反相二维色谱制备甘草中黄酮类化合物
|
摘要
目的构建甘草黄酮类化合物系统性分离制备方法。方法采用特异性吸附材料富集甘草中的黄酮类化合物,以自主研发的制备色谱工厂系统,采用色谱分离专家系统软件优化分离制备条件,通过上样量和富集次数等参数的考察,建立了基于分离富集模式的反相二维色谱制备甘草黄酮有效部位及单体化合物的方法。结果建立了以C18为分离、富集填料,甲醇-水、乙腈-水为一维、二维分离流动相,水为富集稀释液,梯度洗脱体积流量和稀释富集液的体积流量均为21 m L/min,上样量300 mg,富集次数3次的二维色谱分离制备甘草黄酮的方法,其分离过程具有良好的重复性。应用该方法分离制备,可重复获得16个甘草黄酮部位和甘草苷、甘草素、芒柄花黄素、刺甘草查耳酮、7,4′-二羟基黄酮、4′-O-[β-D-apio-D-furanosyl-(1→2)-β-D-glucopyranosyl]liquiritigenin、异甘草素、甘草酚、甘草香豆素共9个单体化合物。结论建立的甘草黄酮的制备方法为甘草资源的综合利用和甘草黄酮活性药物开发奠定了基础。
Objective To develop a systematic chromatography separation method for flavonoids from Glycyrrhiza uralensis Fisch.(GU). Methods A new method for the separation of effective parts and monomers of flavonoids from GU by two-dimensional reversed-phase liquid chromatography was developed using the self-developed preparation chromatography plant system with independent intellectual property rights. Flavonoids compounds were enriched with specific adsorption materials. The separation conditions of the chromatography were optimized by the chromatographic separation expert system software, and the loading weight of samples and the enrichment times of a separation were investigated. Results The process of chromatography separation of flavonoids from GU had good precision and reproducibility with C18 as separation and enrichment solid phase and the methanol/water and acetonitrile/water as mobile phase of one-dimensional and two-dimensional chromatography system. The dilution solution which used for one-dimensional and two-dimensional enrichment chromatography was water. The flow rate of gradient elution and dilution enrichment solution was 21 m L/min. The sample loading amount of chromatography separation was 300 mg each time. A total of 16 flavonoids parts contained stable chemical composition were obtained by the repeatable separation method after three times of enrichment. Nine pure compounds were obtained and identified by NMR and MS, which were liquiritin, liquiritigenin, formononetin, echinatin, 7,4′-dihydroxyflavone, 4′-O-[β-D-apio-D-furanosyl-(1→2)-β-D-glucopyranosyl] liquiritigenin, isoliquiritigenin, glycyrol, and glycycoumarin. Conclusion The study can provide a certain reference value for the systematic separation and cognition for flavonoids from GU.
Objective To develop a systematic chromatography separation method for flavonoids from Glycyrrhiza uralensis Fisch.(GU). Methods A new method for the separation of effective parts and monomers of flavonoids from GU by two-dimensional reversed-phase liquid chromatography was developed using the self-developed preparation chromatography plant system with independent intellectual property rights. Flavonoids compounds were enriched with specific adsorption materials. The separation conditions of the chromatography were optimized by the chromatographic separation expert system software, and the loading weight of samples and the enrichment times of a separation were investigated. Results The process of chromatography separation of flavonoids from GU had good precision and reproducibility with C18 as separation and enrichment solid phase and the methanol/water and acetonitrile/water as mobile phase of one-dimensional and two-dimensional chromatography system. The dilution solution which used for one-dimensional and two-dimensional enrichment chromatography was water. The flow rate of gradient elution and dilution enrichment solution was 21 m L/min. The sample loading amount of chromatography separation was 300 mg each time. A total of 16 flavonoids parts contained stable chemical composition were obtained by the repeatable separation method after three times of enrichment. Nine pure compounds were obtained and identified by NMR and MS, which were liquiritin, liquiritigenin, formononetin, echinatin, 7,4′-dihydroxyflavone, 4′-O-[β-D-apio-D-furanosyl-(1→2)-β-D-glucopyranosyl] liquiritigenin, isoliquiritigenin, glycyrol, and glycycoumarin. Conclusion The study can provide a certain reference value for the systematic separation and cognition for flavonoids from GU.
引文
[1]梁鑫淼,丰加涛,金郁,等.中药质量控制技术发展展望[J].色谱,2008,26(2):130-135.
[2]沈保家,秦昆明,刘启迪,等.二维色谱技术及其在中药领域中的应用[J].中国科学:化学,2013,43(11):1480-1489.
[3]陈学国,孔亮,盛亮洪,等.全二维液相色谱串联质谱用于银杏叶提取物成分分析的研究[J].色谱,2005,23(1):46-51.
[4]高辉,温学森,马小军,等.二维液相色谱技术在药物分析中的应用[J].药物分析杂志,2007(4):616-620.
[5]张艳海,金燕,王峥涛.在线二维多中心切割液相色谱法测定三七、人参及其相关产品中8种人参皂苷[J].中草药,2017,48(5):894-901.
[6]唐涛,张维冰,李彤,等.六昧地黄丸组分的二维液相色谱分离[J].分析化学,2007,35(12):1767-1771.
[7]高雯,宋慧鹏,杨华,等.在线二维液相色谱技术在中药研究中的应用进展[J].色谱,2017,35(1):121-128.
[8]Pan X,Liu H,Jia G,et al.Microwave-assisted extraction of glycyrrhizic acid from licorice root[J].Biochem Eng J,2000,5(3):173-177.
[9]Pasquali R,Ambrosi B,Armanini D,et al.Cortisol and ACTH response to oral dexamethasone in obesity and effects of sex,body fat distribution,and dexamethasone concentrations:a dose-response study[J].JCEM,2002,87(1):166-175.
[10]赵艳敏,刘素香,张晨曦,等.基于HPLC-Q-TOF-MS技术的甘草化学成分分析[J].中草药,2016,47(12):2061-2068.
[11]Wang M,Wey S,Zhang Y,et al.Role of the unfolded protein response regulator GRP78/Bi P in development,cancer,and neurological disorders[J].Antioxid Redox Sign,2009,11(9):2307-2315.
[12]张娟,刘芬,李宁,等.UPLC-TOF-MS法鉴定胀果甘草药渣中黄酮类成分[J].现代药物与临床,2012,27(6):558-561.
[13]李怡佳.中药化学成分系统分离策略及设备研究[D].大连:大连工业大学,2016.
[14]邢倩倩,傅青,金郁,等.亲水/反相二维色谱法制备桔梗中的三萜皂苷[J].色谱,2014,32(7):767-772.
[15]Nakanishi T,Inada A,Kambayashi K,et al.Flavonoid glycosides of the roots of Glycyrrhiza uralensis[J].Phytochemistry,1985,24(2):339-341.
[16]Kuroyanagi M,Ueno A,Hirayama Y,et al.Anti-androgen active constituents from dalbergia cochinchinensis PIERRE[J].Nat Med,1996,50(6):408-412.
[17]Yasuda T,Kano Y,Saito K,et al.Urinary and biliary metabolites of daidzin and daidzein in rats[J].Biol Pharm Bull,1994,17(10):1369-1374.
[18]Wang Y H,Hong B,Dou D Q,et al.Study on the flavonoids constituents of cultivated licorice[J].Northwest Pharm J,2004,19(6):252-253.
[19]Shirataki Y,Yokoe I,Komatsu M.Two new flavone glycosides from the roots of Sophora subprostrata[J].IJAC,1986,10(4):327-334.
[20]Shiozawa T,Urata S,Kinoshita T,et al.Revised structures of glycyrol and isoglycyrol,constituents of the root of Glycyrrhiza uralensis[J].Chem Pharm Bull,2008,37(8):2239-2240.
[21]Zeng L,Zhang R Y,Meng T,et al.Determination of nine flavonoids and coumarins in licorice root by highperformance liquid chromatography[J].J Chromatogr A,1990,4(3):247-254.
[2]沈保家,秦昆明,刘启迪,等.二维色谱技术及其在中药领域中的应用[J].中国科学:化学,2013,43(11):1480-1489.
[3]陈学国,孔亮,盛亮洪,等.全二维液相色谱串联质谱用于银杏叶提取物成分分析的研究[J].色谱,2005,23(1):46-51.
[4]高辉,温学森,马小军,等.二维液相色谱技术在药物分析中的应用[J].药物分析杂志,2007(4):616-620.
[5]张艳海,金燕,王峥涛.在线二维多中心切割液相色谱法测定三七、人参及其相关产品中8种人参皂苷[J].中草药,2017,48(5):894-901.
[6]唐涛,张维冰,李彤,等.六昧地黄丸组分的二维液相色谱分离[J].分析化学,2007,35(12):1767-1771.
[7]高雯,宋慧鹏,杨华,等.在线二维液相色谱技术在中药研究中的应用进展[J].色谱,2017,35(1):121-128.
[8]Pan X,Liu H,Jia G,et al.Microwave-assisted extraction of glycyrrhizic acid from licorice root[J].Biochem Eng J,2000,5(3):173-177.
[9]Pasquali R,Ambrosi B,Armanini D,et al.Cortisol and ACTH response to oral dexamethasone in obesity and effects of sex,body fat distribution,and dexamethasone concentrations:a dose-response study[J].JCEM,2002,87(1):166-175.
[10]赵艳敏,刘素香,张晨曦,等.基于HPLC-Q-TOF-MS技术的甘草化学成分分析[J].中草药,2016,47(12):2061-2068.
[11]Wang M,Wey S,Zhang Y,et al.Role of the unfolded protein response regulator GRP78/Bi P in development,cancer,and neurological disorders[J].Antioxid Redox Sign,2009,11(9):2307-2315.
[12]张娟,刘芬,李宁,等.UPLC-TOF-MS法鉴定胀果甘草药渣中黄酮类成分[J].现代药物与临床,2012,27(6):558-561.
[13]李怡佳.中药化学成分系统分离策略及设备研究[D].大连:大连工业大学,2016.
[14]邢倩倩,傅青,金郁,等.亲水/反相二维色谱法制备桔梗中的三萜皂苷[J].色谱,2014,32(7):767-772.
[15]Nakanishi T,Inada A,Kambayashi K,et al.Flavonoid glycosides of the roots of Glycyrrhiza uralensis[J].Phytochemistry,1985,24(2):339-341.
[16]Kuroyanagi M,Ueno A,Hirayama Y,et al.Anti-androgen active constituents from dalbergia cochinchinensis PIERRE[J].Nat Med,1996,50(6):408-412.
[17]Yasuda T,Kano Y,Saito K,et al.Urinary and biliary metabolites of daidzin and daidzein in rats[J].Biol Pharm Bull,1994,17(10):1369-1374.
[18]Wang Y H,Hong B,Dou D Q,et al.Study on the flavonoids constituents of cultivated licorice[J].Northwest Pharm J,2004,19(6):252-253.
[19]Shirataki Y,Yokoe I,Komatsu M.Two new flavone glycosides from the roots of Sophora subprostrata[J].IJAC,1986,10(4):327-334.
[20]Shiozawa T,Urata S,Kinoshita T,et al.Revised structures of glycyrol and isoglycyrol,constituents of the root of Glycyrrhiza uralensis[J].Chem Pharm Bull,2008,37(8):2239-2240.
[21]Zeng L,Zhang R Y,Meng T,et al.Determination of nine flavonoids and coumarins in licorice root by highperformance liquid chromatography[J].J Chromatogr A,1990,4(3):247-254.