Non-toxic pyrrolizidine alkaloids
from
Eupatorium semialatum
qGu
K
nter Lang
!
, Claus M. Passreiter
!
,
*
, Beatriz Medinilla
"
,
Juan-Jose
H
Castillo#, Ludger Witte$
!Institut fu(r Pharmazeutische Biologie, Heinrich-Heine-Universita(t Du(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 ofEupatorium semialatumwere 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 correspondingb-amino acids. ( 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Eupatorium semialatum; Eupatorieae; Pyrrolidineacetic acid methyl ester; Tussilagin; Isotus-silagin; Neo-tusIsotus-silagin; Neo-isotussilagin
1. Introduction
Eupatorium semialatum Benth. is a medicinal plant 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; CaHceres, 1996). Besides the medicinal interest, E. semialatum is interesting from a taxonomic point of view. Nash and Williams (1976) describe this species as quite
q
Part of a current dissertation of G. Lang, Heinrich-Heine-UniversitaKt, DuKsseldorf.
*Corresponding author: Tel.:#49-211-8114172; fax:#49-211-8111923.
E-mail address:passreit@uni-duesseldorf.de (C.M. Passreiter).
di!erent from the South-Mexican E. ligustrinum, but King and Robinson (1987) treatedE. semialatumandE. ligustrinumas 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 fromE. semialatum(Lang et al., 2000), which were quite di!erent from those found inA. ligustrinaand formerly inE. 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 plants of the Asteraceae 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
254, CH2Cl2}MeOH}NH3 (conc.) 85 : 14 : 1. Detection:
Re-agent of Dann (1960) and Mattocks (1967). (Rf: see Passreiter, 1992; SchuKngel and Passreiter, 2000)
GLC-MS. The GLC-MS system consisted of a Carlo Erba 5160 GC equipped with a 30 m]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; MSm/z: 199 [M]`(8), 168(4), 156(7), 124(5), 83(100),
neo-tussilagin (3b) RI"1388; MSm/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 fromE.
semialatumin the usual way (Passreiter, 1992; Witte, 1993) was analyzed by TLC and
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)
andtrans-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 derivatives1band2bwere found were members of the tribes Senecioneae and mostly Heliantheae (Roeder et al., 1981, 1984, 1993; Passreiter et al., 1992; Sener and Etgun, 1996; Wernery et al., 1997; Passreiter, 1998; SchuKngel 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; SchuKngel and Passreiter, 2000).
E. semialatumis 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., 1979.13C-NMR-Spektren von Monoterpenen. Org. Magn. Reson. 7, 426}432. CaHceres, A., 1996. In: GiroHn, L.M., CaHceres, 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 ofEupatorium 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).
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 inArnicaspecies andTussilago farfara. Phytochemistry 31, 4135}4137.
Passreiter, C.M., 1998. Pyrrolizidine alkaloids fromNeurolaena lobata. Biochem. Syst. Ecol. 26, 839}843. Passreiter, C.M., Willuhn, G., Roeder, E., 1992. Tussilagine and isotussilagine: two pyrrolizidine alkaloids
in the GenusArnica. 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 fromPetasites spurius. Pharmazie 48,
953}954.
Roeder, E., Wiedenfeld, H., Hille, T., Britz-Kirstgen, R., 1984. Pyrrolizidine inEchinacea angustifoliaDC. undEchinacea purpureaM. Dtsch. Apoth. Ztg. 45, 2316}2318.
Roeder, E., Wiedenfeld, H., Jost, E.J., 1981. Tussilagin}ein neues Pyrrolizidin-Alkaloid ausTussilago farfara. Planta Med. 43, 99}102.
Romo, J., Rios, T., Quijano, L., 1968. Ligustrin, a guaianolid isolated fromE. ligustrinumDC. Tetrahedron 24, 6087}6091.
SchuKngel, J., Passreiter, C.M., 2000. Pyrrolizidine alkaloids fromMelampodium divaricatum. Biochem Syst. Ecol., 28, 705}706.
Sener, B., Etgun, F., 1996. Pyrrolizidine Alkaloids fromTussilago 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 fromAgeratinaspecies. 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.
