Sesquiterpene Lactones from Inula montana L. M aría A. González-Romero, Lucinda Villaescusa-Castillo and A na M. D íaz-Lanza* D epartam ento de Farmacologfa, Facultad de Farmacia, Universidad de Alcala, 28871 Alcala de Henares, Madrid, Spain. Fax: 918854679. E-mail: tfamdl@farma.alcala.es * A uthor for correspondence and reprint requests Z. Naturforsch. 55 c, 697-700 (2000); received April 10/May 29, 2000 Asteraceae, Inula montana L., Sesquiterpene Lactones Aerial parts of Inula montana were investigated for its sesquiterpenoid composition. Five sesquiterpene lactones, isoinuviscolide, gaillardin, lß-hydroxy-3ß-acetoxy-eudesm-4(15), ll(13)-dien-12-8ß-olide, pulchellin-C and pulchellin-E were identified for the first time in this plant. One of them, lß-hydroxy-3ß-acetoxy-eudesm-4(15),ll(13)-dien-12-8ß-olide, is a novel natural product. The structures of this compounds were established by ID and 2DNM R spectroscopy. Introduction Inula montana L. (tribe Inuleae, fam. A stera­ ceae) is a perennial species occurring in the east of Spain on alkaline and dry soils, extending to west of France and east of Italy (Tutin, 1976). Sev­ eral species from the genus Inula are traditionally used in folk medicine as antipyretic (Muzaffer et al., 1992), antiinflammatory and hepatoprotective drugs (Kurma and Mishra, 1997). However, there are no data in the literature on the chemical con­ stituents and the possible pharmacological effects of Inula montana. The secondary plant metabolites that mediate these pharmacological effects are mainly sesqui­ terpene lactones. This class of compounds includes over 3000 naturally occurring substances, one of the largest group of plant products described (Fi­ scher, 1991). Sesquiterpene lactones occur in many plant families, but are most widely distributed within the Asteraceae. They include a wide variety of biologi­ cal and pharmacological activities. Antitumor, an­ timicrobial, antifeedant, cytotoxic, antibacterial, antifungal, allergenic contact dermatitic and plant growth regulatory activity of several sesquiterpene lactones has been previously reported (Beckman et al., 1998; Pieman and Towers, 1983; Pie­ m an,1984; Warshaw and Zug, 1996). In a previous paper we have described the antileishmanial activ­ ity of a chloroform extract from the aerial parts of Inula montana against Leishmania infantum (prom astigote forms) (M artin et al., 1998). 0939-5075/2000/0900-0697 $ 06.00 In the present paper, we report the isolation and identification of five sesquiterpene lactones, isoi­ nuviscolide, gaillardin, pulchellin-E, pulchellin-C and the new natural product lß-hydroxy-3ß-acetoxy-eudesm -4(15),ll(13)-dien-12-8ß-olide, for the first time from Inula montana. Results and Discussion In the chloroform extract of the leaves of Inula montana L. the following known sesquiterpene lactones were identified: isoinuviscolide ( 1 ), gail­ lardin (2), pulchellin E (4) and pulchellin-C (5). In addition, we have isolated and identified one new natural product, lß-hydroxy-3ß-acetoxy-eudesm4(15),ll(13)-dien-12-8ß-olide (3). The structures of the known compounds (Fig. 1) were determ ined by comparison of their spectroscopic features with those reported in the literature (Yoshioka et al., 1970; Sanz et al., 1991; Ito and Lida, 1981; Pired, 1977). Com pound 3 was obtained as a yellow syrup and had the m olecular formula Q 7 H 2 2 O 5 (m /z 306) as established by EIMS. The IR spectrum confirmed the presence of the O H group (3600 cm -1) and the acetyl carbonyl (1760 cm -1). The 'H-NM R spectrum (Table I) showed four multiplets be­ tween 6.5 and 4.5 ppm corresponding to two exocyclic m ethylene groups. Two broadened singlets at 64.97 and 4.66 were assigned to the methylene protons at C-4 and the presence of a pair of doublets at 66.15 and 5.61 (1H each) is character- © 2000 Verlag der Zeitschrift für Naturforschung, Tübingen • www.znaturforsch.com • Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschung in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht: Creative Commons Namensnennung 4.0 Lizenz. D This work has been digitalized and published in 2013 by Verlag Zeitschrift für Naturforschung in cooperation with the Max Planck Society for the Advancement of Science under a Creative Commons Attribution 4.0 International License. M. A. Gonzäles-Romero et al. ■Sesquiterpene Lactones from Inula montana 698 OH ■ T 14 8[ v =o Fig. 1. Structures for sesquiterpene lactones 1 -5 . Table I. NMR chemical shift assignments for the isolated sesquiterpene lactones. H 1 2 3 4 5 1 2 2.57 1.72 and 1.93 2.60 5.20 1.32 and 1.94 3.71 1.6 and 1.82 3.43 3 4 5 1.58 and 1.78 1.97 and 1.97 3.49 (7=11.8/4.3) 1.62 and 2.16 (7=11.8) 5.17 (7=1.5) 4.99 3.74 — — — — — 6 1.48 1.45 and 2.50 2.20 1.49 and 2.54 1.93 1.35 and 1.76 1.97 1.05 and 1.82 7 8 9 2.51 4.81 5.80 2.59 4.57 5.89 1.75 (7=1.5) 1.50 and 1.78 (7=11.6/6.2) 2.96 (7=6.2/1.5) 4.54 (7=4.9/2.0) 1.45 and 2.63 (7=15.7) 2.96 4.48 1.50 and 2.26 3.10 4.54 1.60 and 2.20 10 11 12 13 14 15 16 17 - - - - - - - - - - - - - - - 5.90 and 6.10 1.82 1.19 6.14 and 5.64 1.84 1.22 2.05 6.15 and 5.61 0.82 4.97 and 4.66 2.13 5.59 0.82 4.90 and 4.63 2.15 6.07 and 5.71 0.80 5.26 and 4.70 - - - - istic of a methylenic group (C -ll) conjugated with a lactonic carbonyl group. The region 2.2-0-8 ppm showed two methyl singlets at 2.13 and 0.82 ppm. The chemical shift of the methyl singlet at 62.13 indicated that this methyl group should be adja­ cent to an oxygen function (acetate methyl). The 13C-NMR spectrum (Table II) showed two signals (6170.2 and 169.9) of carbonyl groups of the quaternary carbons, one at 6170.2 was clearly due to the lactone carbonyl, while the other at 6169.9 could be assigned to an acetyl group. The multiplicity of each carbon was achieved by the - - - D E PT experiment, which revealed the presence of two methyl, five methylene, five methine and five quaternary carbons. The assignment of proton and carbon signals was achieved from DQCOSY and 2D-TOCSY Hom onuclear Hartm ann-Hahn Spec­ troscopy (H O H A H A ) (mixing time = 80 ms) ex­ perim ents and also from ’H -I3C heteronuclear multiple quantum (HM QC) and multiple bond correlations (HMBC). The compounds 1, 2, 4 and 5 were obtained as white crystalline solids. The 'H NMR spectra of 1 and 2 suggested two guaianolides with an a-meth- M. A. Gonzäles-Romero et al. • Sesquiterpene Lactones from Inula montana Table II. 13C NMR chemical shift assignments for the isolated sesquiterpene lactones. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1 2 3 4 5 47.2 25.9 42.0 80.4 53.5 32.0 47.9 83.7 126.7 143.1 141.6 172.3 119.5 21.4 23.8 51.9 77.5 48.5 79.5 51.5 30.4 47.2 84.4 128.7 138.0 141.4 172.1 119.9 22.8 26.7 21.5 172.6 76.9 36.9 71.0 141.7 41.7 26.3 40.2 76.0 37.8 29.7 144.6 169.9 120.6 11.2 105.6 21.0 170.2 47.9 69.6 80.0 141.5 44.3 27.1 40.8 76.2 40.5 33.5 143.5 170.5 120.7 18.5 106.4 20.9 170.2 49.3 73.1 79.5 150.4 45.2 28.7 41.3 78.7 41.6 34.6 144.0 172.6 121.0 19.1 106.3 - - - - ylene-y-lactone group. The difference between compounds 1 and 2 was the presence of the acetyl group at C-2 in 2. As expected the H-2 signal was shifted downfield (65.20) and an acetate methyl singlet was visible (62.05). The ]H NMR spectra of 4 and 5 suggested two eudesmanolides: 2a-hydroxy-3ß-acetoxy-isoalantolactone and 2a,3ß-dihydroxy-isoalantolactone, respectively. Their structures were determ ined by comparison of their spectroscopic features with those reported in the literature (Yoshioka et al., 1970). Experimental MS spectra were obtained in a Hewlett Packard 5988 A mass spectrometer. Analytical TLC was perform ed on precoated Si gel plates (Kieselgel G-60, F-254, 0.25 mm, Merck) using mixtures of chloroform-methanol. Visualization of the TLC plates was achieved with a long wavelength UV lamp and sulfuric acid spray reagent. All ID and 2D NMR spectra were recorded by a Varian Unity Plus spectrometer in CDC13 or C D 3OD solutions (JH at 500 MHz, 13C at 125 MHz). Multiplicities were assigned through D EPT 699 experiments. The standard pulse sequences from the Varian software were used for homonuclear and heteronuclear correlation experiments (DQCOSY, TOCSY, HM QC and HMBC). Collection, extraction and isolation The aerial parts of Inula montana were collected in San A ndres del Congosto (Guadalajara, Spain) in July 1996 and identified by Professor Carmen Bartolom e Esteban. A voucher specimen is kept in the D epartm ent of Vegetal Biology, Faculty of Sciences, University of Alcala (Madrid, Spain). Air-dried plant material (820 g) was extracted with chloroform. The dry chloroform extract (23.43 g) was subjected to solvent partitioning be­ tween n-hexane and H 20 /M e 0 H (5/95 v/v). The H 20 /M e 0 H portion (12.84 g) was column chro­ m atographed over silicagel, eluted with chloro­ form and mixtures chloroform-methanol of increasing polarity, giving fractions A r A 20. Frac­ tions A i to A 10 were eluted with pure chloroform. Fraction A 2 was subm itted to sucesive flash col­ umn chromatography (CC) over silicagel (chloroform-methanol and toluene-diethyl ether) to give isoinuviscolide (1,14.4 mg). Fraction A 5 was re­ chrom atographed over silicagel (hexane-acetone) to give gaillardin (2, 302.7 mg). Fraction A 8 was subm itted to CC over silicagel (toluene-diethyl ether) to give the sesquiterpene lactones lß-hydroxy-3ß-acetoxy-eudesm -4(15),ll(13)-dien-128ß-olide (3,13.6 mg) and pulchellin-E (4, 29.5 mg). Finally, fraction A 9 was rechrom atographed over silicagel (toluene-diethyl ether) to give pulchellinC (5, 10.7 mg). Acknowledgem ents This work was realized with the financial sup­ port of the following institutions: PICASSO (Acciones Integradas Hispano-Francesas, Refs. H-211 and 98-B), FIS (M inisterio de Sanidad y Consumo, Ref. 94/1671). CAM (Comunidad Autönoma de Madrid, Ref. C101/91), Universidad de Alcala (E001/99), and Consejerfa de Educaciön y Cultura de la Comunidad Autönom a de Madrid (Convocatoria 1998). 700 M. A. Gonzäles-Romero et al. ■Sesquiterpene Lactones from Inula montana Beekman A. C., Wierenga P. K., W oerdenbag H. J., Van Uden W., Pras N., Konings A. W. T., El-Feraly F., Calal A. M. and Wikstrom H. V. (1998), A rtemisinin-de­ rived sesquiterpene lactones as potential antitum or compounds: cytotoxic action against bone marow and tumour cell. Planta Med. 64. 615-619. Fischer N. H. (1991), Sesquiterpene lactones. In: Meth. Plant. Biochem. (Dey P. M. and H arborne J. B.. ed.). Academic Press, New York 7. 187-211. Ito K. and Lida T. (1981), Seven sesquiterpene lactones from Inula britannica var. chinensis. Phytochemistry 20,271-273. Kurma Rao S. and Mishra S. H. (1997), Screening of anti-inflammatory and hepatoprotective activities of alantolactone, isolated from the roots of Inula racemosa. Indian Drugs 34 (10), 571-575. Martin T., Villaescusa L., Gasquet M., Delmas F., B arto­ lome C.. Diaz-Lanza A. M., Ollivier E. and Balansard G. (1998), Screening for protozoocidal activity of Spanish plants. Pharm. Biol. 36, 56-62. Muzaffer A., Susan T., Joy S. and Kundu A. B. (1992), Antiinflammatory and antipyretic activity of vicolides of Vicoa indica. Indian J. Exp. Biol. 30. 38-41. Pieman A. K. (1984), Antifungal activity of sesquiter­ pene lactones. Biochem. System. Ecol. 12, 13-18. Pieman A. K. and Towers G. H. N. (1983). Antibacterial activity of sesquiterpene lactones. Biochem. System. Ecol. 11, 321-327. Sanz J. F., Ferrando C. and Marco J. A. (1991), Oxigenated nerolidol esters and eudesmane acids from Inula viscosa. Phytochemistry 30, 3653-3655. St. Pired J. (1977), Terpenes of Compositae plants. IV. Isolation of gaillardin from flowers of Inula britannica L. L. Rocz. Chem. 51, 1277-1279. Tutin T. G. (1976), Flora Europaea (Tutin T. G., ed.). Vol. 4, 135. Cambridge University Press, Cambridge. Warshaw E. M. and Zug A. K. (1996), Sesquiterpene lactone allergy. Am. J. Contact Derm. 7, 1-23. Yoshioka H., Mabry T. J., Dennis N. and Herz W. (1970), Structure and stereochemistry of pulchellin B, C, E and F. J. Org. Chem. 35 3, 627-630.