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Content of zygacine in Zygadenus venenosus at different stages of growth
Phytochemistry, Vol. 31, No. 10,pp. 3417-3418,1992 Printed in Great Britain.
0031-9422/92$5.00+0.00 Pergamon Press Ltd
CONTENT OF ZYGACINE IN ZYGADENUS VENENOSUS STAGES OF GROWTH WALTER MAJAK,RUTH
E.MC~IARMID,WALTER~RISTOFOLI,*
AT DIFFERENT
FANG SUN* and MICHAEL
BENN*
Research Station, Agriculture Canada, 3015 Ord Road, Kamloops, Canada V2B 8A9; *Chemistry Department, The University of Calgary, Calgary, Alberta Canada T2N lN4 (Received in revisedform 20 March 1992) Xey Word Index-Zygadenus
venenosus; Liliaceae; death camas; fluorescence TLC scanning; steroidal alkaloids.
Abstract-A
TLC-scanning procedure was developed for the quantitative determination of zygacine in death camas. The alkaloid levels increased during the pod stage of development when the veratryl and angelyl esters of zygadenine were also detected at high concentrations.
INTRODUCTION
Zygadenine (l), the steroid alkaloid of death camas (Zygadenus spp.) was first isolated in 1913 but its structure and configuration were not reported until 1959 [I]. However, the toxicity of Z. uenenosus and Z. panicula~~ to livestock [Z, 33 and humans [4] has been mainly attributed to the acetyl (Z), vanillyl and veratryl esters of zygadenine and not to zygadenine itself Cl, 51. Zygacine (2) was reported as the major alkaloid in Z. gramineus [6] which is now considered to be Z. venenosus Wats. var. pamineus (Rybd.) Walsh [fl. To our knowledge, there is no information in the literature on the specific concentration of these alkaloids in death camas. The purpose of this study was to develop a method for the quantitative determination of zygacine in Z. venenosus var. gramineus at different stages of growth and hence to further comprehend and predict the toxicity of the plant. RESULTS AND DISCUSSION
Zygacine was readily isolated from Z. venenosus by differential partitioning of free bases at different pH. When the 0.4 N sulphuric acid extract [S] was adjusted to pH 4, the resultant chloroform extraction yielded a mixture of the veratryl and angelyl esters of zygadenine. The *3C NMR data contained a set of resonances corresponding to that reported for the 3-0-angelate obtained from Verutrum maackii [Q] plus additional resonances for the 3-0-veratroate. At pH 8, nearly pure zygacine was obtained, as evidenced by ‘H and 13C NMR spectroscopy. At pH 10, small quantities of zygadenine were isolated. Only trace amounts of zygadenine were detected in freshly extracted samples or in freeze-dried ones that were stored for less than one month. Freeze-dried samples of Zygadenus stored for more than one year or methanolic concentrates stored without desiccation yielded larger amounts of zygadenine, suggesting that the esters of zygadenine are hydrolysed during storage. Initially, the alkaloids of Z. uenenosus were visualized with a meth~ol~ulphu~c acid (1: 1) spray [6] which, in visible light, yielded red spots on silica gel TLC after
P -
a (1)
R = acetyl
(2)
heating. However, examination of the acid-sprayed plates under UV light showed a bright, whitish-blue Buorescence for the Zygadenus alkaloids, as might be expected for the colour reactions of steroids [lo]. Accordingly, we developed TLC fluorescence scanning conditions which proved to be very sensitive with detection limits for zygacine of 20 ng per spot and quantitation usually performed in the 100-1000 ng range. The acid spray was modified (s~phu~c acid-methanol, 1 :Q) to minimize the sulphuric acid abstraction of atmospheric water that quenched the fluorescence. Attempts to quantify Zygadenus alkaloids by GC (TM%) or HPLC (UV detection) were unsuccessful. When the zygacine concentration was examined at different stages of death camas growth, the alkaloid levels were significantly less (P < 0.001) at the bloom stage than at the vegetative or pod stages of growth (Fig. 1). The lower level at bloom, as compared to the vegetative stage of growth, could be partly attributed to the growth and elongation of the plant and the concomitant dilution of the alkaloid. The average height (k s.e.) of the plant was 21.7-1-0.8 cm at the vegetative stage but it increased to 37.8 + 2.4 cm and 45.0 + 1 cm at the bloom and pod stages
3417
W. MAJAK et al.
3418
r
:
Jwer 5TAGE
Ia
J
OF GROWTH
Fig. 1. Zygacine levels. (% dry matter) in Zygadenus venenosus at different stages of growth during 1991. Means are shown with standard errors. Veg=vegetative stage of growth.
var. gramineus
of growth, respectively. This suggests that the alkaloid increase during the pod stage (Fig. 1) was the result of de nova synthesis, since the biomass of the plant was not extensively enlarged between the bloom and pod stages of growth. In agreement, the veratryl and angelyl esters, which were not present during vegetative stages, yielded detectable levels (< 0.1%) during bloom stages but their concentration increased dramatically (0.25 f 0.02% in equivalents of zygacine) during the pod stage of development. The zygadenine esters were mainly associated with reproductive organs such as flowers and pods where zygacine levels ranged from 0.56 to 0.85%. Although Zygadenus bulbs are often implicated in poisoning [2,4], analysis of a composite sample of bulbs at the early pod stage of growth showed only low concentrations (< 0.05%) of zygacine. However, bulbs could be more toxic at other stages of growth, especially if the alkaloids are translocated as root reserves, possibly to the reproductive organs.
EXPERIMENTAL General. Aerial portions of Z. venenosus were collected near Lac du Bois, 11 km from Kamloops, BC. Plant samples at the vegetative (n = 6), bloom (n = 12) and pod (n = 9) stages were freeze-dried and ground. Each sample (1 g) was for free bases as described previously [S] but the adjusted to 8.5 before the final partition into CHCI,.
of growth extracted pH was A similar
procedure was used for the large scale extractron of 1.5 kg fr. material, which was comminuted m MeOH at room temp. For this extract, the pH was adjusted to 4, 8 and 10 before the final partitions into CHCI,. The pH 8 extract yielded nearly pure (710 mg) and, after prep. silica gel TLC zygacine (CHCl,-MeOH-35% aq NH,, 90:10:1), the pH 4 extract (204 mg) yielded a mixt. of the veratryl and angelyl esters of zygadenine. The pH 10 extract contained small quantities of zygadenine. Quantitattoe determination. Free base extracts were resuspended in 2-10 ml MeOH immediately before TLC, and for subsequent storage they were coned to dryness to minimize conversion of zygacine to zygadenine. Aliquots of 1 or 2 ~1 were applied to silica gel plates (Merck No. 5721 or Macherey-Nagel No. 809033), each plate was calibrated with zygacine standards (100-1000 ng), and determinations were always replicated on more than one plate, usually two, The plate was developed in a well-satd chamber with freshly prepared solvent (90: 10: 1) to a height of 11-13 cm, dried in a fume hood for at least 15 min, and then thoroughly sprayed with cont. H,SO,-MeOH (1:9). The plate was then heated in an oven at loo” for 30min and the whitish-blue fluorescence was visualized under UV light (365 nm). The R, values were 0.14 (zygadenine), 0.40 (zygacine), 0.49 (angelyl ester) and 0.51 (veratryl ester); coordinates were determined for each spot. The TLC fluorescence scanning procedures were similar to those described previously [ll] but a 7-60 primary filter, a 2A secondary filter and a near UV lamp (Turner No. 110-850) were used for quantifying the alkaloids of Zygadenus. When an authentic sample ofzygacine was subjected to the above fractionation procedure, It was recovered in 88% yield.
REFERENCES 1. Kupchan, M. (1959) J. Am. Chern. Sot. 81, 1925. 2. Kingsbury, J. M. (1964) Poisonous Plants of the United States and Canada. Prentice-Hall, Englewood Cliffs, NJ. 3. Panter, K. E., Ralphs. M. H., Smart, R. A. and Duelke. B. (1987) Vet. Hum. Toxicol. 29, 45. 4. Wagstaff, D. J. (1987) Clin. Toxrcol. 25, 361. 5. Kupchan, M. and Deliwala, C. V. (1953) J. Am. Chem. Sot. 75, 1025. 6. Gilbertson, T. J. (1973) Phytochemistry 12, 2079. 7. Hitchcock, C. L. and Cronquist, A. (1981) Flora ofthe Pacific Northwest. University of Washington Press, Seattle. 8. Majak, W., McDiarmid, R. E. and Benn, M. H. (1987) J. Agric. Food Chem. 35, 800. 9. Zhao, W., Tezuka, Y., Kikuchi, K., Chen, J. and Guo, Y. (1989) Gem. Pharm. Bull. (Japan) 37, 2920. 10. Stahl, E. (1969) Thin-Layer Chromatography. A Laboratory Handbook. Springer, New York. 11. MaJak, W. and Bose, R. J. (1977) Phytochemistry 16,749.
0031-9422/92$5.00+0.00 Pergamon Press Ltd
CONTENT OF ZYGACINE IN ZYGADENUS VENENOSUS STAGES OF GROWTH WALTER MAJAK,RUTH
E.MC~IARMID,WALTER~RISTOFOLI,*
AT DIFFERENT
FANG SUN* and MICHAEL
BENN*
Research Station, Agriculture Canada, 3015 Ord Road, Kamloops, Canada V2B 8A9; *Chemistry Department, The University of Calgary, Calgary, Alberta Canada T2N lN4 (Received in revisedform 20 March 1992) Xey Word Index-Zygadenus
venenosus; Liliaceae; death camas; fluorescence TLC scanning; steroidal alkaloids.
Abstract-A
TLC-scanning procedure was developed for the quantitative determination of zygacine in death camas. The alkaloid levels increased during the pod stage of development when the veratryl and angelyl esters of zygadenine were also detected at high concentrations.
INTRODUCTION
Zygadenine (l), the steroid alkaloid of death camas (Zygadenus spp.) was first isolated in 1913 but its structure and configuration were not reported until 1959 [I]. However, the toxicity of Z. uenenosus and Z. panicula~~ to livestock [Z, 33 and humans [4] has been mainly attributed to the acetyl (Z), vanillyl and veratryl esters of zygadenine and not to zygadenine itself Cl, 51. Zygacine (2) was reported as the major alkaloid in Z. gramineus [6] which is now considered to be Z. venenosus Wats. var. pamineus (Rybd.) Walsh [fl. To our knowledge, there is no information in the literature on the specific concentration of these alkaloids in death camas. The purpose of this study was to develop a method for the quantitative determination of zygacine in Z. venenosus var. gramineus at different stages of growth and hence to further comprehend and predict the toxicity of the plant. RESULTS AND DISCUSSION
Zygacine was readily isolated from Z. venenosus by differential partitioning of free bases at different pH. When the 0.4 N sulphuric acid extract [S] was adjusted to pH 4, the resultant chloroform extraction yielded a mixture of the veratryl and angelyl esters of zygadenine. The *3C NMR data contained a set of resonances corresponding to that reported for the 3-0-angelate obtained from Verutrum maackii [Q] plus additional resonances for the 3-0-veratroate. At pH 8, nearly pure zygacine was obtained, as evidenced by ‘H and 13C NMR spectroscopy. At pH 10, small quantities of zygadenine were isolated. Only trace amounts of zygadenine were detected in freshly extracted samples or in freeze-dried ones that were stored for less than one month. Freeze-dried samples of Zygadenus stored for more than one year or methanolic concentrates stored without desiccation yielded larger amounts of zygadenine, suggesting that the esters of zygadenine are hydrolysed during storage. Initially, the alkaloids of Z. uenenosus were visualized with a meth~ol~ulphu~c acid (1: 1) spray [6] which, in visible light, yielded red spots on silica gel TLC after
P -
a (1)
R = acetyl
(2)
heating. However, examination of the acid-sprayed plates under UV light showed a bright, whitish-blue Buorescence for the Zygadenus alkaloids, as might be expected for the colour reactions of steroids [lo]. Accordingly, we developed TLC fluorescence scanning conditions which proved to be very sensitive with detection limits for zygacine of 20 ng per spot and quantitation usually performed in the 100-1000 ng range. The acid spray was modified (s~phu~c acid-methanol, 1 :Q) to minimize the sulphuric acid abstraction of atmospheric water that quenched the fluorescence. Attempts to quantify Zygadenus alkaloids by GC (TM%) or HPLC (UV detection) were unsuccessful. When the zygacine concentration was examined at different stages of death camas growth, the alkaloid levels were significantly less (P < 0.001) at the bloom stage than at the vegetative or pod stages of growth (Fig. 1). The lower level at bloom, as compared to the vegetative stage of growth, could be partly attributed to the growth and elongation of the plant and the concomitant dilution of the alkaloid. The average height (k s.e.) of the plant was 21.7-1-0.8 cm at the vegetative stage but it increased to 37.8 + 2.4 cm and 45.0 + 1 cm at the bloom and pod stages
3417
W. MAJAK et al.
3418
r
:
Jwer 5TAGE
Ia
J
OF GROWTH
Fig. 1. Zygacine levels. (% dry matter) in Zygadenus venenosus at different stages of growth during 1991. Means are shown with standard errors. Veg=vegetative stage of growth.
var. gramineus
of growth, respectively. This suggests that the alkaloid increase during the pod stage (Fig. 1) was the result of de nova synthesis, since the biomass of the plant was not extensively enlarged between the bloom and pod stages of growth. In agreement, the veratryl and angelyl esters, which were not present during vegetative stages, yielded detectable levels (< 0.1%) during bloom stages but their concentration increased dramatically (0.25 f 0.02% in equivalents of zygacine) during the pod stage of development. The zygadenine esters were mainly associated with reproductive organs such as flowers and pods where zygacine levels ranged from 0.56 to 0.85%. Although Zygadenus bulbs are often implicated in poisoning [2,4], analysis of a composite sample of bulbs at the early pod stage of growth showed only low concentrations (< 0.05%) of zygacine. However, bulbs could be more toxic at other stages of growth, especially if the alkaloids are translocated as root reserves, possibly to the reproductive organs.
EXPERIMENTAL General. Aerial portions of Z. venenosus were collected near Lac du Bois, 11 km from Kamloops, BC. Plant samples at the vegetative (n = 6), bloom (n = 12) and pod (n = 9) stages were freeze-dried and ground. Each sample (1 g) was for free bases as described previously [S] but the adjusted to 8.5 before the final partition into CHCI,.
of growth extracted pH was A similar
procedure was used for the large scale extractron of 1.5 kg fr. material, which was comminuted m MeOH at room temp. For this extract, the pH was adjusted to 4, 8 and 10 before the final partitions into CHCI,. The pH 8 extract yielded nearly pure (710 mg) and, after prep. silica gel TLC zygacine (CHCl,-MeOH-35% aq NH,, 90:10:1), the pH 4 extract (204 mg) yielded a mixt. of the veratryl and angelyl esters of zygadenine. The pH 10 extract contained small quantities of zygadenine. Quantitattoe determination. Free base extracts were resuspended in 2-10 ml MeOH immediately before TLC, and for subsequent storage they were coned to dryness to minimize conversion of zygacine to zygadenine. Aliquots of 1 or 2 ~1 were applied to silica gel plates (Merck No. 5721 or Macherey-Nagel No. 809033), each plate was calibrated with zygacine standards (100-1000 ng), and determinations were always replicated on more than one plate, usually two, The plate was developed in a well-satd chamber with freshly prepared solvent (90: 10: 1) to a height of 11-13 cm, dried in a fume hood for at least 15 min, and then thoroughly sprayed with cont. H,SO,-MeOH (1:9). The plate was then heated in an oven at loo” for 30min and the whitish-blue fluorescence was visualized under UV light (365 nm). The R, values were 0.14 (zygadenine), 0.40 (zygacine), 0.49 (angelyl ester) and 0.51 (veratryl ester); coordinates were determined for each spot. The TLC fluorescence scanning procedures were similar to those described previously [ll] but a 7-60 primary filter, a 2A secondary filter and a near UV lamp (Turner No. 110-850) were used for quantifying the alkaloids of Zygadenus. When an authentic sample ofzygacine was subjected to the above fractionation procedure, It was recovered in 88% yield.
REFERENCES 1. Kupchan, M. (1959) J. Am. Chern. Sot. 81, 1925. 2. Kingsbury, J. M. (1964) Poisonous Plants of the United States and Canada. Prentice-Hall, Englewood Cliffs, NJ. 3. Panter, K. E., Ralphs. M. H., Smart, R. A. and Duelke. B. (1987) Vet. Hum. Toxicol. 29, 45. 4. Wagstaff, D. J. (1987) Clin. Toxrcol. 25, 361. 5. Kupchan, M. and Deliwala, C. V. (1953) J. Am. Chem. Sot. 75, 1025. 6. Gilbertson, T. J. (1973) Phytochemistry 12, 2079. 7. Hitchcock, C. L. and Cronquist, A. (1981) Flora ofthe Pacific Northwest. University of Washington Press, Seattle. 8. Majak, W., McDiarmid, R. E. and Benn, M. H. (1987) J. Agric. Food Chem. 35, 800. 9. Zhao, W., Tezuka, Y., Kikuchi, K., Chen, J. and Guo, Y. (1989) Gem. Pharm. Bull. (Japan) 37, 2920. 10. Stahl, E. (1969) Thin-Layer Chromatography. A Laboratory Handbook. Springer, New York. 11. MaJak, W. and Bose, R. J. (1977) Phytochemistry 16,749.