基于药材原产地芍药甘草汤质量评价体系研究

Study on the Quality Evaluation System of Shaoyao Gancao Decoction(芍药甘草汤)Based on the Origin of Medicinal Materials

目的采用高效液相色谱法(HPLC)同时测定芍药甘草汤中没食子酸、芍药苷、甘草苷、甘草素和甘草酸5种指标成分的含量并建立芍药甘草汤指纹图谱,探究芍药甘草汤中间体关键质量属性,为其药材基源筛选和质量控制提供参考。方法制备来自不同产地不同批次的18批芍药甘草汤冻干粉作为中间体样品;采用Agilent ZORBAX Eclipse Plus C_(18)(4.6 mm×250 mm,5μm)色谱柱,以乙腈-0.2%磷酸水溶液为流动相,梯度洗脱,柱温35℃,流速0.8 mL/min,进样量10μL,对指标成分的含量和指纹图谱进行检测,评价多批次芍药甘草汤中间体质量属性差异,为其质量标准的建立提供参考。结果5种成分线性关系(R^(2)>0.999)、精密度(RSD≤0.89%,n=6)、重复性(RSD≤1.35%,n=6)、稳定性(RSD≤1.87%,72 h)均良好,加样回收率为(98.24±1.85)%~(104.88±1.38)%(n=6)。含量测定结果显示在不同批次芍药甘草汤中间体中仅没食子酸的含量波动范围较小(4.74~5.92 mg/g),芍药苷、甘草苷、甘草素和甘草酸在不同产地不同批次中的含量均有较大差异,含量范围分别为29.58~38.24、7.21~12.12、0.18~0.89、12.14~42.83 mg/g;建立芍药甘草汤指纹图谱,确定了14个共有峰,并对各峰进行药材归属,其中1、4、5、10号峰来自白芍,2、3、6、7、8、9、11、12、13、14号峰来自炙甘草,炙甘草对共有峰的贡献最大。其中13批芍药甘草汤中间体的HPLC指纹图谱与对照指纹图谱的相似度均大于0.98,第S4、S8、S16、S17、S18批次的相似度大于0.91,说明不同产地批次的药材质量差异较大。结论该方法准确可靠、灵敏度高,具有良好的重复性,可用于测定芍药甘草汤中5种成分的含量及建立芍药甘草汤的指纹图谱,可为经典名方芍药甘草汤的质量标准建立及评价提供依据。

Objective To simultaneously determine the contents of gallic acid,paeoniflorin,liquiritin,liquiritigenin and ammonium glycyrrhetate in Shaoyao Gancao Decoction(芍药甘草汤,SGD)by HPLC,and establish the fingerprints,to explore the key quality attributes of the intermediates of SGD,and to provide reference for the screening and quality control of its medicinal materials.Methods 18 batches of lyophilized powder of SGD were prepared as intermediate samples.Agilent ZORBAX Eclipse Plus C_(18)(4.6 mm×250 mm,5μm)column was used,with acetonitrile(A)-0.2%phosphoric acid aqueous solution(B)as the mobile phase for gradient elution,column temperature of 35℃,flow rate of 0.8 mL/min,injection volume of 10μL.The contents of the index components were detected to evaluate the quality attribute difference of the intermediate of multiple batches of SGD.Results The linear relationship(R^(2)>0.999),precision(RSD≤0.89%,n=6),repeatability(RSD≤1.32%,n=6)and stability(RSD≤2.79%,72 h)of the five components were good.The recoveries ranged from(98.24±1.85)%to(104.88±1.38)%(n=6).The content determination results showed that only gallic acid fluctuated within a small range(4.74-5.92 mg/g)in different batches of SGD.The contents of paeoniflorin,liquiritin,liquiritigenin and ammonium glycyrrhetate in different production areas and different batches were significantly different.The contents ranged from 29.58-38.24,7.21-12.12,0.18-0.89 and 12.14-42.83 mg/g,respectively.At the same time,the fingerprints of SGD was established,and 14 characteristic peaks were identified,among which peaks 1,4,5 and 10 were from Baishao(Paeoniae Radix Alba),peaks 2,3,6,7,8,9,11,12,13 and 14 were from Zhigancao(Glycyrrhizae Radix Et Rhizoma Praeparata Cum Melle)Fisch,and Zhigancao(Glycyrrhizae Radix Et Rhizoma Praeparata Cum Melle)contributed the most to the characteristic peaks.The similarity between the HPLC fingerprints of 18 batches of SGD and the control fingerprints was greater than 0.98 for most batches,and the similarity between the S4,S8,S16,S17 and S18 batches was greater than 0.91,indicating that the quality of medicinal materials from different producing areas was quite different.Conclusion The method is accurate,reliable,sensitive and reproducible.It can be used to determine the contents of five index components in SGD and establish the fingerprints,which can provide a basis for the establishment and evaluation of the quality standard of famous classic formulas of SGD.