加味黄连膏差异标志物含量测定及其皮肤保护作用

Determination of differential markers of Modified Huanglian Ointment and its skin protective effect

目的:建立加味黄连膏HPLC指纹图谱,结合化学模式识别方法筛选差异标志物,进行定量检测,并研究其对皮肤的保护作用。方法:采用高效液相色谱法考察21批加味黄连膏的相似度并建立指纹图谱,结合化学模式识别,以变量重要性投影(VIP)值>1为标准筛选影响加味黄连膏质量的差异标志物,通过含量测定对其质量进行评价,并以加味黄连膏对B16F10细胞酪氨酸酶抑制活性及黑色素生成的抑制活性为指标评价其皮肤保护作用。结果:加味黄连膏的HPLC指纹图谱中21批样品(S1~S21)的相似度>0.950,共标定17个共有峰,并指认出8个峰,盐酸黄柏碱、芝麻酚、阿魏酸、盐酸黄连碱、盐酸小檗碱、盐酸巴马汀、姜黄素和芝麻素。OPLS-DA结果显示,3号峰(盐酸黄柏碱),5号峰(阿魏酸),15号峰(姜黄素),16号峰(芝麻素)为影响加味黄连膏质量的差异标志物,其含量分别为0.021 0~0.028 7、0.006 7~0.010 0、0.021 5~0.025 8、0.209 5~0.235 5 mg·g^(–1),且加味黄连膏对B16F10细胞酪氨酸酶抑制率为100.33%,黑色素抑制率为59.80%。结论:建立的指纹图谱及含量测定方法为加味黄连膏的质量评价提供了可靠的理论依据,为工业化生产提供了科学的质控依据;加味黄连膏具有一定的皮肤保护作用,可为其临床应用提供参考。

Objective To establish the high performance liquid chromatography(HPLC)fingerprint of Modified Huanglian Ointment,screen for differential markers with chemical pattern recognition,detect them quantitatively and examine its skin protective effect.Methods The similarity of 21 batches of Modified Huanglian Ointment was examined by HPLC fingerprint.With chemical pattern recognition and a variable importance projection(VIP)value>1 as a standard,differential biomarkers affecting the quality of Modified Huanglian Ointment were screened.The quality of Modified Huanglian Ointment was evaluated by content determination and its skin protective effect examined by an inhibition of Modified Huanglian Ointment on tyrosinase and melanin production in B16F10 cells.Results The similarity of 21 batches of samples(S1-S21)was>0.950 in HPLC fingerprint of Modified Huanglian Ointment.Among 17 common peaks,8 peaks were phellodendron hydrochloride,sesamol,ferulic acid,coptis hydrochloride,berberine hydrochloride,palmatine hydrochloride,curcumin and sesamin.OPLS-DA results showed that peak 3(phellodendrine hydrochloride),peak 5(ferulic acid),peak 15(curcumin)and peak 16(sesamin)were differential markers affecting the quality of Modified Huanglian Ointment.And their contents were 0.0210–0.0287,0.0067–0.0100,0.0215–0.0258 and 0.2095–0.2355 mg·g^(–1) respectively.Moreover,the inhibitory rate of Modified Huanglian Ointment on tyrosinase and melanin was 100.33%and 59.80%respectively.Conclusion The established fingerprint and content determination provide a reliable theoretical basis for quality evaluation of Modified Huanglian Ointment.It provides a scientific basis for quality control for industrial production.Modified Huanglian Ointment has some skin protection effect and it provides references for its clinical applications.