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Alkaloids from Strychnos chrysophylla
Journal of Ethnopharmacology, 10 (1984) Elsevier Scientific
243-241
243
Publishers Ireland Ltd.
Short Communication
ALKALOIDS FROM STRYCHlVOS CHRYSOPHYLLA
R. VERPOORTE,
J. AADEWIEL,
J. STR6MBOM’and
A. BAERHEIM
SVENDSEN
Department of Pharmacognosy, Gorlaeus Laboratories, University of Leiden, P.O. Box 9502,230O RA Leiden (The Netherlands) and ‘Department ofPharmaaognosy, University of Uppsala,P.O. Box 579, S75123 Uppsala (Sweden) (Accepted
October 13,1983)
Strychnos chrysophylla Gilg is a large liana found in the rain forest often along river banks - in S. Nigeria, Cameroon, Gabon and Congo (Brazzaville) (Leeuwenberg 1969). In a pharmacological screening, muscle relaxant effects were noted in tertiary alkaloid fractions of the stembark as well as clonic convulsions (Sandberg et al., 1971). In a screening for antimicrobial activity (Verpoorte et al., 1983) a weak activity was noted against Staphylococcus aureus and Escherichia coli. Bisset and Phillipson (1971) detected at least two alkaloids in extracts of leaves, one of these compounds coloured yellow, and was similar to a yellow alkaloid observed in leaf extracts of a number of Strychnos species. Recently we reported on the presence of longicaudatine in S. chrysophylla (Massiot et al., 1983), in connection with its structure elucidation. Here we will report in detail on the alkaloids found in the stembark of the species mentioned. Experimental Plant material
Stembark of Strychnos chrysophylla was collected by A.J.M. Leeuwenberg (collection number Lg 7865) close to in June-July, 1970. The systematic identification of the formed by Dr. A.J.M. Leeuwenberg. A voucher specimen herbarium in Wageningen, the Netherlands.
F. Sandberg and Kribi (Cameroon) plant was peris kept at the
Extraction Ground plant material (2 kg) was made wet with 10% sodium hydrogen carbonate solution. The plant material was then extracted twice, overnight, with 4 1 diethyl ether. The extracts were combined and taken to dryness (5.28 g) (E,). Subsequently, the plant material was extracted twice, over0378-8741/84/$01.80 o 1984 Elsevier Scientific Published and Printed in Ireland
Publishers Ireland Ltd.
244
night, with 4 1 chloroform. Upon evaporation of the combined chloroform extracts 2.96 g of dry extract (C,) was obtained. The two extracts were further treated, separately, in the following way. The extract was partitioned between ethyl acetate and 2% aqueous acetic acid. The ethyl acetate layer was then further extracted twice with 2% aqueous acetic acid. The combined aqueous layers were basified with NaHCO, (pH - 9) and exhaustively extracted with chloroform. The combined chloroform extracts were taken to dryness. In this way the diethyl ether extract (E,) yielded 50 mg purified tertiary alkaloid extract (E,) and the chloroform extract (C,), 263 mg purified extract (C,). Separation of the alkaloids The tertiary alkaloid extracts Cz and E2 were separated on a Merck ready made Silica gel 60 column (Size B), using chloroform~toluene/absolute ethanol saturated with NH, (9 + 9 f 2) as mobil phase. The major alkaloid fractions contained longicaudatine. The other fractions were further purified by means of preparative TLC. TLC The following
TLC-systems
were used for the separation
of the alkaloids:
(1) toluene/absolute ethanol saturated with ammonia in various ratios (2) toluene/chloroform/absolute ethanol saturated with ammonia (9 -I-9 + 2) (3) petroleum ether (60-80”C)/isopropanol/absolute ethanol saturated with ammonia (85 + 10 + 5) (4) petroleum ether (6~8O’C)/chlorofo~/absoiute ethanol saturated with ammonia (7.5 : 1.25: 1.25) (5) ethyl acetate/isopropanol/25% ammonia (9 + ‘7 + 2) (6) cyclohexane/chloroform/diethylamine (6 + 5 f 1) All solvent systems were used in combination with ready made plates Silica gel F254 (Merck) in saturated chromatography tanks. The alkaloids were detected by means of quenching of UV-light at 254 nm, and by the spray reagents iodoplatinate, 0.2 M ferric (III) chloride in 35% perchloric acid and 1% ceric (IV) sulphate in 10% sulphuric acid. With the latter two reagents, colours were observed immediately after spraying as well as after heating with hot air. Characterization of the alkaloids Longicaudatine. UV(MeOH) showed maxima at 310 (sh), 290,283 and 260 nm. FDMS showed a molecular weight of 568. The ‘H-NMR (300 MHz, CDCIB, 6 in ppm) showed characteristic signals at 7.35 (m), 7.30 (m), 7.00 (m), 6.69 (t,H-lo), 6.30 (d,H-la), 6.10 (bt,H-19), 6.08 (s,H-17’) and 5.62 (q,H-19’). With the ferric (III) chloride in perchloric acid and the ceric (IV) sulphate in sulphurlc acid spray-reagents, a blue colour was observed.
245
These colour reactions and the RtvaIues were the same as for the reference compound (Massiot et al., 1983). Wieland Gumlich aldehyde. UV(MeOH) showed maxima at 298 and 246 nm. EIMS (180°C 70 eV) showed characteristic fragments at m/z: 310 (M’, 48), 292(10), 281(10), 267(10), 199(42), 180(100), 144(62), 143(90) and 130(60). With the ceric (IV) sulphate in sulphuric acid spray-reagent an orange colour was obtained. The colour and the R+lues were the same as for the reference compound (Verpoorte and Baerheim Svendsen, 1978). Caracurine V. UV(MeOH) showed maxima at 304 and 262 nm. EIMS (150°C 10 eV) showed characteristic fragments at m/z 584 (M’, loo), 180( 70), 144(25) and 143(13). The ‘H-NMR (300 MHz, CDCIJ, 6 in ppm) showed characteristic signals at 7.13 (t,H-ll,H-ll’), 7.04(d,H-9,H-9’), 6.83(t,H-lO,H-lo’), 6.44(d,H-12,H-12’), 6.08(bm,I-I-19,H-19’) and 4.70(d,H-17,H-17’). With the ferric (III) chloride in perchloric acid spray-reagent a purple-blue colour was observed, with the ceric (IV) sulphate in sulphuric acid spray-reagent a purple colour, which changed to yellow in the centre of the spot. These colour reactions and the Rtvalues were the same as for the reference compound (Verpoorte and Baerheim Svendsen, 1978). Longicaudatine N-oxide. UV(MeOH) showed maxima at 310 (sh), 290, 283 and 260 nm. FDMS showed ions at 584 (M’, 20) and 568 (M’-16,100%). The ‘H-NMR (300 MHz, CDCl,, 6 in ppm) showed characteristic signals at7.46(m),7.20(m),7.07(m),6.81(t,H-lO),6.59(bt,H-19),6.36(d,H-12), 5.97 (s,H-17’) and 5.85 (q,H-19’). With the ferric (III) chloride in perchloric acid and the ceric (IV) sulphate in sulphuric acid spray-reagents the same colour reactions were observed as for longicaudatine Reduction with sulphurous acid. The alkaloid was dissolved in sulphurous acid, left for 2 h at room temperature, the solution subsequently basified and extracted with chloroform. On TLC one major reaction product was observed with the same colour reactions and RfvaIues as longicaudatine. Oxidation of longicaudatine. Longicaudatine was dissolved in methanol and some drops of 3% hydrogen peroxide solution were added. After 24 h two major products were observed on TLC, the least polar one corresponded with the alkaloid identified as longicaudatine N-oxide on TLC. Results and discussion Only small amounts of alkaloid were found to be present in the stembark (0.016% tertiary alkaloids). Column chromatography of the tertiary alkaloid fraction and following preparative TLC yielded four alkaloids. Three of these could be identified by means of their spectral data and comparison with reference alkaloids as Wieland-Gumlich aIdehyde, caracurine V and longicaudatine (Fig. 1). The fourth alkaloid, a minor compound, showed the same colour reactions on TLC and had the same UV-spectrum as longicaudatine. In pure
246
Fig. 1. Longicaudatine,
longicaudatine N,-oxide.
longicaudatine this alkaloid could be observed after some time. The unknown alkaloid showed two ions in the FDMS at m/z 568 and m/z 584. N-oxides are known to readily lose oxygen, thus giving rise to a large M’-16 fragment (Hesse, 1974). The MS thus pointed to a mono-N-oxide of longicaudatine. Indeed, oxidation of longicaudatine with hydrogen peroxide at room temperature gave, as major reaction product, a compound with the same TLC-behaviour as the unknown alkaloid. Reduction of the latter gave a compound with the same TLC-behaviour and colour reaction as longicaudatine. The small amount of material available resulted in a ‘H-NMR of which only the aromatic region was intelligible. The most apparent difference with the spectrum of longicaudatine was the shift of the broad triplet of H-19 from 6.10 ppm to 6.58 ppm in the N-oxide. A similar change is observed in the ‘H-NMR of strychnine N-oxide if compared with that of strychnine (Verpoorte, unpublished results). The Strychnos moiety of the dimer is thus the most likely place of N-oxidation. This agrees with the well-known ease of N-oxidation in the strychnine-type of alkaloids (Phillipson and Bisset, 1972; Verpoorte, 1976). The alkaloid is thus identified as longicaudatine N,-oxide. Due to the use of chloroform in the isolation of the alkaloids this alkaloid could be an artefact. The presence of a tertiary alkaloid of the toxiferine type (caracurine V) does explain the weak muscle relaxation found for the tertiary alkaloid extract (Verpoorte and Baerheim Svendsen, 1977). The antimicrobial activity of caracurine V (Verpoorte et al., 1978) agrees with the effect found in the general screening of the extract. However, according to biograms a further - yet unidentified - non-alkaloidal compound which has antimicrobial activity is also present. References B&et, N.G. and Phillipson, J.D. (1971) The African Species of Strychnos, Part II, The Alkaloids. Lloydia 34, 1. Hesse, M. (1974) Progress in Mass Spectrometry, Vol. 1, Indolalkaloide. Verlag Chemie, Weinheim, p. 5.
247 Leeuwenberg, A.J.M. (1969) The Loganiaceae of Africa VIII - Sttychnos III, Mededelingen Landbouwhogeechool Wageningen 69, 1. Massiot, G., Zeches, M., Mirand, C., Le Men-Olivier, L., Delaude, C., Baser, K.H.C., Bavovada, R., Bisset, N.G., Hylands, P.J., Stromborn, J. and Verpoorte, R. (1983) Occurrence of longicaudatine, a new type of bis-indole base and bisnor-C-alkaloid H in Strychnos species. Journal of Organic Chemistry, in press. Phillipson, J.D. and Bisset, N.G. (1972) Quaternisation and oxidation of strychnine and brucine during plantextraction. Phytochemistry 11, 2547. Sandberg, F., Verpoorte, R. and Cronlund, A. (1971) Screening of African StWChncX species for convulsant and muscle-relaxant effects. Acta Pharmaceutics Suecica 8, 341. Verpoorte, R. (1976) Pharmacognostical studies of some African Strychnos species. Thesis, Leiden. Verpoorte, R. and Baerheim Svendsen, A. (1978) Alkaloids of Strychnos dolichothyrsa Gilg ex Onochie et Hepper. Journal of Pharmaceutical Sciences 67, 171. Verpoorte, R., Beek, T.A. van, Thomassen, P.H.A.M., Aandewiel, J. and Baerheim Svendsen, A. (1983) Screening of antimicrobial activity of some plants belonging to the Apocynaceae and Loganiaceae.. Journal of Ethnopharmacology 8, 287-302. Verpoorte, R., Kodde, E.W., Doorne, H. van and Baerheim Svendsen, A. (1978) Antimicrobial effect of the alkaloids from Strychnos afzelii Gilg. Planta Medica 33, 237.
243-241
243
Publishers Ireland Ltd.
Short Communication
ALKALOIDS FROM STRYCHlVOS CHRYSOPHYLLA
R. VERPOORTE,
J. AADEWIEL,
J. STR6MBOM’and
A. BAERHEIM
SVENDSEN
Department of Pharmacognosy, Gorlaeus Laboratories, University of Leiden, P.O. Box 9502,230O RA Leiden (The Netherlands) and ‘Department ofPharmaaognosy, University of Uppsala,P.O. Box 579, S75123 Uppsala (Sweden) (Accepted
October 13,1983)
Strychnos chrysophylla Gilg is a large liana found in the rain forest often along river banks - in S. Nigeria, Cameroon, Gabon and Congo (Brazzaville) (Leeuwenberg 1969). In a pharmacological screening, muscle relaxant effects were noted in tertiary alkaloid fractions of the stembark as well as clonic convulsions (Sandberg et al., 1971). In a screening for antimicrobial activity (Verpoorte et al., 1983) a weak activity was noted against Staphylococcus aureus and Escherichia coli. Bisset and Phillipson (1971) detected at least two alkaloids in extracts of leaves, one of these compounds coloured yellow, and was similar to a yellow alkaloid observed in leaf extracts of a number of Strychnos species. Recently we reported on the presence of longicaudatine in S. chrysophylla (Massiot et al., 1983), in connection with its structure elucidation. Here we will report in detail on the alkaloids found in the stembark of the species mentioned. Experimental Plant material
Stembark of Strychnos chrysophylla was collected by A.J.M. Leeuwenberg (collection number Lg 7865) close to in June-July, 1970. The systematic identification of the formed by Dr. A.J.M. Leeuwenberg. A voucher specimen herbarium in Wageningen, the Netherlands.
F. Sandberg and Kribi (Cameroon) plant was peris kept at the
Extraction Ground plant material (2 kg) was made wet with 10% sodium hydrogen carbonate solution. The plant material was then extracted twice, overnight, with 4 1 diethyl ether. The extracts were combined and taken to dryness (5.28 g) (E,). Subsequently, the plant material was extracted twice, over0378-8741/84/$01.80 o 1984 Elsevier Scientific Published and Printed in Ireland
Publishers Ireland Ltd.
244
night, with 4 1 chloroform. Upon evaporation of the combined chloroform extracts 2.96 g of dry extract (C,) was obtained. The two extracts were further treated, separately, in the following way. The extract was partitioned between ethyl acetate and 2% aqueous acetic acid. The ethyl acetate layer was then further extracted twice with 2% aqueous acetic acid. The combined aqueous layers were basified with NaHCO, (pH - 9) and exhaustively extracted with chloroform. The combined chloroform extracts were taken to dryness. In this way the diethyl ether extract (E,) yielded 50 mg purified tertiary alkaloid extract (E,) and the chloroform extract (C,), 263 mg purified extract (C,). Separation of the alkaloids The tertiary alkaloid extracts Cz and E2 were separated on a Merck ready made Silica gel 60 column (Size B), using chloroform~toluene/absolute ethanol saturated with NH, (9 + 9 f 2) as mobil phase. The major alkaloid fractions contained longicaudatine. The other fractions were further purified by means of preparative TLC. TLC The following
TLC-systems
were used for the separation
of the alkaloids:
(1) toluene/absolute ethanol saturated with ammonia in various ratios (2) toluene/chloroform/absolute ethanol saturated with ammonia (9 -I-9 + 2) (3) petroleum ether (60-80”C)/isopropanol/absolute ethanol saturated with ammonia (85 + 10 + 5) (4) petroleum ether (6~8O’C)/chlorofo~/absoiute ethanol saturated with ammonia (7.5 : 1.25: 1.25) (5) ethyl acetate/isopropanol/25% ammonia (9 + ‘7 + 2) (6) cyclohexane/chloroform/diethylamine (6 + 5 f 1) All solvent systems were used in combination with ready made plates Silica gel F254 (Merck) in saturated chromatography tanks. The alkaloids were detected by means of quenching of UV-light at 254 nm, and by the spray reagents iodoplatinate, 0.2 M ferric (III) chloride in 35% perchloric acid and 1% ceric (IV) sulphate in 10% sulphuric acid. With the latter two reagents, colours were observed immediately after spraying as well as after heating with hot air. Characterization of the alkaloids Longicaudatine. UV(MeOH) showed maxima at 310 (sh), 290,283 and 260 nm. FDMS showed a molecular weight of 568. The ‘H-NMR (300 MHz, CDCIB, 6 in ppm) showed characteristic signals at 7.35 (m), 7.30 (m), 7.00 (m), 6.69 (t,H-lo), 6.30 (d,H-la), 6.10 (bt,H-19), 6.08 (s,H-17’) and 5.62 (q,H-19’). With the ferric (III) chloride in perchloric acid and the ceric (IV) sulphate in sulphurlc acid spray-reagents, a blue colour was observed.
245
These colour reactions and the RtvaIues were the same as for the reference compound (Massiot et al., 1983). Wieland Gumlich aldehyde. UV(MeOH) showed maxima at 298 and 246 nm. EIMS (180°C 70 eV) showed characteristic fragments at m/z: 310 (M’, 48), 292(10), 281(10), 267(10), 199(42), 180(100), 144(62), 143(90) and 130(60). With the ceric (IV) sulphate in sulphuric acid spray-reagent an orange colour was obtained. The colour and the R+lues were the same as for the reference compound (Verpoorte and Baerheim Svendsen, 1978). Caracurine V. UV(MeOH) showed maxima at 304 and 262 nm. EIMS (150°C 10 eV) showed characteristic fragments at m/z 584 (M’, loo), 180( 70), 144(25) and 143(13). The ‘H-NMR (300 MHz, CDCIJ, 6 in ppm) showed characteristic signals at 7.13 (t,H-ll,H-ll’), 7.04(d,H-9,H-9’), 6.83(t,H-lO,H-lo’), 6.44(d,H-12,H-12’), 6.08(bm,I-I-19,H-19’) and 4.70(d,H-17,H-17’). With the ferric (III) chloride in perchloric acid spray-reagent a purple-blue colour was observed, with the ceric (IV) sulphate in sulphuric acid spray-reagent a purple colour, which changed to yellow in the centre of the spot. These colour reactions and the Rtvalues were the same as for the reference compound (Verpoorte and Baerheim Svendsen, 1978). Longicaudatine N-oxide. UV(MeOH) showed maxima at 310 (sh), 290, 283 and 260 nm. FDMS showed ions at 584 (M’, 20) and 568 (M’-16,100%). The ‘H-NMR (300 MHz, CDCl,, 6 in ppm) showed characteristic signals at7.46(m),7.20(m),7.07(m),6.81(t,H-lO),6.59(bt,H-19),6.36(d,H-12), 5.97 (s,H-17’) and 5.85 (q,H-19’). With the ferric (III) chloride in perchloric acid and the ceric (IV) sulphate in sulphuric acid spray-reagents the same colour reactions were observed as for longicaudatine Reduction with sulphurous acid. The alkaloid was dissolved in sulphurous acid, left for 2 h at room temperature, the solution subsequently basified and extracted with chloroform. On TLC one major reaction product was observed with the same colour reactions and RfvaIues as longicaudatine. Oxidation of longicaudatine. Longicaudatine was dissolved in methanol and some drops of 3% hydrogen peroxide solution were added. After 24 h two major products were observed on TLC, the least polar one corresponded with the alkaloid identified as longicaudatine N-oxide on TLC. Results and discussion Only small amounts of alkaloid were found to be present in the stembark (0.016% tertiary alkaloids). Column chromatography of the tertiary alkaloid fraction and following preparative TLC yielded four alkaloids. Three of these could be identified by means of their spectral data and comparison with reference alkaloids as Wieland-Gumlich aIdehyde, caracurine V and longicaudatine (Fig. 1). The fourth alkaloid, a minor compound, showed the same colour reactions on TLC and had the same UV-spectrum as longicaudatine. In pure
246
Fig. 1. Longicaudatine,
longicaudatine N,-oxide.
longicaudatine this alkaloid could be observed after some time. The unknown alkaloid showed two ions in the FDMS at m/z 568 and m/z 584. N-oxides are known to readily lose oxygen, thus giving rise to a large M’-16 fragment (Hesse, 1974). The MS thus pointed to a mono-N-oxide of longicaudatine. Indeed, oxidation of longicaudatine with hydrogen peroxide at room temperature gave, as major reaction product, a compound with the same TLC-behaviour as the unknown alkaloid. Reduction of the latter gave a compound with the same TLC-behaviour and colour reaction as longicaudatine. The small amount of material available resulted in a ‘H-NMR of which only the aromatic region was intelligible. The most apparent difference with the spectrum of longicaudatine was the shift of the broad triplet of H-19 from 6.10 ppm to 6.58 ppm in the N-oxide. A similar change is observed in the ‘H-NMR of strychnine N-oxide if compared with that of strychnine (Verpoorte, unpublished results). The Strychnos moiety of the dimer is thus the most likely place of N-oxidation. This agrees with the well-known ease of N-oxidation in the strychnine-type of alkaloids (Phillipson and Bisset, 1972; Verpoorte, 1976). The alkaloid is thus identified as longicaudatine N,-oxide. Due to the use of chloroform in the isolation of the alkaloids this alkaloid could be an artefact. The presence of a tertiary alkaloid of the toxiferine type (caracurine V) does explain the weak muscle relaxation found for the tertiary alkaloid extract (Verpoorte and Baerheim Svendsen, 1977). The antimicrobial activity of caracurine V (Verpoorte et al., 1978) agrees with the effect found in the general screening of the extract. However, according to biograms a further - yet unidentified - non-alkaloidal compound which has antimicrobial activity is also present. References B&et, N.G. and Phillipson, J.D. (1971) The African Species of Strychnos, Part II, The Alkaloids. Lloydia 34, 1. Hesse, M. (1974) Progress in Mass Spectrometry, Vol. 1, Indolalkaloide. Verlag Chemie, Weinheim, p. 5.
247 Leeuwenberg, A.J.M. (1969) The Loganiaceae of Africa VIII - Sttychnos III, Mededelingen Landbouwhogeechool Wageningen 69, 1. Massiot, G., Zeches, M., Mirand, C., Le Men-Olivier, L., Delaude, C., Baser, K.H.C., Bavovada, R., Bisset, N.G., Hylands, P.J., Stromborn, J. and Verpoorte, R. (1983) Occurrence of longicaudatine, a new type of bis-indole base and bisnor-C-alkaloid H in Strychnos species. Journal of Organic Chemistry, in press. Phillipson, J.D. and Bisset, N.G. (1972) Quaternisation and oxidation of strychnine and brucine during plantextraction. Phytochemistry 11, 2547. Sandberg, F., Verpoorte, R. and Cronlund, A. (1971) Screening of African StWChncX species for convulsant and muscle-relaxant effects. Acta Pharmaceutics Suecica 8, 341. Verpoorte, R. (1976) Pharmacognostical studies of some African Strychnos species. Thesis, Leiden. Verpoorte, R. and Baerheim Svendsen, A. (1978) Alkaloids of Strychnos dolichothyrsa Gilg ex Onochie et Hepper. Journal of Pharmaceutical Sciences 67, 171. Verpoorte, R., Beek, T.A. van, Thomassen, P.H.A.M., Aandewiel, J. and Baerheim Svendsen, A. (1983) Screening of antimicrobial activity of some plants belonging to the Apocynaceae and Loganiaceae.. Journal of Ethnopharmacology 8, 287-302. Verpoorte, R., Kodde, E.W., Doorne, H. van and Baerheim Svendsen, A. (1978) Antimicrobial effect of the alkaloids from Strychnos afzelii Gilg. Planta Medica 33, 237.