逆流色谱分离黄酮类化合物——大豆、藤茶和白水曲柳皮中黄酮类化合物的分离
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摘要
黄酮类化合物(Flavonoids)是自然界存在的最大的一类酚性物质，具有抗心血管病、抗癌、抗菌等生物活性。本文主要探讨了逆流色谱法分离大豆、藤茶和白水曲柳皮中的黄酮类化合物，结果表明，逆流色谱法是一种非常有效的分离黄酮类化合物的方法。
以两相溶剂系统正己烷：乙酸乙酯：丁醇：冰乙酸：水(1：2：1：5：1，v／v)，用装配260mL和1100mL柱的高速逆流色谱仪分离了大豆异黄酮样品，260mL分离柱可一次分离500mg大豆异黄酮样品获得大豆甙33mg，染料木甙41mg，6’’-丙二酰葡萄糖基大豆甙37mg和6’’-丙二酰葡萄糖基染料木甙24mg。1100mL柱分离可一次分离3g大豆异黄酮样品获得大豆甙203mg，染料木甙241mg，6’’-丙二酰葡萄糖基大豆甙217mg和6’’-丙二酰葡萄糖基染料木甙135mg。
以水：甲醇：乙酸乙酯：正乙烷(4：2：3：1,v／v)构成两相溶剂系统，16g藤茶甲醇提取物在三柱串联高速逆流色谱上经9小时分离，获得了11.3g二氢黄酮醇单体
(1)。用水对藤茶甲醇提取物结晶除去大部分二氢黄酮醇，母液冻干用于杨梅甙的分离制备。500mg母液冻干物，以水：甲醇：乙酸乙酯：正己烷(4：3：6：1,v／v)为两相溶剂系统，在单柱体高速逆流色谱上经7小时分离，获得63mg杨梅甙单体(2)。两种化合物的结构经ESI-MS和~1H-NMR、~(13)C-NMR分析得到了确认。
以水：甲醇：乙酸乙酯：正乙烷(3：2：2：2,v／v)构成两相溶剂系统，成功地分离了白水曲柳皮提取物中的黄酮类化合物。每次分离，可进样1g提取物粗品，分离得到120mg圣草酚(Ⅰ)，29.5mg 5，7-二羟色酮者(Ⅱ)和50mg 4’，5，7—三羟黄烷酮(Ⅲ)。本研究亦是首次从白水曲柳皮提取物中分离获得圣草酚、5，7-二羟色酮和4’，5，7—三羟黄烷酮(柑桔黄素)。三种化合物的结构经ESI-MS-MS和NMR分析获得了确定。
上丫日研
、、.︸.气曰勺︵卜
产.﹄尸‘︸l)、、l
\
4阿1111一l︶
1
O、.、
已
Flavonoids possess several bioactivities such as anticancer, preventing angiocardiopathy and antibiosis. The present paper mainly aims to the preparative separation of the flavoniods in extract of soybean, leaves extract of Ampelopsis grossedentata and bark extract of Salix alba.
Four isoflavone components were purified from soybean extract by high-speed counter-current chromatography (HSCCC). Two types of multilayer coil separation columns were used: a small column made of standard 2.6-mm I.D. PTFE (polytetrafluoroethylene) tubing with a 260-ml capacity and a large column of convoluted PTFE tubing of 5.7-mm average I.D. with a 1200-ml capacity. Separation was performed with a two-phase solvent system composed of hexane-ethyl acetate-1-butanol-methanol-acetic acid-water (1:2:1:5:1, v/v) by eluting the lower aqueous phase at 2ml /min (small column) and 5ml /min (large column) at a revolution speed of 700rpm. From 500 mg of crude sample the small column yielded 33mg of daidzin, 41mg of genistin, 27mg of 60-0-malonyldaidzin and 24mg of 6'-0-malonylgenistin. The large convoluted column separated, from 3g of crude sample, 203mg of daidzin, 241mg of genistin, 158mg of 6'-Omalonyldaidzin and 135mg of 60-O-malonylgenistin all at over 90% purity.
Purification of dihydromyricetin from an extract (16g) of leaves of Ampelopsis grossedentata was performed using a preparative triple-column countercurrent chromatograph. With a solvent system composed of n-hexane-ethyl acetate-methanol-water (1:3:2:4, v/v) 11.3g of dihydromyricetin was obtained at a high purity of over 99% by HPLC at 254 nm in 9h. Separaton of myricetrinin was conducted through two steps. First, the extract of leaves was crystallized with water to remove most of dihydromyricetin in order to obtain the extract with relative myricetrinin content. Then, the extract was separated using HSCCC with water:methanol:ethyl acetate:n-hexane(4:3:6:l, v/v) as solvent system. At the given
OH
OH
OH
O
(1)
(2)
condition, 500mg extract was successfully separated to yield 63mg myricetrinin. The chemical structures of dihydromyricetin and myricetrinin were confirmed by means of ESI-MS-MS, and NMR analysis.
The flavonoids in bark extract of Salix alba were separated at preparative scale using high-speed countercurrent chromatography (HSCCC). In each separation, 1.0 g crude extract was separated to yield pure 120mg eriodictyol (I), 29.5 mg 5,7-dihydroxychromone (II) and 50 mg naringenin (III), respectively, while water-methanol-ethyl acetate-n-hexane (3:2:2:2, v/v) was used for a two-phase solvent system of the HSCCC separation. The chemical structures of three flavonoids
were confirmed by means of ESI-MS-MS, and NMR analysis.
OH
4. OH
以两相溶剂系统正己烷：乙酸乙酯：丁醇：冰乙酸：水(1：2：1：5：1，v／v)，用装配260mL和1100mL柱的高速逆流色谱仪分离了大豆异黄酮样品，260mL分离柱可一次分离500mg大豆异黄酮样品获得大豆甙33mg，染料木甙41mg，6’’-丙二酰葡萄糖基大豆甙37mg和6’’-丙二酰葡萄糖基染料木甙24mg。1100mL柱分离可一次分离3g大豆异黄酮样品获得大豆甙203mg，染料木甙241mg，6’’-丙二酰葡萄糖基大豆甙217mg和6’’-丙二酰葡萄糖基染料木甙135mg。
以水：甲醇：乙酸乙酯：正乙烷(4：2：3：1,v／v)构成两相溶剂系统，16g藤茶甲醇提取物在三柱串联高速逆流色谱上经9小时分离，获得了11.3g二氢黄酮醇单体
(1)。用水对藤茶甲醇提取物结晶除去大部分二氢黄酮醇，母液冻干用于杨梅甙的分离制备。500mg母液冻干物，以水：甲醇：乙酸乙酯：正己烷(4：3：6：1,v／v)为两相溶剂系统，在单柱体高速逆流色谱上经7小时分离，获得63mg杨梅甙单体(2)。两种化合物的结构经ESI-MS和~1H-NMR、~(13)C-NMR分析得到了确认。
以水：甲醇：乙酸乙酯：正乙烷(3：2：2：2,v／v)构成两相溶剂系统，成功地分离了白水曲柳皮提取物中的黄酮类化合物。每次分离，可进样1g提取物粗品，分离得到120mg圣草酚(Ⅰ)，29.5mg 5，7-二羟色酮者(Ⅱ)和50mg 4’，5，7—三羟黄烷酮(Ⅲ)。本研究亦是首次从白水曲柳皮提取物中分离获得圣草酚、5，7-二羟色酮和4’，5，7—三羟黄烷酮(柑桔黄素)。三种化合物的结构经ESI-MS-MS和NMR分析获得了确定。
上丫日研
、、.︸.气曰勺︵卜
产.﹄尸‘︸l)、、l
\
4阿1111一l︶
1
O、.、
已
Flavonoids possess several bioactivities such as anticancer, preventing angiocardiopathy and antibiosis. The present paper mainly aims to the preparative separation of the flavoniods in extract of soybean, leaves extract of Ampelopsis grossedentata and bark extract of Salix alba.
Four isoflavone components were purified from soybean extract by high-speed counter-current chromatography (HSCCC). Two types of multilayer coil separation columns were used: a small column made of standard 2.6-mm I.D. PTFE (polytetrafluoroethylene) tubing with a 260-ml capacity and a large column of convoluted PTFE tubing of 5.7-mm average I.D. with a 1200-ml capacity. Separation was performed with a two-phase solvent system composed of hexane-ethyl acetate-1-butanol-methanol-acetic acid-water (1:2:1:5:1, v/v) by eluting the lower aqueous phase at 2ml /min (small column) and 5ml /min (large column) at a revolution speed of 700rpm. From 500 mg of crude sample the small column yielded 33mg of daidzin, 41mg of genistin, 27mg of 60-0-malonyldaidzin and 24mg of 6'-0-malonylgenistin. The large convoluted column separated, from 3g of crude sample, 203mg of daidzin, 241mg of genistin, 158mg of 6'-Omalonyldaidzin and 135mg of 60-O-malonylgenistin all at over 90% purity.
Purification of dihydromyricetin from an extract (16g) of leaves of Ampelopsis grossedentata was performed using a preparative triple-column countercurrent chromatograph. With a solvent system composed of n-hexane-ethyl acetate-methanol-water (1:3:2:4, v/v) 11.3g of dihydromyricetin was obtained at a high purity of over 99% by HPLC at 254 nm in 9h. Separaton of myricetrinin was conducted through two steps. First, the extract of leaves was crystallized with water to remove most of dihydromyricetin in order to obtain the extract with relative myricetrinin content. Then, the extract was separated using HSCCC with water:methanol:ethyl acetate:n-hexane(4:3:6:l, v/v) as solvent system. At the given
OH
OH
OH
O
(1)
(2)
condition, 500mg extract was successfully separated to yield 63mg myricetrinin. The chemical structures of dihydromyricetin and myricetrinin were confirmed by means of ESI-MS-MS, and NMR analysis.
The flavonoids in bark extract of Salix alba were separated at preparative scale using high-speed countercurrent chromatography (HSCCC). In each separation, 1.0 g crude extract was separated to yield pure 120mg eriodictyol (I), 29.5 mg 5,7-dihydroxychromone (II) and 50 mg naringenin (III), respectively, while water-methanol-ethyl acetate-n-hexane (3:2:2:2, v/v) was used for a two-phase solvent system of the HSCCC separation. The chemical structures of three flavonoids
were confirmed by means of ESI-MS-MS, and NMR analysis.
OH
4. OH
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64. Egger, K. In Thin-Layer Chrom atography. A laboratory Handbook(ed. E. Stahl),p.687. Springer-Verlag, Berlin, 1969.
65. Farmakalidis, E; Hathcock, J.N.; and Murrphy, P.A., Isoflavone estrogenic potency of genistin and daidzin in mice, Food Chem. Toxic, 1985, 23,741-5.
66. Ferrell J.E., Sing P.D.G.C., Loew G. et al. Structure/activity studies of flavonoids as inhibitors of cyclic AMP phosphodiesterase and relationship to quantum chemical indices. Mol. Pharmacol, 1979, 16: 556～568.
67. Fischer, L.(1969).In Laboratory Techniques in Biochemistry and Molecular Biology. Vol. 1, p. 157. North-Holland Publ. Co. Fleckenstein A. Annu. Rer. Pharmacol. Toxical, 1977, 17: 149～166.
68. G C Chen. High performance countercurrent chromatography for quantitative analysis of five flavenoids. J Liq Chromatogr, 1992, 15(15～16): 2857.
69. G J Kapadia, et al. High-speed countercurrent chromatography separation of biflavenoids from Garcinia kola seeds. J Chromatogr, 1994,673:142.
70. Harborne J.B. et al. The Flavonoid. Chapman & Hall, 1975.
71. H Oka, et al. Multilayer coil planet centrifuge for analytical high-speed countercurrent chromatography. J Chromatogr, 1989,479:53.
72. I Slacanin, et al. Modifications to a high-speed countercurrent chromatography for improved separation capability. J Chromatogr, 1989,482:243.
73. I Slacanin, et al. Isolation and determination of flavonol glycosides from Epilobium species. J Chromatogr, 1989,482:243.
74. Jang DS, Park EJ, Hawthorne ME, Vigo JS, Graham JG, Cabieses F, Santarsiero BD, Malterud, K.M., Bremnes, T.E., Faegri, A., Moe, T., Sandanger, Dugstad, E.K., Anthonsen, T., Henriksen, L.M., Flavonoids from the wood of Salix caprea as inhibitors of wood-destroyingfungi. J Nat Prod, 1985, 48: 559-563.
75. Jussi-Pekka Rauha, Susanna Remes, Antimicrobial effects of Finnish plant extracts containing flavonoids and other phenolic compounds, International journal of food microbiology. 2000.56(1):3～12.
76. Kaladas R.S. et al., Reproductive and metabolic effects of phytoestrogens in mammals. Reproductive Toxicology; 1989, 3(2): 81.
77. Konstantinopoulou M, Karioti A, Skaltsas S, Skaltsa H. Sesquiterpene lactones from Anthemis altissima and their anti-Helicobacter pylori activity. J Nat Prod, 2003,66: 699-702.
78. Mabry, T. J. et.al,(1970),The Systematic Identification of Flavonoids, Springer-Verlag, Berlin.
79. Malterud, K.M., Bremnes, T.E., Faegri, A., Moe, T., Sandauger, Dugstad, E.K., Anthonsen, T., Henriksen, L.M., Flavonoids from the wood of Salix caprea as inhibitors of wood-destroyingfungi. J Nat Prod 1985,48: 559-563.
80. Mesecar AD, Fong HH, Mehta RG, Pezzuto JM, Kinghorn AD. Potential cancer chemopreventive constituents of the seeds of Dipteryx odorata (tonka bean). J Nat Prod, 2003, 66:583-587.
81. M Vanhaelen, et al. Countercurrent chromatography for isolation of flavonol glycosides from Gingko biloba leaves. J Chromatogr, 1988,11:2969.
82. Ogata S, Miyake Y, Yamamoto K, Okumura K, Taguchi H. Apoptosis induced by the flavonoid from lemon fruit (Citrus limon BURM. f.) and its metabolites in HL-60 cells. Biosci Biotechnol Biochem, 2000, 64:1075-1078.
83. Robards K., Antolovich M. Analytical chemistry of fruit bioflavonoids. Analyst; 1997, (112):11R～34R.
84. Rowe, J.W. (Ed.), Natural Products of Woody Plants. Springer-Verlag, Berlin, Heidelberg, 1989.
85. Sanchez de Rojas VR, Somoza B, Ortega T, Villar AM. Isolation of vasodilatory active flavonoids from the traditional remedy Satureja obovata. Planta Med, 1996, 62:272-274.
86. Seikel, M. K. In The Chemistry of Flavonoid Compounds(ed. J. B. Harborne), p. 37. Academic Press, London and New York, 1962.1
87. Simon A, Chulia AJ, Kaouadji M, Delage C. Quercetin 3-[triacetylarabinosyl(1-->6)galactoside] and chromones from Calluna vulgaris. Phytochemistry, 1994, 36:1043-1045.
88. Tetsu Akiyama. et al. Genistin, a specific inhibitor of tyrosine-specific protein kinases. The Journal of Biological Chemistry. 1987, 262 (12): 5592.
89. T Y Zhang, et al. Separation of flavonoids and alkaloids in medicinal herbs by high-speed countercurrent chromatography. J Chromatogr, 1988, 435:159.
90. T Y Zhang, et al. Separation of hydroxyanthraquinone derivatives extracted from rheum with analytical high-speed countercurrent chromatography. J Chromatogr, 1988,442:455.
91. T Y Zhang, et al. Rapid separation of flavonoids by analytical high-speed countercurrent chromatography. J Chromatogr, 1988,445:199.
92. Xiao D. Gu Z.L. Bai J.P. et al. Acta Pamarcol. Sin., 1995;16; (3); 223～226
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63. Dapkevicius A, van Beck TA, Lelyveld GP, van Veldhuizen A, de Groot A, Linssen JP, Venskutonis R. Isolation and structure elucidation of radical scavengers from Thymus vulgaris leaves. J Nat Prod, 2002,65:892-896.
64. Egger, K. In Thin-Layer Chrom atography. A laboratory Handbook(ed. E. Stahl),p.687. Springer-Verlag, Berlin, 1969.
65. Farmakalidis, E; Hathcock, J.N.; and Murrphy, P.A., Isoflavone estrogenic potency of genistin and daidzin in mice, Food Chem. Toxic, 1985, 23,741-5.
66. Ferrell J.E., Sing P.D.G.C., Loew G. et al. Structure/activity studies of flavonoids as inhibitors of cyclic AMP phosphodiesterase and relationship to quantum chemical indices. Mol. Pharmacol, 1979, 16: 556～568.
67. Fischer, L.(1969).In Laboratory Techniques in Biochemistry and Molecular Biology. Vol. 1, p. 157. North-Holland Publ. Co. Fleckenstein A. Annu. Rer. Pharmacol. Toxical, 1977, 17: 149～166.
68. G C Chen. High performance countercurrent chromatography for quantitative analysis of five flavenoids. J Liq Chromatogr, 1992, 15(15～16): 2857.
69. G J Kapadia, et al. High-speed countercurrent chromatography separation of biflavenoids from Garcinia kola seeds. J Chromatogr, 1994,673:142.
70. Harborne J.B. et al. The Flavonoid. Chapman & Hall, 1975.
71. H Oka, et al. Multilayer coil planet centrifuge for analytical high-speed countercurrent chromatography. J Chromatogr, 1989,479:53.
72. I Slacanin, et al. Modifications to a high-speed countercurrent chromatography for improved separation capability. J Chromatogr, 1989,482:243.
73. I Slacanin, et al. Isolation and determination of flavonol glycosides from Epilobium species. J Chromatogr, 1989,482:243.
74. Jang DS, Park EJ, Hawthorne ME, Vigo JS, Graham JG, Cabieses F, Santarsiero BD, Malterud, K.M., Bremnes, T.E., Faegri, A., Moe, T., Sandanger, Dugstad, E.K., Anthonsen, T., Henriksen, L.M., Flavonoids from the wood of Salix caprea as inhibitors of wood-destroyingfungi. J Nat Prod, 1985, 48: 559-563.
75. Jussi-Pekka Rauha, Susanna Remes, Antimicrobial effects of Finnish plant extracts containing flavonoids and other phenolic compounds, International journal of food microbiology. 2000.56(1):3～12.
76. Kaladas R.S. et al., Reproductive and metabolic effects of phytoestrogens in mammals. Reproductive Toxicology; 1989, 3(2): 81.
77. Konstantinopoulou M, Karioti A, Skaltsas S, Skaltsa H. Sesquiterpene lactones from Anthemis altissima and their anti-Helicobacter pylori activity. J Nat Prod, 2003,66: 699-702.
78. Mabry, T. J. et.al,(1970),The Systematic Identification of Flavonoids, Springer-Verlag, Berlin.
79. Malterud, K.M., Bremnes, T.E., Faegri, A., Moe, T., Sandauger, Dugstad, E.K., Anthonsen, T., Henriksen, L.M., Flavonoids from the wood of Salix caprea as inhibitors of wood-destroyingfungi. J Nat Prod 1985,48: 559-563.
80. Mesecar AD, Fong HH, Mehta RG, Pezzuto JM, Kinghorn AD. Potential cancer chemopreventive constituents of the seeds of Dipteryx odorata (tonka bean). J Nat Prod, 2003, 66:583-587.
81. M Vanhaelen, et al. Countercurrent chromatography for isolation of flavonol glycosides from Gingko biloba leaves. J Chromatogr, 1988,11:2969.
82. Ogata S, Miyake Y, Yamamoto K, Okumura K, Taguchi H. Apoptosis induced by the flavonoid from lemon fruit (Citrus limon BURM. f.) and its metabolites in HL-60 cells. Biosci Biotechnol Biochem, 2000, 64:1075-1078.
83. Robards K., Antolovich M. Analytical chemistry of fruit bioflavonoids. Analyst; 1997, (112):11R～34R.
84. Rowe, J.W. (Ed.), Natural Products of Woody Plants. Springer-Verlag, Berlin, Heidelberg, 1989.
85. Sanchez de Rojas VR, Somoza B, Ortega T, Villar AM. Isolation of vasodilatory active flavonoids from the traditional remedy Satureja obovata. Planta Med, 1996, 62:272-274.
86. Seikel, M. K. In The Chemistry of Flavonoid Compounds(ed. J. B. Harborne), p. 37. Academic Press, London and New York, 1962.1
87. Simon A, Chulia AJ, Kaouadji M, Delage C. Quercetin 3-[triacetylarabinosyl(1-->6)galactoside] and chromones from Calluna vulgaris. Phytochemistry, 1994, 36:1043-1045.
88. Tetsu Akiyama. et al. Genistin, a specific inhibitor of tyrosine-specific protein kinases. The Journal of Biological Chemistry. 1987, 262 (12): 5592.
89. T Y Zhang, et al. Separation of flavonoids and alkaloids in medicinal herbs by high-speed countercurrent chromatography. J Chromatogr, 1988, 435:159.
90. T Y Zhang, et al. Separation of hydroxyanthraquinone derivatives extracted from rheum with analytical high-speed countercurrent chromatography. J Chromatogr, 1988,442:455.
91. T Y Zhang, et al. Rapid separation of flavonoids by analytical high-speed countercurrent chromatography. J Chromatogr, 1988,445:199.
92. Xiao D. Gu Z.L. Bai J.P. et al. Acta Pamarcol. Sin., 1995;16; (3); 223～226
93. Yang Y. Studies on the chemical constituents of Viscum liquidambaricolum. Zhong Cai Yao, 1998, 21:22-23.
94. Zhang SX, Tani T, Yamaji S, Ma CM, Wang MC, Cai- SQ, Zhao YY. Glycosyl flavonoids from the roots and rhizomes of Asarum longerhizomatosum. J Asian Nat Prod Res, 2003,5:25-30.