Purely olefinic alkamnides in Heliopsis longipes and Acmella (Spilanthes) oppositifolia

BiochemicalSystematicsand Ecology, Vol. 24, No. 1, pp. 43-47, 1996 Copyright0 1996 ElsevierScience Ltd Printedin Great Britain.All rights reserved 0305-1978/96 $15.00+0.00

Pergamon 0305-1978(95)00099-2

Purely Olefinic Alkamides in Heliopsis longipes and Acme//a (Spilanthes) oppositifolia JORGE MOLINATORRES,” RAFAEL SALGADO-GARCIGLIA,’ ENRIQUE RAMIREZ-CHAVEZ* and ROSA E. DEL RlOt ‘Departamento de Biotecnologia y Bioquimica de Plantas, CINVESTAV-IPN U. IRAPUATO, Mexico; tlnstituto de lnvestigaciones Quimico Biolbgicas Universidad Michoacana de San Nicol& de Hidalgo, Morelia, Mich. Mexico

Key Word Index-Heliopsis longipes; Acmella (Spilanthes) oppositTo/ia; Compositae; lamides; medicinal plants; flavouring and insecticidal compounds; chemotaxonomy.

unsaturated

al ky-

AbstractAcmella (Spilanthes) oppositifolia and Heliopsis longipes are collected from the wild and used as medicinal, flavouring and insecticidal plants. These species contain purely olefinic alkamides (not containing acetylenic bonds) with affinin 1 as the main lipidic component and N-2-methylbutyldeca2E,GZ,8E_trienamide 2, in lower quantities. In A. opposittioka N-isobutyl-dodeca-2E,4E,8Z,l OE-tetraenamide 3 was detected with the above mentioned alkamides. For this species the same components can be found in roots and aerial parts. Heliopsis longipes alkamides are present only in roots. A comparison of the purely olefinic alkamide structures observed in the Heliantheae is discussed briefly.

Introduction Heliopsis longipes and A. oppositifolia are both used as spice, insecticide and medicinal plants. Heliopsis longipes is traded in Mexico under the Nahuatl names of

chilcuague and chilmecatl. Due to the presence of the insecticidal amide affinin 1 (Nisobutyl-deca-2f,GZ,8E_trienamide), a large supply of plant roots collected from the wild was necessary to satisfy international demand (Fisher, 1957). Nowadays, local demand for the flavouring and anaesthetic root is fulfilled by limited cultivation. The isomeric structure of affinin 1 was debated for a long time, but no detailed studies of other amide components in this species have been been performed. Another

NH

1

N-isobutyl-deca-2E,GZ,BE-trienamide

(Received 25 May 1995; accepted 11 August 1995)

43

J. MOLINA-TORRESETAL.

44

member of the same tribe, A. oppositifolia, is a pantropical species traditionally used in tropical America with similar applications as H. Iongipes. This species has been employed as a model to study root growth under in vitro conditions (Rores et al., 1993). Our interest was to study the presence of alkamides accounting for the use of these species as flavouring and insecticidal and medicinal plants.

Materials and Methods Sample specimens of Heliopsis Iongipes (Gray) Blake and Acmel/a (Spilanthes) oppositifolia Jansen were

collected from Xichu, SierraGorda, Gto. and Sierrade Alvarez,SLP. Mexico, respectively.Voucher specimens were deposited in the Instituto de Ecologia AC, P,~tzcuaro,Mich. Taxonomic characterizationwas kindly accomplishedby Dr J. Rzedowskiat the above mentioned institution. Heliopsis Iongipes and A. oppositifolia underground and aerialtissue were freeze-driedand macerated separately,using a mortar and pestle,then extractedovernight with ethyl acetate.Theseextractswere filtered, evaporated under reduced pressure,and then repeatedlypurified by TLC precoated Si gel plates (Sigma) developedwith n-hexane-ethylacetate. Purifiedfractions were analyzedby low resolution mass spectroscopy (Hewlett Packard Mod. 5972 MSD) coupled to capillary gas chromatography (Hewlett Packard GC Mod 5890, with a column HP Ultra-2; 25 m x 0.02 mm i.d.; 0.33 #m film thickness). Finally, compounds were isolatedby HPLC on a Waters RP-18 column eluted with acetonitrile-water40q30% as described by Baueret al. (1988). 1H and 13C NMR spectrawere recorded in CDCI3at 200 MHz in a Varian model Gemini 200. IR spectrawere obtained with a Perkin-Elmermodel 599P.

Results and Discussion The following amides were isolated and characterized. From/-L Iongipes: affinin (Nisobutyl-deca-2E,6Z,8E-trienamide 1) 7.3 mg/g dry underground tissue, and N - 2 methyl-butyl-deca-2E,6Z,8E-trienamide 2, 0.5 mg/g dry underground tissue. From A. oppositifolia: affinin, 0.351 mg/g in dry underground tissue and 0.172 mg/g dry green tissue; 2, 0.060 mg/g in dry underground tissue and 0.026 mg/g in dry green tissue; and N-isobutyl-dodeca-2E,4E,8Z, lOE-tetraenamide 3, 0.028 mg/g in dry underground tissue and 0.0055 mg/g in dry green tissue. Natural alkamides are chiefly distributed in the Compositae, characteristically in the Heliantheae and the Anthemideae (last reviewed by Greger, 1984). These amides have been isolated from roots and flower heads and, less frequently, from leaves. Even when purely olefinic alkamides are associated with acetylenic alkamides, the olefinic alkamides should be considered as a distinct class of natural products. Within the Heliantheae, from about 500 species in close to 100 genera studied chemically, over 250 acetylene and related compounds have been reported (for a review on the subject see Christensen and Lam, 1991a). However, pure olefinic alkamides in this tribe are found only in 13 species belonging to six genera in five subtribes (Table 1): Ecliptinae (Wedelia): Galinsoginae (Acmella); Helianthinae (Echinacea); Verbesininae

0

\

\--I

I

2

N 2-methylbutyr-deca2E,6Z,8E-trienamide

PURELY OLEFINIC ALKAMIDES IN H. LONGIPES

45

o

\

\

/

/

/

N- isobutyl-dodeca - 2E,4E,8Z,10E-tetraenamide

(Salmea); Zinnininae (Heliopsis and Sanvitalia). Even in these genera not all species examined yielded alkamides. The most abundant sources of purely olefinic alkamides are the Heliantheae (13 species containing 15 different structures in total) and the Anthemideae (33 species with 99 different structures). For a review on related structures in the Anthemideae see Christensen and Lam (1991b). All purely olefinic amide structures reported in the Heliantheae to date contain either a C10 or C12 olefinic chain, with two notable exceptions: one in Acmella (Spilanthes) ciliata with two octadien (isobutyl and phenylethyl) amides (Martin and Becker, 1985); and another in Sanvitalia ocymoides with two tetradeca (tetraen and pentaen) isobutylamides (Dominguez et al., 1987). All have an even number of carbons (seeTable 1). In contrast, the acetylenic amides in the same tribe, contain a wider range of the olefinic chains from C9 to C18, including both even and odd number of carbons (Christensen and Lam, 1991a). Heliantheae purely olefinic alkamides are characteristic to this tribe. Two genera, Acmella and Echinacea, with four and five species respectively, contain most of the species in which purely olefinic alkamides have been detected in this family. The alkamide pattern in Echinacea is very conserved. In the four species, the fatty acid chain is C12 and the amide moiety present is isobutylamide. There are variations only in the presence of double bonds and isomerism in positions 10. The more unsaturated homologues: 2E,4E,8Z and either isomer (E and Z) in position 10 are present in the four species. However, for E. purpurea and E. simulata the less unsaturated homoIogues have not been reported (Table 1). For Acmella, the olefinic alkamide chemistry is more heterogeneous. The chain length includes C8, C10 and C12, with a wider range of double bonds; the amide moiety includes isobutyl, 2-methyl butyl and phenyl-ethyl. The latter amide moiety is more frequently found in acetylenic alkylamides. The most widely distributed olefinic alkamides in the Heliantheae are: N-isobutyldodeca-2E,4E, BZ,lOE-tetraenamide in nine species, followed by affinin 1 in six species, N-2-methylbutyl-deca-2E,6Z,8E-trienamide and N-isobutyl-dodeca2E,4E,8Z,lOE-tetraenamide in five species each. Affinin 1 and 2 are often found together, where the former is frequently the main lipidic component. This is true for Acmella ciliata (additionally to other alkamides), forA. oleracea and now conclusively for A. oppositifolia, and H. Iongipes, but has not been reported conclusively for Wedelia parivceps. Most of the deca-alkamides described in the studied species have the three double

J. MOLINA-TORRES ETAL.

46 TABLE 1 PURELY OLEFINIC ALKAMIDES IN THE HELIANTHEAE Heliantheae

Chain

Chain bonds

Amide

Reference

C10

2E,6Z.8e

Isobutyl

JoneseZal (1982)

C12 C08 C08 C10 C10 C10 C10 C10 C12 C12 C12 C12 C10 C10 C10 C10 C12

2E,4E.BZ.IOE 2E,4Z 2Z.4E 3E.6Z,8E 2E, 6Z,8E 6Z.8E 2E,6Z,8E 2E,6Z,8E 2E,4E,8Z,10E 2E,4Z,8Z,IOE 2E,4E,8Z,IOE 2E,4E,8E,10Z 2E,6Z,8E 2E,6Z,8E 2E,6Z,8E 2E,6Z,8E 2E,4E,8Z,IOE

Isobutyl Isobutyl 2 Phenylethyl 2 Phenylethyl 2-Phenylethyl Isobutyl Isobutyl 2me Butyl Isobutyl Isobutyl 2me-Butyl Isobutyl Isobutyl 2me- Butyl Isobutyl 2me- Butyl Isobutyl

BohlmannetaL (1980) Martin and Becker (1985 Martin and Becker (1985 Martin and Becker (1985 Martin and Becker (1984 Martin and Becker (1984 Martin and Becker (1984 Martin and Becker (1984 Martin and Becker (1985 Martin and Becker (1985 Martin and Becker (1985 Jendiko (1986) Greger et al. (1985) Gregeretal. (1985) CalleetaL (1988) This paper This paper

C12 C12 C12 C12 C12 C12 C12 C12 C12 C12 C12 C12

2E,4E,8Z,IOE 2E,4E,8Z,IOZ 2E,4E 2E.4E,8Z,IOE 2E,4E,8Z.IOZ 2•4E 2E.4E,8Z,IOE 2E,4E,8Z.10Z 2£,4E,8Z 2E,4E 2E,4E,8Z,IOE 2F,4E,8Z,IOZ

Isobutyl Isobutyl Isobutyl Isobutyl Isobutyl Isobutyl Isobutyl Isobutyl Isobutyl Isobutyl Isobutyl IsobutyI

Bauer et aL (1989) Bauer et aL (1989) BaueretaL (1989) Bauer and Reminger (1989) Bauer and Reminger (1989) Vauer and Reminger (1989) Bauer and Reminger (1989) Bauer and Reminger (1989) Bauer and Rerninger (1989) Bauer and Reminger (1989) Bauer and Foster (1991) Bauer and Foster (1991)

C12 C12

2~.4E,8Z,10E 2E.4E,8Z,10Z

Isobutyl Isobutyl

Bohlmanneta/. (1985) Herz and Kulanthaivel (1985)

C10 C10

2E,6Z,8E 2E,6Z,8E

Isobutyl 2me Butyl

CrombieetaL (1963) This paper

C14 C14

2E,4E,8Z,IOE 2E,4E,8Z

Isobutyl Isobutyl

Dominguez et aL (1987) Dominguez et aZ (1987)

Eeliptinae Less Wedelia parviceps Blake Galinsoginae B. and H Acmella (Spilanthes) a/ba ciliata H.B.K (=Acme/la ciliata Cass)

rnaufitiana o/efacea L oppositifo/ia (americana R&B) (Lamark) Jansen

Helianthinae Drumordt Echinacea angustifoha D C

pa/lida (Nutt.) Nutt

purpurea (L) Monech

simulata Verbesininae B. and H Sa/mea scandens (L) D.C Zinniinae B. and H Hefiopsis Iongipes (Gray) Blake Sanvitalia ocymoides D. C

bonds found in affinin I (2E,6Z,8E), while the dodecamides have four double bonds (2E,4E, most 8Z, but also 8Eand either 10 Eor Z). One ester homologue to the deca2E,6Z,8E-trienoic acid amides, the bornyl ester, has been reported present in /-/ Iongipes roots (Molina-Torres et al, in press). Considering published results, just one atkamide structure from the Heliantheae is also found in the Anthemideae, the dodeca 2E,4E-isobutylamide in Leucocyclus formosus Boiss: ssp. formosus (Greger et al., 1981). In relation with other families, there are only two purely olefinic alkamide structures that the Compositae have in common with any of them. These are: the dodeca 2E,4E,8Z, IOZ and the dodeca

PURELY OLEFINICALKAMIDES IN t4. LONG/PES

47

2E,4E,8Z,IOE isobutylamides, which also occur in the sole species of the ArisIolochiacea family containing alkamides: Asiasarum heterotropoides Maek. var. mandshuricum Maek. (Yasuda et al., 1981). On the other end, the dienoic-2E,4E unsaturation, widely distributed in olefinic amides with variable chain length in other tribes (e.g. 20 structures in the Anthemideae) is present in Heliantheae solely in one structure, namely in Echinacea (seeTable 1). Acknowledgement--Thiswork was supported by Consejo Nacional de Ciencia y Tecnologia, Mexico (Grant 3118N).

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