补骨脂炮制品水提和醇沉物化学成分及肝毒性研究

Study on chemical compositions and hepatotoxicity of water extract and ethanol precipitate of processed products of Psoralea corylifolia

目的研究补骨脂不同炮制品水提及醇沉物中5种化学成分的含量变化情况,并初步评价其肝毒性。方法制备补骨脂生品和雷公法、流水漂洗法、盐炙法炮制品的水提样品,以及雷公法、盐炙法炮制品的醇沉样品,采用高效液相色谱法测定其中补骨脂苷、异补骨脂苷、补骨脂素、异补骨脂素、补骨脂酚的含量并进行比较;以不同质量浓度补骨脂生品、流水漂洗法、雷公法、盐炙法水提样品和雷公法、盐炙法醇沉样品作用72 h,计算各样品对野生型斑马鱼的半数致死浓度(LC50)和最大非致死浓度(MNLC);以对乙酰氨基酚为阳性对照,以上述样品(MNLC)作用72 h,观察野生型斑马鱼的基本形态和转基因斑马鱼的肝脏表型。采用Pearson相关性分析评价成分含量与肝毒性的相关性。结果与生品水提物比较,不同炮制品水提样品中补骨脂苷、异补骨脂苷的含量均显著降低(P<0.05),而雷公法、盐炙法醇沉样品中补骨脂素、异补骨脂素、补骨脂酚的含量均显著升高(P<0.05)。生品、流水漂洗法、雷公法、盐炙法水提样品和雷公法、盐炙法醇沉样品的LC50分别为2.45、5.00、5.38、1.55、2.36、0.64 g/L(以生药量计),MNLC分别为2.21、4.53、5.02、1.37、2.13、0.53 g/L(以生药量计);与空白对照组比较,各样品处理组斑马鱼幼鱼均表现出不同程度的畸形,肝脏相对荧光强度均显著减弱(P<0.05或P<0.01)。补骨脂酚、异补骨脂素、补骨脂素等脂溶性成分与肝脏相对荧光强度的相关性较高(R2>0.7)。结论补骨脂炮制品经水提后以补骨脂苷、异补骨脂苷为主,经醇沉后补骨脂素、异补骨脂素、补骨脂酚的含量增加;醇沉样品的肝毒性更强,且肝毒性与补骨脂脂溶性成分的含量呈正相关。

OBJECTIVE To study the content changes of 5 chemical compositions in water extract and ethanol precipitate of different processed products of Psoralea corylifolia,and to preliminarily evaluate its hepatotoxicity.METHODS The water extracts from crude product of P.corylifolia and processed products by Leigong method,running water rinsing method,and salt stir-frying method were prepared,as well as the ethanol precipitates of processed products by Leigong method and salt stir-frying method were prepared.The contents of psoralenoside,isopsoralenoside,psoralen,isopsoralen and bakuchiol were determined by high-performance liquid chromatography and compared.The median lethal concentration(LC50)and maximum non-lethal concentration(MNLC)of each sample to wild-type zebrafish juveniles were calculated after 72 h of treatment with different concentrations of water extracts from raw product and processed products by running water rinsing method,Leigong method and salt stir-frying method,different concentrations of ethanol precipitates from processed products by Leigong method and salt stir-frying method,and the acetaminophen was used as the positive control.The basic morphology of wild-type zebrafish juveniles and the liver phenotype of transgenic zebrafish juveniles were observed after 72 h of treatment with the above samples(MNLC).Pearson correlation analysis was used to evaluate the correlation between component content and hepatotoxicity.RESULTS Compared with the water extract of raw products,the contents of psoralenoside and isopsoralenoside in the water extract of different processed products were generally decreased(P<0.05),while the contents of psoralen,isopsoralen and bakuchiol in the ethanol precipitate of Leigong method and salt stir-frying products were significantly increased(P<0.05).The LC50 of water extracts of crude product and processed products by running water rinsing method,Leigong method,salt stir-frying method,and ethanol precipitates of processed products by Leigong method and salt stir-frying method were 2.45,5.00,5.38,1.55,2.36,0.64 g/L(calculated by crude drug),and MNLC were 2.21,4.53,5.02,1.37,2.13,0.53 g/L(calculated by crude drug).Compared with the blank control group,the zebrafish juveniles in each sample treatment group showed different degrees of deformity,the liver relative fluorescence intensity was significantly weakened(P<0.05 or P<0.01).Fat-soluble components such as bakuchiol,isopsoralen and psoralen were highly correlated with liver fluorescence intensity(R2>0.7).CONCLUSIONS The processed products of P.corylifolia mainly compose of psoralenoside and isopsoralenoside after water extraction,the contents of psoralen,isopsoralen and bakuchiol increase after alcohol precipitation,and the hepatotoxicity is positively correlated with the contents of liposoluble compositions in P.corylifolia.