Composition of the essential oil of Acanthopanaxtrifoliatus (L.) Merr. (Araliacaea) from Vietnam

Alain Muselli,1 Tran Minh Hoi,2 Luu Dam Cu,2 La Dinh Moi,2 Jean-Marie BessieÁ re,3 Ange Bighelli1

and Joseph Casanova*1

1Universite de Corse, Equipe Chimie et Biomasse, CRES-URA CNRS 2053, Route des Sanguinaires, 20000 Ajaccio, France2Institut d'Ecologie et des Ressources Biologiques (IERB), Nghia do, Tu Liem, Hanoi, Vieà tnam3Universite de Montpellier, Laboratoire de Phytochimie, Ecole Nationale Supe rieure de Chimie, 34075 Montpellier cedex, France

Received 20 February 1998

Revised 20 May 1998

Accepted 21 May 1998

ABSTRACT: The essential oil of Acanthopanax trifoliatus (L.) Merr. from Vietnam, was investigated usingcombination of capillary GC, GC±MS and carbon-13 NMR spectroscopy after fractionation by columnchromatography. More than 60 compounds, representing 97.4% of the total amount were identi®ed. The main

components were a-pinene (23.9%), sabinene (14.9%), terpinen-4-ol (9.0%), b-pinene (7.7%) and p-cymene(5.8%). Carissone, an eudesmane derivative, was identi®ed as a minor component by carbon-13 NMR. Copyright# 1999 John Wiley & Sons, Ltd.

KEY WORDS: Acanthopanax trifoliatus (L) Merr.; essential oil composition; GC±MS; 13C-NMR

Introduction

The genus Acanthopanax, which belongs to the familyAraliaceae, is widespread in South-East Asia. InVietnam, ®ve species have been detected and amongthese, Acanthopanax trifoliatus (L.) Merr has been usedin folk medicine.1 A. trifoliatus is a rigid shrub 1±4 mhigh, growing wild in the subtropical forests of NorthVietnam. It is a spiny shrub with alternate palmateleaves with toothed margins. The whitish or yellow-greenish ¯owers are present in a terminal panicle ofumbels. The fruits are globular and blacken duringripening. Decoctions of root-bark and stem-bark areused in the treatment of rheumatism, lumbago,ostealgia and impotence.2 Five studies have reportedthe identi®cation of steroid derivatives3,4 and triter-penoid carboxylic acids5,7 extracted from stems andleaves of A. trifoliatus.

In the present work we studied the chemical com-position of the essential oil obtained from leaves andbranches of Acanthopanax trifoliatus (L.) Merr., byconventional techniques (GC, GC±MS) as well as by13C-NMR spectroscopy.

Materials and Methods

Plant Material, Oil Isolation and Fractionation

The stems and leaves were collected in autumn in LangSon Province and were submitted to hydrodistillationfor 4 h using a Clevenger-type apparatus. The essentialoil yield was 0.1% (w/w vs. fresh material). A voucherspecimen was deposited at the herbarium of theInstitute of Ecology and Biological Resources, Hanoi,Vietnam.

The essential oil (600 mg), was submitted to chroma-tography (silica gel 0.2±0.5 mm). Two fractions wereeluted with pentane and diethyl ether, respectively.The ®rst fraction (F1, 211 mg) contained hydrocar-bons, the second (F2, 285 mg) contained all oxygenatedcompounds.

GC and GC±MS Analysis

GC analysis was carried out using Shimadzu GC-A14apparatus equipped with a DB-1 capillary column(30 m� 0.25 mm, ®lm thickness 0.25 mm). Oven temp-erature was programmed from 508C (3 min) to 2208C at38C/min; injector temperature, 2008C; detector temp-erature, 2508C. The relative proportions of individualcomponents were expressed as percentage obtained by

FLAVOUR AND FRAGRANCE JOURNAL

Flavour Fragr. J., 14, 41±44 (1999)

*Correspondence to: J. Casanova, Universite de Corse, Equipe Chimie etBiomasse, CRES-URA CNRS 2053, Route des Sanguinaires, 20000 Ajaccio,France.

Contract/grant information: Collectivite Territoriale de Corse.Contract/grant information: MinisteÁ re des A�aires EtrangeÁ res.Contract/grant information: Caisse des De poà ts et Consignations.Contract/grant information: Socie te Montier (Ghisonaccia, Corsica).

Copyright # 1999 John Wiley & Sons, Ltd. CCC 0882±5734/99/010041±04$17.50

peak-area normalization, all relative response factorsbeing taken as one.

GC±MS analysis was performed on a Hewlett-Packard type 8972. The chromatograph was equipped

with a BPX5 column (20 m� 0.20 mm) and wasprogrammed from 508C to 2208C at 38C/min; injectortemperature, 2008C; detector temperature, 2408C. Thedetector ionization potential was 70 eV.

Table 1. Chemical composition of essential oil of Acanthopanax trifoliatus (L.) Merr.

Constituents RI (%) Identi®cation

a-Thujene 917 0.7 RI, GC±MS 13C-NMRa-Pinene 925 23.9 RI, GC±MS 13C-NMRCamphene 930 0.2 RI, GC±MS 13C-NMRSabinene 958 14.9 RI, GC±MS 13C-NMRb-Pinene 962 7.7 RI, GC±MS 13C-NMRMyrcene 980 0.1 RI, GC±MS 13C-NMRp-Cymene 1009 5.8 RI, GC±MS 13C-NMRLimonene 1018 1.8 RI, GC±MS 13C-NMRg-Terpinene 1048 0.3 RI, GC±MS 13C-NMRtrans-Sabinene hydrate 1051 2.4 RI, GC±MS 13C-NMRp-Cymenene 1069 0.1 RI, GC±MSa-Pinene oxide 1075 0.1 RI, GC±MScis-Sabinene hydrate 1081 2.4 RI, GC±MS 13C-NMRLinalool 1085 0.7 RI, GC±MS 13C-NMRcis-Thujone 1091 0.1 RI, GC±MSa-Campholenal 1098 0.4 RI, GC±MS 13C-NMRcis-Pinene hydrate 1103 0.5 RI, GC±MStrans-Pinocarveol 1121 2.2 RI, GC±MS 13C-NMRtrans-Sabinol 1129 0.2 RI, GC±MStrans-Verbenol 1131 2.3 RI, GC±MS 13C-NMRPinocamphone 1143 0.2 RI, GC±MSCryptone 1150 0.2 RI, GC±MS 13C-NMRTerpinen-4-ol 1159 9.0 RI, GC±MS 13C-NMRp-Cymen-8-ol 1163 1.9 RI, GC±MSMyrtenal 1166 0.6 RI, GC±MS 13C-NMRa-Terpineol 1172 1.2 RI, GC±MS 13C-NMRVerbenone 1177 1.3 RI, GC±MSMyrtenol 1190 0.6 RI, GC±MS 13C-NMRPiperitol 1205 0.3 RI, GC±MStrans-Carveol 1208 0.4 RI, GC±MS 13C-NMRCarvone 1211 0.2 RI, GC±MSPiperitone 1221 0.1 RI, GC±MStrans-Sabinene hydrate acetate 1226 0.1 RI, GC±MScis-Sabinene hydrate acetate 1236 0.2 RI, GC±MSGeraniol 1250 0.3 RI, GC±MS 13C-NMRMyrtanol 1258 0.1 RI, GC±MSCarvenone 1260 0.4 RI, GC±MSThymol 1287 0.2 RI, GC±MStrans-Sobrerol 1350 0.6 RI, GC±MSGeranyl acetate 1358 0.1 RI, GC±MSa-Copaene 1363 0.3 RI, GC±MS 13C-NMRb-Bourbonene 1369 0.3 RI, GC±MSb-Elemene 1377 1.1 RI, GC±MS 13C-NMRb-Caryophyllene 1403 0.2 RI, GC±MS 13C-NMRb-Gurjunene 1457 0.1 RI, GC±MSb-Selinene 1465 0.1 RI, GC±MSa-Muurolene 1480 0.2 RI, GC±MSd-Cadinene 1502 0.1 RI, GC±MSElemol 1523 1.7 RI, GC±MS 13C-NMRNerolidol 1541 0.3 RI, GC±MSSpathulenol 1547 1.0 RI, GC±MS 13C-NMRCaryophyllene oxide 1549 1.6 RI, GC±MS 13C-NMRHumulene oxide 1570 0.5 RI, GC±MSg-Eudesmol 1597 0.4 RI, GC±MSepi-a-Cadinol (t-Cadinol) 1605 0.4 RI, GC±MS 13C-NMRepi-a-Muurolol (t-Muurolol) 1606 0.6 RI, GC±MS 13C-NMRa-Muurolol 1608 0.3 RI, GC±MSb-Eudesmol 1612 0.6 RI, GC±MS 13C-NMRSelin-11-en-4-ol 1614 0.1 RI, GC±MSa-Eudesmol 1615 0.4 RI, GC±MSa-Cadinol 1617 1.2 RI, GC±MS 13C-NMRCarissone 1665 1.1 13C-NMR

Retention indices (RI), order of elution and percentages are given on the apolar (DB-1) column.

42 A. MUSELLI ET AL.

Copyright # 1999 John Wiley & Sons, Ltd. Flavour Fragr. J., 14, 41±44 (1999)

13Carbon NMR Analysis

Spectra was recorded on a Bruker AC 200 Fouriertransform spectrometer operating at 50.323MHz,equipped with a 10 mm probe, a deuterated chloro-form, with all shifts referred to internal tetramethyl-silane (TMS). Other parameters were: pulse width(PW); 5.0ms (¯ip angle 458); acquisition time, 1.3 s for32K data table with a spectral width (SW) of 12,500Hz(250 ppm); CPD mode decoupling; digital resolution,0.763Hz/pt. The number of accumulated scans was5000 for each sample (200 mg of the oil in 2 ml CDC13).An exponential multiplication of the free inductiondecay with the line broadening of 1.0Hz was appliedbefore Fourier transformation.

Identification of Components

Identi®cation of the components was based (a) on theirGC retention indices (RI) on apolar column, deter-mined relative to the retention time of a series ofn-alkanes with linear interpolation with those ofauthentic compounds; (b) on computer matching withmass spectral libraries8 and comparison of spectra withliterature data;9,10 and (c) by 13C-NMR following themethodology reported by Forma cek and Kubeczka11

and improved in our laboratory.12 The identi®cation isbased on comparison of the resonances in the mixturewith those of the reference spectra compiled in ourspectral library with the help of laboratory-producedsoftware. Each compound is unambiguously identi®edtaking into account: the number of identi®ed carbons;the number of overlapping signals; and the di�erence ofchemical shift of each resonance in the mixturespectrum and in the reference. This technique is well-suited for chemical polymorphism studies13,14 as well asfor identi®cation of stereosomers or compounds whichexhibit insu�ciently resolved mass spectral patterns,15

or compounds which co-elute on the polar and apolarcolumns conventionally used for essential oil analysis.16

The whole essential oil was investigated by GC,GC±MS and 13C-NMR and both fractions (F1 andF2) obtained after column chromatography weresubmitted separately to 13C-NMR analysis.

Results and Discussion

The composition of the leaf and stem essential oil ofAcanthopanax trifoliatus (L.) Merr. is reported inTable 1. More than 60 compounds, which represented97.4% of the total amount of the oil, have beenidenti®ed. Among them, 18 were hydrocarbons(57.9%) and 45 oxygenated compounds (39.5%). Thisoil was characterized by the presence of a high content

of monoterpenes (85.0%) while sesquiterpenes repre-sented 12.4% of the total amount. The main con-stituents were a-pinene (23.9%), sabinene (14.9%),terpinen-4-ol (9.0%), b-pinene (7.7%) and p-cymene(5.8%). Fourteen other components amounted to2.4±1.0% each. In contrast with the ®ve majorcompounds, all were oxygenated terpenes, exceptlimonene and b-elemene.

Computer-aided analysis of the 13C-NMR spectrumof the whole oil allowed the identi®cation of 13components (a-pinene, sabinene, b-pinene, p-cymene,limonene, trans-sabinene hydrate, cis-sabinene hydrate,trans-pinocarveol, trans-verbenol, terpinen-4-ol,elemol, caryophyllene oxide, a-cadinol). From theanalysis of the spectra of the hydrocarbon fraction(F1) and oxygenated fraction (F2), other compoundswere identi®ed: a-thujene, camphene, myrcene, g-terpi-nene, a-copaene, b-elemene and b-caryophyllene on theone hand; linalool, a-campholenal, cryptone, myrtenal,a-terpineol, myrtenol, trans-carveol, geraniol, spathu-lenol, t-cadinol, t-muurolol, b-eudesmol and carissoneon the other hand. 13C-NMR determined the stereo-chemistry of oxygenated monoterpenes such as cis- andtrans-sabinene hydrate, trans-pinocarveol, trans-verbe-nol and trans-carveol, as well as the identi®cation ofsesquiterpene alcohols with a cadinane and muurolaneskeleton. The spectrum of the oxygenated fraction (F2)also exhibited several resonances which were unas-signed. Computed searching in the literature datashowed that 15 signals belonged to carissone, asesquiterpenic hydroxyketone bearing the eudesmaneframework and reported in Persea japonica Sieb.17 andCarissa edulis18 methanolic extracts.

This study is a good example of the complementarityof GC±MS and NMR for the analysis of complexessential oils.

Acknowledgements Ð The authors are indebted to the CollectiviteÂTerritoriale de Corse, the MinisteÁ re des A�aires EtrangeÁ res and theCaisse des De poà ts et Consignations for ®nancial support (Pro-gramme de coope ration de centralise e Corse, Vieà t-nam), and theSocie te Montier (Ghisonaccia, Corsica) for a research grant.

References

1. Ministry of Science, Technology and Environment, Red DataBook of Vietnam, Vol. 2 Ð Plants, Science and TechniquesPublishing House, Hanoi (1996).

2. Agence de Coope ration Culturelle et Technique, MeÂdecineTraditionnelle et PharmacopeÂe: Les Plantes MeÂdicinales au VieÃt-nam, Paris Tome 1, 23 (1996).

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7. M. Lischewski, P. H. D. Ty, L. Kutschabsky, D. Pfei�er, H. V.Phiet, A. Preiss, T. V. Sung and G. Adam, Phytochemistry, 24,2355 (1985).

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