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目的建立不同栽培模式滇龙胆的HPLC指纹图谱,对马钱苷酸,獐牙菜苦苷,龙胆苦苷和当药苷进行定性、定量分析;结合化学计量学对林药复合栽培滇龙胆资源进行评价。方法色谱柱为岛津Shim-pack VP-ODS液相色谱柱(4.6 mm×150mm,5μm),流动相:0.1%甲酸水溶液(A)和乙腈(B),梯度洗脱,流速1.00 m L·min-1,柱温30℃,检测波长241 nm,进样量10μL。化学计量学分析方法包括:系统聚类分析、偏最小二乘判别分析和变量投影重要性准则。结果龙胆苦苷、獐牙菜苦苷、当药苷和马钱苷酸在10~4 000μg·m L-1内线性良好,相关系数为0.999 2~0.999 9;4种化学成分精密度的RSD分别为0.87%,2.1%,0.90%和2.4%;重复性实验的RSD分别为0.97%,1.9%,2.0%和2.2%;稳定性实验的RSD均小于2.2%;加样回收率在97.12%~103.81%之间,RSD在0.71%~2.0%之间。指纹图谱相似性分析显示多数样品相似性系数大于0.970,不同栽培模式的70份样品化学成分种类相似,含量有明显变化;栽培药材龙胆苦苷>獐牙菜苦苷>马钱苷酸>当药苷,龙胆苦苷计量特征与其余3种活性成分差别较大。滇龙胆与尼泊尔桤木复合栽培模式下,药材4种化学成分总含量最高;与核桃树复合栽培有利于马钱苷酸的积累,与桉树复合栽培有利于獐牙菜苦苷的积累,与杉木复合栽培有利于当药苷的积累。以上结果表明,针对不同化学成分,应有多种最佳栽培模式。偏最小二乘判别分析和变量投影重要性准则研究显示,龙胆苦苷(VIP=1.513),獐牙菜苦苷(VIP=1.208)和保留时间在10 min后的化学成分对药材栽培模式的区分具有显著贡献。结论 HPLC指纹图谱结合化学计量学可为滇龙胆林药复合栽培最佳模式的选择及质量控制提供方法和理论依据。
Objective To establish a HPLC fingerprint of Brassostrantia longipes with different cultivation modes and qualitative and quantitative analysis of linmic acid, swertiamarin, gentiopicrin and Danggui glycosides; Courage resources for evaluation. Methods The chromatographic column was Shimadzu Shim-pack VP-ODS liquid chromatography column (4.6 mm×150 mm, 5 μm). The mobile phase consisted of 0.1% formic acid aqueous solution (A) and acetonitrile (B). The gradient elution was performed at a flow rate of 1.00 m L· Min-1, column temperature 30 °C, detection wavelength 241 nm, injection volume 10μL. The chemometric analysis methods include: systematic cluster analysis, partial least squares discriminant analysis, and variable projection importance criteria. Results The linearity of gentiopicrin, swertiamarin, fenglucoside, and vinacin was good in the range of 10~4 000 μg·m L-1. The correlation coefficient was 0.999 2 to 0.999 9; the precision of the four chemical components was RSD were 0.87%, 2.1%, 0.90%, and 2.4%, respectively; RSDs for repeatability experiments were 0.97%, 1.9%, 2.0%, and 2.2%, respectively; RSDs for stability experiments were all less than 2.2%; sample recovery was 97.12. Between 10% and 103.81%, the RSD is between 0.71% and 2.0%. The similarity analysis of fingerprints showed that the similarity coefficient of most samples was greater than 0.970, and the chemical composition of 70 samples in different cultivation patterns was similar, and the content had significant changes. Gentiopicrin> swertiapicrin> tigbolic acid> The measurement characteristics of glycosides and gentiopicroside differ significantly from the other three active ingredients. The total content of four chemical constituents of Medicinal Gentian was the highest under the compound cultivation mode of Pteris gentiana and Nepalus communis. The compound cultivation with walnut trees was beneficial to the accumulation of tricartenic acid, and compound cultivation with Eucalyptus was beneficial to the accumulation of pinellitrin. The compound cultivation of Chinese fir is beneficial to the accumulation of aglycone. The above results indicate that there should be a variety of optimal cultivation modes for different chemical components. Partial Least Squares Discriminant Analysis and Variable Projection Importance Criteria Studies showed that the chemical composition of gentiopicroside (VIP=1.513), swerpine glycoside (VIP=1.208) and retention time after 10 min on the cultivation mode of medicinal herbs The distinction has a significant contribution. Conclusion HPLC fingerprint combined with chemometrics can provide the method and theoretical basis for the selection and quality control of the best mode for the compound cultivation of Gentiana grandiflorum.