Rev. bras. farmacogn. vol.26 número3

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w ww.e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Antioxidant,

DNA

damage

protective,

neuroprotective,

and

␣

-glucosidase

inhibitory

activities

of

a

flavonoid

glycoside

from

leaves

of

Garcinia

gracilis

Chonlakan

Supasuteekul

a,b

_,

_Wanroong

_Nonthitipong

b

_,

_Sarin

_Tadtong

c

_,

_Kittisak

_{Likhitwitayawuid}

b

_,

Parkpoom

Tengamnuay

a

_,

_Boonchoo

_Sritularak

b,∗

a_Department_of_{Pharmaceutics}_and_Industrial_Pharmacy,_Faculty_of_{Pharmaceutical}_Sciences,_{Chulalongkorn}_University,_Bangkok,_Thailand b_Department_of_{Pharmacognosy}_and_{Pharmaceutical}_Botany,_Faculty_of_{Pharmaceutical}_Sciences,_{Chulalongkorn}_University,_Bangkok,_Thailand c_Department_of_{Pharmacognosy,}_Faculty_of_Pharmacy,_{Srinakharinwirot}_University,_{Nakhon-nayok,}_Thailand

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received2November2015 Accepted24January2016 Availableonline5March2016

Keywords:

Antioxidant DNAprotective Flavonoidglycoside

Garciniagracilis

␣-Glucosidase Neuroprotective

a

b

s

t

r

a

c

t

TheleavesofGarciniagracilisPierre,Clusiaceae,havebeenusedasflavouringmaterialsinfood,with

nopreviousreportsoftheirbiologicalactivitiesandchemicalconstituents.Inthisstudy,themethanolic

extractofG.gracilisaffordedthreecompoundsnamelyapigenin-8-C-␣-l-rhamnopyranosyl-(1_→2)-␤-d

-glucopyranoside(1),5-hydroxymethyl-2-furaldehyde,andvanillicacid.Alloftheisolateswereinitially

evaluatedforsuperoxideanionradicalscavengingactivityand␣-glucosidaseinhibitoryeffects.

Com-pound1,whichwasthemajorcomponent,showedthemostpotentactivitiesamongthesethreeisolates.

Furtherbiologicalevaluationsrevealedthatcompound1couldpreventthepBR322plasmidDNAdamage

inducedbythephotochemicalreactionofriboflavinandprotectP19-derivedneuronsfromtheoxidative

stressconditioninducedbyserumdeprivation.Itwasconcludedthatthepotentbiologicalactivitiesof

G.graciliscouldbeattributedtothesynergisticeffectofcompound1withotherconstituentsfoundin

theplant.

Introduction

Ageingisanunavoidablephenomenoninlivingorganismsthat resultsinmorphological,biochemical,functionaland psychologi-calchangesintheorganism(Moreiraetal.,2014).Accordingtoa UnitedNationsreport,theaveragelifespanoftheworld popula-tionhasbeenincreasing,anditisestimatedthatthepercentage ofelderlypeople(thoseaged60yearsorover)willcontinueto growand willreach21.1% by2050due tolow birth rates and longevity(Rahman,2007;UnitedNations,2013).Thisincreasewill resultinanincreaseinage-relatedchronicdiseases,including car-diovasculardisease,cancer,diabetes,andneurologicaldisorders, suchasAlzheimer’sdiseaseandParkinson’sdisease.Therefore,itis importanttosearchforanti-ageingmedicines,foods,anddietary supplementsthataresafeandeffectivetoreducemorbidityand provideagoodqualityoflifeforelderlyindividuals(Rahman,2007). Theexcessiveproductionofreactiveoxygenspecies(ROS)and freeradicalsis consideredtobea significantcause ofoxidative

∗ Correspondingauthor.

E-mail:boonchoo.sr@chula.ac.th(B.Sritularak).

damageinbiomolecules,suchasproteins,lipids,andDNA, eventu-allyleadingtonumerousdegenerative diseases.However,these unfavourableeffectscouldbepreventedby theconsumption of antioxidantstoprotectthecellsfromROSandmaintainROS con-centrationsat a lowlevel (Gulet al.,2011; Meng etal., 2012). Variousmedicinalandfoodplantsarerich sourcesoffree radi-calscavengingmolecules,whichhavestrongantioxidantactivities (Kuateetal.,2011).Inlightofthesehealthbenefits,thesearchfor antioxidantcompoundsfromnaturalproductshasattracted inter-est.

Moreover,ROSandfreeradicalsarealsogeneratedby hypergly-caemia,andmaybeassociatedwiththemetabolicabnormalities thatoccurinpatientswithdiabetesmellitus(Tiwarietal.,2013). Onecurrentapproachtothetreatmentofdiabetesandobesityis tocontrolbloodglucoselevels.␣-Glucosidaseisakeyintestinal enzymeincarbohydratedigestion.Theinhibitionofthisenzyme coulddelaythecarbohydratehydrolysis process,leading tothe preventionof excessglucoseabsorption inthegut.Acarbose is awell-known␣-glucosidaseinhibitorthatisusedtotreat type-II diabetes mellitus, but this inhibitors appears to have major side effects, including gastrointestinal disturbance and weight gain (Hollander, 2007). Therefore, it is important to find new

http://dx.doi.org/10.1016/j.bjp.2016.01.007

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C.Supasuteekuletal./RevistaBrasileiradeFarmacognosia26(2016)312–320 ␣-glucosidaseinhibitorswithfewersideeffects,andhigherpatient

approval(Kimetal.,2000;Yinetal.,2014).

ThegenusGarciniabelongstothefamilyClusiaceaeandincludes 390speciesthatarewidelydistributedintropicalAsia,Australia, Polynesia, and southern Africa. Twenty-ninespecies have been reportedinThailand.Thesespeciesareevergreentreesthatrange fromsmalltomediuminsize,andsomespeciescangrowupto 30minheight(Ritthiwigromet al.,2013;Semwal etal.,2015). PreviousstudiesreportedthatGarciniaplantscontainmany sec-ondarymetabolitesandpossessvariouspharmacologicaleffects, includingantitumour, antioxidant,anti-inflammatory, and anti-immunosuppressiveeffects(Serujietal.,2013).

Garciniagracilis Pierre,whichis alsoknownasCha-mangor Mak-paeminThai,isoneoftheGarciniaspeciesthatwere discov-eredinThailand.TheripefruitsandleavesofG.gracilisareedible. Theleavesofthisplanthavetraditionallybeenusedasflavouring materialsinfoods(Suksrietal.,2005).Therootsarealsousedas antipyreticsfolkmedicine(Chuakul,2009).However,nostudies have investigated thechemical constituentsand pharmacologi-calactivitiesofthisplanttodate.Inthis study,ourpreliminary screeningof a methanolextractprepared fromtheleavesofG. gracilisshowedavarietyofpotentbiologicalactivities,including superoxidescavengingeffects (70.65%inhibitionata concentra-tionof100␮g/ml),protectionagainstDNAdamage(76.46%ata concentrationof100␮g/ml),andneuroprotectiveeffects(100%cell viabilityataconcentrationof100ng/ml).Thisextractalso exhib-ited␣-glucosidaseinhibitoryactivity(99.49%ataconcentrationof 2mg/ml).Theseresultspromptedustoinvestigatetheextractto identifythecompoundsresponsiblefortheseactivities.

Inthepresentstudy,wedescribetheisolationofcompounds1–3 fromtheleavesofG.gracilis,aswellastheevaluationof apigenin-8-C-␣-l-rhamnopyranosyl-(1_→2)-␤-d-glucopyranoside (1), the majorisolatedcompound,forantioxidant,DNAprotective, neu-roprotective,and␣-glucosidaseinhibitoryactivities.Compound1 wasinitiallyidentifiedfromtheleavesofBambusatextilis(Wang etal.,2012).However,thispaperisthefirstreporttodescribethe presenceofaflavonoidglycosideinG.gracilis.

Materialsandmethods

Plantmaterial

Leaves of Garcinia gracilis Pierre, Clusiaceae, were collected fromPrincessMahaChakriSirindhornHerbalGardeninMueang RayongDistrictinRayong,ThailandinFebruary2011. Authenti-cationwasperformedbycomparisonwithherbariumspecimens in the National Park, Wildlife and Plant Conservation Depart-ment oftheMinistry ofNatural Resources and Environment.A voucherspecimen(GG-022554)wasdepositedintheDepartment ofPharmacognosy andPharmaceuticalBotany intheFaculty of PharmaceuticalSciencesatChulalongkornUniversityinThailand.

Chemicals

The P19 cell line (ATCC CRL-1857) was obtained from ATCC®_, _USA. _Foetal _bovine _serum _(FBS), _new-born _calf _serum (NCS), alpha minimal essential medium (␣-MEM), and an antibiotic-antimycotic solution were purchased from Gibco®_, USA. All trans-retinoic acid (RA), cytosine-1-␤-d-arabinoside, 1:250 porcine trypsin, poly-l-lysine (MW>300,000), 2,3-bis(2-methoxy-4-nitro-5-sulphonyl)-2H-tetrazolium-5-carboxanilide sodium (XTT), ␣-glucosidase from Saccharomyces cerevisiae, p -nitrophenyl-␣-d-glucopyranoside(pNPG)phenazinemethosulfate (PMS),1,1-diphenyl-2-picrylhydrazyl(DPPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), quercetin, and

nitrobluetetrazolium(NBT)wereobtainedfromSigma–Aldrich®_, USA. Riboflavin and acarbose were purchased from Fluka Analytical®_,_Switzerland._{Ethylenediaminetetraacetic}_acid_(EDTA) wasprocuredfromMay&Baker®_,_England._pBR322_plasmid_DNA was obtainedfromVivantis TechnologiesSdn. Bhd.®_, _Malaysia. Allsolvents wereanalyticalgrade and purchasedfromMerck®_, Germany.

Extractionandisolation

DriedandpowderedleavesofG.gracilis(1kg)weremacerated withMeOHtoyield268.16gofmethanolextractaftersolvent evap-oration.ThiscrudeextractwassuspendedinH₂Oandpartitioned withEtOAcandBuOH togenerateanEtOAcextract(60.23g),a BuOHextract(119.76g),andanaqueousextract(42.08g).

TheBuOHextractwasthenfractionatedbycolumn chromatog-raphy(CC)(MCIgel,MeOH-H2Ogradient)togeneratefivefractions (I–V).FractionII(13.53g)wasseparatedbyCC(silicagel, EtOAc-MeOHgradient)togenerateninefractions(II-AtoII-I).FractionII-E (3.41g)wasfurtherpurifiedonSephadexLH-20(MeOH)to gen-erate1(781.2mg).TheEtOAcextractwasseparatedbyvacuum liquidchromatographyVLC(CH2Cl2-Acetonegradient)togenerate sevenfractions(I–VII).FractionIV(7.58g)waschromatographed onasilicagelcolumn(CH2Cl2-EtOAcgradient)toyieldseven frac-tions(IV-AtoIV-G).FractionIV-C(0.94g)wasfurthersubjected toSephadex LH-20 (acetone)to generate 2(20mg). FractionV (5.13g)wasthenseparatedbysilicagel(CH2Cl2-EtOAcgradient) togeneratethreefractions(V-AtoV-C).FractionV-B(0.57g)was subsequently purifiedby SephadexLH-20(acetone)toobtain3 (20.2mg).Allorganicsolvents usedforextractionand isolation werecommercialgradeandredistilledpriortouse.Theisolates (1–3)showedmorethan98%purityintheNMRspectrum.

Apigenin-8-C-˛-l-rhamnopyranosyl-(1_→2)-ˇ-d -glucopyranoside(1₎

Yellowamorphoussolid;C27H30O14;HR-ESI-MSm/z601.1536 [M+Na]+_;_IR_max_:_3367,_2935,_1654,_1360,₈₃₇_cm−1_;_UV_max_:₂₁₅ and333nm;1_H_NMR_(DMSO-_d₆_,₃₀₀_MHz)_and13_C_NMR_(DMSO-_d₆_, 75MHz),asshowninTable1.

5-Hydroxymethyl-2-furaldehyde(2₎

Brownishoil;C6H6O3;HR-ESI-MSm/z149.0216[M+Na]+; IR

max:3390,1673,1521cm−1;1HNMR(acetone-d6,300MHz):ı 9.59(1H,s,H-1),7.38(1H,d,J=3.3Hz,H-3),6.58(1H,d,J=3.3Hz, H-4),4.65(2H,s,H-6);13_C_NMR_(acetone-_d

6,75MHz):ı177.2(C-1), 162.0(C-5),152.5(C-2),122.9(C-3),109.3(C-4),56.6(C-6).

Vanillicacid(3₎

Whitepowder;C8H8O4;HR-ESI-MSm/z191.0321[M+Na]+;IR

max: 3467,2917, 2847, 1671, 1433,1110, 763cm−1; 1H NMR (acetone-d6,300MHz):ı7.62(1H,dd,J=8.4,1.5Hz,H-6),7.57(1H, s,H-2),6.92(1H,d,J=8.4Hz,H-5),3.91(3H,s,3-OCH3);13CNMR (acetone-d6,75MHz): ı166.7(COOH),151.2(C-4),147.2 (C-3), 124.0(C-6),122.0(C-1),114.6(C-5),112.6(C-2),55.4(3-OCH₃).

Determinationofantioxidantactivity

AssayofDPPHradicalscavengingactivity

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C.Supasuteekuletal./RevistaBrasileiradeFarmacognosia26(2016)312–320

Table1

1_H₍₃₀₀_MHz)_and13_C_NMR₍₇₅_MHz)_chemical_shifts_of_compound₁_(DMSO-_d 6). Position 1_H₍_J_in_Hz) 13_C _HMBC_(correlation_with1_H)

2 – 164.2 2′_and₆′

3 6.78 s 102.6 –

4 – 182.3 –

5 – 160.8 5-OH and 6

6 6.27s 98.5 5-OH

7 – 162.5 6and1′′

8 – 104.3 6and1′′

9 – 156.0 1′′

10 – 104.6 3 and 6

1′ – 121.7 3′, 5′and 3 2′ 8.03d(8.7) 129.1 6′ 3′ _6.91_d_(8.7) _116.1 ₅′

4′ _– _161.4 ₂′_,₃′_,₅′_and₆′ 5′ _6.91_d_(8.7) _116.1 ₃′

6′ 8.03 d (8.7) 129.1 2′

5-OH 13.12 s – –

Glucose

1′′ _4.75_d_(9.9) _71.8 ₂′′ 2′′ 4.06 dd (8.7, 9.1) 75.2 1′′and 1′′′ 3′′ 3.51 m 80.0 2′′and 4′′ 4′′ 3.43m 70.8 3′′and6′′

5′′ _3.24_m _81.9 ₁′′

6′′ _3.75_br_d_(12.0) _61.3 _– 3.53m

Rhamnose

1′′′ _4.97_s _100.5 ₂′′ 2′′′ _3.60_m _70.6 ₄′′′ 3′′′ _3.08_dd_(9.0,_2.3) _70.4 ₁′′′_and₄′′′ 4′′′ 2.90 t (9.0) 71.6 2′′′, 3′′′and 6′′′ 5′′′ 2.10 m 68.4 1′′′and 6′′′ 6′′′ 0.46d(6.0) 17.9 4′′′

performedforIC50determination.Thereactionmixture(200␮l)

ineachwellcontained20␮lofthesamplesolutionand180␮lof 50␮MDPPHina96-wellmicrotiterplate.Thereactionmixture wasthenincubatedfor30min,andtheabsorbanceat510nmwas measuredwithamicroplatereader.ThepercentageofDPPHradical scavengingactivitywasthencalculatedasfollows:

%DPPHradicalscavengingactivity=Ac−As Ac ×100,

whereAcistheabsorbanceofthecontrolandAsistheabsorbanceof

thesamples.Theexperimentwasperformedintriplicate(n=3),and eachexperimentconsistedofthreerepetitions.MeOHwasusedas anegativecontrol.Troloxwasusedasapositivecontrolandtreated underthesameconditionsasthesamples.

Assayofsuperoxideanion(O2•−)scavengingactivity

Thisassaymeasurestheabilityof thetestsample toinhibit thereductionofNBTtoblueformazanbyO2−(Chatsumpunetal.,

2010).Thesamplesolutionswerepreparedbydissolvingthetest sampleinasolutionof30%MeOHinpotassiumphosphatebuffer. Thereaction(200␮l)wasperformedbyadding40␮lofsample solutionand20␮lof750␮MNBTtoamixtureof20␮lof50mM potassiumphosphatebuffer,100␮lof266␮Mriboflavin,and20␮l of1mMEDTAina96-wellmicrotiterplate.Thereactionmixture wasthenilluminatedwithafluorescentlampfor10minatroom temperature.Theformationofblueformazanwasthenmonitored basedontheincreaseintheabsorbanceat570nm.Asimilar reac-tionmixturewaskeptinthedarkandservedastheblank.The percentageofO2−radicalscavengingactivitywasthencalculated asfollows:

%O−

2radicalscavengingactivity=

Ac−As

Ac ×100,

whereAcistheabsorbanceofthecontrolandAsistheabsorbanceof thesamples.Theexperimentwasperformedintriplicate(n=3),and

eachexperimentconsistedofthreerepetitions.Asolutionof30% MeOHwasusedasanegativecontrol.Troloxwasusedasapositive controlandtreatedunderthesameconditionsasthesamples.

AssayofDNAprotectiveactivity

Theinhibitoryeffectof thetestsamplesonsupercoiledDNA breakage was assessed using the agarose gel electrophoresis method(Chatsumpun etal.,2010).Briefly,thetest sample was initiallyevaluatedataconcentrationof100␮g/ml,andtwo-fold serialdilutionwasperformedforIC50 determination.Each reac-tionmixture(10␮l)contained2␮lofthesamplesolution,1␮lof 50mMpotassiumphosphatebuffer,5␮lof266␮Mriboflavin,1␮l of1mMEDTA,and1␮lof100ng/␮lpBR322plasmidDNA.The mix-turewasthenilluminatedwithafluorescentlampfor30min.The sameexperimentwaskeptinthedarkasablank.Themixturewas subsequentlytreatedwith2␮lofloadingdye(0.25% bromophe-nolblue,0.25%xylenecyanol,and40%sucroseinwater),andload onto0.7%agarosegel.Electrophoresiswasconductedat100Vina Tris–aceticacid–EDTAbuffer.Then,thegelwasstainedwith ethid-iumbromide(0.5␮g/mlindeionizedwater)andvisualizedunder ultravioletlight.ImageswereobtainedusingaMiniBISGel Doc-umentationsystemand analysedwithGelQuantAnalysis(DNR BioImagingSystems,Jerusalem,Israel).Theexperimentwas per-formedintriplicate(n=3),andeachexperimentconsistedofthree repetitions.Asolutionof30%MeOHwasusedasanegative con-trol.Troloxandquercetinwereusedaspositivecontrolsandtreated underthesameconditionsasthesamples.

AssayofneuroprotectiveeffectsonculturedP19-derivedneurons

Cellculture

P19cellswereculturedinP19GM(␣-MEMsupplementedwith 7.5%NCS,2.5%FBS,and1%antibiotics-antimycoticsolution)ina humidified5%CO2atmosphereat37◦C.Cellsinmonolayercultures weremaintainedintheexponentialgrowthphasebysubculturing every2days(Jones-Villeneuveetal.,1982).

DifferentiationofP19cellsintoP19-derivedneurons

Exponentiallygrownculturesweretrypsinisedandseparated intoindividualcells.ThedifferentiationofP19cellswasperformed byseeding2×106cells/mlontoa100-mmbacteriologicalculture dishcontaining10mlofP19IM(␣-MEMsupplementedwith5%FBS and1%antibiotics-antimycoticsolution)and0.5␮MRA.Abulky aggregateofcellswasformedinsuspension.Thecellclusterswere thendissociatedafter4daysofRAtreatmentusinga5-mlglass measuringpipetteand resuspendedonpoly-l-lysine-pre-coated multi-well plates (platescoated with 50␮g/ml of poly-l-lysine dissolvedinaphosphate-bufferedsaline(PBS)solutionovernight and sterilized underUV light for 30min) ata concentrationof 7×104cells/ml (150␮l/well) in P19SM (␣-MEM supplemented with10%FBS,and1%antibiotic-antimycoticsolution). Thecells werethen incubatedfor anadditional day.Theproliferation of non-neuronalcellswasinhibitedby theaddition of cytosine-1-␤-d-arabinosideorAra-C (10␮M) onthefirstdayafterplating, andthemediumwasrenewedeveryfewdays.Thedifferentiated P19-derivedneuronswereusedafterday14ofthedifferentiation process(Jones-Villeneuveetal.,1982;Jones-Villeneuveetal.,1983; MacPhersonandMcBurney,1995;Tadtongetal.,2012).

Neuronalviabilityassay

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C.Supasuteekuletal./RevistaBrasileiradeFarmacognosia26(2016)312–320 1000,and10,000ng/ml.DMSOwasaddedtotheculturesata

con-centrationof0.5%(v/v)asasolventcontrol.P19SMsupplemented with10␮MAra-Cwasaddedtothecontrolwells.Thecellswere keptat37◦_C_for₁₈_h._Then,₅₀_␮_l_of_an_XTT_solution₍₁_mg/ml_XTT in60◦_C_␣_-MEM_with₂₅_␮_M_PMS)_was_added,_and₁₅₀_␮_l_of_the mediumwastakenout.After4hofincubation,100␮lofPBS,pH 7.4wasaddedtoeachwell.Theopticaldensity(OD)wasmeasured at450nmusingmicroplatereader.Theexperimentwasperformed intriplicate(n=3),andeachexperimentconsistedofthree repe-titions,withmediumat100%cellviabilityasacontrol(Tadtong etal.,2012;Tadtongetal.,2007).Theconcentrationthatpromoted bettersurvivaloftheculturedneuronsthanthecontrolwasfurther evaluatedforneuroprotectiveactivity.

Neuritogenicityassay

TheassaywasconductedwithP19-derivedneuronscultured ina96-wellplateusingtheserumdeprivationmethod(Iacovitti etal.,1997;López-Maderueloetal.,2001;Tadtongetal.,2013). TheP19SMsupplementedwith10␮MAra-Cwasremovedafter 14 days of the differentiation process, and the sample solu-tionsinDMSOdiluted withP19SMcontaining10␮MAra-C,the ␣-MEMsupplementedwith10␮MAra-C,andthe1% antibiotic-antimycoticsolutionwithoutFBSwereaddedtogenerateafinal sampleconcentrationthatenhancedthesurvivalofcultured neu-rons more than the control. DMSO wasadded to the cultures at a concentration of 0.5% as a solvent control, followed by P19SMwith10␮MAra-C in thecontrol wells.␣-MEM supple-mentedwith10␮MAra-Cand1%antibiotic-antimycoticsolution withoutFBS wereused togenerate the oxidative stress condi-tion. The cells were kept at 37◦_C _for ₁₈_h. _Cell _viability _was assayed usingthe XTT reduction method.The experiment was performed in triplicate (n=3), and each experiment consisted of 3 repetitions, with medium at 100% cell viability as a con-trol.

Assayof˛-glucosidaseinhibitoryactivity

The␣-glucosidaseinhibitoryeffectwasevaluatedasdescribed previously,withslightmodifications(Sunetal.,2014).Thisassay measurestheenzyme activitybyinvestigating therelease ofp -nitrophenolfromthepNPGsubstrate.Eachsamplewasinitially evaluatedataconcentrationof2mg/ml,andtwo-foldserial dilu-tionwasperformedforIC50determination.Thereactionmixturein a96-wellmicrotiterplateinitiallycontained10␮loftestsample and40␮lof0.1U/ml␣-glucosidaseandwaspre-incubatedat37◦_C for10min.Then,50␮lof2mMpNPGwereaddedtothemixture andfurtherincubatedat37◦_C_for₂₀_min._The_reaction_was_then terminatedbytheadditionof100␮lofa1MNa2CO3solution.The amountofp-nitrophenolreleasedwasmeasuredusingamicroplate readertodeterminetheabsorbanceat405nm.Thepercentageof ␣-glucosidaseinhibitoryactivitywasthencalculatedasfollows:

%˛-glucosidaseinhibitoryactivity= Ac−As

Ac ×

100,

whereAcistheabsorbanceofthecontrolandAsistheabsorbance ofthesamples.Theexperimentwasperformedintriplicateand eachexperimentconsistedofthreerepetitions.FivepercentDMSO wasusedasanegativecontrol.Acarbosewasusedasapositive control and treated under the same conditions as the sam-ples.

Statisticalanalysis

Allanalyseswerecarriedoutintriplicate(n=3).Thedatawere presentedasthemean±standarddeviation(SD).One-way analy-sisofvariance(ANOVA)withtheleastsignificantdifference(LSD) testwascarriedouttoidentifysignificantdifferencesbetweenthe

control andexperimentalgroups using SPSSversion18.0 (SPSS Inc., Chicago, IL).Differenceswere considered significantwhen p<0.05.

Resultsanddiscussion

Isolationandidentificationofisolatedcompounds

ThephytochemicalinvestigationoftheMeOHextractfromG. gracilisleavesyielded aglycosidicflavonecompound (1)as the majorconstituent,alongwith5-hydroxymethyl-2-furaldehyde(2) andvanillicacid(3).The1_H_and13_C_NMR_assignments_of_compound 1thatwerereportinapreviousstudywerebasedon1DNMR exper-iments(Wangetal.,2012).ThepreviouslyreportedNMRdataare differentfromourresults,especiallywithrespecttothe13_C_NMR dataforthesugarmoieties.In thisstudy,thestructure of com-pound1wasidentifiedbasedon1DNMR(1_H,13_C_NMR),_2D_NMR (1_H–1_H_COSY,_HSQC,_and_HMBC),_HR-ESI-MS,_FTIR,_and_UV spec-troscopy.Compound1wasobtainedasayellowamorphoussolid. TheHR-ESImassspectrumofthatcompoundshowedapeakforthe [M+Na]+_ion_at_m_/_z_601.1536_(calculated_for_601.1533),_indicating amolecularformulaofC27H30O14.TheIRspectrumofcompound1 exhibitedthebroadabsorptionofahydroxylgroupat3367cm−1 andacarbonylgroupat1654cm−1_._These_results,_along_with_UV absorptionmaximaat215and333,suggestedaflavoneglycoside structureforcompound1.

Accordingtothe1_H_and13_C_NMR_spectra_of₁_that_are_listed inTable1,the13_C_NMR_data_for_compound₁_revealed_the pres-enceoffifteenaromaticcarbonresonancesfortheaglyconepart ofthecompoundandtwelvesugarsignals,whichweredetected asonehexoseandonedeoxyhexoseunit.Basedonthe1_H_NMR data,theapigeninskeletonwasidentifiedfromthepresenceofone protonsingletatıH6.78(H-3)inthearomaticregion,aswellas twoprotonsignalsatıH 6.91andıH 8.03(each2H,d,J=8.7Hz) whichindicatedaparasubstituentatC-4′_of_ring_B._From_the_HMBC spectra,thesignalatıH8.03wasassigned totwoorthoprotons ofringB(H-2′ _and_H-6′₎_based_on_the_correlation_of_these pro-tonswithC-2ofringC.Ahydrogen-bondedhydroxylproton(s, 5-OH)inringAwasassignedtothesharpsingletpeakatıH13.12 inthe1_H _NMR_spectrum._Based _on_the_HMBC_spectra,_the pro-tonat5-OHexhibitedcorrelationswithC-6atıC98.5,C-5atıC 160.8andC-10atı_C104.6.Consequently,theıH6.27(1H,s)of H-6wasindicatedbyitscorrelationwiththecarbonsignalofC-6in HSQC.

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C.Supasuteekuletal./RevistaBrasileiradeFarmacognosia26(2016)312–320 (ıH 4.75, d,J=9.9Hz)and H-6(ıH 6.27,s)in theHMBC

experi-ment.

Accordingtotheabovespectroscopicdata,compound1was identified as apigenin-8-C-␣-l-rhamnopyranosyl-(1_→2)-␤-d -glucopyranoside.

OH

OH OH

OH

OH O

1

O O O O HO

HO HO

Theother knowncompounds 2 and 3 wereidentified as 5-hydroxymethyl-2-furaldehyde(2)andvanillicacid(3)basedona comparisonoftheirNMRspectraldatawithvaluesavailableinthe literature(Espinozaetal.,2008;Changetal.,2009).

HO

OH

2

3

CHO

OCH₃ CO₂H

O

Preliminaryscreeningofisolatedcompounds

Based ona primary screen for superoxideanion scavenging activityat100␮g/ml,compound1and3exhibitedpotent scav-enger activity, with values of 96.45% and 82.61%, respectively, whilstcompound2displayednoactivity.TheIC50ofcompound1 was23.91±5.37␮Mandthatofcompound3was19.88±1.34␮M. Afterscreening for␣-glucosidase inhibitoryactivityat2mg/ml, onlycompound1showedactivity,with96.90%inhibitionandan IC50of0.56±0.01mM.Compounds2and3wereinactive. Prelim-inarystudiesofthebiologicalactivitiesoftheisolatedcompounds demonstratedtheantioxidantpotentialandrelatedeffectsof com-pound1.Moreover,thisflavonoidwithan8-Csubstitutioninthe A-ringhasnotbeenreportedpreviouslyinG.gracilis.Therefore,the presenceofan8-Cflavonoidglycosidedeservedfurther chemotax-onomicattentionandadditionalinvestigationofbiologicalactivity inthisstudy.

Antioxidantactivity

DPPHradicalscavengingactivity

DPPHisacolouredandstablenitrogenfreeradical.Thisassay determinesthereducingcapacityofanantioxidantbymeasuring thechangeofcolourfromviolettoyellowbasedonitsabsorbance. Antioxidantscaneliminatethisfreeradicalviatheprocessof hydro-genatomtransferorelectrondonation(Mohammedetal.,2015).

Based on the results, the highest scavenging activity was observedfor Trolox(IC50=7.7±1.74␮M). Compound1 showed a dose-dependent but weaker scavenging effect in this model

(IC50=117.47±14.14␮M)(Table2,Fig.1).Theweakactivityof theapigeninglycosidewasconsistentwithapreviousreportbyLu andFoo(2001)andindicatedtheimportanceofthe3′_,4′_-dihydroxy groupoftheB-ring,whichisakeyfactorforscavengingDPPH(Lu andFoo,2001;Lietal.,2008;Mohammedetal.,2015).In addi-tion,thesubstitutionofahydrogenatomattheC-8positioninthe flavoneAringbythetwosugarmoietiesalsodecreasedthe antioxi-dantactivityduetosterichindrance,asthosebulkygroupsreduced accessthecentreoftheDPPHradical(Prioretal.,2005;Zengetal., 2013).

Superoxideradicalscavengingactivity

Thesuperoxideradicalisasignificantcellularfreeradicaland isassociatedwithanincreaseinoxidativedamageinbiomolecules duetotheproductionofmorepowerfulreactivespecies.In our model,O2−wasgeneratednon-enzymaticallybythephotoreaction ofriboflavinandassayedbasedonthereductionofNBTtogenerate blueformazan.However,thisprocesscanbeinhibitedwhenO2− scavengersarepresent(Chatsumpunetal.,2010).

The O₂− _scavenging _activity _of _compound ₁ _is _shown _in Table2 and Fig.1.Compound 1was foundtoexhibit stronger scavengingactivitythanTrolox,withIC50values of23.91±5.37 and 95.66±9.83␮M,respectively. Many studies reported vari-ableresultsfortheO2−scavengingactivityofflavonoidglycosides dependingonthepositionofglycosylation,theattachedhydroxyl group, and the type and number of sugars in the structures (Yokozawaetal.,1997;Zengetal.,2013;Xiaoetal.,2014;Materska, 2015).BasedonthestudiesofLuandFoo(2001),thecatecholand pyrogallolintheBringareresponsibleforstrongantioxidant activ-ity,butthescavengingactivitiesoftheflavoneglycosideswereall higherthanthatofTrolox.Thoseresultswereconsistentwiththe findingofYokozawaet al.(1997)thatapigenincaninhibitROS species,evenintheabsenceof6-or3′_-OH._Those_authors_also indi-catedthatthelinkedrhamnosesugaryieldedbetterpropertiesthan glucoseintheiraglycone.Therefore,thehighpotencyofcompound 1inthescavengingofsuperoxideradicalsmightalsoberelatedto thelinkedsugar.

DNAprotectiveactivity

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Table2

IC50values(␮M)ofcompound1isolatedfromGarciniagracilisforDPPHradicalscavenging,superoxideanionradicalscavenging,DNAprotective,and␣-glucosidaseinhibitory activities.

Sample DPPHa₍_␮_M) _Superoxide_aniona₍_␮_M) _DNA_protectivea₍_␮_M) _␣_-Glucosidasea_(mM)

Compound1 117.47±14.14a 23.91±0.23a 23.40±3.37a 0.56±0.01a

Trolox 7.7±1.47b 95.66±9.83b 125.75±29.91b –

Quercetin – – 21.01±1.24a –

Acarbose – – – 0.90±0.06b

Dissimilarlettersinthesamecolumnindicatesignificantlydifferentvaluesforeachparameteratp<0.05usingone-wayanalysisofvariance(ANOVA)withtheleastsignificant difference(LSD)test.

a_The_data_values_are_expressed_as_the_mean_±_SD_of_triplicate_experiments₍_n₌_3),_and_each_experiment_consists_of₃_repetitions.

80

120

100

80

60

40

20

0

A

B

C

DPPH r

adical sca

ve

nging

activity

, %

Concentration (µg/ml) Concentration (µg/ml)

Concentration (µg/ml)

Supero

xide anion r

adical

sca

ve

nging activity

, %

α

-glucosidase inhibitor

y

activity

, %

100

80

60

40

20

0 60

40

20

0

1 10 100 1000 1 10 100

50 500 5000

Fig.1. Concentration-dependentinhibitoryeffectsofcompound1on:(A)theDPPHradical,(B)thesuperoxideanion(O2−),and(C)the␣-glucosidaseenzyme.Thedataare

expressedasthemean±SDoftriplicateexperiments(n=3),andeachexperimentconsistsofthreerepetitions.

Compound 1

Trolox OC

1 2 3 4 5 6 7

120

Inhibition, %

Concentration (µg/ml) 100

80

60

40

20

0

1 10 100

1 2 3 4 5 6 7

SC

OC

SC

OC

SC

Quercetin

D

A

B

C

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Neurite

Neuron cell

Neurite

Neuron cell

A

C

B

Fig.3.Phase-contrastmicrographsoftheneuritogenicityofP-19-derivedneuronsafter18hofincubationin:(A)P19SM(␣-MEM+10%,v/vFBS+10␮MAra-C)without treatment(control),(B)serumdeprivationconditions(␣-MEM+10␮MAra-C)withouttreatment(toxicconditions),and(C)serumdeprivationconditions(␣-MEM+10␮M Ara-C)treatedwithcompound1at100ng/ml;scalebar=10␮m.

flavonoidstoprotectagainstDNAdamagemaybeassociatedwith freeradicalscavengingactivity(Boyleetal.,2000).

Neuroprotectiveactivity

OxidativedamagecausedbyROSspeciesalsooccursinthebrain dueitslargeoxygenconsumption,itslargequantityoffattyacids, anditslowlevel ofantioxidantenzymes.Furthermore,neurons cannotpromptlyrecoverviamitosisand celldivision afterthey aredamagedduetotheirpostmitoticstatus(Tangsaengvitetal., 2013).P-19cells,whichareawell-knowninvitromodelderived frommurineembryonalcarcinoma,weredifferentiatedinto neu-ronsusingretinoicacid.TheP-19-derivedneuronswerefoundto beirreversiblypostmitoticandtocontainparticular neurotrans-mitters,suchas␥-aminobutyricacidandacetylcholine,whichare similartothosefoundinmatureCNSneurons(Tadtongetal.,2012). The viability of P-19-derived neurons in the presence of compound 1 was investigated using theXTT assay. Compound 1 showed 100% neuron viability at a nontoxic concentration (100ng/ml).Accordingly,theneuroprotectiveabilityofcompound 1at100ng/mlwasthenevaluatedinaserumdeprivationmodel. Serum is a mixture that contains a large amount of proteins and somevital growth factorsthat are required for the prolif-erationof cells inculture.The lackof seruminduced oxidative stressconditionsforthecells,eventuallyresultingincell apopto-sis(Tangsaengvitet al., 2013). Interestingly,at a concentration of 100ng/ml, compound 1 significantly protected the cultured neuronsagainstROStoxicity duringserumdeprivation-induced oxidativestress, asshown inFig. 3.Treatment withcompound 1 increased the neuriteoutgrowth of the cultured neurons by approximatelyfour-fold in comparison to theuntreated condi-tion(Table3).Manyreportsrevealedthatatalowconcentration, flavonoidsactasneuroprotectivesubstancesviatheactivationof themitogen-activatedprotein kinase(MAP kinase)pathway. In

Table3

Neuroprotectiveactivityduringserumdeprivation.

Sample Neuronal viabilitya_(%)

Compound1(100ng/ml) 65.74±9.41b

␣-MEM+0.5%DMSO 16.34±7.73

␣-MEM 16.59±8.13

P19SMc₊_0.5%DMSO _100.30_±_0.52

P19SMc _100.00_±_0.00

a_The_data_are_expressed_as_the_mean_±_SD_of_triplicate_experiments₍_n₌_3),_and

eachexperimentconsistsof3repetitions.

b_p_<_0.05_when_compared_with_the_toxic_condition₍_␣_-MEM)_and_the_solvent

con-trolofthetoxiccondition(␣-MEM+0.5%DMSO)usingone-wayanalysisofvariance (ANOVA)withtheleastsignificantdifference(LSD)test.

c _P19SM_is_composed_of_␣_-MEM₊_10%_(v/v)_FBS.

contrast,athighconcentrations,flavonoidsactivatethecaspase pathway,leadingtoapoptosis(MandelandYoudim,2004;Tadtong etal.,2013;Williamsetal.,2004).

˛-Glucosidaseinhibitoryactivity

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120

∗

Neuronal viability

, %

100

80

60

40

20

0

Compound 1

a-MEM+0.5%DMSO P19SM+0.5%DMSO

a-MEM P19SM

Fig.4. Theeffectofcompound1ontheneuronalviabilityofP-19-derivedneurons intheserumdeprivationmodel.Thehistogramshowsthepercentageofcellviability relativetovehicle-treatedcontrolcultures.Eachbarexpressesthemean±SDof

trip-licateexperiments(n=3),andeachexperimentconsistsof3repetitions.Significant differenceswerefoundforthecomparisonsofcompound1withthetoxiccondition (␣-MEM)andthesolventcontrolofthetoxiccondition(␣-MEM+0.5%DMSO)using one-wayanalysisofvariance(ANOVA)withtheleastsignificantdifference(LSD) test.*p<0.05.

ThisstudyisthefirstreportthatflavoneglycosidesfromG. gra-cilisexhibit␣-glucosidaseinhibitoryactivity,suggestingthatthis plantcouldbeapotentialsourceof␣-glucosidaseinhibitorsforthe treatmentofdiabetes(Fig.4).

Inconclusion, chromatographicseparationof themethanolic extractfromtheleavesofG.gracilisledtotheisolationand identi-ficationofthreecompounds,apigenin-8-C-␣-l -rhamnopyranosyl-(1→2)-␤-d-glucopyranoside(1),5-hydroxymethyl-2-furaldehyde (2),andvanillicacid(3).Amongtheseisolates,compound1was obtainedinthelargestquantityandexhibitedpotential superox-ideanionradicalscavengingactivity,aprotective effectagainst pBR322 plasmid DNA damage, a protective effect against P19-derivedserumdeprivation,and␣-glucosidaseinhibitoryactivity.

Authors’contributions

CS(PhDstudent)contributedtotheisolationandpurification ofthecompounds,therunningofthelaboratorywork,the anal-ysisofthedata,andthedraftingofthepaper.WNcontributedto isolationandpurificationofthecompounds.STcontributedtothe cell-basedassayofneuroprotectiveactivity.PTandKLcontributed tothecriticalreadingofthemanuscript.BScontributedtotheplant collection,thesupervisionofthelaboratoryworkandcritical read-ingofthemanuscript.Allauthorshavereadthefinalmanuscript andapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

This study was supported by the 100th _Anniversary Chu-lalongkorn University Fund for Doctoral Scholarship of Chula-longkornUniversity. The authors would also like tothank The ResearchInstrumentCenteroftheFacultyofPharmaceutical Sci-encesatChulalongkornUniversityforprovidingresearchfacilities.

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