泽泻提取物三萜类成分含量测定研究

目的: 建立紫外-可见分光光度法和高效液相色谱法测定泽泻提取物中三萜类成分含量。方法: 采用紫外-可见分光光度法,以5%香草醛(用冰醋酸配制)-高氯酸显色,在555 nm波长处测定吸光度,计算泽泻提取物总三萜含量;应用高效液相色谱法同时测定泽泻提取物中12个三萜类成分,采用Ultimate XB-C₁₈ 色谱柱(4.6 mm×150 mm,5 μm),以乙腈-5%甲醇水为流动相梯度洗脱,流速1.0 mL · min^-1,检测波长208 nm。结果: 紫外-可见分光光度法测定总三萜含量,以23-乙酰泽泻醇B计,质量浓度在5.9~15.6 μg · mL^-1 范围内与吸光度值呈良好线性关系(r=0.999 1),平均加样回收率99.05%,RSD为2.3%;高效液相色谱法测定12个泽泻三萜类成分含量,在考察的浓度范围内,线性关系良好(r>0.999 0),回收率均在95.89%~99.33%范围内,RSD为2.0%~3.4%。泽泻提取物中12个三萜类成分的含量:泽泻醇A 5.71%~5.95%、泽泻醇B 3.47%~3.99%、泽泻醇C 2.53%~2.97%、泽泻醇G 3.07%~3.93%、泽泻醇L 3.65%~3.99%、23-乙酰泽泻醇A 2.39% ~3.02%、23-乙酰泽泻醇B 10.56%~11.32%、23-乙酰泽泻醇C 9.44%~9.86%、16-羰基-23-乙酰泽泻醇A 0.63% ~3.14%、16-羰基-24-乙酰泽泻醇A 1.08%~1.21%、11-去氧泽泻醇B 2.29%~2.85%、24-乙酰泽泻醇A 4.04% ~5.02%。结论: 经方法学验证,本文建立的紫外-可见分光光度法可以用于检测泽泻提取物中总三萜含量,高效液相色谱法可用于检测泽泻提取物中12个泽泻三萜类成分含量。

Objective: To establish ultraviolet-visible(UV-Vis)spectrophotometry and HPLC method for determination of triterpenoids in Rhizoma Alismatis extracts. Methods: With 5% vanillin-acetic acid and perchloric acid as the developer, the colorimetric method was applied.Total triterpenoids in extracts was determined at 555 nm wavelength by UV-Vis spectrophotometry, and 12 triterpenoids was determined by HPLC with Ultimate XB-C₁₈ column(4.6 mm×150 mm, 5 μm)and the mobile phase consisting of acetonitrile-water(5% methanol)at a flow rate of 1.0 mL · min^-1.The detection wavelength was set at 208 nm. Results: With 23-acetate alisol B as the standard, the UV-Vis spectrophotometry method was applied.There was a good linear relationship between the concentration and absorbance of 23-acetate alisol B in the range of 5.9-15.6 μg · mL^-1 (r=0.999 1);the average recovery rate was 99.05% and the RSD was 2.3%.By HPLC, the linear range of 12 detected compounds were fine(r>0.999 0), the overall recoveries ranged from 95.89%-99.33%, with the RSD ranging from 2.0%-3.4%.The contents of 12 triterpenoids including alisol A, alisol B, alisol C, alisol G, alisol L, 23-acetate-alisol A, 23-acetate-alisol B, 23-a cetate-alisol C, 16-oxo-23-acetate-alisol A, 16-oxo-24-acetate-alisol A, 11-deoxy alisol B, 24-a cetate-alisol A were 5.71%-5.95%, 3.47%-3.99%, 2.53%- 2.97%, 3.07%-3.93%, 3.65%-3.99%, 2.39%-3.02%, 10.56%-11.32%, 9.44%-9.86%, 0.63% -3.14%, 1.08%-1.21%, 2.29%-2.85% and 4.04%-5.02%, respectively. Conclusion: The two methods are in accordance with the requirements of the methodology validation, indicating that the ultraviolet-visible(UV-Vis)spectrophotometry method can be used for the detection of the total triterpenoids and the HPLC method can be used for detection of 12 triterpenoids.