Antioxidant activity of methanolic extract of Bombax ceiba

Please download to get full document.

View again

of 6
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Similar Documents
Information Report
Category:

Science

Published:

Views: 37 | Pages: 6

Extension: PDF | Download: 0

Share
Description
Antioxidant activity of methanolic extract of Bombax ceiba
Tags
Transcript
  Redox Report  2009 Vol 14 No 1  41 Redox Report  2009 Vol 14 No 1  41 Antioxidant activity of methanolic extract of  Bombax ceiba Tiago O. Vieira 1 , Ataa Said 2 , Elsayed Aboutabl 3 , Mona Azzam 2 , Tânia B.Creczynski-Pasa 1 1 Departamento de Ciências Farmacêuticas, Centro de Ciências da Saúde, Universidade Federal de SantaCatarina, Florianópolis, Brazil 2 Department of Pharmacognosy, National Research Center, Cairo, Egypt   3 Department of Pharmacognosy, Faculty of Pharmacy, Cairo, Egypt  The antioxidant activity of a methanolic extract of  Bombax ceiba   was evaluated using severalantioxidant assays, in terms of its:(i) ability to scavenge DPPH (1,1-diphenyl-2-picryl-hydrazyl) andhydroxyl free radicals; (ii) action against lipid peroxidation (in rat liver microsomes and soy beanphosphatidylcholine liposomes), induced by ascorbyl radicals and peroxynitrite; and (iii) effect onmyeloperoxidase activity. The cytotoxicity was monitored through the mitochondrial activity in theVero cell line. The extract showed antioxidant activity in all assays, the EC 50  ( µ g/ml) for DPPH was87 and for lipid peroxidation of microsomes and soy bean liposomes induced by ascorbyl radicalswere 141 and 105, respectively, and by peroxynitrite were 115 and 77, respectively. The K 0.5  valuefor myeloperoxidase activity inhibition by the extract was 264  µ g/ml. The extract showed very lowtoxicity toward Vero cells. Keywords:  Bombax ceiba  , antioxidant, free radical, myeloperoxidase, deoxyribose, lipid peroxidation Introduction Bombaxceiba (syn. Bombaxmalabaricum DC., Salamaliamalabarica DC.Schott&Endl.and Gossampinusmalabarica DC.Merr.)knownasthesilkcottontreeandasSemul,belongstothefamilyBombacaceae.ItisanimportantmedicinalplantwidelycultivatedinPakistan,India,ChinaandAustralia. 1 Theplantiscommonlyusedforthetreatmentofdiarrhea,fever,chronicinflammation,catarrhalaffectionandulcerationofthebladderandkidneyintraditionalsystemsofmedicine. 2 Allpartsoftheplanthavemedicinalsignificance;forexample,itsrootsandflowersareregardedashavingdiuretic,laxative,tonicandrestorativeproperties,whiletheleavesarereportedtohaveapplicationinthetreatmentofskineruptions. 1,2 Also,themethanolicextractofleavesandmangiferin(axanthoneobtaineddirectlyfromthemethanolicextract)haveantioxidantandanalgesicproperties. 3 Phytochemicalinvestigationhasrevealedthepresenceof naphthoquinones,polysaccharides,anthocyanins,shamimin,lupeolandmangiferininthisplant. 4,5 Manyantioxidantcompoundsfromnaturallyoccurringplantsourceshavebeenidentifiedasfreeradicaloractiveoxygenandnitrogenscavengers.Theflavonoidsarethemostimportantgroupamongthenaturalantioxidants.Theirbiologicaleffectsareattributedprincipallytofreeradicalscavengingormetalionchelation.Metalionsaredirectlyinvolvedinthegenerationofreactivespecies. 6 Animbalancebetweentheconcentrationofreactiveoxygenspecies(ROS)andreactivenitrogenspecies(RNS)andtheantioxidantdefensemechanismsofthebodyplaysapivotalroleinmanypathologies. Research article Correspondence to: Tânia Beatriz Creczynski-Pasa, Departamento deCiências Farmacêuticas, Centro de Ciências da Saúde, UniversidadeFederal de Santa Catarina, C.P. 476, CEP 88040-900, Florianópolis, SC,Brazil. Tel: +55 48 37218057; Fax: +55 48 37219542;E-mail: taniabcp@gmail.comReceived 3 March 2008, revised manuscript accepted 5 December 2008 © W. S. Maney and Son Ltd 2009DOI 10.1179/135100009X392485  42  Redox Report  2009 Vol 14 No 1 Vieiraetal. Antioxidantactivityofmethanolicextractof  B.ceiba Inbiologicalsystems,theprimarysourceofallRNSisnitricoxidegeneratedbynitricoxidesynthases.Acombinationwiththesuperoxideanionradicalresultsintherapidproductionofperoxynitritewhichisevenmorereactivethanitsprecursors.AlthoughONOO  –  isnotafreeradical,itsoxidizingpotentialissufficienttoinitiatelipidperoxidationreactionsdirectlybyabstractingahydrogenatomfromapolyunsaturatedfattyacidandalsotoformnitratedlipids. 7 Also,ONOO  –  mayinduceDNAdamage, 8 enzymeandionchannelinactivationviaproteinoxidationandnitration, 9 andinhibitionofmito-chondrialrespiration. 10 Additionally,atlowconcent-rations,peroxynitritetriggersapoptoticcelldeath,whereasathighconcentrationsitinducesnecrosis. 10 Myeloperoxidaseexertsanimportantroleininnatehostdefenseresponseandalsoininflammatoryprocesses. 11 Theantimicrobialactionofmyelo-peroxidasehasbeenattributedtoitsuniquepropertytoproducehypochlorousacidandothertoxicagentsthatcreateanenvironmentwithinthephagolysosomeofneutrophilsthatinhibitsordestroysforeignmicro-organisms.Antioxidantdefenses(enzymaticand/ornon-enzymatic)protectorganismsagainsttheoxidativestresscausedbytheinvasionofmicro-organisms.Theoverproductionofhypochlorousacidanditsderivativescandamagehosttissues.Inthisstudy,weinvestigatedthe invitro anti-oxidantpotentialofamethanolicextractof  B.ceiba including,forthefirsttime,actionagainstlipidperoxidationinducedbyascorbylradicalsandperoxynitriteintwolipidmembranemodels– liposomesandmicrosomes.ThescavengingpropertiesintermsofhydroxylandDPPHradicals,andtheinhibitoryeffectregardingmyeloperoxidaseactivity,werealsoanalyzed.Additionally,wereportthelowcytotoxicityoftheextractstowardtheVerocellline. Materials and methods Reagents Hexadecyltrimethyl-ammoniumbromide(HTMAB), θ -dianisidine,2-deoxy- D- ribose,1,1-diphenyl-2-picryl-hydrazyl(DPPH),thiobarbituricacid(TBA),[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide](MTT)andsoybeanphosphatidylcholinewerepurchasedfromSigmaChemicalCompany(EUA).Allotherreagentswereofanalyticalgrade. Plant material Samplesof  B.ceiba flowerswerecollectedfromplantsgrowingintheOrmanGarden,Giza,Egypt,inApril–May2003andauthenticatedbyProf.DrK.El-Batanony(ProfessorofTaxonomy,Facultyof Science,CairoUniversity).AvoucherspecimenwasdepositedintheNRCherbarium(number3241).Sampleswereairdriedandthenpowderedandkeptinawell-sealedcontainer. Extraction Thedriedpowder(380g)of  B.ceiba flowerswascontinuouslyextractedwithlightpetroleumether(40–60ºC)andthenwithetherinasoxhletapparatus;thedefattedpowderwasexhaustivelyextractedwithchloroformfollowedby70%methanol.Theextractwasevaporatedtodrynessunderreducedpressuretogivearesidue.Thephytochemicalanalysisofthisplantextractshowedtracesoftannins,alkaloids,flavonoids,sterols,triterpenes,saponinsandcoumarins.Theplantconstituentshavebeenpreviouslydescribedintheliterature. 4,5 DPPH radical scavenging activity  DPPHscavengingactivityoftheextractswasmeasuredusingapublishedmethodwithslightmodifications. 12 Theassayisbasedontheincubationofthereactionmediumfor30minat37ºCinanethanolicsolutionof150  µ MDPPHandtheopticaldensityisthenmeasuredat515nm.TheantioxidantactivityoftheplantextractwasexpressedasEC 50 ,whichwasdefinedastheextractconcentrationrequiredtoreduce50%oftheDPPHmolecules. Liposome preparation Bilayerliposomeswerepreparedbyvesicledispersion.Soybeanphosphatidylcholine(50mg/ml)wasdissolvedinchloroformanddriedunderastreamof nitrogengas,followedbyaperiodundervacuum.Thedriedlipidfilmswerehydratedwith10mMTricineand2.5mMMgCl 2 bufferatpH7.4. 13 Microsome preparation Microsomeswerepreparedfromratliverbydifferentialcentrifugationwithcalciumaggregation. 14 Thefractionsobtainedwerestoredat–84ºC.TheproteinconcentrationwasdeterminedaccordingtotheLowrymethod. Lipid peroxidation induced by ascorbyl radicals and  peroxynitrite Lipidperoxidationwasinducedbytheadditionof25 µ MFeSO 4 and500  µ Mascorbicacid,forascorbylradicalsor2.7mMperoxynitrite 15 inareactionmediumcontaining2mgmicrosomalprotein/mlorliposomes(lipidsat12.5mg/ml),and0.1MTris-HCl,  Redox Report  2009 Vol 14 No 1  43 Vieiraetal. Antioxidantactivityofmethanolicextractof  B.ceiba pH7.4.Thesampleswereincubatedfor30minat37ºCandtheextentoflipidperoxidationwasdeterminedbythethiobarbituricacidmethod. 16 Theamountof thiobarbituricacidreactivesubstances(TBARS)wascalculatedusinganextinctioncoefficientof1.56×10 5 M  –1 cm  –1 .Inallcases,ablankrunwasincludedusingonlytheorganicsolvent(inthesameamount)toassessitsinterferenceintheassays.LipidperoxidationinhibitoryactivitywasexpressedasIC 50 . Production and detection of hydroxyl radical ThehydroxylradicalwasproducedusingavariationoftheFentonreaction,throughthemixtureof hydrogenperoxidewithaFeCl 3  –NTAsystem.Thereactionmediumcontaining25  µ MFeCl 3 ,100  µ Mnitrilotriaceticacid(NTA),100mMphosphatebufferpH7.4,2.8mMdeoxyriboseand1.4  µ Mhydrogenperoxidewasincubatedat37ºCfor20minunderdifferentextractconcentrations.Subsequently,2.8%TCAand1%TBAwereaddedtothereactionandkeptat100ºCfor15minfollowedbyimmersioninanicebath.Productsofdeoxyriboseoxidationweredeterminedspectrophotometricallyat532nm. 17 Fordeoxyriboseoxidationstudies,theextractsweredissolvedin0.05MNaOHandthepHwasadjustedto7.4with0.1MHCl.Organicsolventswerenotusedforpreparingsolutionssincetheyinterferewithhydroxylradicaldetermination. Myeloperoxidase activity  Ratlungswerehomogenizedinanice-cold50mMphosphatebufferatpH6.0,containing0.5%HTMABaspreviouslydescribedandfreeze-thawedthreetimes. 18 Thesampleswerecentrifugedat12,000  g  at4ºCfor20min.Thesupernatantwasassayedinareactionmediumcontaining50mMphosphatebuffer,pH6.0at25ºC,  θ -dianisidine(0.167mg/ml)andH 2 O 2 (0.0006%). 19 Theenzymeactivitywasdeterminedfromtheslopeoftheabsorptioncurveat450nm,followingthechangesinabsorptionforthefirst30sineachsampleconcentrationoftheextracts.Astandardcurveofmyeloperoxidaseactivitywasobtainedusingacommercialenzymepreparation.Sodiumazide(100 µ M)wasusedtoinhibitmyeloperoxidaseactivityasacontrolfortheassay. Cytotoxicity  Thecytotoxiceffectoftheextractwasexpressedascellviability.Verocells(monkeyepithelialkidneycells;ATCC:CCL81)weregrowninDulbecco’sModifiedEagle’sMedium(DMEM),supplementedwith10%fetalbovineserum,100U/mlpenicillin,100  µ g/mlstreptomycin,2mM L- glutamine,1.5g/lsodiumbicarbonateand10mMHEPES(pH7.4).Cellswereplatedatadensityof6.0×10 4 perwellin96-wellplates.After24hofincubationat37ºCinahumidified5%CO 2 atmosphere,themediumwasremovedfromeachwellandreplenishedwithdifferentconcentrationsofextractssolubilizedinDMSO(maximally2%).Thecellswereexposedfor24htothemediumwhichwasthenremoved,0.5mg/mlofMTTwereaddedandthecellswereincubatedfor2hat37ºC.TheMTTsolutionwasremovedandisopropylalcohol/0.04MHClwasaddedtodissolvetheformazancrystals.Theabsorbancewasmeasuredinamultiwellspectrophotometerat540nm.Tocalculatethecellviabilityvalues,theresultsweretransformedtopercentageofcontrols.Forthecellcultureexperi-ments,statisticalsignificancewasassessedbyANOVAfollowedbyBonferroni’s t -test,and P <0.05wastakenasstatisticallysignificant( n =3). Results and discussion DPPHiscommonlyusedasatooltoevaluatethefreeradicalscavengingactivityofnewcompounds.Thisisastablefreeradicalwhichisreducedwhenitreceivesanelectronorhydrogenatom.Hydrogendonatingabilityisanindexoftheprimaryantioxidants. 20 ThecapabilityofreducingtheDPPHradicalisdeterminedbythedecreaseinabsorbanceinducedbythecompounds.Aconcentration-responserelationshipwasobservedfortheDPPHradicalscavenging Figure1DPPHradicalscavengingeffectofmethanolicextractof  B. ceiba  .Theassaywascarriedoutspectrophotometricallyinthepresenceof150  µ MDPPH. The results are the mean ± SEM oftriplicates from three independent experiments  44  Redox Report  2009 Vol 14 No 1 Vieira et al. Antioxidant activity of methanolic extract of B. ceiba activity (Fig. 1). The methanolic extract of B. ceiba showed an EC 50 value of 87 ± 3 µ g/ml. It has beenfound that cysteine, glutathione, ascorbic acid,tocopherol, flavonoids, tannins and aromatic aminesreduce and decolorize DPPH through their hydrogendonation ability. 21 Phenolic compounds in themethanolic extract of B. ceiba are probably involved intheir antiradical activity.Lipid membranes are the principal targets of reactive species and it is well known that suchmembranes vary in lipid composition and theirresponse to ROSand RNSaction. We tested whetherthe methanolic extract of B.ceiba was able to inhibitlipid peroxidation induced by ascorbyl radicals andperoxynitrite in rat liver microsomes and soy bean PCliposomes.Figure 2 shows the IC 50 values for ascorbylradicals, which were 141 ± 5 µ g/ml and 105 ± 2 µ g/ml,respectively, while those for peroxynitrite were 115 ± 3 µ g/ml and 77 ± 4 µ g/ml, respectively. Membrane lipidsare rich in unsaturated fatty acids that are mostsusceptible to oxidative processes, an important causeof cell membrane destruction and cell damage.However, lipid peroxidation cannot be used as a singlecriterion of oxidative stress. 22 The inhibition of lipidperoxidation by antioxidants may be due to their freeradical scavenging activity and the fact that flavonoidscan interact with lipids in liposomes, intercalatingbetween the acyl chains of lipids and stabilizingmembranes by decreasing membrane fluidity. Thus,they can reduce the diffusion of free radicals and,thereby,decrease the kinetics of free radicalreactions. 23 The literature shows, for example, thatsome flavonoids are scavengers of peroxynitrite. 24 The extract tested in this study was also found to be apotent scavenger of hydroxyl radicals, one of the mostdestructive oxidants known, produced from the high-energy ionization of water, principally through theFenton and Haber–Weiss reactions. 25 In this regard, theplant extract was effective in avoiding the oxidation of deoxyribose at very low concentration (Fig.3). Theconcentration dependence of deoxyribose on itsoxidative degradation by Fenton reagents in the absenceor presence of 200 µ g/ml of extract was investigated. Toverify this effect, the concentration of deoxyribose in thereaction medium was gradually increased and the effectof the extracts in preventing the deoxyribose oxidationby HO • decreased in a concentration-dependent way.This result indicates that the extracts and deoxyribosecompete in terms of HO • trapping.Although our study did not clarify whether theplant extract only scavenges HO • or if it also acts as an Figure 2Comparison of the effect of the methanolic extractof B. ceiba  in different lipid membrane models. Theresults are expressed as percentage of lipidperoxidation induced by ascorbyl radicals andperoxynitrite in liver microsomes (circles) and soybean liposomes (squares). The value consideredas 100% of peroxidation in microsomes inducedby ascorbyl radicals was 24.6 ± 1.1 µ mol/mg ofprotein and by peroxynitrite 18.3 ± 1.4 µ mol/mg ofprotein. The results are the mean ± SEM oftriplicates from three independent experiments   Figure 3Effect of deoxyribose concentration on theoxidative degradation of deoxyribose by hydroxylradicals in the absence or presence of B. ceiba  methanolic extract. Inset: decrease in protectionagainst deoxyribose degradation with an increasein deoxyribose concentration. Re-plot of the dataobtained directly, in the presence of plant extract.The results are the mean ± SEM of triplicates fromthree independent experiments  Redox Report  2009 Vol 14 No 1  45 Vieira et al. Antioxidant activity of methanolic extract of B. ceiba Fe 2+ chelator, 17 it did indicate that there is competitionbetween the adductor (deoxyribose) and the extracts,suggesting scavenging action.Myeloperoxidase is an enzyme present inneutrophils which plays a central role in infection andinflammation. The principal physiological action of this enzyme is to convert hydrogen peroxide andchloride ions to HOCl. The results of a previous studysuggested that myeloperoxidase is a catalyst for lipidperoxidation via tyrosyl radical formation. 26 Themethanolic extract of B.ceiba inhibited the enzymeactivity, as can be seen in Figure 4 (inset), which showsthe kinetics of enzyme inhibition and the re-plotteddata giving the K 0.5 value (264 ± 6 µ g/ml). The enzymeactivity was determined from the slope of theabsorption curve measured at 450 nm for the first 30 s ineach concentration of the plant extract. Kato andcollaborators 27 demonstrated a strong myeloperoxidaseinhibition by phenolic antioxidants such as quercetin,curcumin, and ferulic, caffeic and gallic acids. Previousstudies have demonstrated that certain anti-inflammatory drugs are able to inhibit myelo -peroxidase activity and this inhibition may account fortheir anti-inflammatory action, 28 having a beneficialeffect against oxidative stress.The cytotoxicity of the methanolic extract of B.ceiba was also investigated. The cytotoxicity of acompound can be determined by measuring cytotoxicparameters, such as the integrity of the cell and theeffect on cell growth. Since the latter is a more sensitivecytotoxic parameter and more appropriate forpharmacological studies, the cytotoxicity of this plantextract was investigated on growing Vero cells using anMTT assay. This is because, although flavonoids arefrequently described as not being toxic, some studieshave shown that many flavonoids have cytotoxicpotential due to their activity as topoisomerase IIinhibitors 29 and they are able to influence a variety of cell functions by cell signaling modulation. 30 In theassay carried out here, the maximal non-toxicconcentration was defined as the highestconcentration of the plant extract that did not affectcell growth compared with the controls. Theconcentration that induced 30% of the cell death rateor above was considered cytotoxic. Up to 500 µ g/ml of the extract the cell viability was maintained at 97 ±5.8% (results not shown). The methanolic extract of B.ceiba started to show toxicity toward cells at 750 µ g/ml, i.e . at a much higher concentration than thosewhich showed antioxidant activity. Conclusions The methanolic extract of B. ceiba showed significantantioxidant potential against ROS and RNS and thisproperty may be related to its pharmacological activities.The antioxidative effect of the plant extract is possiblydue to the presence of phenolic components. Furtherstudies will be aimed at the isolation and character -ization of the substances responsible for the antioxidantpotential of the plant extract.  Acknowledgements This work was supported by Conselho deDesenvolvimento Científico e Tecnológico (CNPq),Fundação de Fomento à Pesquisa de Santa Catarina(FAPESC) and Coordenaçao de Aperfeiçoamento dePessoal de Nivel Superior (CAPES) in Brazil; and ThePharmacognosy Department of The NationalResearch Center, in Egypt. References 1.Chanda YR. The Wealth of India, Raw Material  . New Delhi: CSIR,1962; 175–183.2.Nadkarni KM. The Indian Materia Medica, Popular Parkashan,Bombax . 1976; 207–209.3.Dar A, Faizi S, Naqvi S et al  . Analgesic and antioxidant activity of  Figure 4Inhibition of myeloperoxidase activity by themethanolic plant extract of B. ceiba  . Kinetics ofenzyme inhibition calculated from the data shownin the inset to obtain the K 0.5 value. The valueconsidered as 100% of enzyme activity was 4.0 ± 1.0 U/min/mg of protein. The activity wasmeasured spectrophotometrically at 450 nm
Recommended
View more...
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks
SAVE OUR EARTH

We need your sign to support Project to invent "SMART AND CONTROLLABLE REFLECTIVE BALLOONS" to cover the Sun and Save Our Earth.

More details...

Sign Now!

We are very appreciated for your Prompt Action!

x