Evaluation Of Antioxidant Activity In Different Parts Of .
Int.J.Curr.Microbiol.App.Sci (2015) 4(9): 372-379ISSN: 2319-7706 Volume 4 Number 9 (2015) pp. 372-379http://www.ijcmas.comOriginal Research ArticleEvaluation of Antioxidant Activity in Different Parts ofSyzygium cumini (Linn.)Elizabeth Margaret1*, A. M. Shailaja2 and V. Venugopal Rao312Department of Botany, St. Ann s College for Women, Hyderabad, A.P., IndiaDepartment of Biochemistry, St. Ann s College for Women, Hyderabad, A.P., India3Department of Genetics, St. Ann s College for Women, Hyderabad, A.P., India*Corresponding authorABSTRACTKeywordsAntioxidants,Syzygium,Anti- microbialactivityThe present study was aimed at evaluation of antioxidant activity in methanolicextract of leaves, fruit pulp and seeds of Syzygium cumini (L.). Another aspect ofthe study was to evaluate the anti microbial activity of the different parts of theplant, keeping in view its pharmacological potential. The quantitativedetermination of compounds viz., phenolics and flavonoids supposed to beantioxidants was made and overall antioxidant activity was measured usingstandard methods. The results demonstrate that the total phenolic and flavonoidcontent of S. cumini leaves is greater than the content found in pulp and seedextracts. A linear correlation between total phenolic content and antioxidantactivity (r2 0.464529) has been reported. The results suggested that the phenoliccompounds contribute effectively to the antioxidant activity. The highestantioxidant property of leaves is noteworthy as compared to seed and pulp in thepresent study. Our study showed antibacterial activity against both the gramnegative (E. coli) and gram- positive (Staphylococcus aureus) cultures grown in theleaf extract of S. cumini.Introductionof important nutritional compositions.Syzygium cumini (syn. Eugenia jambolana)commonly known as a Jamun containsvarious phytoconstituents such as tannins,alkaloids, steroids, flavonoids, terpenoids,fatty acids, phenols, minerals, carbohydratesand vitamins.Antioxidants are vital substances, whichpossess the ability to protect the body fromdamages caused by free radical-inducedoxidative stress. Much attention has beenfocused on the activity of the naturalantioxidants present in fruits and vegetables,because potentially these components mayreduce the level of oxidative stress. Dietaryantioxidants include ascorbate, tocopherols,carotenoids and bioactive plant phenols.Amongst the plant sources Syzygium cuminifruit is one of those which contain a varietySyzygium cumini (L.) is an evergreentropical tree belonging to the familyMyrtaceae and native to Bangladesh, India,Nepal, Pakistan, Sri Lanka, Philippines and372
Int.J.Curr.Microbiol.App.Sci (2015) 4(9): 372-379Indonesia. Syzygium cumini is an evergreentree to a height of 25 m, with grayish whiteyoung stems and lower bark coarse anddiscolored. Leaves are exstipulate, petiolate,simple, elliptic to broadly oblong, smooth,glossy, somewhat leathery, 5 10 cm long,acuminate tip with entire margin and onopposite phyllotaxy. Flowers white topinkish, about 1 cm across, in branchedclusters at stem tips, calyx cuplike and 4petals fused into a cup with many stamens.Fruits are dark purplish red, shiny, withwhite to lavender flesh, ovoid, single seededberry measuring 2.2 4.5 cm length and 1.53cm in diameter. The seed weighs1 3gmsand an average sized fruit is said to contain68 86mg of pulp.acid (1.21%) Glucose, Fructose, mannoseand galactose are the principal sugars.Jamun has also been reported to beprotective in liver disease which could playan important role in prevention of liverdamage. In addition, studies also show ananti-cancer potential of fruit extract. Thesecould be possibly due to several bioactivephytochemicalsincludingpolyphenolswhich have the purple pigment calledanthocyanin. Many scientists have studiedthe pharmacological activity of Syzygiumcumini like antidiarrhoel, antioxidant,gastro-protective, anti-allergic, astringent,analgesic, anti-inflammatory, anti-plaque,antimicrobial and the most important antidiabetic activity.Phytochemical constituentMost pharmacological work on diabeteswith seedswas carried out butpharmacological potential on other parts ofthe plant needs to be explored for discoveryof safer drugs keeping in view the multidimensional functionalities of the plant. Thepresent study was aimed at evaluation ofantioxidant activity in methanolic extract ofleaves, fruit pulp and seeds. Another aspectof the study was to evaluate the antimicrobial activity of the different parts ofthe plant.The principle component Jambolan containschemical constituents like anthocyanins,glucoside, ellagic acid, isoquercetin,kaemferol and myrecetin. Seed alkaloid,jambosine, and glycoside jambolin orantimellin are said to have retarding effect tothe diastatic conversion of starch into sugar.Ellagic acid of the seed extract has propertyto control the blood pressure levels (Morton,1987). The seeds have been reported to berich in flavonoids, a well-knownantioxidant, which accounts for thescavenging of free radicals and protectiveeffect on antioxidant enzymes (Ravi et al.,2004).Material and MethodsPlant material usedTotal phenolics of the seeds said have animportant antioxidant activity (Bajpai et al.,2005). The fruit is rich in sugar, mineralsalts, vitamins C. Fruit of Syzygium cuminicontain malic acid and a small quantity ofoxalic acid is also reported to be present.Gallic acid and tannins account forastringency of the fruit. The purple color ofthe fruit is due to presence of cyanidingdiglycosides. Fruit contain sugar (8.09%),non reducing sugar (9.26%) and sulfuricThe fully mature fruits and leaves ofSyzygium cumini L., were collected from asingle tree during the study period August2013-14 (Hyderabad, India). The methanolicextracts of the pulp, seed and leaves werethe samples used for the study.Determination of total phenolicsThe total phenolics of the different sampleextracts were determined by the Folin373
Int.J.Curr.Microbiol.App.Sci (2015) 4(9): 372-379Ciocalteu method. The diluted aqueoussolution of extract (0.5 ml) was mixed withFolin Ciocalteu reagent (0.2N, 2.5ml). Thismixture was allowed to stand at roomtemperature for 5 min and then sodiumcarbonate solution (75 g/l in water, 2ml) wasadded. After 2 hr of incubation, theabsorbance was measured at 760 nm againstwater blank. A standard calibration curvewas plotted using Gallic acid.Thin layer chromatography of crudeextracts of leaf, pulp and seed in S. cuminiThe separation and identification wasperformed using TLC. Samples for TLC onSilica gel were prepared with vaporizationof 5 ml of sample up to 1ml. Solutions ofStandard substance tannic acid, ascorbicacid, salicylic acid, benzoic acid, catechol,resorcinol were prepared by dissolving10mg in 1ml of distilled water.TLCseparation was carried out on silica gel.Solvent system used was benzene: glacialacetic acid: water (125:72:3)Estimation of total flavonoidsTotal flavonoid content was estimatedfollowing aluminium chloride colorimetricmethod (Chang et al., 2002). 2 ml of eachextract (1:10 g ml-1) in methanol wasseparately mixed with 1.5 ml of methanol,0.1 ml of 10% aluminum chloride, 0.1 ml of1M potassium acetate and 2.8ml of distilledwater. It was left at room temperature for 30min after which the absorbance of thereaction mixture was measured at 415nmwithadoublebeamUV/Visiblespectrophotometer. The calibration curvewas plotted by preparing the quercetinsolutions at concentrations 40 to 200 µg/100µl in methanol.Determination of antimicrobial activityThe antibacterial activity was measured byAgar well diffusion assay (Perez et al.,1990). The plant extracts were allowed todiffuse out into the medium and interact in aplate freshly seeded with the test organisms.All the plates are incubated at 37 C for24hrs. The test cultures used wereEscherichia coli (Gram negative rods) &Staphylococcus aureus (Gram positivecocci). The pure cultures were obtainedfrom National collection of IndustrialMicroorganisms (NCIM), NCL, CSIR lab,Pune. The bacteria were maintained onnutrient Agar plates (Himedia, India) slopesat 4 C and sub cultured as per therequirement. The antibacterial spectrum ofthe test sample was determined in terms ofzone sizes around each well i.e. diameter ofinhibition zones. Each result is a mean ofthree replicates.Estimation of total antioxidant activityThe ability of the extracts to reduce iron (III)was assessed by the method of Oyaizu, M.(1986). In the reducing power assay, thepresence of antioxidants in the sampleswould result in the reduction of Fe3 to Fe2 by donating an electron.Amount of Fe2 complex can then bemonitored by measuring the formation ofPerl's Prussian blue at 700 nm. Increasingabsorbance at 700 nm indicates an increasein the reductive ability. Ascorbic acid atvarious concentrations (10 100µg/ml) wasused as standard. Increased absorbance ofthe reaction mixture indicates increase inreducing power.Statistical analysisResults are presented as the mean SD.Correlation between analysis of antioxidantactivity and the total phenolic and flavonoidcontents were carried out using thecorrelation and regression applications in theMicrosoft Excel.374
Int.J.Curr.Microbiol.App.Sci (2015) 4(9): 372-379highest (324.67 mg ASE/ml (Fig.3). Seedextract value was 292.5 mg ASE/ml washigher than pulp extract value of 249.5 mgASE / ml. The highest antioxidant propertyof leaves is noteworthy as compared to seedand pulp in the present study. The presenceof polyphenolic compounds in methanolextracts of seed, leaf & pulp of S. cuminimight be responsible for the antioxidantactivity.Results and DiscussionPhenols are very important plantconstituents because of their antioxidantactivity. The antioxidant activities of theplant extracts are often explained by theirtotal phenolics and flavonoid contents.There is a wide range of total phenol contentin the extracts of the plants under study.Ocimum sps showed high phenolic contentaccording to the results by Veeru et al.,(2009) and Asparagus racemosus also hadcomparablehighphenoliccontentscontributing to the medicinal uses. Ghafar etal. (2010) found the flavonoid content ofCitrus to be the highest. The standard curvegenerated with gallic acid for total phenolcontent determination is presented in (Fig.1). The total phenolic content in themethanolic extract of S. cumini leaf was17.6mg/g, seed 16.1.mg/g and the pulp8.7mg/g respectively. While the flavonoidcontent in leaf 43.24mg/g, seed 19.1/g andpulp was 12.27mg/g (Fig. 2). These resultsdemonstrated that the total phenolic contentof S. cumini leaves were greater than thecontent found in pulp and seed extracts. Apositive correlation was observed betweentotal antioxidant activity and total phenoliccontent in a study made by Iuliana et al.(2011) in the herbal plants. In our presentstudy a similar linear correlation betweentotal phenolic content and antioxidantactivity (r2 0.464529) has been reported.The results suggested that the phenoliccompounds contribute effectively to theantioxidant activity. The antioxidantcapacity of a compound can be measured bythe ability of the compound to intercept freeradicals by scavenging or trapping methods(Huang et al., 2005).Phenols exhibit variable absorption in theUV or UV/VIS region (Constantine D.Stalikas et al., 2007). Phenolic acids withbenzoic acid carbon framework have theirmaxima in the 200-290 nm range. Onlyexception is Gentisic acid which has anabsorbance at 355nm. The cinnamatederivatives due to additional conjugationshow a broad absorbance band in the region,270 to 360nm.All flavonoid glycones contain at least onearomatic ring and efficiently absorb UVlight. The first maximum, which is found inthe 240-285 nm is due to the A ring andsecond maxima which is in 300 550nmrange is attributed to the substitution patternand conjugation of the C ring.It is evident that phenolics absorb well inUV range and UV detection is thereforeconvenient method to localize a phenol inthe effluent of a column. However no singlewavelength is sufficient for theirsimultaneous monitoring in various naturalplant extracts. Detection at 280nm is oftenused for simultaneous separation of mixturesof phenolic acids. In our study the leafextract showed six peaks (Fig. 4) with thefirst two peaks having maximum absorptionin the range 365 450nm. The first peak at365 nm could be referred to as presence ofQuercetin and it was reported that chalconesabsorb at 365 390nm (Pawar and Salunkhe,2013).Reducing power assay value (RPA)expressed in ascorbic acid equivalents, wasused to determine the antioxidant ability ofdifferent extracts in the present study. TheRPA value for S. cumini leaf extract was the375
Int.J.Curr.Microbiol.App.Sci (2015) 4(9): 372-379Table.1 Antibacterial activity in different parts of the plantS.NoType of Extract123Leaf ExtractPulp ExtractSeed ExtractE.coliZone ofinhibition(mm)15-StaphylococcusZone of Inhibition(mm)21-Fig.1 Standard graph of gallic acidy 0.031893x .017143 r2 0.9949308Fig.2 Standard graph of quercetiny 0.00514x 0.1314 r2 0.998905422Fig.3 Ferric reducing power determination of standard ascorbic acid376
Int.J.Curr.Microbiol.App.Sci (2015) 4(9): 372-379Fig.4 UV Spectrum of leaf sampleFig.5 UV Spectrum of seed sampleFig.6 UV Spectrum of pulp sample377
Int.J.Curr.Microbiol.App.Sci (2015) 4(9): 372-379Fig.7 TLC of phenols and flavinoidsThe third peak at 508 nm, fourth one at536nm, fifth one at 607nm, and sixth one at663.6 nm. UV Spectra was obtained byscanning the sample in the range, 200400nm. The spectra showed gradual rise inabsorbance from 280nm to 400nm.the leaf, seed and pulp extracts. The otherstandards could not be detected in theextracts but the extracts showed other bands,one among which was common in all theextracts (Rf value 0.77) corresponding toflavonols.The seed extract showed maximumabsorbance at 369 nm (Fig. 5) in visiblespectrum which indicates the presence ofrutin in the sample and also at 417nm wherepolyphenolic acids show absorbance. TheUV spectrum shows gradual rise inabsorbance from 280nm to 365 nm. Thepeak value of absorbance at 365 nm couldbe referred to presence of Quercetin (Pawarand Salunkhe, 2013).Determination of antimicrobial activityThe antimicrobial activity of the S. cuminileaves may be due to tannins and otherphenolic constituents. S. cumini is known tobe very rich in gallic and ellagic acidpolyphenol derivatives. Most antibacterialmedicinal plants are more effective againstgram-negative bacteria (Lin et al., 1999;Srinivasan et al., 1989). Our results showedantibacterial activity against both the gramnegative (E. coli) and gram- positive(Staphylococcus aureus) cultures grown inthe leaf extract of S. cumini but pulp andseed extracts did not show any antibacterialactivity. The results of the extract of thesamples are presented in the table 1.The Pulp extract showed two majorabsorption peaks in visible spectrum withfirst peak at 386nm (Fig. 6). This could bereferred to presence of catechol. The secondabsorption band was obtained at 532nm andis characteristic of anthocyanins. Alsomaximum absorption between 365 to 390nmsuggests presence of chalcones as they arereported to absorb in this range. The UVspectra showed gradual rise in absorption inthe range 280-400nm.In conclusion, Syzygium is widely used bythe traditional healers for the treatment ofvarious diseases especially diabetes andrelated complications. The plant has manyimportant compounds which confer mostcharacteristics of the plant. High radicalscavenging activity was observed in all partsof the plant, especially leaves. FurtherThe TLC chromatogram of phenols andflavonoids in our study (Fig. 7) showed thepresence of tannic acid and ascorbic acid in378
Int.J.Curr.Microbiol.App.Sci (2015) 4(9): 372-379investigations are needed from HPLC data,to support with specific identifiable fractionsof compounds, so that this plant could beexploited as an antioxidant additive or anutritional supplement.Morton, J. 1987. Fruits of warm climates.creativeresourcesystems,Winterville, North Carolina. Pp.304 307.Oyaizu, M. 1986. Studies on products ofbrowning reactions: antioxidativeactivities of products of browningreaction prepared from glucosamine.Jpn. J. Nutr., 103: 413 419.Pawar, N.P., Salunkhe, V.R., 2013.Development and validation of U.V.Spectrophotometric method forsimultaneous estimation of Rutin &Gallic Acid in Hydro alcoholicextract of Triphala churna. JPRIF,5(2): 724 729.Perez, C., Pauli, M., Bazerque, P. 1990. Anantibiotic assay by the agar-welldiffusion.Radomir, V., Malba a, Eva, S. Lon ar,Ljiljana A. Kolarov, 2004. TLCAnalysisofsomephenoliccompounds in Kombucha Beverage.APTEFF, 35: 1 280.Ravi, K., Ramachandran, B., Subramanian,S. 2004. Protective effect of Eugeniajambolana seed kernel on tissueantioxidantsinstreptozotocininduced diabetic rats. Biol. Pharm.Bull., 27: 1212 1217.Srivastava, T. 2013. Study of composition,activity and phenolic content ofherbal products. Int. J. Eng. Sci.Technol., 4(4): 1412 1420.Veeru, P., Kishore, M.P., Meenakshi, M.2009. Screening of medicinal plantextracts for antioxidant activity. J.Med. Plants Res., 3(8): 608 612.ReferencesBajpai, M., Pande, A., Tewari, S.K.,Prakash, D. 2005. Phenolic contentsand antioxidant activity of some foodand medicinal plants. Int. J. FoodSci. Nutr., 56: 287 291.Chang, C., Yang, H., Wen, Chern, J. 2002.Estimation of total flavonoid contenton propolis by two complementarycolorimetric methods. J. Food DrugAnalysis, 10: 178 182.Constantine D. Stalikas, 2007. Extraction,separation and detection methods forphenolic acids and flavonoids. J. SepSci., 30: 3268 3295.Ghafar, M.F.A., Prasad, K.N., Weng, K.K.,Isamil,A.2010.Flavinoid,hesperdine, total phenolic contentsand antioxidant activities from Citrussps. Afr. J. Biotechnol., 9(3): 326330.Huang, D., Qu B., Prior, R.L. 2005. Thechemistrybehindantioxidantcapacity assays. J. Agric. FoodChem., 53: 1841 1856.Iuliana Spiridon, Ruxanda Bordirlau,Carmen-Alice Teaca, 2011. Totalphenolic content and antioxidantactivity of plants used in traditionalRomanian herbal medicine. Cent.Eur. J. Biol., 6(3): 388 396.Lin, J., Opoku, A.R., Geheeb-Keller, M.,Hutchings, A.D., Rerblanche, S.E.,Jagar, A.K., Van Staden, J. 1999.Preliminary screening of sometraditional zulu medicinal plants thanopharmacol., 68: 267 274.379
antioxidant, which accounts for the scavenging of free radicals and protective effect on antioxidant enzymes (Ravi et al., 2004). Total phenolics of the seeds said have an important antioxidant activity (Bajpai et al., 2005). The fruit is rich in sugar, mineral salts, vitamins C. Fruit of Syzygium cumini contain malic acid and a small quantity of
The antioxidant parameters including Superoxide dismutase (SOD), glutathione peroxidase and glutathione levels were evaluated [16,17]. Then the animals were sacrificed under anaesthesia using diethyl ether and the tissue samples of liver, kidney and heart were collected for evaluation of antioxidant levels in tissues. Antioxidant Assays
evaluation of the free radical scavenging activity of the drug sample. All eight marketed hepatic formulation showed the capacity of scavenging of free radical. The lowest antioxidant activity is shown by Udiliv i.e. 8.97% and silybon shows the highest antioxidant activity i.e. 60.86%.
Antioxidant property In this experiment, methanol extract of Aporosa wallichii Hook.f. leaves were tested properly through DPPH assay and TPC to determine the antioxidant property of this plant ( Pavithra et al., 2009). Antioxidant activity is very important in preventing free radical reactions because they can neutralize free radical by their
The antioxidant activity of the different solvent extracts of T. erecta flower and its fraction was evaluated by DPPH free radical scavenging activity, superoxide anion radical scavenging activity, ABTS cation free radical scavenging activity, ferric reducing antioxidant power and reducing capacity assessment by the
Theoretical Evaluation of Antioxidant Activity of Tea Catechins N.S. Labidi1*, L. Guerguer 1, A. Kacemi 1 1. Institut des Sciences, Département des Sciences de la Matière, Centre Universitaire de Tamanrasset, BP (10034) Sersouf- Tamanrasset (11000)-Algeria Abstract To quickly evaluate the antioxidant activity of tea catechins epicatechin (EC), -
commonly used stable free radical, to test the potential of compounds as free radical scavengers of hydrogen donors and to investigate the antioxidant activity of plant extracts.[17] Antioxidant molecules when incubated, react with DPPH and convert it into 2, 2 -diphenyl-1-picryl hydrazine, which is a measure of the scavenging
Evaluation of antioxidant activities of flower extract (fresh and dried) . eliminate free radicals and other reactive oxygen and nitrogen species, and these . Table.1 Phytochemical and antioxidant activity analysis of water and ethanol extract of fresh flowers of Saraca indica
the standard three-rail shear test, as described in ‘‘ASTM D 4255/D 4255M The standard test method for in-plane shear properties of polymer matrix composite materials by the rail shear method’’. This setup, however, requires drilling holes through the specimen. In this study, a new design based on friction and geometrical gripping, without the need of drilling holes through the .
