Biochemical Systematics and Ecology 31 (2003) 155–166 www.elsevier.com/locate/biochemsyseco Chemotaxonomic analysis of the genus Cryptolepis A. Paulo a,∗, P.J. Houghton b a b Faculty of Pharmacy, Centro de Estudos de Ciências Farmacêuticas, Av. Prof. Gama Pinto, University of Lisbon, Lisbon 1649-003, Portugal Pharmacognosy Research Laboratories, King’s College London, Franklin-Wilkins Building 150 Stamford Street, SE1 8WA London, UK Received 10 July 2001; accepted 4 February 2002 Abstract A chemotaxonomic study is made on the basis of a phytochemical screening of the leaves of 60% of the African Cryptolepis species and the review of the secondary metabolites identified in the genus Cryptolepis, subfamily Periplocoideae and related families Asclepiadaceae and Apocynaceae. Our study indicates that the chemistry of the genus Cryptolepis is in agreement with its taxonomic position within the taxon Periplocaceae/Periplocoideae and also that the chemical evidence obtained so far is consistent with the idea that the taxon Periplocaceae/Periplocoideae is an evolutionary link between the families Apocynaceae and Asclepiadaceae. Our observations also strengthens the argument that the taxon Periplocaceae/Periplocoideae should be considered an independent family.  2002 Elsevier Science Ltd. All rights reserved. Keywords: Cryptolepis; Periplocaceae; Asclepiadaceae; Steroids; Alkaloids; Chemotaxonomy 1. Introduction The genus Cryptolepis R. Br. belongs to the Periplocoideae subfamily of the Asclepiadaceae (Forster, 1990; Liede and Albers, 1994) or according to other botanists (Hutchinson and Dalziel, 1963) to the family Periplocaceae. Although the taxo∗ Corresponding author. Tel.: +351-1-7946473. E-mail address: mapaulo@ff.ul.pt (A. Paulo). 0305-1978/03/$ - see front matter  2002 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0305-1978(02)00075-3 156 A. Paulo, P.J. Houghton / Biochemical Systematics and Ecology 31 (2003) 155–166 nomic hierarchy of the taxon Periplocaceae/Periplocoideae is in question all botanists agree on the differences and similarities between this taxon, the Apocynaceae family and the Asclepiadaceae subfamilies: Asclepiadoideae and Secamonoideae. A list of 74 genera ascribed to the Periplocoideae subfamily, in which Cryptolepis is included, was published by Liede and Albers (1994) although no attempt was made to establish synonymies. For instance, according to Forster (1990), the genera Leposma Blume, Ectadiopsis Benth., Gymnolaema Benth., Batesanthus N. E. Br., Stomatostemma N. E. Br. and Streptomanes Schumann should all be considered synonyms of Cryptolepis R. Br. The Periplocaceae/Periplocoideae are confined to the tropics of the ‘Old World’ and so the genus Cryptolepis, comprising ca. 20 species, can be found in Africa, Madagascar, Asia, Australia and Papua New Guinea. Bullock (1955) considered only 7 African species: C. capensis Schltr., C. hypoglauca K. Schum., C. apiculata K. Schum., C. sanguinolenta (Lindl.) Schltr., C. cryptolepioides (Schltr.) Bullock, C. obtusa N. E. Br. and C. sinensis (Lour.) Merr. subsp. africana Bullock. Later Forster (1990) transferred into Cryptolepis 3 other African taxa: C. pendulina (Venter and D. V. Field) P. Forster, C. purpureus (N. E. Br.) P. Forster and C. newii (Benth.) P. Forster. Two species are considered to be endemic of Madagascar: C. grandidieri Roxb. ex. R. Br. and C. albicans Jumelle and Perrier (Verhoeven and Venter, 1994). Four species were reported from Asia: C. sinensis (Lour.) Merr. (synonymy of C. elegans Wall. ex. G. Don.), C. buchananii Roem. and Schult., C. grandiflora Wight and C. javanica Blume (Kew Records, 1993; Bullock, 1955). One Australian species: C. grayi P. Forster and three species from Papua New Guinea: C. nymanii (Schuman) P. Forster, C. papillata P. Forster, C. lancifolia P. Forster were found in the Oceania continent (Forster, 1990, 1991). When the distribution of Cryptolepis species in Africa is considered, it is seen that they are all found near the east coast with the exception of C. sanguinolenta which is widely distributed in the coastal countries of West Africa and C. purpureus, found in Cameroon. C. sanguinolenta grows in countries from Senegal to Nigeria including Gambia, Guinea Bissau, Guinea, Ghana, Ivory Coast and Sierra Leone. Although there are no reports of this species in Cameroon or Sudan, it was reported from Zaire (Cimanga et al., 1991, 1996a, 1996b, 1997), Angola (Malange and Cazengo districts) and there is one record of its occurrence in East Africa in Uganda (Bullock, 1955). C. obtusa is the species most widely distributed in Southeastern Africa. This species is found in South Africa (Transvaal), Mozambique and Tanzania (Bullock, 1955). Two other species are reported from South Africa: C. cryptolepioides from Transvaal and C. capensis from Natal and Transkei districts. C. sinensis subsp. africana was recorded as occurring only in Kenya. C. hypoglauca was found in Tanzania and Kenya and C. apiculata also in these two countries and in Zimbabwe (Bullock, 1955). C. pendulina was found in Mozambique and C. newii in the Kilimanjaro mountain area of Tanzania/Kenya (Forster, 1990). In respect to the chemistry of the genus Cryptolepis only three species have been phytochemically studied. The roots of C. sanguinolenta were found to be rich in bioactive indole alkaloids (Paulo et al., 2000a). In the leaves and roots of C. buchananii the main secondary metabolites found are cardenolides and pseudo-alkaloids A. Paulo, P.J. Houghton / Biochemical Systematics and Ecology 31 (2003) 155–166 157 (nicotinoyl glycosides) (Purushothaman et al., 1988; Dutta et al., 1980) and the roots of C. apiculata also afforded cardenolides (Hegnauer, 1964). The aim of this paper is to analyse the chemical evidence that may improve the existing classification of the species within the genus Cryptolepis and to contribute to the discussion on the taxonomic rank of the taxon Periplocaceae/Periplocoideae to which the genus belongs. 2. Material and methods 2.1. Plant material Eleven leaf samples of 6 African and 1 Madagascan Cryptolepis species were used for phytochemical screening purposes (Table 1). Table 1 Plant materials (leaves) Sample no. Species voucher specimen Place of collection Time of collection Presence of Herbarium flowers/fruits 1 C. albicans Jumelle and Perrier Phillipson 3425 C. capensis Schltr. Hilliard & Burtt 7575 C. apiculata K. Schum. Wild Goldsmith & Mulles 6631 C. obtusa N. E. Br. Vollesen 3699 C. obtusa N. E. Br. Phiri 184 Madagascar (South West) South Africa (Natal) Zimbabwe February 1990 January 1975 December 1964 June 1976 Fruits Kew Flowers Kew Flowers Kew Flowers Kew April 1968 Flowers Kew July 1976 Flowers Kew February Flowers 1966 Guinea Bissau October Fruits (Cacheu) 1995 Nigeria 1971 Mozambique October Flowers (Maputo) 1993 Mozambique April 1996 (Maputo) Kew 2 3 4 5 6 9 10 C. hypoglauca K. Schum. Faulknes 4914 C. cryptolepoides Bullock Simon 670 C. sanguinlenta Schltr. M.A. Diniz et al. 995 C. sanguinlenta Schltr. C. obtusa N.E. Br. 11 C. obtusa N.E. Br. 7 8 Tanzania (South East) Zambia (Luanga Valley) Tanzania (Tango) Zimbabwe LISC a a a a—The identification of plant materials was confirmed by the specialists of the Royal Botanic Gardens of Kew Herbarium 158 A. Paulo, P.J. Houghton / Biochemical Systematics and Ecology 31 (2003) 155–166 2.2. Phytochemical screening Dried plant material of all samples (100 mg) were powdered and put in contact with 10 mL of EtOH 96% for 24 h, protected from light. Then, plant material and the solution were refluxed for 10 min. After filtration through G4 glass filters, the extract was evaporated to dryness, weighed and kept in dry and dark conditions at room temperature. Extracts obtained are listed in Table 2. The dried extracts were redissolved in the necessary amount of CHCl3:MeOH (1:1) to make solutions of 5 mg/ml and 20 µl of each solution was applied onto silica gel thin layer chromatography (TLC) plates (20 × 20cm, 0.25 cm thickness) with an automatic applicator in a narrow uniform line of 8 mm width distancing 20 mm from the bottom edge and 32 mm from the side edge. Bands were at 8 mm from each other. An ascending development of 160 mm was carried out in presaturated developing chambers. The presence of phenolic compounds (flavonoids and phenolic acids) in extracts was detected by spraying the TLC plates with Natural Products-Polyethyleneglycol reagent (no.28 in Wagner et al. (1984) under UV366nm light, after development with AcOEt: HCOOH:H2O (6:1:1). Alkaloids were screened with Dragendorff’s and Iodoplatinate reagents (no. 96 and 147 in Stahl, 1969), after development with CHCl3:MeOH:NH3 (90:10:1). For the detection of cardenolides, the TLC plates were developed with AcOEt:MeOH:H2O (77:15:8) and sprayed with Kedde reagent (no. 23 in Wagner et al. (1984). Steroids and terpenes were visualised after spraying the TLC plates with Anisaldehyde -sulphuric acid (no. 2 in Wagner et al. (1984) and heating them for 10 min at 105 °C. For the development of saponins the system AcOEt: MeOH:H2O (77:15:8) was used and the non-polar steroids and terpenes were developed with hexane:AcOEt (8:2). Table 2 Ethanolic extracts of leaf samples of Cryptolepis Sample no. Extract Amount (mg) 1 2 3 4 5 6 7 8 9 10 11 CabL-90 CcaL-75 CapL-64 CoL-76 CoL-68 ChL-76 CcyL-66 CsL-95E CsL-71E CoL-93E CoL-96E 14.0 12.5 12.0 12.7 6.0 11.0 20.2 13.6 17.6 22.1 25.1 A. Paulo, P.J. Houghton / Biochemical Systematics and Ecology 31 (2003) 155–166 159 3. Results and discussion 3.1. Phytochemical screening of the leaves of African Cryptolepis species The ethanolic extracts of the leaves of 6 African and 1 Madagascan Cryptolepis species (Tables 1 and 2) were screened by TLC for phenolic compounds, alkaloids, cardenolides and non-polar steroids and terpenes (Table 3). The phytochemical screening performed, although not designed to be an exhaustive chemotaxonomic study, incorporated the minimum necessary requirements which allowed a chemotaxonomic analysis of the results (Smith, 1976). These requirements can be summarised as follow: (i) the African section of the genus Cryptolepis was well represented in the sample since it included 6 of the 7 African species recognised by Bullock (1955) and not contested by Forster (1990); (ii) some of the species were represented by more than one sample collected in different geographical areas and at different months; and (iii) the plant material was well identified (Table 1) and the chemical techniques chosen adequate to the task. It could be concluded that the main secondary metabolites present in the leaf ethanolic extracts of African Cryptolepis species are phenolic compounds. They were detected in all extracts except in extracts of C. sanguinolenta (CsL-95E, CsL-71E) and the extract (CoL-68) of one sample (no. 5) of C. obtusa. The lack of phenolic compounds in the extract CoL-68 is maybe due to the use of ethanol to preserve the herbarium sample although this hypothesis could not be proved, because the herbarium did not have any information on the way of sample preservation at the time of collection used with each herbarium sample. The other two reasons that could explain the observed lack of polar secondary metabolites in extract CoL-68 Table 3 Main secondary metabolites detected in the ethanolic extracts of the leaves of African Cryptolepis species Main secondary metabolites Species Extract Phenolic compounds Alkaloids Non-polar steroids Cardenolides and terpenes C. C. C. C. CabL-90 CcaL-75 CapL-64 CoL-76 CoL-68 CoL-93E CoL-96E ChL-76 CcyL-66 CsL-95E CsL-71E ++ ++ + + – ++ + +/– ++ – – – – – – – – – + – + ++ + + + + ++ +/– + + +/– + + albicans capensis apiculata obtusa C. hypoglauca C. cryptolepoides C. sanguinolenta ++ strong reaction; + evident reaction; +/– very weak reaction; – no reaction. – – – – – – – – – – – 160 A. Paulo, P.J. Houghton / Biochemical Systematics and Ecology 31 (2003) 155–166 (Table 3), i.e. sample no. 5 was collected from a different geographical area and at a different time of the year, which would imply a different stage in the plant life cycle, were discounted because: (i) other samples of C. obtusa collected in different geographical areas did show similar chromatographic profiles; (ii) the 3 samples of C. obtusa (nos. 4, 5 and 10) were at similar stages of the life cycle since they were all flowering (Table 1); and (iii) sample no. 11 collected in a non-flowering stage also showed a similar chromatographic profile when compared with the flowering sample no. 10. The Retention Factor (Rf) values and colour of spots under UV366nm light after reaction with NP/PEG spray reagent, by comparison with literature data (Wagner et al., 1984), indicated that caffeoyl-quinic acids such as chlorogenic acid (1) (light blue spots; Rf⬇0.5) and quercetin (2) glycosides (orange spots) could be the main phenolic compounds present in the leaf extracts of African Cryptolepis species. This supposition was confirmed by the isolation of the mentioned compounds from sample no. 10 (Paulo et al., 1997). Non-polar steroids and terpenes were detected in all extracts as expected since many of these compounds are chemical constituents of all plants, where they have a structural role in organelle membranes, although they are often considered as secondary metabolites (Mann, 1987). Alkaloids were detected in the leaves of two species: C. sanguinolenta and C. hypoglauca whereas cardenolides were not found in any ethanolic extract of the leaves of the species screened. 3.2. The position of C. sanguinolenta within the genus Cryptolepis The results of the phytochemical screening of the leaves of 60% of the African Cryptolepis species and of the isolation and identification of indole alkaloids from C. sanguinolenta (Paulo et al., 1995, 2000a) led to the conclusion that this species is chemically different from the other African Cryptolepis species studied, since no phenolic compounds were detected in the leaf extracts of the two samples studied. However, there is no report in the literature of any substantial difference in the botanical characters of C. sanguinolenta that could justify a separation of this species from the genus Cryptolepis or even the creation of subgenera. One must not forget that the species C. sanguinolenta is the only species of those studied that grows in the west of Africa. Therefore no final conclusion can be drawn from the results since through the centuries this species has been under different physical and chemical external factors (soil and climate) and different environmental stress (e.g. predators) which could lead to the blockage of the flavonoid biosynthetic route and favoured the alkaloid one (Swain, 1963). Finally, more work, especially chemical studies, must be done on the genus Cryptolepis. This work should include: (i) a screening of the chemical composition of the African species not included in this study; (ii) the phytochemical study of organs other than the leaves; (iii) the phytochemical study of species from other continents; and (iv) a more detailed examination of the botanical characters of species showing a complete different pattern of secondary metabolites, such as C. sanguinolenta. A. Paulo, P.J. Houghton / Biochemical Systematics and Ecology 31 (2003) 155–166 161 After these studies, there may be enough data to support a division of the genus Cryptolepis into subgenera. 3.3. The genus Cryptolepis within the taxon Periplocaceae/Periplocoideae As with the genus Cryptolepis, the chemistry of the taxon Periplocaceae/Periplocoideae is poorly known. Only 3 of the 74 genera of this taxon (Liede and Albers, 1994) have been studied for their chemical composition. They are the genera Periploca, Cryptostegia and Cryptolepis. The type of compounds that is common to the 3 genera are steroid glycosides, in which cardenolides are included due to their common biosynthetic pathway. From the analysis of Table 4, which includes not only our results but also results published by others, one must conclude that the chemical evidence from the genus Cryptolepis concerning: (i) the steroid nucleus including the pattern of oxidation, (ii) type of sugars and (iii) presence of cardenolides in the Asiatic species of C. buchananii, supports the taxonomic position of the genus Cryptolepis within the taxon Periplocaceae/Periplocoideae. 3.4. Relationship of the taxon Periplocaceae/Periplocoideae with Apocynaceae and Asclepiadaceae families To make chemical comparisons between these three taxa the type of compounds that can lead to a conclusion must be selected. The phenolic compounds found so far in the genus Cryptolepis (Paulo et al., 1997), and also for the first time identified in the Periplocaceae/Periplocoideae, are of common distribution in the plant kingdom and so they are unlikely to have chemotaxonomic utility in this case. According to Hegnauer (1964) the families Apocynaceae and Asclepiadaceae are chemically identical in cardenolides, but very different in their alkaloidal composition. This author also believed that the development of the cardenolides in the Apocynaceae family led to pregnane alkaloids, whereas in the Asclepiadaceae the cardenolides evolved to polyhydroxy-C11-steroids. So, it is only on the basis of the structure of alkaloids, steroidal alkaloids and hydroxysteroids that the chemotaxonomic relationships can be discussed. The type of alkaloids isolated from the species C. sanguinolenta, indole alkaloids (Paulo et al., 1995, 2000a and references within) are very common in Apocynaceae family but have never been found in Asclepiadaceae (Hegnauer, 1964, 1988, 1989). In the Asclepiadaceae, alkaloids are rare although some of the type phenanthroindolizidine and phenanthroquinolizidine were found in the genera Tylophora and Cynanchum (Hegnauer, 1964; Rao et al., 1970a, 1970b; Wiegrebe et al., 1970; Ali and Bhutani, 1992; Abe et al., 1995) and these are biogenetically very different from the indole alkaloids (Mann, 1987). The steroidal alkaloids 3 and 4 isolated from C. obtusa (Paulo et al., 2000b) also differ markedly from those identified in the Asclepiadaceae where they are basically polyhydroxysteroids esterified with nicotinic acid at C12 or C20 (Hegnauer, 1964, 1989; Summons et al., 1972; Aquino et al., 1995, 1996; Ma and Fang, 1997). The 162 A. Paulo, P.J. Houghton / Biochemical Systematics and Ecology 31 (2003) 155–166 Table 4 Chemical characteristics of the steroid glycosidesa isolated from Periplocaceae/Periplocoideae species Genus and species Genin (oxygenated positions) Cryptolepis C. obtusa C. buchananii Steroidal alkaloids 5⌬-Pregnene (3β, 15α, (16β-N), 17β, 20(R)) (3β, (16β-N), 17β, 20(R)) Cardenolides sarmentogenin (3β, 11α) sarverogenin (3β, 7β, 8β, 11α, 12, 14β) Periploca P. sepium P. calophyla P. nigrescens Steroid esters 5⌬-Pregnene (3β, 16α/β, 20(R/S)) (3β, 20(S)) (3β, 17α, 20(R/S)) (3β, 14β, 20(R/S)) Sugars References 2,6-Dideoxy-3-Omethylhexose, 6-deoxy-3-Omethylhexose sterified at C20. Paulo et al., 2000b glucose and 2,6-dideoxy-3-Omethylhexose Shah and Khare, 1981 Purushothaman et al., 1988 2,6-dideoxy-3-Omethylhexose, 6-deoxy-3-Omethylhexose sterified at C3 and/or C20. Itokawa et al., 1988; Umehara et al., 1995; Srivastava et al., 1982; Sethi et al., 1988 Cardenolides periplogenin (3β, 5β, 16β) glucose and 2,6 strophanthidol (3β, 5β, -dideoxy-3-O16β, 19) methylhexose Cryptostegia Cardenolides C. grandiflora gitoxigenin (3β, 14β, 16β) C. madagascariensis 16-Acetyl-gitoxigenin 16-Propionyl-gitoxigenin Gaignault and Bidet, 1988;Umehara et al., 1995 Doskotch et al., 1972; Sanduja et al., 1984 a The compound β-sitosteryl-3-O-β-glucopyranoside is excluded from this Table since it has no chemotaxonomic value due to its widely distribution in the plant kingdom (Buckingham, 1994). steroidal alkaloids 3 and 4 are of a pregnane type like the ones identified in Apocynaceae, but they are also different from those, since in the steroidal alkaloids of Apocynaceae the transamination occurs at C3 or C20 (Hegnauer, 1964). If one considers the hypothesis of Hegnauer (1964) that the pregnane alkaloids in the Apocynaceae evolved from cardenolides, it can be concluded that steroidal alkaloids 3 and 4 evolved from 16-acetyl-gitoxigenin (5), a cardenolide already identified in Periplocaceae/Periplocoideae (Sanduja et al., 1984). When steroid glycosides of other than the cardenolide and alkaloidal type are considered, it can be concluded that in the Periplocaceae/Periplocoideae (see Table 4) they are not so oxygenated/hydroxylated as in Asclepiadaceae where the genin sarcostin (3β, 8β, 12β, 14β, 17β, 20-hexahydroxy-pregn-5-ene) is common A. Paulo, P.J. Houghton / Biochemical Systematics and Ecology 31 (2003) 155–166 163 (Summons et al., 1972; Warashina and Noro, 1995, 1996; Lin and Lin, 1995; Aquino et al., 1995, 1996; Ma and Fang, 1997). The sugars 2,6-dideoxy-3-O-methylhexoses and 6-deoxy-3-O-methylhexoses are common both in Asclepiadaceae and Apocynaceae (Hegnauer, 1964). Considering all the above observations, we think that the chemical evidence obtained so far is consistent with the idea that the taxon Periplocaceae/Periplocoideae is an evolutionary link between the families Apocynaceae and Asclepiadaceae (Omlor, 1996; Cronquist, 1981) due to its chemical similarities with both families and strengthens the argument that the taxon Periplocaceae/Periplocoideae should be considered an independent family because its chemistry is markedly different from Asclepiadaceae if alkaloids and steroidal alkaloids are considered. However, this latter conclusion is based on data obtained from only two Cryptolepis species (C. sanguinolenta and C. obtusa) and so more studies must be done with other species and genera of the Periplocaceae/Periplocoideae group. 164 A. Paulo, P.J. Houghton / Biochemical Systematics and Ecology 31 (2003) 155–166 Acknowledgements The authors acknowledge David Goyder from the Royal Botanic Garden Herbarium of Kew and Adélia Diniz from the Centro de Botânica do Instituto de Investigação Tropical de Lisboa, for their kind collaboration in the provision and identification of plant material. References Abe, F., Iwase, Y., Yamauchi, T., Honda, K., Hayashi, N., 1995. Phenanthroindolizidine alkaloids from Tylophora tanakae. Phytochemistry 39, 695–699. Ali, M., Bhutani, K.K., 1992. Alihirsutine A, a new phenanthroquinolidine alkaloid from Tylophora hirsuta. Fitoterapia 63, 243–244. Aquino, R., Pizza, C., Tommasi, N., Simone, F., 1995. New polyoxypregnane ester derivatives from Leptadenia hastata. J. Nat. Prod. 58, 672–679. Aquino, R., Peluso, G., Tommasi, N., Simone, F., Pizza, C., 1996. New polyoxypregnane ester derivatives from Leptadenia hastata. J. Nat. Prod. 59, 555–564. Bullock, A.A., 1955. Notes on African Asclepiadaceae. 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