Biochemical Systematics and Ecology, VoL 19, No. 8, pp. 673-675, 1991. 0305-1978/91 $3.00 + 0.00 Printed in Great Britain. © 1991 Pergamon Press plc.

Exudate Flavonoids in Three Persian Asteraceae Species

ECKHARD WOLLENWEBER* and ABDOLHOSSEIN RUSTAIYANt *lnstitut ~ r Botanik der Technischen Hochschule, Schnittspahnstref,Z,e 3, D-6100 Darmstadt, Germany;

tDepartment of Chemistry, Faculty of Science, University of Shahid Beheshty, Eveen, Tehran, Iran

Key Word Index--Artemisia o/iveriana; Pu/icaria gnaphalodes; Tanaceturn polycepha/urn; Asteraceae; Anthemideae; Inuleae; exudate flavonoids.

Abstract--Aereal parts of three Asteraceae from Iran have been analysed for exudate flavonoids. Artemisia oliveriana, Pulicaria gnaphalodes and Tanaceturn polycephalum are shown to accumulate mostly 6-methoxylated flavonols and flavones as external aglycones.

Introduction Previous studies on the occurrence of flavonoid aglycones in exudates of higher plants have shown that Asteraceae is one of the most important families with regard to external accumulation of flavonoid aglycones [1 ]. We have now had the opportunity to analyse three species collected in Iran, namely Arternisia oliveriana J. Gay ex Bess. in DC, Pulicaria gnaphalodes (Vent.) Boiss., and Tanaceturn polycephalum Sch.Bip. These species had previously been analysed for terpenoids [2-4], which are supposedly excreted by the characteristic Asteraceae glandular trichomes. The accompanying flavonoids, however, had not been taken into account.

Materials and Methods Artemisia oliveriana was collected in Khorassan province, Iran. Pu/iceria gnapha/odes and Tanaceturn poly- cephalurn were collected in the Alborz Mountains, 40-60 km north of Tehran, Iran on August 28, 1987 and August 3, 1988, respectively. Vouchers are deposited in the Herbarium of the Department of Botany at Shahid Beheshty University in Tehran. Dry aerial parts were rinsed with Et20-petroI-MeOH to dissolve the exudate material. The syrup residue obtained after solvent evaporation was dissolved in boiling MeOH, Fatty and waxy material precipitated on cooling and was eliminated by centrifugation. The solution was then passed over Sephadex LH-20 to separate flavonoid aglycones from the bulk of terpenoids. Fractions with similar flavonoid content were combined and most products were then directly identified by comparison with markers. TLC was on polyamide DC-11 (solvents: A, petrol, bp 100-140°-toluene-MeCOEt-MeOH, 12:6:6:1; B, toluene-petrol, bp 100-140°-MeCOEt-MeOH, 12:6:2:1; C, toluene-dioxane-MeOH, 8:1:1; D, toluene-MeCOEt-MeOH, 12:5:3) and on silica (solvents: A, toluene-MeCOEt, 9:1; B, toluene-clioxane-HOAc, 18:5:1). Mass spectra were obtained at 70 eV by direct inlet.

A small quantity of compound 1 was obtained from Artemisia oliveriana and was crystallized from ethyl acetate. The yellow crystals have mp 229-231°. Its mass spectrum indicates a flavone or flavonol with 2 OH and 30Me groups ([M] + 344), with one of the methoxyls at C-6 ([M]+-15<[M]+). Direct comparison with markers showed it to be 6-hydroxyluteolin 6,3',4'-trimethyl ether (eupatilin) (isomers, 6,7,3'-triMe and 6,7,4'-triMe are discernable on polyamide TLC). MS m/z(rel, int.) 344 ([M] ÷, 100), 329 ([M-15], 77), 326 (65), 301 ([M--43], 61).

A good amount of compound 2 was obtained from some combined fractions of Pulicana gnaphalodes. It crystallized from ethanol as fine yellow needles, mp 181-182 °. Its molecular mass (rn/z 374) and chromato- graphic behaviour indicated a flavone or flavonol with 2 OH and 40Me groups, the important mass fragment at M-15 pointing to a 6-methoxy flavonoid. By direct comparison with several authentic samples of flavonoids meeting this proviso it was shown to be quercetagetin 3,6,7,3'-tetramethyl ether (chrysosplenetin). (Identity with the isomers, 3,6,7,4'tetraMe=casticin, and 3,6,3',4'-tetraMe=bonanzin, was excluded by direct comparison on polyamide.) The mp agrees with literature data [5]. MS m/z (rel. int.) 374 ([M] +, 100), 373 (40), 359 ([M--15], 91), 331 ([M--43], 22), 181 (9), 151 (16). Quercetagetin 3,6,3'-triMe is discernible from the isomer 3,6,4'-triMe on polyamide TLC by a different colour reaction with Nat A.

Results Results of flavonoid analysis are presented in Table 1.

(Received 11 February 1991)

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674 E. WOLLENWEBER AND A. RUSTAIYAN

TABLE 1. FLAVONOID AGLYCONES FROM THE EXUDATES OF THREE PERSIAN ASTERACEAE

Artemisia oliveriana Pulicaria gnaphalode$ Tanacetum polycephalum

Apigenin Apigenin Ap 7-Me Ap 4 '-Me Scutellarein 6-Me Scut 6,7-diMe Scut 6,4"-diMe

Luteotin 7,3'-diMe

6-OH-Lut 6,3'-diMe 6-OH-Lut 6,7,3'-triMe 6-OH-Lut 6,3',4'-tdMe

Luteolin Lut 3'-Me

6-Hydroxyluteolin 6-Me 6-OH-Lut 6,7-diMe

6-OH-Lut 6,3",4'-triMe 6-Hydroxykaempferol 3,6,7-triMe

Quercetin Qu 3'-Me Quercetagetin 6,7-diMe Queg 3,6,7-triMe Queg 3,6,7-triMe Queg 3,6,3'-triMe Queg 3,6,7,3'-tetraMe

Queg 3,6,7,4"-tetraMe Eriodictyol Eriod 7-Me

Artemisia oliveriana From the leaf exudate of Artemisia oliveriana, compound 1 was obtained in crystal-

line form. It was identified as 6-hydroxyluteolin 6,3',4'-trimethyl ether (eupatilin). Further exudate flavonoids of A. oliveriana include apigenin and methyl ethers of apigenin, scutellarein and 6-hydroxyluteolin. There were nine flavones in total.

Pulicaria gnaphalodes Compound 2 was crystallized from the leaf exudate of Pulicaria gnaphalodes, where

it is the major flavonoid. It was identified as quercetagetin 3,6,7,3'-tetramethyl ether (chrysosplenetin). In addition, this species exhibits three further methyl derivatives of quercetagetin as well as small amounts of quercetin and isorhamnetin and traces of eriodictyol and eriodictyol-7-methyl ether.

Tanacetum polycephalum None of the flavonoids excreted by Tanacetum polycephalum could be isolated.

However, a series of aglycones were identified by comparison with authentic samples. They comprise six flavones and three flavonols.

Discussion Flavonoid aglycones have already been reported for many species of Artemisia (c.f. [6]), but their localization was normally ignored. External flavonoid accumulation in eight species was stressed recently [7]. The flavonoid aglycones now found in A. oliveriana are in accordance with the flavonoid profiles known for other species of the genus, where 6-methoxylated flavones and flavonols are often dominating.

In Pulicaria, the European species R dysenterica has been studied previously (see [7] and Refs therein). It was found to exhibit methyl derivatives of quercetagetin and of 6-hydroxykaempferol, along with traces of quercetin and kaempferol methyl ethers. R paludosa, a species native to western Spain, has been found to produce 6-hydroxy-

EXUDATE FLAVONOIDS IN THREE PERSIAN ASTERACEAE SPECIES 675

kaempferol 3,6-dimethyl ether, scutellarein 7,4'-dimethyl ether and 5,6,8-trihydroxy- 7,4'-dimethoxyflavone [8]. Methyl ethers of quercetagetin have been reported for Pulicaria arabica [9, 10]. R incisa is exceptional in that it lacks 6-rnethoxy flavonoids: quercetin 3-rnethyl and 3,7-dimethyl ethers, kaempferol 3-rnethyl ether and taxifolin 7-methyl ether were recently reported for this species [11].

The presence of two highly methylated derivatives each of quercetagetin and of 6-hydroxyluteolin has been reported for Tanacetum santolinoides [12]. Methyl ethers of quercetagetin, of 6-hydroxyluteolin and of scuttelarein have been found as exudate flavonoids in T. chiliophyllum and in T. vulgare [7]. Now T. polycephalum is added with a very similar flavonoid profile.

Acknowledgements--Thanks are due to Ing. A. Mozafarian, the Research Institute of Forests and Rangelands, Tehran, for collecting the plant material. Financial support by the Deutsche Forschungsgemeinschaft to labora- tory work at Darmstadt (E.W.) is gratefully acknowledged.

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p. 233. Chapman & Hall, London. 7. Wollenweber, E., Mann, K. and Valent-Vetschera, K. M. (1989) Fitoterapia 60, 460. 8. San Feliciano, A., Medarde, M., Gordaliza, M., del Olmo, E. and del Corral, J. M. M. (1989) Phytochemistry

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