二黄汤半仿生法提取液指纹图谱归属研究

目的：对二黄汤方药半仿生法（SBE法）≤1 000 Da提取液的指纹图谱各特征峰的来源进行归属研究。方法：采用SBE法分别制备二黄汤全方和各单味药黄芩、黄连、甘草的提取液，绘制相应的HPLC指纹图谱，通过比对，确定其特征峰的来源；通过对照品比对，鉴定部分特征峰的成分。色谱条件：采用Diamonsil C₁₈（250 mm×4.6 mm，5 μm）色谱柱，0.02 mol·L^-1乙酸铵溶液（冰乙酸调pH 4.0）（A）-乙腈（B）为流动相，梯度洗脱，流速0.5 mL·min^-1，检测波长265 nm，柱温25 ℃，进样量20 μL。结果：二黄汤的SBE法≤1 000 Da提取液的HPLC指纹图谱有32个特征峰，其中峰9、11、13、16、17、25、26、27、30、31来自黄芩，峰15、20、21来自黄连，峰3、10、12、23、24、32来自甘草，峰2、18、19来自黄芩、黄连和甘草，峰14、28、29来自黄芩和甘草，峰1、4、5、6、7、8、22来自黄连和甘草。以保留时间和紫外吸收曲线为判断依据，进行对照品比对，鉴别出甘草苷、黄芩苷、巴马汀、小檗碱、甘草素、甘草酸、黄芩素、甘草次酸共8个已知成分，其中峰22是含小檗碱和甘草素的混合峰，与归属结果相同。结论：二黄汤SBE法提取液指纹图谱的建立符合要求，归属研究方法合理。与对照品比对，二黄汤SBE法≤1 000 Da提取液中各特征峰的归属结果准确，可为特征峰的成分鉴定和谱效关系研究提供依据。

Objective: To study the ownership of HPLC fingerprints of Erhuang decoction extracts with Semi-bionic extraction method.Methods: SBE method was used to prepare Erhuang decoction extracts of prescription,Scutellariae Radix,Coptidis Rhizoma and Glycyrrhizae Radix et Rhizoma,respectively.The HPLC fingerprint was recorded and the ownership of the characteristic peaks of SBE≤1 000 Da extraction was analyzed.Compared with the reference,components of the characteristics peaks were identified.Chromatography conditions were optimized as follows:Diamonsil C₁₈(250 mm×4.6 mm,5 μm)column was adopted,the mobile phase was 0.02 mol·L^-1 ammonium acetate solution(glacial acetic acid adjusted pH 4.0)(A)-acetonitrile(B)with gradient elution at a flow rate of 0.5 mL·min^-1,the detection wavelength was 265 nm,the column temperature was 25 ℃,and the injection volume was 20 μL.Results: The HPLC fingerprint of the SBE≤1 000 Da extraction showed 32 peaks:peak 9,11,13,16,17,25,26,27,30,31 originated from the fingerprint of Scutellariae Radix;peak 15,20,21 originated from Coptidis Rhizoma;peak 3,10,12,23,24,32 originated from Glycyrrhizae Radix et Rhizoma;peak 2,18,19 originated from Scutellariae Radix,Coptidis Rhizoma and Glycyrrhizae Radix et Rhizoma;peak 14,28,29 originated from Scutellariae Radix and Glycyrrhizae Radix et Rhizoma;peak 1,4,5,6,7,8,22 originated from Coptidis Rhizoma and Glycyrrhizae Radix et Rhizoma.Glycyrrhizin,baicalin,palmatine,berberine,glycyrrhizin,glycyrrhizic acid,baicalein,glycyrrhetinic acid were already identified based on the retention time and UV absorption curve.The 22th peak was mixed with berberine and glycyrrhizin.In addition,the comparison results were consistent with the ownership results.Conclusion: The established fingerprint of SBE extracts met the requirement of analysis,and the attribution method was reasonable.Results of the ownership of each characteristic peak of Erhuang decoction≤1 000 Da extracts with SBE method were accurate.This study can provide evidence for the research of component identification and spectrum effect relationship.