鲜干品组方六神曲发酵前后指标成分的LC-MS测定及指纹图谱分析

Determination of Index Components and Fingerprint About Medicated Leaven Composed of Fresh or Dry Medicines Before and After Fermentation by LC-MS

目的：建立六神曲的指纹图谱质控方法,比较鲜干品各组样品发酵前后槲皮苷、槲皮素、木犀草素等成分的变化状况。方法：经拆方研究,按前期优选的工艺制备六神曲基本组及鲜干品各组,分别为基本组、鲜品煎汁组、干品1/3量煎汁组及干品全量煎汁组,并于发酵前后取样检测。采用LC-MS检测,Ulitimate XB-C18色谱柱（4.6 mm×250 mm,5μm）,流动相甲醇-0.2%甲酸水溶液梯度洗脱,检测波长254 nm,电喷雾离子源（ESI）,正离子模式检测。结果：确定鲜干品组方六神曲指纹图谱共有峰15个,其中10,13,15号峰分别为槲皮苷、木犀草素、槲皮素。基本组仅含木犀草素,发酵前后质量分数分别为18.5,21.3μg·g^-1,明显低于其他3组。在发酵前,鲜品煎汁组样品的3种成分含量均显著高于其他3组,与发酵前相比,发酵后木犀草素、槲皮素质量分数分别增长54.84%,4.53%;槲皮苷质量分数降低了0.76%。干品全量煎汁组发酵后3种成分含量较发酵前显著增加,其中槲皮苷、木犀草素、槲皮素质量分数分别增加了59.9%,161.7%,75.5%。结论：该质控检测方法精密、准确、重复性好,适用于六神曲的量化质量评价。发酵法可促进六神曲部分药效成分的溶出度增加及其生物转化反应的发生,初步揭示了六神曲发酵前后药效物质变化状况及鲜品入药优于干品的药效物质基础。

Objective： To establish a fingerprint quality control method of medicated leaven,and to compare the change of effective components（ such as quercitin,luteolin,quercetin,and so on） about medicated leaven composed of fresh or dry medicines before and after fermentation. Method： By studying on the decomposed recipes of medicated leaven,samples of four groups were prepared. Each group was fermented separately under the prior optimum conditions,taking samples before fermentation and after the completion of the fermentation,and then samples dried at 40 ℃ for testing. LC-MS analysis was performed on a Ulitimate XB-C18 column（ 4. 6 mm ×250 mm,5 μm） with the mobile phase consisting of methanol-0. 2% formic acid aqueous solution in gradient mode,the flow rate was 1. 0 m L · min^-1,the detection wavelength was set at 254 nm,electrospray ionization source（ ESI） was adopted in positive ion mode. Result： A total of 15 common peaks were confirmed in the fingerprint of medicated leaven composed of fresh medicines or dry ones; among them,peak 10,13,15 were quercitin,luteolin, quercetin, respectively. The base group contained only a very small amount of luteolin determined by LC-MS, the content of this ingredient was 18. 5 μg·g^-1 and 21. 3 μg·g^-1 before and after fermentation,it was significantly lower than the other groups. The contents of quercitin,luteolin,quercetin in the second group（ composed of fresh medicines） were higher and had obvious advantages than the other groups before the fermentation, after fermentation, the amounts of luteolin and quercetin had grown by 54. 8% and4. 53%,respectively,but the content of quercitrin was slightly reduced by 0. 76%. The contents of quercitin,luteolin,quercetin in the 4 thgroup（ composed of dry ones） were significantly increased after fermentation.Conclusion： The method is precise,accurate with high repeatability,which is suitable for quantitative evaluation of medicated leaven. In addition,the fermentation method can increase the solubility of active ingredients and the occurrence of biological transformation reactions about medicated leaven, the results show that the changes of pharmacological substances of medicated leaven before and after fermentation and pharmacodynamic material basis of fresh medicines is better than dry ones be used as medicine.