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金银花的HPLC指纹图谱建立及其抗炎作用谱-效关系研究
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| 篇名： | 金银花的HPLC指纹图谱建立及其抗炎作用谱-效关系研究 |
| TITLE： | Establishment of HPLC Fingerprint of Lonicera japonica and Study on Its Anti-inflammatory Spectrum-effect Relationship |
| 摘要： | 目的：建立金银花指纹图谱，并研究其抗炎作用的谱-效关系。方法：采用高效液相色谱法（HPLC）。色谱柱为DiamonsilC18，流动相为0.1％甲酸水溶液-乙腈（梯度洗脱），流速为1.0mL/min，柱温为30℃，检测波长为238nm，进样量为10μL。以绿原酸为参照，采用《中药色谱指纹图谱相似度评价系统（2012版）》建立10批不同产地金银花药材样品的HPLC指纹图谱；通过对照品比对，指认共有峰对应的化学成分，并进行相似度分析。采用二甲苯、角叉菜胶及棉球诱导的急、慢性炎症小鼠模型评价10批金银花药材水提物对耳、足、肉芽肿的肿胀抑制率，并计算平均值，作为综合药效指标；基于共有峰峰面积和综合药效指标，采用灰色关联度分析法（GRA）和偏最小二乘回归分析法（PLSR）联合分析金银花指纹图谱与抗炎作用的谱-效关系，选择GRA关联度大于0.7且PLSR模型回归系数大于0的色谱峰为特征峰，计算10批金银花药材中特征峰峰面积占共有峰峰面积总和的百分比（即“峰占比”）。结果：10批金银花药材样品的HPLC指纹图谱中有共有峰25个，相似度为0.775～0.994；指认出其中9个成分，分别为芦丁（峰18）、金丝桃苷（峰20）、异绿原酸B（峰22）、木犀草素（峰21）、绿原酸（峰9）、马钱苷（峰10）、新绿原酸（峰2）、异绿原酸C（峰25）、异绿原酸A（峰23）。10批金银花对小鼠耳肿胀、足肿胀、肉芽肿均具有抑制作用，抑制率平均值为47.95％～56.52％；GRA分析显示，关联度由大到小依次为峰8＞12＞18＞16＞3＞11＞20＞22＞19＞21＞1＞9＞10＞13＞24＞14＞2＞17＞25＞23＞5＞4＞15，且其关联度均大于0.7；峰2、4、5、7、8、10、12、13、14、15、16、17、18、20、21、22、24的PLSR模型回归系数均大于0，与抗炎作用成正相关；除峰7外，其余均为特征峰，且峰5、8、10、16、18、20、24的变量重要性投影值均大于1。10批金银花药材特征峰的峰占比为58.61％～71.19％。结论：成功建立金银花的HPLC指纹图谱，10批样品组分相似，抗炎成分含量相对较高；暂定金银花特征峰峰占比不得低于51.8％。 |
| ABSTRACT： | OBJECTIVE：To establish fingerprint of Lonicera japonica ，and to study its anti-inflammatory spectrum-effect relationship. METHODS ：HPLC was adopted. The determination was performed on Diamonsil C 18 column with mobile phase consisted of 0.1％ formic acid solution-acetonitrile （gradient elution ）at the flow rate of 1.0 mL/min. The column temperature was 30 ℃，and detection wavelength was 238 nm. The sample size was 10 μL. Using chlorogenic acid as reference，HPLC fingerprint of 10 batches of L. japonica from different production areas was established according to TCM Chromatographic Fingerprint Similarity Evaluation System （2012 edition）. By comparing with reference substance ，chemical constituents corresponding to common peaks were identified ，and the similarity analysis was conducted. Acute and chronic inflammatory models of mice induced by xylene ，carrageenan and cotton ball were used to evaluate inhibition rate of 10 batches of L. japonica to ear，foot and granuloma swelling； the average value was calculated as the comprehensive pharmacodynamic index. The spectrum-effect relationship with HPLC fingerprint of L. japonica and anti-inflammatory effect was analyzed by grey relational analysis （GRA）and partial least squares regressiosn （PLSR）based on common peak area and comprehensive pharmacodynamic index . Chromatographic peaks with correlation＞0.7 and regression coefficient of PLSR model ＞0 were characteristic peaks. The percentage of peak areas of characteristic peaks to peak areas of common peak was calculated in 10 batches of L. japonica （e.g.“peak ratio ”）. RESULTS ： There were 25 common peaks in HPLC fingerprints of 10 batches of L. japonica ，with similarity of 0.775-0.994. Totally 9 peaks were confirmed ，i.e. rutin （peak 18），hyperoside（peak 20），isochlorogenc acid B （peak 22），galuteolin（peak 21），chlorogenc acid（peak 9），loganin（peak 10），neochlorogenic acid （peak 2），isochlorogenic acid C （peak 25），isochlorogenic acid A （peak 23）. All 10 batches of L. japonica had inhibitory effects on ear swelling ，foot swelling and granuloma ，with average inhibitory rate of 47.95％-56.52％. The correlation by GRA was peak 8＞12＞18＞16＞3＞11＞20＞22＞19＞21＞1＞9＞10＞13＞24＞14＞2＞ 17＞25＞23＞5＞4＞15，and all of correlations were greater than 0.7. The regression coefficient of PLSR for peaks 2，4，5，7，8， 10，12，13，14，15，16，17，18，20，21，22，24 were all greater than 0；those peaks were positively correlated with anti-inflammatory effect and were characteristic peaks except for peak 7； among them ，VIP values of peaks 5，8，10，16，18， 20，24 were greater than 1. The peak ratio of 10 batches of L. japonica was 58.61％-71.19％. CONCLUSIONS ：HPLC fingerprint of 10 batches of L. Japonica is successfully established. 10 batches of samples have similar components ，and the content of anti-inflammatory components is relatively high. The proportion of characteristic peaks to common peaks should not be less than 51.8％. |
| 期刊： | 2020年第31卷第20期 |
| 作者： | 缪艳燕，徐帮会，徐剑，张永萍，刘杰，刘耀 |
| AUTHORS： | MIAO Yanyan ，XU Banghui ，XU Jian，ZHANG Yongping ，LIU Jie，LIU Yao |
| 关键字： | 金银花；高效液相色谱法；指纹图谱；抗炎作用；谱-效关系；灰色关联度；偏最小二乘法 |
| KEYWORDS： | Lonicera japonica ；HPLC；Fingerprint；Anti-inflammatory effect ；Spectrum-effect relationship ；Grey relational |
| 阅读数： | 269 次 |
| 本月下载数： | 7 次 |
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