Biochemical Systematics and Ecology 29 (2001) 143}147

Non-toxic pyrrolizidine alkaloids from Eupatorium semialatumଝ GuK nter Lang!, Claus M. Passreiter! *, Beatriz Medinilla", Juan-JoseH Castillo#, Ludger Witte$ !Institut fu( r Pharmazeutische Biologie, Heinrich-Heine-Universita( tDu( sseldorf, Universita( tsstrasse 1, Geb. 26.23, D-40225 Du( sseldorf, Germany "Facultad Ciencias Quimicas y Farmacia, Universidad de San Carlos, Guatemala Ciudad, Guatemala #Facultad de Agronomia, Universidad de San Carlos, Guatemala Ciudad, Guatemala $Institut fu( r Pharmazeutische Biologie, Technische Universita( t Braunschweig, D-38106 Braunschweig, Germany Received 4 January 2000; accepted 22 March 2000

Abstract

The leaves of Eupatorium semialatum were investigated for the occurrence of pyrrolizidine alkaloids. Although this type of alkaloids generally occurs in the Eupatorieae, only unusual non-toxic pyrrolizidines of the tussilagin type were identi"ed. All compounds are methyl esters of the corresponding b-amino acids. ( 2001 Elsevier Science Ltd. All rights reserved.

Keywords: Eupatorium semialatum; Eupatorieae; Pyrrolidineacetic acid methyl ester; Tussilagin; Isotus- silagin; Neo-tussilagin; Neo-isotussilagin

1. Introduction

Eupatorium semialatum Benth. is a medicinal from the Alta Verapaz/ Guatemala (Nash and Williams, 1976). It is mainly used against malaria, but other uses are also recorded in the literature (Clewell, 1975; Medinilla, 1978; Morton, 1981; CaH ceres, 1996). Besides the medicinal interest, E. semialatum is interesting from a taxonomic point of view. Nash and Williams (1976) describe this species as quite

ଝPart of a current dissertation of G. Lang, Heinrich-Heine-UniversitaK t, DuK sseldorf. * Corresponding author: Tel.: #49-211-8114172; fax: #49-211-8111923. E-mail address: [email protected] (C.M. Passreiter).

0305-1978/01/$- see front matter ( 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 5 - 1 9 7 8 ( 0 0 ) 0 0 0 3 7 - 5 144 G. Lang et al. / Biochemical Systematics and Ecology 29 (2001) 143}147 di!erent from the South-Mexican E. ligustrinum, but King and Robinson (1987) treated E. semialatum and E. ligustrinum as one. Moreover, as a result of their studies, E. ligustrinum and subsequently E. semialatum must be a species of Ageratina and should therefore be named A. ligustrina (A.P. Decandolle, R.M. King and H. Robinson). Recently, we reported the isolation and structure elucidation of sesquiterpene lactones of the eudesmanolide type from E. semialatum (Lang et al., 2000), which were quite di!erent from those found in A. ligustrina and formerly in E. ligustrinum (Romo et al., 1968; Tamayo-Castillo et al., 1988). Since pyrrolizidines, a common feature of the Eupatorieae (Culvenor, 1978; Rizk, 1991), can also be used as chemotaxonomic markers, we now report on this type of secondary metabolites. The occurrence of such compounds, especially D-1,2-unsaturated derivatives, is additionally important as such unsaturated pyrrolizidines are responsible for numerous acute and chronic toxicities of medicinal of the and Boraginaceae (Mattocks, 1986).

2. Materials and methods

2.1. Plant material

Leaves of Eupatorium semialatum Benth. were collected in the Alta Verapaz, Guatemala. A voucher (JC2541) is on deposit in the herbarium AGUAT, Herbario Ernesto Carrillo, Universidad de San Carlos, Apartodo Postal 1545, Zona 12, Guatemala Ciudad, Guatemala.

2.2. Extraction and purixcation

The dried, powdered plant material (725 g) was exhaustively extracted with dichloromethane and then with methanol in a Soxhlet apparatus. 94 g of the methanolic extract was puri"ed as previously described (Passreiter, 1992; Witte et al., 1993). The obtained alkaloid extract (8 mg) was analyzed by GLC-MS and TLC. TLC. Silica gel 60 F,CHCl}MeOH}NH (conc.) 85 : 14 : 1. Detection: Re- agent of Dann (1960) and Mattocks (1967). (Rf: see Passreiter, 1992; SchuK ngel and Passreiter, 2000) GLC-MS. The GLC-MS system consisted of a Carlo Erba 5160 GC equipped with a30m;0.32 mm fused silica capillary column coated with the methyl silicone stationary phase DB-1 (J&W Scienti"c, California). Helium was used as carrier gas. Conditions: injector 2503C, split 1 : 20; temperature program 100}3003C, 63C/min. The capillary column was directly coupled to the quadrupole mass spectrometer Finnigan MAT 4515. EI-spectra were recorded at 40 eV in combination with the Incos data system. tussilagin (1b)RI"1407; MS m/z : 199 [M]> (7), 168(5), 156(7), 124(6), 83(100), 70(3), 55(22) isotussilagin (2b)RI"1379; MS m/z : 199 [M]> (8), 168(4), 156(7), 124(5), 83(100), 70(3), 55(22) G. Lang et al. / Biochemical Systematics and Ecology 29 (2001) 143}147 145

neo-tussilagin (3b)RI"1388; MS m/z : 199 [M]> (7), 168(6), 156(8), 124(5), 83(100), 70(6), 55(21) neo-isotussilagin (4b)RI"1417; MS m/z : 199 [M]> (5), 168(5), 156(9), 124(6), 83(100), 70(3), 55(22) pyrrolidine-2-acetic acid methyl ester (5b)RI"1098; MS m/z : 143 [M]> (1.5), 128(1.6), 115(2.5), 110(4), 70(100), 56(14), 43(14).

3. Results and discussion

The alkaloid phase obtained after puri"cation of the methanolic extract from E. semialatum in the usual way (Passreiter, 1992; Witte, 1993) was analyzed by TLC and GC/MS. From this phase tussilagin (1b), isotussilagin (2b), neo-tussilagin (3b) and neo-isotussilagin (4b), and pyrrolidine-2-acetic acid methyl ester (5b) were identi"ed by their mass spectra at their respective retention times in comparison with authentic compounds. Additionally, all compounds gave violet spots in TLC after detection with the reagent of Dann and Mattocks (Dann, 1960; Mattocks, 1967). All methyl ester derivatives (1b}5b) were again found to be artifacts formed during Soxhlet extraction with methanol from the corresponding acids (1a}5a), as previously de- scribed (Passreiter, 1992, 1998; SchuK ngel and Passreiter, 2000). 146 G. Lang et al. / Biochemical Systematics and Ecology 29 (2001) 143}147

In addition to the violet spots for the non-toxic pyrrolizidines, we found two blue spots, which normally indicate the presence of toxic D-1,2-unsaturated derivatives (Mattocks, 1986). Surprisingly, these compounds were identi"ed as trans-terpin (6) and trans-sobrerol (7) (Barnes, 1958; Bohlmann and Zeisberg, 1979). This means that not only toxic pyrrolizidines give blue reaction products with the reagent of Dann and Mattocks, but also these monoterpenes. There was no further evidence for any other toxic pyrrolizidine by TLC or GLC/MS. The derivatives of tussilagin di!er from all other pyrrolizidines by the presence of a C-2 methyl group and a carboxyl group at C-1 (Mattocks, 1986; Passreiter, 1992). Moreover, the underlying acids have rather to be seen as b-amino acids than as alkaloids. All 13 plants of the Asteraceae in which the acid derivatives 1b and 2b were found were members of the tribes and mostly Heliantheae (Roeder et al., 1981, 1984, 1993; Passreiter et al., 1992; Sener and Etgun, 1996; Wernery et al., 1997; Passreiter, 1998; SchuK ngel and Passreiter, 2000). From Arnica species, Tussilago farfara, Neurolaena lobata and Melampodium divaricatum both b-amino acids were reported together with their possible biosynthetic precursor pyrrolidine-2-acetic acid (5a), its methylester (5b) as well as their C-1 epimers neo-tussilagin (3b) and neo- isotussilagin (4b) (Passreiter, 1992, 1998; SchuK ngel and Passreiter, 2000). E. semialatum is the "rst species of the Eupatorieae in which derivatives of tussilagin were found. The previously reported occurrence of these compounds in the Senecioneae and the compounds "rst found in the Eupatorieae suggest that these b-amino acids are probably more widely distributed in the Asteraceae than previously expected. Further investigations on the occurrence of these alkaloids in other plants are in progress.

References

Barnes, C.S., 1958. The properties and structures of the 1,8-terpins and some related compounds. Aust. J. Chem. 11, 134}146. Bohlmann, F., Zeisberg, R., 1979. C-NMR-Spektren von Monoterpenen. Org. Magn. Reson. 7, 426}432. CaH ceres, A., 1996. In: GiroH n, L.M., CaH ceres, A. (Eds.), Plantas de uso medicinal en Guatemala. Editorial Universitaria, Guatemala, pp. 89}90. Clewell, A.F., 1975. Las compuestas de Honduras. Ceiba 19, 119}244. Culvenor, J.J.C., 1978. Pyrrolizidine alkaloids } Occurrence and systematic importance in angiosperms. Bot. Not. 131, 473}486. Dann, A.T., 1960. Detection of N-oxides of the pyrrolizidine alkaloids. Nature 186, 1051. King, R.M., Robinson, H., 1987. The Genera of the Eupatorieae (Asteraceae). Monographs in Systematic Botany, Vol. 22, Missouri Botanical Garden. Allen Press, Lawrence, pp. 1}581. Lang, G., Passreiter, C.M., Medinilla, E., Castillo, J.-J., 2000. Eudesmanolides and further terpenes from the leaves of Eupatorium semialatum. Z. Naturforsch., in press. Mattocks, A.R., 1967. Spectrophotometric determination of unsaturated pyrrolizidine alkaloids. Anal. Chem. 39, 443}447. Mattocks, A.R., 1986. Chemistry and Toxicology of Pyrrolizidine Alkaloids. Academic Press, London. Medinilla, J.C., 1978. Aspectos de la Medicina Popular en el Area Rural de Guatemala. Guatemala Indigena 13, 13 (appendix). Morton, J.F., 1981. In: Atlas of Medicinal Plants of Middle America: Bahamas to Yucatan. Charles C. Thomas, Spring"eld, p. 933. G. Lang et al. / Biochemical Systematics and Ecology 29 (2001) 143}147 147

Nash, D.L., Williams, L.O., 1976. Flora of Guatemala. Fieldiana: Botany, Vol. 24, Part XII. Field Museum of Natural History, Chicago, p. 96. Passreiter, C.M., 1992. Co-occurence of 2-Pyrrolidineacetic acid with the pyrrolizidines tussilaginic acid and isotussilaginic acid and their 1-epimers in Arnica species and Tussilago farfara. Phytochemistry 31, 4135}4137. Passreiter, C.M., 1998. Pyrrolizidine alkaloids from Neurolaena lobata. Biochem. Syst. Ecol. 26, 839}843. Passreiter, C.M., Willuhn, G., Roeder, E., 1992. Tussilagine and isotussilagine: two pyrrolizidine alkaloids in the Genus Arnica. Planta Med. 58, 556}557. Rizk, A.-F.M., 1991. Naturally Occurring Pyrrolizidine Alkaloids. CRC Press, Boca Raton, FL. Roeder, E., Eckert, A., Bourauel, T., 1993. Pyrrolizidine Alkaloids from spurius. Pharmazie 48, 953}954. Roeder, E., Wiedenfeld, H., Hille, T., Britz-Kirstgen, R., 1984. Pyrrolizidine in Echinacea angustifolia DC. und Echinacea purpurea M. Dtsch. Apoth. Ztg. 45, 2316}2318. Roeder, E., Wiedenfeld, H., Jost, E.J., 1981. Tussilagin } ein neues Pyrrolizidin-Alkaloid aus Tussilago farfara. Planta Med. 43, 99}102. Romo, J., Rios, T., Quijano, L., 1968. Ligustrin, a guaianolid isolated from E. ligustrinum DC. Tetrahedron 24, 6087}6091. SchuK ngel, J., Passreiter, C.M., 2000. Pyrrolizidine alkaloids from Melampodium divaricatum. Biochem Syst. Ecol., 28, 705}706. Sener, B., Etgun, F., 1996. Pyrrolizidine Alkaloids from Tussilago farfara L. J. Fac. Pharm. Gazi Univ. 13, 171}173. Tamayo-Castillo, G., Jakupovic, J., Bohlmann, F., Rojas, A., Castro, V., King, R.M., 1988. Germacranolides and other constituents from Ageratina species. Phytochemistry 27, 2893}2897. Wernery, U., Bourauel, T., Roeder, E., Meier, U., Wiedenfeld, H., 1997. Toxic Diterpene Glycosides from Iphiona aucheri. Fitoterapia 68, 278}280. Witte, L., Rubiolo, P., Bicchi, C., Hartmann, T., 1993. Comparative analysis of pyrrolizidine alkaloids from natural sources by Gas}Chromatography}Mass Spectrometry. Phytochemistry 32, 187}196.