基于毒性成分转化的甘遂醋炙工艺优选

Optimization of parameters for stir-frying of Kansui Radix with vinegar based on conversion of toxic components

优选基于代表性毒性二萜成分转化的醋甘遂最佳炮制工艺,为醋甘遂的标准化生产提供参考。以甘遂中毒性较大的代表性二萜成分3-O-(2′E,4′Z-癸二烯酰基)-20-O-乙酰基巨大戟二萜醇(3-O-EZ)和大戟萜酯C(KPC)及其醋炙转化产物巨大戟二萜醇(ingenol)和20-去氧巨大戟二萜醇(20-deoxyingenol)为研究对象,分别采用人正常结肠黏膜上皮细胞(NCM460)和人结肠癌细胞(HT-29)进行肠毒性和泻水药效评价,并建立毒性成分转化的HPLC评价方法,探究甘遂醋炙转化规律。基于该转化规律,以转化产物ingenol和20-deoxyingenol总含量为目标,以炮制温度、炮制时间和醋用量为关键工艺参数,运用Box-Behnken响应面法优选醋甘遂的最佳炮制工艺并进行验证。结果显示甘遂醋炙后,毒性较大的双酯型3-O-EZ和KPC经酯键断裂先转化为单酯型3-O-(2′E,4′Z-癸二烯酰基)巨大戟二萜醇(3-EZ)和5-O-苯甲酰基-20-去氧巨大戟二萜醇(5-OBen),最终转化为几乎无毒性的ingenol和20-deoxyingenol,同时,其泻水药效得以保留。所建立的毒性成分转化含量测定方法中6个成分在相应的浓度范围内与峰面积的线性关系良好(R2≥0.9998),平均加样回收率为98.20%~102.3%(RSD≤2.4%)。甘遂醋炙后,其代表性二萜类及中间产物成分含量明显减少,相比生甘遂降低了14.78%~24.67%;转化产物总量明显增加,相比生甘遂升高了14.37%~71.37%。工艺优选结果显示炮制温度对产物总量的影响最显著,炮制时间次之。最佳炮制工艺为炮制温度为210℃,炮制时间为15 min,醋用量为30%。验证实验结果与预测值的相对误差为1.68%,表明该工艺合理稳定,具有良好的重复性。基于毒性成分转化优选的甘遂醋炙工艺有利于在保证饮片减毒存效基础上提高醋甘遂生产的稳定性,为同类有毒中药的工艺优选提供借鉴。

This study aims to optimize the parameters for stir-frying of Kansui Radix with vinegar based on the conversion of representative toxic diterpenes,which is expected to serve as a reference for the standardized production of Kansui Radix stir-fried with vinegar.To be specific,the toxic components[3-O-(2′E,4′Z-decadienoyl)-20-O-acetylingenol(3-O-EZ),kansuiphorin C(KPC)]in Kansui Radix and the products(ingenol,20-deoxyingenol)after the stir-frying with vinegar were selected.The toxicity to intestine and water-draining activity were evaluated with NCM460(normal human colon mucosal epithelial cell line)and HT-29(a human colorectal adenocarcinoma cell line).An HPLC method was then developed to assess the conversion of toxic components.On this basis,temperature,time,and amount of vinegar for the processing of Kansui Radix were optimized with the Box-Behnken design and the content of ingenol and 20-deoxyingenol as evaluation index.The results showed that after the stir-frying of Kansui Radix with vinegar,3-O-EZ and KPC were first converted to monoester 3-O-(2′E,4′Z-decadienoyl)ingenol(3-EZ)and 5-O-benzoyl-20-deoxyingenol(5-O-Ben)and finally to almost non-toxic ingenol and 20-deoxyingenol,respectively.Meanwhile,the water-draining activity was retained.Six compounds had a good linear relationship with the peak area in the corresponding concentration ranges(R2≥0.9998),and the average recovery fell in the range of 98.20%-102.3%(RSD≤2.4%).The content of representative diterpenes and intermediate products was 14.78%-24.67%lower in the Kansui Radix stir-fried with vinegar than in the Kansui Radix,while the content of the conversed products was 14.37%-71.37%higher.Among the process parameters,temperature had significant influence on the total content of products,followed by time.The optimal parameters were 210℃,15 min,and 30%vinegar.The relative error between the experimental results and the predicted values was 1.68%,indicating that the process was stable and reproducible.The strategy of screening optimal parameters for stir-frying of Kansui Radix with vinegar based on the transformation of toxic components can help improve the production stability,reduce the toxicity,and ensure the efficacy of Kansui Radix stir-fried with vinegar,which can serve as a reference for the process optimization of similar toxic Chinese medicinals.