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Sterol constituents in seeds of Ornithopus sativus
Phyrochemrstry, Vol. 33,No 2,pp.506-507, 1993 Printed in Great Britain.
003l-9422/93 S6.00+ 0.00 Q 1993Pergamon Press Ltd
STEROL CONSTITUENTS
IN SEEDS OF ORNITHOPUS
SATIVUS
J~RGEN SCHMIDT, BARBARASPENGLER, G~NTER ADAM and HERBERT BUDZIKIEWICZ* Institute of Plant Biochemistry, Weinberg 3, O-4050HaIle/S., Germany; *Institute for Organic Chemistry, University of Cofogne,
GreinstraDe 41, W-5ooOKiiln, Germany
Key Word Index-Omithopus hydroxysterols.
sativus;
Leguminoseae;
seeds; B-amyrin; sterols; 7-oxosterols;
7-
Abstract-The phytosterols of the seeds of Ornithopus sativus were identified by capillary GC and GC-MS. Several 7oxo- and the corresponding 7a- and 7/?-hydroxysterols and the pentacyclic triterpene p-amyrin were identified.
Table 1. Sterol composition in the seeds of 0. sutious Recently, we reported on the occurrence of the brassinosteroids castasterone and 24-epi-castasterone in the seeds of Ornithopus sativus [l]. Phytosterols are assumed to be the biogenetic precursors of brassinosteroids [2]. Therefore, we were interested to know the sterol pattern of this plant material. This paper describes the isolation and identification of the sterol constituents of the cultural crop Serradella (Omithopus sativus Brot.).
Sterol
$2)
RR, (as acetates)
Composition (%)
Cholesterol Campesterol Stigmasterol Sitosterol
1.55 1.91 2.03 2.28
2.00 2.47 2.64 2.95
5 11 I 71
*Relative retention time with respect to 5a-cholestane. RESULTS AND DISCUSSION
The seeds of O~itho~us sativus (100 g) were powdered and extracted with ether in a Soxhlet for 72 hr. The etherextract (8.7 g) was partitioned between water and chloroform. The chloroform fraction (8.4 g) was chromatographed on silica gel using a n-hexane-ether-methanol gradient system. Further purification was achieved by preparative TLC. The fraction eluted with nhexane-ether (7:3) (30 mg) was purified by preparative TLC using chloroform-methanol (99 : 1) as the developing solvent. The compound thus obtained (R/ 0.7,21 mg after crystallization from methanol) was identified as the pentacyclic triterpene /?-amyrin (MS, IR, ‘H NMR, [orIn, R, mp). The fraction eluted with n-hexane-ether (1: 1) (14 mg) was separated by preparative TLC (chloroform-methanol, 97: 3). Cholesterol, campesterol, stigmasterol and sitosterol (Rs 0.66, IO mg) were identified by capillary gas chromatography and GC-MS by comparison with authentic samples [3] (Table 1). The fraction eluted with n-hexane-ether (1:9) (13 mg) was further purified by preparative TLC (chloroformmethanol 19: 1) and the obtained material (R/ 0.65) was shown by GC and GC-MS to be a mixture of the 7oxoderivatives of campesterol, stigmasterol and sitosterol by comparison with authentic samples (Table 2). From the fraction eluted with methanol (137 mgf a mixture of six 7-hydroxysterols was obtund by preparative TLC
Table 2. 7-Oxosterols in the seeds of 0. safivus Sterof (free)
RR:
Composition (%)
7-Oxocampesterol 7-Oxostigmasterol 7-Oxositosterol
3.33 3.57 4.OI
23 14 63
*Relative retention time with respect to Sa-cholestane.
with chlorofo~-methanol(19: 1, R, 0.35, 11 mg). Using ether-cyclohexane (9: 1) as a developing solvent [4] the 7a- and 7@-isomers with R, 0.25 (2 mg) and 0.4 (1.5 mg), respectively, were separated. The identification of the 7hydroxysterols was carried out by mass spectrometry and capillary gas chromatography of the corresponding TMS ethers by comparison with authentic samples (Table 3). The origins of sterols oxidized at C-7 either by a nonselective autoxidation [S, 63 or by a non-selective biological oxidation [7] have been considered. These compounds show antitumour activity [S, 93, whereas 7/?hydroxycampesterol was shown to be the most active compound isolated from Euphorbia fischeriuna [73. With regard to a possible co-occurrence of brassinosteroids and their hypothetic precursor sterols in seeds of
Short Reports Table 3. ‘I-Hydroxysterols in the seeds of 0. sativus Sterol (as TMS ether)
RR+*
Composition (%)
7a-Hydroxycampesterol 7a-Hydroxystigmasterol 7a-Hydroxysitosterol 7p-Hydroxycampesterol 7B-Hydroxystigmasterol II/I-Hydroxysitosterol
2.06 2.14 2.44 2.69 2.75 3.18
18 14 68 22 17 61
507
The GC-MS measurements were performed with an KRATOS M 25 instrument coupled with a Carlo Erba MFC 500 gas chromatograph (column SE-52, 25 m x 0.32 mm, film thickness 0.25 pm); temp. program: 210” for 2 min and then raised to 280” at a rate of 15” min-‘; carrier gas He, flow rate 1 mlmin- ‘; splitless injection. Acknowledgement-The authors are indebted to Mrs C. Kuhnt for the GC measurements and the Deutsche Forschungsgemeinschaft for financial support.
*Relative retention time with respect to Sa-cholestane. REFERENCES
1. Schmidt, J., Spengler, B., Yokota, T. and Adam, G.
Ornithopus satious it is of special interest, that compared to the observed sterol composition, only brassinosteroids with a C,,-skeleton (castasterone and its 24epimer) could be detected [ 11. Campesterol is the only identified C,,-sterol in seeds of Ornithopus satious. EXPERIMENTAL
Plant material. The seeds of Ornithopus sativus Brot. var. Mecklenburger were obtained from Saat und Pllanz.en Wittenberg, Germany. Instrumentation. The capillary GC measurements were carried out on a Carlo Erba MEGA 5160 instrument under the following conditions: column PB-1 (WGA, Werner Giinther Analysentechnik, 50 m x 0.32 mm, 0.25 w film thickness); inj. temp. 225”, column temp. 270” (isothermal); detection FID (temp. 290”); carrier gas N,, flow rate 1.5 mlmin-‘, split injection (split ratio 1:20). The acetylation of sterols was carried out with Ac,O-pyridine at room temp. for 12 hr. The dihydroxysterols were silylated with Sil-Prep (pyridine-HMDSTMCS 9: 3: 1, SERVA) for 60 min at room temp.
(1993) Phytochemistry 32 (in press). 2. Yokota, T., Ogino, Y., Suzuki, H., Takahashi, N., Saimoto, H., Fujioka, S. and Sakurai, A. (1991) in Brassinosteroids-Chemistry, Bioactioity, Application (Cutler, H. G., Yokota, T. and Adam, G., eds), p.86. ACS Symposium Series 474, American Chemical Society, Washington DC. 3. Goad, L. J. (1991) in Methods in Plant Biochemistry Vol. 7, Chap. 11, p. 369. 4. Daly, G. G., Finocchiaro, T. and Richardson, T. (1983) J. Agric. Food Chem. 31, 46. 5. Van Lier, J. E. and Smith, L. L. (1970) J. Org. Gem. 35, 2627. 6. Teng, T. J., Kuhlig, M. J., Smith, L. L. and Kang, G. (1973) J. Org. Chem. 38, 119. 7. Schroeder, G., Rohrner, M., Beck, J. P. and Anion, R. (1980) Phytochemistry 19, 2213. 8. Cheng, K. P., Nagano, H., Bang, L. and Ourisson, G. (1977) J. Chem. Res. (S) 217; J. Chem. Res. (M) 2501. 9. Nagano, H., Poyser, J. P., Cheng, K. P., Bang, L. and Ourisson, G. (1977) J. Chem. Res. (s) 218; J. Chem. Res. (M) 2522.
003l-9422/93 S6.00+ 0.00 Q 1993Pergamon Press Ltd
STEROL CONSTITUENTS
IN SEEDS OF ORNITHOPUS
SATIVUS
J~RGEN SCHMIDT, BARBARASPENGLER, G~NTER ADAM and HERBERT BUDZIKIEWICZ* Institute of Plant Biochemistry, Weinberg 3, O-4050HaIle/S., Germany; *Institute for Organic Chemistry, University of Cofogne,
GreinstraDe 41, W-5ooOKiiln, Germany
Key Word Index-Omithopus hydroxysterols.
sativus;
Leguminoseae;
seeds; B-amyrin; sterols; 7-oxosterols;
7-
Abstract-The phytosterols of the seeds of Ornithopus sativus were identified by capillary GC and GC-MS. Several 7oxo- and the corresponding 7a- and 7/?-hydroxysterols and the pentacyclic triterpene p-amyrin were identified.
Table 1. Sterol composition in the seeds of 0. sutious Recently, we reported on the occurrence of the brassinosteroids castasterone and 24-epi-castasterone in the seeds of Ornithopus sativus [l]. Phytosterols are assumed to be the biogenetic precursors of brassinosteroids [2]. Therefore, we were interested to know the sterol pattern of this plant material. This paper describes the isolation and identification of the sterol constituents of the cultural crop Serradella (Omithopus sativus Brot.).
Sterol
$2)
RR, (as acetates)
Composition (%)
Cholesterol Campesterol Stigmasterol Sitosterol
1.55 1.91 2.03 2.28
2.00 2.47 2.64 2.95
5 11 I 71
*Relative retention time with respect to 5a-cholestane. RESULTS AND DISCUSSION
The seeds of O~itho~us sativus (100 g) were powdered and extracted with ether in a Soxhlet for 72 hr. The etherextract (8.7 g) was partitioned between water and chloroform. The chloroform fraction (8.4 g) was chromatographed on silica gel using a n-hexane-ether-methanol gradient system. Further purification was achieved by preparative TLC. The fraction eluted with nhexane-ether (7:3) (30 mg) was purified by preparative TLC using chloroform-methanol (99 : 1) as the developing solvent. The compound thus obtained (R/ 0.7,21 mg after crystallization from methanol) was identified as the pentacyclic triterpene /?-amyrin (MS, IR, ‘H NMR, [orIn, R, mp). The fraction eluted with n-hexane-ether (1: 1) (14 mg) was separated by preparative TLC (chloroform-methanol, 97: 3). Cholesterol, campesterol, stigmasterol and sitosterol (Rs 0.66, IO mg) were identified by capillary gas chromatography and GC-MS by comparison with authentic samples [3] (Table 1). The fraction eluted with n-hexane-ether (1:9) (13 mg) was further purified by preparative TLC (chloroformmethanol 19: 1) and the obtained material (R/ 0.65) was shown by GC and GC-MS to be a mixture of the 7oxoderivatives of campesterol, stigmasterol and sitosterol by comparison with authentic samples (Table 2). From the fraction eluted with methanol (137 mgf a mixture of six 7-hydroxysterols was obtund by preparative TLC
Table 2. 7-Oxosterols in the seeds of 0. safivus Sterof (free)
RR:
Composition (%)
7-Oxocampesterol 7-Oxostigmasterol 7-Oxositosterol
3.33 3.57 4.OI
23 14 63
*Relative retention time with respect to Sa-cholestane.
with chlorofo~-methanol(19: 1, R, 0.35, 11 mg). Using ether-cyclohexane (9: 1) as a developing solvent [4] the 7a- and 7@-isomers with R, 0.25 (2 mg) and 0.4 (1.5 mg), respectively, were separated. The identification of the 7hydroxysterols was carried out by mass spectrometry and capillary gas chromatography of the corresponding TMS ethers by comparison with authentic samples (Table 3). The origins of sterols oxidized at C-7 either by a nonselective autoxidation [S, 63 or by a non-selective biological oxidation [7] have been considered. These compounds show antitumour activity [S, 93, whereas 7/?hydroxycampesterol was shown to be the most active compound isolated from Euphorbia fischeriuna [73. With regard to a possible co-occurrence of brassinosteroids and their hypothetic precursor sterols in seeds of
Short Reports Table 3. ‘I-Hydroxysterols in the seeds of 0. sativus Sterol (as TMS ether)
RR+*
Composition (%)
7a-Hydroxycampesterol 7a-Hydroxystigmasterol 7a-Hydroxysitosterol 7p-Hydroxycampesterol 7B-Hydroxystigmasterol II/I-Hydroxysitosterol
2.06 2.14 2.44 2.69 2.75 3.18
18 14 68 22 17 61
507
The GC-MS measurements were performed with an KRATOS M 25 instrument coupled with a Carlo Erba MFC 500 gas chromatograph (column SE-52, 25 m x 0.32 mm, film thickness 0.25 pm); temp. program: 210” for 2 min and then raised to 280” at a rate of 15” min-‘; carrier gas He, flow rate 1 mlmin- ‘; splitless injection. Acknowledgement-The authors are indebted to Mrs C. Kuhnt for the GC measurements and the Deutsche Forschungsgemeinschaft for financial support.
*Relative retention time with respect to Sa-cholestane. REFERENCES
1. Schmidt, J., Spengler, B., Yokota, T. and Adam, G.
Ornithopus satious it is of special interest, that compared to the observed sterol composition, only brassinosteroids with a C,,-skeleton (castasterone and its 24epimer) could be detected [ 11. Campesterol is the only identified C,,-sterol in seeds of Ornithopus satious. EXPERIMENTAL
Plant material. The seeds of Ornithopus sativus Brot. var. Mecklenburger were obtained from Saat und Pllanz.en Wittenberg, Germany. Instrumentation. The capillary GC measurements were carried out on a Carlo Erba MEGA 5160 instrument under the following conditions: column PB-1 (WGA, Werner Giinther Analysentechnik, 50 m x 0.32 mm, 0.25 w film thickness); inj. temp. 225”, column temp. 270” (isothermal); detection FID (temp. 290”); carrier gas N,, flow rate 1.5 mlmin-‘, split injection (split ratio 1:20). The acetylation of sterols was carried out with Ac,O-pyridine at room temp. for 12 hr. The dihydroxysterols were silylated with Sil-Prep (pyridine-HMDSTMCS 9: 3: 1, SERVA) for 60 min at room temp.
(1993) Phytochemistry 32 (in press). 2. Yokota, T., Ogino, Y., Suzuki, H., Takahashi, N., Saimoto, H., Fujioka, S. and Sakurai, A. (1991) in Brassinosteroids-Chemistry, Bioactioity, Application (Cutler, H. G., Yokota, T. and Adam, G., eds), p.86. ACS Symposium Series 474, American Chemical Society, Washington DC. 3. Goad, L. J. (1991) in Methods in Plant Biochemistry Vol. 7, Chap. 11, p. 369. 4. Daly, G. G., Finocchiaro, T. and Richardson, T. (1983) J. Agric. Food Chem. 31, 46. 5. Van Lier, J. E. and Smith, L. L. (1970) J. Org. Gem. 35, 2627. 6. Teng, T. J., Kuhlig, M. J., Smith, L. L. and Kang, G. (1973) J. Org. Chem. 38, 119. 7. Schroeder, G., Rohrner, M., Beck, J. P. and Anion, R. (1980) Phytochemistry 19, 2213. 8. Cheng, K. P., Nagano, H., Bang, L. and Ourisson, G. (1977) J. Chem. Res. (S) 217; J. Chem. Res. (M) 2501. 9. Nagano, H., Poyser, J. P., Cheng, K. P., Bang, L. and Ourisson, G. (1977) J. Chem. Res. (s) 218; J. Chem. Res. (M) 2522.