Rev. bras. farmacogn. vol.25 número5

RevistaBrasileiradeFarmacognosia25(2015)526–528

w w w. s b f g n o s i a . o r g . b r / r e v i s t a

Short

communication

Chemical

constituents

of

Cycas

vespertilio

Consolacion

Yasa ˜

na

Ragasa

_,

_Vincent

_Antonio

_Santos

_Ng

_,

Esperanza

Maribel

Guiao

Agoo

_,

_Chien-Chang

_Shen

a_{ChemistryDepartment,DeLaSalleUniversityScience&TechnologyComplex,LeandroV.LocsinCampus,BinanCity,Laguna,Philippines} b_{ChemistryDepartment,DeLaSalleUniversity,Manila,Philippines}

c_{BiologyDepartment,DeLaSalleUniversity,Manila,Philippines} d_{NationalResearchInstituteofChineseMedicine,Taipei,Taiwan}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received23January2015 Accepted16June2015 Availableonline8July2015

Keywords: Cycadaceae Cycasvespertilio Lariciresinol Paulownin Pinoresinol Sesamin

a

b

s

t

r

a

c

t

ChemicalinvestigationofCycasvespertilioA.Lindstr.&K.D.Hill,Cycadaceae,aplantendemictothe Philippines,yieldedpinoresinol(1),sesamin(2),paulownin(3),amixtureof␤-sitosterolandstigmasterol, andtriacylglycerolsfromtheconebase;1,3,␤-sitosterol,stigmasterol,triacylglycerols,andlariciresinol (4)fromthecataphylls;␤-sitosterolfromthemegasporophylllamina;␤-sitosterolandamixtureof

trans-4-hydroxycinnamatefattyacidesters(5)andcis-4-hydroxycinnamatefattyacidesters(6)from theunripesarcotesta;and␤-sitosterolandtriacylglycerolsfromtheripesarcotesta.Thestructures1–6

wereelucidatedbyextensive1Dand2DNMRspectroscopy.

©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.

Introduction

Cycas,theonlycurrentlyknowngenusoftheFamilyCycadaceae, isconsideredasfossilplantsthoughtheymayhaveevolvedonly about12millionyearsago(Nagalingumetal.,2011).Thecycads resemblepalmsinmorphologyandarecommonlycalledsagopalm. Thesearewidely distributedin theTropics,withspeciesfound inAsia,Africa, SoutheastAsia,Pacific,andAustralia(Donaldson, 2003).Theyalsogrowonvolcanic,limestone,ultramafic,sandy,or evenwater-loggedsoilsingrasslandandforesthabitats(Madulid andAgoo,2009).

InthePhilippines,there areelevencycadspeciesnamely, C. aenigmaK.D.Hill&Lindstrom,C.curranii(J.Schust.)K.D.Hill,C. edentatadeLaub.,C.lacrimansLindstrom&K.D.Hill,C.nitidaK.D. Hill&Lindstrom,C.riuminianaPorteexRegel,C.saxatilisK.D.Hill&

∗ Correspondingauthor.

E-mail:[email protected](C.Y.Ragasa).

Lindstrom,C.sancti-lasalleiAgoo&Madulid,C.wadeiMerr.,C. ves-pertilioLindstrom&K.D.Hill,andC.zambalensisMadulid&Agoo (MadulidandAgoo,2009;Lindstrometal.,2008;AgooandMadulid, 2012).Allspecies,exceptforC.edentata,areendemictothe Philip-pines(Lindstrometal.,2008).

Thisstudyispartofourresearchonthechemicalconstituents ofCycasspeciesendemic tothePhilippines.Inanearlierstudy, wereportedtheisolationofsterols,triacylglycerols,anda diter-pene fromthe different parts of Cycassancti-lasallei (Ng et al., 2015).Wereporthereintheisolationorfractionationand iden-tificationofpinoresinol(1),sesamin(2),paulownin(3),amixture of␤-sitosterolandstigmasterolandtriacylglycerolsfromthecone base; 1, 3, triacylglycerols, and lariciresinol (4) from the cata-phylls;␤-sitosterolfromthemegasporophylllamina;␤-sitosterol andamixtureoftrans-4-hydroxycinnamatefattyacidesters(5) andcis-4-hydroxycinnamatefattyacidesters(6)fromtheunripe sarcotesta;and␤-sitosterolandtriacylglycerolsfromtheripe sar-cotesta.Thestructures1–6wereelucidatedbyextensive1Dand 2DNMRspectroscopy.Thisisthefirststudyonthechemical con-stituentsofC.vespertilio.

http://dx.doi.org/10.1016/j.bjp.2015.06.002

C.Y.Ragasaetal./RevistaBrasileiradeFarmacognosia25(2015)526–528 527

Materialsandmethods

NMR spectrawere recordedonaVarianVNMRS spectrome-terinCDCl3 at600MHzfor1H NMRand150MHzfor13C NMR spectra.SolventswereevaporatedundervacuumusingHeidolph WB2000.Columnchromatographywasperformedwithsilicagel 60(70–230mesh).TLCwasperformedwithplasticbackedplates coatedwithsilicagelF254andtheplateswerevisualizedby spray-ingwithvanillin/H2SO4solutionfollowedbywarming.

CycasvespertilioA.Lindstr.&K.D.Hill,Cycadaceae,conebase, cataphylls,megasporophylllamina,unripesarcotesta,andripe sar-cotestawerecollectedfromIloilo,PanayIsland,PhilippinesinApril 2013.Voucherspecimenswerecollectedandauthenticatedbyone oftheauthors(EMGA)anddepositedintheDeLaSalle University-ManilaHerbarium(DLSUH3112).

Theair-driedconebase(100g),cataphylls(123.5g), megasporo-phylllamina(92g),andfreeze-driedunripesarcotesta(19g)and ripesarcotesta(51.6g)ofC.vespertiliowereseparatelygroundin ablender,soakedinCH2Cl2 atroomtemperatureforthreedays andthenfiltered.Thesolventwasevaporatedundervacuumto yieldcrudeextractsof1.1g,1.2g,0.5g,0.3g,and0.6gforcone base,cataphylls,megasporophyll lamina,unripe sarcotesta,and ripesarcotesta,respectively.Theseextractswerechromatographed using increasing proportions of acetone in CH2Cl2 at 10% increment.

The10%acetoneinCH2Cl2fractionfromthechromatographyof thecrudeconebasewasrechromatographed(3×)using10%EtOAc inpetroleumethertoyieldtriacylglycerols(8mg).The20% ace-toneinCH2Cl2 fractionwasrechromatographed(3×)using15% EtOAcin petroleum etherto yield2 (4mg)after washing with petroleumether.The30%acetoneinCH2Cl2fractionwas rechro-matographedusing15%EtOAcinpetroleumether,followedby20% EtOAcinpetroleum ether.Thefractions elutedwith15%EtOAc inpetroleumetherwerecombinedandrechromatographedusing 15%EtOAcinpetroleumethertoyieldamixtureof␤-sitosteroland stigmasterol(7mg)afterwashingwithpetroleumether.The frac-tionselutedwith20%EtOAcinpetroleumetherwerecombined andrechromatographed(4×)usingCH3CN:Et2O:CH2Cl2(0.5:0.5:9 byvolume)toyield3(3mg)afterwashingwithpetroleumether. The60%acetoneinCH2Cl2fractionwasrechromatographed(5×) usingCH3CN:Et2O:CH2Cl2(1:1:8byvolume)toyield1(5mg)after washingwithpetroleumether.

The30%acetoneinCH2Cl2fractionfromthechromatography ofthecrudecataphyllextractwasrechromatographedusing15% EtOAcinpetroleumether,followed by20%EtOAcinpetroleum ether.Thefractionselutedwith20%EtOAcinpetroleumetherwere combinedandrechromatographed(5×)usingCH3CN:Et2O:CH2Cl2 (0.5:0.5:9 by volume) to yield 3 (3mg) after washing with

petroleum ether.The 70–80%acetonein CH2Cl2 fractions were combinedandrechromatographed(4×)usingCH3CN:Et2O:CH2Cl2 (2:2:6byvolume)toyield4(3mg)afterwashingwithpetroleum ether.The30%acetoneinCH2Cl2fractionfromthe chromatogra-phyofthecrudeunripesarcotestaextractwasrechromatographed (3×)using15%EtOAcinpetroleumethertoyieldamixtureof5and 6(3mg).

Pinoresinol(1)

13_{CNMR(150MHz,CDCl}

3):ı85.86(C-1),54.15(C-2),71.66 (C-3),132.90(C-1′_),108.56(C-2′_),146.68(C-3′_),145.22(C-4′_),114.24 (C-5′_),118.96(C-6′_),55.95(3′_-OCH

3).

Sesamin(2₎

1_{HNMR(600MHz,CDCl}

3):ı4.70(2H,d,J=4.2Hz,H-1,H-4), 3.03(2H,m,H-2,H-5),3.85(2H,dd,J=3.6,9.0Hz,H-3,H-6),6.83 (2H,d,J=1.2Hz,H-2,2′_{),6.75–6.79(4H,H-5}′_,H-5′′_,H-6′_,H-6′′_),5.93 (2× OCH2O ),4.22(2H,dd,J=6.6,9.0Hz,H-3, H-6);13CNMR (150MHz,CDCl3):ı85.76(C-1,C-4),54.31(C-2,C-5),71.70(C-3, C-6),135.03(C-1′_,C-1′′_),106.48(C-2′_,C-2′′_{), 147.96(C-3}′_,C-3′′_), 147.10(C-4′_,C-4′′_),108.18(C-5′_,C-5′′_),119.35(C-6′_,C-6′′_),101.06 (2× OCH2O ).

Paulownin(3)

1_{H NMR (600MHz, CDCl}

3): ı 6.91 (2H, dd, J=1.8, 16.8Hz), 6.77–6.86 (m, 4H), 5.97 (s, 2H, OCH2O ), 5.94 (s, 2H, OCH2O ),4.80(1H,s,H-1),4.03(1H,d,J=9.0Hz,H-3),3.92(1H, d,J=9.6Hz,H-3),4.82(1H,d,J=4.8Hz,H-4),3.03(1H,mH-5),3.82 (1H,dd,J=6.0,9.0Hz,H-6),4.50(1H,dd,J=8.4,9.0Hz,H-6);13_C NMR(150MHz,CDCl3)ı87.48(C-1),91.64(C-2),74.74(C-3),85.76 (C-4),60.35(C-5),71.66(C-6),134.57(C-1′_),129.09(C-1′′_),107.38, 106.90(C-2′_,C-2′′_{),148.12,148.01(C-3}′_,C-3′′_{),147.94,147.28} (C-4′_,C-4′′_{),108.61,108.21(C-5}′_,C-5′′_),120.09(C-6′_),119.81(C-6′′_), 101.26,101.11(2× OCH2O ).

Lariciresinol(4₎

13_{CNMR(150MHz,CDCl}

3):ı82.81(C-1),52.60(C-2),42.40 (C-3),72.89(C-4),33.33(C-5),60.94(C-6),134.76(C-1′_),108.26 (C-2′_),146.61(C-3′_),145.02(C-4′_),114.24(C-5′_),118.75(C-6′_),132.26 (C-1′′_),121.19(C-2′′_),146.50(C-3′′_),143.97(C-4′′_),114.39(C-5′′_), 111.17(C-6′′_),55.93(OCH

3).

trans-4-Hydroxycinnamatefattyacidesters(5)

13_{CNMR(150MHz,CDCl}

3):ı127.89(C-1),115.81(C-2,C-6), 129.90(C-3,C-5),157.47(C-4),144.10(C-7),115.87(C-8),167.43 (C-9),64.45/64.61(C-1′_),28.24(C-2′_{),130.02,130.23( CH),27.20,} 27.27(allylicCH2′),22.68,26.05,29.02–29.76(CH2′)n,31.91(CH2′), 14.11/14.06(CH3′terminal).

cis-4-Hydroxycinnamatefattyacidesters(6)

13_{C NMR (CDCl}

528 C.Y.Ragasaetal./RevistaBrasileiradeFarmacognosia25(2015)526–528

Resultsanddiscussion

Thestructuresof1–6wereelucidatedbyextensive1Dand2D NMRspectroscopyandconfirmedbycomparisonoftheir13_CNMR datawiththosereportedforpinoresinol(1)(Ragasaetal.,2000), sesamin(2)(Leeetal.,2002),paulownin(3)(Angleetal.,2008), lariciresinol(4)(Ragasa etal., 2000),trans-4-hydroxycinnamate fattyacidesters(5)(RagasaandAlimboyoguen,2013),and cis-4-hydroxycinnamatefattyacidesters(6)(Nishimuraetal.,2009).

TheseresultsindicatethatCycasvespertiliosharessimilar chem-icalcharacteristicswithothermembersofthefamilyCycadaceae: Cycasbeddomeiwhichcontainedpinoresinol(1)(Dasetal.,2006); Cycascircinalis L.which yielded lariciresinol(4)(Ferreiraet al., 2009);andC.micronesicaK.D.Hill.whichyielded␤-sitosterol,and stigmasterol(Marleretal.,2006).Toourknowledge,thisisthefirst reportontheoccurrenceof2–3and5–6inthegenusCycasand thefamilyCycadaceae.Thus,2–3and5–6maybecome chemotaxo-nomicmarkersforCycasvespertilioandcouldbeusedtodistinguish amongCycasspecies.

Authors’contributions

EMGAcollectedandidentified thesample andwrotepartof the introduction. VASN worked on the isolation and purifica-tionofthechemicalconstituentsofC.vespertilio.CYRworkedon thepurificationofthechemicalconstituents,conductedliterature search,elucidatedthestructuresofthecompoundsandwrotethe manuscript.CCSobtainedtheNMRspectra,conductedliterature searchandreviewedthemanuscript.Alltheauthorshavereadthe finalmanuscriptandapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgment

AresearchgrantfromtheCommissiononHigherEducation– PhilippineHigherEducationResearchNetwork(CHED–PHERNet) ofthePhilippinesisgratefullyacknowledged.

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