基于高分辨质谱分析的经典名方百合地黄汤中生地黄汁的制备工艺

Preparation of Rehmanniae Radix Juice for Baihe Dihuangtang Based on UPLC-Q-TOF-MS

目的:对参照历代本草和医籍中记载的生地黄汁4种制备方法进行研究,以期找到最符合百合地黄汤方义的生地黄汁制备方法。方法:经古籍文献考证,已知制备生地黄汁的4种方法分别是鲜地黄捣汁、鲜地黄蒸汁、鲜地黄煮汁、生地黄煮汁。采用超高效液相色谱-四级杆-飞行时间串联质谱技术(UPLC-Q-TOF-MS),分析4种方法制备得到的生地黄汁的化学成分,采用主成分分析技术(PCA)对其质谱数据进行分析。结果:(1)从4种生地黄汁共鉴定出27个化学成分,包括10个环烯醚萜苷类、14个苯乙醇苷类、2个酚酸和1个紫罗兰酮。4种生地黄汁中共有的化学成分有15个,包括7个环烯醚萜苷类、7个苯乙醇苷类和1个酚酸。(2)4种生地黄汁共有的化学成分,经对照品比对的有5个,分别为梓醇、桃叶珊瑚苷、地黄苷D、益母草苷和洋地黄叶苷C。(3)毛蕊花糖苷和异毛蕊花糖苷在鲜地黄捣汁中未检测到,但在鲜地黄蒸汁、鲜地黄煮汁、生地黄煮汁中均检测到,说明毛蕊花糖苷和异毛蕊花糖苷是鲜地黄受热后产生的,并非鲜地黄的原有成分。(4)通过对洋地黄叶苷C和毛蕊花糖苷的一级、二级碎片离子比对分析,结合两者及异毛蕊花糖苷3种化学成分的空间结构异同,认为毛蕊花糖苷是由洋地黄叶苷C受热断裂糖苷键,脱去一分子甘露糖转化而来。(5)毛蕊花糖苷可异构化生成异毛蕊花糖苷,异毛蕊花糖苷的相对含量随着鲜地黄受热时间的增加而增加,但毛蕊花糖苷和洋地黄叶苷C的相对含量却并未相应减少,推测洋地黄叶苷C也是由其上游成分转化而来。结论:鲜地黄捣汁与其他3种方法制备得到的“生地黄汁”在成分方面有显著区别,其他3种方法制备的“生地黄汁”化学成分组成较相似。虽然4种方法均可用于制备“生地黄汁”,但以鲜地黄蒸汁、鲜地黄煮汁和生地黄煮汁作为百合地黄汤中“生地黄汁”的制备方法更合适。

Objective:To compare the four preparation methods of Rehmanniae Radix juice described in ancient literature and find the method that is most suitable for the preparation of Rehmanniae Radix juice used in Baihe Dihuangtang.Method:The ancient medical books record four methods for preparing Rehmanniae Radix juice:crushing fresh Rehmanniae Radix for juice,steaming fresh Rehmanniae Radix for juice,boiling fresh Rehmanniae Radix for juice,and boiling dry Rehmanniae Radix for juice.Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS)was employed to detect the compounds in the four juice samples,followed by principal component analysis(PCA).Result:(1)Totally 27compounds were identified in the juice samples,including 10 iridoid glycosides,14 phenylethanoid glycosides,2 phenolic acids,and 1 irisone.Among them,15 common compounds were shared by the four juice samples,including 7 iridoid glycosides,7 phenylethanoid glycosides,and 1 phenolic acid.(2)Five common compounds in the four juice samples can be matched with the reference standards,which were catalpol,aucubin,rehmannioside D,ajugol,and purpureaside C.(3)Verbascoside and isoacteoside were not detected in the juice prepared by crushing fresh Rehmanniae Radix,while it was detected in the other three juice samples,which indicated that the two components were produced after heating rather than being the original components in fresh Rehmanniae Radix.(4)The comparison of the ion fragments demonstrated that verbascoside was produced from purpureaside C after the cleavage of the glycosidic bond and removal of a molecule of mannose.(5)Isoacteoside could be isomerized from verbascoside,and its relative content increased with the extension of heating time.However,the relative content of verbascoside and purpureaside C did not decrease significantly.Therefore,it was hypothesized that purpureaside C was produced from its upstream component.Conclusion:The juice prepared by crushing fresh Rehmanniae Radix has the chemical composition significantly different from the juice samples prepared with the other 3 methods,while the latter 3 juice samples had similar chemical composition.Although all the four methods can be used,it is more suitable to prepare Rehmanniae Radix juice by steaming fresh Rehmanniae Radix,boiling fresh Rehmanniae Radix,and boiling dry Rehmanniae Radix.