Antiplasmodial and Cytotoxic Activity of Piper Piedecuestanum Trel. and Yunck

OPEN ACCESS Research Journal of Medicinal Plants ISSN 1819-3455 DOI: 10.3923/rjmp.2018.57.64 Research Article Antiplasmodial and Cytotoxic Activity of Piper Piedecuestanum Trel. and Yunck 1,2Ana María Mesa Vanegas, 1Jhon Fredy Toro Suaza, 1Ana María Vásquez Cardona, 1Silvia Blair Trujillo, 2Carlos Peláez Jaramillo, 3,4Santiago Díaz Oltra, 3César Augusto Pachón and 3Miguel Carda 1Malaria Research Group, University Research Headquarters (SIU), Carrera 62 52-59, Tower 1, Lab. 610 SIU, Faculty of Medicine, University of Antioquia, A.A 1226, Medellín, Colombia 2Interdisciplinary Group of Molecular Studies GIEM, Faculty of Exact and Natural Sciences, University of Antioquia, A.A 1226, Medellín, Colombia 3Department of Inorganic and Organic Chemistry, Universitat Jaume I, 12071, Castellón, Spain 4Department of Education, Universitat Jaume I, 12071, Castellón, Spain Abstract BackgroundandObjective:Plasmodiumresistancetoantimalarialdrugshasexpandedandintensified,makingnewandeffective antimalarialdrugsurgently.Theobjectiveofthisworkwasthe invitro evaluationofantiplasmodialactivityofextractsofdifferentpolarity andcompoundsofthespecies P.piedecuestanum .MaterialsandMethods:Theplantmaterialswereobtainedthroughsuccessive extractionsusingsolventsofdifferentpolaritysuchashexane(H),dichloromethane(D),ethylacetate(A)andmethanol(M)and separationstechniquesforfractionationandisolationofcompounds.Theantiplasmodialactivitiesoftheextractsandcompoundswere evaluatedbySYBRGreenI®methodandevaluatedthecytotoxicityinthecelllinesU-937,HUVECbytheMTTmethod.Results:The antiplasmodialandcytotoxicactivityoftheextractsofdichloromethane(PPD)andethylacetate(PPAE)withantiplasmodialactivity ofIC50=17.93µgmLG1;IS=2.093andIC50=19.5µgmLG1;IS=0.791,respectivelyarereportedforthefirsttime.Inaddition,from P.piedecuestanum specieswereisolationandcharacterizationfivemetabolites5,8-Hydroxy-7-methoxyflavone(1),6,7-dimethoxy-5,8dihydroxyflavone(2),6,7-dimethoxy-5-hydroxyflavone(mosloflavone)(3),5,6-dihydroxy-7-methoxyflavone(negletein)(4),5-hydroxy-7methoxyflavone(5)andabrominatedderivativefrom(5)named6,8bromo-5-hydroxy-7-methoxyflavone(7).Compound(1)presented promisingantiplasmodialactivitywithanIC50=7.325µgmLG1(25.69µM);ISHUVEC=13.65.Conclusion:Chemicalanalysisofextractsand compoundsfrom P.piedecuestanum spices  willplayacentralroleinthedevelopmentandmodernizationofanantimalarialherbal traditionalinColombia. Keywords:  Piperpidecuestanum ,antiplasmodialactivity,cytotoxicactivity, Plasmodiumfalciparum Citation: AnaMaríaMesaVanegas,JhonFredyToroSuaza,AnaMaríaVásquezCardona,SilviaBlairTrujillo,CarlosPeláezJaramill,SantiagoDíazOltra,César AugustoPachón,MiguelCarda,2018.AntiplasmodialandcytotoxicactivityofpiperpiedecuestanumTrel.andYunck.Res.J.Med.Plants,12:57-64. CorrespondingAuthor:AnaMaríaMesaVanegas,UniversityofAntioquia,FacultyofExactandNaturalSciences,InstituteofBiology,UniversityofAntioquia, A.A1226,Medellín,ColombiaTel:+573017947846 Copyright: ©2018AnaMaríaMesaVanegas etal .ThisisanopenaccessarticledistributedunderthetermsofthecreativecommonsattributionLicense, whichpermitsunrestricteduse,distributionandreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. CompetingInterest: Theauthorshavedeclaredthatnocompetinginterestexists. DataAvailability: Allrelevantdataarewithinthepaperanditssupportinginformationfiles. Res.J.Med.Plants,12(2):57-64,2018 INTRODUCTION Malaria is a disease caused by protozoan parasites belongingtothefamilyPlasmodiidae,genus Plasmodium, whicharetransmittedbyfemalemosquitoesofthegenus Anopheles1.Currently,malariaisapressinghealthproblemin many parts of the world, particularly in Africa and Latin America, whicharetheregionswiththehighestmortality rates.Recentdataindicatethatmalariaispresentin 97countriesandanestimated3.2billionpeopleareatriskof contractingthedisease2.Intheyear2015,214millioncasesof malariawerereportedand438.000peoplediedmostofthem childrenunder5yearsofage.Mostofthesecasesoccurred inAfrica3.Atpresent,thesituationisbecomingevenmore complicated by the spread of drug resistant parasites, especially in areas where Plasmodium falciparum and Plasmodiumvivax areendemicandofhigherprevalence. Few alternative drugs are under development and urgent measuresarerequiredtoidentifynewclassesofantimalarial agents, many of which have their origins from natural products4. Piper,thenominalgenusofthefamilyPiperaceae,isone ofthemostdiversegeneraofbasalangiosperms.Itiscurrently considered to have about 1500 species and its greatest diversity is found in the humid forests of tropical regions around the world5. Ecologically, they are important and dominantcomponentsinthehumidforests,especiallyinthe neotropicsandtheyconstituteanimportantpartofthedietof some families of American bats, insects and birds6-8. Few speciesofPiperareeconomically important,amongthem Pipernigrum fromwhichthepepperisobtained,condiment popularlyusedallovertheworld.ForthePipergenus,awide rangeoftraditionalfoodshasbeenreportedinthetreatment ofvariousdiseasessuchasmalaria,anemia,cholera,diabetes, asthma,bronchitis,pneumonia,influenza,rheumatismand  arthritis. In this study, plants were used as condiments, aphrodisiacs,stimulantsandhallucinogens9-11.Chemically,in Piperaceae,lignansandneolignanshavebeenfound,tannins, saponins, phenolic compounds, terpenes, flavonoids and alkaloids among others12. Many of these compounds, especiallyalkaloids,terpenesandlignans,areresponsible fortheantiplasmodialactivityofmanyspeciesreportedin other parts of the world13,14. A large variety of Piperaceae speciesareusedbytraditionalmedicinetotreatmalaria,some ofwhichhavebeenthesubjectofantiplasmodialactivityboth invitro and invivo 15.Regardingtheantiplasmodialpotential ofextractsandcomponentsof P.piedecuestanum Trel.and Yunck.,therearenotreportsintheliteratureandthereisa singlereportinvestigatingtheantioxidantactivityofthese extracts16. Thus,thepresentstudywasaimedtoobtainextractsof differentpolarityofthespecies P.piedecuestanum Trel.and Yunck,isolatedandcharacterizedtheirmajorcomponents. Evaluate antiplasmodial activity in  vitro   on continuous culturesof P.falciparum chloroquine-sensitivestrainNF-54 and evaluated the cytotoxicity activity of theextracts and compoundsinthecelllinesU-937andHUVEC. MATERIALSANDMETHODS AllexperimentswererealizedinLaboratoryofMalaria Research Group. University Research Headquarters (SIU), UniversityofAntioquia,Medellin,ColombiaandDepartment of Inorganic and Organic Chemistry, Universitat Jaume I, Castellón,Spain. Chemical and solvents: hexane, dichloromethane, ethyl acetate,methanol,chloroform,ethanol,dimethylsulphoxide (DMSO),Silica-gel60,sulfuricacid,N,N-Dimethylformamide (DMF),N-Bromosuccinimide(NBS),RPMI-1640,HEPES, SYBR Green I®, 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT)and standard chloroquine were used.Analyticalgradereagents/chemicalswerealsousedin thisexperiment. Collectionandidentificationofplantmaterial:Threetypes ofsampleswerecollectedfromtheplantmaterial:Asample forherbariumspecimen,samplesascontrolsofspecimens collected and samples of leaves and stems to obtain the extracts. The specimen for herbarium was processed, depositedandtaxonomicallycharacterizedintheherbarium oftheUniversityofAntioquia(HUA)anddeterminedas Piperpiedecuestanum Trel.andYunck.(Voucherf.191.1950) collectedinPiedecuesta,Santander-Colombia. Preparationofextractsandisolationofcompounds: Piperpiedecuestanum (PP)plantmaterialwassubjectedto adesiccationprocessatroomtemperaturewithaerationand withoutexposuretosunlightfor10days.About2.36gof mixtureofleavesandstemsofthegroundvegetablematerial wasinitiallytakentoapercolationprocessuntilexhaustion (5days/3times)usingethanol(E)thatwasthenfilteredand concentratedonarotaryevaporator.Ontheotherhand, 0.36kgofgroundmaterialwassubjectedtoextractionwith solventsofupwardpolarity(500mL)byapercolationprocess toexhaustionwitheachofthefollowingsolvents:hexane(H), dichloromethane(D),ethylacetate(A)andmethanol(M).After 3days,theextractwasconcentratedunderreducedpressure inarotaryevaporator.Thefiveextractsoftheplantwere 58 Res.J.Med.Plants,12(2):57-64,2018 codedwiththeinitialsofthespeciesnameandthetypeof extractandaccordingtothepolarityofthesolvent,starting with the petroleum ether (H) (PPH), dichloromethane (D) extract(PPD),ethylacetate(EA)(PPAE),methanol(M)(PPM) and ethanolic extract encoded as (PPE). All extracts were monitoredbythinlayerchromatography(CCD)supported withMerck®Silica-gel60GF254usingdifferentelutionsystems. Theextractionpercentagesoftheextractswerecalculated accordingtoEq.1: (1) Extracts weight deExtraction (%) 100 Weight of plant material  Theextractsconsideredasactivewereprocessed byfractionationandisolationofthemajorsubstances. Theextractofdichloromethane(PPD)ofthespeciesof P.piedecuestanum Trel.andYunck.(4.89g)wasactiveand wasfractionatedbycolumnchromatographyusingaseluent gradientsofpetroleumether:ethylacetate,EtOAcandMeOH. Thirtyfractionswereobtainedfromwhichfraction10was taken and column chromatography was performed using petroleumether,petroleumether:DCM(1:1),DCM,gradients of DCM: EtOAc and finally MeOH as eluent. Twenty three fractionswereobtainedwhichwerepooledbetween1-7and preparativeplatechromatographywasperformedusingDCM astheeluentand20mgofthecompounddesignatedas(4) were purified. On the other hand, to section 25 of the dichloromethane extract column, preparative layer chromatographywasperformedandtwocompoundswere isolated,oneofwhichwasayellowamorphoussolidnamed (2)(31.8mg)andtheotheracrystallineorangesolidreferred toas(1)(183.9mg).Theremainingfractionsofthepetroleum etherextractanddichloromethanewerecombinedtoperform column chromatography again using petroleum ether gradients: DCM, DCM: EtOAc, EtOAc: Methanol (MeOH) gradients and finally MeOH and obtained 14 fractions, fractionsof6-10weretakenandcolumnchromatography usingeluentpetroleumether,petroleumether:DCM(1:1), DCM and DCM: MeOH gradients as eluents, whereby 30 fractions,fractions1-9and15-17weretakenforpreparative platechromatographyelutingwithpetroleumether: DCM25:3andDCMtoisolate39mgofthecompound(4), 59mgofcompound(3)and58mgofcompound (5). Halogenated atoms were introduced into the isolated compoundsbytheproductionofbrominatedderivatives.Only asuccessfulreactionwasperformedfromcompound(5). The experimental procedure is briefly described below: a solutionof(5)(20mg,0.71mmol)wasdissolvedinN, N-Dimethylformamide(DMF)andN-BromosuccinimideNBS (0.71mmol)wasadded.Thereactionmixturewasleftat0EC for2hundernitrogen.Subsequently,thetemperaturewas gradually raised to 80EC for 24 h. The crude mixture was diluted in dichloromethane (10 mL) and washed with saturated aqueous ammonium chloride (3×10 mL). The organic layer was dried (Mg2SO4) and concentrated under reduced pressure to give compound (6). All extracts and compounds were stored at room temperature for the biologicalassays. Structural characterization: IR spectra were obtained by using KBr pellets on a Jasco FT/IR-6200 spectrometer, spanning the region 4000-600 cmG1. Mass spectra were measured on a Q-TOF mass spectrometer (Waters, Manchester,UK)withelectrospray-typecombinedionization sourceandZ-spraydesignAPCI;thecapillaryvoltageof3.5KV wasusedinthepositivedirectionandtheconevoltagewas set at 20V. NMR spectra were recorded on Varian Unity spectrometers of 300 and 500 (approximate operating frequencies,300and500MHzfor1H,125and75MHzfor13C). The nature of the carbon signals (C, CH, CH2, CH3) was determined using the APT or DEPT techniques. Signal assignments were performed using two-dimensional heteronuclearcorrelations(COSYandHMQC/HMBC).Unless otherwise indicated, the spectra were measured in CDCl3 solution.Chemicalshifts(δ)arereportedinppmusingthe residualsolventsignals(δ7.27ppmfor1Hand77.0ppmfor 13C)asreference.Asfarasthereferencingofthespectrawhen itcomestomultiplets,therangetheyoccupyisincluded. Compound (1): 5,8-Hydroxy-7-methoxyflavone, yellow amorphoussolid.C16H12O5.TOFMSES+[M+H]:285.0760. 1H-NMR-300MHz δ(chloroform-d1)(ppm):12.50(1H,s, C5-OH),7.89(2H,dd,J=2.2,8.0Hz,Ar-H),7.56-7.52(3H,m, Ar-H),6.69(1H,s,C6-H),6.63(1H,s,C3-H),4.018(3H,s,CH3O). 13C-NMR-75 MHz δ (chloroform-d1) (ppm): 183.06, 165.59, 162.15,153.33,146.04,132.19,131.88,130.048,129.49 (CH×2),126.67(CH×2),105.91,105.85,90.95,56.8817. Compound(2):6,7-dimethoxy-5,8-dihydroxyflavone,yellow amorphoussolid.C17H14O5.TOFMSES+[M+OH]:315.0869. 1H-NMR-300MHz δ(chloroform-d1)(ppm):12.63(1H,s, C5-OH),8,20(2H,dd,J=2.2,8.0Hz,Ar-H),7.53-7.51(3H,m, Ar-H),6.82(1H,bs,C8-OH),6.56(1H,s,C3-H),3.98(3H,s,CH3O), 3.94(3H,s,CH3O).13C-NMR-75MHzδ(chloroform-d1)(ppm): 197.36,174.85,159.77,152.88,151.92,136.96,132.52,130.72, 129.04 (CH×2), 128.03 (CH×2), 96.95, 91.36, 86.65, 61.37, 56.8017. 59 Res.J.Med.Plants,12(2):57-64,2018 Compound (3): 6,7-dimethoxy-5-hydroxyflavone (mosloflavone),yellowamorphoussolid.C17H14O5.TOFMSES+ [M+H]:299.0919.1H-NMR-500MHzδ(chloroform-d1)(ppm): 12.68(1H,s,C5-OH),7.88(2H,dd,J=2.2,8.0Hz,Ar-H), 7.54-7.52(3H,m,Ar-H),6.66(1H,s,C3-H),6.56(1H,s,C8-H), 3.97 (3H, s, CH3O), 3.93 (3H, s, CH3O). 13C-NMR-125 MHz δ (chloroform-d1)(ppm):182.66,163.90,158.87,153.27,152.99, 132.66,131.80,131.25,129.05(CH×2),126.20(CH×2),106.24, 105.56,90.62,60.80,56.2917. Compound(4):5,6-dihydroxy-7-methoxyflavone(negletein), yellow amorphous solid. C16H12O5. TOF MS ES+ [M+H]: 285.0767.1H-NMR-300MHzδ(chloroform-d1)(ppm):11.72 (1H,bs,C5-OH),8.20(2H,dd,J=2.2,8.0Hz,Ar-H),7.53-7.48 (3H,m,Ar-H),6.51(1H,s,C3-H),6.39(1H,s,C8-H),3.90(3H,s, CH3O). 13C-NMR-75 MHz δ (chloroform-d1) (ppm): 194.62, 166.54,166.0,161.55,145.36,131.97,130.314,129.49,129.46 (CH×2),129.08(CH×2),114.04,104.95,94.04,57.6017. Compound (5): 5-hydroxy-7-methoxyflavone, yellow amorphoussolid.C17H14O5.TOFMSES+[M+H]:269.0810. 1H-NMR-500MHz δ(chloroform-d1)(ppm):12.72(1H,s, C5-OH),7.88(2H,dd,J=2.2,8.0Hz,Ar-H),7.53-7.52(3H,m, Ar-H),6.67(1H,s,C3-H),6.50(1H,s,C6-H),6.37(1H,s,C8-H),3.88 (3H, s, CH3O). 13C-NMR-125 MHz δ (chloroform-d1) (ppm): 182.46,165.59,162.15,157.76,131.80,131.28(CH×2),129.05 (CH×2),126.64,105.83,105.67,98.17,92.65,55.7817. Compound (6,7): 6,8 bromo-5-hydroxy-7-methoxyflavone, crystalyellowcolor.C16H11O4Br2.TOFMSES+[M+H]:426.9007. 1H-NMR-500MHz δ(chloroform-d1)(ppm):12.72(1H,s, C5-OH),7.89(2H,dd,J=2.2,8.0Hz,Ar-H),7.53-7.52(3H,m, Ar-H),6.67(1H,s,C3-H),3.88(3H,s,CH3O)17. Biologicalactivitytest Invitro determinationofantiplasmodialactivity: Invitro antiplasmodialactivityassayswereperformedonthesensitive (NF-54)chloroquinestrain. P.falciparum strains(NF-54)were culturedandmaintainedaccordingtothemethodof TragerandJensen18,usingasuspensionof5%human A+erythrocytesinRPMI-1640culturemedium(SigmaR6504) dissolvedinsterilewaterwith25mMHEPES,5.0%NaHCO3, 10%freshhumanA+serum(inactivatedat56ECfor30min) incubatedin5%O2,5%CO2and90%N2anatmosphere.Fresh red blood cells were added twice a week. In vitro antiplasmodial activity by the SYBR Green I® method was performedintheMalariaGroup,accordingtothe methodologydescribedbySmilkstein etal .19.Assayswere performedonFalcon®96-wellflatbottomplates.Asuspension ofparasitizedredbloodcellswithahematocritof2.5%anda parasitemiaof1%wereprepared.Cultivationwithtreatments andchloroquine(CQ)positivecontrolwereincubatedat37EC for48hin5%CO 2, 5% O2 and 90% of N2 atmosphere. Subsequently,thecontentsofeachwellweretransferredto GreinerProonedishesandtheparasiteswerelabeledwitha solutionofSYBR®GreenI2Xinlysisbuffer.Theplateswere incubatedatroomtemperatureinthedarkforonehourand the relative fluorescence units (RFU) were read on a spectrofluorometerat485nmexcitationwavelengthand 538nmemissionwavelength.Treatmentswerepreparedtoa stocksolutionof10mgmLG1inpureDMSOandsonicatedto facilitatedissolution,firstdilutionwas1%andshowedtobe non-toxictotheparasite.Fromthissolution,50µLweretaken andadjustedto1000µLwithcompleteRPMI-1640medium, obtaining a final concentration of 0.5 mg mLG1. Seven concentrations of each extract were evaluated in a range between100-1.56µgmLG1.Eachconcentrationwasevaluated intriplicateontheplateandthreeindependentassayswere performed.TheCQcontrolwasevaluatedinarangebetween 150-4.7nMandthecontrolofPeruvianquinaextract(MeOH: H2O;70:30)wasevaluatedintherangeof0.01-10µgmLG1. Datafromthreetrialswereanalyzedtofindtheinhibitory concentrationinµgmLG1(IC50).Inhibitoryconcentrations 50(IC50±SD)werecalculatedforeachcompoundfroma non-linearlogisticregressionmodel.Thedatawereanalyzed andplottedusingGraphPadPrism4forMacintoshversion 4.0b which outputs the adjustment value (r) (GraphPad Software, San Diego, California, USA). To classify the antiplasmodialactivityofanextract,theMalariaGroupofthe University of Antioquia established a consensus for the extractsevaluated:highlyactive<5µgmLG1,promising 6-15µgmLG1,moderateactivity16-30µgmLG1,lowactivity 31-50µgmLG1andnon-active>50µgmLG120-22.Toclassifythe antiplasmodial activity of a compound, the Malaria Group estimatedthatacompoundispromisingiftheIC50is<10µM23. Cytotoxicitytestingandcalculationoftheselectivityindex (SI):Themethodof3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide(MTT)accordingtoMosmann24,which revealscellulardamageatthemitochondriallevel,toevaluate thecytotoxicactivityoftheextractsandcompounds.U-937 andHUVECcellslineswereused,whichweremaintainedin continuousculturesintheMalariaGroupLaboratory.These cells were cultured at 37EC and 5% CO2inRPMImedium supplementedwith10%inactivatedFetalBovineSerum(FBS). Themediachangeswereassessedevery48horaccordingto 60 Res.J.Med.Plants,12(2):57-64,2018 pHchangesofthemedium,with10mincentrifugationat 1000 rpm and by replacement with fresh medium25. In Neubauer'schamber,U-937cellswerecountedand platedina96-wellflatbottomplate,200.000cellsmLG1in RPMI1640mediumwith10%FBS.Forothertheassay,the HUVEC cells are counted in  a  Neubauer  chamber  and seededina96-wellflatbottomplate2×105cellswellG1in 100µLofRPMI-1640mediumwith10%fetalbovine serum.Theywereincubatedat37ECwith5%CO2for72hin thepresenceofeachof the sevenconcentrationsofeach extractand/orcompoundandevaluatedinarangebetween 100-1.56 µg mLG1.Eachconcentrationwasevaluatedin triplicate in the dish and three independent trials were performed. Subsequently, mitochondrial dehydrogenase activitywasmeasuredbyadding20µLwellG1ofMTTtoa concentrationof5mgmLG1andincubatedfor3hat37EC under5%CO2.Todissolvetheformedcrystals,100µLwellG1 ofa50%solutionofisopropanoland10%SDSwereadded and the absorbance read at 595 nm in an ELISA reader (BioRad).Forcytotoxicityassays,thepositivecontrolwasthe culturemediumandthenegativecontrolwasamphotericin whichshowedtoxicityintheU-937cellsandHUVEGcellline. Datafromthreeindependenttrialswereanalyzedusingthe GraphPadPrism5programtofindthetoxicconcentration inµgmLG1(CC50)usinganon-linearlogisticregressionmodel. The coefficient of variation (% CV) was also estimated to estimatethedegreeofdispersionoftheCC50obtainedfrom thethreeindependentassays26.Toclassifythecytotoxicityof theextract,theMalariaGroupoftheUniversityofAntioquia established a consensus for thesamplesevaluated:highly toxic <10 µg mLG1, cytotoxic 10-40 µg mLG1, moderately cytotoxic41-100µgmLG1andnocytotoxic>100µgmLG127.In addition,theselectivityindex(IS),whichindicatesselectivity towards the parasite, was calculated as the relationship betweencytotoxicCC50activityandantiplasmodialactivity IC50.FortheU-937celllineitwasestimatedthatISvalues above2areconsideredpromisingextractstobeevaluatedin theHUVECcellline(endothelialcellsisolatedfromthehuman umbilicalcordvein).FortheHUVECcellline,ISvaluesabove 5wereconsideredaspromisingextracts. Statistical analysis: Measurements were performed in triplicateandtheresultswerepresentedasthemeanandits standarddeviation(DS).Thedatawereanalyzedandplotted usingGraphPadPrism4forMacintoshversion4.0bwhich outputs the adjustment value (r) (GraphPad Software,San Diego,California,USA)andallcalculationswereperformedin thestatisticalprogramSTATGRAPHICSCENTURIUNXVI. RESULTSANDDISCUSSION The percentages of extractable material, in vitro antiplasmodialactivityinchloroquine-sensitive Plasmodium falciparum strainNF-54,cytotoxicityinU-937promonocytes, HUVECendothelialcellsanddeterminationoftheselectivity indexofextractsandcompoundsof P.piedecuestanum Trel.  and Yunck were presented in Table 1 and 2. They were classified according to the antiplasmodial potential established by the Malaria Group of the University of Antioquia.Thepercentagesofextractablematerialwiththe different solvents showed better yields for the dichloromethaneextractsofthestemsandleavesmixtureof the P.piedecuestanum (PPD)specieswith1.75%,howeverno association was found between extraction yields and the biologicalteststhatwereperformed.Forallextractsofthe species P. piedecuestanum  Trel. and Yunck. found an adequate concentration-response relationship with statisticallysignificantcorrelationcoefficients(R²)forallthe samplesevaluated.Thechloroquinepositivecontrolhada meanvalueofIC50=26.1±5.4nM andthecontrolofthe Peruvian quinine extract (MeOH: H2O; 70:30) presented a meanvalueofIC50=0.32±0.16µgmLG1. Table1:Cytotoxicityandantiplasmodialactivityoftheextractsofdifferentpolaritiesof P.piedecuestanum Trel.andYunck Performanceof extraction(gof IC50(µgmLG1)X±SD* CC50(µgmLG1)X±SD CC50(µgmLG1)X±SD extract/gofdry ---------------------------------------- -------------------------- ---------------------------- Code Extractbypercolation,leavesandstems plantmaterial)(%) Strainof P.falciparum NF-54** CelllineU-937 IS*** CelllineHUVEC***** IS*** PPH Petroleumether,25ECZP*:5mm10days 0.7 >50 24.9±4.1 ND NE ND PPD Dichloromethane,25ECZP*:5mm10days 1.7 17.9±2.02 37.6±1.7 2.1 101.4±2.7 5.7 PPAE Ethylacetate,25ECZP*:5mm10days 1.1 19.5±1.8 15.4±2.6 0.8 80.7±1.6 4.1 PPM Methanol,25ECZP*:5mm10days 1.6 >50 42.3±0.23 ND NE ND PPE Ethanol25ECZP*:5mm24h 0.9 25.2±0.3 12.4±0.33 0.5 NE NE *ZP:Particlesize,**Dataofasinglereplica,***X(Average)+SD(Standarddeviation),****ChloroquinepositivecontrolIC50=26.10±5.37nM,peruvianmachineextract 0.32±0.16µgmLG1,*****Selectivityindex(IS)=CC50(µgmLG1)/IC50(µgmLG1),******ND:NotdeterminedvalueofIC50>50µgmLG1,Classificationofantiplasmodial activity:highlyactive<5µgmLG1,promising6-15µgmLG1,moderateactivity16-30µgmLG1,lowactivity31-50µgmLG1andnon-activeactivity>50µgmLG1. Classificationofcytotoxicactivity:highlytoxic<10µgmLG1,cytotoxic10-40µgmLG1,moderatelycytotoxic41-100µgmLG1andnon-cytotoxic>100µgmLG1 61 Res.J.Med.Plants,12(2):57-64,2018 120 100 80 60 40 20 0 Inhibitory concentration 50 (IC y CC µg mL ) 50 50 G 1 IC 50 CC U-937 50 CC HUVEG 50 PPHExtH PPHTExtD PPHTExtA PPHTExtM PPHTExtE Extracts of different polarity (PP) P. pidecuestanum * + Table2:Antiplasmodialactivityofisolatedcompoundsof P.piedecuestanum IC50(µgmLG1)X±SD* CC50(µgmLG1)X±SD ---------------------------------------- --------------------------- Code Structure Strainof P.falciparum NF-54** CelllineHUVEC IS*** 1 7.3±0.4or25.7µM >100 13.7 HCO 3 OH O OOH 2 >50 >100 ND HCO 3 HCO 3 O O OH OH 3 >50 60.57 ND HCO 3 HCO 3 O O OH 4 >50 >100 ND HCO 3 HCO 3 O O OH 5 >50 >100 ND HCO 3 O O OH 6 >50 >100 ND HCO 3 HCO 3 O O OH Br *X(Average)+SD(Standarddeviation),**ChloroquinepositivecontrolIC50=0.008±2.78µgmLG1orIC50=26.1±5.4nM,***Selectivityindex(IS)=CC50(µgmLG1)/IC50 (µgmLG1)IS>2confirmsefficacyandsafety,ND:Notdetermined.Thedefinitionoftheantiplasmodialactivityusedwas:IC50<5µgmLG1-strongactivity,6-15µgmLG1moderateactivity,16-30µgmLG1-slightlyactiveandIC50>30µgmLG1-inactive Fig.1: Comparison  of the antiplasmodial andcytotoxic activityofextractsofdifferentpolarityof P.piedecuestanum Thebiologicalactivityfoundfortheextractsofthespecies P.piedecuestanum Trel.AndYunckshowthatthepetroleum ether extract (PPH) with non-polar components and the methanolic extract (PPM) with polar components did not presentantiplasmodialactivity(IC50>50µgmLG1).However, extracts of dichloromethane and ethyl acetate showed moderateantiplasmodialactivitywithIC50=17.9µgmLG1; ISHUVEG=2.09andIC50=19.5µgmLG1;ISHUVEG=0.79,even presentingbetterpharmacologicalactivitythantheethanolic extractwithIC50=25.2µgmLG1;ISHUVEG=0.49(Fig.1).Itcanbe inferredthattheeffectoftheseextractsisduetocomponents withmoderatelypolarandnon-toxiccharacteristics,sincefor theextractofdichloromethane,whichwasthemostactive andselective,aselectivityindex>2waspresented,indicating thespecificityofextracttowardstheparasite.Thefactthatthe cytotoxicityofthisextractwillincreaseCC50=37.63µgmLG1 with respect to the other extracts indicates that the componentspresentinthisextractarepoorlycytotoxic.In 62 Res.J.Med.Plants,12(2):57-64,2018 addition, when these extracts were evaluated in HUVEG metabolicallyactivecellsaninterestingISwasfound,sinceit wasshownthattheextractsdidnotaffectthistypeof cells,sincetheircytotoxicconcentrationswereCC50=101.35; ISHUVEG=5.65andCC50=80.7;ISHUVEG=4.14,fortheextractsof dichloromethaneandethylacetaterespectively. For species of Piper genus, promising activity against Plasmodium in P.capense L.specieshasbeen demonstrated.(Piperaceae)withanIC50=7.0µgmLG1activity in P.hostmannianum strain(W2)chloroquineresistant28 with an IC50=8.0µgmLG1, P.umbellatum with70% inhibitionat40µgmLG1and P.sarmentosum withan IC50=0.05µgmLG129. Invivo antimalarialactivityofthe Piperbetle   leaf  methanolic  extract  evaluated in Plasmodium berghei  (NK65) infected mice has been demonstratedoverarangeofconcentrations (50-400mgkgG1)30.Theactivityof(-)-methylenderatinwas confirmed in vivo  in mice infected with P. vinckei petteri , showingan80%reductioninparasitemiaatadoseof 20mgkgG1dayG131.Itisthefirsttimethatthemetabolites have beenreported 5,8-Hydroxy-7-methoxyflavone(1),6,7dimethoxy-5,8-dihydroxyflavone(2), 6,7-dimethoxy-5hydroxyflavone (mosloflavone) (3), 5,6-dihydroxy-7methoxyflavone(negletein)(4),5-hydroxy-7-methoxyflavone (5) from P. piedecuestanum  species and a brominated derivative from (6) named 6,8 bromo-5-hydroxy-7methoxyflavone(7)inthespecies P.piedecuestanum .The compound(1)wastheonlyonethatpresentedantiplasmodial activitywithanIC50=7.325µgmLG1(25.69µM);ISHUVEC=13.65. The brominated derivative of (5) also did not exhibit antiplasmodialactivity,indicatingthatforthiscompoundthe presence of halogenated groups does not significantly influence the therapeutic response. Other species of this genushavereportedvariousflavonoidswithantiplasmodial activity. The compound 6-prenyl-3'-methoxyethyliodethiol showednoactivityonthe P.falciparum strain.Analysisofthe structure-activityratioshowsthatthepresenceofadjacent methoxy and hydroxyl groups or the absence of adjacent hydroxylgroupscouldcontributetotheinactivityofthe 6-prenyl-3'-methoxyethyliodethiol compound and the substituentsintheflavanonebackboneclearlyinfluencethe antiplasmodial activity32. The antiplasmodial activity of flavonoids is arousing a great interest in the chemical medicine,sincetheyareorientingstudiesofstructurerelation SARofchalconesandderivativesofflavonoidswithdiverse substituentsinringB,AandC.Chalconesaremoreselective thanmethoxyflavones,chalconederivativecompoundshave amoreselectiveactivity(6.7-16.9µM)over P.falciparum (W2) than flavonoid derivatives (5-33 µM)32; methoxyflavones presentthebestactivities,eventheseresultscomplementthe validityoftheresultsobtainedinthisworkfortheisolated compound5,8-hydroxy-7-methoxyflavone(1). CONCLUSIONANDRECOMMENDATION Theantiplasmodialandcytotoxicactivityofthespecies P.piedecuestanum wasreportedforthefirsttimeinwhich themoderatelyactiveextractswerethoseofdichloromethane andethylacetatewiththebesteffectofantiplasmodial andcytotoxicactivitywithIC50=17.93µgmLG1;IS=2.093and IC50 = 19.5 µg mLG1; IS = 0.791, respectively. The antiplasmodial effect of  these  extracts  is  due  to components with mild  polar  characteristics  and  little toxicity,whichmotivatescontinuingwiththestudyofthe SARstructure-activityrelationshipofflavonoidswithvarious substituentsinringB,AandC. ACKNOWLEDGMENTS The authors are grateful for the financial  support providedbytheColombianMinistryofAgriculture (No.009-2007-V7552-38-07),ColcienciasthroughtheJoséde CaldasfranchisesandtheUniversityofAntioquia. REFERENCES 1. 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