eiochemicalSysternaticsa~lEcology, Vol. 12, No~2, pp. 179-181, 1984. 0305-1979/84 $3.00 + 0.00 Printed in Great Britain. Pergamon Press Ltd.

Distribution of Chromenes and Benzofurans in Encelia califomica

PETER PROKSCH* and ELOY RODRIGUEZt *Botanilches Institut der Universitiit zu K61n, Gyrhofi~. 15, D-5000 K61n 41, West Germany;

tPhytochernical laboratory, Departmem Of Ecology end Evolutionary Biology, University of California, Irvine, CA 92717, U.S.A.

Key Word Index- Ence//a cM/forn/cs; Heilanthese; Asterecese; chromenes; benzofurans; __.J~,~_~uiterpana lactones; organ specific distribution. ~ c t - Two populations of Encel~ cMffomica (Asteracwe) were analysed for chromenes and benzofurans on an organ specific basis using HPLC. Both classes of compounds were present in all parts of the plant studied including roots, stems, leaves, capitula and achenes. The distribution patterns were less complex in roots and achenes when compared to stems. leaves and capitula which yielded three chromenes and two banzofurans. In addition to the chromenes and banzofurans the capitula afforded a sescluiterpene lactona of the eudesmanolide type which was absent in the other parts of the plant.

Introduction The genus Ence//a Adans. (Asteraceae, tribe Heli- antheae) [1] is one of the dominant perennial genera of the southwestern United States and Mexico [2]. Our previous phytochemical studies on Encelia had established that chrornenes (benzo- pyrans) and benzofurans are among the prominent natural products present [3, 4]. Chromenes and benzofurans are common in the Heliantheae, but are also found in other tribes of the Asteraceae, including the Astereae, Eupatorieae, Inuleae and Senecioneae [5]. Although these natural products have been reported in the leaves and stems of many Asteraceae we have shown that in Encelia californica Nutt. they are also present in roots, capitula and achenes.

Results and Discussion The genus Encelia consists of 16 species (or sub species), most of which inhabit the arid and semiarid areas of the Southwestern United States and Mexico, with E. fatJnosa A. Gray ex Tort. con- sidered as a dominant element of the Sonoran desert. E. californica, however, is primarily restric- ted to the coastal shrub zones of Southern California ranging down to Baja California, Mexico [2].

A previous phytochemical study by Geissman and coworkers [6] reported the isolation of the

(Rece/ved 24 July 1983)

chromene encecalin (3) and of two benzofurans, euparin (6) and euparone methyl ether (2,5-acetyl- 6-hydroxybenzofuran) from the leaves of E. californica; essq uiterpene lactones we re previously reported to be absem in the species [6].

As part of our studies on Ence/~ we have reinvestigated E. californica with emphasis on the organ specific accumulation of the dominant chromenes and benzofurans. Two populations of the species, one from the Irvine area (California, U.S.A.) and one from Baja California (Mexico), were analysed using HPLC and spactroscopical methods. In the population from Irvine we found chrornenes and benzofurans to be present in all parts of the plant analysed, including roots, stems, leaves, capitula and achenes (Fig. 2). The patterns of chromenes and benzofurans were less complicated in the roots and achenes, with one or two compounds missing, which were additionally present in stems, leaves and capituia. In addition to encecalin (3) and euparin (6), we identified two further chromenes (2 and 4) and one benzofuran (5). Contrary to an eadier report [6], we could not isolate euparone methyl ether from E. califomica.

The capitula yielded, in addition to the chromenes and benzofurans, a seSCluiterpene lactone (1). The 1H NMR spectrum revealed that this lactone was an eudesrnanolide with two exo- cyclic methylenes. The signals at d 5.6 and 6.2 were indicative of the exocyclic methylene of the lactone ring as could be confirmed by decoupling

179

180 PETER PROKSCH AND BOY RODRIGUEZ

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experiments, whereas the broad singlets at (5 5.1 and 6.1 represent the exocyclic methylene at C-4, being in a-position to the carbonyl group at C-3. The spectral data suggested further the fusion of the lactone ring at C-7 and C-8. The ~H NMR data of this compound were very similar to encelin, another 7,8-1actonized eudesmanolide that had previously been isolated from E. farinosa [7]. The major difference is the magnitude of the coupling constant J7.~3, which is only 1.5 Hz for the lactone from E. californica and 3 Hz for encelin [7]. The smaller coupling constant J = 1.5 Hz is indicative for cis-lactones whereas J = 3 Hz reflects the trans- lactone [8, 9]. Thus the sesquiterpene lactone from E. californica is the cis-somer of encelin.

From the second population of E. califomica, collected at San Quintin, Baja California, Mexico, only leaves and stems were available and showed no qualitative differences in regard to the chromenes and benzofurans present compared to the population from the Irvine area:

In a previous study we could demonstrate that several chromenes and benzofurans (3, 4 and 5), present in several species of Encelia, are a new dase of natural photosensitizers and exhibit phototoxic activity against microorganisms [10]. Of the compounds screened, 6-methoxyeuparin (5) was the most active. In this study we found 6- methyleuparin present only in the aerial parts of

E. californica, suggesting a possible defensive role against pathogens where UV radiation is required. Further experiments on the ecological significance of chrornenes and benzofurans from Ence//a are underway.

Expedmenlal Encehb cal/fom/ca was collected at the Back Bay, Irvine, Southern Califomle, end at San Quintin, Baja California, Mexico, in the spring of 1982. Voucher specimens are deposited at the Botanical Institute, University of K6in.

The plants from the Irvine population were separated into roots, sterns, leaves, capitule end echenas, dried, ground and extracted with MeOH. From the second population from San Quintin only items and leaves were available in sufficient amounts for analysis. The crude MeOH extracts were injected into a Waters liquid chromatograph, equipped with a solvent pump Model M 45, a solvent programmer Model 660, a universal injector Model U6K end a UV detector Model 440. Detection was at 254 nm. The hplc column was LiChrosorb RP-8 (250 x 4 mm), pore size 5/an (AIItech, Los Altos, CA). The MeOH extracts were separated using a linear gradient of A (H20) and B (ecatonitHle-AcOH, 98:2), starting from 40% B in A to 100% B in 30 min, flow rate 1.5 ml/min [3]. Identification of the peaks was done by Jimultenaous injection of the isolated and identified compounds.

Compounds 2-6 (Fig. 1) were isolated end identified by their spectroecopical properties according to [3]. The sesqui- terpene lectone was isolated by column chromatography on Sephadex LH-20, eluent MeOH, and by repeated prep. TLC on silica gel, solvent system CHCI3-1ight petrol ether (9:1 ).

Spectra were recorded on the following instruments: 1H NMR Varian EM 390 (spectra recorded at 90 MHz in CDCI 3 with TMS as internal standard); Finnigan Quedrupole MS 4000 GC-Ms with Tektronix 4010-1 date system and Data General Nova 3 (spectrum is given by heated probe).

Compound1 (Rg. 1 ). 1H NMR: (I 1.0 (3H, $, C-14), 3.0 (1H), m, C-7); 4.5 (1H, m, C-8), 5.1 (1H, hrs, C-15), 5.6 (1H, d, JT,13= l.5Hz, C-13),6.0(1H, d, Jt.2=9Hz, C-2);6.1 (1H, brs, C-15), 6.2 (1H, d, J 7 13= 1.5Hz, C-13); 6.7(1H, d, J1.2= 9 Hz, C-1). MS (CI, iso-'butsn, 100 eV) m/z (rel. int. %): 245 [M + 1] +, (100), 91 (55), 81 (60).

References 1. Stuessy, T. F. (1977) in The Biology and Chemistry of the

Compositae (Heywood, V. H., Harborne, J. B. end Turner, B. L , eds.), p. 621. Academic Press, London.

2. Jepson, W. L. (1966) A Manual of the Flowering Plants of California, p. 1081. University of California Press, Berkeley.

3. Prokech, P. and Rodriguez, E. (1982) J. Chromatogr. 240, 543.

4. Prokech, P., AreguIlin, M. and Rodriguez, E. (1083) Plants Med. 47, 224.

5. Proksch, P. end Rodriguez, E. (1983) Phytochemistry 22, ~35.

6. Bjeldanes, L. F. and Geissman, T. A. (1969) Phyto- chemist~/ 8, 1293.

7. Geissman, T. A. and Mukherjee, R. (1968) J. Org. Chem. 33,656.

CHROMENES AND BENZOFURANS IN E. CALIFORNICA 181

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RG. 2. HPLC CHROMATOGRAMS (1.0 a.u.f.s.) OF AUQUOTS FROM CRUDE MeOH EXTRACTS OF (a) ACHENES; (b) CAPITULA; (c) STEMS; (d) LEAVES; (e) ROOTS FROM ENCELIA CALIFORNICA. Numbering of the peaks follows Fig. 1.

8, Samek, Z. (1978) Tetrahedron Letters 43, 3210. 9. Rscher, N. H., Olivier, E. J. and Fischer, H. D. (1979)

in Progress in the Chemistry of Organic Natural Products

(Herz, W., Gdsebach, H. and Kirby, G. W., ads.) Vol. 38, p. 148. Springer Verlag, Wien.

10. Proksch, P., Pmksch, M., Towers, G. H. N. end Rodriguez, E. (1983)J. Nat. Prod. 46, 331.