PHYTOCHEMISTRY Phytochemistry 66 (2005) 469–480 www.elsevier.com/locate/phytochem Calystegines as chemotaxonomic markers in the Convolvulaceae q Thomas Schimming a, Kristina Jenett-Siems a, Petra Mann a, Britta Tofern-Reblin a, Jenny Milson b, Robert W. Johnson c, Thierry Deroin d, Daniel F. Austin e, Eckart Eich a,* a Institut fu¨r Pharmazie (Pharmazeutische Biologie), Freie Universita¨t Berlin, Ko¨nigin-Luise-Straße 2-4, D-14195 Berlin, Germany b The State of Queensland, Department of Primary Industries, Longreach, Qld., Australia c Queensland Herbarium, Brisbane Botanic Gardens Mt. Coot-tha, Toowong, Qld., Australia d Laboratoire de Phane´rogamie, Muse´um National dÕHistoire Naturelle, 16, rue Buffon, F-75005 Paris, France e Conservation & Science Department, Arizona-Sonora Desert Museum, 2001 N. Kinney Road, Tucson, AZ 85743, USA Received 8 September 2004; received in revised form 20 December 2004 Available online 26 January 2005 Abstract An extended GC–MS study of 129 convolvulaceous species belonging to 29 genera (all 12 tribes) including the results of a previous survey (65 spp.) revealed the occurrence of one to six polyhydroxy alkaloids of the nortropane type (calystegines) in 62 species belong- ing to 22 genera of all tribes except the unique parasitic Cuscuteae. The large genus Ipomoea turned out to comprise calystegine-posi- tive species in at least eight out of ten sections checked. The number of the calystegines used as reference compounds has been increased from seven (previous survey) to 11 (present study). Furthermore, the results concerning these additional four alkaloids could also be completed for all species of the previous survey. The plant material (epigeal vegetative parts and/or roots, flowers, fruits/seeds) was obtained from collections in the wild from a wide range of tropical, subtropical, and temperate locations of all con- tinents as well as from cultivation in the greenhouse. All plant organs turned out to be potential locations for the occurrence of these metabolites though they are detectable often only in certain organs of a given species. Three genera (Cuscuta, Operculina, Polymeria) might have lost the ability to synthesize these plesiomorphic characters in the course of the evolution since the examination of several different organs and/or provenances of five species each failed to show calystegines as constituents. Nevertheless, the present data clearly demonstrate that the occurrence of calystegines is an almost consistent trait in the Convolvulaceae in principle, from basal to most advanced tribes. Ó 2005 Published by Elsevier Ltd. Keywords: Convolvulaceae; GC–MS analysis; Tropane alkaloids; Polyhydroxy alkaloids; Calystegines; Castanospermine; Swainsonine; Glycosidase inhibitors; Chemotaxonomy 1. Introduction sidases inhibiting the enzymes. Thus, they have aroused increasing interest as, e.g., potential antiviral, Polyhydroxy alkaloids, e.g., the calystegines (nor- anticancer, and antidiabetic agents. From the ecolog- tropanes) bind specifically to the active sites of glyco- ical point of view such compounds may cause intox- ications in cattle (Asano et al., 2000 and literature q Part 17 in the series ‘‘Phytochemistry and Chemotaxonomy of the therein) and are also toxic for vertebrates, insects, Convolvulaceae’’. For part 16, see Jenett-Siems et al. [Phytochemistry, and microbes (Fellows et al., 1989 and literature 66 (2005) 223–231]. * Corresponding author. Tel.: +49 30 838 53720; fax: +49 30 838 therein). Furthermore, they exhibit antifeedant effects 53729. against lepidopterans (Simmonds et al., 1990). How- E-mail address: [email protected] (E. Eich). ever, it is not yet clear if there is a physiological role 0031-9422/$ - see front matter Ó 2005 Published by Elsevier Ltd. doi:10.1016/j.phytochem.2004.12.024 470 T. Schimming et al. / Phytochemistry 66 (2005) 469–480 of polyhydroxy alkaloids in general and of calyste- the consequence that all continents are represented gines in particular for the producing plant species it- now. (2) Again for the first time the rare genera Cardio- self (Dra¨ger, 2004; Hoeke and Dra¨ger, 2004). chlamys, Humbertia, Rapona (all endemic to Madagas- The calystegines seem to be confined to the two major car), and Hildebrandtia (East Africa/Madagascar/ Solanales families, Convolvulaceae and Solanaceae, as Arabia) altogether classified as basal (Stefanovic´ et al., well as to the unrelated Moraceae. To date the struc- 2003), as well as the more advanced genera Polymeria tures of 15 calystegines have been elucidated including (endemic to Australia) and Astripomoea (endemic to five trihydroxynortropanes (A3,A5–A8), six tetrahydr- Africa) are involved. (3) We have been able to include oxy congeners (B1–B6), and two pentahydroxy deriva- species from not yet investigated sections of the large tives (C1,C2) (see Fig. 1). Besides free calystegines, genera Ipomoea and Merremia, respectively. (4) The several glycosides could be isolated from solanaceous number of species in certain genera which had turned species. In addition, the N-methylated congeners of out already in the previous study to be calystegine-posi- the calystegines B2 and C1, respectively, have been found tive has been increased disproportionately (the largest again in solanaceous species (Asano et al., 2000; Dra¨ger, genus Ipomoea by additional 21 species, comprising 2004). The occurrence of calystegines in the family Sola- now in total 38 species; the large genus Convolvulus by naceae is documented in the literature for 15 genera (out 13 (now in total: 18), the basal genus Erycibe by 3 of 92) covering 32 species (out of 2300) (Table 3). In the (now in total: 5) in order to confirm these previous find- sister family Convolvulaceae these alkaloids were found ings. (5) In our first study two genera (Jacquemontia, in Calystegia sepium and Convolvulus arvensis (Tepfer Operculina) did not show any occurrence of calystegines et al., 1988), two further Calystegia spp. (Molyneux in different samples of several species. By including fur- et al., 1995) and seven Ipomoea taxa (Asano et al., ther species and/or organs of these genera we wanted to 2001). In our previous study on the occurrence and distri- find out if these will support this previous finding or not. bution of these compounds in the Convolvulaceae com- (6) Our former study was based only on seven calyste- prising 65 species (out of 1850) from 22 genera (out of gines whereas now we could integrate four additional 55) after all 30 species belonging to 15 genera were found congeners. This also means that we are able to enhance to be calystegine-positive (Schimming et al., 1998). the results of the former 65 species by these additional Our present study has been extended considerably in polyhydroxylated alkaloids. Thus, we want to report several respects: (1) It comprises another 64 species on the occurrence/the lack of all these metabolites in including for the first time also Australian taxa with 129 species belonging to 29 genera which include HN HN HN HN OH 5 4 1 2 OH HO HO OH OH HO HO HO HO 3 HO 6 7 HO Calystegine A Calystegine A3 Calystegine A5 6 Calystegine A7 HN HN HN OH HN OH OH OH HO HO OH HO HO OH HO OH HO HO OH HO Calystegine B1 Calystegine B2 Calystegine B3 Calystegine B4 HN CH3 OH CH3 N N HO HO OH OH OH HO HO HO OH HO HO OH HO Calystegine C1 N -Methylcalystegine B N -Methylcalystegine C 2 1 Fig. 1. Structure of the calystegines and N-methylcalystegines included in this study. T. Schimming et al. / Phytochemistry 66 (2005) 469–480 471 members of all 12 convolvulaceous tribes recently estab- erature are involved in the numbers (22 and 42, respec- lished on the basis of molecular cladistic phylogenetic tively): four species which have not been included in our analysis (Stefanovic´ et al., 2002), afterwards correctly study and in addition I. pes-caprae which has been nega- aligned with the traditional phylogenetic point of view tive in our study (for details see Table 1). This genus (Stefanovic´ et al., 2003)(Table 1). turned out to comprise calystegine-positive species in at There are a few reports on the co-occurrence of both, least eight of ten sections checked. For the remaining calystegines and swainsonine, a polyhydroxylated indo- two sections (Leptocallis, Orthipomoea) only one and lizidine alkaloid well-known from certain fabaceous two species, respectively, have been available. Thus, it is species, in the seeds of two Australian Ipomoea taxa, equivocal if these sections are calystegine-negative in gen- I. polpha and I. sp. Q6 (aff. calobra, Weir Vine), the lat- eral. In the case of Merremia the four sections involved in ter supposed to be a subspecies of the former (Molyneux this study seem to contain calystegine-positive species in et al., 1995) and from the leaves of a pantropical toxic contrast to the closely related genus Operculina (see be- Ipomoea species of American origin, I. carnea ssp. fistul- low). Jacquemontia has been the strangest genus since osa (de Balogh et al., 1998; Asano et al., 2001; Haragu- the assay of 20 samples taken from altogether six species chi et al., 2003). Therefore swainsonine as well as its resulted in 19 negative findings; only one sample turned congener castanospermine have been also integrated in out to be positive: the roots of J. tamnifolia with at least our present study. five calystegines. This is especially remarkable because the aerial parts from two provenances and the fruits of this species were also negative. The number of compounds in calystegine-positive 2. Results and discussion species (a few taken from the literature included; see above and Table 1) varied between one and six. Only Since polyhydroxylated alkaloids cannot be isolated one compound each could be detected in the samples by conventional alkaloid separation techniques, ion-ex- of 24% of these species, followed by two and three com- change chromatography was employed for separating pounds each (22% of the species each), four (20%), five this polar alkaloid fraction from neutral and acidic con- (9%), and six compounds (3%).