世界中医药

目的:采用HPLC(高效液相)指纹图谱分析研究葛根芩连汤中药物的成分。方法:在指纹图谱研究方法中采用HPLC-DAD(高效液相-二极管阵列检测器),HPLC指纹图谱试验色谱条件:进样量为20 μL,色谱柱选择直径5 μm的Ultimate AQ C18柱,柱温30 ℃,流动相A是0.02 mol/L醋酸铵+0.3%三乙胺+1%冰醋酸混合而成,最终PH值为4.3;流动相B是乙腈,流速统一为0.7 mL/min。检测波长通过预实验不断调整,最终葛根素、大豆苷、大豆苷元、染料木素、甘草酸铵和甘草次酸的检查波长为250 nm;黄芩苷、汉黄芩苷、黄芩素、汉黄芩素和甘草苷检测波长为280 nm;余下对照品检测波长为346 nm;并对HPLC精密度试验、重复性试验、稳定性试验、专属性试验、加样回收率试验、线性回归试验等方法学进行严谨性考察,通过比较混合对照品溶液指纹图谱,葛根芩连汤单味药材指纹图谱和葛根芩连汤指纹图谱,分析研究葛根芩连汤中的14个药物成分。结果:1)精密度试验平均相对峰面积RSD为0.38%,重复性试验平均相对峰面积RSD为1.14%,稳定性试验平均相对峰面积RSD为1.03%,专属性试验结果良好。2)14个药物成分线性回归良好,且加样回收试验中平均回收率的范围是96.45%～104.69%,RSD的范围是为0.65%～2.11%。3)本研究根据不同的检测波长250 nm、280 nm和346 nm,将混合对照品指纹图谱分为3张图,便于后续分析,同时标注14个对照品成分的峰号。4)在检测波长为250 nm时,葛根指纹图谱有11个特征峰,而甘草有2个特征峰;在检测波长280 nm时,黄芩有6个特征峰,而在检测波长346 nm时,黄连有4个特征峰。5)在葛根芩连汤的指纹图谱中显示波长250 nm时有12个特征峰,其中1~11主要体现葛根的药物成分特征,而12主要体现甘草的药物成分特征;波长280 nm时有7个特征峰,主要体现甘草和黄芩的药物成分特征;波长346 nm时有4个特征峰,主要体现黄连的药物成分特征。结论:从整体性解析葛根芩连汤剂HPLC指纹图谱的药物成分来源,为葛根芩连汤中含有的药物成分等物质基础、药理相关性研究提供实验基础。

English Summary:

To study the composition of the drugs in Gegen Qin Lian Decoction by HPLC (high performance liquid) fingerprint.Methods:HPLC-DAD was used in the fingerprint research method (high performance liquid chromatography with diode array detector),HPLC fingerprint chromatography test conditions: sample volume of 20 L,Ultimate AQ C18 column with diameter of 5 m column,the column temperature was 30 degrees,the mobile phase is A 0.02 mol/L +0.3% three +1% ammonium acetate ethylamine acetic acid mixture,the final pH was 4.3;the mobile phase is B acetonitrile,0.7 mL/min uniform velocity.The detection wavelength by constantly adjusting the final pre experiment,puerarin,daidzin,daidzein,genistein,ammonium glycyrrhizinate and glycyrrhetinic acid test wavelength was 250 nm; baicalin,wogonoside,baicalein,wogonin and liquiritin detection wavelength was 280 nm; the remaining standard detection wavelength was 346 nm;and the HPLC precision test,repeatability test,stability test,specificity test,recovery test,linear regression test methodology for rigorous investigation,through the comparison of mixed standard solution of fingerprint,gegenqinliantang single fingerprint and gegenqinliantang fingerprint analysis of Radix Puerariae Qinlian Decoction of 14 ingredients.Results:1)The average relative peak area RSD of precision test was 0.38%,the average relative peak area RSD of repeatability test was 1.14%,the average relative peak area RSD of stability test was 1.03%,and the specificity test result was good.2)The linear regression of 14 drug components is good,and the range of average recovery in the sample recovery test is 96.45%～104.69%,and the range of RSD is 0.65%～2.11%.3)According to different detection wavelengths 250 nm,280 nm and 346 nm,the fingerprint of mixed reference products was divided into 3 diagrams,which was convenient for subsequent analysis,and 14 peaks of reference compounds were also labeled.4)At the detection wavelength of 250 nm,there were 11 characteristic peaks in the fingerprint of radix puerariae,while 2 characteristic peaks in Glycyrrhiza uralensis Fisch.There were 6 characteristic peaks in Scutellaria baicalensis Georgi at the detection wavelength 280,while there were 4 characteristic peaks in Rhizoma Coptidis at the detection wavelength of 346 nm.5)There are 12 characteristic peaks in Gegenqinlian Decoction fingerprint displayed in the wavelength of 250 nm,characteristics of the 1~11 drug ingredients mainly puerarin and 12 among them,mainly licorice ingredient characteristics;280 nm wavelength of 7 peaks,mainly reflected the drug composition characteristics of liquorice and Radix Scutellariae; wavelength 346 nm there are 4 characteristic peaks,mainly reflected the drug composition characteristics of Coptis chinensis.Conclusion:The source of drug composition of Gegen Qin Lian Decoction HPLC fingerprint is analyzed integrally,providing experimental basis for the material basis and pharmacological correlation research of Gegen Qin Lian decoction.

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