基于HPLC指纹图谱结合化学模式识别法评价白花蛇舌草质量

黄岚; 罗镭; 李正; 依泽; 陈碧莲

doi:10.13748/j.cnki.issn1007-7693.20243024

基于HPLC指纹图谱结合化学模式识别法评价白花蛇舌草质量

Quantitative Evaluation of Hedyotis Diffusa Willd Quality Based on HPLC Fingerprint Combined with Chemical Pattern Recognition

摘要

摘要:
目的建立白花蛇舌草Hedyotis diffusa Willd HPLC指纹图谱，对不同产地、采收期、野生与栽培白花蛇舌草及混淆品进行对比分析。
方法采用Kromasil C₁₈色谱柱(4.6 mm×250 mm，5 µm)，流动相为乙腈-0.1%磷酸水溶液，梯度洗脱，流速为1.0 mL·min^–1，波长为330 nm，柱温为 30 ℃，进样量为10 μL。结合相似度评价、聚类分析(cluster analysis，CA)、主成分分析(principal component analysis，PCA)、正交偏最小二乘法-判别式分析(orthogonal partial least squares discriminant analysis，OPLS-DA)比较不同产地、采收期、野生与栽培白花蛇舌草及其混淆品的差异。
结果 HPLC指纹图谱共标定了白花蛇舌草12个共有峰，结合超高效液相色谱-四极杆-飞行时间质谱仪(UPLC-Q-TOF-MS/MS)定性鉴别技术指认了绿原酸、水晶兰苷、白麻苷、槲皮素-3-O-桑布双糖苷、山柰酚-3-O-龙胆二糖、芦丁、皮素-3-O-2-O-(6-O-E-芥子酰基)-β-D-吡喃葡萄糖基-β-D-吡喃半乳糖苷、槲皮素-3-O-2-O-(6-O-E-阿魏酰基)-β-D-吡喃葡糖基-β-D-吡喃半乳糖苷、(E)-6-O-对香豆酰鸡屎藤次苷甲酯、山柰酚-3-O-2-O-(6-O-E-阿魏酰)-β-D-葡萄糖基-β-D-半乳糖苷、(E)-6-O-阿魏酰鸡屎藤次苷甲酯、(E)-6-O-对香豆酰鸡屎藤次苷甲酯异构体。通过相似度分析发现，白花蛇舌草的不同产地和采收期、野生与栽培样品均存在一定的差异，与混淆品样品有明显差异；通过CA、PCA发现，30批白花蛇舌草样品存在产地交叉的现象，说明产地不是影响其质量的关键因素；通过CA、PCA、OPLS-DA 可将40批样品根据不同采收期、野生与栽培、基原进行区分，通过 OPLS-DA 项下的投影中的变量重要性投影(variable importance in projection，VIP)各筛选出6个差异性标志物。
结论建立的指纹图谱方法能准确有效区分白花蛇舌草不同采收期、野生与栽培及其混淆品，为白花蛇舌草的质量评价及鉴别提供参考。

Abstract:
OBJECTIVE To establish high performance liquid chromatography(HPLC) fingerprint for Hedyotis diffusa Willd and analyze them with different regions, harvesting periods, wild and cultivated and adulterants.
METHODS The HPLC analysis was performed on a Kromasil C₁₈ column(4.6 mm × 250 mm, 5 µm) by using a mobile phase of acetonitrile-0.1% phosphoric acid aqueous solution in a gradient elution at a flow rate of 1.0 mL·min^–1, with a detection wavelength of 330 nm, and a column temperature of 30 ℃ and the injection volume of 10 µL. Similarity evaluation, cluster analysis(CA), principal component analysis(PCA), and orthogonal partial least squares discriminant analysis(OPLS-DA) were utilized to compare the differences among different regions and harvesting periods and wild and cultivated of Hedyotis diffusa Willd and its adulterants.
RESULTS A total of 12 common peaks of Hedyotis diffusa Willd were identified by HPLC fingerprinting, and by the qualitative identification technique of ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry(UPLC-Q-TOF-MS/MS) identified chlorogenic acid, monotropein, baimaside, quercetin-3-O-sambubioside, kaempferol 3-O-gentiobioside, rutin, quercetin-3-O-2-O-(6-O-E-sinapoyl)-β-D-glucopyranosyl-β-D-galactopyranoside, quercetin-3-O-2-O-(6-O-E-feruloyl)-β-D-glucopyranosyl-β-D-galactopyranoside, (E)-6-O-Coumaroylscandoside methyl ester, kaempferol 3-O-2-O-(6-O-E-feruloyl)-β-D-glucopyranosyl-β-D-galactopyranoside, (E)-6-O-Feruloylscandoside methyl ester, (E)-6-O-Feruloylscandoside methyl ester isomer. Similarity analysis found that there were some differences between the wild and cultivated samples in different places and harvesting periods, and there were obvious differences between the samples and the confused ones. But CA and PCA found that 30 batches of samples had cross-origin phenomenon, indicating that origin was not the key factor affecting its quality. CA, PCA, and OPLS-DA could differentiate 40 batches of samples according to different harvesting periods, wild and cultivated,and botanical origins, and OPLS-DA identified six differential markers through the variable importance in projection(VIP).
CONCLUSION The established fingerprint method can accurately and effectively differentiate between different harvesting periods and wild and cultivated Hedyotis diffusa Willd, as well as its adulterants, which provides reference for quality evaluation and identification of Hedyotis diffusa Willd.

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