Rev. bras. farmacogn. vol.27 número5

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

Original

Article

Perillanolides

A

and

B,

new

monoterpene

glycosides

from

the

leaves

of

Perilla

frutescens

Yang

Liu,

Xiao-Hong

Liu,

San

Zhou,

Hua

Gao,

Guo-Liang

Li,

Wei-Jie

Guo,

Xin-Yu

Fang,

Wei

Wang

SchoolofPharmacy,QingdaoUniversity,Qingdao266021,People’sRepublicofChina

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received15April2017 Accepted8June2017 Availableonline29July2017

Keywords:

Monoterpeneglycoside PerillanolideA PerillanolideB Xanthineoxidase

a

b

s

t

r

a

c

t

Twonewmonoterpeneglycosides,perillanolidesAandB,togetherwithaknowncompoundreported fromthegenusPerillaforthefirsttimewereisolatedandcharacterizedfromtheleavesofPerillafrutescens

(L.)Britton,Lamiaceae,agarnishandcolorantforfoodsaswellascommonlyusedfortraditionalmedicine. Thestructuresoftheisolatedcompoundswereelucidatedonthebasisofextensivespectroscopic evi-dencesderivedfromnuclearmagneticresonanceexperiments,massspectrometryandbycomparing theirphysicalandspectroscopicdataofliterature.Thesecompounds,togetherwiththepreviously iso-latedsecondarymetabolitesofthisspecies,wereinvestigatedfortheirinhibitoryeffectsonxanthine oxidaseinvitro.Ofthecompounds,luteolinshowedthestrongestinhibitoryactivitywithanIC50valueof

2.18␮M.Esculetinandscutellareinmoderatelyinhibitedtheenzyme,whileperillanolidesAandB,and 4-(3,4-dihydroxybenzoyloxymethyl)phenyl-O-␤-d_{-glucopyranosideexertedweakactivities.}

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

Introduction

Perilla frutescens (L.) Britton, a member of the mint family Lamiaceae,is widelydistributed worldwide, especially in east-ern Asia (Heci, 2001). As an edible crop, the cultivation of P. frutescens has more than 2000 years of history in China as wellas in other Asian countries (Lee and Kim, 2007). In addi-tion to its edible usages, P. frutescens leaf is commonly used in traditional medicines for relieving exterior syndrome and cold-dispelling, promotingthecirculation ofQi, and harmoniz-ingthestomach(Yuet al.,2016).Pharmacological researchhas been reported indicating that P. frutescens leaf possesses anti-allergic (Shin et al., 2000), anti-inflammatory (Makino et al., 2002), anti-oxidative (Zekonis et al., 2008), anticancer (Wang et al., 2013), antibacterial (Choi et al., 2010), and antidepres-sant activities(Takedaet al., 2002). Otherphysiological effects ofP. frutescens leafin severaldiseases have also beenrecently described in the literatures. For example, ultraviolet radiation-inducedextracellularmatrixdamageinhumandermalfibroblasts and hairless mice were significantly protected by P. frutescens leaves extract (Bae et al., 2017). Water-extracted P. frutescens

∗ Correspondingauthor.

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

leavesandstemsincreasedthelevelofleukemiainhibitory fac-tor(LIF),amajorcytokineregulatingendometrialreceptivity,and LIF receptor in human endometrial Ishikawa cells (Kim et al., 2016).

Inoursearchingforxanthineoxidase(XO)inhibitorsfrom edi-ble and medical Chinese natural plants, we foundtheaqueous extractsofP.frutescensleavesshowedXOinhibitoryactivityfor thefirsttime.Bioactivity-guidedfractionationledtotheisolation of caffeic acid, vinyl caffeate, rosmarinicacid, methyl rosmari-nate,andapigeninwithXOinhibitoryactivity(Huoetal.,2015). Furthermore,twelvecompoundswerealsoisolatedfromthe aque-ousextractofP.frutescensleaf(Huoetal.,2016).Ourcontinued interestin discoveringnewcompoundsfromthis sourceledus toisolatetwonewmonoterpeneglycosides,namedperillanolides A (1) and B (2), together with a known compound, 4-(3,4-dihydroxybenzoyloxymethyl)phenyl-O-␤-d-glucopyranoside (3). The structures of theisolated compounds weredetermined by spectroscopicmethods,including1Dand2DNMR,HR-ESI-MS,and chemicalmodification.Here,wealsoreporttheevaluationofXO inhibitory activitiesof thecompoundsisolated by usfrom this plant.

http://dx.doi.org/10.1016/j.bjp.2017.06.003

O O 3' 3 1 5' 1' 1 O OH OH HO OH O 3' 3 1 5' 1' 2 O OH OH HO OH O O O O HO HO O OH OH HO OH 3

Materialsandmethods

Generalexperimentalprocedures

The experimentalprocedures, includingHPLC, MS and NMR instrumentation, were previously described (Gao et al., 2017). Allopurinol,XO,and xanthinewereboughtfromSigma–Aldrich Chemicals (St. Louis, MO, USA). Compounds loliolide, isolo-liolide (Kimura and Maki, 2002), dehydrovomifoliol (Park et al., 2011), trans-p-hydroxycinnamic acid (Fu et al., 2011), esculetin (Zhang et al., 2014), luteolin, scutellarein, scutellarin, p-hydroxybenzaldehyde(Liangetal.,2015),negletein(Yoonetal., 2011),p-hydroxyacetophenone(Fengetal.,2013),andsericoside (Ponouetal.,2010)wereisolatedandidentifiedfromP.frutescens leavesinourlaboratory.

Plantmaterial

TherawmaterialsofPerillafrutescens(L.)Britton,Lamiaceae, leaveswerecollectedfromChangchun suburb,JilinProvinceof China,inOctober2011.Botanicalauthenticationwasperformed byBao-MinFeng,CollegeofLifeScienceandTechnology,Dalian University.Avoucher specimen(ZS20111001)wasdepositedat theInstituteofPhytochemistry,JilinAcademyofChineseMedicine Sciences.

Extractionandisolation

Theair-driedleavesofP.frutescens(4kg)wereextractedthree timeswithboilingwaterfor2handthenfilteredthrougha two-layermesh.Thewaterfiltrateswereconcentratedunderreduced pressuretoaffordadarkbrownresidue(583g).Theextractwas sus-pendedinwater(2l)andpartitionedwithn-butanol(2l×4times), followedbyconcentrationtoyield125gthen-butanolextract.Part ofthen-butanolextract(60g)wassuspendedinwater(4l)andthen passedthroughaopencolumnchromatographyonHPD600 macro-porousresinandsuccessivelyelutedwithwater,20%ethanol,50% ethanol,and70%ethanoltogive20%ethanolelutedpart(14.3g), 50%ethanolelutedpart(27.5g),and70%ethanolelutedpart(8.0g). The20%ethanolelutedpart(4g)waschromatographedfurther onaRP-18silicagelcolumnandelutedwithagradientincreasing methanol(30–100%)inwatertoyieldeightfractions(Frs. 20-1–20-8)onthebasisofTLCanalyses.Compounds1(2.97mg,Rt=99min)

and2(2.71mg,Rt=108min)wereobtainedfromFr.20-7by

prepar-ativeHPLCemployingMeOH/H2O(55:45)asthemobilephaseata flowrateof2.0ml/min.Thefractionelutedwith50%ethanol(15g) wasfractionatedbycolumnchromatographyonsilicageleluting withasolventsystem(CHCl3:MeOH=40:1,19:1,9:1,8:2)toobtain sixfractions(Frs.50-1–50-6).Fr.50-1wasseparated chromato-graphicallyona RP-18silica gelcolumn withasolvent system

(MeOH:H2O=5:5,10:0) to give subfractions, which were puri-fiedbypreparativeHPLCusingMeOH:H2O(40:60)asthemobile phaseataflowrateof2.0ml/mintoyieldcompound3(9.65mg, Rt=79min).

Spectraldata

PerillanolideA(1):colorlessgum;[␣]D20−21.7(c0.23,MeOH); UV(MeOH)_max(logε):202(3.72)nm;1_H(CD

3OD,500MHz)and 13_CNMR(CD

3OD,125MHz)seeTable1;HR-ESI-MSm/z353.1565 [M+Na]+_(calcd.forC

16H26O7Na,353.1570).

PerillanolideB(2):colorlessgum;[␣]D20−46.7(c0.15,MeOH); UV(MeOH)_max(logε):202(3.94)nm;1_H(CD

3OD,500MHz)and 13_CNMR(CD

3OD,125MHz)seeTable1;HR-ESI-MSm/z353.1600 [M+Na]+_(calcd.forC

16H26O7Na,353.1570).

Acidichydrolysisandsugaridentification

Compounds1and2(each1mg)wereacidichydrolyzedwith 1ml of 7% HCl heated at 60◦_{C for 2h. Aftercooling, the} reac-tionmixturewasneutralizedwithanAmberliteIRA400column, and the eluate was concentrated. The sugar residues obtained fromthehydrolysisweredissolvedinanhydrouspyridine(0.5ml) andl-cysteinemethylesterhydrochloride(2mg)wasadded.The mixture was stirred at 60◦_{C for 1.5h. After the reaction} mix-turewasdriedinvacuo,theresiduewastrimethylsilylatedwith 1-trimethylsilylimidazole(0.1ml)for2h.Themixturewas parti-tionedbetweenhexane and H2O (1ml each),and organiclayer (1␮l)wasanalyzedbyGC–MS.Identificationofd_{-glucopyranosides} for 1 and 2 was detected in each case by co-injection of hydrolysatewithstandardsilylatedsamples,givingsinglepeaks atd_{-glucopyranose(13.5and13.5min)for1and2,respectively.} Retention timesof authentic samplestreated in the sameway with1-trimethylsilylimidazoleinpyridinewered-glucopyranose (13.5min).

XOinhibitoryactivityassayandXOinhibitorymodeofaction assay

The XO inhibition assays and the inhibitory mode assay of theisolatedcompoundswereperformedaccordingtothemethod modifiedbyourgroup(Liuetal.,2016).Anoverviewaboutthe effectsofthesesubstancesonXOactivityisgiveninTable2.

Resultsanddiscussion

Compound1wasisolatedasacolorlessgum,andshowed posi-tiveLiebermann–Burchardreaction,suggestingittobeaglycoside. The molecularformula wasdeterminedto beC16H26O7 onthe basisofthequasimolecularionpeakatm/z353.1565[M+Na]+_in thepositiveHR-ESI-MS(calcd.forC16H26O7Na,353.1570),which wasfurthersupportedbythe1_HNMRand13_{CNMRspectraldata} (Table1).The13_{CNMRandDEPTspectrarevealed16carbon} sig-nals due to two methylcarbons, three methylene carbons, ten methinecarbons,andonenonprotonatedcarbon,ofwhichten car-bons wereassigned to theaglyconepartincluding two methyl carbonsat ı_{C 22.9and 23.0, two methylenecarbons at}ı_{C 35.4}

Table1

1_HNMRand13_{CNMRspectraldataofcompounds1and2(500and125MHz,CD}

3OD,ı ppm,JinHz).

Position 1 2

ı_H ı_C ı_H ı_C

Aglycone

1 4.83,t,(7.5) 72.6(d) 2.69,dd(14.7,4.6) 30.5(d)

2.64,dd(14.7,6.6)

2 1.86,m 35.4(t) 4.01,m 77.4(d)

1.71,m

3 1.27,m 35.8(t) 1.44,m 44.8(t)

1.10,m 1.22,m

4 1.53,m 29.1(d) 1.84,m 25.2(d)

5 0.87,d(6.7) 23.0(q) 0.87,d(6.7) 23.8(q)

6 0.87,d(6.7) 22.9(q) 0.87,d(6.7) 22.5(q)

2′ _{7.51,brs 142.5(d) 7.36,brs 141.6(d)}

3′ _{126.6(s) 122.4(s)}

4′ _{6.47,d(1.7) 110.0(d) 6.42,d(1.6) 112.9(d)}

5′ _{7.46,d(1.7) 144.7(d) 7.38,d(1.6) 143.7(d)}

Sugar

1′′ _{4.16,d(7.4) 100.5(d) 4.40,d(7.8) 102.4(d)}

2′′ _{3.21,m 75.1(d) 3.18,m 75.3(d)}

3′′ _{3.24,m 78.1(d) 3.35,m 78.3(d)}

4′′ _{3.25,m 71.9(d) 3.31,m 71.9(d)}

5′′ _{3.14,m 77.9(d) 3.24,m 77.8(d)}

6′′ _{3.87,dd(11.8,2.3) 63.0(t) 3.86,dd(11.7,2.4) 63.0(t)}

3.66,dd(11.8,6.1) 3.68,dd(11.7,5.4)

Table2

Xanthineoxidaseinhibitoryactivitiesoftheisolatedcompounds.

Compounds Concentration(␮M) Inhibitionratio(%) IC50(␮M) Modeofinhibition

PerillanolideA(1) 200 1.10±0.08 >200 –

PerillanolideB(2) 200 1.46±0.13 >200 –

4-(3,4-Dihydroxybenzoyloxymethyl)phenyl-O-␤-d-glucopyranoside(3) 200 35.46±0.59 >200 –

Loliolide 200 14.53±1.47 >200 –

Isololiolide 200 1.23±0.32 >200 –

Dehydrovomifoliol 200 1.45±1.08 >200 –

trans-p-Hydroxycinnamicacid 200 10.52±1.00 >200 –

Esculetin

100 78.85±0.23

32.56 Competitive

50 64.40±0.75

25 44.39±1.60

5 9.96±2.36

Luteolin

5 66.07±0.29

2.18 Competitive

4 62.21±0.28

3 56.46±1.44

2 49.37±1.06

Scutellarein

100 65.96±0.19

48.66 Mixed

75 60.59±0.43

50 51.71±0.56

25 30.05±1.04

Scutellarin 200 40.06±1.23 >200 –

p-Hydroxybenzaldehyde 200 10.11±0.78 >200 –

Negletein 200 40.91±0.65 >200 –

p-Hydroxyacetophenone 200 8.54±0.94 >200 –

Sericoside 200 1.05±0.24 >200 –

Allopurinol – – 2.07 Competitive

J=7.5Hz),and3-furanylringprotonsat6.47(1H,d,J=1.7Hz),7.46

(1H,d,J=1.7Hz)and7.51(1H,brs).InspectionoftheDQFCOSY

andHMBCcorrelations allowedassembleof thealiphaticchain

andfuranylringstartingfromthemethylsignalsatı_H0.87.The

aglyconemoietyof1wasproposedtobe1-(3′

-furanyl)-4-methyl-pentan-1-ol,whichwassimilartoperillaketone,themostabundant

volatilecompoundfromP.frutescensleaves(Bassolietal.,2013).

Thecarbonylcarboninthestructureofperillaketonewasabsent inthat of1, instead,signalsfor anoxymethine(ı_H/C 4.83/72.6) wereobserved.TheDQFCOSYcorrelationsfromH-1(ı_H 4.83)to H-2a(ı_H1.86)andH-2b(ı_H1.71)aswellastheHMBCcorrelations fromH-1(ı_H4.83)toC-2(ı_C35.4),C-3′₍ı_C122.4),C-3(ı_C35.8),

C-2′ ₍ı_C 141.6),andC-4′ ₍ı_C110.0)supportedtheabove deduc-tion(Fig.1).Theremaining1_{H NMRand}13_{C NMRspectraldata} wereascribedtothesugarmoiety.Usingtheanomericprotonat

O

O

O

OH

OH

OH

OH

O

O

O

HO

OH

OH

OH

Fig.1.KeyDQFCOSY(—)andHMBC(→)correlationsofcompounds1and2.

couplingsobservedforalloftheoxymethineprotonsimplyingtheir axialposition.Thepositionofglucosewasunambiguously estab-lishedbyanHMBCexperiment,inwhichalong-rangcorrelation wasobservedbetweentheH-1′′₍ı_H4.16)ofd-glucoseandtheC-1 (ı_C72.6)ofaglycone(Fig.1).Acidhydrolysisof1afforded␤-d -glucopyranose,whichwasidentifiedbyGCanalysiswithauthentic sugars.Tofigureouttheconfigurationoftheonlychiralcenter (C-1)fortheaglyconemoietyof1,theopticalrotationoftheaglycone wasexperimentallyrecorded([␣]D20−30.0,c0.10,MeOH).By com-parisonwiththatof (S)-1-(3′_{-furyl)-3-buten-1-ol([␣]}

D25−30.7, c1.72,CH2Cl2)(Bierstedtetal.,2001),theS-configurationatC-1 wastentativelydeducedtothiscompound.Thus,thestructureof 1wasconfirmedas(S)-1-(3′_{-furanyl)-4-methyl-pentan-1-O-␤-}d -glucopyranosideandthecompoundwasnamedperillanolideA.

Compound 2 was isolated as a colorless gum. It showed a quasimolecularionpeakatm/z353.1600[M+Na]+_{inthepositive} HR-ESI-MS.Acomparativeanalysisof1_Hand13_{CNMRspectraldata} of2(Table1)withthoseof1suggestedthepresenceofanaliphatic chain,afuranylring,andaglucosemoiety.However,thenuanced spectraldataandthedifferentretentiontimeinthe preparative-HPLCindicatedthedistinctconstructionofthetwocompounds. DetailedanalysisoftheaboveNMR spectraldataaswellas2D NMRcorrelations(Fig.1),thesignificantdifferencebetween1and 2,liedin theglycosylationsite.TheDQFCOSYcorrelationsfrom H-2(ı_{H 4.01)toH-1a(}ı_H2.69),H-1b(ı_{H 2.64),H-3a(}ı_{H 1.44),}

andH-3b(ı_H 1.22)aswellasHMBCcorrelationsfromH-1a(ı_H

2.69)andH-1b(ı_H2.64)toC-2(ı_C77.4),C-3(ı_C44.8),C-2′ ₍ı_C

141.6),C-3′₍ı_{C122.4),andC-4}′₍ı_{C112.9),fromtheanomeric}

pro-tonatı_H4.40(1H,d,J=7.8Hz)totheoxymethineC-2(ı_C77.4) supportedtheglycosylationsitetobeC-2(Fig.1).Acidhydrolysisof 2afforded␤-d_{-glucopyranoseandaglycone.Bycomparisonofthe} aglyconeopticalrotation([␣]D25−50.0,c0.10,MeOH)withthatof (R)-methyl3-hydroxyhexanoate([␣]Drt−23.3,c1.16,CHCl3)(Jiang etal.,2010)and(S)-ethyl4-hydroxy-5-phenylpentanoate([␣]D25 +14.5,c1.00,CHCl3)(Kotkaretal.,2007),theR-configurationatC-2 wastentativelydeducedtothiscompound.Thestructureof2was thusdeterminedas(R)-1-(3′_{-furanyl)-4-methyl-pentan-2-O-␤-}d -glucopyranosideandatrivialnameperillanolideBwasassigned.

Compound3wasidentifiedbycomparingthe1_Hand13_CNMR, aswellasMSspectrawiththatreportedintheliterature.Itwas determined tobe 4-(3,4-dihydroxybenzoyloxymethyl)phenyl-O

-␤-d-glucopyranoside(3)(NakataniandKikuzaki,1987).

Inthisstudy,compounds1–3andtwelvepreviouslyisolated compoundswereevaluatedforXOinhibitoryactivity.Allopurinol wasusedasapositivecontrol.Whenxanthinewasaddedtothe mixtureofXOandincreasingconcentrationsofesculetin,luteolin, scutellarein,orallopurinol,theproductionofuricacidshoweda concentration-dependentdecreasecomparedtocontrol.Luteolin significantlyinhibitedXOactivitywithanIC50valueof2.18␮M, whichisclosetoallopurinol’sIC50valueof2.07␮M.IC50valuesfor esculetinandscutellareinwere32.56␮Mand48.66␮M, respec-tively,indicatingweakinhibitionofXOactivity.TheIC50valuefor othercompoundswastoohightodetermine.TheLineweaver–Burk plotswereperformedtounderstandtheenzymeinhibitionmodes ofesculetin,luteolin,andscutellarein.Esculetinandluteolin exhib-itedcompetitiveinhibitions,whileamixedinhibitionmodewas observedforscutellarein.

Conclusions

Phytochemical investigation of the leaves of P. frutescens led to the isolation of two new monoterpene and a known compound.Thesecompounds,togetherwiththepreviously iso-lated secondary metabolites of this species, were investigated for their inhibitory effects on xanthine oxidase in vitro. Of

thecompounds,luteolin showedthe strongestinhibitory activ-ity with an IC50 value of 2.18␮M. Esculetin and scutellarein moderately inhibited the enzyme, while perillanolides A (1) andB(2),and4-(3,4-dihydroxybenzoyloxymethyl)phenyl-O-␤-d -glucopyranoside(3)exertedweakactivities.Theobtainedresults werebenefitforthesubsequentresearchesofgenusPerilla.

Authors’contributions

YL,XHL,GLL,andWJGperformedtheextraction,isolation,and elucidationoftheconstituents.SZandXYFwereinvolvedinthe biologicalevaluations.HGconfirmedalltheisolationand struc-turalelucidationprocedures.WWdesignedthestudy,supervised the laboratorywork, and contributed to critical reading of the manuscript. Alltheauthors haveread thefinalmanuscript and approvedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgment

ThisprojectwassupportedbytheFosteringPlanforOutstanding YoungTeachersinCollegeofMedicineofQingdaoUniversityunder Grant2015001.

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