Pergamon BiochemicalSystematicsand Ecology, Vol.25. No. 4. pp. 305-308, 1997 © 1997 ElsevierScience Ltd All rights reserved.Printed in Great Britain 0305-1978/97 $17.00+0.00 PII: S0305-1978(97)00004-5 Quinolizidine Alkaloids from the East-African Legume Dicraeopetalum stipulare Harms KALEAB ASRES,* ANDREAS TEIT and MICHAEL WINKTt *School of Pharmacy, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia; llnstitut f0r Pharmazeutische Biologie, Universit~t Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany Key W o r d Index--Dicraeopetalum stipulare; Leguminosae; quinolizidine alkaloids; capillary gas chromatography-mass spectrometry. Abstract--The alkaloid composition of leaves of Dicraeopetalum stipulate Harms was examined by capillary gas chromatography (G/C) and GLC--mass spectrometry. 5,6-Dehydrolupanine, anagyrine and cytisine figured as major and baptifoline, N-methylcytisine, lupanine, ammodendrine, N-formylcytisine and N-acetylcytisine as minor alkaloids. Sparteine, rhombifoline and 6J]-hydroxylupanine were detected in trace amounts. Two new alkaloids were tentatively identified as 10-oxolupanine and 17-oxolupanine. © 1997 Elsevier Science Ltd Introduction Dicraeopetalum stipulate Harms represents a monotypic genus of the tribe Sophoreae (family Leguminosae). The plant grows in the south-eastern province of Ethiopia and the adjoining areas of Somalia and Kenya (Polhil, 1981). Although the name of the plant has been recorded in the literature as Hangelelo in the Somali vernacular (Thulin, 1989), it is known as Saban Sable in the area from which it was collected for the present study. D. stipulare is a tree which grows up to a height of 5-8 m and has white flowers and indehiscent flat pods which contain one or two seeds (Thulin, 1983). It belongs to the Cadia group of plants along with the genera Amphimas, Acosmium, Myrocarpus, Holocalyx, Lovanafia and Cadia. The genus Acosmium is closely related to Dicraeopetalum and, formerly, the latter was included in the Acosmium genus (Thulin, 1983). Among the chemically investigated plants belonging to this group, the genera Acosmium and Cadia were shown to produce quinolizidine alkaloids; however, no information is available in the literature regarding the chemical constituents of D. stipulare. In the present study, the alkaloid composition of the leaves of D. stipulate was analysed by capillary GLC and G C - M S and the chemotaxonomic implication of this finding is briefly discussed. M a t e r i a l s and M e t h o d s Plant material The plant material was collected in December 1993 from the surrounding areas of Filtu, a small village town in Sidamo region about 720 km south-east of Addis Ababa, Ethiopia. The plant was identified by Dr Insermu Kelbesa (The National Herbarium, Department of Biology, Addis Ababa University, where voucher specimens were deposited). tCorresponding author (Fax: + 49 6221 544884; E-mail; F01 @urz.uni-heidelberg.de). (Received 17 October 1996; accepted 25 November 1996) 305 306 K. ASRES ETAL. Alkaloid extraction. Dried powdered leaves (150g) were defatted with n-hexane in a Soxhlet apparatus for 48 h and further extracted with 80% MeOH for 72 h. The dark green residue remaining after removal of the aqueous MeOH under reduced pressure was taken up in 2% H2SO4 (40 ml) and filtered. The acidic aqueous extract was washed with Et20 until the washings were colourless, basified with conc. NH4OH (pH 9) and extracted with CH2CI2 ( 4 x 4 0 ml). The combined CH2CI2 extracts were dried (anhydrous Na2SO4) filtered and concentrated in vacuo. The acid base purification procedure was repeated three times to give a light brown semi-solid (0.104 g, 0.07%). Alkaloid analysis. Alkaloid extracts were subjected to high resolution gas chromatography under the following conditions: DB1 (J & W) fused silica capillary column (15 mx0.25 mm), carrier gas He; FID detector, detection temp., 300°; injection temp., 250°; split 1:25; oven temp. prog.: initial temp. 150 ~', 2 min isothermal, 150-250 ° at 15° min 1,250_300 ° at 25° min -1, 300 ° 15min isothermal. Capillary G C - M S was performed on an Ohio Valley OVl (15mxO.25mm) column coupled directly to a quadrupole Finnigan Mat 4500 mass spectrometer, ElMS were recorded at 45 eV. Conditions: carrier gas He; splitless, injection temp. 250 °, oven 120 ° 2min isothermal, 120-250 ° at 10°min -1, 250-300" at 15°min 1. RIs were calculated using cochromatographed standard hydrocarbons. Results and Discussion The alkaloid profile of D. stipulare has been studied by the use of GLC (Fig. 1 ) and GCMS (Table 1 ). A total of 14 quinolizidine alkaloids have been detected in the leaves of this plant. Unambiguous identification of 12 of these alkaloids was achieved by comparing their mass spectra and Kovats retention indices with those reported in the literature (Wink, 1993; Wink et al., 1995). 5,6-Dehydrolupanine, anagyrine, cytisine, baptifoline and N-methylcytisine were shown to be the major alkaloids while lupanine, ammodendrine, N-formylcytisine and/V-acetylcytisine occurred as minor components. Rhombifoline, sparteine, 6~-hydroxylupanine, RI = 2204 and RI = 221 8 could only be detected in trace amounts. The quantitative pattern of these alkaloids is given in Table 2. Further analysis of the remaining alkaloids, RI = 2204 and RI = 221 8 was not possible due to their occurrence in trace amounts and also because of paucity of plant material. However, from their molecular ion at m / z - - 2 6 2 and their respective El M S fragmentation 5 12 4 14 FIG. 1. SEPARATION OF QUINOLIZIDINE ALKALOIDS FROM DICRAEOPETALUMSTIPULAREBY CAPILLARY GLC. 307 QUINOLIZIDINE ALKALOIDS TABLE 1. IDENTIFICATION OF ALKALOIDS OFDICRAEOPETALUMSTIPULAREHARMS BY GC-MS Alkaloid RI M+ Abundant ions (relative abundance %) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1780 1873 1959 1995 2132 2155 2165 2204 2218 2334 2344 2390 2457 2696 234 208 204 190 246 (244) 248 262 262 218 232 244 (264) 260 234(24) 208(46) 204(15) 190(70) 246(25) 203 (84) 248(45) 262(23) 262(94) 218(78) 232(42) 244(32) 246(16) 260(46) Sparteine Amrnodendrine N- Methylcytisine Cytisine 5,6-Dehydrolupanine Rhombifoline Lupanine N- Formylcytisine N-Acetylcytisine Anagyrine 6~-Hydroxylupanine Baptifoline 193(37) 137(100) 136(44) 98(93) 165(100) 136(56) 123(62) 109(85) 160(4) 146(5) 73(5) 58(100) 160(25) 147(84) 146(100) 134(22) 148(6) 134(9) 98(100) 97(38) 160(12) 146 (4) 77(11 ) 68(100) 150(38) 149(58) 136(100) 98(28) 234(14) 138(51 ) 136(64) 84(100) 150(42) 136(83) 98(55) 97(100) 160(20) 147(47) 146(100) 134(15) 190(10) 160(16) 147(83) 146 (100) 160(9) 146 (13) 136(10) 98(100) 148(8) 134(18) 98(100) 97(20) 243(6) 160(17) 146(29) 114(100) TABLE 2. ALKALOID COMPOSITION OF DICRAEOPETALUM STIPULARE HARMS AS DETERMINED BY GLC (TOTAL ALKALOI DS = 100%) Alkaloid % 1 Sparteine trace 2 3 4 5 Ammodendrine N- Methylcytisine Cytisine 5,6-Dehydrolupanine 1.7 7.9 16.0 30.2 6 Rhornbifoline trace Lupanine 4.1 7 8 9 10 11 12 N- Formylcytisine N-Acetylcytisine Anagyrine 2.5a 2.5a 25.6 13 6~- Hydroxylupanine trace 14 Baptifoline 9.5 trace trace aAIkaloids coeluted. The value refers to the sum of the two alkaloids. pattern, it can be deduced that the alkaloids are dioxosparteines. Furthermore, owing to the fact that both of these alkaloids contain a prominent ion at m/z= 1 36, the possibility of having a 10,17-dioxosparteine structure can be ruled out since such compounds do not show any fragment ion at m/z= 136 (Schumann et al., 1968). This together with biosynthetic considerations led to the assumption that the alkaloids contain a 2,10and/or a 2,17-dioxosparteine structure. The MS of RI = 2204 exhibits a significant peak at m / z = 2 3 4 ( M + - 2 8 ) which arises from the elimination of a CO group typical of 17oxosparteines (Schumann et al., 1968). The presence of prominent fragment ions such as at m/z=150, 136, 97 and 84 is also indicative of a 17-oxo substitution. It can therefore be proposed that RI = 2204 is a 17-oxolupanine isomer. RI = 2218 showed a strong molecular ion peak at m/z = 262 (abundance 94%) indicating that it is a highly stable compound. The ions at re~z=234 ( M + - 2 8 ) , 233 308 K ASRES ETAL. ( M + - 2 9 ) and 205 ( M + - 5 7 ) correspond to the elimination of CO, C2H5 and CH3CH2-CO, respectively, characteristic of the fragmentation pattern of 10-oxosparteines (Schumann et al., 1 968). The MS of RI = 2218 also shows similarities with those of 1 0oxosparteines in that it exhibits strong ions at m / z = 1 5 0 (abundance 42%), 136 (abundance 83%) and 97 (abundance 1 00%). Thus it is likely that the compound is one of the isomers of 1 0-oxolupanine. The occurrence of the dipiperidine alkaloid, ammodendrine, in D. stipulare is of chemotaxonomic significance. Outside the genus A m m o d e n d r o n (tribe Sophoreae), dipiperidine alkaloids are known to coexist with quinolizidine alkaloids mainly in plant species belonging to the tribe Genisteae. Such an observation has led to a suggestion that A m m o d e n d r o n should be transferred to the tribe Genisteae (Mears and Mabry, 1971). However, the present finding clearly shows that plants belonging to the tribe Sophoreae already have the biosynthetic capacity to produce dipiperidine alkaloids such as ammodendrine indicating that the aforementioned view is not relevant. Furthermore, the occurrence of ammodendrine in other plants belonging to the tribe Sophoreae has also been reported previously (Wink and Witte, 1 987). It may also be worthwhile to note that among the Cadia group of plants that have already been investigated so far, the alkaloid profile of D. stipulare appears to be similar to that of A c o s m i u m in that they both contain the tetracyclic sparteine and lupanine-type alkaloids supporting their botanical similarities. However, the chemistry of the t w o genera is different from each other by the occurrence of pentacyclic quinolizidine alkaloids in A c o s m i u m (Fitzgerald et al., 1964; Balandrin and Kinghorn, 1981 ). Thus, the present finding is consistent with the current taxonomic classification of Dicraeopetalum as a separate genus. Acknowledgements--The DeutscherAkademischerAustauschdienst (DAAD) are thanked for their financial support to one of us (K.A.). We are also most gratefulto Dr Insermu Kelbesafor identification of plant material. References Balandrin, M. F. and Kinghorn, A. D. (1981 ) Characterizationof sweetinine, a constituent of Sweetia e/egans, as the ormosia alkaloid, (+) -6-epipodopetaline.J. Nat. Prod. 44, 619-622. Fitzgerald,T. J., LaPidus,J. B. and Beal,J. L. (1964) Sweetinine,an alkaloidfrom Sweetia panamensis. L/oydia 27, 107-110. Mears, A. J. and Mabry, T. J. (1971) Alkaloids in the Leguminosae. In Chemotaxonomy of the Leguminosae (Harborne, J. B., Boulter, D. and Turner, B. L., eds), p. 88. Academic Press, London. Polhil, R. M. (1981) Sophoreae. In Advances in Legume Systematics (Polhil, R. M and Raven, P. H., eds), p. 218. Royal Botanic Garden, London. Schumann, D., Neuen-Jehle, N. and Spiteller, G. (1968) Schlesselbruchstucke in den Massenspektrenyon Alkaloiden. Mh. Chem. 99, 390-408. Thulin, M. (1983) Leguminosae of Ethiopia, p. 65. AiOPrint, Odense, Copenhagen. Thulin, M. (1989) The Leguminosae. In Flora of Ethiopia, Vol. 3 (Heidberg, I. and Edwards, S. eds). The National Herbarium, Addis Ababa University,Addis Ababa. Wink, M (1993) Quinolizidinealkaloids. In Methods in Plant Biochemistry, Vol. 8, Alkaloids and Sulphur Compounds (Waterman, P., ed.), pp. 197-239. Academic Press, London. Wink, M. and Witte, L. (1987) Cell-freesynthesisof the alkaloidsammodendrineand smipine. Z. Naturforsch. 42c, 197-204. Wink, M., Meil~ner, C. and Witte, L. (1995) Patterns of quinolizidine alkaloids in 56 species of the genus Lupinus. Phytochemistry 38, 139-153.