Rev. bras. farmacogn. vol.26 número4

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

Original

Article

Cholinesterase

inhibitory

activity

and

structure

elucidation

of

a

new

phytol

derivative

and

a

new

cinnamic

acid

ester

from

Pycnanthus

angolensis

Taiwo

O.

Elufioye

_,

_Efere

_M.

_Obuotor

_,

_Joseph

_M.

_Agbedahunsi

_,

_Saburi

_A.

_Adesanya

b_{DepartmentofBiochemistry,ObafemiAwolowoUniversity,IleIfe,Nigeria}

c_{DrugResearchandProductionUnit,FacultyofPharmacy,ObafemiAwolowoUniversity,IleIfe,Nigeria} d_{DepartmentofPharmacognosy,FacultyofPharmacy,ObafemiAwolowoUniversity,IleIfe,Nigeria}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received29September2015 Accepted27January2016 Availableonline1April2016

Keywords:

Pycnanthusangolensis

Alzheimer’sdisease Cholinesteraseinhibitors Bioactivecompounds

a

b

s

t

r

a

c

t

TheleavesofPycnanthusangolensis(Welw.)Warb.,Myristicaceae,areusedasmemoryenhancerand anti-ageinginNigerianethnomedicine.Thisstudyaimedatevaluatingthecholinesteraseinhibitory propertyaswellasisolatesthebioactivecompoundsfromtheplant.Theacetylcholinesteraseand butyryl-cholinesteraseinhibitorypotentialsofextracts,fractions,andisolatedcompoundswereevaluatedby colorimetricandTLCbioautographicassaytechniques.Theextractinhibitedbothenzymeswith activ-ityincreasingwithpurification,ethylacetatefractionbeingmostactivefractionat65.66±1.06%and 49.38±1.66%againstacetylcholinesteraseandbutyrylcholinesterase,respectivelywhilethesupernatant had77.44±1.18inhibitionagainst acetylcholinesterase. Twonew bioactivecompounds,(2E,18E )-3,7,11,15,18-pentamethylhenicosa-2,18-dien-1-ol(namedeluptol)and [12-(4-hydroxy-3-methyl-oxo-cyclopenta-1,3-dien-1yl)-11-methyl-dodecyl](E)-3-(3,4-dimethylphenyl)prop-2-enoate (named omi-foateA)wereisolatedfromtheplantwithIC50of22.26␮g/ml(AChE),34.61␮g/ml(BuChE)and6.51␮g/ml (AChE),9.07␮g/ml(BuChE)respectively.Theresultsshowedthattheplanthascholinesteraseinhibitory activitywhichmightberesponsibleforitsmemoryenhancingaction,thusjustifyingitsinclusionin traditionalmemoryenhancingpreparations

©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Alzheimer’sdisease(AD)isaneurodegenerativedisorder affect-ingseveralpeopleandisyet incurable(Prinzetal.,2013).This makes an urgent need for thedevelopment of highly effective medicationsandtherapiesveryimperative,eventhoughthe mul-tifactorial nature of the disease, involving several unbalanced networkofreceptorsandenzymeshasmadebothdiagnosisand treatmentverydifficult(Ballardetal.,2011).

Nevertheless,currentmanagementstrategiesforADarebased on N-methyl-d-aspartate receptor (NMDA) antagonist

meman-tineandacetylcholineateraseinhibitors(AChEI)suchasdonopesil, rivastigmine and galanthamine (Prinz et al., 2013). Although memantinecanslowdowntherateofneurodegenerationinAD, itdoesnotprovideacureforthedisease(MassoudandGauthier, 2010). Cholinesterase inhibitors on the other hand improve

∗ Correspondingauthor.

E-mail:[email protected](T.O.Elufioye).

cholinergicactivityinthebrainofADpatientandstillremaingood treatmentoption.

It isa knownfactthat theuseof alternativemedicineis on theincreaseallovertheworldwiththemostincreaseinvolving theuseofherbalmedicine,folkmedicine,homoeopathyand

mas-sage(Eisenbergetal.,1998;Ernst,2000).Theremaybeseveral

reasonsforthisincreasebutthreebasictheories:patient dissatis-factionwithconventionaltreatmentasaresultofineffectiveness, adverseeffectsandcost,patientsneedformorepersonalcontrol over theirhealthand bettercompatibilitywithpatients’values, spiritual/religiousbeliefandworldview,havebeenproposedto providesomeexplanations(Astin,1998).Ithasalsobeensuggested thatwithtime,thiscontinuingdemandforalternativetherapies willhavegreateffectonhealthcaredelivery(Kessleretal.,2001). Littlewonderwhygreatresearcheffortsarebeingconcentratedin thisareawithparticularemphasisonherbalmedicineand medic-inalplants.

Medicinalplantshavealsobeengoodsourcesofclinicaldrugs ingeneralformany years(LiandVederas,2009;Silvermanand

Holladay,2014).Manydrugsinclinicalpracticetodayareeither

http://dx.doi.org/10.1016/j.bjp.2016.01.010

directlyfrommedicinalplantsorhavetheirbasictemplatefrom compoundsderivedfromplants(Lazarus,2008)andplantshave alsocontributedsignificantlyinprovidingdrugsforthetreatment ofCNSdisorders.Theseincludetropanealkaloidsfrom Erythrox-ylun coca, opium alkaloids from Papava somniferum, and the cholinesteraseinhibitorphysostigminefromPhysostigma

veneno-sum(Burger,2003)aswellasgalantaminefromNarcissusspecies

(Berkovetal.,2009).Severalothercholinesteraseinhibitorsin

par-ticularhavebeenisolatedfrommedicinalplants(Mukherjeeetal.,

2007;Ahmedetal.,2013).

Pycnanthusangolensis(Welw.)Warb.,Myristicaceae,commonly calledAfricannutmeg,is anevergreen treeabout25–35mhigh and60–100cmindiameter(Orwaetal.,2009).Theuseofdifferent partsoftheplantinfolkloreiswelldocumented(Acheletal.,2012). Theleafjuice hasbeenusedfororalthrushinchildren(Abbiw, 1990)while adecoctionof theleaveshasbeenfoundusefulin ulcer,woundhealing,andhaemorrhoids(Agyareetal.,2009).The stemhasalsobeenreportedusefulinjaundice,coatedtongueand tuberculosis(Fortet al.,2000;Tsaassietal.,2010; Ashidietal., 2010).Severalbioactivecompounds,someofwhicharepotential drugleadshavebeenisolatedfromtheplant.Thecytotoxiceffect offlavonoidsisolatedfromtheplanthasbeenreported(Mansoor et al., 2011). Analgesic and anti-inflammatory fattyacids have alsobeenreportedintheplant(Brilletal.,2004).Otherreported activitiesincludeantioxidant(OladimejiandAkpan,2015), anti-helminthic(OnochaandOtunla,2010),antimalarial(Ancolioetal., 2002)andcholesterol lowering(Leonard, 2004), antinociceptive andantiulcer(SofidiyaandAwolesi,2015).

Wehavealsopreviouslyreportedthecholinesterase,bothacetyl and butyryl, inhibitory activity of crude extracts of this plant

(Elufioyeetal.,2010).Inthisstudy,weisolatedandcharacterized

thecholinesteraseinhibitoryconstituentfromtheplant.

Materialsandmethods

Chemicals

The chemicals used include electric eelacetylcholinesterase (EC 3.1.1.7, type VI-s) and Horse butyxylcholinesterase (EC 3.1.1.8)whichwereproductsofFlukaCo.,Germany.Acetylcholine iodide(ATCHI),buthrylcholinechloride(BuCHCl), 5,5-dithio-bis-nitrobenzeneacid(DTNB),andphysiostigmine(eserine)salicylate werefromSigmaCo.,UK.Reagentsfor bufferincludedisodium hydrogenorthophosphatedihydrate(Na2HPO4·2H2O)andsodium

dihydrogenphosphate(NaH2PO4·12H2O),bothofwhichwereof

analyticalgrade.Alsousedweresilicagelforcolumn(ASTM),and pre-coatedTLCplates,G60PF254(Merck).

Plantmaterialcollectionandauthentication

TheplantPycnanthusangolensis(Welw.)Warb.,Myristicaceae, wasidentifiedbyMr.OladeleoftheDepartmentofPharmacognosy, FacultyofPharmacy,andauthenticatedbyDr.H.IllohoftheBotany Department,ObafemiAwolowoUniversity,Ile-Ifewithherbarium numberIFE13039.TheleaveswerecollectedformRoad7,O.A.U CampusinAugust2005.

Preparationofextractandfractions

Thepowderedleavesweremaceratedwith80%methanolfor 72handextractwasconcentratedinvacuotodrynessat40◦_C.The

methanolicextractwaspartitionedintohexane,ethylacetateand water.Boththeextractandthevariousfractionswerescreenedfor theirAChEandBuChEinhibitoryactivity.

Ethylacetateextractionandprecipitationstudies

PowderedleavesofP.angolensiswerebulkextractedwith100% ethylacetateandtheextractconcentrated.Lipidconstituentwere precipitatedout by gradual addition of methanol. The precipi-tateswerefilteredandweighed.Bothsupernatantsandprecipitates werethentestedforcholinesteraseinhibitoryactivity.

Phytochemicalandcholinesteraseanalysis

The TLC of both the precipitatesand the supernatant were carried out using chloroform:hexane (7:3) as solvent system. Somedevelopedplatesweresprayedwithdifferentphytochemical screeningreagentslikevanillin/sulphuricacid,antimony trichlo-ride,Dragendorff’sreagentandanisaldehydesprayreagents.The other developed plates were subjected to TLC bioautographic enzymeassay.

Cholinesteraseinhibitoryassay

Thecholinesterase(bothAChEandBuChE)inhibitoryactivities ofthecrudeextract,fractions,precipitate,supernatantandisolated compoundswerecarriedoutusinga96wellmicro-platereader accordingtothemodifiedmethodofEllman(Ellmanetal.,1961;

Houghtonetal.,2004;Elufioyeetal.,2013).

The reaction mixture contained2000ml 100mM phosphate bufferatpH8.0,100mloftestsamplestocksolutioninmethanol atafinalconcentrationof42.5␮g/ml,100mloftheenzyme,either acetylcholinesterase(AChE) or butyrylcholinesterase(BuChE) at afinal concentrationof0.003␮/mland0.001␮/mlrespectively. 100␮l of di-thio-nitrobenzoate (DTNB) (0.3mM) dissolved in 100MphosphatebufferpH7.0containing120mMsodium bicar-bonate.Theassaymixturewaspre-incubatedonwaterbathat37◦_C

for30minafterpropermixing.Thereactionwasstartedbyadding of100␮lofacetylthiocholineiodide(ATChI)orbutyrylthiocholine chloride(BTChCI)atafinalconcentrationof0.5mM.Methanoland eserin((−)physiostigmine)wereusedas negativeand positive controlsrespectively.Changeinabsorbanceatmax412was mea-suredevery30soveraperiodof5minatambienttemperature.All assayswerecarriedoutintriplicateandthepercentageinhibition calculatedas:

Percentage inhibition=a−b

a ×100

where a=A/min of control; b=A/min of test sample;

A=changeinabsorbance.

ActivespotswerealsomonitoredbyTLCbio-autographicassay methodaccordingtoRheeetal.(2001).Thesampleswerespotted onpre-coated(G60PF254)TLCaluminiumplateanddeveloped inappropriatesolventsystem.Thedevelopedplateswerethenair driedandsprayedwith2.55×10−3_{units/mlofthecholinesterase}

enzymeuntilsaturated.Theplateswerethenincubatedat37◦_Cfor

atleast20minbeforesprayingwith0.5mMofthesubstrate(ATChI orBTChCI)andthenDTNB.Whitespotsonayellowbackground indicatepositiveresult.

Isolationofbioactivecomponents

Spectroscopicanalysis

Spectroscopicanalysis,both1Dand2DNMRwerecarriedout. Structureelucidationwasdonebasedon1_Hand13_CNMR,COSY,

HMQC,andHMBCspectradata.

Resultsanddiscussion

Theprecipitateand supernatant werespottedonpre-coated silica gel plates and subjected to preliminary phytochemical screening by spraying withvanillin/H2SO4, Dragendoff reagent,

antimonytrichloride, and anisaldehydesprayreagentusingthe samesolventsystem(hexane:chloroform(3:7)).TheTLCplateafter sprayingwithvanillin/H2SO4showedthatsupernatantgavebetter

colourreactiontothesprayingreagent.Thespotsgavedifferent colourstothereagentandthiscouldbeindicativeofthenatureof theconstituentsintheplants.Concentratedsulphuricacidisused inthegeneraldetectionoforganiccompounds(Harborne,1973).It isalsousefulinthedetectionofsteroids,terpenes,lipidsand

essen-tialoils(Pothier,2000).Positivedetectionisindicatedbyanumber

ofcolours,blueforlinalol,redorvioletforthymol,yellow–brown foreugenol,etc.(Pothier,2000).Theaboveplatewhichshowed dif-ferentcolourswithvanillin/H2SO4indicatesthepresenceoforganic

compoundssuchasterpenes,steroidsoressentialoils.

TheTLCplatesprayedwithDragendoffreagentindicatedthe presenceofalkaloidintheprecipitatesofP.angolensis,andC.jagus

withpositivereactionisindicatedforalkaloidsbyorange-brown zonesagainstayellowbackground(Pothier,2000).Thepresence ofalkaloidmaybetruebecausealkaloidshavebeenfoundtohave AChEinhibitoryactivity(Houghtonetal.,2004).Botheserinfrom

PhysostigmavenenosumandgalanthaminefromCrinumare alka-loidswhichhavebeenimplicatedasAChEinhibitors.

Antimonytrichloride isusedin thedetectionof cardiac gly-cosides and saponins (Pothier, 2000). Positive result is usually indicated bycoloured zones forterpenoids andflavonoids. The platesshowedsomecoloured(yellow)zoneswhichcouldbe ter-penoids or flavonoids in the precipitate and supernatant of P. angolensis.

Spraying with anisaldehyde was used for the detection of terpenoids (usually purple,blue or red) and someother com-poundse.g.lignands,sugarandflavonoids(Pothier,2000).Theplate showedthepresence ofterpenoidal compoundsin both super-natantandprecipitate.

Severalcompoundsbelongingtovariousclasseshavebeen pre-viouslyreportedinP.angolensis.Theseincludeflavonoids(Mansoor et al., 2011), fattyacids (Brill et al., 2004), terpenoidquinones (Waboetal.,2007),lignands(Abrantesetal.,2008;Ericetal.,2010) andsteroids(Connolly,2006)thussupportingthevalidityofthe phytochemicalscreening.

Comparingthephytochemicalanalysesusingthevariousspray reagentsandtheTLCAChEandBuChEinhibitoryactivities,itcould beobservedthatsupernatantsgavemoreactivespots.Thesespots couldbesteroids,terpenoids,orterpenes.

Boththequantitativeandqualitativecholinesteraseinhibitory assaysoftheprecipitateandsupernatantshowedthattheactivity washigherinthesupernatantwhencomparedwiththeprecipitate

(Table1).ItwasalsoobservedfromtheplatethatBuChEappeared

nottoshowasmanyactivezonesasAChE.Mostofthese com-poundsarelikelytobeterpenoidalinnaturebecauseoftheirpurple colourinvanillin/H2SO4.Thus,thesupernatantwassubjectedto

furtherphytochemicalanalysis.

TLCbioautographicAChE assayof fractionsfrom theVLCas wellasparallelsprayingwithvanillin/H2SO4 revealedtheactive

spots. Theactive sub-fractions were subjected torepeatedVLC separately followed by PTLC to isolate compounds 1 and 2.

Table1

AnticholinesteraseactivityofextractandfractionsofPycnanthusangolensis.

Pycnanthusangolensis %Inhibition(AChE) %Inhibition(BuChE)

Methanolextract 43.96±3.04 43.59±1.77

Hexanefraction 23.94±2.24 11.49±1.97

Ethylacetatefraction 65.66±1.06 49.38±1.66

Aqueousfraction 48.80±1.15 42.17±1.44

Precipitate 72.60±3.34 ND

Supernatant 77.44±1.18 ND

Eserin 92.63±3.66 89.30±2.11

ND,notdetermined.

Spectroscopicanalysisoftheisolatedcompoundswasdoneto con-firmtheirstructures.

Compound1wasobservedasoilwithbrownishyellowcolour. The1_{HNMRspectrum,(CDCl}

3,300Hz)showedsignalsatı6.4(s), ı5.4(t),ı4.1(d),ı2.0(d),ı1.4(m),ı0.85(m),ı0.87(m),ı0.9(m),ı 1.70(s)andı1.60(s).The13_CNMR(CDCl

3,300Hz)dataare:ı59.63 (C-1),123.30(C-2),140.50(C-3),40.08(C-4),26.93(C-5),37.51 (C-6),33.90(C-7),37.64(C-8),25.35(C-9),39.95(C-10),33.01(C-11), 39.58(C-12),25.00(C-13),37.50(C-14),28.19(C-15),29.91(C-16), 36.88(C-17),135.50(C-18),123.48(C-19),24.68(C-20),16.23 (C-21),16.38(C-22),19.96(C-23),22.83(C-24),22.92(C-25),19.93 (C-26).

Thesignalat5.4(t)representstheolefinicprotonsassignedto theprotonsonC-2andC-19.Thesignalatı4.1(d)representsan alcoholprotonandisassignedtotheprotonresidingonC-1.The multipletsignalsatı1.40toı1.35representthemethylene pro-tonsonC-7,C-11andC-15.Themultipletsatı1.30toı1.00were assignedtoprotonsonC-6,C-8,C-9,C-10,C-12,C-13,C-14,C-16, andC-17.Thesignalatı1.60(s)wasassignedtothemethyl pro-tonsonC-22andC-26whilethesignalatı1.70wasassignedto theOHgroup.Theremainingsignalsatı0.85(m),ı0.87(m)andı 0.9(m)wereassignedtothemethylprotonsonC-21,C-23,C-24and C-25.

The13_{Cspectrumshowedthattherewere6CH}

3,13CH2,5CH and2C.Thus,compound1appearstobeaC-26carboncompound. Diagnosticaretheoxygenatedterminalmethylenecarbon resonat-ingatı59.39(C-1),themethinecarbonsresonatingatıc123.39and ıc123.48(C-2andC-19),respectively,andthequaternarycarbons C-3andC-19resonatingatıc140.50andıc 135.50respectively. Thetertiarymethylgroups(C-22andC-26)onC-3andC-18 res-onatedatıc16.38andıc19.93,respectively;thesecondarymethyl groups(C-23,C-24andC-25)resonatedatıc19.96,ıc22.83and ıc 22.92whiletheterminalmethylgroup(C-21)resonatedatıc 16.23.

Onanalyzingthespectraofcompound1,itappearstobean extensionofphytolbyadditionaldoubleboundandmethylgroups. WhilephytolisC-20(Arigonietal.,1999),compound1isaC-26 compoundwithadditionalCH3atC-22,CH2atC-16,C-17andC-20,

Spectradataforcompound2areasfollows:

1_HNMR(CDCl

3,300Hz):ı6.6(s),ı6.4(s),ı6.0(t),ı5.1(m),ı

4.2(t),ı3.1(d),ı2.6(dd),ı2.2(m),ı2.0(m),ı1.6(m),andı1.2(m).

13_CNMR(CDCl

3,300Hz)ı132.30(C-1),124.73(C-2),132.42(C-3),

134.77(C-4),139.94(C-5),123.74(C-6),145.69(C-7),118.32(C-8), 173.52(C-9),68.33(C-10),28.11(C-11),26.56(C-12),28.38(C-13), 29.29(C-14),34.76(C-15),29.91(C-16),29.58(C-17),27.76(C-18), 39.26(C-19),25.85(C-20),39.79(C-21),146.10(C-22),133.33 (C-23),130.91(C-24),148.68(C-25),188.18(C-26),16.11(C-27),17.88 (C-28),16.15(C-29),16.28(C-30).

The13_{Cspectrumofcompound2showedthattherewere4CH} 3,

11CH2,7CHand8C.Thus,compound2appearstobeaC-30

com-pound.Particularare thecarbonylcarbonsC-9andC-26 which resonatedatıc173.52andıc188.18,respectively.Alsodiagnostic istheoxygenatedmethylenecarbonatC-10whichactsasabridge betweenthetwoaromaticringsystemsandresonatedatıc68.33. DiagnosticalsoisthemethinecarbonsC-7andC-8resonatingatıc

145.69andıc118.32,respectivelywhichareinHMBCcorrelating

withthecarbonylatC-9.ThequaternarycarbonsC-25(ıc148.68)

wasdifferentiatedfromthatatC-24(ıc130.91)duetothehydroxyl grouponC-25whichmadeitabsorbatahighervalue.Thetertiary methylgroupsC-27,C-29andC-30resonatedatıc16.11,ıc16.15

andıc16.28whilethesecondarymethylcarbonC-28resonatedat

ıc17.88.

TheprotonNMRsignalatı6.6(s)representsthemethyene pro-tonresidingonC-23resonatingatı133.33intheHMQCwhilethat atı6.4(s)residesonC-3atı132.42.Thetripletatı6.0wasshown toresideonthecarbonatı145.69whichwasassignedasC-7.The multipletatı5.1showedcorrelationwiththecarbonsatı124.73 (C-2),␦123.74(C-6),andı118.32(C-8)intheHMQCspectra.The signalatı4.2(t),showedcorrelationwiththediagnosticOCH2

car-bonatı68.33andisassignedtoC-10whilethesignalatı3.1(d) correlatedwiththecarbonatı27.76assignedtoC-18.The mul-tipletatı2.2toı2.0correlatedwithcarbonsignalsatı26.56,ı

34.76,ı29.58,andı39.79andwereassignedtocarbonsC-12,C-15, C-17andC-21whilethemultipletatı1.6toı1.2wereassigned tothemethylgroupsatC-27,C-28,C-29andC-30.IntheHMBC, thediagnosticCH2atC-10showedcorrelationwiththeCH2signal

atı28.11whichwasassignedtoC-11.AlsotheCH2atı39.79

(C-21)iscoupledtothequaternarycarbonatı146.10(C-22)while thecarbonylcarbonatı188.18(C-26)iscoupledtothecarbon resonatingatı133.33(C-23).TheHMBCspectraalsoshowedthat theCHatı145.69(C-7)coupledwiththequaternarycarbonatı

173.52(C-9).

HMBCcorrelationsofcompound2

When compared with literature (Renmin et al., 2004;

Venkateswaraetal.,2011), itwasobservedthatcompound 2is

acinnamicacidderivative.Thereare,however,differencesat C-4andC-5oftheisolatedcompoundandcinnamic acidbecause ofthe4,5-dimethylsubstitutiononPA-3whichmadethese car-bonsabsorbatslightly higherıvalues (ı134.77andı 139.94), respectively. Derivatives of cinnamic acid in the literature are mostly2,3-dimethoxyor2,3-dihydroxyunlikecompound2which isa2,3-dimethylderivative.Also,thegroupattachedtothe cin-namic acidthrough theester linkagehas never been reported

as such in the literature. Thus compound 2,[12-(4-hydroxy-3-methyl-oxo-cyclopenta-1,3-dien-1yl)-11-methyl-dodecyl] (E )-3-(3,4-dimethylphenyl)prop-2-enoate,namedomifoateAappearsto benewaswellanditisbeingreportedascholinesteraseinhibitor forthefirsttime.

Inconclusion,extractsoftheleavesofP.angolensisinhibited cholinesteraseenzymesandtwonewcompoundswithsignificant cholinesterase inhibitoryactivitywereisolated fromthe super-natantofthemostactiveethylacetatefraction.

Author’scontribution

TOEperformedtheexperiments,didtheliteraturesearches,and wrotethemanuscript.EMOmanagedtheexperimentalprocessand providedtechnicalassistance.AuthorJMAcontributedtothe pro-tocolandprovidedgeneralsupervisionofthestudy.AuthorSAA designedthestudyandwrotetheprotocol.Allauthorshaveread andapprovedthesubmissionofthemanuscript.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.bjp.2016.01.010.

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