds extracts along with thestandard antioxidants, ascorbic acid (AA) and BHTComparative reducing power of Calotropis gigantea flowers extracts along with thestandard antioxidants, ascorbic acid and BHTThe free radical scavenging activity of many plant based extracts and the standardantioxidants like ascorbic acid and quercetin were dependent on their concentration [37however such free radical scavenging antioxidant properties of Calotropis gigantextracts on free radicals like that of DPPH were found to be saturating (for concentrationsbeyond 5µg/ml) as shown in the Figures 1-6. The details of the results were discussed asCalotropis giganteaLeaves extractsCalotropis gigantea (Figures 1 & 4) reached the saturationvalues of free radical scavenging activity by 5µg/ml concentration whereas the standardantioxidant, ascorbic acid followed a gradually increasing free radical scavenging activity tothe saturation level at around 15µg/ml concentration. Conversely, the quercetin, the otherstandard antioxidant was found (Figure 4) to reach the saturation levels of free radicalInternational Journal of Advanced Research in Engineering and Technology (IJARET), ISSNJune (2013), © IAEMEbuds extracts along with theflowers extracts along with theThe free radical scavenging activity of many plant based extracts and the standardantioxidants like ascorbic acid and quercetin were dependent on their concentration [37-41],Calotropis gigantea solventextracts on free radicals like that of DPPH were found to be saturating (for concentrations6. The details of the results were discussed as(Figures 1 & 4) reached the saturationvalues of free radical scavenging activity by 5µg/ml concentration whereas the standardscavenging activity tothe saturation level at around 15µg/ml concentration. Conversely, the quercetin, the otherstandard antioxidant was found (Figure 4) to reach the saturation levels of free radical
  • 9. International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME176scavenging activity on hydrogen peroxide by 5µg/ml concentration, but then its saturationfree radical scavenging activity was only of about half the free radical scavenging activitysaturation values of ascorbic acid (Figure 1). Similarly the Calotropis gigantea leavesextracts recorded the higher (twice) free radical scavenging saturation activities in case ofethanol, methanol & chloroform leaves extracts on DPPHradicals compared to that ofhydrogen peroxide radicals; whereas the aqueous leaves extracts exhibited higher free radicalscavenging activity on hydrogen peroxide compared to that of DPPH radicals (Figures 1, 4, 7& 8). These results were also supported by the increasing reducing power of Calotropisgigantea leaves extracts as shown in Figure 9 where the leaves extracts reducing powerprofiles against the concentration were gradually increasing but then these profiles were verylow in reducing power in comparison to that of the standard antioxidants, ascorbic acid andBHT. The effective dosage for 50% saturating free radical scavenging activity (ED50) asshown in the Figures 7 and 8 for various Calotropis gigantea solvent extracts, brought outethanol leaves extract with lowest ED50 concentration of 2µg/ml on DPPHfreeradicals andaqueous leaves extract with highest ED50 concentration of 4µg/ml on hydrogen peroxide freeradicals.4.2 Antioxidant properties of Calotropis giganteaBuds extractsThe free radical scavenging activity of Calotropis gigantea buds extracts were shownin the Figures 2, 7 & 4, 8 with respect to DPPH and hydrogen peroxide free radicals wherethe methanol buds extract recorded higher percentage of free radical scavenging activity onDPPH radicals (80%) and the least ED50 concentration was found as 2.2µg/ml with respectto hydrogen peroxide radicals. However the ethanol buds extract has shown decreasingactivity on DPPH radicals beyond 5 µg/ml concentration whereas its converse profile wasobserved on hydrogen peroxide radicals, otherwise all other buds extracts exhibited thesaturating free radical scavenging activity for the respective concentrations (>5µg/ml). Theseresults were also supported by the moderate and decreasing reducing power of the Calotropisgigantea solvent extracts of leaves, buds and flowers as shown in the Figure 10.4.3 Antioxidant properties of Calotropis giganteaFlowers extractsThe Calotropis gigantea flowers extract free radical scavenging activity with respectto variation in their respective concentrations in the solvents were shown in the figures 3 & 6where ethanol flowers extract exhibited higher and lower scavenging activities on DPPH(76%) and hydrogen peroxide (15%) radicals respectively. On the other hand aqueousflowers extracts had the least scavenging activity on DPPH (10%) free radicals and the higherscavenging activity on hydrogen peroxide (41%) free radicals. Conversely, the chloroformflowers extracts recorded moderately saturating free radical scavenging activity on bothDPPH and hydrogen peroxide free radicals whereas these chloroform flower extractsreducing power profiles were found to exhibit lowest saturating reducing powers as shown inthe Figure 11. In another interesting finding, the methanol flowers extracts recordedincreasing free radical scavenging activity on hydrogen peroxide free radicals beyond theconcentration of 5 µg/ml. However the reducing power of methanol flowers extract wasfound to be decreasing with increasing concentration whereas the reverse effects were foundin case of aqueous flowers extracts. These observed free radical scavenging activities of thesolvent extracts of Calotropis gigantea leaves, buds and flowers may be due to the bioactivecompounds like tannins, polyphenols and alkaloids [42-43] etc. present as the residualcomponents in these solvent extracts.
  • 10. International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME1775. CONCLUSIONSThe study has provided an evidence for the antioxidant free radical scavengingactivities of Calotropis gigantea leaves, buds and flowers extracts of ethanol, methanol,chloroform and aqueous solvent extracts. These results also demonstrated the comparablefree radical scavenging activities for some of the solvent extracts like, ethanol leaves extract,methanol leaves extract, ethanol buds extract, methanol buds extract, ethanol flowers extractand methanol flowers extract on DPPH radicals with that of the standard antioxidant, ascorbicacid. Similarly, methanol, chloroform and aqueous extracts of leaves, buds and flowers alsoexhibited the comparable free radical scavenging activity on hydrogen peroxide radicals withthat of the standard antioxidant, quercetin. However the reducing power of these solventextracts of Calotropis gigantea leaves, buds and flowers were very low in comparison to thatof the standard antioxidants like ascorbic acid and BHT. So these results support the use ofCalotropis as part of the traditional medicine to a considerable extent as these extractsrecorded the moderate antibiotic and wound healing properties [44-48] that may aid as part ofthe treatment to terminate the pathogens and their effects on animals and humans.ACKNOWLEDGEMENTDr. M Lakshmi Narasu, Professor & Dr. ArchanaGiri, Assistant Professor, Faculty ofBiotechnology, Jawaharlal Nehru Technological University, Hyderabad, Dr.ArchanaGajbhiye, Head, Department of Biotechnology, Sir Padampat Singhania University,Udaipur were acknowledged for their suggestions and critical comments on the researchwork. Mr. Ashok Ghosh, President, Ms. Rinu Ghosh, Vice President, Prof. P.C. Deka, ViceChancellor and Prof. AchintyaChaudary, Dean, School of Engineering of Sir PadampatSinghania University, Udaipur were also acknowledged and thanked for their financialsupport and approvals for the research work.REFERENCES[1] J Chaudiere, and R. Ferrari-Iliou, Intracellular antioxidants: From chemical tobiochemical mechanisms, Food and Chemical Toxicology, 37, 1999, 949–962.[2] T Finkel, and N.J. Holbrook, Oxidants, oxidative stress and the biology of ageing,Nature,408, 2000, 239–247.[3] M Valko, D. Leibfritz, J. Moncol, M.T. Cronin, M. Mazur, and J. Telser, Freeradicals and antioxidants in normal physiological functions and human disease, TheInternational Journal of Biochemistry and Cell Biology,39, 2007, 44–84.[4] Y Christen, Oxidative stress and Alzheimer disease, The Americal journal of clinicalnutrition, 71, 2000, 621S–629S.[5] L.A del Río, L.M. Sandalio, J.M. Palma, P. Bueno, and F.J. Corpas, Metabolism ofoxygen radicals in peroxisomes and cellular implications, Free Radical Biology &Medicine,13, 1992, 557–580.[6] D.C Hooper, G.S. Scott, A. Zborek, T. Mikheeva, R.B. Kean, H. Koprowski, andS.V. Spitsin, Uric acid, a peroxynitrite scavenger, inhibits CNS inflammation, blood-CNSbarrier permeability changes, and tissue damage in a mouse model of multiple sclerosis,TheFASEB Journal,14, 2000, 691–698.
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