Flavonol triglycosides of leaves from Maytenus robusta with acetylcholinesterase inhibition.

Mini

review

Flavonol

triglycosides

of

leaves

from

Maytenus

robusta

with

acetylcholinesterase

inhibition

Grasiely

F.

de

Sousa

a

,

Mariana

G.

de

Aguilar

a

,

Jacqueline

A.

Takahashi

a

,

Tânia

M.A.

Alves

b

,

Markus

Kohlhoff

b

,

Sidney

A.

Vieira

Filho

c

,

Grácia

D.F.

Silva

a

,

Lucienir

P.

Duarte

a,

*

a

DepartamentodeQuímica,UniversidadeFederaldeMinasGerais,AvenidaPresidenteAntônioCarlos,6627,Pampulha,31270-901,BeloHorizonte-MG,Brazil

b

LaboratóriodeQuímicadeProdutosNaturais,FundaçãoOswaldoCruz,AvenidaAugustodeLima,1715,BarroPreto,30190-002,BeloHorizonte-MG,Brazil

c

EscoladeFarmácia,UniversidadeFederaldeOuroPreto,RuaCostaSena,171,Centro,35400-000,OuroPreto-MG,Brazil

ARTICLE INFO

Articlehistory: Received28July2016

Receivedinrevisedform27October2016 Accepted31October2016

Availableonline15November2016

Keywords: Maytenusrobusta Celastraceae Flavonolglycosides Acetylcholinesteraseinhibition ABSTRACT

AlthoughMaytenusrobustaaqueousinfusionsofleavesareusedinBraziliantraditionalmedicinefor

stomachdiseasetreatment,onlyafewchemicalstudiesofthisspeciesarefoundinliterature.The

phytochemicalinvestigationofmethanolextractfromM.robustaleavesyieldedtheknowncompound

kaempferol (3) and two new flavonol glycosides: kaempferol-3-O-b-D-glucopyranosyl-(1!3)-a

-L-rhamnopyranosyl-(1!2)-b-D-glucopyranoside(1)andquercetin-3-O-b-D-glucopyranosyl-(1!3)-a

-L-rhamnopyranosyl-(1!2)-_b-D-glucopyranoside(2).Thechemicalstructuresof1and2wereelucidated

by1D/2DNMR,ESI–MSandESI–MS2_{spectraldata.Itisthefirsttimeflavonoidshavebeenreportedfrom}

M.robusta.Flavonols1and2showed66%and80%acetylcholinesterase(AChE)inhibition,comparedto

93%ofthestandardeserine,bytheEllman’smethod.Thesesubstancesareoneofthefewactiveflavonols

linkedtoatrisaccharidechainintheliteraturepresentingthisactivity,andcontributetothescreeningfor

newtypesofnaturalAChEinhibitors.

ã2016PublishedbyElsevierLtdonbehalfofPhytochemicalSocietyofEurope.

Contents

1. Introduction ... 34

2. Resultsanddiscussion ... 35

3. Experimental ... 37

3.1. Generalexperimentalprocedures ... 37

3.2. Plantmaterial ... 37

3.3. Extractionandisolation ... 37

3.4. Compoundcharacterization ... 37

3.4.1. Kaempferol-3-O-b-D-glucopyranosyl-(1!3)-a-L-rhamnopyranosyl-(1!2)-b-D-glucopyranoside(1) ... 37

3.4.2. Quercetin-3-O-_b-D-glucopyranosyl-(1!3)-a-L-rhamnopyranosyl-(1!2)-b-D-glucopyranoside(2) ... 37

3.5. Invitroacetylcholinesteraseinhibition ... 37

4. Conclusions ... 37

Acknowledgements ... 38

References ... 38

1.Introduction

MaytenusisoneofthelargestgeneraoftheCelastraceaefamily. About300Maytenusspeciesareidentifiedandfoundmainlyin tropicaland subtropical regions(McKenna etal., 2011).Several speciesareusedintraditionalmedicinein theformofaqueous infusionsforstomachdiseasetreatment(Leiteetal.,2001;Niero *Correspondingauthor.

E-mailaddress:lucienir@ufmg.br(L.P. Duarte).

http://dx.doi.org/10.1016/j.phytol.2016.10.024

1874-3900/ã2016PublishedbyElsevierLtdonbehalfofPhytochemicalSocietyofEurope.

ContentslistsavailableatScienceDirect

Phytochemistry

Letters

etal.,2011;Queirogaetal.,2000).Moreover,avarietyofbiological activitieswas foundto Maytenus genussuch as: antimicrobial, antiulcer, antiinflammatory, antinociceptive, antioxidant (Niero etal.,2011)andpotentialcentralnervoussystem(CNS)stimulate (Omena, 2007). Studies suggested a relationship between the biologicalactivitiesandMaytenussecondarymetabolites,suchas flavonoids(Jorgeetal.,2004;Leiteetal.,2001;Nieroetal.,2011; Souza-Formigonietal.,1991).

Maytenus robusta Reissek occurs in the Brazilian Atlantic rainforest and is known as “espinheira santa” or “cancerosa”. Ourpreviousstudiesfromhexaneextractsofleavesandbranches furnishedasteroidtogetherwithfriedelaneand hopene penta-cyclictriterpenes(Sousaetal.,2012a,2014;Sousaetal.2012b).

Nieroetal.(2006)studiedthemethanolextractoftheaerialparts of this plant and only found triterpenes and a steroid. These researchersdissolvedthemethanol extract,previouslydried,in water and partitioned it successively with hexane and ethyl acetate;however,justthelatterwaschromatographedonsilicagel column.Therefore, tothebestof ourknowledge,noflavonoids havebeenfoundsofarinthisplant.

Inthiswork,leaveswereexhaustivelymaceratedwithhexane, chloroform,ethylacetateandmethanolatroomtemperature.The methanolextractwasfractionedemployingnormaland reversed-phase chromatography furnishing kaempferol and two new flavonolglycosideswithatrisaccharidechain(compound1and 2).Thelasttwowerecharacterizedby1D/2DNMR,ESI–MSand ESI–MS2_{spectraldata.}

Flavanoidsarebioactivecompoundsthatpresentactionsonthe brainandcognitiveperformance, highantioxidantactivity,high metal-chelating property and low toxicity (Uriarte-Pueyo and Calvo,2011;Rendeiroetal.,2015).Uriarte-PueyoandCalvo(2011), intheirreview,foundonly128flavonoidsactivefor acetylcholin-esterase (AChE) inhibition, in which only 35 were flavonols. Analyzingthegroupofflavonols,only16containasugarmoiety but none of them contain a trisaccharide chain. In fact, no publications,tothebestofourknowledge,aboutAChEinhibitors duetoflavonolswithatrisaccharidechainwerefoundsofar.The

newcompounds1and2showedAChEinhibitionwhenevaluated invitrobyEllman’sbioassayandareoneofthefewexamplesofthe flavonolslinkedtoatrisaccharidechainpresentingthisactivity. 2.Resultsanddiscussion

ThemethanolextractofleavesfromM.robustafurnishedthe twonewflavonolglycosideskaempferol-3-O-

b

-D -glucopyranosyl-(1!3)-

a

-L-rhamnopyranosyl-(1!2)-

b

-D-glucopyranoside (1) and quercetin-3-O-

_b

-D-glucopyranosyl-(1!3)-

a

-L -rhamnopyra-nosyl-(1!2)-

b

-D-glucopyranoside (2), along with the known compoundkaempferol(3)(Fig.1).

Compound 1 was obtained as a light-yellow solid and its molecularformula,C33H40O20,wasestablishedbyHR-ESI–MS(m/

z:757.2185 [M+H]+,calc.757.2183).TheIRspectrumpresented absorption bands correspondent to hydroxyl (3416 and 1074cm1), carbonyl (1656cm1), and phenyl (1610 and 1178cm1)groups. Its 1H NMR spectrum showed four doublet signalsofaromaticprotons:twometa-coupled,at

_d

H6.19(1H,d,

J=2.0Hz, H-6) and 6.38(1H, d, J=2.0Hz, H-8), and two ortho-coupled, at

d

H6.89(2H,d,J=8.8Hz,H-3’/H-5’)and8.04(2H,d,

J=8.8Hz,H-2’/H-6’).Thisinformationidentifiescompound1asa kaempferolderivative.Thesignalsofthreeanomericprotonsand carbonatomsinthe1_Hand13_{CNMRspectrasuggestedcompound}

1asatrisaccharide.Basedintheaglyconecarbonchemicalshifts, we concluded the trisaccharide is linked toC-3. The anomeric protonsignalscorrelatedat

d

H5.70(1H,d,J=7.6Hz,H-1”)and

d

C

100.4;at

d

H5.25(1H,d,J=1.5Hz,H-1”’)and

d

C102.4;andat

d

H

4.55(1H,d,J=7.8Hz,H-1””)and

_d

C105.8intheHSQCspectrum.

HSQC-TOCSYspectrumsecuredtheassignmentsforonerhamnose andtwoglucosemolecules.Thelarge3JH1-H2couplingconstants

(JH1”-H2”=7.6 and JH1””-H2””=7.8Hz) established the

b

-con

figu-rationsfortheglucoseunits.Thesmall3_J

H1-H2couplingconstant

(JH1”’-H2”’=1.5Hz)establishedthe

a

-configurationforthe

rham-noseunit(Sannomiyaetal.,1998).Thesaccharidechain (glucose-rhamnose-glucose)was establishedconsideringthecorrelations observed between H-1””/C-3”’ and H-1”’/C-2” in the HMBC

O OH HO O OH O O O HO HO HO 1'' 2'' 3' 5' 1' 2 4 5 7 9 10 6'' 1''' 3''' 1'''' 6''' 6'''' O OH O HO O OH OH OH OH R O O OH HO OH OH 2 4 10 9 7 5 1' 2' 4' 6'

1 R = H

2 R = OH

3

spectrum.Finally,compound1wasidentifiedas

kaempferol-3-O-b

-D-glucopyranosyl-(1!3)-

a

-L-rhamnopyranosyl-(1!2)-

b

-D -glucopyranoside(Fig.1).Table1showsthecompleteNMRdatafor thisflavonol.TheESI–MS2_{(m/z:757.2183[M+H]}+_)spectrumof1

exhibited the fragment ions at m/z 449.1080 [Mglucosyl rhamnosyl+H]+_{andatm/z287.0551[Mglucosylrhamnosyl}

glucosyl+H]+_{correspondingtothelossofsugars.}

Compound 2 was isolated as a dark-yellow solid and its molecularformula,C33H40O21,wasdeterminedbyHR-ESI–MS(m/

z:773.2134[M+H]+_{,calc.773.2140).TheIRspectrumof2showed}

absorptionsbandsat3418cm1(OH),1658cm1(C=O),1610cm1 (C=C), 1204cm1 (phenol C-O) and 1074cm1 (alcohol C-O). Differentfromcompound1,the1_{HNMRspectrumof2exhibited}

twobroad signals (

d

H 6.25,1H; and

d

H 6.45,1H), two doublet

signals(

d

H6.93,1H,d,J=8.2Hz;and

d

H7.66,1H,d,J=1.6Hz),anda

doubletof doublet (

_d

H 7.63,1H, d, J=1.6e8.8Hz), attributed to

aromaticprotonsofaquercetinderivative.The1_Hand13_CNMR

spectraof2showedthreesignalsofanomericprotonsandcarbon. TheHSQCspectrumexhibitedcorrelationsbetweentheanomeric atoms:

d

H5.70(1H,d,J=7,6Hz)and

d

C100.3;

d

H5.23(1H,broad

signal)and

d

C102.2;and

d

H4.56(1H,d,J=7,9Hz)and

d

C105.9.We

concludedthatcompound2presentsthesamesugarmoleculesof compound 1 after analysing its HSQC-TOCSY spectrum. In the HMBCspectrum,correlationswereobservedbetweenH-1””/C-3”’ andH-1”’/C-2”(Table2).Therefore,compound2displaysthesame trisaccharidesequencethan1withadifferentaglycon,andwas identified as quercetin-3-O-

b

-D-glucopyranosyl-(1!3)-

a

-L -rhamnopyranosyl-(1!2)-

b

-D-glucopyranoside (Fig. 1). Its ESI– MS2_{(m/z:773.2134[M+H]}+_{)spectrumshowedfragmentionsat}

m/z465.1038[Mglucosylrhamnosyl+H]+_{andatm/z303.0499}

[Mglucosylrhamnosylglucosyl+H]+ corresponding to the lossofsugars.

The13_{CNMRspectraldataof3weresimilartotheonerelated}

byAgrawaletal.(1989)anditwasidentifiedaskaempferol(Fig.1). Compounds1and2wereevaluatedinvitrofor acetylcholines-terase(AChE)inhibitionusingthecolorimetricmethodestablished byEllmanbutadaptedfor96-wellmicroplate(Rheeetal.,2001). The assay using Ellman’s method is relatively simple, fast and straightforward (Ingkaninan et al., 2000). Flavonoids 1 and 2 demonstrated663%and802%ofAChEinhibition,respectively. TheinhibitionofAChEhasbeenoneofthemostusedstrategiesfor Alzheimer's disease (AD). Alkaloids such as galantamine or alkaloid-relatedsyntheticcompounds, suchas rivastigmine,are considered useful drugs to alleviate cognitive symptoms in patientswith AD.However, this class of substances provides a

Table1

NMRspectraldataofcompound1.

Atom dC Type dH HMBC HSQC-TOCSY

2 158.6 C 3 134.5 C 4 179.5 C 5 163.3 C 6 99.8 CH 6.19,d, J=2.0 5,7,8,10 8 7 165.8 C 8 94.6 CH 6.38,d, J=2.0 6,7,9,10 6 9 158.5 C 10 106.0 C 10 123.1 C 20 132.2 CH 8.04,d, J=8.8 2,40,60 30 30 _{116.2 CH 6.89,d,} J=8.8 10_,40_,50 ₂0 40 _{161.4 C} 50 116.2 CH 6,89;d; J=8.8 10,30,40 60 60 _{132.2 CH 8.04,d,} J=8,8 2,20_,40 ₅0 10_{’ 100.4 CH 5.70,d,} J=7.6 2”,3”,4”,5” 20_{’ 80.0 CH 3.61 1”,3”} 30’ 78.9a CH 3.57b 40’ 71.6 CH 3.28 5” 50_{’ 78.5}a _{CH 3.22}b _4”,6” 60_{’ 62.7 CH2 3.50} 3.72 10_{” 102.4 CH 5.25,d,} J=1.5 2”,3”’,2”’,5”’ 2”’ 20” 71.6 CH 4,27–429,m 1”’ 30” 83.3 CH 3.89 40_{” 72.8 CH 3.51 5”’} 50_{” 69.7 CH 4.08–4.13,m} 60_{” 17.7 CH3 0.98,d,J=6.3 4”’,5”’ 3”’,4”’,5”’} 10_{”’ 105.8 CH 4.55,d,} J=7.8 3”’ 2””,3””,4””,5”” 20”’ 75.5 CH 3.28 3”” 30_{”’ 77.8 CH 3.36 1””,4””} 40_{”’ 71.2 CH 3.36} 50_{”’ 77.8 CH 3.36 1””,4””} 60_{”’ 62.4 CH2 3.59} 3.87 CD3OD,100or400MHz,dppm,J=Hz. a,b

Thesignalscouldbeexchanged.

Table2

NMRspectraldataofcompound(2).

Atom dC Type dH HMBC HSQC-TOCSY

2 158.2 C 3 134.6 C 4 179.3 C 5 163.2 C 6 99.9 CH 6.25,sl 5,7,8,10 8 7 165.8 C 8 94.7 CH 6.45,sl 6,7,9,10 6 9 158.4 C 10 105.9 C 10 _{123.2 C} 20 117.3 CH 7.66,d, J=1.6 10,30,40,60 30 146.2 C 40 _{149.7 C} 50 _{116.3 CH 6.93,d,} J=8.4 10_,30_,40_,60 ₆0 60 _{123.2 CH 7.63,dd} J=1.6e8.4 2,10_,40_,20 ₅0 1” 100.3 CH 5.70;d, J=7.6 2”,5” 2” 79.7 CH 3.65 1”,3”,1”’ 1” 3” 78.9a CH 3.58b 1” 4” 71.8 CH 3.36 5” 78.6a CH 3.24b 6” 62.6 CH2 3.56c 3.74c 1”’ 102.2 CH 5.23,sl 2”,3”’,2”’,5”’ 2”’ 2”’ 71.4 CH 4.29,sl 3”’ 83.4 CH 3.88 6”’ 4”’ 72.7 CH 3.51 6”’ 5”’ 69.6 CH 4.07–4.11,m 6”’ 6”’ 17.9 CH3 0.98,d, J=6.1 4”’,5”’ 1”” 105.9 CH 4.58;d, J=7.9 3”’ 2””,3””,4””,5”” 2”” 75.6 CH 3.29 1””,3”” 1”” 3”” 77.9 CH 3.37 4”” 1”” 4”” 71.2 CH 3.32 1”” 5”” 77.8 CH 3.42 6”” 62.4 CH2 3.74c 3.88c CD3OD+DMSO-d6,100or400MHz,dppm,J=Hz. a,b,c

seriesofside-effects,suchashepatotoxicityandgastrointestinal disorders (Xieet al., 2014).Compared to alkaloids, only a few flavonoidsare documented as AChE inhibitors(Williams et al., 2011).InastudydevelopedbyOrhanetal.(2007),theflavonols quercetinandkaempferol-3-O-galactosidewerescreenedfortheir in vitro AChE inhibitory activity employing Ellman’s method adapted for 96-well microplate. Only quercetin was found to inhibitAChE76.2%(1mgmL1),whilekaempferol-3-O-galactoside showed no inhibition. Compound 2, a quercetin derivative, demonstratedaninhibition of 80%of AChEand compound1, a kaempferoltrisaccharide,aninhibitionof66%ofthisenzymeat thesameconcentration.Thesesubstancesareoneofthefewactive flavonolslinkedtoatrisaccharidechainintheliteraturepresenting this activity, and contribute tothe screening for new types of naturalAChEinhibitors.

3.Experimental

3.1.Generalexperimentalprocedures

OpticalrotationswereobtainedusinganADP220Bellinghan+ StanleyLtdpolarimeter.UVspectraweremeasuredonaThermo ScientificTM

MultiskanGOspectrometer.IRspectrawererecorded ona ShimadzuIR-408spectrometer, inKBrdiscs. NMR spectra wererecordedonBrukerDRX400Avancespectrometer,usingTMS asan internal standardand CD3OD orCD3ODand DMSO-d6 as

solvent. LC–MS analyses were performed ona Nexera UHPLC-system(Shimadzu)hyphenatedtoamaXisETDhight-resolution ESI-QTOFmassspectrometer(Bruker)operatinginapositiveion modefromm/z40–1000.Sampleswereinjectedona Shimadzu SupelcoTitancolumn(C18, 1.9

m

m,2.1100mm)underaflowrate of200

m

L/minwithphasesof0.1%(v/v)formicacidinbothwater andacetonitrile.UV-chromatogramswererecordedat

_l

214and

_l

254nm.Columnchromatography(CC)processeswerecarriedout usingsilicagel60(70–230mesh)orSephadexLH-20(Pharmacia). Reversed-phase medium pressure chromatography was per-formedonIsoleraOneBiotage1 apparatusequippedwithaUV/ VISdetector(set at

l

270 and350nm)and a KP-C18-HSSNAP column.Thinlayerchromatography(TLC)wascarriedoutusing precoatedsilicagelplates.

3.2.Plantmaterial

Leaves from M. robusta were collected in June 2010 at the ParqueEstadualdoItacolomi,OuroPretoCity,MinasGerais,Brazil. ThespecieswasidentifiedbyDra.MariaCristinaTeixeiraBraga Messias(UniversidadeFederaldeOuroPreto).Avoucherspecimen (OUPR: 25559) was deposited in the Herbário Professor José Badini,UniversidadeFederaldeOuroPreto.

3.3.Extractionandisolation

M.robusta leavesweredried atroomtemperatureand then powdered. The powder material (888.8g) was subjected to exhaustivesubsequentextractionwithhexane,chloroform,ethyl acetateandmethanol(3Lofeachsolvent,5days,room tempera-ture,2times).Afterthesolventremoval,theMeOHextract(130g) wasobtained.Aportionoftheextract(25g)waschromatographed onsilicagelCCelutedwithpurechloroformandingradientmode withmethanol,addingthelast10%by10%untiltheuseofpure methanol.Fractionswerecombinedingroupsaccordingtotheir TLCanalysis.Group1(fractions15_–22;chloroform-methanol8:2; 355.6mg) was chromatographedon a Sephadex LH-20 column elutedwithmethanol,providingayellowsolid(3.0mg)whichwas identified as kaempferol (3). Group 2 (fractions 36–58;

chloroform-methanol 1:1; 6.3g) was chromatographed on a SephadexLH-20columnelutedwithmethanolandthefractions werecombinedinto2groups,2aand2b.Group2a(fractions14– 18;2.7g)and2b(fractions19–26;2.1g)werepurifiedbymedium pressure chromatographyona reversed-phaseKP-C18-HSSNAP column. The mobile phase consisted of two solvents: water containing0.1%(v/v)formicacid(A),andmethanol(B).Group2a waselutedingradientmode(10–100%ofB)toobtain kaempferol-3-O-

_b

-D-glucopyranosyl-(1!3)-

a

-L-rhamnopyranosyl-(1

!2)-b

-D-glucopyranoside (1) (fractions 159–169; 51–56% of B; 128.3mg).Group2b was eluted in gradient mode(20–100% of B)toobtainquercetin-3-O-

_b

-D-glucopyranosyl-(1!3)-

a

-L -rham-nopyranosyl-(1!2)-

b

-D-glucopyranoside (2) (fractions 22–27; 35–40%ofB;48.9mg).

3.4.Compoundcharacterization

3.4.1.Kaempferol-3-O-

b

-D-glucopyranosyl-(1!3)-

a

-L -rhamnopyranosyl-(1!2)-

b

-D-glucopyranoside(1)

Light-yellowsolid;mp186Cdec.;½a23

D 76.47(c0.68,MeOH); UV(MeOH):

l

max289,350nm;IR(KBr)

n

/cm13416,1656,1610,

1178,1074;NMR(CD3OD)dataof1H(400MHz)and13C(100MHz),

seeTable 1; HR-ESI–MS (positive-ionmode): 757.2185 [M+H]+_,

calc.757.2191.MS2_{ofm/z757.2183:449.1080(m/zcalc.449.1084)}

[Mglucosylrhamnosyl+H]+_{, 287.0551 (m/z calc. 287.0556)}

[Mglucosylrhamnosylglucosyl+H]+_.

3.4.2.Quercetin-3-O-

b

-D-glucopyranosyl-(1!3)-

a

-L -rhamnopyranosyl-(1!2)-

b

-D-glucopyranoside(2)

Dark-yellowsolid;mp187Cdec.;½a23

D 65.96(c0.94,MeOH); UV(MeOH):

l

max282,354nm;IR(KBr)

n

/cm13418,1658,1610,

1204,1074;NMR(CD3OD+DMSO-d6)dataof1H(400MHz)and13C

(100MHz),seeTable2;HR-ESI–MS(positive-ionmode):773.2134 [M+H]+,calc.773.2140.MS2ofm/z757.2134:465.1038(m/zcalc. 465.1033) [Mglucosylrhamnosyl+H]+_{, 303.0499 (m/z calc.}

303.0505)[Mglucosylrhamnosylglucosyl+H]+_.

3.5.Invitroacetylcholinesteraseinhibition

The AChEinhibition ofthe newcompounds 1and 2 (25

m

L, 10mgmL1inDMSO,resultinginaconcentrationof0.25mgper well or 1mgmL1)was measuredusing a 96-well microplates readerbasedonanadaptedEllman’smethod(Rheeetal.,2001). Theassaywasperformedinquintuplicate.Physostigmine(eserine; 0.25mgperwell)wasusedaspositivecontrolandDMSOasblank. UsinganElisathermoplatemicroplatereader,theabsorbancewas measuredat

l

405nmbeforeandafteradditionof acetylcholines-terase, every 60s for eight and ten times, respectively. The absorbanceincreaserelativetosubstratehydrolysiswascorrected byreactionratevariationbeforeandafteradditionoftheenzyme. Inhibitionpercentagewascalculatedcomparingtheratesofthe samplewiththeblank.

4.Conclusions

Weisolatedandelucidatedtwonewflavonolglycosidesanda known_flavonoidfromM.robustamethanolleafextract: kaemp-ferol-3-O-

_b

-D-glucopyranosyl-(1!3)-

a

-L -rhamnopyranosyl-(1!2)-

b

-D-glucopyranoside(1),quercetin-3-O-

b

-D -glucopyrano-syl-(1!3)-

a

-L-rhamnopyranosyl-(1!2)-

b

-D-glucopyranoside (2)andkaempferol(3).Thisisthefirstworkthatreportsflavonoids from this plant. In vitro assays demonstrated significant AChE inhibition for flavonols 1 and 2. This data highlights these compoundsasoneofthefew,ifnottheunique,flavonolslinked toatrisaccharidechainpresentingthisactivity.

Acknowledgements

The authors thank Conselho Nacional de Desenvolvimento CientíficoeTecnológico(CNPq),CoordenaçãodeAperfeiçoamento dePessoaldeNívelSuperior(CAPES)andFundaçãodeAmparoà Pesquisa do Estado de Minas Gerais (FAPEMIG) for financial support. The authors also thank Salomão B. V. Rodrigues for revisingtheEnglishandcriticalmanuscriptreviews.

AppendixA.Supplementarydata

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. phytol.2016.10.024.

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