Two New Antiinflammatory Elemanolides from Centaurea Chilensis

.,...... Joumalof <.:.- ETHNO-- PHARMACOLOGY Journa1 of Ethnopharmacology 40 (1993) 149-153

Two new antiinflammatory elemanolides from Centaurea chilensis

Rosa E. Negretea, Nadine Backhousea, Ivan Cajigala, CarIa Delportea, Bruce K. Cassels*b, Eberhard Breitmaierc, Gert EckhardtC

apharmacognosy Laboratory, Faculty o/ Chemical and Pharmaceutical Sciences, hDepartment o/ Chemistry, Faculty o/ Sciences, University o/ Chile, Casilla 653, Santiago 1, Chile cOrganic Chemistry lnstitute, Bonn University, Bonn, Germany

(Received 19 March 1992; revision received 8 Ju1y 1993; accepted 8 Ju1y 1993)

Abstraet

Two previously undescribed elemanolide esters, the 2-methylpropanoate and 2-methyl-2-propenoate of 11,13• dehydromelitensin, were iso1ated in the course of a bioassay-guided fractionation from the aeria1 parts of Centaurea chilensis Hook, et Am" used traditionally to treat 'gout and rheumatism', The mixture ofboth substances exhibits anti• inflammatory activity in the carrageenan-induced paw edema assay.

Key words: Antiinflammatory; E1emanolides; Centaurea chilensis; Asteraceae

l. Introduction (Negrete et al., 1988b), and 11{3-H-II,13-dihydro• desacyl-cynaropicrin 8{3-D-glucoside (Negrete et The genus· Centaurea (Asteraceae, tribe al., 1988a). Cynareae) is a well-known source of sesquiterpene No elemanolides have been reported previously lactones. As part of a study of species growing in from C. chilensis Hook. et Am., but other Cen• Chile, we have now obtained two new elemanolide taurea species have afforded melitensin, dehydro• esters from C. chilensis Hook. et Am. Earlier melitensin, and several ester s of these sesquiter• chemical studies on this plant had afforded {3• pene lactones: melitensin was obtained originally sitosterol and stigmasterol (Negrete et al., 1984), from C. melitensis L. (González et al., 1971) and the flavonoids apigenin 7-0-glucoside, chrysoeriol later from C. aspera varo stenophyl/a (Dufour) 7-0-glucoside, chrysoeriol, luteolin, kaempferol, Nyman (Picher et al., 1984); its structure and quercetin, and hispidulin (Negrete et al., 1988a), as stereochemistry have been confirmed by X-ray well as the guaianolides dehydrocostus lactone analysis (Tortajada et al., 1988); this elemanolide and 8a-hydroxydehydrocostus lactone (Negrete has al so been synthesized via Cope rearrangement et al., 1984), 8a-acetoxydehydrocostus lactone of cnicin to 11,13-dehydro-melitensin (González et al., 1974a), which was first isolated from C. pul/ata • Corresponding author.

L. (González et al., 1974b); C. melitensis afforded ing, respectively 50, 300, and 400 g of residue after melitensin and 11, 13-dehydromelitensin ¡3-hyd• removing the solvents in vacuo. All 3 extracts were roxybutyrates (González et al., 1975); C. arbuti• administered to guinea pigs (see below) to monitor folia Svent. gave 11,13-dehydromelitensin iso• potential antiinflammatory activities. The chloro• valerate (isoarbutifolin) (González et al., 1981), C. form extract proved to be the most active in the phrygia L. yielded the 8a-(5-hydroxy)-angeloyl carageenan-induced paw edema assay, and was ester (Tsankova and Ognyanov, 1985) and C. therefore chromatographed on a silica gel G cineraria L. ssp. umbrosa (Lacaita) Pign. (syn. C. column, eluting with petroleum ether-CH2Cl2 ucriae Lacaita ssp. ucriae) gave the 2-methyl-3,4• mixtures of increasing polarity as shown in dihydroxybutyrate (Bruno and Herz, 1988). Table 1. C. chilensis has been used in folk medicine for 11,13-Dehydromelitensin 2-methylpropanoate 'gout and rheumatism', although the treatment (1) and 2-methyl-2-propenoate (I1) from fractions advocated in recent years only involves external B, C and D (see Table 1) were purified by further use (Muñoz et al., 1981). Several guaianolides are chromatography on silica gel G with petroleum known to possess anti-inflammatory activity, ether-CH2Cl2 (3:2) increasing the CH2CI2 propor• which has been related to the presence of the tion up to 100%. The major constituent obtained a-methylene-')'-Iactone moiety (Hall et al., 1979, in this way (300 mg) seemed to be homogeneous 1980), and this suggested a possible relationship on TLC (silica gel; light petrol-EtOAc 7:3; between the chemistry of this plant and its tradi• RF = 0.35) and had the appearance of a slightly tional use. In this paper we describe the bioassay• greenish lacquer, insoluble 'in petroleum ether, guided isolation, structure elucidation, and anti• somewhat soluble in CHCI3 and in EtOAc, very inflammatory activity of a mixture of two soluble in MeOH, giving a purple Lieber• previously undescribed elemanolide esters: 11,13• mann-Burchard reaction. In CHCI3, the mixture dehydromelitensin 2-methylpropanoate (1) and 2• apparently polymerizes, giving a white precipitate methyl-2-propenoate (I1), from C. chilensis. which cannot be redissolved. EIMS m/z data were: (relative abundance) 264.1366 (0.26%; calculated 2. Materials and methods ClsH2004, 264.1361), 246.1265 (7.65%; calculated ClsHIS03, 246.1256), 228.1149 (8.43%; calculated Aerial parts of Centaurea chilensis were col• C'SH'60b 228.1149). Complete lH_ and l3C_ lected in the flowering season (December) in Las NMR assignments are shown in Table 2. Tacas, La Serena (near sea level, 300S latitude), Anti-inflammatory activity was tested against Chile. A voucher specimen (L. Arancibia 16078) is carrageenan-induced edema in the guinea pig on deposit in the Botany Laboratory of the Facul• hindpaw. A-Carrageenan (1% in saline, 0.1 mI) was ty of Chemical and Pharmaceutical Sciences, Uni• injected into the left plantar aponeurosis of groups versity of Chile, Santiago (SQF). Melting points are uncorrected and were de ter• mined on a Kofler hot stage. IR spectra (KBr Table 1 discs) were recorded utilizing a Leitz IIIG infrared Fraetionation of the ehloroform extraet of Centaurea chilensis spectrometer. NMR spectra in CDCI3 with TMS as internal standard were recorded at 400 MHz Fraetion 25:75Solvent75:2550:50100%1000;',100'10petroleumCH2Cl2MeOHpetroleumeompositionether/CH2Cl2ethernone+Anti-+++++ B ('H) and 100.5 MHzCFED (l3C) on a Bruker WM-400 inf1ammatoryaetivity A spectrometer, using commercially available pulse programs for DEPT and 2D spectra ('H_1H and 'H-l3C COSY, COLOC). The HREIMS was recorded on a Kratos MS-30 instrument. The air-dried, ground plant material (4.0 kg), was extracted successively (at room temperature) with petroleum ether, CHCI3, and MeOH, yield- R.E. Negrete et al. / J. Ethnopharmacol. 40 (1993) /49-153 151

of 10-15 guinea pigs of both sexes (Pirbright) by the paw, immersed up to a mark made weighing 200-300 g, fasted for 24 h, with water ad previously just above the ankle, before injecting libitum, and kept at 18-20°C. The volume of the carrageenan, and 3 h later. Drugs or vehicle (pro• injected paw was determined with a plethysmo• pyleneglycol) were administered orally 1 h before meter by measuring the volume of water displaced injection. The percent inhibition of edema· was

Table 2 l3c and IH chemical shifts from the 100 MHz 13C and 400 MHz IH NMR spectra - assignments based on the 100/400 MHz FI• decoupled CH correlation

CHCCCH147.87113.28112.97145.59613C46.15171.40173.72119.5970.3780.03139.1351.8153.1342.9535.1966.6819.3119.1119.03 C Atoms CH3CH2CH2CHn147.85113.2546.05119.63167.6780.0070.96139.08137.43126.9551.7453.0342.9219.2618.41 61H 3JHH coupling 153a 17aIOb14a14b1218a13b13a11b19b16blOa17b18b19a49b7b6b8b8a3b29a7a5a5blb1a (ppm) b for compound 11. (ppm) (H) a11a6afor compound16al.

6.03; 5.58; 5.54 1.96; 6.14; 5.69 5.83 2.49; 5.00; 4.90; 1.71; 1.16 5.83 2.51; 5.00; 4.90; 1.68; 1.19 a2.49; 4.05; 3.94; 5.40 b 2.51; 4.05; 3.94; 5.40 2.90; 4.46; 6.03 3.05; 4.48; 6.03 6.14; 1.96 6./4; 5.69 1.96 6.03; 5.54 3.05 6.03; 5.58 2.90 5.40; 5.00 4.05; 3.94; 2.49 5.40; 5.00 4.05; 3.94; 2.51 a5.00; 4.90 b5.02; 5.02 4.46 2.90; 2.49 4.48 3.05; 2.51 5.27 3.05; 1.88; 1.68 5.22 2.90; 1.88; 1.71 4.05; 3.94 5.40; 5.00 2.90 5.22; 6.03; 5.58 3.05 4.48; 5.27; 6.03; 5.54 2.49 4.46; 1.16; 1.88; 5.00; 5.40 2.51 4.48; 1.19; 1.88; 5.00; 5.40 1.88; 1.71 1.16 1.88; 1.68 1.19 2.49; 5.00; 4.90; 1.16; 1.88; 1.71 2.51; 5.02; 5.02; 1.19; 1.88; 1.68 2.61 1.16 2.49; -1.71 1.19 2.51; 1.68 1.17 1.19 1.19 1.17 1.96 6.14; 5.69 152 R.E. Negrete et al. /1. Ethnopharmacol. 40 (1993) 149-153 calculated for each group with respect to its made on the basis of DEPT, homo- and hetero• vehicle-treated control group, and analyzed nuclear COSY, and COLOC spectra, and are sum• statistically using Student's t-test. marized in Table 2.

3. Results 4. Discussion and conclusion

A 20% infusion of the aerial parts of this plant, The fact that germacradienolides undergo facile similar to that recommended by folk healers, thermal isomerization to elemadienolides has led administered orally to guinea pigs, exhibited weak to the suggestion that some of these latter products (20%) but significant (P < 0.05) anti-inflamma• may be artefacts (Barrero et al., 1989). The drying tory activity at 4 ml!kg in a carrageenan-induced of the plant material, the extraction and all purifi• paw edema assay (Winter et al., 1962).The residue cation steps relating to compounds 1 and n, of the methanol extract of C. chilensis, dissolved in however, were carried out at room temperature propyleneglycol, showed similar activity which without adding any reagents which might pre• attained 29% at 400 mglkg and 49% at 600 mglkg sumably catalyze such a reaction. We therefore (P < 0.05). The residue of the chloroform extract conclude that live plants of C. chilensis probably was the most potent, giving a significant 35% in• contain these elemanolides, regardless of the hibition of edema at 200 mglkg, and 65% at 600 possibility that they may be produced in vivo, via mglkg. Bioassay-guided fractionation by column non-enzymatic Cope rearrangement of saloniten• chromatography of the chloroform extract (Table olide or some of its esters. To the best of our 1) led to the purification of an active fraction knowledge, compounds 1 and n have not been which appeared to be homogeneous on TLC and described in the literature. Furthermore, the which gave a significant 41% inhibition of edema previously unreported anti-inflammatory activity at 12 mg/kg, 60% at 33 mglkg, and 67% at 64 of elemanolides suggests that this may be a com• mg/kg (ED65= 18.6 mg/kg). In the same assay, mon property of a-methylene-'Y-lactones with ade• naproxen sodium salt, a commercially available quate absorption and distribution characteristics, antiinflammatory agent, gave 58% inhibition at 4.3 as shown previously for guaianolides (Hall et al., mglkg (ED50= 1.65 mg/kg). Consequently, the 1979,1980). Finally, the presence of orally active material from C. chilensis, although several times anti-inflammatory sesquiterpene lactones in the less potent than naproxen, is at least as effective as aerial parts of Centaurea chilensis (at concentra• this synthetic drug in reducing inflammation. tions greater than 0.0075%, w/w) may provide a The active fraction displayed an IR spectrum rationale for the traditional use of this plant in in• showing the presence of hydroxyl groups, C=C flammatory diseases. double bonds, 'Y-lactone and unsaturated ester functions. The HREIMS exhibited an apparent 5. Acknowledgements molecular ion at m/z 264.1366 (0.26%; calculated C15H2004,264.1361) indicative of a sesquiterpene This work was possible because of financial lactone. The lH-NMR spectrum could be assign• assistance from the University of Chile (DTI Q ed, for the most part, to an elemanolide structure, 2945 8923), International Foundation for Science but the signals due to the ester moieties (doublets (F/1494-1) and Deutsche Forschungsgemeinschaft at 6.14 and 5.69 ppm assigned to H-18 and -18' of (Germany). We are especiallY grateful to Mr the methacrylic acid; septuplet at 2.61 ppm Eduardo Johnson, ISP, Santiago, for providing attributable to the isopropyl methine of the isobu• the animal s and facilities for the biological tests. tyric acid) showed that the sample was an approx• 6. References imately 1:1 mixture of two substances, I and n. This was confirmed by the l3C_NMR spectrum Barrero, A.F., Sánchez, J.F. and Rodríguez, 1. (1989) Ger• which exhibited characteristic peaks for both macranolides from Centaurea melitensis. Phytochemistry 28, esters. The complete 1H and l3C assignments were 1975. R.E. Negrete et al./ J. Ethnopharmacol. 40 (1993) 149-153 153

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