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以黑刺菝葜为试材,应用紫外可见分光光度法,研究测定其6-酮型甾体皂苷类化合物含量的方法;以LG-C为标准品,依据显色剂对LG-C反应的专属性和灵敏度,确定紫外可见分光光度法的显色体系和最大吸收波长;在单因素试验的基础上,通过星点设计-响应面法,确定最适显色反应条件。结果表明:紫外可见分光光度法的显色体系为茴香醛-硫酸法Ⅱ,最大吸收波长为429nm;最佳显色温度82.4℃,反应时间24min,显色剂用量1.2mL。优化反应条件后,在7.51~50.90μg范围内,LG-C与吸光度值呈良好线性关系,r=0.996 9;平均加样回收率为103.37%,RSD 2.2%。该方法灵敏度高、专属性强,重复性好,可以作为黑刺菝葜中6-酮型甾体皂苷含量的测定方法。
The method of determining the content of 6-keto steroidal saponins with UV-Vis spectrophotometry was studied by using the black stinging mullet as test material. Based on LG-C as reference material and LG-C reagent Specificity and sensitivity to determine the colorimetric system and the maximum absorption wavelength of UV-Vis spectrophotometry. On the basis of single-factor experiments, the optimal color reaction conditions were determined by the method of apical design-response surface method. The results showed that the colorimetric system of UV-visible spectrophotometry was anisaldehyde-sulfuric acid method Ⅱ, the maximum absorption wavelength was 429nm, the optimal color temperature was 82.4 ℃, the reaction time was 24min and the amount of reagent was 1.2mL. After optimizing the reaction conditions, LG-C has a good linear relationship with the absorbance value in the range of 7.51 ~ 50.90μg, r = 0.996 9; The average recovery was 103.37%, RSD 2.2%. The method has the advantages of high sensitivity, strong specificity and good repeatability, and can be used as a method for determining the content of 6-ketosteroid saponins in the black thorn gall.