Rev. bras. farmacogn. vol.25 número2

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

Original

Article

Constituents

of

Corynaea

crassa

“Peruvian

Viagra”

Gonzalo

R.

Malca

Garcia

,

Lothar

Hennig

,

Joachim

Sieler

,

Rainer

W.

Bussmann

a_{InstitutfürOrganischeChemieandInstitutfürAnorganischeChemie,FakultätfürChemieundMineralogie,UniversitätLeipzig,Johannisallee29,04103Leipzig,Germany} b_{WilliamL.BrownCenter,MissouriBotanicalGarden,P.O.Box299,St.Louis,MO63166-0299,USA}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received9September2014

Accepted24February2015

Availableonline23March2015

Keywords:

␤-Amyrone

␤-Amyrine

Corynaeacrassa Huanarpo Lupenone

Lupeol/␤-sitosterol

a

b

s

t

r

a

c

t

Aphytochemicalinvestigationofmethanolandn-hexaneextractsoftuber/rootsofCorynaea crassa

Hook. f., Balanophoraceae, led to the isolation and characterization of ␤-sitosterol, lupenone, ␤-amyrone, lupeol,and␤-amyrine.Unusualcomplex1:1mixturesoflupenone/␤-amyroneandlupeol/␤-amyrine obtainedfromtheextractswereidentifiedbyNMRandHR-MSexperiments.Thestructureofthe1:1 lupenone/␤-amyronemixturewasconfirmedbyX-rayanalysis.Thesetriterpeneketonederivatives, onlydistinguishedeitherby5-or6-memberedEring,co-crystallizeinonecommonunitcellinthesolid state.

© 2015 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. All rights reserved.

Introduction

Medicinal plants with aphrodisiac activity are used for the treatmentoferectiledysfunctionallovertheworld.Avarietyof botanicalssuchas TribulusterrestrisL., Zygophyllaceae, Aframo-mum melegueta (Roscoe) K. Schum., Zingiberaceae, Eurycoma longifoliaJack,Simaroubaceae,Cnidiummonnieri(L.)Cusson, Api-aceae,FerulahermonisBoiss.,Apiaceae,Mucunapruriens(L.)DC., Fabaceae,Lepidiummeyenii Walp.,Brassicaceae,Passiflora incar-nataL.,Passifloraceae,andsomecompoundslikeyohimbinewere reportedtohave beneficial effects onsexualfunction, support-ing older claims and offering new hope (Hosseinzadeh et al., 2008).

Likewise,incontemporaryPeruvianfolkmedicineaverylarge numberofmedicinalplantsareusedforaphrodisiacalpurposes,a fewhundredofthemtomodulatefertility,ortoinducesterility (Pachacuti-Yamqui, 1992). Plant mixtures and magic are com-monlyappliedtoincrease ortodecreaselibido(Elferink,2000). Oneoftheplantsusedintheseaphrodisiacmixturesiscommonly knownasChutarpo(orhuanarpo),forwhichthereisafemaleand maledesignation. JatrophamacranthaMüll.Arg., Euphorbiaceae (BussmannandSharon,2007;Pardo,2002;DeFeo,1992;Brack Egg, 1999; Desmarchelier et al., 1996a,b; Oshima et al., 2003; Tincoetal.,2011;Benavidesetal.,2006;Okuyamaetal.,1996;

∗ _{Correspondingauthor.}

E-mail:[email protected](G.R.MalcaGarcia).

Schultes, 1980) and Corynaea crassa Hook. f., Balanophoraceae (BussmannandSharon,2006,2007;BussmannandGlenn,2010) areboth underthemale designation“Huanarpo macho”. “Hua-narpomacho”istraditionallyusedstrictlyasamaleaphrodisiac, whilethe“Huanarpohembra”Cnidoscolusperuvianus(Müll.Arg.) J.F.Macbr.,Euphorbiaceae,thefemaledesignation,isusedstrictly asafemaleaphrodisiac.Theuseofanyofthesebytheopposite sexisthoughttohaveanti-aphrodisiaceffect(Pachacuti-Yamqui, 1992).

AdetaileddescriptionofpreparationanduseofC.crassa “Hua-narpomacho”inanalcoholicbeverageisgivenin(Bussmannetal., 2011a,b).InPeruthisplantcanbefoundintheregionsofCajamarca (San Ignacio, Chota and Jaén), Amazonas (Bagua), Lambayeque (Chiclayo),LaLibertad(Trujillo),Pasco(Oxapampa)andCusco(La Convención)(BrakoandZarucchi,1993;Tropicos).

C. crassa is a hemi-parasitic plant, which grows on roots of other species. Likewise Balanophoraceae family consists of 17 genera and approximately 50 species (Tupac Otero et al., 2009). The natural distribution reaches from Costa Rica to Bolivia and theplant grows at altitudes from 1250 to 3600m (Tupac Otero et al., 2009). C. crassa is not only an aphro-disiac plant there are also reports from ethanolic extracts which have shown biological activity against Staphylococcus aureus (Bussmann et al., 2010, 2011a,b). The ethanolic extract exhibit a toxicity of 116␮g/ml using a brine-shrimp lethality assay however, the water extract was found to be non-toxic (<10,000␮g/ml) for the same lethality assay (Bussmann et al., 2011a,b).

http://dx.doi.org/10.1016/j.bjp.2015.02.007

Currently,there is littleinformation aboutconstituentsofC. crassa(Duffand Nickrent,1997;Nickrentetal.,1997).A phyto-chemicalinvestigationofthewholeplantsofC.crassawasthus performedaspartofourworkonthediscoveryofbioactive com-poundsfromPeruvianherbalmedicines.Thisledtoisolationand characterizationof␤-sitosterol,lupenone,␤-amyrone,lupeol,and ␤-amyrine.

Inthis work,theisolation andstructure elucidationofthese constituentsaredescribed.

Materialsandmethods

UsedNMR,MSandHPLCmachinesandmethods

NMRspectra(1_H,13_{C,APT,H,H-COSY,HSQC,andHMBC)were} recordedonaVarianMercury400plus(400MHzfor1_H,100MHz for13_{C)andaVarianMercury300plus(300MHzfor}1_H,75MHz for13_{C)spectrometer,respectively,at26}◦_{CandtheuseofCDCl}

3as asolvent.Thechemicalshiftsarereportedrelativetothe resid-ualsolventpeak,which wasusedas aninternalreference (1_H: 7.26ppm,13_{C:77.16ppm).Chemicalshiftsaregiveninıvalues,} couplingconstantsJinHz;HR-MSspectrawereperformedwitha BrukerDaltonicsESI-FT-ICR-MSAPEXII[7T]andHPLCVarianPro star360,vp250/2Nucleosilcolumn50-7.

Preparationofextracts

Isolationandstructuralelucidationofsteroids

Thetubers/rootsofCorynaeacrassaHook.f.,Balanophoraceae, wereboughtattheMayoristaMarketinTrujilloandtheModelo MarketinChiclayoinNorthernPeru.Driedandpowderedroots (200g)ofC.crassawereextractedfor4daysatroomtemperature, usingsolventsofincreasingpolarity,namely,1.5Ln-hexaneand 1.5Imethanol.Thesolventwasthenevaporated underreduced pressure,then-hexaneextractyielded1.062gandthemethanol extract4.240gofcrudeproduct.Liebermann’sreagentenabledus todetermineremainingsteroidsandtriterpenesinthe methano-licextract,which wasre-dissolvedinwater andextracted with dichloromethaneyielding906mg.Bothextractswerecombined forasaponificationprocesswith5%NaOHandweobtainedintotal 209mgofsteroidsandtriterpenes(TS1).

TheplantmaterialwastakenfromBussmannandSharon’s col-lection(Voucher:Supportinginformation,p.19).

Isolationbychromatography

TS1(209mg)wasfractionatedbycolumnchromatographyon silicagelelutingwithn-hexane/dichloromethanegradientsystem (10:0.5→ 10:1→ 10:1.5→ 10:2→ 10.2.5→ 10:3)togivefive frac-tions.Fraction1containedtracesoftwosteroids(5.3mg)seemed ontheTLCplateand1_{HNMRbuttheywerenotpossibletoseparate} byCCandHPLC.

Fraction 2 (20.1mg) was subjected to further HPLC with pure n-hexane (Rf: 0.81); After several purifications a lot of material was lost. Then the finally material was recrys-tallized from methanol/chloroform to yield ␤-amyrenone/ lupenone (10.8mg). Fractions 3 and 4 (62.9mg) were fur-ther purified (Silica gel, n-hexane/DCM=5:0.06→ 5:0.1→ 5:0.2→ 5:0.3→ 5:0.4) to yield 12.5mg of ␤-sitosterol. And finallyfraction5 (18.8mg)waspurified byflash CC(n-hexane/ DCM=10:0.1→10:0.3→10:0.4→10.5→10:6→10:0.7) and HPLC (n-hexane/isopropanol=9:0.0–9:0.02) then recrystallized frommethanol/chloroformtoyield15.3mgof␤-amyrine/lupeol (Rf: 0.24). Finally, 2.8mg of lupeol (1) was isolated by HPLC

(n-hexane/isopropanol=9:0.1).

HO

1

23 24 8 25

27 18

28 19

22 21 29 30

1 2

4 ₅ 6

7 9

11 12

13 14

15 16 17

3 10

26

Lupeol (1): White powder; MP. 215◦_{C; [˛]}

D=+26,4 (c=0.4, CHCl3);1H NMR(400MHz,CDCl3):ı(ppm)=0.69 (1H,m,H-5), 0.77(1H,s,H-24),0.80(1H,s,H-28),0.84(1H,s,H-25),0.94(1H, m,H-1b),0.96(1H,m,H-15b),0.96(1H,s,H-27),0.97(1H,s,H-23), 1.04(1H,s,H-26),1.10(1H,m,H-12b),1.19(1H,m,H-22),1.27(1H, m,H-21),1.29(1H,m,H-11b),1.30(1H,m,H-9),1.38(1H,m,H-7), 1.39(1H,m,H-6b),1.39(1H,m,H-18),1.43(1H,m,H-11a),1.48 (1H,m,H-16),1.52(1H,m,H-6a),1.61(1H,m,H-15a),1.61(1H,m, H-2),1.62(1H,m,H-13),1.64(1H,m,H-1a),1.70(1H,m,H-12a), 1.70(1H,s,H-30),2.38(1H,m,H-19),3.18(1H,dd,J=11.0;5.3Hz, H-3),4.56(1H,s,H-29b),4.68(1H,s,H-29a).

13_{C NMR (100MHz,CDCl}

3): ı(ppm)=14.8(CH3, C-27), 15.6 (CH3,C-24),16.1(CH3,C-26),16.2(CH3,C-25),18.1(CH3,C-28), 19.0(CH2,C-6),19.8(CH3,C-30),21.2(CH2,C-11),25.3(CH2,C-12), 27.2(CH2,C-15),27.5(CH2,C-2),28.4(CH3,C-23),30.1(CH2,C-21), 34.2(CH2,C-7),35.9(CH2,C-16),37.2(C,C-10),38.5(CH,C-13), 38.7(CH2,C-1),39.8(C,C-4),40.3(CH2,C-22),41.1(C,C-8),42.8(C, C-14),43.2(C,C-17),47.8(CH,C-19),48.5(CH,C-18),50.9(CH, C-9),55.5(CH,C-5),79.3(CH,C-3),109.5(CH2,C-29),151.2(C,C-20); HR-MS:m/z449.43[M+Na]+_,forformulaC

30H50O. ˇ-SitosterolHR-MS:m/z437.41[M+Na]+_,forformulaC

29H50O.

Lupenone/ˇ-amyroneHR-MS:m/z447.40[M+Na]+_,forformula C30H48O.

Lupeol/ˇ-amyrine HR-MS: m/z 449.43 [M+Na]+_{, for formula} C30H50O.

X-raycrystallography

Recrystallization of the lupenone/␤-amyrone mixture from methanol/chloroform(1:0.3)gives colorlessprisms.A crystalof thedimensions0.3mm× 0.15mm× 0.15mmwasusedfor anal-ysis.TheintensitiesweremeasuredonanIPDS1diffractometer (Fa.STOE)andwerecorrectedforLorentzandpolarizationeffects. Thestructurewassolvedbydirectmethods,andtherefinement was performed with SHELX-97 (Sheldrick, 1997). Crystal data:

a=11.203 ˚A, b=14.956(2) ˚A, c=30.513(6) ˚A, V=5112.5 ˚A3_{, space} group P2(1)2(1)2(1), Z=8, Dcalc=1.103mg/m3, =0.71073 ˚A, (MoK␣)=0.064mm−1_{,F(000)=1888,andT=213(2)K,thetotalof} 38,852reflections(9556independent,Rint=0.082)intherangeof 2.2◦_{≤ ≤ 26.1}◦_{and559refinedparameters.Thefinalparameters} wereR1=0.0442,R2=0.0948for3195observedreflections with

I>2(I)andagoodness-of-fit=0.558.

Crystallographicdataforthestructuredeterminedinthispaper have beendepositedatCambridge CrystallographicData center (CCDCreferencenumberisCCDC1008248)andcanbeobtained freeofchargefromCambridgeCrystallographicDataCentreCCDC, 12UnionRoad,CambridgeCB21EZ,UK;fax:+4401223336033;

[email protected].

Resultsanddiscussion

150.80 145.29 121.58 109.51 4000 3800

3600

3400

3200

3000

2800

2600

2400

2200

2000

1800

1600

1400

1200

1000

800

600

400

200

220 215 210 205 200 195 190 185 180 175 170 165 160 155

f1 (ppm) lupenone β-amyrone

b a

a′

b′

c′

c, c′ a′

a b′

b

c

o o

150 145 140 135 130 125 120 115 110 0

Fig.1.Characteristicalpartofthe13_{CNMRspectrumofthe1:1lupenone/␤-amyronemixture.}

151.04 145.30 121.86 109.47 79.12 79.10 3400

3200

3000

2800

2600

2400

2200

2000

1800

1600

1400

1200

1000

800

600

400

200

160 155 150 145 140 135 130 125 120 115 110 105 100 95 90 85 80 f1 (ppm)

lupeol

β-amyrine

HO a

a′

b′

b

c

HO

b

0

b′ a′ a c

c′

c′

A

A

C3A

C23A

C4A C5A

C6A C24A

C2B

C1B

C25B

C10B

C11B

C12B

C13B

C19B C30B C29B

C20B C18B

C17B

C21B

C22B C28B C16B C15B

C9B C1B

C3B

C23B

C4B

C5B

C24B

C6B C7BC26B

C27B

C14B C8B

C8A

C14A C27A

C15A

C16A

C28A C22A

C17A C21A

C19A C20A

C29A C30A

C18A C13A C12A C11A

C9A C10A C1A C2A

C25A

C7AC26A

B

B

A

C

D

B

C

D

Fig.3. Molecularstructuresoflupenone(A)and␤-amyrone(B)(ellipsoidswith50%probability).

describedin(Dominguez,1973;Harborne,1984).Wehave iden-tifiedsteroids, triterpenes, flavonoids, cardiotonics,tannins and anthocyanines,whereasalkaloidshavenotbeendetected. Accord-ing toLiebermann–Burchard’s tests, the experimentsalso have shown anintensegreen darkcoloration for steroidsand triter-penes,indicatingthatthesesecondarymetabolitesareinthecrude extract.

Thesteroid␤-sitosterol,wasoneofthemainproductsisolated fromthe n-hexaneextract and purificationby chromatography withn-hexane/dichloromethane.␤-Sitosterolitselfiswidely dis-tributedintheplantkingdomandusedinherbaltherapy,especially forbenignprostatichyperplasia(BPH)(Bergesetal.,1995).The structurewasconfirmedbyNMR,HR-MSandcomparisonwiththe literatureresults(Wrightetal.,1978;Faizietal.,2001).

Ina nextstep,then-hexaneextractfurnisheda1:1mixture ofthepentacyclicisomerictriterpeneslupenoneand␤-amyrone. It was not possible to separate both compounds using chro-matographicmethods.Finally,weweresuccessfultoidentifythe structureofthecomplexmixturebyuseof1Dand2DNMR spec-troscopy,X-rayandHR-MS.Ourresultsareinagreementwiththe literaturedata(Wenkertetal.,1978;Carpenteretal.,1980).These isomerictriterpeneketonederivativesareonlydistinguishedinits structurebytheir5-and6-memberedEring,respectively,andgive thesamehighresolutionmassspectrum.Inthe1_HNMRspectrum ofthemixtureweobservedthreesignalsofolefinicprotonsina ratio1:1:1.Thesignalat5.18ppmwasassignedtothemethine pro-tonin␤-amyrone(Fig.1),whilethesignalsat4.66and4.54ppm, respectively,resultfromthemethyleneprotonsoflupenone.

The 13_{C NMR spectrumshows typical signals at218.03 and} 217.65ppm corresponding to thecarbonyl groups. The olefinic carbon atoms appear at 109.51 ( CH2) and 150.80ppm ( C ) (lupenone), and at 121.59 ( CH ) and 145.29ppm ( C ) (␤ -amyrone)(Fig.1).

We were able to recrystallize the triterpene mixture using methanol/chloroform(1:0.2)togivecrystalsenoughsuitablefor X-rayanalysis.Thecrystalstructureunambiguouslyconfirmsthe interpretation of the spectra and the structure of the isolated products.Intheorthorhombicunitcelltherearetwostructural con-stitutionisomersoftheformulaC30H48O:lupenoneand␤-amyrone (Fig.3).

Theformationofa1:1mixedcrystalisduetotheclose resem-blanceoftheconformationwiththeringsA,B,CandD.Acalculation ofthepuckeringparametersaccordingto(CremerandPople,1975) givesparameterswhichagreewithaC(chair)–conformation.Only theC-ringof␤-amyronehasanE(envelope)–configuration.The reasonforthisdifferenceisthedoublebondbetweenC12B–C13B (1.335(5)Å).Afittingofbothmolecularstructuresshowsthebroad consistencybetweentheringsA,B,CandD(Fig.4).

OurX-raydataareinagreementwiththeX-raystructuresof lupenoneand␤-amyroneobtainedasseparatedcompoundsby iso-lationfromotherplants(Dampawanetal.,1977;Yanetal.,1989; Jaiswaletal.,2004).

01A

C1A

C10A

C5A

C4A C24A

C23A

C25A

C6A C7A _C8A

C26A

C14A

C15A _C16A

C17A

C22A C21A C19A

C18A C13A

C12A

C20A

C30A C29A

C27A C11A

C9A

C28A C3A

C2A

Fig.4.Fittingofthemolecularstructuresoflupenoneand␤-amyrone.Inthecrystalstructurethetwodifferentmoleculesalsoformseparatehelicalarrangementsalongthe

b-axis.

techniques,MSmeasurementsandcomparisonwiththeliterature results(Wenkertetal.,1978;Carpenteretal.,1980).Inthe1_HNMR spectrumtheolefinicprotonsappearat5.18( CH of␤-amyrine), 4.68and4.56( CH2oflupeol),andadditionallythetypicalCH O absorptionsat3.20ppm.Inthe13_{Cspectrumtheolefiniccarbons} appearnearlyunchanged(151.03,145.29,121.85and109.45ppm) incomparisontothespectrumofthelupenone/␤-amyrone mix-ture.ButinsteadofcarbonylsignalsnowtwocloselyrelatedCH O signalsappearat79.12and79.10ppm.

AfteradditionalHPLCpurificationofthismixtureitwas possi-bletoisolatelupeolasnearlypurecompoundforseparateNMR characterization.

Interestingly, the common appearance of the combinations lupenone/␤-amyroneandlupeol/␤-amyrinewasobservedforfirst time.Incontrast,thecombinationoflupanone/lupeolalreadywas foundinTamarindusindicaL.,Fabaceae(Mathur,2012),andthe combinationof␤-amyrone/␤-amyrinewasisolatedand character-izedfromDiospyrosmorrisianaHance,Ebenaceae(Yanetal.,1989). The formation of both lupenone/␤-amyrone and lupeol/␤ -amyrineeach in a 1:1ratio wasunexpected becauselupenone andlupeolwereformedviathesamelupenylcation,whereas␤ -amyrineand␤-amyronewereformedviatheoleanylcationinthe proposedbiosyntheticalpathway(GalloandSarachine,2009;Seo etal.,1981).

Acomplexmixtureofallthese4pentacyclictriterpenesfromthe sameplanthasneverbeenobservedbefore.Otherwise,the individ-ualcompoundshavebeenisolatedanddescribedasconstituentsin differentaphrodisiacplants(Mathur,2012;Mazumderetal.,1999; Corrêaetal.,2009;Immanetal.,2007).

Conclusion

Thepresentpaperdescribestheisolationandcharacterization offiveconstituentsfromC.crassa:␤-sitosterolandtwointeresting 1:1mixtures oftriterpenes:lupenone/␤-amyroneand lupeol/␤ -amyrine.Theresultsmaybehelpfulinfurtherinvestigationsofthe biologicalactivityofthesenaturalcompounds.

Authors’contribution

GRMG(PhDstudent)contributedincollectingplantsampleand identification,runningthelaboratorywork,anddraftedthepaper; LHdidtheNMRinvestigations;JSperformedtheX-rayanalysis;

RWBcontributedincollectingplantsampleandidentification.All theauthorshavereadthefinalmanuscriptandapprovedthe sub-mission.

Conflictsofinterest

Theauthorsdeclarenoconflictsinterest.

Acknowledgments

TheauthorsaregratefultoMrs.RamonaOehme(Universityof Leipzig,InstituteofAnalyticalChemistry,Germany)forthe mea-surementoftheMSspectra.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.bjp.2015.02.007.

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