高速逆流色谱技术在天然产物分离制备中的探索性研究
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摘要
目的：(1)建立单独配制溶剂系统的固定相和流动相的新方法,并将之用于天然产物的高速逆流色谱(High-speed counter-current chromato-graphy, HSCCC)分离纯化；(2)采用J型HSCCC系统和反胶团溶剂体系对活性天然蛋白进行分离纯化。
方法：(1-1)气相色谱(GC)辅助单独配制溶剂系统。采用GC色谱和内标法测定传统配制的固定相和流动相中各有机溶剂的含量,利用质量参数计算水的含量。根据测定结果分别计算固定相和流动相中各溶剂的体积分数,再单独配制两相溶剂,并将该法用于黄连(Coptis chinensis Franch)生物碱的分离纯化。(1-2) UNIFAC数学模型-逆流色谱溶剂系统选择(CCC-SSS)软件单独配制溶剂系统。根据UNIFAC数学模型计算各溶剂在溶剂系统上下相中的活度系数和摩尔百分比,再结合各试剂的密度和分子量计算出各试剂在上下相中的体积含量,以此来单独配制两相溶剂。并将该法用于穿山龙(Dioscorea nipponica Makino)和竹叶(Lophatherum gracile)中活性成分的分离纯化。(2)采用两性表面活性剂和有机溶剂建立的反胶团溶液为固定相,磷酸钠盐缓冲液为流动相,对来自菠萝和螺旋藻中的菠萝蛋白酶和藻蓝蛋白进行分离纯化。
结果：(1-1)在HSCCC分离纯化黄连生物碱的过程中,使用GC辅助单独配制的氯仿-甲醇-水(2：1：1,v／v／v)两相溶剂系统成功分离出黄连碱、小檗碱、巴马汀、表小檗碱和药根碱五种生物碱,其纯度和回收率均分别大于98.5%和92.0%。在进行等效HSCCC分离时,与传统的溶剂配制方法相比,可以节约134 mL氯仿、336 mL甲醇和452 mL水。(1-2)采用UNIFAC数学模型单独配制的正己烷-乙酸乙酯-乙醇-水(2：5：2：5,v／v／v／v)成功用于穿山龙中薯蓣皂苷的分离制备；用此法单独配制的乙酸乙酯-正丁醇-水(2：1：3,v／v／v)成功的从竹叶中分离纯化了槲皮素-3-O-葡萄糖苷。此法与传统的溶剂系统配制方法相比,分离效率相当,但节省了有机溶剂。(3)采用(0.1 mol/L CTAB/正辛烷-正己醇)反胶团溶液做固定相,以流动相A (0.05 mol/L NaH2PO4, pH=7.0,0.2 mol/L KCl)进行溶剂系统的动态平衡,流动相B (0.05 mol/L NaH2PO4, pH=7.0,0.4 mol/L KCl)进行洗脱,成功的从菠萝粗蛋白中分离出达到电泳纯和质谱纯的菠萝蛋白酶,回收率高达88.4%；同样采用反胶团溶液(0.1 mol／L CTAB／正辛烷-正己醇)做固定相,流动相A(0.05 mol/L NaH2PO4,pH=5.0,0.2 mol/LKCl)和流动相B (0.05 mol/L NaH2PO4, pH=8.0,0.4 mol/L KCl)成功的从螺旋藻粗蛋白中分离出藻蓝蛋白,产物纯度为A620／A280=4.25,回收率约85%。
结论：(1)我们对新建的单独配制模式配制的溶剂系统与传统模式配制的溶剂系统在HSCCC分离过程中的固定相保留率、分离时间、固定相和流动相的用量进行比较发现：在HSCCC分离过程中,单独配制的溶剂系统在保证较好分离效果的情况下可节省大量的有机溶剂,不仅减少了资源浪费,而且环保。(2)利用反胶团溶剂系统成功分离纯化了菠萝蛋白酶和藻蓝蛋白,表明反胶团溶剂系统在HSCCC用于生物大分子的分离纯化方面有着较大的应用前景和研究价值,这将为蛋白质化学和组学提供新的研究技术平台。
Objective:(1) To develop new methods for single preparing stationary and mobile phases used in high-speed counter-current chromatography (HSCCC). (2) To separate and purify active proteins from natural resources by J-type HSCCC apparatus using reverse micelles solvent systems.
Method:(1-1) Gas chromatography (GC)-aid single preparation of solvent system. The content of each solvent in stationary and mobile phases of a solvent system can be determined and calculated by GC analysis. According to the volume ratios of solvents in each phase, the mobile and stationary phases were parepared, respectively. Then, the new developed method was used to separate alkaloids from Coptis chinensis Franch by HSCCC. (1-2) UNIFAC mathematic model for single preparation of solvent system. According to UNIFAC mathematic model, the activity coefficient and molar ratio of solvents in each phase were calculated. Then, the density and molecular mass of each solvent were used to calculate the volume ratios for single preparing the stationary and mobile phases. The examples of separating the active compounds from Dioscorea nipponica Makino and Lophatherum gracile were carried out to demonstrate the pragmatic of the new approach. (2) The reverse micelles solvent system was used to separate proteins by J-type-HSCCC. The stationary phase was reverse micelles phase (0.1 mol/L CTAB/isooctane-hexylalcohol), and the mobile phase was aqueous phase consisted of Na-phosphate buffers and adjusted by HCl and KC1 to afford suitable pH value and ionic strength. Bromelain from pineapple and c-phycocyanin from Spirulina were separated by the method.
Result:(1-1) Five alkaloids including palmatine, berberine, epiberber- ine, jatrorrhizine and coptisine were separated from C. chinensis by HSCCC using a solvent system composed of chloroform-methanol-water (2:1:1, v/v/v) single prepared by GC-aid method. The purities and recoveries of all products were over 98.5% and 92%. However,134 mL chloroform,336 mL methanol and 452 mL water were saved in HSCCC procedure with the two phases single prepared. (1-2) Dioscin from D. nipponica Makino and quer-cetin-3-O-glucoside from L. gracile were successfully isolated using the solvent systems composed of n-hexane-ethyl acetate-ethanol-water (2:5:2: 5, v/v/v/v) and ethyl acetate-n-butanol-water (2:1:3, v/v/v), which were single prepared by UNIFAC mathematic model coupled with counter-current chromatography solvent selection software (CCC-SSS). The comparative study between the developed single preparation methods and traditional together preparation method were carried out, and there were no difference. But solvent consume was significant different. Especially, when the solvent system single prepared by UNIFAC mathematic model, much organic solvent were saved. For quercetin-3-O-glucoside purification,356 mL ethyl acetate,182 mL n-butanol and 13 mL water were saved. For dioscin isolation,136 mL n-hexane,315 mL ethyl acetate and 58 mL methanol were saved, but 54 mL water was more used, when the new approach was carried out. (2) Pure bromelain (electrophoresis purity and MS purity,100%; recovery,88.4%) from pineapple and high pure c-phycocyanin (purity, A620/A280=4.25; recovery,85%) from Spirulina were successfully separated by HSCCC using reverse micelles solvent systems.
Conclusion:(1) The comparative study of single and together preparation of the two phases of solvent system proved that the single preparation methods are convenient, economic and reliable for natural product separation by HSCCC. And importantly, they can save much solvents, and friendly to our living conditions and natural resource. (2) The HSCCC technique using reverse micelles solvent system can be widely employed in the filed of protein separation and purification.
方法：(1-1)气相色谱(GC)辅助单独配制溶剂系统。采用GC色谱和内标法测定传统配制的固定相和流动相中各有机溶剂的含量,利用质量参数计算水的含量。根据测定结果分别计算固定相和流动相中各溶剂的体积分数,再单独配制两相溶剂,并将该法用于黄连(Coptis chinensis Franch)生物碱的分离纯化。(1-2) UNIFAC数学模型-逆流色谱溶剂系统选择(CCC-SSS)软件单独配制溶剂系统。根据UNIFAC数学模型计算各溶剂在溶剂系统上下相中的活度系数和摩尔百分比,再结合各试剂的密度和分子量计算出各试剂在上下相中的体积含量,以此来单独配制两相溶剂。并将该法用于穿山龙(Dioscorea nipponica Makino)和竹叶(Lophatherum gracile)中活性成分的分离纯化。(2)采用两性表面活性剂和有机溶剂建立的反胶团溶液为固定相,磷酸钠盐缓冲液为流动相,对来自菠萝和螺旋藻中的菠萝蛋白酶和藻蓝蛋白进行分离纯化。
结果：(1-1)在HSCCC分离纯化黄连生物碱的过程中,使用GC辅助单独配制的氯仿-甲醇-水(2：1：1,v／v／v)两相溶剂系统成功分离出黄连碱、小檗碱、巴马汀、表小檗碱和药根碱五种生物碱,其纯度和回收率均分别大于98.5%和92.0%。在进行等效HSCCC分离时,与传统的溶剂配制方法相比,可以节约134 mL氯仿、336 mL甲醇和452 mL水。(1-2)采用UNIFAC数学模型单独配制的正己烷-乙酸乙酯-乙醇-水(2：5：2：5,v／v／v／v)成功用于穿山龙中薯蓣皂苷的分离制备；用此法单独配制的乙酸乙酯-正丁醇-水(2：1：3,v／v／v)成功的从竹叶中分离纯化了槲皮素-3-O-葡萄糖苷。此法与传统的溶剂系统配制方法相比,分离效率相当,但节省了有机溶剂。(3)采用(0.1 mol/L CTAB/正辛烷-正己醇)反胶团溶液做固定相,以流动相A (0.05 mol/L NaH2PO4, pH=7.0,0.2 mol/L KCl)进行溶剂系统的动态平衡,流动相B (0.05 mol/L NaH2PO4, pH=7.0,0.4 mol/L KCl)进行洗脱,成功的从菠萝粗蛋白中分离出达到电泳纯和质谱纯的菠萝蛋白酶,回收率高达88.4%；同样采用反胶团溶液(0.1 mol／L CTAB／正辛烷-正己醇)做固定相,流动相A(0.05 mol/L NaH2PO4,pH=5.0,0.2 mol/LKCl)和流动相B (0.05 mol/L NaH2PO4, pH=8.0,0.4 mol/L KCl)成功的从螺旋藻粗蛋白中分离出藻蓝蛋白,产物纯度为A620／A280=4.25,回收率约85%。
结论：(1)我们对新建的单独配制模式配制的溶剂系统与传统模式配制的溶剂系统在HSCCC分离过程中的固定相保留率、分离时间、固定相和流动相的用量进行比较发现：在HSCCC分离过程中,单独配制的溶剂系统在保证较好分离效果的情况下可节省大量的有机溶剂,不仅减少了资源浪费,而且环保。(2)利用反胶团溶剂系统成功分离纯化了菠萝蛋白酶和藻蓝蛋白,表明反胶团溶剂系统在HSCCC用于生物大分子的分离纯化方面有着较大的应用前景和研究价值,这将为蛋白质化学和组学提供新的研究技术平台。
Objective:(1) To develop new methods for single preparing stationary and mobile phases used in high-speed counter-current chromatography (HSCCC). (2) To separate and purify active proteins from natural resources by J-type HSCCC apparatus using reverse micelles solvent systems.
Method:(1-1) Gas chromatography (GC)-aid single preparation of solvent system. The content of each solvent in stationary and mobile phases of a solvent system can be determined and calculated by GC analysis. According to the volume ratios of solvents in each phase, the mobile and stationary phases were parepared, respectively. Then, the new developed method was used to separate alkaloids from Coptis chinensis Franch by HSCCC. (1-2) UNIFAC mathematic model for single preparation of solvent system. According to UNIFAC mathematic model, the activity coefficient and molar ratio of solvents in each phase were calculated. Then, the density and molecular mass of each solvent were used to calculate the volume ratios for single preparing the stationary and mobile phases. The examples of separating the active compounds from Dioscorea nipponica Makino and Lophatherum gracile were carried out to demonstrate the pragmatic of the new approach. (2) The reverse micelles solvent system was used to separate proteins by J-type-HSCCC. The stationary phase was reverse micelles phase (0.1 mol/L CTAB/isooctane-hexylalcohol), and the mobile phase was aqueous phase consisted of Na-phosphate buffers and adjusted by HCl and KC1 to afford suitable pH value and ionic strength. Bromelain from pineapple and c-phycocyanin from Spirulina were separated by the method.
Result:(1-1) Five alkaloids including palmatine, berberine, epiberber- ine, jatrorrhizine and coptisine were separated from C. chinensis by HSCCC using a solvent system composed of chloroform-methanol-water (2:1:1, v/v/v) single prepared by GC-aid method. The purities and recoveries of all products were over 98.5% and 92%. However,134 mL chloroform,336 mL methanol and 452 mL water were saved in HSCCC procedure with the two phases single prepared. (1-2) Dioscin from D. nipponica Makino and quer-cetin-3-O-glucoside from L. gracile were successfully isolated using the solvent systems composed of n-hexane-ethyl acetate-ethanol-water (2:5:2: 5, v/v/v/v) and ethyl acetate-n-butanol-water (2:1:3, v/v/v), which were single prepared by UNIFAC mathematic model coupled with counter-current chromatography solvent selection software (CCC-SSS). The comparative study between the developed single preparation methods and traditional together preparation method were carried out, and there were no difference. But solvent consume was significant different. Especially, when the solvent system single prepared by UNIFAC mathematic model, much organic solvent were saved. For quercetin-3-O-glucoside purification,356 mL ethyl acetate,182 mL n-butanol and 13 mL water were saved. For dioscin isolation,136 mL n-hexane,315 mL ethyl acetate and 58 mL methanol were saved, but 54 mL water was more used, when the new approach was carried out. (2) Pure bromelain (electrophoresis purity and MS purity,100%; recovery,88.4%) from pineapple and high pure c-phycocyanin (purity, A620/A280=4.25; recovery,85%) from Spirulina were successfully separated by HSCCC using reverse micelles solvent systems.
Conclusion:(1) The comparative study of single and together preparation of the two phases of solvent system proved that the single preparation methods are convenient, economic and reliable for natural product separation by HSCCC. And importantly, they can save much solvents, and friendly to our living conditions and natural resource. (2) The HSCCC technique using reverse micelles solvent system can be widely employed in the filed of protein separation and purification.
引文
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