仙人掌总皂苷提取纯化工艺的研究
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摘要
仙人掌(Opuntia dillenii Haw.)为仙人掌科(Cactaceae)仙人掌属植物,在世界各地有广泛的分布。近年来研究表明,仙人掌总皂苷是仙人掌属植物中含有的一类重要的生物活性成分。目前,国内对仙人掌皂苷的提取分离纯化工艺未见任何系统的报导。因此,其提取、纯化工艺的研究显得尤为重要。故本研究旨在此方面作一种摸索,为今后仙人掌资源的综合利用和开发奠定基础。针对仙人掌皂苷的研究基础与现状,结合具体实验条件,本文系统地研究了比色法测定仙人掌总皂苷含量的方法;用正交设计法优选了仙人掌总皂苷的醇回流提取工艺条件;研究了D101大孔吸附树脂富集、纯化仙人掌总皂苷的吸附性能和洗脱参数等。其研究的主要结果如下:
1、确定了测定仙人掌总皂苷含量的比色-紫外分光光度法:以齐墩果酸(OleanolicAcid,OA)为对照,确定出仙人掌总皂苷的最大吸收波长为548 nm,测定仙人掌总皂苷含量的标准曲线方程为A=6.425 C -0.0016,加标回收率为98.47%,相对标准偏差RSD为3.29%,该方法快捷、精确、重现性良好。
2、研究了仙人掌总皂苷的醇回流提取工艺,在单因子实验的基础上,采用L_9(3~4)正交实验确定了醇回流提取仙人掌总皂苷的适宜工艺参数:提取剂乙醇浓度为80%、固液比为1:15、提取温度70℃、提取时间6h,仙人掌总皂苷的平均得率0.1159%。
3、探讨了大孔吸附树脂法对仙人掌总皂苷分离纯化的影响。实验研究和分析了D101大孔树脂对仙人掌总皂苷的吸附和解吸性能。结果表明:D101大孔树脂适于仙人掌总皂苷的分离和精制,饱和吸附量达9.922 mg/g干树脂;吸附条件为:溶液pH值为3.0、上样流速2BV/h、解吸液流速2BV/h;通过旋转正交实验及方差分析确定了仙人掌总皂苷分离富集的优化操作条件为:色谱柱的径高比1:11.57,柱温28.83℃。采用约5BV/h水溶液以2BV/h流速淋洗后,再以5BV 50%的乙醇溶液以2BV/h流速解吸,可较好地分离仙人掌总皂苷。
4、用优化的工艺流程提取分析了经发酵的仙人掌8个样品中总皂苷的含量。
综上所述,采取乙醇回流提取、大孔吸附树脂等工艺技术可以有效的提高仙人掌总皂苷的的提取分离效率。
Opuntia dillenii Haw. is a plant belonging to genus Opuntia in Cactaceae family. The plants of Opuntia have a wide distribution in the world. Recent studies show that total of Opuntia saponins is an important biologically active component in genus Opuntia plant. There is no systemic report on the research of extraction and purification technique of total of Opuntia saponins in China. The objective of this paper is to lay a foundation for compositive utilization and exploitation of Cactaceae. On the bases of previous research of total of Opuntia saponins and the facilities of the laboratory, this thesis systematically studied a colorimetric method for quantitative analysis of total of Opuntia saponins , optimized the parameters of ethanol reflux extraction which was screened by orthogonal design. The purification processes of total of Opuntia saponins with D101 macroporous resin was generally studied by taking adsorption quantities, eluting ratio and so on. The major results are shown as follows:
1. The content of total of Opuntia saponins was determined by UV-VIS spectrophotometry after it has been colored. Using oleanolic acid as a standard substance, assured detective wavelength of total of Opuntia saponins was 548 nm and standard curve equation was A=6.425C-0.0016, the average recovery was 98.47% with RSD of 3.29%. The method is rapid, accurate and repeatable.
2. Ethanol reflux extraction processes on total saponins from Opuntia were studied. Single element test and L_9(3_4) orthogonal design test were performed about alcohol concentration, ratio of solid to liquid, extraction temperature and time. As a result, the optimal conditions were obtained as follows: the solvent is 80% ethanol, the temperature is 70℃, the extracting time is 6h and solid-liquid ration is 1: 15. The result shows that the average extraction rate of total of Opuntia saponins is 0.1159%.
3. The separation and purification of total of Opuntia saponins were carried out with the method of macroporous adsorption resin. The absorption and desorption of D101 macroporous resin to total of Opuntia saponins were studied. The result indicated that D101 macroporous resin fits for the separation of total of Opuntia saponins and its static state adsorption ratio reaches 9.922mg/g dried resin. The optimal purification parameters of D101 were: pH of 3, flowing rate of adding sample and adsorption solution were 2BV/h and 2BV/h respectively. Through response orthogonal test and statistic software STATGRAPHICS plus 5.1 and variance analysis, the optimization operational condition was established. The ratio diameter to high of the chromatographic column was 1:11.57, and temperature was 28.83℃. It is desired to elute with 5BV at the flow rate of 2BV/h then desorpt with 5BV and 50% ethanol at the same flow rate, to effectively separate total of Opuntia saponins.
4. Content of total of Opuntia saponins of 8 fermentation Opuntia analysed by optimal technique flow.
To sum up, the results of the whole experiments demonstrated that techniques such as ethanol reflux extraction and macroporous resin purification could effectively enhance the extraction and purification rates of total of Opuntia saponins.
1、确定了测定仙人掌总皂苷含量的比色-紫外分光光度法:以齐墩果酸(OleanolicAcid,OA)为对照,确定出仙人掌总皂苷的最大吸收波长为548 nm,测定仙人掌总皂苷含量的标准曲线方程为A=6.425 C -0.0016,加标回收率为98.47%,相对标准偏差RSD为3.29%,该方法快捷、精确、重现性良好。
2、研究了仙人掌总皂苷的醇回流提取工艺,在单因子实验的基础上,采用L_9(3~4)正交实验确定了醇回流提取仙人掌总皂苷的适宜工艺参数:提取剂乙醇浓度为80%、固液比为1:15、提取温度70℃、提取时间6h,仙人掌总皂苷的平均得率0.1159%。
3、探讨了大孔吸附树脂法对仙人掌总皂苷分离纯化的影响。实验研究和分析了D101大孔树脂对仙人掌总皂苷的吸附和解吸性能。结果表明:D101大孔树脂适于仙人掌总皂苷的分离和精制,饱和吸附量达9.922 mg/g干树脂;吸附条件为:溶液pH值为3.0、上样流速2BV/h、解吸液流速2BV/h;通过旋转正交实验及方差分析确定了仙人掌总皂苷分离富集的优化操作条件为:色谱柱的径高比1:11.57,柱温28.83℃。采用约5BV/h水溶液以2BV/h流速淋洗后,再以5BV 50%的乙醇溶液以2BV/h流速解吸,可较好地分离仙人掌总皂苷。
4、用优化的工艺流程提取分析了经发酵的仙人掌8个样品中总皂苷的含量。
综上所述,采取乙醇回流提取、大孔吸附树脂等工艺技术可以有效的提高仙人掌总皂苷的的提取分离效率。
Opuntia dillenii Haw. is a plant belonging to genus Opuntia in Cactaceae family. The plants of Opuntia have a wide distribution in the world. Recent studies show that total of Opuntia saponins is an important biologically active component in genus Opuntia plant. There is no systemic report on the research of extraction and purification technique of total of Opuntia saponins in China. The objective of this paper is to lay a foundation for compositive utilization and exploitation of Cactaceae. On the bases of previous research of total of Opuntia saponins and the facilities of the laboratory, this thesis systematically studied a colorimetric method for quantitative analysis of total of Opuntia saponins , optimized the parameters of ethanol reflux extraction which was screened by orthogonal design. The purification processes of total of Opuntia saponins with D101 macroporous resin was generally studied by taking adsorption quantities, eluting ratio and so on. The major results are shown as follows:
1. The content of total of Opuntia saponins was determined by UV-VIS spectrophotometry after it has been colored. Using oleanolic acid as a standard substance, assured detective wavelength of total of Opuntia saponins was 548 nm and standard curve equation was A=6.425C-0.0016, the average recovery was 98.47% with RSD of 3.29%. The method is rapid, accurate and repeatable.
2. Ethanol reflux extraction processes on total saponins from Opuntia were studied. Single element test and L_9(3_4) orthogonal design test were performed about alcohol concentration, ratio of solid to liquid, extraction temperature and time. As a result, the optimal conditions were obtained as follows: the solvent is 80% ethanol, the temperature is 70℃, the extracting time is 6h and solid-liquid ration is 1: 15. The result shows that the average extraction rate of total of Opuntia saponins is 0.1159%.
3. The separation and purification of total of Opuntia saponins were carried out with the method of macroporous adsorption resin. The absorption and desorption of D101 macroporous resin to total of Opuntia saponins were studied. The result indicated that D101 macroporous resin fits for the separation of total of Opuntia saponins and its static state adsorption ratio reaches 9.922mg/g dried resin. The optimal purification parameters of D101 were: pH of 3, flowing rate of adding sample and adsorption solution were 2BV/h and 2BV/h respectively. Through response orthogonal test and statistic software STATGRAPHICS plus 5.1 and variance analysis, the optimization operational condition was established. The ratio diameter to high of the chromatographic column was 1:11.57, and temperature was 28.83℃. It is desired to elute with 5BV at the flow rate of 2BV/h then desorpt with 5BV and 50% ethanol at the same flow rate, to effectively separate total of Opuntia saponins.
4. Content of total of Opuntia saponins of 8 fermentation Opuntia analysed by optimal technique flow.
To sum up, the results of the whole experiments demonstrated that techniques such as ethanol reflux extraction and macroporous resin purification could effectively enhance the extraction and purification rates of total of Opuntia saponins.
引文
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