Rev. bras. farmacogn. vol.27 número1

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

Original

Article

Paniculatumoside

G,

a

new

C

21

steroidal

glycoside

from

Cynanchum

paniculatum

Hua

Gao,

Wei

Wang

_,

_Wenxi

_Chu,

_Kun

_Liu,

_Yang

_Liu,

_Xiaohong

_Liu,

_Huili

_Yao,

_Qi

_Gao

SchoolofPharmacy,QingdaoUniversity,Qingdao,People’sRepublicofChina

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received16March2016 Accepted30June2016

Availableonline15September2016

Keywords:

Asclepiadaceae

Cynanchumpaniculatum

C21steroidalglycoside

NeocynapanogeninH 3-O-ˇ-d-oleandropyranoside

a

b

s

t

r

a

c

t

AnewC21steroidalglycoside,paniculatumosideG,togetherwithneocynapanogeninCisolatedforthe

firsttimefromthenaturalsourceandtwoknowncompoundswereisolatedandcharacterizedfromthe rootsandrhizomesofCynanchumpaniculatum(Bunge)Kitag.exH.Hara,Apocynaceae,acommonlyused TraditionalChineseMedicine.Onthebasisofspectroscopicanalysis,includingHR-ESI-MS,1Dand2D NMRspectraldata,thestructureofthenewC21steroidalglycosidewaselucidatedasneocynapanogenin

H3-O-ˇ-d_{-oleandropyranoside.}

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

Introduction

Cynanchumpaniculatum(Bunge)Kitag.exH.Hara,Apocynaceae, a perennial herb native to east Asia, is commonly called ‘Xu ChangQing’inChinese,andhasbeenusedasa Traditional Chi-neseMedicinefor thetreatmentofperatodynia, gastroenteritis, venomoussnake bite,and ascites(Jiangand Li,1977).Previous phytochemicalinvestigationsonC.paniculatumhaverevealedthe presenceofphenolicderivatives,alkaloids,flavonoids, polysaccha-rides,triterpenoids,andC21steroidalglycosides(Niuetal.,2015;

Fuetal.,2015).Thereportedbioactivitiesoftheplantextractsand isolatedconstituentsincludeanti-adipogenic (Jangetal., 2014), neuroprotective(Weonetal.,2013),anti-tumor(Kimetal.,2012), anti-inflammatory,anti-nociceptive,sedative(Choiet al.,2006), araricidal (Kim et al., 2013a), and herpes simplex encephalitis inducingimpairmentpreventiveactivities(Lietal.,2012).Our pre-viousphytochemicalinvestigationonethanolextractofthissource resultedintheisolationofnineC21steroidalaglyconesand

glyco-sides(Chuetal.,2015).Inourcontinuingstudyonthissource,one newsteroidalglycoside(1)togetherwiththreeknowncompounds (2–4)wereisolatedandidentified.Itshouldbenotedthat com-pound2wasisolatedforthefirsttimefromthenaturalsource.

∗ _{Correspondingauthor.}

E-mail:qddxwangwei@qdu.edu.cn(W.Wang).

TheirstructureswereelucidatedbydetailedinterpretationofNMR andMSdata.

Materialsandmethods

Generalexperimentalprocedures

OpticalrotationsweremeasuredbyusingaJASCOP-1020 auto-maticdigitalpolarimeter(JASCOCorporation,Tokyo,Japan).The

NMR spectral datawererecorded ona BrukerAV-500FT-NMR

(500MHzfor1_{Hand125MHzfor}13_C)inC

5D5N,usingvisualC5D5N

resonances(1_{Hı7.21,7.58,and8.73,}13_{Cı123.5,135.5,and149.0)}

for internalreference. Allchemical shifts (ı) aregiven in ppm.

HR-ESI-MSandESI-MSwereobtainedwitha BrukermicroTOFQ

massspectrometer(BrukerDaltonics,Bremen,Germany).Column

chromatographywasperformedwithmacroporousresinHPD100

(CangzhouBonAdsorberTechnologyCo.,Ltd,Cangzhou,China)and RP-18reversed-phasesilicagel(S-50mm,YMC,Kyoto,Japan).TLC analysiswascarriedoutonpre-coatedTLCplateswithsilicagel RP-1860F254 (Merck,Darmstadt,Germany,0.25mm).Detection

wasachievedbysprayingwith10%H2SO4inMeOHfollowedby

heating.PreparativeHPLCwasperformedonaNP7005Cpump con-nectedwithaSHODEXRI-102detector(ShokoScientificCo.,Ltd,

Tokohama,Japan),usingMegresODScolumn(250mm×10mm,

i.d.,5␮m,HanbangSci.&Tech.,Haian,China).HPLC-gradeMeOH waspurchasedfromMerck.HPLC-gradewaterwaspurifiedusing

http://dx.doi.org/10.1016/j.bjp.2016.06.010

aMilli-Qsystem(millipore,Boston,MA,USA).Allsolventsusedfor thechromatographicseparationsweredistilledbeforeuse.

Plantmaterial

The roots and rhizomes of Cynanchum paniculatum (Bunge)

Kitag.exH.Hara,Apocynaceae,wereobtainedinJingde Pharma-ceuticalCompany,Bozhou,AnhuiProvinceofChina,andidentified byProf.BaominFeng,DalianUniversity,China.Avoucher speci-men(CPXCQ-2014-03)wasdepositedattheCollegeofPharmacy, QingdaoUniversity,China.

Extractionandisolation

Theroots andrhizomesofC.paniculatum(10kg)werereflux extractedtwicewith90%ethanol for1.5hand thesolventwas evaporatedunderreducedpressuretogiveanEtOHextract(1.5kg). TheEtOHextract(1.2kg)wasdissolvedwithwaterandsubjected

tocolumnchromatographyonHPD-100macroporousresin and

elutedwithEtOH-H2O(0:100,30:70,70:30,and95:5),successively.

Thefractionelutedwith70%ethanol(100g)waschromatographed overaD941macroporousresincolumn,elutingwith95%ethanol andatotalof15gresiduewascollected.Theresiduewas chro-matographed further on a RP-C18 silica gel and eluted with a

gradient increasing MeOH (30–80%) in water to give sixteen

subfractions (Fr.C1–C16) on the basis of TLC analyses. Fr.C14 waspurified bypreparativeHPLCusingMeOH/H2O(60:40)ata

flow rate of 2ml/min (Megres C18 column, 250mm×10.0mm, 5␮m)toyieldcompound1(4.91mg,tR=41.0min).Compound2

(5.60mg,tR=16.0min)and compound 3(8.25mg, tR=60.0min)

wereobtainedfromFr.C13andFr.C12bypreparativeHPLC(Megres C18 column, 250mm×10.0mm, 5␮m; flow rate, 2.0ml/min)

employing MeOH/H2O (55:45) and MeOH/H2O (52:48) as the

mobilephase,respectively.Thefractionelutedwith95%ethanol (10g)wasseparatedchromatographicallyonaRP-C18silicagelto

getfivesubfractions(Fr.C1′_–C5′_{)onthebasisofTLCanalysis.Fr.C4}′

wasisolatedbypreparativeHPLCusingMeOH/H2O(60:40)ata

flowrateof1.6ml/min(MegresC18column,250mm×10.0mm, 5␮m)toyieldcompound4(62.29mg,tR=140min).

Spectraldata

NeocynapanogeninH3-O-ˇ-d-oleandropyranoside(1):An amor-phouspowder;[␣]D25+45.7(c0.01,MeOH);1H-(C5D5N,500MHz)

and 13_{C-NMR (C}

5D5N, 125MHz) see Table 1; HR-ESI-MS m/z

573.2667[M+Na]+_(calcdforC

29H42NaO10,573.2676).

NeocynapanogeninC(2):Anamorphouspowder;[␣]D25−65.4

(c 0.01, MeOH); 1_{H- (C}

5D5N, 500MHz) and 13C-NMR (C5D5N,

125MHz)seeTable2;HR-ESI-MSm/z399.1783[M+Na]+_(calcdfor

C21H28NaO6,399.1784).

Resultsanddiscussion

Compound1wasobtainedaswhiteamorphouspowder,and

showed positive Liebermann–Burchard and Keller–Kiliani reac-tions,suggestingittobeasteroidalglycosidewitha2-deoxysugar moiety(Zhuetal.,1999).Itsmolecularformulawasdeterminedas C29H42O10onthebasisofpositiveHR-ESI-MSadduction[M+Na]+at m/z573.2667(calcdforC29H42NaO10:573.2676),whichwas

fur-thersupportedbythe1_H-and13_{C-NMRspectraldata(Table1).}

The13_{C-NMRand DEPTspectrarevealed29 carbonsignals due}

tofivemethylcarbons,sixmethylenecarbons,thirteenmethine

carbons, and five nonprotonated carbons, of which 22 carbons

wereassignedtotheaglyconepartincludingtwotertiarymethyl carbons(ıC20.6 and 24.3),onemethoxylcarbon (ıC55.0),one

oxygenatedmethylenecarbon(ıC70.4),fouroxygenatedmethine

carbons(ıC70.0,78.1,84.8,and104.3),fourolefiniccarbons(ıC

120.4,131.0,139.4and142.3),oneacetaliccarbon(ıC114.6),and

onecarbonylcarbon(ıC179.3),whichexhibitedthecharacteristics

of13,14:14,15-disecopregnane-type steroidalglycoside.The1

H-NMRspectrumoftheaglyconemoietyshowedtwoangularmethyl protonsatıH1.09(3H,s)and1.73(3H,s),twogeminalcoupled

oxygenated-methyleneprotonsatıH4.14(1H,dd,J=10.0,4.8Hz) and4.42(1H,dd,J=9.9,7.4Hz),fouroxygen-substitutedmethine protonsatıH3.69(1H,m),4.02(1H,ddd,J=12.6,9.0,4.6Hz),5.62

(1H,s),and5.74(1H,ddd,J=8.1,7.4,4.8Hz),togetherwithtwo olefinicprotonsatıH5.43(1H,m)and5.47(1H,m).Inaddition,

onemethoxygroupresonatedatıH3.50(3H,s)wasobservedin

Table1

1_H-NMRand13_{C-NMRspectraldataofcompound1(500and125MHz,C}

5D5N,ıppm,JinHz).

Position 1 PaniculatumosideAa

ıH ıC ıC

Aglycone

1˛ 1.40(t,J=12.2Hz) 45.5(t) 37.2(t)

1ˇ 2.42(dd,J=13.0,4.6Hz)

2 4.02(ddd,J=12.6,9.0,4.6Hz) 70.0(d) 29.7(t)

3 3.69(m) 84.8(d) 77.0(d)

4˛ 2.65(m) 37.5(t) 39.1(t)

4ˇ 2.59(m)

5 – 139.4(s) 140.3(s)

6 5.43(m) 120.4(d) 120.1(d)

7˛ 2.62(m) 29.2(t) 30.4(t)

7ˇ 2.50(m)

8 2.47(m) 40.9(d) 41.4(d)

9 2.13(td,J=11.4,5.2Hz) 51.9(d) 52.1(d)

10 – 38.6(s) 37.8(s)

11˛ 2.55(m) 30.4(t) 30.3(t)

11ˇ 2.29(ddd,J=11.9,7.4,4.4Hz)

12 5.47(m) 131.0(d) 133.2(d)

13 – 142.3(s) 139.4(s)

14 – 179.3(s) 179.4(s)

15˛ 4.42(dd,J=9.9,7.4Hz) 70.4(t) 70.5(t)

15ˇ 4.14(dd,J=10.0,4.8Hz)

16 5.74(ddd,J=8.1,7.4,4.8Hz) 78.1(d) 78.0(d)

17 3.29(d,J=8.1Hz) 56.1(d) 56.0(d)

18 5.62(s) 104.3(d) 107.3(d)

19 1.09(s) 20.6(q) 19.6(q)

20 – 114.6(s) 115.1(s)

21 1.73(s) 24.3(q) 24.3(q)

18-OCH3 3.50(s) 55.0(q)

Sugar

1′_{(Ole) 4.84(dd,J=9.8,1.8Hz) 99.3(d) 98.3(d)}

2′_{˛ 2.59(m) 37.3(t) 37.5(t)}

2′_{ˇ 1.78(ddd,J=12.0,9.8,4.5Hz)}

3′ _{3.51(m) 81.5(d) 81.7(d)}

4′ _{3.46(m) 76.1(d) 76.5(d)}

5′ _{3.65(m) 73.1(d) 72.9(d)}

6′ _{1.56(d,J=6.1Hz) 18.4(q) 18.8(q)}

3′_{-OCH3 3.49(s) 57.1(q) 57.1(q)}

a_{DatafromLietal.(2004).}

aglyconespectraldataof1withthoseofneocynapanogeninC,the aglyconeofpaniculatumosideB(Lietal.,2004),themain differ-enceswerethepresenceofsignalforanadditionalmethoxyl(ıH/C

3.50/55.0)andthechangesofthechemicalshiftsinC-1(+8.2ppm), C-2(+39.7ppm),andC-3(+7.7ppm),aswellasinC-18(+5.6ppm) andC-13(−3.4ppm)intheNMRspectraof1.Theaglyconemoiety

ofcompound 1wastherefore proposedtobea

2-hydroxyl-18-methoxylderivativeofneocynapanogeninC,whichwereproved bytheHMBCcorrelationsfromıH1.40and2.42(H-1)toıC70.0

(C-2),84.8(C-3),139.4(C-5),38.6(C-10),20.6(C-19),fromıH2.59 and2.65(H-4)toıC70.0(C-2),84.8(C-3),139.4(C-5),120.4(C-6),

38.6(C-10),andfromıH3.50(18-OCH3)toıC104.3(C-18)(Fig.1).

Therelativeconfigurationoftheaglyconewaselucidatedbythe NOESYspectrumandthevicinalproton-protoncouplingconstant. ThecouplingconstantbetweenH-2andH-3(9.0Hz)wastypicalfor

Fig.1.KeyHMBCcorrelationsofcompound1.

trans-diaxialprotons,indicatingthatbothoxygenatedsubstituents wereequatorial.Observed1,3-diaxialNOEcorrelationsfor H-2/H-4␤,H-2/H-19,H-4␤/H-19andH-1␣/H-3(Fig.2)furthersupported the␤-orientationofH-2and␣-orientationofH-3andrevealedthe chairconformationoftheAring.Thetrans-diaxialrelationshipof H-8andH-9,namely,the␤-orientationofH-8and␣-orientation ofH-9,wassuggestedbythesplittingpatternofH-9(td,J=11.4, 5.2Hz)andtheNOESYcorrelationsforH-8/H-19andH-1␣/H-9(Bai etal.,2005).Inaddition,theNOEcorrelationfromthemethoxyl groupatC-18toH3-21confirmedthemethoxylgroupatC-18as ˛-orientation.Thusthestructurefortheaglyconeofcompound1

wasdeducedandatrivialnameneocynapanogeninHwasassigned. Protonsignalswerealsoassignedtoonesecondarymethylgroup atıH1.56(d,J=6.1Hz),onemethoxylgroupatıH 3.49(s),and

oneanomericprotonatıH4.84(dd,J=9.8,1.8Hz),whose

mul-tiplicities suggestedthepresenceof one2,6-dideoxy-sugarin a saccharidechainand␤-configurationofthehexoseunit.The13_C

NMRandDEPTdataindicatedtheexistenceofone oleandropyra-nosylunit.ItwasconfirmedbytheobservedDQFCOSYandHMBC correlations.Forthedeoxysugars,sinceonlyd-formauthentic sam-plescouldbeobtained,theirabsoluteconfigurationscouldnotbe assignedbyGCanalysis,butdeterminedtobed_-formsby compar-isonoftheir13_{C-NMRspectroscopicdatawiththosereporteddata.}

Themostsignificantdifferencesinthe13_{C-NMRdatabetweend}

-andl_{-configurationoleandropyranosylinvolvetheresonancesof} C-2.ThechemicalshiftofC-2inthel_{-oleandropyranosylisless} than35ppm,butthatofC-2inthed-oleandropyranosylappears above 36ppm. Therefore, the oleandropyranosyl unit of 1 was determinedtobed_{-configurationbasedonits}13_{C-NMRchemical} shiftofC-2at37.3ppm(Table1)(Lietal.,2004;Maetal.,2007; Yangetal.,2011;Kimetal.,2013b),anditslocationwas deter-minedtobeC-3bytheH-1′_{/C-3HMBCcorrelation(Fig.1).Thus,}

thestructureof1wasfinallyestablishedasneocynapanogeninH 3-O-ˇ-d-oleandropyranoside.

Compound2wasobtainedaswhiteamorphouspowder,and

showed positive Liebermann–Burchard reaction. Its molecular

formulawasdeterminedasC21H28O6onthebasisofpositive

HR-ESI-MSadduction[M+Na]+_{atm/z399.1783(calcdforC}

21H28NaO6:

399.1784),whichwasfurthersupportedbythe1_H-NMRand13

C-NMRdata(Table2).The1_{H-NMRdatashowedtwoolefinicprotons}

atıH5.34(1H,brd,J=4.6Hz)and5.55(1H,d,J=11.0Hz),three

oxygen-substitutedmethineprotonsatıH3.82(1H,m),5.77(1H,

ddd,J=8.1,7.7,5.2Hz),and6.33(1H,brd,J=6.0Hz),twogeminal coupledoxygenated-methyleneprotonsatıH4.16(1H,dd,J=9.8,

5.0Hz)and4.39(1H,dd,J=9.8,7.2Hz),twomethylsignalsatıH

1.04(3H,s)and1.84(3H,s).The13_{C-NMRspectrumshowed21}

carbonsignals,includingtwotertiarymethylcarbons(ıC19.8and

25.0),anoxygenatedmethylenecarbon(ıC70.0),threeoxygenated

Table2

1_H-NMRand13_{C-NMRspectraldataofcompound2(500and125MHz,C}

5D5N,ı

ppm,JinHz).

Position ıH ıC

1 1.17(m) 37.6(t)

1.83(m)

2 1.74(m) 32.5(t)

2.08(m)

3 3.82(m) 70.7(d)

4 2.54(m) 43.1(t)

2.62(m)

5 – 141.1(s)

6 5.34(brd,J=4.6Hz) 119.4(d)

7 2.58(m) 29.1(t)

2.90(q,J=12.2Hz)

8 2.52(m) 41.6(d)

9 2.08(m) 52.2(d)

10 – 37.7(s)

11 2.27(m) 30.4(t)

2.51(m)

12 5.55(d,J=11.0Hz) 130.2(d)

13 – 145.5(s)

14 – 179.6(s)

15 4.16(dd,J=9.8,5.0Hz) 70.0(t)

4.39(dd,J=9.8,7.2Hz)

16 5.77(ddd,J=8.1,7.7,5.2Hz) 78.3(d)

17 3.38(d,J=8.1Hz) 56.8(d)

18 6.33(brd,J=6.0Hz) 98.7(d)

19 1.04(s) 19.8(q)

20 – 113.6(s)

21 1.84(s) 25.0(q)

methinecarbons(ıC70.7,78.3,and98.7),fourolefiniccarbons(ıC 119.4,130.2,141.1and145.5),anacetaliccarbon(ıC113.6),anda carbonylcarbon(ıC179.6),whichexhibitedthecharacteristicsof 13,14:14,15-disecopregnane-typesteroidalglycoside.Comparison ofthespectraldataof2withthoseofpaniculatumosideB,anewC21 steroidalglycosideisolatedfromthedriedrootofC.paniculatum(Li etal.,2004),thechangesofthechemicalshiftsinC-2(+2.5ppm), C-3(−6.4ppm),C-4(+4.0ppm)showedthatithasnolinkageofthe sugarmoietyattheC-3hydroxylgroupoftheaglycone.Thus,the structureof2wasestablishedasneocynapanogeninC,the agly-coneofpaniculatumosideB.Itshouldbenotedthatcompound2

wasisolatedforthefirsttimefromthenaturalsource.

Compounds3and4wereidentifiedbycomparingthe1_H-and 13_{C-NMR,aswellasMSspectrawiththosereportedinthe}

liter-atures.TheyweredeterminedtobecynapanosideA(3)(Sugama etal.,1986)andcynatratosideA(4)(Zhangetal.,1985).

Authors’contributions

HG,WXC,HLY,andQGperformedtheextraction,isolation,and elucidation of theconstituents. KL, YL,and XHL contributed to checkingandconfirmingalloftheproceduresoftheisolationand identification.WWdesignedthestudy,supervisedthelaboratory work,andcontributedtocriticalreadingofthemanuscript.Allthe authorshavereadthefinalmanuscriptandapprovedthe submis-sion.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

This projectwassupported by theNational Natural Science

FoundationofChinaunderGrant81273396;ShandongProvince

HigherEducationalScienceandTechnologyProgramunderGrant J15LM12.

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