Flavonoids and Terpenoids from the Resinous Exudates of Madia Species (Asteraceae, Helenieae) Eckhard Wollenwebera*, Marion Dörra, Marco Dörsama, Abu El-Hamed Hassanb,d, Ahmed A. Ahmedc, M. F. Hegazyc, and Klaus-Peter Zellerd a Institut für Botanik der Technischen Universität, Schnittspahnstrasse 3, D-64287 Darmstadt, Germany. Fax: 0049-6151/164630. E-mail: [email protected] b Department of Chemistry, Aswan-Faculty of Science, South Valley University, Aswan, Egypt c Department of Chemistry, Faculty of Science, El-Minia University, El-Minia 61519, Egypt d Institut für Organische Chemie, Universität Tübingen, D-72076 Tübingen, Germany * Author for correspondence and reprint requests Z. Naturforsch. 58c, 153Ð160 (2003); received October 10/November 14, 2002 The resinous material accumulated on aerial parts of Madia species is shown to consist mainly of diterpenes, containing a series of flavonoid aglycones. A6- and/or 8-O-substitution is characteristic for many of these flavonoids. Three known rare diterpenes were found and the structure elucidation of a diterpene with a new carbon skeleton, named madiaol, is re- ported. Key words: Madia, Flavonoid Aglycones, Diterpenes Introduction Material and Methods Madia is an Asteraceae genus (tribus Hele- Madia anomala (seed: Botanischer Garten Bay- nieae-Madiinae Benth.) that comprises 18 species reuth), M. capitata (seed: Botanischer Garten of annual or perennial herbs, growing in the West- Halle), M. citrigracilis (seed: Botanischer Garten ern United States and in Chile (2 species). Within Bayreuth), M. dissitiflora (seed: Botanischer Gar- the subtribe, they are characterized by sophisti- ten Dijon) and M. elegans (seed: natural habitat in cated features of their inflorescences (see Bremer, CA, coll. G. Yatskievych) were cultivated in the 1994, for further details). Depending on the spe- Botanischer Garten der TU Darmstadt. Voucher cies, glandular trichomes and resinous excretions specimen are kept in the herbarium of the Institut are obvious on stems, leaves, and bracts, hence the fuer Botanik der TU Darmstadt. Aerial parts were trivial name tarweed. Little is known so far about collected during the flowering period (JuneÐ the external accumulation of flavonoid aglycones August) and briefly rinsed with acetone while and terpenoids, a phenomenon that is widespread fresh. The concentrated solutions were defatted in the family (Wollenweber and Valant-Vetschera, (MeOH, Ð 10 ∞C; centrifugation) and passed over 1996). Bohm and co-workers previously reported Sephadex LH-20, eluted with methanol, to sepa- the occurrence of flavonoid aglycones in the leaf rate the flavonoids from the prevailing terpenoids. exudate of Madia sativa (Bohm et al., 1992). Wol- Flavonoid portions were subjected to column lenweber et al. (1997) studied the resinous leaf ex- chromatography on silica and/or on polyamide udate of Madia sativa once more, and also that of SC-6, eluted with toluene and increasing amounts M. elegans. They found a wide array of flavonoids of methylethyl ketone and methanol. The leaf and for the first species, but very few for the latter. In stem exudates of Madia anomala and M. citrigraci- the present study, we analyzed the exudate flavo- lis exhibited the same flavonoid patterns and were, noids and terpenoids of M. anomala Greene, M. therefore, combined and subjected directly to SC capitata Nutt., M. x citrigracilis Keck, and M. dis- on polyamide. Fractions were monitored and com- sitiflora Torr. & Gray as well as the terpenoids of parisons with markers were made by TLC on poly- M. capitata and M. dissitiflora. amide DC-11 with the solvents toluene-petrol 0939Ð5075/2003/0300Ð0153 $ 06.00 ” 2003 Verlag der Zeitschrift für Naturforschung, Tübingen · www.znaturforsch.com · D 154 E. Wollenweber et al. · Flavonoids and Diterpenes from Madia ether 100Ð140 ∞ÐMeCOEt-MeOH 12:6:2:1, to- Results and Discussion luene-dioxane-MeOH 8:1:1 v/v/v, and toluene- Structure elucidation MeCOEt-MeOH 12:5:3 v/v/v, and on silica with the solvents toluene-MeCOEt 9:1 v/v and toluene- Flavonoids dioxane-HOAc 18:5:1 v/v/v. Chromatograms were From the lipophilic exudates accumulated on viewed under UV (366 nm) before and after aereal parts of of four Madia species we identified spraying with “Naturstoffreagenz A” (1% of di- a series of flavonoid aglycones (for structural for- phenyl-boric acid -ethanolamine complex in mulae see Fig. 1). The following compounds were MeOH). identified by a combination of APCI-MS and co- Atmospheric pressure chemical ionization TLC with authentic markers: Apigenin-4Ј-O- (APCI) and electrospray mass spectra were re- methyl ether (MH+ at m/z 285), isoscutellarein-8- corded by J. F. Stevens at OSU (Corvallis, OR) on O-methyl ether (MH+ at m/z 301), isoscutellarein- a PE Sciex API III-plus triple quadrupole instru- 8,4Ј-dimethyl ether (MH+ at m/z 315), 8-methoxy- ment (PE Sciex, Thornhill, Ontario, Canada) as luteolin (MH+ at m/z 317), 5,3Ј,4Ј-trihydroxy-6,7,8- described elsewhere (Stevens et al., 1999). trimethoxyflavone (MH+ at m/z 361), 5,7,3Ј,4Ј-te- NMR spectra of compound 14 (acerosin) were trahydroxy-3,6,8-trimethoxyflavone (MH+ at m/z run in Halle (A. Porzel and J. F.Stevens) on a Var- 377), 5,3Ј,4Ј-trihydroxy-3,6,7,8-tetramethoxyfla- ian UNITY instrument at 500 MHz (1H) and vone (MH+ at m/z 391). The remaining flavonoids 125 MHz (13C) in DMSO-d6 at room temper- (except No 14) were identified by co-TLC with δ δ ature. The solvent resonances ( H 2.50 and C authentic markers available in E. W.’s lab. 39.51) were used as internal shift references. 2-Di- Compound 14 was identified by mass spectro- mensional experiments (1H-1H COSY, 1H-13C metry and NMR spectroscopy as 5,7,3Ј-trihydroxy- HSQC and HMBC) were carried out using stan- 6,8,4Ј-trimethoxy flavone, trivially known as acer- dard Varian pulse sequences. 1 δ osin. H NMR (500 MHz, DMSO-d6) H 12.78 (s, The terpenoid portions of the five Madia species OH-5), 10.43 and 9.58 (both br s, OH-7 and now studied showed the same TLC features in dif- OH-3Ј), 7.56 (1H, dd, J = 8.4, 2.2 Hz, H-6Ј), 7.47 ferent solvents, thus indicating that the major com- (1H, d, J = 2.2 Hz, H-2Ј), 7.11 (1H, d, J = 8.6 Hz, ponents were identical. They were, therefore, com- H-5Ј), 6.79 (1H, s, H-3), 3.88 (3H, s, 8-OMe), 3.87 bined to increase the starting amount. Separation (3H, s, 4Ј-OMe), 3.77 (3H, s, 6-OMe); 13C of the crude materials by Sephadex LH-20 (n-he- δ (125 MHz, DMSO-d6) C 182.0 (C-4), 163.1 (C-2), xane-CH2CH2, 7:2 ) gave compounds 4 and 5 as 151.0 (C-4Ј), 150.7 (C-9), 148.2 (C-5), 146.6 (C-3Ј), major components (ca. 3 g each), compounds 2 145.2 (C-7), 131.4 (C-6), 127.8 (C-8), 122.9 (C-1Ј), (7 mg) and 3 (12 mg) as minor constituents. Terpe- 118.5 (C-6Ј), 112.7 (C-2Ј), 112.1 (C-5Ј), 102.89 and noids were visualized by spraying silica plates with 102.85 (C-3 and C-10), 61.3, 60.1 and 55.7 (OMe-6, MnCl2 reagent, followed by heating (Jork et al., OMe-8 and OMe-4Ј). The position of the methoxy 1989). groups was determined by 1H-13C HMBC spectro- EI mass spectra of terepenoids were recorded scopy. on a TSQ 70 quadruple mass spectrometer (Fin- negan) at 70 eV and 200 ∞C ion source temper- Terpenoids ature. Exact mass measurements were performed using an AMD Intectra modified MAT 711A in- Fractionation, by silica gel column chromatogra- strument with fragments from perfluorotributy- phy, of the combined terpenoid portions yielded lamine as reference ions. the new diterpene 2, in addition to three known NMR spectra were recorded on a Bruker diterpenes, 3Ð5 (For structures see Fig. 2). Com- AMX 400 instrument in solvents given in the text. pound 2 was isolated as a yellow material, α[D] Ð1 2-Dimensional spectra were measured by means Ð 10.54 (c = 0.0023, CH2Cl2). IR cm : 3400, of standard Bruker puls sequences. 1610,895. HMBC correlations: H-18,19 correlated with C-2, C-4, C-5, H-20 correlated with C-1, C-5, C-10, exomethylene protons correlated with C-9, C-11, H-3 correlated with C-19, H-1 correlated E. Wollenweber et al. · Flavonoids and Diterpenes from Madia 155 Flavones: R =H 5,7,5Ј-triOH: Apigenin 8 3´ Ј HO O 5,7,8,4 -tetraOH: Isoscutellarein B OH 5,7,3Ј,4Ј-tetraOH: Luteolin 7 A C 4´ 5,6,7,3Ј,4Ј-pentaOH: 6-Hydroxyluteolin 3 Ј Ј 6 5´ 5,7,8,3 ,4 -tetraOH: 8-hydroxyluteolin (Hypolaetin) 5 R Flavonols: R =OH OH O 3,5,7,4Ј-tetraOH: Kaemperol 3,5,7,3Ј,4ЈpentaOH: Quercetin 3,5,6,7,3Ј,4Ј-hexaOH: Quercetagetin 3,5,7,8,3Ј,4Ј-hexaOH: Gossypetin 8 3´ Flavonones: R =H HO O Ј B OH 5,7,4 -triOH: Naringenin 7 A C 4´ 5,7,3Ј,4Ј-etraOH: Eriodictyol 3 5´ 6 5 R Flavanonols: R =OH 3,5,7,4Ј-tetraOH: Aromadendrin OH O 3,5,7,3Ј,4Ј-pentaOH: Taxifolin Fig. 1. Flavonoid stuctures. 16 20 12 14 1 10 13 15 16 20 11 OH 2 11 12 O OH HO 17 3 5 OH 1 9 4 6 7 8 9 13 14 2 10 8 O 15 OH 3 5 7 4 6 18 19 17 OH 18 19 1 2 HO HO OH OH O OH OH Fig.