盾叶薯蓣中薯蓣皂甙元提取新工艺的研究
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摘要
薯蓣皂甙元是合成多种甾体类药物的重要原料，其主要来源是从薯蓣属植物中提取获得。盾叶薯蓣是薯蓣皂甙元提取的重要原料，目前已实现工业化生产，而生产过程中大量使用酸水，造成了严重的环境污染。本文采用新的工艺进行薯蓣皂甙元的提取，为实现盾叶薯蓣综合利用提供理论依据。
本论文的研究工作由以下几个部分组成：
(1)建立了一种准确、快速分析测定薯蓣皂甙元含量的反相高效液相色谱方法，对提取物中薯蓣皂甙元与其它杂质的色谱分离条件进行优化，确定流动相为甲醇-水(95∶5，V／V)，流速1.0mL／min；UV检测波长208nm；柱温25℃。结果表明薯蓣皂甙元在0.10～2.00g／L范围内峰面积与其质量浓度具有很好的线性关系(r=0.9998)，样品平均加标回收率为99.76％，精密度RSD为1.76％。该方法具有较高的精密度和准确性，且分析周期短，适用于薯蓣皂甙元含量的精确分析。同时，采用显色法，建立了分光光度法测定薯蓣皂甙元含量，并与色谱法进行比较；
(2)研究了从盾叶薯蓣中预分离薯蓣总皂甙和淀粉、纤维素，再进行水解，提取薯蓣皂甙元的新工艺。通过正交实验对薯蓣皂甙元的提取工艺进行优选，选用L_(25)(5~6)正交表进行实验，以薯蓣皂甙元收率为评价参数，考察了6个因素对薯蓣皂甙元提取量的影响。得到最佳的工艺条件为：甲醇回流提取4h，回流速度为10分钟／次，用2.5mol／L的硫酸水解6h，120号溶剂汽油回流提取2h，回流速度为15分钟／次。占盾叶薯蓣95％以上的淀粉与纤维素实现了与薯蓣总皂甙的分离，大大减少了水解阶段的酸水用量。
(3)研究了盾叶薯蓣提取薯蓣皂甙元后废渣发酵制取酒精的最佳实验指标，以酒精产量为评价参数，采用5因素4水平的正交实验，用可见分光光度法对16种制取方法所得到的酒精进行比较分析。结果表明，酒精与重铬酸钾溶液经加热显色后，在585nm波长有最大吸收波长，酒精体积分数在0.028％～0.24％范围内线性关系良好(r=0.9998)；发酵时间对于酒精产量影响最大，在实验室条件下，采用50g原料使用7U／g原料淀粉酶，110U／g原料纤维素酶，110U／g原料糖化酶，0.04g／g原料酵母，发酵4天，可使酒精产量达到最大。
Diosgenin is a steroidal compound very useful in pharmaceutical industry as a natural source of steroidal hormones. Dioscorea Zingiberensis C. H. Wright is an important raw material for extracting diosgenin, whose industrial extraction has been carried out. However, mass pollution is generated in the process because of acid hydrolysis. A novel technology for diosgenin extraction was studied in this paper. The work in this paper is described as follows:
(1) An accurate and rapid reversed-phase high performance liquid chromatography was established for the determination of diosgenin. The chromatographic conditions were studied for the separation diosgenin from other component. Chromatographic separation was carried out on VP-ODS( 150×4.6 mm, 5μm)column using MeOH:H_2O(95:5, V/V) as the mobile phase at a flow rate of 1.0 mL/min. The UV detection was performed at 208 nm. Results showed that the linear range of calibration curve was 0.1～2.0 g/L (r=0.9998), the average recovery was 99.76% with RSD of 1.76%(n=3). It could be concluded that the method was accurate, reproducible and precision could be applied rapidly determine the diosgenin. At the same time, spectrophotometric analysis of diosgenin was studied for comparison with chromatography.
(2) A novel technology for extracting diosgenin from Dioscorea Zingibersis C. H. Wright was studied. Several important factors were analyzed in the orthogonal study of six factors and five levels, and the content of diosgenin was valued as an evaluation index using spectrophotometry. The results indicated that the concentration of sulfuric acid had an obvious effect on the content of diosgenin extracted. And experiments show the optimal technology for diosgenin extraction is under the conditions shown as followings: 20 g sample is refluxed for 4 h with methanol, and 10 minutes for each reflux operation; then 100 mL 2.5 mol/L H_2SO_4 is added for 6 hours of hydrolysis; at last is refluxed for 2 h with gasoline, and 15 minutes for each reflux operation, which can be used as a rapid and clean extraction technology. Preextraction of starch and cellulose, as main compound ofDioscorea Zingibersis C. H. Wright, would reduce acid consumption obviously comparing to traditional technology.
(3) The optimum fermentation conditions in alcohol production by residue from diosgenin processing were established. Several important factors were analyzed in the orthogonal study of five factors and four levels. The content of alcohol was valued as an evaluation index using spectrophotometry. The results indicate that the linear range of calibration curve was 0.028%～0.24% (r=0.9998) after color reaction, and optimal fermentation conditions were determined as follows: the use level ofα-amylase as 7 U/g, cellulase 110 U/g, glucoamylase 110 U/g, yeast 0.04 g/g, and 4 days fermentation time.
本论文的研究工作由以下几个部分组成：
(1)建立了一种准确、快速分析测定薯蓣皂甙元含量的反相高效液相色谱方法，对提取物中薯蓣皂甙元与其它杂质的色谱分离条件进行优化，确定流动相为甲醇-水(95∶5，V／V)，流速1.0mL／min；UV检测波长208nm；柱温25℃。结果表明薯蓣皂甙元在0.10～2.00g／L范围内峰面积与其质量浓度具有很好的线性关系(r=0.9998)，样品平均加标回收率为99.76％，精密度RSD为1.76％。该方法具有较高的精密度和准确性，且分析周期短，适用于薯蓣皂甙元含量的精确分析。同时，采用显色法，建立了分光光度法测定薯蓣皂甙元含量，并与色谱法进行比较；
(2)研究了从盾叶薯蓣中预分离薯蓣总皂甙和淀粉、纤维素，再进行水解，提取薯蓣皂甙元的新工艺。通过正交实验对薯蓣皂甙元的提取工艺进行优选，选用L_(25)(5~6)正交表进行实验，以薯蓣皂甙元收率为评价参数，考察了6个因素对薯蓣皂甙元提取量的影响。得到最佳的工艺条件为：甲醇回流提取4h，回流速度为10分钟／次，用2.5mol／L的硫酸水解6h，120号溶剂汽油回流提取2h，回流速度为15分钟／次。占盾叶薯蓣95％以上的淀粉与纤维素实现了与薯蓣总皂甙的分离，大大减少了水解阶段的酸水用量。
(3)研究了盾叶薯蓣提取薯蓣皂甙元后废渣发酵制取酒精的最佳实验指标，以酒精产量为评价参数，采用5因素4水平的正交实验，用可见分光光度法对16种制取方法所得到的酒精进行比较分析。结果表明，酒精与重铬酸钾溶液经加热显色后，在585nm波长有最大吸收波长，酒精体积分数在0.028％～0.24％范围内线性关系良好(r=0.9998)；发酵时间对于酒精产量影响最大，在实验室条件下，采用50g原料使用7U／g原料淀粉酶，110U／g原料纤维素酶，110U／g原料糖化酶，0.04g／g原料酵母，发酵4天，可使酒精产量达到最大。
Diosgenin is a steroidal compound very useful in pharmaceutical industry as a natural source of steroidal hormones. Dioscorea Zingiberensis C. H. Wright is an important raw material for extracting diosgenin, whose industrial extraction has been carried out. However, mass pollution is generated in the process because of acid hydrolysis. A novel technology for diosgenin extraction was studied in this paper. The work in this paper is described as follows:
(1) An accurate and rapid reversed-phase high performance liquid chromatography was established for the determination of diosgenin. The chromatographic conditions were studied for the separation diosgenin from other component. Chromatographic separation was carried out on VP-ODS( 150×4.6 mm, 5μm)column using MeOH:H_2O(95:5, V/V) as the mobile phase at a flow rate of 1.0 mL/min. The UV detection was performed at 208 nm. Results showed that the linear range of calibration curve was 0.1～2.0 g/L (r=0.9998), the average recovery was 99.76% with RSD of 1.76%(n=3). It could be concluded that the method was accurate, reproducible and precision could be applied rapidly determine the diosgenin. At the same time, spectrophotometric analysis of diosgenin was studied for comparison with chromatography.
(2) A novel technology for extracting diosgenin from Dioscorea Zingibersis C. H. Wright was studied. Several important factors were analyzed in the orthogonal study of six factors and five levels, and the content of diosgenin was valued as an evaluation index using spectrophotometry. The results indicated that the concentration of sulfuric acid had an obvious effect on the content of diosgenin extracted. And experiments show the optimal technology for diosgenin extraction is under the conditions shown as followings: 20 g sample is refluxed for 4 h with methanol, and 10 minutes for each reflux operation; then 100 mL 2.5 mol/L H_2SO_4 is added for 6 hours of hydrolysis; at last is refluxed for 2 h with gasoline, and 15 minutes for each reflux operation, which can be used as a rapid and clean extraction technology. Preextraction of starch and cellulose, as main compound ofDioscorea Zingibersis C. H. Wright, would reduce acid consumption obviously comparing to traditional technology.
(3) The optimum fermentation conditions in alcohol production by residue from diosgenin processing were established. Several important factors were analyzed in the orthogonal study of five factors and four levels. The content of alcohol was valued as an evaluation index using spectrophotometry. The results indicate that the linear range of calibration curve was 0.028%～0.24% (r=0.9998) after color reaction, and optimal fermentation conditions were determined as follows: the use level ofα-amylase as 7 U/g, cellulase 110 U/g, glucoamylase 110 U/g, yeast 0.04 g/g, and 4 days fermentation time.
引文
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