区分3种法定基源麻黄的酚类成分HPLC特征图谱中指标成分的确定及4种成分的含量测定

Determination of markers from characteristic HPLC chromatogram of phenols in three official origins of Ephedrae Herba and quantitative analysis of four phenols

针对麻黄中酚类成分建立HPLC特征图谱,寻找其中指标成分,并对其在草麻黄、中麻黄、木贼麻黄草质茎中的分布及含量规律进行分析。利用HPLC-DAD建立酚类成分特征图谱,采用YMC-Pack ODS-A(4.6 mm×250 mm,5μm)色谱柱,以0.01%甲酸水溶液-乙腈为流动相,梯度洗脱,流速1.0 m L·min^(-1),柱温40℃,检测波长330,350 nm。对46批次法定基源麻黄自采样品进行测定,所得图谱可实现基源间有效区分。结合主成分分析、峰面积分布分析,细致探讨图谱中12个主要成分峰对3种法定基源麻黄特征及差异的贡献:其中,10,11,12号峰峰面积在木贼麻黄中显著高于另两基源,利用其总峰面积值(木贼麻黄中高于146 m AU),可实现木贼麻黄与另两基源的有效区分;1号峰峰面积在草麻黄、4号峰峰面积在中麻黄中显著高于其他法定基源,分别为两基源麻黄中重要的差异成分峰;8,9号峰为3种基源麻黄中的共有特征成分峰;其余各峰在不同基源麻黄中的分布存在不同程度差异,辅助上述成分,实现3种法定基源麻黄的有效区分。进一步利用上述色谱条件,首次测定了法定基源麻黄中异牡荆素-2″-O-α-L-鼠李糖苷(1)、牡荆素(2)、茶花粉黄酮B(5)、草棉黄素-7-O-β-D-葡萄糖苷(6)4种指标成分的含量,在检测范围内线性良好,平均加样回收率为97.8%~102.5%。所测样品中4种成分的含量范围分别为trace^1.55(1),trace^0.160(2),trace^0.284(5),trace^0.620(6)mg·g^(-1),且不同基源麻黄中,各成分含量存在差异。其中,1在草麻黄中质量分数(0.670±0.88)mg·g^(-1)显著高于另两基源麻黄(除Ei-060630-2-2外低于0.16 mg·g^(-1));6在木贼麻黄中平均含量水平(0.260±0.039 2)mg·g^(-1)达草麻黄(0.120±0.270)mg·g^(-1)与中麻黄(0.136±0.485)mg·g^(-1)中的2倍,此二成分对法定基源麻黄间区分有重要贡献。所建酚类成分HPLC特征图谱与含量测定方法较为简便、准确,选定指标成分为区分麻黄基源、完善药材质量标准,提供了新的科学依据。

This study is to establish the characteristic HPLC chromatogram of phenols in Ephedrae Herba, from which to pick out the marker peaks, followed by the analysis of the regularity of their distribution and content in the herbaceous stems of Ephedra sinica, E. intermedia and E. equisetina. The HPLC-DAD method for the characteristic ehromatogram as well as quantitative analysis was established. The separation was carried out on a YMC-Pack ODS-A column（4.6 mm×250 mm,5μm） , eluted with the mobile phases as 0.01% formic acid aqueous solution（A） and acetonitrile（B） in a linear gradient（0-10 min, 17% B; 10-25 rain, 17%-19% B; 25- 33 rain, 19%-48% B; 33-35 rain, 48%-51% B; 35-44 min, 51% B）. The flow rate was kept at 1.0 mL·min-1. The cohmm temperature was 40℃, and the detection wavelength was set at 350 nm（0-16 min） and 330 nm（ 16-44 min）. Forty-six batches of collected samples from three official origins of Ephedrae Herba were detected, whose liquid chromatograms proven to be helpful to the difthr- entiation of different origins. With principal component analysis and the analysis of distribution of peak area, twelve key peaks from lille chromatogram were discussed in details on their contributions to the characteristics and differences of three official origins of the herb： peak area of peak 10, 11, 12 were found out to be significantly higher in E. equisetina than in other two origins, whose stun（ higher than 146 mAU in E. equisetina） was useful for the discrimination between E. equisetina and the other two origins; peak area of 1 and 4 were respectively higher in E. sinica and E. intermedia than in other official origins, indicating their important effect on the differentiation of corresponding origins ; peak 8 and 9 were picked out as two characteristic common peaks in three official origins of the herb, whose peak area showed little difference among different origins ; further, peak area of other key peaks in the chromatogrmn also showed some difference among three origins, which make contributions to the differentiation of origins as well. Then, four phenols as 2＇＇-O-α- L-rhamnosyl-isovitexin （ 1 ）, vitexin （ 2 ）, pollenitin B （ 5 ） and herbacetin-7-O-β-D-glucoside （ 6 ） were quantitative analyzed with the above-mentioned method, with good linear relationship and accuracy （recoveries in a range of 97.8%-102. 5% ）. The content of the four phenols were firstly reported in Ephedrae Herba from official origins, which were respectively trace-1.55 （1） , trace-0. 160 （2） , trace-0. 284（5） and trace-0. 620（6） mg·g-1 in all of the tested samples. In addition, the content of these phenols showed differ ences in three official origins, especially 1, whose content in E. sinica[ （0. 670 ±0. 88） mg·g-1] were significantly higher than in other two origins （lower than 0. 16 mg·g-1 besides sample Ei-060630-2-2）, and 6, whose average content in E. equisetina [（0. 260 ±0.0392） mg·g-1] were twice as high as inE. sinica [（0.120±0.270） mg· g-1] andE. intermedia [（0.136±0.485） mg·g-1], indicating the important effects of the two constituents on the differentiation among three official origins of the herb. The method established for the characteristic HPLC chromatogram and quantitative analysis of phenols was simple and accurate, and the marker constituents selected may provide new guides for the discrimination of official origins as well as the improvement of quality criteria of EphedraeHerba.