黑果小檗果实中锦葵素-3-O-葡萄糖苷的分离纯化及安全性研究

Isolation and purification of malvidin 3-O-glucoside from Berheris heteropoda Schrenk fruits and its safety studies

中文关键词:  黑果小檗  锦葵素-3-O-葡萄糖苷  柱层析  急性毒性

英文关键词:Berheris heteropoda Schrenk  malvidin 3-O-glucoside  column chromatography  acute toxicity

中文摘要:

      目的 建立并验证黑果小檗果实中锦葵素-3-O-葡萄糖苷(malvidin 3-O-glucoside, Ma-3G)的分离纯化方法, 并评估其安全性。方法 利用超声辅助浸提法提取黑果小檗果实中的花色苷(Berberis atrocarpa Schneid anthocyanin, BHSA)粗提物, 将BHSA粗提物用乙酸乙酯萃取, AB-8大孔吸附树脂、聚酰胺树脂纯化, 采用Sephadex LH-20凝胶柱对纯化后BHSA进行分离, 分别考察洗脱液浓度、上样量对花色苷不同组分分离效果的影响, 并筛选出最佳分离条件, 将最佳条件下分离得到的Ma-3G进行紫外可见光谱扫描及高效液相色谱法检测。采用固定剂量法对小鼠以2000 mg/kg Ma-3G的单次口服剂量进行安全性实验。结果 经乙酸乙酯萃取、AB-8大孔吸附树脂、聚酰胺树脂纯化后得到BHSA粗品纯度为50.76%, Sephadex LH-20凝胶柱层析的最佳分离条件为上样量20 mg, 洗脱液为含有0.5% HCl的20%甲醇溶液, 在此条件下成功获得了Ma-3G单体。使用面积归一化法进行纯度测定, 结果显示其纯度为99.48%。黑果小檗果实中Ma-3G的安全性实验表明小鼠给药后14 d精神状态良好, 对小鼠的死亡率、体重、脏器系数、血常规和病理均无显著影响。结论 通过组合柱层析法对BHSA进行分离纯化, 成功获得了高纯度的Ma-3G单体。这种方法为工业应用中高效、低成本地分离黑果小檗果实中的花色苷提供了一种实用的解决方案。安全性实验表明黑果小檗果实中Ma-3G可引起急性毒性的毒性剂量范围>2000 mg/kg, 为后期体内药理实验奠定研究基础。

英文摘要:

      Objective To establish and validate a method for the isolation and purification of malvidin 3-O-glucoside (Ma-3G) from the fruits of Berheris heteropoda Schrenk, and evaluate its safety. Methods The crude extract of Berberis atrocarpa Schneid anthocyanin (BHSA) was extracted by ultrasound-assisted leaching. The crude extract of BHSA was extracted with ethyl acetate, purified by AB-8 macroporous adsorbent resin and polyamide resin, and then separated from the purified BHSA on a Sephadex LH-20 gel column, and the effects of the concentration of the eluent and the amount of sample were investigated on the separation of the different components of the anthocyanin and the optimum conditions were screened out, respectively. The Ma-3G obtained by isolation under optimum conditions was subjected to ultraviolet-visible spectrum scanning as well as high performance liquid chromatography. The safety test was carried out using a single oral dose of 2000 mg/kg Ma-3G in mice using the fixed-dose method. Results The purity of the crude BHSA was 50.76% after extraction with ethyl acetate, purification with AB-8 macroporous adsorbent resin and polyamide resin. The optimum separation conditions of Sephadex LH-20 gel column chromatography were as follows: The sample size was 20 mg and the eluent was 20% methanol solution containing 0.5% HCl, under which Ma-3G monomer was successfully obtained. The purity of Ma-3G monomer was determined by area normalisation method, and the result showed that the purity was 99.48%. The safety test of Ma-3G in the fruit of Berheris heteropoda Schrenk showed that the mice were in good mental state 14 d after the administration of Ma-3G, and there was no significant effect on the mortality, body weight, organ coefficients, blood counts and pathology of the mice. Conclusion High purity Ma-3G monomer has been successfully obtained by separation and purification of BHSA by composite column chromatography, which provides a practical solution for the high efficiency and low cost separation of anthocyanins from Berberis heteropoda Schrenk fruits in industrial applications. The safety test has showed that Ma-3G in Berberis heteropoda Schrenk fruits can cause acute toxicity in the dose range of >2000 mg/kg, which lays the foundation for the in vivo pharmacological study.