Gurjenenewwe2014年7月21日

ChemicalComposition,Antioxidant,AntimicrobialandAnti-inflammatoryActivitiesoftheStemandLeafEssentialOilsfromPiperflaviflorumfromXishuangbanna,SWChinaRenLia,b,*,Jing-jingYanga,b,Yuan-feiWangc,QianSuncandHua-binHua,*aKeyLaboratoryofTropicalPlantResourceandSustainableUse,XishuangbannaTropicalBotanicalGarden,ChineseAcademyofSciences,Menglun,Mengla,Yunnan666303,PRChinabUniversityofChineseAcademyofSciences,Beijing100049,PRChinacYunnanTraditionalChineseMedicalCollege,Kunming650500,PRChinahuhb@xtbg.
ac.
cn;liren@xtbg.
ac.
cnReceived:April11th,2014;Accepted:May4th,2014Thepresentstudyisthefirstinvestigationofthechemicalcomposition,antioxidant,antimicrobialandanti-inflammatoryactivitiesofthestemandleafessentialoilsfromPiperflaviflorumC.
DC(SEOPandLEOP),aplantthathasbeenconsumedasawildvegetable,andusedasmedicine,andspicebytheethnicgroupsinXishuangbanna,SWChina.
AnalyzedbyGC-MS,42and30componentswereidentifiedrepresenting90.
1%and95.
3%oftheSEOPandLEOP,with(E)-nerolidol(16.
7%and40.
5%),β-caryophyllene(26.
6%and14.
6%)andelixene(5.
3%and12.
3%)astheirmainconstituents,respectively.
OurresultsindicatethatSEOPandLEOPhavegoodanti-inflammatoryactivitybysignificantlyinhibitingNOproductioninducedbyLPSinRAW264.
7cellsat0.
04‰withouteffectoncellviability,andnegligibleantioxidantactivityinbothABTSandFRAPassays.
Moreover,theLEOPshowedcomparableactivitywiththepositivecontrol(tigecycline)againstAspergillusfumigatus,withMICandMBCvaluesrangingfrom256to1024g/mL.
Theanti-inflammatoryactivityinLPS-inducedRAW264.
7cellsisworthyoffurtherinvestigationtodiscoverthepossiblemechanismsoftheNOproductioninhibitioneffectoftheseessentialoils.
Keywords:Piperflaviflorum,Essentialoil,Antioxidant,Antimicrobial,Anti-inflammatory,Xishuangbanna.
Piperaceaeplants,distributedmostlyinNorthandSouthAmericaandratherfewerinAsia,areusuallyaromaticherbs,shrubs,orclimbers,and68ofthemhavebeenfoundinChina[1].
Ithasbeenreportedthattheseplantshaveantioxidant,antiproliferative,antimicrobial,antinociceptive,anti-inflammatory,antiulcerandlarvicidalactivities[2a-2e,3a].
EthnobotanicalstudieshaveshownthatmanyspeciesofPiperaceaewerealsocultivatedandusedbytheethnicpeopleinXishuangbanna,SWChina.
Forexample,theleavesandstemsofPiperflaviflorumC.
DCarecommonlyconsumedasvegetablesandspices[3b,4a],anditsstemsarealsousedasanindigenousremedyforinnerheat,stomachdisorder,andreliefofpainanditching[4b,5].
Phytochemicalinvestigationsfoundthattheaerialpartsofthisplantarerichinalkaloids,lignansandsterols[6a,b].
Arecentreportshowedthatanewapiofuranoside(flaviflosideA),isolatedfromthestem,hasmoderatecytotoxicityagainsttheCCRF-CEMcancercellline[7a].
However,studiesontheessentialoilsofthisplantwereneglected.
Therefore,theaimofthisstudywastoinvestigatethechemicalcomposition,antioxidant,antimicrobialandanti-inflammatoryactivitiesofthestemandleafessentialoilsofP.
flaviflorum(SEOPandLEOP).
ThechemicalcompositionofSEOPandLEOParegiveninTable1.
ThecomponentsofthetwoessentialoilsarelistedaccordingtotheirretentiontimesonaHP-5MScolumn.
Thetwoessentialoilspresentedasimilarcompositionwiththesamedominantcomponents.
Specifically,44componentswereidentified(42inSEOPand30inLEOP),representing90.
1%and95.
3%ofthetotaloil,with(E)-nerolidol(16.
7%and40.
5%),β-caryophyllene(26.
6%and14.
6%)andelixene(5.
3%and12.
3%)asthemainconstituentsoftheSEOPandLEOP,respectively.
OurresultsagreedwithpreviousstudiesthattheessentialoilsofPiperaceaespeciesarerichin(E)-nerolidolandβ-caryophyllene[7b-d,8a].
However,therewerealsosomedifferences,forexamplethemajorcomponentsoftheleafessentialoilofP.
aduncumsubsp.
ossanum(C.
CD.
)fromCubawerecamphor(18.
1%)andcamphene(15.
6%)[8b],andthoseofP.
aduncumwerepiperitone(34%),camphor(17.
1%)andcamphene(10.
9%).
ThemaincomponentsofP.
auritumandP.
umbellatumweresafrole(71.
8and26.
4%,respectively)[9].
AllthepresentedresearchhasshownthatthechemicalcompositionsoftheessentialoilsfromPiperaceaespeciesshowwidevariations,andnodiscerniblechemotaxonomictrendsbetweenthespecieshavebeenfiguredoutasyet.
OurresultsalsosupporttheviewthatmoredataareneededfromPiperaceaespeciesessentialoilstosortouttheirchemicaldifferencesandsimilarities[10].
TheantioxidantactivityoftheSEOPandLEOPweremeasuredbyABTSandFRAPassays.
Gallicacidequivalentantioxidantcapacity(GAEAC)atvariousconcentrations(2.
5-20mg/mL)oftheSEOPandLEOPareshowninTable2.
TheSEOPandLEOPdisplayedverylowantioxidantactivityinbothassays.
Theantioxidantactivityisprobablyduenotonlytothepresenceof(E)-nerolidolandβ-caryophyllene,whichhavebeenprovedtohaveantioxidantactivityinpreviousstudies[11a,b],butalsotootherconstituents.
IntheABTSassay,the50%inhibitionconcentrations(IC50)ofSEOP,LEOPandgallicacidwere21.
3±0.
04(mg/mL),23.
3±0.
05(mg/mL)and29.
9±0.
04(g/mL),respectively.
SEOPshowedgenerallystrongerantioxidantpotentialthanLEOP.
TheMICandMBCvaluesofSEOPandLEOPareshowninTable3.
Theessentialoilsshowedantimicrobialactivityagainstsomeofthetestedpathogensandspoilageorganisms.
Forinstance,thetwoessentialoilswerebothactiveagainstKlebsiellapneumonia,acommonpathogeninhospitals,withMICandMBCvaluesrangingfrom2048to5120g/mL.
TheSEOPwasactiveagainstNPCNaturalProductCommunications2014Vol.
9No.
71011-10141012NaturalProductCommunicationsVol.
9(7)2014Lietal.
Table1:ChemicalcompositionofSEOPandLEOP.
NoRIcalRIlitCompoundSEOP(%)LEOP(%)1930929α-Pinene0.
70.
92974971β-Pinene2.
13.
23991991β-Myrcene0.
20.
2410271030D-Limonene0.
50.
6510431149Benzeneacetaldehyde0.
1611021103Linalool1.
84.
4711771175Terpinen-4-ol0.
10.
2811911187α-Terpineol0.
30.
5912851289Bornylacetate0.
0610129412942-Undecanone0.
61.
31113371339δ-EIemene0.
11213501351α-Cubebene1.
00.
61313661370Cyclosativene0.
41413781376α-Copaene7.
53.
11513851384β-Bourbonene0.
071613901390β-Cubebene1.
40.
31713931395L-β-Elixene0.
40.
41814101409α-Gurjenene0.
80.
41914251422β-Caryophyllene26.
614.
62014391436Aromandendrene0.
31.
12114541456α-Humulene2.
01.
02214581457Z-4-Dodecenol0.
32314611462Alloaromadendrene1.
20.
82414741471(+)epi-Bicyclosesquiphellandrene0.
22514781476γ-Muurolene0.
40.
52614821480GermacreneD2.
51.
42714891482Eremophilene0.
12814981492Elixene5.
312.
32915011500α-Muurolene0.
50.
63015101509β-Bisabolene0.
13115141514γ-Cadinene0.
43215251525δ-Cadinene3.
22.
13315331528Cadinadiene-1,40.
23415721573(E)-nerolidol16.
740.
53515841578(-)-Spathulenol3.
71.
43615851580(-)-Globulol0.
63715861583Caryophylleneoxide1.
80.
93815881590Viridiflorol1.
30.
33915951593Ledol0.
84016011602Guaiol0.
34116541647τ-Muurolol1.
40.
44216591654δ-Cadinol0.
54316671668α-Cadinol2.
30.
34416791683Bulnesol0.
4Totalidentified(%)9095.
3Oilyield(%)0.
060.
3GroupedcompoundsMonoterpenehydrocarbons3.
44.
9Oxygenatedmonoterpenes2.
25.
1Sesquiterpenehydrocarbons54.
239.
6Oxygenatedsesquiterpenes29.
444.
0Others0.
71.
3RIcalreferstotheretentionindexexperimentallycalculatedusingC7-C30alkanes.
RIlitreferstotheretentionindextakenfromNISTdatabase.
Table2:Gallicacidequivalentantioxidantcapacity(ggallicacid/mL)ofSEOPandLEOP(mg/mL)byABTSandFRAPassaya.
ABTSassayFRAPassayConcentrationSEOPLEOPSEOPLEOP2.
52.
9+0.
33.
2+0.
110.
6±0.
28.
1±0.
254.
3±0.
36.
0+0.
315.
4+0.
314.
4±0.
3108.
4±0.
211.
9±0.
329.
3+0.
126.
5±0.
31516.
1±0.
418.
1+0.
250.
8+0.
230.
1±0.
22027.
0±0.
222.
0+0.
457.
0+0.
340.
0±0.
2aAllvalueswereperformedintriplicateandexpressedasmeans±SD.
Staphylococcusaureus,acommonfood-bornepathogen,withMICandMBCvaluesrangingfrom1024to3072g/mL.
LEOPhadactivitycomparablewiththatofthepositivecontrol(Tigecycline)againstAspergillusfumigatus,whichcancauselungdiseaseandfoodspoilage,withMICandMBCvaluesrangingfrom256to1024g/mL.
Table3:MICandMBC(μg/ml)oftheSEOPandLEOPa.
SEOPLEOPPositivecontrolbMicrobialstrainsMICMBCMICMBCMICMBCS.
aureus10243072NANA0.
250.
5A.
baumannii20484096NANA0.
51E.
coliNANA307230720.
050.
25K.
pneumoniae2048512020484096256512A.
fumigatusNANA25610245121024aAllvaluesareexpressedasmeansofthreeindependentexperiments.
bPositivecontrol:VancomycinforS.
aureus;FluconazoleforA.
baumannii;CefotaximeforE.
coli;AmikacinforK.
pneumonia;TigecyclineforA.
fumigatus.
NAmeans"notactive"Figure1:EffectsofSEOPandLEOPontheviabilityofRAW264.
7cellsbyMTSassay.
ResultswereexpressedaspercentagesofMTSproductionbynon-treatedcells(blankgroup).
Alltestswereperformedintriplicateandexpressedasmeans±SD.
Figure2:EffectofSEOPandLEOPonNOproductioninRAW264.
7cellsbytheGriessreagentsystem.
ResultsareexpressedaspercentagesofNObytheLPS-treatedcells(LPSgroup).
Alltestswereperformedintriplicateandexpressedasmeans±SD.
*P99.
9%killingofthebacteriumrelativetotheconcentrationofbacteriumthatwaspresentintestwellsat0h[14].
TheMICsandMBCsofAmikacin,Fluconazole,Vancomycin,TigecyclineandCefotaximewerealsodeterminedinparallelexperimentsinordertocontrolthesensitivityofthestandardtestorganisms.
Alltestswereperformedintriplicate.
Cellculture:ThemurinemacrophagecelllineRAW264.
7wasobtainedfromKunmingInstituteofZoology,ChineseAcademyofSciences(KCB200603YJ)andculturedinDMEM(ThermoScientific,Logan,UT,USA)containing10%fetalbovineserum,1%penicillin-streptomycinand1%L-glutamine(Sigma-Aldrich,St.
Louis,MO,USA)at37°Cina5%CO2incubator(ThermoScientific,Forma371,Steri-cycle,USA),andsubculturedevery2days.
Cellviabilityassay:ThecellviabilitywasevaluatedbyMTSassayfollowingthemanufacturer'sinstructions.
IntheMTSassay,100μLcellsuspension(1*105cells/well)wascultivatedin96-cellplatesfor18hasdescribedabove.
Thencellswerepre-treatedwithvariousconcentrationsoftheessentialoilsfor30minpriortofurtherincubationinthepresenceof1μg/mLLPSforanother24h.
Finally,20LofCellTiter96AQueousOneSolutionReagent,preparedbyMTS(3-[4,5,dimethylthiazol-2-yl]-5-[3-carboxymethoxy-phenyl]-2-[4-sulfophenyl]-2H-tetrazolium,innersalt)inthepresenceofphenazineethosulfate(PES),wasaddedtoeachwellandincubatedfor1hat37°Cinthe5%CO2incubator.
Theabsorbanceofeachwellwasmeasuredat490nmdirectlyusingaMultifunctionalMicroplateReader(ThermoScientificVarioSkan,USA).
ResultswereexpressedasapercentageofMTSproductionbynon-treatedcontrolcells(blankgroup).
Valueswerepresentedasmean±standarddeviation(SD)of3independenttests.
MeasurementofNOproduction:NOproductionbyLPS-stimulatedRaw264.
7cellswasmeasuredbytheaccumulationofnitriteintheculturesupernatants,usingtheGriessreagentsystem1014NaturalProductCommunicationsVol.
9(7)2014Lietal.
(Promega,Madison,WI,USA)andfollowingthemanufacturer'sinstructions.
Cellswereseededin96-wellplatesat1*106cells/wellandthenincubatedwithculturemedium(control)for18h.
Thencellswerepre-treatedwithvariousconcentrationsoftheessentialoilsfor30minbeforetheywerestimulatedwith1μg/mLLPSandfurtherculturedforanadditional24h.
Inbrief,50μLofculturesupernatantswerecollectedandmixedwiththeGriessreagentsystem(1%sulfanilamidein0.
1mol/LHCland0.
1%N-(1-naphthyl)ethylenediaminedihydrochloride).
Thentheplateswereincubatedfor10minat37°Cinthe5%CO2incubator.
Theabsorbancewasmeasuredat550nminaMultifunctionalMicroplateReader(ThermoScientificVarioSkanFlash,USA).
Nitriteconcentrationwasdeterminedfromasodiumnitritestandardcurve.
ResultswereexpressedaspercentagesofNObyLPS-treatedcells(LPSgroup).
Valueswerepresentedasmean±standarddeviation(SD)of3independenttests.
Statisticalanalysis:Allexperimentswereperformedintriplicateandexpressedasmeanvalues±standarddeviation(SD).
The50%inhibitionconcentration(IC50)wascalculatedbyProbitregressionanalysisofSPSS14.
0forWindows(SPSSInc.
Chicago,USA).
Theone-wayANOVAanalysis,withDunnett'smultiplecomparisontests,wasperformedusingSPSS14.
0forstatisticalevaluation.
SignificanceofdifferencewasacceptedatP<0.
05.
Acknowledgments-TheauthorsgratefullyacknowledgethefinancialsupportoftheSpecialGrantforBasicWorkofScienceandTechnologyfromtheMinistryofScienceandTechnology,China(grantNo.
2012FY110300).
WealsothanktheCentralLaboratoryofXishuangbannaTropicalBotanicalGarden,ChineseAcademyofSciencesfortechnicalsupportofthisstudy.
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