白芍药汁炙黄药子的炮制减毒工艺及其机制研究

Toxicity attenuation processing technology and mechanism of Rhizoma Dioscoreae Bulbiferae stir-fried with Paeoniae Radix Alba decoction

该研究首次探究白芍药汁炙黄药子的炮制减毒工艺,并进一步探索其减毒机制。采用3因素3水平正交试验法制备白芍药汁炙黄药子炮制品9个,以高效液相测定黄药子炮制前后主要肝毒性成分黄独素B含量下降为依据进行减毒工艺的初步筛选,在此基础上,选取黄药子生品及代表性的炮制品以2 g·kg^(-1)(相当于临床等效剂量)灌胃给予小鼠21 d,末次给药24 h后,取血清和肝组织,结合反映肝功能的血清生化指标及肝脏组织病理进一步筛选验证工艺,然后借助试剂盒法检测肝组织脂质过氧化及抗氧化指标,蛋白免疫印迹法检测小鼠肝脏醌氧化还原酶(NADPH quinone oxidoreductase 1,NQO1)和谷氨酸半胱氨酸连接酶(glutamate-cysteine ligase,GCLM)蛋白表达进一步探究炮制减毒机制。结果表明白芍药汁炙黄药子各炮制品均能不同程度降低黄独素B的含量并改善黄药子诱导的肝损伤,其中A_(2)B_(2)C_(3)炮制工艺能使黄药子生品诱导的过高的谷丙转氨酶(alanine transaminase,ALT)和谷草转氨酶(aspartate transaminase,AST)水平分别降低50.2%和42.4%(P<0.01,P<0.01),白芍药汁炙黄药子炮制品能不同程度逆转黄药子生品诱导的小鼠肝脏NQO1和GCLM的蛋白表达水平的降低(P<0.05或P<0.01),并能不同程度逆转小鼠肝脏丙二醛(malondialdehyde,MDA)水平的升高以及谷胱甘肽(glutathione,GSH)、谷胱甘肽过氧化物酶(glutathione peroxidase,GPX)、谷胱甘肽S-转移酶(glutathione S-transferase,GST)水平的降低(P<0.05或P<0.01)。综上,该研究表明白芍药汁炙黄药子的最佳炮制减毒工艺为A_(2)B_(2)C_(3)组合,即“10%的白芍药汁闷润黄药子饮片,在130℃炮制11 min”,并且减毒机制涉及增强肝脏NQO1和GCLM抗氧化蛋白表达及相关抗氧化酶水平。

This study explored toxicity attenuation processing technology of Rhizoma Dioscoreae Bulbiferae stir-fried with Paeoniae Radix Alba decoction for the first time,and further explored its detoxification mechanism.Nine processed products of Rhizoma Dioscoreae Bulbiferae stir-fried with Paeoniae Radix Alba decoction were prepared by orthogonal experiment with three factors and three levels.Based on the decrease in the content of the main hepatotoxic component diosbulbin B before and after processing of Rhizoma Dioscoreae Bulbiferae by high-performance liquid chromatography,the toxicity attenuation technology was preliminarily screened out.On this basis,the raw and representative processed products of Rhizoma Dioscoreae Bulbiferae were given to mice by gavage with 2 g·kg^(-1)(equival to clinical equivalent dose)for 21 d.The serum and liver tissues were collected after the last administration for 24 h.The serum biochemical indexes reflecting liver function and liver histopathology were combined to further screen out and verify the processing technology.Then,the lipid peroxidation and antioxidant indexes of liver tissue were detected by kit method,and the expressions of NADPH quinone oxidoreductase 1(NQO1)and glutamate-cysteine ligase(GCLM)in mice liver were detected by Western blot to further explore detoxification mechanism.The results showed that the processed products of Rhizoma Dioscoreae Bulbiferae stir-fried with Paeoniae Radix Alba decoction reduced the content of diosbulbin B and improved the liver injury induced by Rhizoma Dioscoreae Bulbiferae to varying degrees,and the processing technology of A_(2)B_(2)C_(3) reduced the excessive levels of alanine transaminase(ALT)and aspartate transaminase(AST)induced by raw Rhizoma Dioscoreae Bulbiferae by 50.2%and 42.4%,respectively(P<0.01,P<0.01).The processed products of Rhizoma Dioscoreae Bulbiferae stir-fried with Paeoniae Radix Alba decoction reversed the decrease protein expression levels of NQO1 and GCLM in the liver of mice induced by raw Rhizoma Dioscoreae Bulbiferae to varying degrees(P<0.05 or P<0.01),and it also reversed the increasing level of malondialdehyde(MDA)and the decreasing levels of glutathione(GSH),glutathione peroxidase(GPX),and glutathione S-transferase(GST)in the liver of mice(P<0.05 or P<0.01).In summary,this study shows that the optimal toxicity attenuation processing technology of Rhizoma Dioscoreae Bulbiferae stir-fried with Paeoniae Radix Alba decoction is A_(2)B_(2)C_(3),that is,10%of Paeoniae Radix Alba decoction is used for moistening Rhizoma Dioscoreae Bulbiferae and processed at 130℃for 11 min.The detoxification mechanism involves enhancing the expression levels of NQO1 and GCLM antioxidant proteins and related antioxidant enzymes in the liver.