w w w . s b f g n o s i a . o r g . b r / r e v i s t a
Original
Article
In
vitro
alpha
glucosidase
inhibition
and
free-radical
scavenging
activity
of
propolis
from
Thai
stingless
bees
in
mangosteen
orchard
Boonyadist
Vongsak
a,∗,
Sumet
Kongkiatpaiboon
b,
Sunan
Jaisamut
a,
Sasipawan
Machana
a,
Chamnan
Pattarapanich
aaFacultyofPharmaceuticalSciences,BuraphaUniversity,Chonburi,Thailand
bDrugDiscoveryandDevelopmentCenter,ThammasatUniversity,PathumThani,Thailand
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received12April2015 Accepted6July2015 Availableonline26July2015
Keywords:
Alphaglucosidase
Freeradicalscavengingactivity Mangosteen
Propolis Stinglessbee
a
b
s
t
r
a
c
t
Thechemicalcomponentandbiologicalactivityofpropolisdependonfloraareaofbeecollectionand beespecies.Inthestudy,thepropolisfromthreestinglessbeespecies,LepidotrigonaventralisSmith,
LepidotrigonaterminataSmith,andTetragonulapagdeniSchwarz,wascollectedinthesameregionof mangosteengardenfromThailand.Totalphenoliccontent,alphaglucosidaseinhibitoryeffect,and free-radicalscavengingactivityusingFRAP,ABTS,DPPHassaysweredetermined.Themostpotentactivityof propolisextractwasinvestigatedforbioactivecompoundsandtheirquantity.TheethanolextractofT. pagdenipropolishadthehighesttotalphenoliccontent12.83±0.72gofgallicacidequivalentsin100g oftheextract,andthestrongestalphaglucosidaseinhibitoryeffectwiththeIC50of70.79±6.44g/ml. Thefree-radicalscavengingactivityevaluatedbyFRAP,ABTS,DPPHassaysshowedtheFRAPvalueof 279.70±20.55molFeSO4equivalent/gextractandtheIC50of59.52±10.76and122.71±11.76g/ml, respectively.Gamma-andalpha-mangostinfromT.pagdenipropolisextractwereisolatedand deter-minedforthebiologicalactivity.Gamma-mangostin exhibitedthestrongestactivityforbothalpha glucosidaseinhibitory effectand free-radicalscavengingactivity. UsingHPLC quantitativeanalysis method,thecontentofgamma-andalpha-mangostinintheextractwasfoundtobe0.94±0.01and 2.77±0.08%(w/w),respectively.ThesefindingssuggestedthatT.pagdenipropolismaybeusedasamore suitablerawmaterialfornutraceuticalandpharmaceuticalproductsandthesemangostinderivativesas markers.
©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.
Introduction
Overproductionoffreeradicalspeciescanbedamagingtohost cellsandleadtovariousailmentssuchasinflammatory,agingand diabetes(Zhouetal.,2015).Inthecaseofdiabetes,alpha glucosi-daseisavitalenzymeessentialforcleavageofmaltosetoglucose fortheabsorptioninto theblood streamin thesmallintestine. Therefore,alphaglucosidaseinhibitorscouldregulateabnormally highstagesofplasmaglucoseaftercarbohydrateingestion(Ryu etal.,2011).Nowadays,␣-glucosidaseinhibitors,suchasacarbose, voglibose and miglitol, have been accepted for clinical use in the treatmentof type 2 diabetes. Nonetheless, some synthetic alphaglucosidase inhibitorshave undesirable side effects, such as abdominal cramping, flatulence and diarrhea (Zhang et al., 2015).Asaresult,manyscientistshaveturnedtheirattentionto
∗ Correspondingauthor.
E-mail:boonyadist@buu.ac.th(B.Vongsak).
natural alphaglucosidase inhibitors including mangosteen and beeproducts,whichareutilizedtodevelopnutraceuticalsorlead compoundsforantidiabeticmanagement(Matsuietal.,2004;Ryu etal.,2011;Juárez-Rojopetal.,2014).
Naturalproductsfrombeeshavebeenextensivelyemployed sinceancienttimebecauseofitsbroadpharmacologicalactivity (Dantasetal., 2014; Kustiawanetal., 2014).Propolisis oneof thebeeproductsthatexhibitsnumerousbiologicalactivitiessuch as antioxidant,anti-inflammatory, antitumor, antiviral, antibac-terial, antifungal,antidiabetic activitiesandis alsolistedin the LondonPharmacopoeiasandChinesePharmacopoeias(Sforcinand Bankova,2011;Zhangetal.,2015).However,thechemicaland bio-logicalactivitiesofpropolisvarydependingonbeespeciesandthe floraatsiteofbeecollection.Forinstance,propolisfromEurope andNorthAmericaregions’maincompoundscomprisesmostly fla-vanones,flavones,cinnamicacidsandtheiresters,whilethatfrom Brazilcomprisesmainlyprenylatedp-coumaricacids,diterpenic acids(Bankova,2005;Sforcin,2007).Also,differentracesof hon-eybeescollectedatthesameareademonstratedvaryingpotency.
http://dx.doi.org/10.1016/j.bjp.2015.07.004
Apismelliferacaucasicashowedasuperiorantibacterialactivityto ApismelliferacarnicaandApismelliferaanatolica;stinglessbees– Trigonaincisa,Timiaapicalis,Trigonafusco-balteataandTrigona fus-cibasis–fromIndonesiarevealeddifferentdegreesofcytotoxicity (SiliciandKutluca,2005;Kustiawanetal.,2014).
Stinglessbees, whose propolis is utilized for medicinal and nutraceuticalpurposes,arealargegroupofeusocialinsectthatplay apartinplantpollinationintropicalregions(Choudharietal.,2012; daCunhaetal.,2013).InThailandandIndia,stinglessbee propo-lisispopularlyappliedforthetreatmentofmaladiessuchasacne, diabetesandinflammation(Umthongetal.,2011;Choudharietal., 2012).Antimicrobial,antiproliferativeandantioxidantactivitiesof severalspeciesofstinglessbeepropoliswerealsoinvestigated(da Cunhaetal.,2013;Dutraetal.,2014).Nevertheless,thestudyof propolisfromThaistinglessbees,(LepidotrigonaventralisSmith, Lepidotrigonaterminata Smith,and TetragonulapagdeniSchwarz (Apidae)),whicharecommerciallycultivatedinartificialhivesin fruitgardensand marketedin severalpreparationsinThailand, islimited.Thus, theobjective ofthepresentworkwasto com-parealphaglucosidaseinhibitoryeffectandfree-radicalscavenging activityofpropolisfromthreestinglessbeespeciesinthesame areaofThaimangosteenorchard.Theactivecompoundsfromthe speciesthatdemonstratedthestrongest activitywereidentified andquantified.
Materialsandmethods
Chemicalproducts
1,1-Diphenyl-2-picrylhydrazyl(DPPH) radical, 2,2′
-azino-bis-(3-ethylbenzothiazoline-6-sulfonicacid)diammoniumsalt(ABTS), 2,4,6-Tris(2-pyridyl)-s-triazine(TPTZ),␣-glucosidasefrom
Saccha-romycescerevisiae,acarbose, 4-nitrophenyl␣-d-glucopyranoside (pNPG), iron(III)chloride hexahydrate, ferrous sulfate, iron(II) sulfate heptahydrate, were obtained from Sigma–Aldrich® (St. Louis,MO, USA), aluminum chloride, acetic acid, ascorbic acid, Folin–Ciocalteureagent,gallicacidwerepurchasedfromMerck® (Darmstadt,Germany). Sodiumbicarbonateand potassium per-sulfate werepurchased fromAjax Finechem® (NSW, Australia). Allreagentswereofanalyticalgrade.Deionizedwaterwas puri-fiedbyUltraClearTMsystem(SiemenWaterTechnologiesCorp®). Ethanol,ethylacetate,dichloromethane,hexane,formicacidand HPLCgrademethanolwerepurchasedfromLabscan®(Thailand). Standardgamma-mangostin(1)and alpha-mangostin(2)purity morethan98%werepurchased fromChengduBiopurify Phyto-chemicalsLtd,Sichuan,China.
Propolissampleandpreparation
PropolisofLepidotrigonaventralisSmith,Lepidotrigonaterminata Smith,and TetragonulapagdeniSchwarzwerecollectedfroman apiaryinthemangosteengardeninDecemberfromMakham dis-trict,Chanthaburiprovince,easternofThailandin2014,andkept inthedarkat4◦Cuntiluse.Thestinglessbeeswereidentifiedby
Dr.ChamaInson,DepartmentofEntomology,Facultyof Agricul-ture,KasetsartUniversity.Thevoucherspecimens(Lepidotrigona ventralisNo.1214001,Lepidotrigonaterminata No.1214003,and TetragonulapagdeniNo. 1214003) weredeposited at Faculty of PharmaceuticalSciences,BuraphaUniversity,Thailand.
Propolisfromdifferentbeespecies(10g)wasseparatelycleaned andcutintosmallpiecesandwasthensonicatedwith80%ethyl alcohol (200ml) at 40◦C for 30min. The suspension was
cen-trifugedat3000×gfor5minat20◦C.Thesupernatantwaskept
whilethepelletwasre-extractedusingthesameprocedure.The supernatantswere pooled together and evaporated in a rotary
evaporator.Eachextractwasdewaxedbysonicationwith100mlof hexaneat40◦Cfor20minandcentrifugedat2000×gfor5minat
20◦C.Thesupernatantwasdiscardedwhilethecruderesiduewas
keptandstoredinthedarkat0◦C.
Separationofactivecompounds
Thepropolisextractthatexhibitedthestrongestactivitywas selectedtoseparatethebioactivecompounds.Thecruderesidue wassubjectedtocolumnchromatography(3cm×20cm,silicagel (0.063–0.200mm,Merck 7734))with20% ethyl acetatein hex-aneasamobilephase.Thesub-fractionswerepooledtogetherto obtaintwomainfractionsasmonitoredbythin-layer chromatog-raphy.Thefirstfractionwassubjectedtopreparativethin-layer chromatography(pTLC)with50%ethylacetate/hexanetoobtain compound1.ThesecondfractionwasalsoappliedtopTLCwith dichloromethane (triplerun)asa mobilephase to obtain com-pound2.Eachpurecompoundwasdissolvedin99.98%CDCl3orin 99.8%methanol-d4(ca.5mgin0.7ml)andtransferredinto5mm NMRsample tube(Promochem,Wesel,Germany). Spectrawere recordedbytheBrukerTopspinsoftwareonaBrukerAVANCE400 spectrometer(Bruker,Rheinstetten,Germany).
Biologicalassay
Determinationofcontentsoftotalphenoliccompounds
TotalphenoliccontentwasdeterminedusingFolin–Ciocalteu reagentfollowing methoddescribed by Vongsak etal. (2013b). Eachsample(1000g/ml),200lwasmixedwith500lofthe Folin–Ciocalteureagent(diluted1:10withdeionizedwater)and 800lof sodiumbicarbonatesolution(7.5%,w/v). Themixture wasallowedtostandatroomtemperaturefor30minwith inter-mittentshaking.Theabsorbancewasmeasuredat765nmusinga UV–Visiblespectrophotometer(Hitachi®,Japan).Thesame proce-durewasrepeatedforthegallicacidstandardsolution(500,250, 125, 62.5,31.25and 0g/ml)and thequantificationwasmade basedonastandardcurvegeneratedwithofgallicacid.Thecontent oftotalphenoliccompoundswascalculatedasmean±SD(n=3) andexpressedasgramsofgallicacidequivalents(GAE)in100gof theextract.
Alphaglucosidaseinhibitionassay
The ␣-glucosidase inhibitory activity was carried out spec-trophotometricallyusingpNPG assubstrate (Zhouet al.,2015). Samples, standard solutions (gamma- and alpha-mangostin) or positive control(acarbose) withdifferentconcentrations (50l,
Tables 1and 3), ␣-glucosidase (50l,2unit/ml)and phosphate buffer (50l, pH 7.0) were mixed and pre-incubated at 37◦C for 10min, and then pNPG (50l,20mM) was added to start thereaction.Afterincubationat37◦Cfor30min,theabsorbance
was measured at 405nm using UV–Visible microplate reader (Metertech®
, Taiwan). The percent inhibition of ␣-glucosidase activitywascalculatedasfollows:
Inhibition(%)=(A1−A2)
A1 ×
100,
whereA1 istheabsorbanceofcontrol,andA2 istheabsorbance ofsamples.Theextentofinhibitionwasexpressedasmean±SD (n=3)oftheenzymaticactivity(IC50).
Ferricreducingantioxidantpower(FRAP)assay
600nmusingUV–Visiblemicroplatereader(Metertech®,Taiwan). Ferroussulfate(FeSO4·7H2O,2.0,1.0,0.5,0.25,0.125,0.063mM) wasusedasreferencestandardandtheresultswereexpressedas molFe2+equivalent/gextract.Standardsolutions(gamma-and alpha-mangostin)andpositivecontrol(ascorbicacid)weretreated under thesame conditionas thesamples,and then mean±SD (n=3)wascalculated.
ABTSradicalscavengingassay
ABTSradicalscavengingactivitywasmeasuredbythemethod describedbyAl-Mansoubetal.,2014.ABTSradicalcation(ABTS•+) solutionwaspreparedbymixing14mMABTSand4.9mM potas-sium persulfate solutions in equal volume. The solution was allowedtoreactinthedarkatroomtemperaturefor16–20hbefore use.Then,1mlofthesolutionwasdilutedwith40mlethanolto yieldworkingABTSsolutionwithanabsorbanceof0.70±0.02at 734nm.To750lABTSworkingsolution,750ltestsampleswere added.Theabsorbanceofeachsolutionwasdeterminedat734nm usingUV–Visiblespectrophotometer(Hitachi®,Japan)after6min. Thecorresponding blankreadingswere alsotaken andpercent inhibitionwasthencalculatedasfollows:
Inhibition(%)=(A1A−A2)
1 ×
100,
whereA1istheabsorbanceofcontrol,andA2istheabsorbanceof samples.Eachdeterminationwasdoneintriplicate,andthe aver-ageIC50 value wascalculatedas mean±SD. Standard solutions (gamma-andalpha-mangostin)andpositivecontrol(ascorbicacid) weretreatedunderthesameconditionsasthesamples.
DPPH-scavengingassay
The free radical scavenging activity of the extracts, stan-dard solutions (gamma- and alpha-mangostin) and positive control (ascorbic acid) was investigated using 1,1-diphenyl-2-picrylhydrazyl(DPPH)radicalscavengingmethod(Vongsaketal., 2013b).Atotalof750loftheextractorstandard/positive con-trolwasaddedto750lofDPPHinmethanolsolution(152M). Afterincubationat37◦Cfor20min,theabsorbanceofeachsolution
wasdeterminedat517nmusingUV–Visiblespectrophotometer (Hitachi®, Japan). The corresponding blank readings were also takenandpercentinhibitionwasthencalculatedasfollows:
Inhibition(%)=(A1−A2)
A1 ×
100,
whereA1istheabsorbanceofcontrol,andA2istheabsorbanceof samples.Eachdeterminationwasdoneintriplicate,andtheaverage IC50valuewascalculatedasmean±SD.
Determinationofbioactiveconstituentcontents
Thepropolisextractwiththehighestactivitywasselectedfor investigatingthequantityofbioactivecompoundsusingvalidated HPLCmethod(Vongsaketal.,2014).Theinstrumentusedwasan Agilent1260Series®
(AgilentTechnologies)equippedwitha1260 QuatpumpVLquaternarypump,1260ALSautosampler,1260TCC columnthermostat,and1260DADVLdiodearraydetector.The chromatographicseparationwascarriedoutonaHypersilBDS® C18column(4.6mm×100mmi.d.,3.5m)withaC18guard col-umn.Themobilephaseswere(A)0.2%formicacidinwaterand(B) methanol.Gradientelutionwasused:75%BinAto90%BinAfor 10min;90%BinAto100%Bfor5min;100%Bfor10min.The col-umnwasre-equilibratedwith75%BinAfor10minpriortoeach analysis.Theflowratewas1.0ml/minat25◦C.UV–Visdetector
wasmonitoredatthewavelengthof245nmandinjectionvolume was5lforallsamplesandstandards.
T.pagdenisamplewaspreparedbyaccuratelyweighing30mg
ofextractanddissolvinginmethanol(5ml).Toenablecomplete Table
Table2
NMRdataforcompounds1and2.
Compounds 1HNMR 13CNMR
Gamma-mangostin(1) (400MHz,CDCl3/methanol-d4),ı(ppm)=6.65(s,1H,ArH), 6.20(s,1H,ArH),5.28–5.23(m,2H,vinylic-H×2),4.13(d, J=6.61Hz,2H,allylic-H),3.34(d,J=7.06Hz,2H,allylic-H), 1.82(s,3H,CH3),1.78(s,3H,CH3),1.66(s,2×3H,2×CH3)
(100MHz,CDCl3/methanol-d4),ı(ppm)=182.3(C O), 161.4(ArC-OAr),160.3(ArC-OH),154.9(ArC-OAr),152.9 (ArC-OH),150.6(ArC-OH),139.9(ArC-OH,132.4(ArC), 132.3[CH C(CH3)2],128.1[CH C(CH3)2],122.8(CH C), 122.3(CH C),111.6(ArC),109.6(ArC),103.4(ArC),100.2 (ArCH),92.3(ArCH),25.7(2×CH3),25.6(CH2),21.3(CH2), 17.9(CH3),17.6(CH3)
Alpha-mangostin(2) (400MHz,CDCl3),ı(ppm)=13.80(s,1H,OH),6.86(s,1H, ArH),6.32(s,1H,ArH),6.28(s,1H,OH),6.11(s,1H,OH), 5.34–5.28(m,2H,vinylic-H×2),4.12(d,J=6.20Hz,2H, allylic-H),3.83(s,3H,OCH3),3.48(d,J=7.12Hz,2H, allylic-H),1.87(s,3H,CH3),1.86(s,3H,CH3),1.80(s,3H, CH3),1.72(s,3H,CH3)
(100MHz,CDCl3),ı(ppm)=182.1(C O),161.6(ArC-OAr), 160.7(ArC-OH),155.8(ArC-OAr),155.1(ArC-OH),154.5 (ArC-OH),142.6(ArC-OCH3),137.1(ArC),135.7 [CH C(CH3)2],132.1[CH C(CH3)2],123.2(CH C),121.4 (CH C),112.3(ArC),108.4(ArC),103.7(ArC),101.5(ArCH), 93.3(ArCH),62.0(OCH3),26.6(CH2),25.8(CH3),25.7 (CH3),21.5(CH2),18.2(CH3),17.9(CH3)
dissolution,each sample wassonicatedfor 60min.Priortothe injection,eachsolutionwasfilteredthrougha0.22mnylon
mem-branefilter.
Stocksolutionsofstandardcompounds(1)and(2)were pre-paredbyaccuratelyweighing and dissolvingthecompounds in methanoltoobtainthefinalconcentrationof1000g/ml.
Work-ingsolutionsofstandard compoundswereobtainedbydiluting thestockstandardsolutionswithmethanoltoachievethedesired concentrations.Calibrationcurveswereconstructedfromthepeak areaversustheamountofthestandardsbyleastsquare regres-sionacrosstherangeof0.78–100g/mlfor compounds(1)and
1.5–1000g/mlforcompound(2).
Statisticalanalysis
Theresultswerereportedasmean±standard deviation(SD) (n=3).Theaveragecontentsoftotalphenolics,FRAPvalues and IC50ofthedifferentpropolisbeespeciesextractwerestatistically investigatedusingone-wayanalysisofvariance(ANOVA)withleast significantdifference(LSD)bySPSSforWindows®16.0.A statis-ticalprobability(pvalue)less than0.05indicated astatistically significantdifferencebetweengroups.
Resultsanddiscussion
Phenoliccompounds aretheprincipal bioactive materialsin propoliswhichhavebeenreportedtohavenumerous pharmaco-logicaleffects,includinganti-oxidationandanti-diabetes(Matsui et al., 2004). Total phenolic contents in various stingless bees propolis extracts are presented in Table 1. T. pagdeni propolis extractpromotedsignificantlyhigher contentoftotal phenolics than propolisextract of other species. Totalphenolics in these differentpropolisextractsrangedfrom2.16to12.83gGAE/100g extract.Theresultsindicatedthepossibleinfluencesofdiverse stin-glessbeespeciesondifferentcontentsofphenolicsinpropolis.The alphaglucosidaseinhibitoryeffectofvariouspropolisextractsis
displayedinTable1.TheIC50 valuesofpropolisextractsranged from70.79to469.66g/mlagainstbaker’syeastalpha glucosi-dase,andthatofT.pagdenipropolisextractwassignificantlylower than155.82g/mlofacarbose,apositivecontrol.Thus,T.pagdeni propolisextractpossessedthehighestinhibitoryeffectonalpha glucosidasecomparedtotheotherspeciesandpositivereference. Thiseffectwaspossiblyduetophytochemicals,whichinclude phe-noliccompoundssuchasxanthonesandphenolicacids(Ryuetal., 2011).
Free radicals canbe deleterious tocells and lead tovarious chronicdiseasessuchasdiabetes,arthritis,andasthma(Vongsak etal.,2013a).Thefreeradicalscavengingactivityofstinglessbees propolis extractswas determined onthe basis of the scaveng-ingactivityofFRAP,ABTSandDPPHassays(Table1).Theresults showedthattheextractspossessedreducingactivityintherange of 61.14–279.70mol FeSO4 equivalent/g extract while that of ascorbicacidwas7698.02molFeSO4 equivalent/gextract.The extractswereabletoscavengeABTSandDPPHradical,especially thatofT.pagdenipropolis.ItpromotedstrongerABTSandDPPH radicalscavengingactivity thanthe otherextractswith IC50 of 59.52and122.71g/ml,respectively,whileascorbicacid,a pos-itivereference,showedtheactivityatIC50of6.07and7.33g/ml, respectively.Theseexperimentsindicatedthatalphaglucosidase inhibitionandfree-radicalscavengingactivityofpropolisextracts fromT.pagdeniexhibitedstrongeractivitythanthatofL.ventralis andL.terminata.Thus,T.pagdenipropolisextractwasselectedto identifyandquantifythebioactivecompounds.
Gamma-mangostin(1)andalpha-mangostin(2)wereseparated and identified as the active constituents in T. pagdenipropolis extractfrommangosteenorchard(Table2).Inalphaglucosidase inhibitionassay,eachcompounddemonstratednoteworthyalpha glucosidaseinhibitoryeffect(IC504.22and29.27M)while acar-bosedisplayedtheIC50of241.36M(Table3).Thesechemicals werepreviouslyreportedinseedcasesofGarciniamangostanaand hadbeenreportedtodisplayalphaglucosidaseinhibitoryeffect (Ryu etal.,2011).In regardstofreeradicalscavengingactivity,
Table3
ThecontentsofbioactivecompoundsandtheirbiologicalactivitiesinTetragonulapagdenipropolisextractfrommangosteenorchard.
Bioactivecompounds Contentsofmajor compoundsin80% ethanolicextracts(%w/w)*
Alphaglucosidase inhibitoryassay IC50(M)*
FRAPassay(molFeSO4
equivalent/gramextract)*
ABTSassay IC50(M)*
DPPHassay IC50(M)*
Gamma-mangostin(1) 0.94±0.01 4.22±0.58a 5366.82±54.71a 160.92±1.23a 39.94±1.16a
alPha-mangostin(2) 2.77±0.08 29.27±7.02c 15.32
±3.89c n.d. n.d.
Dissimilarlettersinthesamecolumnindicatesignificantlydifferentforeachparameteratp<0.05usingone-wayanalysisofvariance(ANOVA)withleastsignificantdifference (LSD).
* Expressedasmean±SD(n=3),n.d.=notdetectable.Inalphaglucosidaseinhibitoryassay,theconcentrationsofgamma-mangostin(1)were12.5,6.25,3.125,1.56,0.77
and0g/ml,whilealpha-mangostin(2)were100,50,25,12.5,6.25and0g/ml.InABTSassayandDPPHassaytheconcentrationsofgamma-andalpha-mangostinwere
mU 700
600
500
400
300
200
100
0
0 2.5 5 7.5 10 12.5 15 17.5
(1)
(2)
20 22.5 min
Fig.1.HPLCfingerprintsofTetragonulapagdenipropolisextractfrommangosteenorchardinThailand,gamma-mangostin(1)andalpha-mangostin(2).
gamma-mangostin(1)exhibitedastrongactivitycomparableto thatofascorbicacidintheDPPHassaywhilealpha-mangostin(2) showedweaktonon-detectableactivityinABTSandDPPHassays (Table3).
RO
HO O
OH O
OH
1R=H
2R=CH3
HPLCwasusedtoquantifybioactivecompounds,gamma-and alpha-mangostinintheT.pagdenipropolisextractbecauseofthe strongestactivities.Thismethodhasbeenvalidatedforits linear-ity,precision,andaccuracy(Vongsaketal.,2014).Theamountsof bioactivecompounds,gamma-mangostin(1)andalpha-mangostin (2),intheT.pagdenipropolisextractwere0.94and2.77%(w/w), respectively (Table 3) at retention times of 7.46and 9.98min, respectively (Fig. 1), respectively, in theHPLC fingerprints.The presenceofthesemangostinderivativesisconsideredtobevital forthealphaglucosidaseinhibitoryeffectofT.pagdenipropolis.
Inconclusion,thestudyprovidesdatatosupporttheusageof differentstinglessbeepropoliscultivatedinmangosteenorchard asrawmaterialfornaturalantioxidantandanti-diabeticagents. Based on this study, the propolis extract of T. pagdeni should beutilizedasabettersourceofrawmaterialforalpha glucosi-dase inhibitory effect and anti-oxidativepurposes than others. Themangostinderivatives,especiallygamma-mangostin,themost activeagent,couldpossiblybeappliedasmarkersfor standardiza-tion.
Authors’contributions
BVcontributionincludedcollectingsamples,designingand per-forminglaboratorywork,analyzingtheresults,andpreparingthe paper. SK contribution included HPLC analysis. SJ contribution includedtheisolationandpurificationofthecompounds.SMand CPcontributionincludedcollectionofsamplesandsupervisionof thelaboratorywork.Alltheauthorshavereadthefinalmanuscript andapprovedthesubmission.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgments
ThisworkwassupportedbytheHigherEducationResearch Pro-motionandNationalResearchUniversityprojectofThailand,Office oftheHigherEducationCommission.
TheauthorsalsowouldliketothankFacultyofPharmaceutical Sciences,BuraphaUniversityforfinancialsupportand Mr.Wisit Tanooard,Ms.NipathaIssaroaswellasThailocalbeekeeperofKon ChanChannarong(stinglessbee)inChantaburiprovince,Thailand forfacilitiesandpropoliscollection.WealsothankDrug Discov-eryandDevelopmentCenter,ThammasatUniversityforlaboratory facilitiesandMr.PanuponKhumsupanforhiskindproofreadingof themanuscript.
References
Al-Mansoub,M.A.,Asmawi,M.Z.,Murugaiyah,V.,2014.Effectofextraction sol-ventsandplantpartsusedontheantihyperlipidemicandantioxidanteffects ofGarciniaatroviridis:acomparativestudy.J.Sci.FoodAgric.94,1552–1558.
Bankova,V.,2005.Chemicaldiversityofpropolisandtheproblemofstandardization. J.Ethnopharmacol.100,114–117.
Choudhari,M.K.,Punekar,S.A.,Ranade,R.V.,Paknikar,K.M.,2012.Antimicrobial activityofstinglessbee(Trigonasp.)propolisusedinthefolkmedicineof West-ernMaharashtra,India.J.Ethnopharmacol.141,363–367.
daCunha,M.G.,Franchin,M.,deCarvalhoGalvao,L.C.,deRuiz,A.L.,deCarvalho, J.E.,Ikegaki,M.,deAlencar,S.M.,Koo,H.,Rosalen,P.L.,2013.Antimicrobialand antiproliferativeactivitiesofstinglessbeeMeliponascutellarisgeopropolis.BMC ComplementAltern.Med.13,23.
Dantas,C.G.,Nunes,T.L.G.M.,Nunes,T.L.G.M.,daPaixão,A.O.,Reis,F.P.,Júnior,W.d.L., Cardoso,J.C.,Gomes,M.Z.,Gramacho,K.P.,2014.Pharmacologicalevaluationof beevenomandmelittin.Rev.Bras.Farmacogn.24,67–72.
Dutra,R.P.,Abreu,B.V.,Cunha,M.S.,Batista,M.C.,Torres,L.M.,Nascimento,F.R., Ribeiro,M.N.,Guerra,R.N.,2014.Phenolicacids,hydrolyzabletannins,and antioxidantactivityofgeopropolisfromthestinglessbeeMeliponafasciculata Smith.J.Agric.FoodChem.62,2549–2557.
Juárez-Rojop, I.E., Tovilla-Zárate, C.A., Aguilar-Domínguez, D.E., Fuente, L.F.R.-d.l.,Lobato-García, C.E.,Blé-Castillo, J.L.,López-Meraz,L.,Díaz-Zagoya,J.C., Bermúdez-Oca ˜na,D.Y.,2014.Phytochemicalscreeningandhypoglycemic activ-ityofCaricapapayaleafinstreptozotocin-induced diabeticrats.Rev.Bras. Farmacogn.24,341–347.
Kustiawan,P.M.,Puthong,S.,Arung,E.T.,Chanchao,C.,2014.Invitrocytotoxicityof Indonesianstinglessbeeproductsagainsthumancancercelllines.AsianPac.J. Trop.Biomed.4,549–556.
Ryu,H.W.,Cho,J.K.,Curtis-Long,M.J.,Yuk,H.J.,Kim,Y.S.,Jung,S.,Kim,Y.S.,Lee, B.W.,Park,K.H.,2011.␣-Glucosidaseinhibitionandantihyperglycemic activ-ityofprenylatedxanthonesfromGarciniamangostana.Phytochemistry72, 2148–2154.
Sforcin,J.M.,Bankova,V.,2011.Propolis:isthereapotentialforthedevelopmentof newdrugs?J.Ethnopharmacol.133,253–260.
Sforcin,J.M.,2007.Propolisandtheimmunesystem:areview.J.Ethnopharmacol. 113,1–14.
Silici,S.,Kutluca, S.,2005. Chemical compositionandantibacterial activityof propoliscollectedbythreedifferentracesofhoneybeesinthesameregion.J. Ethnopharmacol.99,69–73.
Umthong,S.,Phuwapraisirisan,P.,Puthong,S.,Chanchao,C.,2011.Invitro antipro-liferativeactivityofpartiallypurifiedTrigonalaevicepspropolisfromThailand onhumancancercelllines.BMCComplementAltern.Med.11,37.
Vongsak,B.,Gritsanapan,W.,Wongkrajang,Y.,Jantan,I.,2013a.Invitroinhibitory effectsofMoringaoleiferaleafextractanditsmajorcomponentson chemi-luminescenceandchemotacticactivityofphagocytes.Nat.Prod.Commun.8, 1559–1561.
Vongsak,B.,Sithisarn,P.,Mangmool,S.,Thongpraditchote,S.,Wongkrajang,Y., Grit-sanapan,W.,2013b.Maximizingtotalphenolics,totalflavonoidscontentsand antioxidantactivityofMoringaoleiferaleafextractbytheappropriateextraction method.Ind.CropsProd.44,566–571.
Vongsak,B.,Kongkiatpaiboon,S.,Machana,S.,Weerakul,T.,Pattarapanich,C., Lai-wattanaphaial,J.,Issaro,N.,2014.HPLCquantitativeanalysisofstinglessbee propolisextracts.In:BuraphaUniversityInternationalConference2014.Global WarmingandItsImpacts,ChonBuri,Thailand.
Zhang,H.,Wang,G.,Beta,T.,Dong,J.,2015.Inhibitorypropertiesofaqueousethanol extractsofpropolisonalpha-glucosidase.Evid.BasedComplementAltern.Med.,
http://dx.doi.org/10.1155/2015/587383,ArticleID587383.
