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0971-4693/94 AltelopathyJournal t (2) :95-104 (1994\ @ , International Alie lopathy Foundatio

Identification of naturally occurring,chemicals from Artemis, princeps var, .orientalis

KYEONG WON YUN*. BONG-SEOP KILI AND JONG_SEI PARK2 Departmentof Oriental Medicine Resources SunchonNational University, Sunchon,540-742, Republic of Korea

(Receivedin revised'form: June3, 199{)

ABSTRACT )

The GCIMS method wasemployed for analysis and identificationof phytotoxic I substancesfrom wormwood plants (Artemisiaprinceps var. orientalis). Benzoicacid I and 16 phenolic compounds were detectedin wormwood leaf water extract. Terpe- noids suchas n-hexanal,(a*B)-phellandrene, ({)-camphene, phenylethylalcohol,ben- I zaldehyde,a-pipene, B-pinene, 3-carene, a-terpinene, p-cymene,D-limonene, cineole, li phenylacetaldehyde,y-terpinene, trans-2-octanal, (-)-thujone, camphor,terpinen-4- 'a ol, geranylacetate, carveol, iso-phorone,nerol, iso-bornyl acetate, eugenol, B-caryo- n phyllene, B-myrcene, a-humulene, cadinene, terpinolene, linalyl fornrate, trans- caryophyllene,furfural, acetoin, dipentene,geraniol and cedrol were identifiedfrom 0 wormwoodessential oil. Keywords: Artemisiaprincepsvar.orientalis, phytotoxic substances,water extract, wormwoodessential oil

INTRODUCTION I Different parts ofl plants have been bioassayedand inhibitors have h found in them (ll). Chemicals from leaves of Artemisiaprincepsvar. orien t suppressedgermination and seedlinggrowth of selectedplants (13, 2l). Most kn allelopathic compounds are secondary plant metabolitesviz., phenolic a flavonoids, terpenoids,steroids, alkaloids and organic cyanides(5, 9, 14, 15, The chemical composition of essentialoil of Artemisia has been investig (2,8, l8). The objectiveof the presentstudy was to determinewhichof chemical componentsof Artemisia princeps var. orientalis exhibitedallelopi effects(21).

MATERIALS AND METHODS Water-soluble Substances

The extractionof phenolic compoundsfrom wormwood plants was car out by the method of Kil and Yim (13). A gaschromatograph (Hewlett pacl

*Correspondenceauthor. rDepartment of BiologyEducation, Wonkwang University, Iri, S'70_i49,Republic of Korea. 2Doping control center, KAlsr, seoul, 136-791,Republic of Korea. 96 Yun, Kil and Park

5890,U. S.A.) using J & W fused silica capillary and nrethylsiliconebanded column(B-l;60mx0.25mm i. d ) wasemployed for gas chromatography. Oven temperaturerange was 100-300'C at 4'C/min. The head pressureof column was 30psi and split ratio was I : 50. The integratorwas a Hewlett Packard3392 A and thcvolume <-rf sample injected was 0.5-1.0 pl. Identiiicationof eachpeak was made bycomparing the retentiontimes of the peaks with thoseof commercialcompounds obtainedfrom Sigmaand Aldrich chemicalcompanies.

VolatileSubstances

Karlsruker'sapparatus was usedto preparevolatile substancesfrom worm- wood plants. The volatile substanceswere analysed by gas chromatography (Hewlett-Packard5890) using SE-54 column (:0mx0.33 ltmx 0.2 mm i. d.). Temperaturewas programmed from 45'C (5 min) to 300"C (3 min) at 4"C/min. Carriergaswas helium, flow rate 0,5 ml/min, with FlD. Injector temperaturewas 250'C. Splitratio u,asl: l0 and headpressure was 34 psi. Injection volumeof allsamples was 0.20 pl. Identificationof eachpeak was made by the comparison of retentiontimes and mass spectra of the peakswith those of commercialcom- poundsobtained from Sigma,Aldrich and Fluka chemicalcompanies.

RESULTS AND DISCUSSION

Wrter-solublePhytotoxins

Phytotoxicchemicals present in wormwr-,odleaf extract were identifiedby gas chromatography.The identity of thesecompounds was confirmed by comparing theretention time of the standards. Phenolicacids such as salicylic,gentisic, caffeic acidand protocatechuic acid werethe major chemicalsidentified in wormwood leaf extract(Fig. l).

VolatilePhytotoxins

Figures2,3 and 4 show the chromatogramsof wormwood essentialoil extractedfrom leaf, root and flower, respectively. The identification of each componentwas made by comparisonof the retenticntime and mass spectra with thoseof commercialauthentic samples.

Twenty-sixcomponents were identified in leaf essentialoil, 28 in flower oil and23 in root oil. The major constituentsof the essentialoil in leavesand flowers werecineole and camphor,and thclsein roots were (a-t-p)-phellandreneand iso- bornvlacetate. Identification of allelochenicals in Artemisia 9',,

Fig. 1. GC chromatogram of the combined ether extracts of A. princeps var. orientalis leaf. 1, benzoic acid;2, phenylacetic acicl;3, catechol;4, salicylic acid; 5, t-cinnamic acid; 6, z-hydroxybenzoic acidl'7,p-hydroxybenzoic acid: 8, phlorogluicinol;9,p-phenylbenzaldehyde; 10, vanillic acid; ll, gentisic acid; 72, o-lty

i t,, rl!i' iit/

]( 36 l8 *l '.2 rJ -5

trME (mra,

Fig.2. GC chromatogramof the essentialoil of A. princepr var. orientalis leaf. l, n-hexanal; 2, (a*9)-phellandrene; 3, (+)-camphene; 4, phenyl ethyl '1, alcohol; 5, benzaldehyde;6, e,-pinene; B-Rinene; 8, 3-carene; g, a-terpinene; 10, p-cymene; 11, cineole; 12,phenyl-+thyl acetaldehyde;13, T-terpinene;14, terpinolene; 15, Iinalyl formate;16, (-)-thujone; 17,camphor; 18, terpinen-4-ol; 19, carveol; 20, iso-bornyl acetate;21, eugenol;22, B- caryophyllene:23, oc-humulene:24, cadinene;25, trans-caryophylleneand 26, farnesol. Yun, Kil and Park

a l/. 16 18 20 72 7L 26 28 30 32 y 36 38 40 L? U 46 L8 50 52 REtE\rra'\IrMI lrua.)

Fig. 3. GC chromatogramof the essentialoil of A. princepr var. ori,nto | flower. 1, n-hexana|'2.16g r p1*phellandrene;3, (+ )-camphene;4, phenyl ethyl -carene; alcohol;5, benzaldehyde;6, a-pinene;7, B-pinene;8, 3 9, a-terpinene; 10,p-cyrnene; ll, D-limonene;12,cineole: 13, phenyl acetaldehyde:14, ^y- terpinene;15, trans-2-octanal; 16, (-)-thu.ione; 17,camphor; 18, terpinen-4- ol; 19,geranyl acetate;20,carveol;21, iso-phorone; 22,nerol;23, iso-bornyl acetate:-24,eugenol;25, B-caryophyllene; 26, B myrcene;27,e-humelene and 28,cadinene. ROOI 6

(b 12 1L 16 18 20 22' u 26 28 30 n 34 36 30 40 LZ u t.u 50

RETENTI0N TIME (mrq.l

Fig.4. GC chrornatogramof the essentialoil of 1. f rircef, var. or:entalis root. l, n-hexanal;1;furfural: 3, acetoin;4, (a|p)-phellandrene; 5, bezaldehyde; 6, a-pinene; 7, g-pinene; 8, p-cymene;9, dipentene; l0' cineole; 11,Y. terpin'ene:tZ t-)-fhujone; 13, camphor; 14, terpinen-4^ol; 15, geranyl acetate;16, carveol: 17,geraniol; 18, iso-bornyl acetate;19. p-ca_ryo-phyllene; 20. a-humulene;21, cadinene;22, ttans-caryophylleneand 23, cedrol. I Identifcation of allelochemicals in Artemisia

Various components of essentialoil from several species of Artemisia h been documented in the literature (Tablc l).

TABLE 1. Volatilecompounds in differentArtemisia species

Compound Artemisiaprinceps A.g.tzl A. r.1r) A.p.(rl A. h.$l A.".( var, orientalis* Leaf Flower Root I 234 n-Hexanal + + Benzaldehyde + + a-Pinene + + + + 3-Carene + + af p-Phellandrene + + a-Terpinene + + + + + (-)-Thujone + + Terpinene-4-ol + + + + + + Camphor + + + + + Carveol + ++ Cineole + ++ + + Linalyl acetate + Bornyl acetate + + iso-Bornyl acetate + + Eugenol -L + + B-Caryophyllene + + + + Geranyl acetate + + + trans-Caryophyllene + + + Farnesol + + (*)-Camphene + + + + + Nerol + Cadinene + + p-Pinene + + + + Cedrol D-Limonene + + a-Thujene + Myrcene +-f p-Cymene +++++ 8-Terpinene + Con t00 Yum, Kil and Park

Table I contd.

Terpinolene + + trans-Pinocarveol t- Pinocarvone -T-I Benzylacetate + a-Terpineol + + Myrtenol -r Phellandral + cis-Piperitol + Cuminicaldehyde + p-Isopropil-Phenol + p-Menth-2-en-7-ol + 1,S-p-Menthadien-7-ol + Ca'vaool + Benzylbutyrate + l-p-Menthen-9-ol + a-Copaene + Methyleugenol + 3,?-DimethYloctYlacetate + + p-Sesquiphellandrene +

ftCubebene + SabinylproPionate + p-Farnesene + Sabinylacetate + l-Dodecanol + Nerolidol + Lhamazulene + iso-ButylPhthalate + + Hexadecanoicacid + + Phytol + + 2-Methyl-butyl-2-methyl-butyrate + Borneol + + + Citronellol + a-Terpinylacetato + + Citronellylacetate +

Contd. Identifcationof allelochemicolsin Artemisia

Toblc I contd.

B-Elemene + a-Guaience + + p-Charmigrene + Artemone + + Citronel lyli-valerate + L inalyl 3-methyl-butyrate + Davanone + Guaiol + Bazzaneol + Tetradecanoicacid J- 4,6,I G-Trimethyl-2-pentadecanone + l-Hexadecanol + Pentadecanoicacid +

l-Otadecanol f Linoleic acid +

2-Methyl-l-butanol I cis-3-Hexenyl butyrate + trans-3-Hexenyl butyrate + 2-Methylbuty-2-methylbutyrate + n-Amyl-3-methyl butyrate + trans-Pinocarreol + 8-Terpinol -r

a-Terpinol -T.I Myrtenol + iso-Amyl tigliate + lrans-p-Farnesene + B--Phenytethyl-2-methylbu tylate + B-Phenylethyl-n-va lerate + p-Phenylethyl tigtiate + Hdroxy davanone + bo-Butyl Phthalate + p-Cymen-8-ol + Piperitol + Cumin atdehyde + 102 Yun, Kil and Park

TableI contd.

Terpenylacetate + Piperitone + Thymol + t "ans-Sabinenehydrat e + Bornylpropionate + Chrysanthenone + B-Elemenene + 8-Cadinen + ot-Httmulene + a-Cedrene + a-Guainene + p-Guainene + Curcumene + ArtemisiaKetone + iso-Thujone +

*Presentstudy; A.g.-Artemisiaglabello, A. r.-A. rupestris,A. p.-A. persica,A. h.-A. herba- alba, A. c. -A . californica.

Inhibition of seedgermination and seedlingelongation by wormwood plants in thc absence of physical competition shows that the inhibition is due to allelo- chemicalsof the 2lanti (21) The identification of benzoic acid. phenylacetic, salicylic,l-cinnarnic, rn-hydroxybenzoic. p-hydroxybenzoic, rar;illic, gentisic, a-hydroxycinnamic, protocatechuic,syringic, p-coumaric, frrulic, caffeic acid, cat.'chol,phlrroglucinol and p-benzaldehydeby GC in worrnwood leaf also supports its allel,rpathicpotential. There is considerableevidence that allelopathy is probably due to complex interactionsof all compotindspresent (1,3,4,6,7, 12, 16,20),

Halligan (lO; reported that the volatile toxins from A. califurnicawere monoterpenoids,sesquiterpenoids and sesquiterpenehydrocarbons, the major compoirndsbeing l, 8-cineole,artemisia ketone, a-thujone, camphor. Th.-essential oils were fractionatedfrttm A. abrotanum leaves by dry column chromatography and about 40 different chemicalsincluding l, 8-cineole were identified by gas chromatographyand massspectrometry (17). The steam--distilledessential essential oil of the three Asiatic Artemisia spp. were a pinene pinene,a-terpinene, terpinen-4-ol, 1, 8-cineoleand (*)-camphene.

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