经典名方大秦艽汤HPLC指纹图谱及含量测定方法研究

HPLC fingerprint and content determination method of classical prescriptions Daqinjiao Decoction

目的制备10批大秦艽汤标准煎液,建立其HPLC指纹图谱并计算指标成分含量、转移率等数据,为建立大秦艽汤的质量控制标准提供参考。方法依据古方要求还原至现代煎煮方式制备大秦艽汤标准煎液。采用岛津Inertsil ODS-3 C_(18)色谱柱(250 mm×4.6 mm,5μm);流动相为乙腈-0.1%磷酸水溶液,梯度洗脱;体积流量为1.0 m L/min;柱温为30℃;检测波长为237 nm;进样量为10μL。建立10批大秦艽汤HPLC指纹图谱,使用中药色谱指纹图谱相似度评价系统软件(2012A版)进行相似度分析,最后对共有峰进行鉴定和药材归属,并定量测定大秦艽汤中马钱苷酸、龙胆苦苷、升麻素苷的含量。结果建立了10批大秦艽汤的指纹图谱,相似度介于0.902~0.953,确认36个共有峰,指认出7、15、18~21、23、27、31、33、35号峰分别为马钱苷酸、龙胆苦苷、芍药苷、升麻素苷、阿魏酸、甘草苷、5-O-甲基维斯阿米醇苷、黄芩苷、黄芩素、甘草酸、汉黄芩素共11个成分;其中4~8、10~12、15号峰归属于秦艽,19、23号峰归属于防风,17、18号峰归属白芍,30号峰归属于独活,21、25、33号峰归属于甘草,22、27~29、35号峰归属于黄芩,1号峰归属于当归和独活,2号峰归属于秦艽、当归和细辛,3号峰归属于川芎和白芍,13号峰归属于甘草和独活,16号峰归属于秦艽和白芍,20号峰归属于川芎、当归和羌活;以马钱苷酸、龙胆苦苷、升麻素苷3项指标建立其质量控制标准,10批次样品中马钱苷酸、龙胆苦苷、升麻素苷质量分数分别为0.158~0.103、0.475~0.373、0.029~0.012mg/g。结论所建立的汤剂制备方法稳定可行,质量控制方法简单、专属性强,结果准确,可用于大秦艽汤的质量评价。

Objective To establish fingerprints and calculate index component content,transfer rate and other data of 10 batches of Daqinjiao Decoction(DD)by HPLC,in order to provide reference for establishing the quality control standard of DD.Methods According to the requirements of the ancient recipe,the standard decoction of DD was prepared by the modern decoction method.Using Shimadzu inertsil ODS-3 C_(18) column(250 mm×4.6 mm,5μm)for gradient elution with acetonitrile-0.1%phosphoric acid aqueous solution;Flow rate 1.0 mL/min,column temperature 30℃,and detection wavelength 237 nm;The injection volume is 10μL.The HPLC fingerprint of 10 batches of DD were established and evaluated by the similarity evaluation system of the Chinese medicine chromatographic fingerprint similarity evaluation system software(version 2012 A),identify and assign the common peaks,and quantitatively determine the content of loganic acid,gentiopicroside,and prim-O-glucosylcimifugin in DD.Results HPLC fingerprint of 10 batches of DD were established.The similarity was ranged from 0.902 to 0.953 and 36 common peaks were identified as 11 chemical components:loganic acid,gentiopicroside,paeoniflorin,prim-O-glucosylcimifugin,ferulic acid,liquiritin,4-O-beta-glucopyranosyl-5-O-methylvisamminol,baicalin,baicalein,glycyrrhizic acid,and wogonin(corresponding to peaks 7,15,18,19,20,21,23,27,31,33,and 35),peaks 4—8,10—12,and 15 belonged to Gentianae Macrophyllae Radix(GMR),peaks 19 and 23 belonged to Saposhnikoviae Radix(SR),peaks 17 and 18 belonged to Paeoniae Radix Alba(PRA),peak 30 belonged to Angelicae Pubescentis Radix(APR),peaks 21,25,and 33 belonged to Glycyrrhizae Radix et Rhizoma(GRR),peaks 22,27—29,and 35 belonged to Scutellariae Radix,peak 1 belonged to Angelicae Sinensis Radix(ASR)and APR,peak 2 belonged to GMR,ASR and Asari Radix et Rhizoma(ARR),peak 3 belonged to Chuanxiong Rhizoma(CR)and PRA,peak 13 belonged to GRR and APR,peak 16 belonged to GMR and PRA,peak 20 belonged to CR,ASR and Notopterygii Rhizoma et Radix(NRR).Among them,three indicators of loganic acid,gentiopicroside and prim-O-glucosylcimifugin were used to establish theirs quality control standards,the results of content determination respectively were 0.158—0.103,0.475—0.373,and 0.029—0.012 mg/g.Conclusion The preparation method of established decoction is stable and feasible,and the quality control method is simple,specific and accurate.It can be used for the quality evaluation of DD.