Rev. bras. farmacogn. vol.27 número4

RevistaBrasileiradeFarmacognosia27(2017)471–474

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

Original

Article

Maytenus

distichophylla

and

Salacia

crassifolia:

source

of

products

with

potential

acetylcholinesterase

inhibition

Fernanda

L.

Ferreira

_,

_Vanessa

_G.

_Rodrigues

_,

_Fernando

_C.

_Silva

_,

_Bibiane

_L.G.

_Matildes

_,

Jacqueline

A.

Takahashi

_,

_Gracia

_D.F.

_Silva

_,

_Lucienir

_P.

_Duarte

_,

_Djalma

_M.

_Oliveira

_,

_Sidney

_A.V.

_Filho

a_{DepartamentodeQuímica,InstitutodeCiênciasExatas,UniversidadeFederaldeMinasGerais,BeloHorizonte,MG,Brazil}

b_{UniversidadedoEstadodeMinasGerais,UnidadedeDivinópolis,Divinópolis,MG,Brazil}

c_{DepartamentodeQuímicaeExatas,UniversidadeEstadualdoSudoestedaBahia,Jequié,BA,Brazil}

d_{DepartamentodeFarmácia,EscoladeFarmácia,UniversidadeFederaldeOuroPreto,OutoPreto,MG,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received21July2016 Accepted16December2016 Availableonline29March2017

Keywords:

Celastraceae Acetylcholinesterase Naturalproducts Pentacyclictriterpenes Physostigmine

a

b

s

t

r

a

c

t

ThephytochemicalstudyoftheextractleavesfromMaytenusdistichophyllaMart.andSalaciacrassifolia

(Mart.exSchult.)G.Don,Celastraceae,resultedintheisolationof3-oxofriedelane,3␤-hydroxyfriedelane, 3␤,24-dihydroxyfriedelane,3-oxo-28,29-dihydroxyfriedelane,twomixturesofpentacyclictriterpenes (␣-amyrin with ␤-amyrin and 3␤-stearyloxy-urs-12-ene with 3␤-stearyloxy-olean-12-ene), 3␤ -palmityloxy-urs-12-ene,thesteroid␤-sitosterolanditsglycosylatedderivative␤-glucosyl-␤-sitosterol, tritriacontanoicacidandthenaturalpolymerguttapercha.Thechemicalstructuresoftheseconstituents wereestablishedbyIR,1_{H and}13_{CNMRspectraldata.Crudeextracts,themixturesoftriterpenes}

and theisolatedconstituentsweresubjectedtoinvitro acetylcholinesteraseinhibitory evaluation. Acetylcholinesterase inhibitory effect was observed for crude chloroform extract leaves from M. distichophylla(100%)andS.crassifolia(97.93±5.63%)andforthetriterpenes3␤,24-dihydroxyfriedelane (99.05±1.12%), 3-oxo-28,29-dihydroxyfriedelane (90.59±3.76%) and 3␤-palmityloxy-urs-12-ene (97.93±1.47%).Thepercentinhibitionsinducedbythesenaturalproductswereverysimilartothose producedbyphysostigmine(93.94±2.10%)astandardacetylcholinesteraseinhibitor.Therefore,these resultsopenperspectivesfortheuseofthesespeciesassourceofcompoundswithsimilarphysostigmine pharmacologicaleffect.

©2017SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Neurodegenerativediseasesresultfromchronicbreakdownand progressivefunctionalor structurallossofneurons,particularly thoseofthecentralnervoussystem(CNS).Theaccumulationof aggregatedproteinsatneuronshasbeencorrelatedtothesetypes ofdiseases(Park,2010).Theneurodegenerationprocessobserved inAlzheimer′_{sdisease(AD)hasbeencharacterizedby} progres-sivedementia and memoryloss.Elevated levelsof thepeptide ␤-amyloid(A␤)are associated withalterations of the synaptic functionandneuralnetworkactivitythatprobablyunderliesthe cognitivedeficitsthatoccurin AD.Furthermore,the accumula-tionofthis toxicpeptideleadstodepositionofA␤intoplaques andis thoughttodriveapathologic cascade,which culminates in neuronaldeath(Cramer etal., 2012).The lossof cholinergic

∗ Correspondingauthor.

E-mail:[email protected](F.C.Silva).

cells is accompanied bya decreasein the concentrationof the acetylcholine(ACh).Thisendogenouscompoundishydrolyzedby acetylcholinesterase (AChE), a hydrolytic enzyme of theserine classthatisofmajorimportancetophysiologyofthecholinergic synapsesofthesomaticsystem,theautonomicnervoussystemand thecentralnervoussystem(CNS)(Triggleetal.,1998).Therefore, oneofthecurrentacceptedstrategiesinpharmacotherapyofAD hasbeentheuseofAChEinhibitors(Yangetal.,2012).As exam-ple,physostigmine(eserine)exertsastereoselectiveinhibitionof cholinesteraseenzymes,suchasAChEandbutyrylcholinesterase (BuChE)byactingasacompetitororpseudosubstrateand transfer-ringacarbamateresiduetotheenzyme’sactivesite.Spontaneous hydrolysisregeneratesthenativeenzymeanditsfunction(Triggle etal.,1998).

ThecurrentdrugsthatactinhibitingAChEproducelimited ther-apeuticresultsagainstAD,however,primarilyprovideashort-term alleviationofthesymptoms,withoutblockingtheprogressionof disease(Parketal.,2012).Untilthismoment,thedevelopmentof moreefficientAChEinhibitors,whichactmainlyinbrain,hasbeen

http://dx.doi.org/10.1016/j.bjp.2016.12.006

472 F.L.Ferreiraetal./RevistaBrasileiradeFarmacognosia27(2017)471–474

consideredasaneffectiveapproachtobeappliedfortreatingAD (Liuetal.,2013).

Thenatureisarichsourceofbiologicalandchemicaldiversity. Complexchemicalstructuresisolatedfromnaturalproducts can-notbeeasilyobtainedbysynthesisorsemisynthesisinlaboratories (Filho etal.,2006).Thenatural compounds,representedbythe classofpentacyclictriterpenes(PCTT),aresecondaryplant metabo-litesthathave apotentialinhibitory propertyofAChE (Gurovic etal.,2010).ThePCTTwithskeletonlupaneandfriedelanecanbe includedamongstthecompoundstobeusedtotreatCNSdisorders observedinAD(Rodriguesetal.,2014).

TheCelastraceaefamilyrepresentsagoodsourceofPCTTthat areofgreatinterest,duetotheirwiderangeofbiologicalactivities (Silva et al., 2013).Species of this family, like Maytenus ilicifo-lia, have beenused in traditional medicineof different regions ofBrazil,for thetreatmentofgastric ulcers(De Andradeet al., 2007),inflammations,anddiarrhea(Santosetal.,2007).Inaddition, thepharmacologicalpotentialofsomeCelastraceaespecieshave beingevidencedthroughitstraditionaluseinNortheastofBrazil, asCNS stimulant,and totreat insomnia and migraine(Omena, 2009).

Inthepresentworkextractsandconstituentsfromtwospecies oftheCelastraceaefamily,MaytenusdistichophyllaandSalacia cras-sifolia,wereinvestigatesinrelationtotheinvitroAChEinhibitory activity.

Materialsandmethods

1_H(400MHz)and13_{C(100MHz)NMRexperimentswerecarried}

outonaBrukerAvanceDRX-400,operatingat300K.The chem-ical shifts assignments (ı) were expressedin parts-per-million (ppm)andcouplingconstants(J)registeredinHertz(Hz). Tetram-ethylsilane(TMS)wasusedasinternalstandard(ıH=ıC=0).The

infraredspectra(IR)(1%KBrsoln,400–4000cm−1_{)wereobtained}

on Shimadzu IR408 spectrometer. Melting points were

deter-mined on MQAPF-302 apparatus (Microquímica Equipamentos

Ltda).

Columnchromatography(CC)processeswereperformedusing silicagel60[0.063–0.200mm(70–230meshASTM)],as station-aryphase, and organicsolventpure, orin mixtures ofcrescent polarity, as mobile phase. Silica gel 60 (Merck) was used to prepareplates(0.25mm)foranalyticthinlayerchromatography (TLC).

TheleavesofMaytenusdistichophyllaMart.,Celastraceae,were collected in Jequié, Bahia, Brazil, and the species was identi-fiedby Dra. Guadalupe Licona deMacedo of Departamento de BotânicaofUniversidadeEstadualdoSudoestedaBahia(UESB),

Brazil. A voucher specimen (No. HUESB 2093) was deposited

in the Herbarium of Departamento de Botânica of UESB. The leavesofSalaciacrassifolia(Mart.exSchult.)G.Don,Celastraceae, were collectedin Montes Claros, Minas Gerais, Brazil, and the species wasidentified by Dra. Maria Olívia Mercadante-Simões of Universidade Estadual de Montes Claros, Brazil. A voucher specimen(No. HBCB144624) is preserved in theHerbarium of InstitutodeCiênciasBiológicas,UFMG.Theplantmaterialswere collectedinaccordancewithauthorization(Process:

010119/2014-0) to access to the genetic patrimony emitted by Conselho

Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil.

TheleavesofM.distichophyllaandS.crassifoliaweredriedat roomtemperatureandfragmentedonamill,separately.Each pow-deredmaterialwassequentiallyextractedwithhexane,chloroform andethylacetate.

Thechloroformextract(35.6g)fromM.distichophyllawas sub-jectedtosilicagelCC,initiallyelutedwithMeOHandthenwith

CHCl3,thelastsolventyieldedcompound13(6g).Afterremoval

thesolvent,thefractionselutedwithMeOH weresubmittedto successivesilica gel CC,furnishing the constituents7 and 8 as mixture(178mg;hexane–CHCl370:30),1(55mg;hexane–CHCl3

70:30),2(23mg;hexane–CHCl360:40),5and6asmixture(35mg;

hexane–CHCl315:85),10(18mg;hexane–EtOAc95:05),3(272mg;

hexane–EtOAc 70:30), 12 (35mg;CHCl3–EtOAc 95:05),and 11

(87.3mg;hexane–EtOAc30:70).

Thechloroformextract(44g)fromS.crassifoliawassubjected to silica gel CC eluted with MeOH followed by CHCl3

allow-ingtheisolation ofcompound 13 (30g).Thefractions obtained withmethanol,aftertheremovalthesolvent,weresubmittedto successivesilicagelCC,furnishingtheconstituents,9(11.2mg; hexane–CHCl380:20),1(10.2mg;hexane–CHCl325:75),2(9.7mg;

hexane–CHCl315:85),5and6asmixture(3.7mg;hexane–CHCl3

10:90),10(11.8mg;hexane–CHCl35:95),4(24.4mg;CHCl3–EtOAc

27:75)and12(167.3mg;CHCl3–EtOAc10:90).

The in vitro AChE inhibitory activity of extracts and con-stituentswasevaluatedusinga96-wellmicrotiterplatefollowing theEllman′_{smethod(Ellmanetal.,1961).ThebufferA(50mM} Tris–HCl,pH8,containing0.1MNaCland0.02MMgCl2·6H2O),B

(50mMTris–HCl,pH8,containing0.1%bovineserumalbumin), andC(50mMTris–HCl,pH8)werepreparedandusedinthisassay. Thevolumesof25␮lofAChiodide(15mMinwater),125␮lof 5,5-dithiobis-(2-nitrobenzoicacid)(3mMinbufferA),50␮lofbufferB, and25␮lofsample(10mg/mlinMeOHdilutedby10timeswith bufferC,providingafinalconcentrationof1mg/ml)wereadded intoeachwellofa96-wellmicrotiterplate.Insteadoftheaddition ofsamplesolution,25␮lofbufferCwasusedtopreparetheblank sample.Thepositive controlphysostigminewaspreparedusing similarprocedure.Eachassaywascarriedoutinquintuplicate.The absorbancewas measuredat 405nm every60sbyeight times usingan ElisaThermoplate microplatereader. Afteradditionof 25␮lofAChEsolution(0.226U/mlinbufferB),theabsorbancewas againmeasuredevery60s,for10times.Theincreaseinabsorbance relativetospontaneoushydrolysisofsubstratewascorrectedby reactionratevariationbeforeandafteradditionoftheenzyme. Theinhibitionpercentagewascalculatedbycomparingtheresults

produced by the samples and physostigmine, in relation to

blank.

Resultsanddiscussion

Using phytochemical methods the following known

com-pounds were isolated and identified of the leaves from M.

distichophyllaand S.crassifolia:3-oxofriedelane(1)(Mahatoand Kundu, 1994), 3␤-hydroxyfriedelane (2) (Mahato and Kundu, 1994),3␤,24-dihydroxyfriedelane(3)(CostaandCarvalho,2003) and 3-oxo-28,29-dihydroxyfriedelane (4) (Weeratunga et al., 1982), mixtureof ␣-amyrin(5)and ␤-amyrin (8)(Mahatoand Kundu,1994),mixtureofderivatesofPCTT3␤ -stearyloxy-urs-12-ene(6)(Mirandaetal.,2006)and3␤-stearyloxy-olean-12-ene(9) (Vieira-Filhoetal.,2003),3␤-palmityloxy-urs-12-ene(7) (Vieira-Filho et al., 2003), the steroid ␤-sitosterol (10) (Lendl et al., 2005)anditsglycosylatedderivative␤-glucosyl-␤-sitosterol(11) (Lendl et al., 2005), tritriacontanoic acid (12) (Hamdan et al., 2014),andthenaturalpolymerguttapercha(13)(Oliveiraetal., 2006).Eventhoughoccurring indistinct biomes, locatedabout 1100km far from one another, both species, M. distichophylla

F.L.Ferreiraetal./RevistaBrasileiradeFarmacognosia27(2017)471–474 473

R

R

R

R

R

1

_R

_{=O; R}

_=R

_=R

_=H

2

_R

_=OH;

_R

_=R

_=R

_=H

3

_R

_=R

_{=OH; R}

_=R

_=H

4

_R

_{=O; R}

_{=H; R}

_=R

_=OH

5

_R=

_OH

6

_R=

_OC=O(CH

₎

_CH

7

_R=

_OC=O(CH

₎

_CH

1

3

23 10

25 7 26 12

2719 30 29

21

28

15

3

23 24 25

27

28 30

29

R

3

8

_R=O

_H

9

_R=O

_C=O

_(CH

₎

_CH

R

10

_R=OH

11

_R=

O

_-

_-glucose

12

31

CO

H

13

H

Inthe1_{HNMRspectraofPCTT1–4,adoubletsignalatı} H0.87

(J=6.80Hz)wasobservedanditisinagreementtohydrogenof methylC-23ofthefriedelaneskeleton.The1_Hand13_CNMR

spec-traofconstituent5presentsignalsatıH5.13(J=3.60Hz)andat

ıC124.45andıC139.61,whicharecharacteristicofursane

skele-ton.Thesignalofamultipletat5.30,inthe1_{HNMR,associated}

tothesignalsofcarbinolic(80.60)andcarbonyl(173.69)carbons, observedinthe13_{CNMRspectraofcompounds6and7,indicatean}

esterchainattachedtoC-3ofthePCTT.ThepresenceofsignalsatıC

124.30andıC139.50corroboratestoidentifytheseestersasbeing

derivativesof5.ThesignalsatıC121.60andıC145.20,

correspon-denttoolefincarbonatoms,indicatecompound8and9asoleanane derivatives.ThesignalsatıC80.61,correspondenttocarbinolic

car-bon,andatıC173.69,ofcarbonylcarbon,suggestcompound9as

anoleananeesther.Inthe1_{HNMRspectrumofcompound10,the}

signalatıH5.35wasattributedtoolefinhydrogenandamultiplet

atıH3.56tohydrogenofhydroxylatedcarbon.The13CNMRspectra

oftriterpenes10and11presentsimilarprofilesandtheobserved signalatıC101.6typicalofglycoside,indicated11asa

glycosy-latedsteroid.ThesignalsatıC176.06andatıC14.12,observedin

the13_{CNMRspectrumof12,wereassociatedtocarbonylof}

car-boxylicacidandthemethylgroup-endchain,respectively.Based onthesignalsofatripletatıH5.12(J=6.40Hz),ıC134.90andatıC

124.20,thatarecharacteristicofolefinhydrogen,observedinthe NMRspectra,togetheritsappearanceofasemi-solidcompound

13wasidentifiedasguttapercha.Theconstituents1–13isolated fromleavesofM.distichophyllaandS.crassifoliaareinagreement withchemicalprofileofotherspeciesoftheCelastraceaefamily (Silvaetal.,2013).Thecompounds3,10,11,and12,andmixtures of5and6,7and8fromM.distichophylla,andallconstituentsfrom

S.crassifoliahavebeenisolatedforthefirsttimeinthesespecies. ExtractsandcompoundsfromCelastraceaespecieshavebeen showedpotentialinvitroAChEinhibitoryactivity(Alarcónetal., 2008,2015;Yangetal.,2012;Rodriguesetal.,2014;Sousaetal., 2016).Forexample,sesquiterpeneof␤-agarofuran (epoxyeudes-mane) skeleton, isolated fromMaytenus disticha and Euonymus japonicas(Alarcónetal.,2008,2015),pentacyclictriterpenes,from

Maytenussp.(Rodriguesetal.,2014)andflavonoltriglycosidesof leavesfromMaytenusrobusta,showedhighAChEactivity.Alarcón et al. (2008) showed that in polyhydroxy dihydroagarofuran sesquiterpenoidwithanucleusofthealatol-typearemostactive thanotherssesquiterpenes.Onthesecompoundsthepresenceofa freehydroxygroupatC-15,andoftheOAc(C-2)groupnexttoOBz (C-1)couldberesponsiblefortheactivity.Theseterpenoidspossess mixedoruncompetitivemechanismsofinhibition ofAChE,and wereconsideredasmodelsforthedevelopmentofnewnaturally occurringdrugsformanagementstrategiesforneurodegenerative diseases(Alarcónetal.,2015).Sousaetal.(2012)andRodrigues etal.(2014)reportedthephytochemicalandthebiological stud-iesofMaytenusgonocladaMart.andM.imbricataMart.exReissek.

Table1

AChEinhibitoryactivitytochloroformextractsandcompoundsoftheleavesfromMaytenusdistichophyllaandSalaciacrassifolia.

Samples AChEactivity

Inhibition(%) Standarddeviation Coefficientofvariation

ChloroformextractfromM.distichophylla 100 Nd Nd

ChloroformextractfromS.crassifolia 97.93 5.63 0.06

3␤,24-Dihydroxyfriedelane(3) 99.05 1.12 0.01

3-Oxo-28,29-dihydroxyfriedelane(4) 90.59 3.76 0.04

3␤-Palmityloxy-urs-12-ene(7) 94.90 1.47 0.02

Physostigmine(Eserine)a _{93.94 2.10 0.02}

Nd,notdetected.

474 F.L.Ferreiraetal./RevistaBrasileiradeFarmacognosia27(2017)471–474

InthisworkswereobservedhighAChEinhibitioninducedbythe pentacyclictriterpenes3-oxo-11␣-hydroxylup-20(29)-ene, 3-oxo-29-hydroxyfriedelaneand 3,7-dioxofriedelane (Rodrigues et al., 2014) andfor 3␤-hydroxy-21␤-H-hop-22(29)-ene, and 3␤,11␤ -dihydroxyfriedelane(Sousaetal.,2012).Therefore,sesquiterpenes andthetriterpenesshowedAChEinhibitoryactivity,opening pos-sibilitiestotheemploymentofthesecompoundsasdrugleadsto beusedinthetreatmentofAlzheimer’sdisease.

Herein,thechloroformextractsfromleavesofM.distichophylla

andS.crassifolia,andconstituents1–11wereassayedtoinvitro

AChE inhibition. For both chloroform extracts, and for 3␤ ,24-dihydroxyfriedelane(3),3-oxo-28,29-dihydroxyfriedelane(4)and 3␤-palmityloxy-urs-12-ene (7) were observed AChE inhibitory activity(Table1).The otherconstituents didnot present AChE inhibition.TheextractfromM.distichophyllafurnishedtheactive compound3andtheS.crassifoliaextractthecompounds4and9. Theextractsand compounds3␤,24-dihydroxyfriedelane(3)and 3␤-palmityloxy-urs-12-ene (7)showed the same in vitro AChE inhibitoryactivitythan physostigmine, usedas positive control (Table1).Theresultsfoundin thepresentworkconfirmedthat thePCTThaveAChEinhibitionpropertyandopenperspectivesto theemploymentofthesecompoundsinresearchesinvolving phar-macologicalactivitiessimilartothosepresentedbyphysostigmine.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare thatnoexperimentswereperformedonhumansoranimalsfor thisstudy.

Confidentialityofdata. Theauthorsdeclarethattheyhave fol-lowed theprotocolsof theirworkcenter onthepublication of patientdata.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat nopatientdataappearinthisarticle.

Authors’contributions

FLF,VGR,FCS,GDFSandLPDcontributedtotheexperimental work, extraction and isolation of the metabolites from plants, theinterpretation of all these data and wrote the manuscript. SAVFandDMOwroteandrevisedthemanuscript.BLGMandJAT conductedthetest invitroAChE inhibitoryactivity.Allauthors readandapprovedthefinalmanuscript.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorsthanktoFAPEMIG,CAPESandCNPqforfinancial support.

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