反相离子对-高效液相色谱法同时测定葛根芩连汤中13种有效成分及其在配伍机制研究中的应用

Simultaneous determination of 13 active components in Gegen Qinlian Decoction by reversed-phase ion pair-high performance liquid chromatography and its application in compatibility mechanism exploration

目的研究葛根芩连汤不同组方中活性成分含量的差异,揭示其药效物质的变化规律,进而从化学层面探究葛根芩连汤的配伍规律。方法采用反相离子对-高效液相色谱法测定葛根芩连汤不同组方配伍中13种有效成分(葛根素、大豆苷元、黄芩苷、汉黄芩苷、汉黄芩素、小檗碱、巴马汀、药根碱、黄连碱、甘草素、异甘草苷、甘草酸和甘草次酸)的含量。色谱柱为Diamonsil C18柱(250 mm×4.6 mm,5μm);流动相为3.5 mmol/L庚烷磺酸钠溶液(含体积分数0.1%甲酸)-乙腈(含体积分数0.1%甲酸),梯度洗脱,体积流量为1 mL/min;检测波长为270 nm;柱温35℃;进样量为20μL。结果葛根芩连汤中13种有效成分均基线分离、峰形对称,经方法学验证各项效能指标均符合定量分析要求。不同组方配伍中活性成分的含量变化规律:葛根与黄芩、黄连配伍时,葛根中的葛根素与大豆苷元以及黄芩中的黄芩苷含量均显著增加;黄芩、黄连与炙甘草配伍时,其中的黄芩苷、汉黄芩苷、汉黄芩素和小檗碱等有效成分均有增加,同时甘草中的甘草酸亦有显著增加;全方配伍时各有效成分均有不同程度增加。结论所建立的方法可用于葛根芩连汤及其组方的配伍规律研究。葛根芩连汤全方配伍对葛根与黄芩、黄连及炙甘草中有效成分的溶出均起到促进作用。其中,臣药黄芩、黄连能显著促进君药葛根中有效成分的溶出,起到辅佐君药的作用;而佐使药炙甘草不仅可促进臣药中有效成分的溶出,同时可在一定程度上增强臣药对君药的辅佐作用,起到协调全方、增效减毒的作用。

Objective To study the content differences of active ingredients among various formula in Gegen Qinlian Decoction(GQD,葛根芩连汤)to reveal the change law of pharmacodynamic substance and explore the compatibility rule of GQD based on chemistry level.Methods Reversed-phase ion pair-high performance liquid chromatography was established to determinate the content of 13 active components(puerarin,daidzein,baicalin,wogonoside,wogonin,berberine,jatrorrhizine,palmatine,coptisine,liquiritigenin,isoliquiritin,glycyrrhizic acid,glycyrrhetinic acid)in various formula of GQD.Separation was performed on Diamonsil C18 column(250 mm×4.6 mm,5μm).Mobile phase consisted of 3.5 mmol/L sodium heptane sulfonate solution(containing 0.1%formic acid by volume)and acetonitrile(containing 0.1%formic acid by volume)and followed by gradient elution at the flow rate of 1 mL/min.The detection wavelength was 270 nm,and the column temperature was 35℃;The injection volume was 20μL.Results All the 13 components in GQD were separated from baseline and symmetrical in peak shape.The effectiveness indexes were verified by the methodology to meet the requirements of quantitative analysis.Changes rules for content of active ingredients in different combinations:The contents of puerarin and daidzein in Gegen(Puerariae Lobatae Radix,PLR)and baicalin in Huangqin(Scutellariae Radix,SR)were significantly increased when PLR was compatible with SR and Huanglian(Coptidis Rhizoma,CR).When SR and CR were combined with Zhigancao(Glycyrrhizae Radix et Rhizoma Praeparata cum Melle,GRRPM),the active components such as baicalin,wogonoside,wogonin and berberine were increased,and the glycyrrhizic acid in GRRPM was also significantly increased.All the active components were increased in different degrees when the formula was combined with the whole formula.Conclusion The established method can be used to study the compatibility of GQD and its formulations.The combination of GQD can promote the dissolution of active components in PLR,SR,CR and GRRPM.Among them,SR and CR could significantly promote the dissolution of the effective components in PLR,and play the role of adjutant to PLR;And assistant medicine GRRPM not only can promote the dissolution of effective components from the minister medicine,at the same time can be enhanced to a certain extent minister medicine adjuvant role of monarch medicine,have the effect of coordination of all parties to improve effect and decrease toxicity.