Biochemical Systematics and Ecology 29 (2001) 219}222 Gynocardin and cyclopentenylglycine in Rawsonia lucidaq Lise Andersen!, Vicki Clausen!, Hellen A. Oketch-Rabah", Matthias Lechtenberg#, Anne Adsersen!, Adolf Nahrstedt#, Jerzy W. Jaroszewski!,* !Department of Medicinal Chemistry, Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen, Denmark "Department of Pharmacology and Pharmacognosy, University of Nairobi, College of Health Sciences, P.O. Box 19676, Nairobi, Kenya #Institut fu( r Pharmazeutische Biologie und Phytochemie, Westfa( lische Wilhelms-Universita( t Mu( nster, Hittorfstrasse 56 D-48149 Mu( nster, Germany Received 21 January 2000; accepted 22 March 2000 Keywords: Rawsonia lucida; Flacourtiaceae; Cyanohydrin glucoside; Gynocardin; Cyclopentenylglycine; Chemotaxonomy; Cyanogenesis 1. Subject and source Rawsonia lucida Harw. & Sond. is the type species of Rawsonia, a small genus of Flacourtiaceae represented in tropical Africa. Leaves of the plant were collected in April 1997 in Kakamega Forest in Western Kenya. Voucher specimen (DFHJJ1) was deposited in Herbarium C (Botanical Museum, University of Copenhagen, Copenhagen). q Part 18 in the series `Natural Cyclopentanoid Cyanohydrin Glycosidesa. For Part 17 see Andersen et al., 1998. * Corresponding author. Tel.: #45-3530-6372; fax:#45-3530-6040. E-mail address: jj@dfh.dk (J.W. Jaroszewski). 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 4 8 - X 220 L. Andersen et al. / Biochemical Systematics and Ecology 29 (2001) 219}222 2. Previous work R. lucida was reported as strongly cyanogenic in an extensive survey of Flacourtiaceae by Spencer and Seigler (1985), and later by Burrows et al. (1992). However, the exact nature of the cyanogenic glycoside present was not elucidated. Apart from these studies, no phytochemical investigations of the genus Rawsonia have yet been reported. 3. Present study Dried leaves of R. lucida liberated 5.6 l mol HCN/g, as determined according to Nahrstedt (1977). The material (300 g) was extracted with boiling MeOH and fractionated on silica gel using a step gradient of MeOH in AcOEt, the fractions being monitored for cyanogenesis using picrate sandwich assay (Brimer et al., 1983). Final puri"cation of the cyanogenic fractions was achieved by MPLC (Orpegen HD Sil RP-18, 4% aqueous MeOH), to give a total of 0.22 g of gynocardin (1). The identity was con"rmed by comparison of 300 MHz 1H and 75 MHz 13C NMR spectra of the isolated glycoside and its hexaacetate (prepared by overnight treatment with Ac O/ 2 pyridine) with those of authentic (1) and its hexaacetate (Jaroszewski and Olafsdottir, 1987) and with literature data (HuK bel et al., 1981; Jensen and Nielsen, 1986). In a parallel experiment, 30 g of the plant material was extracted twice, each time by overnight stirring with 300 ml H O. The combined extracts were freeze-dried and the 2 residue subjected to ion exchange on Dowex-50 W (H`), from which the total amino acids were eluted with 2 M aqueous ammonia. The amino acid fraction was chromatographed on silica gel using t-BuOH/2-butanone/acetone/MeOH/H O/ 2 conc. NH 40 : 20 : 20 : 1 : 14 : 15, using synthetic cyclopentenylglycine (Olafsdottir 3 et al., 1992; Dennis et al., 1955) as a reference. Appropriate fractions were pooled, freeze-dried, dissolved in D O, and subjected to 1H NMR analysis (400 MHz) at pH 2 6.7. The 1H NMR spectrum of the isolate showed characteristic resonances of the ole"nic protons (d 5.66 and 6.03) of (2S,1@R)-2-(2@-cyclopentenyl)glycine (2) (cf. Katagiri et al., 1997; Cramer et al., 1980), further identi"ed by spiking with synthetic cyclopentenylglycine (mixture of both diastereoisomers). The amount of (2) isolated was approx. 0.2 mg, as estimated from the 1H NMR spectra by the standard addition method. L. Andersen et al. / Biochemical Systematics and Ecology 29 (2001) 219}222 221 4. Chemotaxonomic signi5cance In addition to Rawsonia lucida investigated in this work, the veri"ed examples of the occurrence of gynocardin in Flacourtiaceae include Gynocardia odorata (Kim et al., 1970), Pangium edule (De Jong, 1909), Carpotroche brasiliensis (Spencer et al., 1982) and Kiggelaria africana (Jaroszewski and Olafsdottir, 1987). Moreover, a cyclopentanoid amide with the same oxygenation pattern as gynocardin was isolated from Lindackeria dentata (Gibbons et al., 1998). Cyclopentenylglycine, the possible biosynthetic precursor of gynocardin (Olafsdottir et al., 1992), has so far been isolated only from Hydnocarpus anthelmintica and Caloncoba echinata (Cramer et al., 1980). The Flacourtiaceae consists of two distinct groups (cf. Bernhard and Endress, 1999), a notoriously cyanogenic group consisting of Oncobeae s. l. and Pangieae, and the non-cyanogenic group including mainly Casearieae, Flacourtieae, Homalieae and Scolopieae. Paropsieae, which appears to be only sporadically cyanogenic (Spencer and Seigler, 1985), links to the Passi#oraceae. The previously identi"ed sources of gynocardin all belong to Oncobeae}Pangieae. Rawsonia has been either placed together with Streptothamnus and Berberidopsis in Berberidopsideae (Hutchinson, 1967), included in Oncobeae (Gilg, 1925), or placed in Erythrospermeae (Lemke, 1988). While Streptothamnus}Berberidopsis clearly constitutes a separate group (Lemke, 1988; Bernhard and Endress, 1999), also from the point of view of the cyanogenic constituents that are present (Jaroszewski et al., 1988), R. lucida is the "rst member of the Erythrospermeae sensu Lemke in which a cyanogenic constituent has been identi"ed. The presence of gynocardin suggests a close relationship of Rawsonia with Pangieae and Oncobeae s. str. References Andersen, L., Adsersen, A., Jaroszewski, J.W., 1998. Phytochemistry 47, 1049. Bernhard, A., Endress, P.K., 1999. Pl. Syst. Evol. 215, 141. Brimer, L., Christensen, S.B., M+lgaard, P., Nartey, F., 1983. J. Agric. Food Chem. 31, 789. Burrows, G.E., Drumond, R.B., Basudde, C.D.K., 1992. In: James, L.F., Keeler, R.F., Bailey Jr., E.M., Cheeke, P.R., Hegarty, M.P. (Eds.), Poisonous Plants. Iowa State University Press, Iowa, p. 561. Cramer, U., Rehfeldt, A.G., Spener, F., 1980. Biochemistry 19, 3074. De Jong, A.W.K., 1909. Rec. Trav. Chim. Pays-Bas 28, 24. Dennis, R.L., Plant, W.J., Skinner, C.G., Sutherland, G.L., Shive, W., 1955. J. Am. Chem. Soc. 77, 2362. Gibbons, S., Gray, A.I., Waterman, P.G., 1998. Phytochemistry 49, 2395. Gilg, E., 1925. In: Engler, A., Prantl, K. (Eds.), Die NatuK rlichen P#anzenfamilien, Vol. 21. W. Engelmann, Leipzig, pp. 377. Hutchinson, J., 1967. The Genera of Flowering Plants, Vol. 2, Clarendon Press, Oxford, p. 201. HuK bel, W., Nahrstedt, A., Wray, V., 1981. Arch. Pharm. 314, 609. Jaroszewski, J.W., Olafsdottir, E.S., 1987. Phytochemistry 26, 3348. Jaroszewski, J.W., Jensen, P.S., Cornett, C., Byberg, J.R., 1988. Biochem. Syst. Ecol. 16, 23. Jensen, S.R., Nielsen, J., 1986. Phytochemistry 25, 2349. Katagiri, N., Okada, M., Morishita, Y., Kaneko, C., 1997. Tetrahedron 53, 5725. 222 L. Andersen et al. / Biochemical Systematics and Ecology 29 (2001) 219}222 Kim, H.S., Je!rey, G.A., Panke, D., Clapp, R.C., Coburn, R.A., Long, Jr. L., 1970. Chem. Commun., 381. Lemke, D., 1988. Aliso 12, 29. Nahrstedt, A., 1977. Dtsch. Apoth. Ztg. 117, 1357. Olafsdottir, E.S., J+rgensen, L.B., Jaroszewski, J.W., 1992. Phytochemistry 31, 4129. Spencer, K.C., Seigler, D.S., 1985. Biochem. Syst. Ecol. 13, 421. Spencer, K., Seigler, D.S., Fikenscher, L.H., Hegnauer, R., 1982. Planta Med. 44, 28.