七叶皂苷A～D体外转化规律研究

Study on isomerization law of aescins A,B,C,and D

目的建立HPLC法同时测定七叶皂苷A^D量的方法,考察不同因素水平影响下七叶皂苷A^D的体外转化规律,并优化出最佳转化条件,为调控生产中七叶皂苷A^D的比例提供参考。方法采用Diamonsil C18色谱柱(250 mm×4.6 mm,5μm),以乙腈-0.05%磷酸水溶液(34∶66)为流动相,柱温为35℃,体积流量为1.0 m L/min,检测波长为200 nm,外标法定量;以七叶皂苷A^D的转化率及有关物质的量为指标,通过单因素实验设计,考察p H值、温度、时间3个因素对七叶皂苷A^D体外转化的影响趋势,再采用中心组合设计-响应面法优选转化条件,利用Design Expert 8.0.5软件分析预测最佳转化条件并进行验证试验。结果七叶皂苷A^D分别在0.39~3.49 mg/m L(r=0.999 5)、0.28~2.55 mg/m L(r=0.999 5)、0.19~1.67 mg/m L(r=0.999 7)、0.07~0.59 mg/m L(r=0.999 7)与峰面积呈良好的线性关系;单因素实验表明,p H值、温度、时间对七叶皂苷A和B、C和D转化率的影响呈现相似的趋势,在p H值5.0~10.0,温度45~85℃,时间0.25~8 h时,七叶皂苷A、B部分转化成七叶皂苷C、D,其他条件下七叶皂苷A^D均转化为有关物质;中心组合设计-响应面法研究得到最佳转化条件为p H值6.9,温度54℃,时间7.4 h,在此条件下,七叶皂苷C、D转化率的综合评分为11.50%,与预测值的偏差<5%。结论不同p H值、温度、时间影响下,七叶皂苷A^D的体外转化呈现出一定的规律,响应面法优选出的最佳转化条件稳定可行,可为调控生产中七叶皂苷A^D的比例提供科学的依据。

Objective To investigate the isomerization law of aescins A, B, C, and D under different conditions and optimize the isomerization process. Methods Using the conversion of aescin C and D, and the content of impurity as index, single factor experiment was applied to evaluate the effect trends of isomerization from three parameters including p H value, reaction temperature and time. Then, central composite design-response surface method was used to estimate the relationship between the dependent and independent variables and to validate the optimal conditions. Results According to the single factor experiment, the effect trends of three parameters on conversion of aescins C and D were analogous, and the isomerization happened under the conditions as follows： p H value of 5.0—10.0, reaction temperature of 45—85 ℃, and reaction time of 0.25—8 h. Based on the central composite design, the optimal conditions were predicted as follows： p H value was 6.9, reaction temperature was 54 ℃ and reaction time was 7.4 h. Under such conditions, the experimental values showed on significant difference from the predicted values. Conclusion The isomerization of aescins A, B, C, and D could show a certain law under different conditions, and the optimal isomerization process is stable and available, which could provide a scientific basis of regulating the proportion of aescins A, B, C, and D during industrial production.