光叶丁公藤及其潜在代用品指纹图谱和多成分定量分析比较研究

Comparison between Guangye Dinggongteng(Erycibe schmidtii Craib)and Potential Substitutes by HPLC Fingerprint and Multicomponent Quantitative Analysis

目的建立20批光叶丁公藤及其潜在代用品的高效液相色谱(high performance liquid chromatography,HPLC)指纹图谱及8种有效成分的含量测定方法,并筛选光叶丁公藤最佳代用品。方法Agilent 5 TC-C_(18)色谱柱(250 mm×4.6 mm,5μm),乙腈-0.1%磷酸作流动相进行梯度洗脱;进样量10μL,检测波长345 nm,柱温为30℃,流速为1.0 mL·min^(-1),建立20批样品HPLC指纹图谱,结合相似度评价、聚类热图分析对不同属不同产地的光叶丁公藤及其潜在代用品进行质量评价和比较分析。结果建立的HPLC指纹图谱方法符合方法学要求,20批光叶丁公藤药材及其潜在代用品有10个共有峰,相似度在0.454~0.975之间,蒙自飞蛾藤和飞蛾藤共3批样品与其他样品有较大的差异性。经对照品比对指认了新绿原酸、东莨菪苷、绿原酸、隐绿原酸、东莨菪内酯、异绿原酸B、异绿原酸A、异绿原酸C共8个成分,8个成分的线性范围分别为5.44~544.00、7.86~786.00、8.65~865.00、8.24~824.00、6.45~645.00、8.38~838.00、9.12~912.00、7.74~774.00μg·mL^(-1)(r均大于0.9990);精密度、稳定性、重复性试验的RSD均小于2%;平均加样回收率为98.16%~100.49%(RSD为1.03%~2.62%,n=6);聚类热图分析结果显示,20批样品可聚为4类,其中S5和S16聚为一类,S9、S10、S20聚为一类,S2~S4、S6~S7、S12~S15、S17聚为一类,S1、S8、S18~S19、S11聚为一类。结论所建立的HPLC指纹图谱和8个成分的含量测定方法灵敏度高、稳定性强,可用于光叶丁公藤及其近缘植物的质量评价,为光叶丁公藤代用品筛选提供新的科学依据。

Objective To establish the high performance liquid chromatography(HPLC)fingerprints analysis method of 20 bat-ches of Guangye Dinggongteng(Erycibe schmidtii Craib)and potential substitutes,simultaneously determine the contents of 8 ef-fective constituents and the best substitute of Dinggongteng(Erycibe schmidtii Craib)was selected.Methods HPLC was used with Agilent 5 TC-C_(18)column(250 mm×4.6 mm,5μm),the mobile phase was acetonitrile-0.1%phosphate acid water for gradi-ent elution,the injection volume was 10μL,the detection wavelength was 345 nm,the column temperature was 30℃,and the flow rate was 1.0 mL·min^(-1).HPLC fingerprints of 20 batches medicinal materials was established,the quality evaluation and com-parative analysis of 20 batches of Dinggongteng(Erycibe schmidtii Craib)and potential substitutes from different genera and dif-ferent producing places were carried out combined with similarity evaluation and cluster heatmap analysis.Results The estab-lished fingerprint method met the method requirements.The 20 batches of Dinggongteng(Erycibe schmidtii Craib)and potential substitutes had 10 common peaks in the HPLC fingerprint,and the similarity were ranged from 0.454~0.975.Three batches of samples Feieteng(Porana racemosa Roxb.Hort.Beng.)and Mengzi Feieteng(Porana dinetoides Schneid.)were great different from other samples.A total of 8 components,including neochlorogenic acid,scopoletin,chlorogenic acid,cryptochlorogenic acid,scopoletin,isochlorogenic acid B,isochlorogenic acid A and isochlorogenic acid C were identified by comparison with reference substances.The linear range of neochlorogenic acid,scopoletin,chlorogenic acid,cryptochlorogenic acid,scopoletin,isochlorogen-ic acid B,isochlorogenic acid A and isochlorogenic acid C were 5.44~544.00,7.86~786.00,8.65~865.00,8.24~824.00,6.45~645.00,8.38~838.00,9.12~912.00,7.74~774.00μg·mL^(-1)(r>0.9990),respectively.RSDs of precision,sta-bility(24 h)and repeatability tests were all less than 2%.The recoveries were 98.16%~100.49%(RSD values were.03%~2.62%,n=6).Results of cluster heatmap analysis showed that 20 batches of samples could be divided into 4 catego-ries,including S5 and S16 as one category;S9,S10 and S20 as one category;S2~S4,S6~S7,S12~S15,S17 as one category;S1,S8,S18~S19 and S11 as one category and S7~S10 as one category.Conclusion The established HPLC fingerprint and the content method of 8 components had high sensitivity and stability,which could be used for the quality evaluation of Dinggongteng(Erycibe schmidtii Craib)and its related plants,and provide a new scientific basis for the substitutes screening of Dinggongteng(Erycibe schmidtii Craib).