不同产地金钗石斛醇提物HPLC指纹图谱的建立及其抗炎作用的谱效关系研究

冯月 段飞鹏 李一圣 邱书奇

摘 要 目的：建立不同产地金钗石斛醇提物的高效液相色谱法（HPLC）指纹图谱，并进行其抗炎作用的谱效关系研究。方法：制备12批不同产地金钗石斛（S1～S12）的醇提物样品。采用HPLC法检测，并结合《中药指纹图谱相似度评价系统（2012年版）》建立12批金钗石斛醇提物的指纹图谱并进行共有峰指认和相似度评价。以炎症因子[白细胞介素4（IL-4）、IL-6]含量为抗炎指标，采用酶联免疫吸附试验考察12批金钗石斛醇提物对脂多糖诱导的炎症模型RAW264.7巨噬细胞的抗炎作用;采用灰色关联度分析法分析金钗石斛醇提物指纹图谱中各共有峰与抗炎指标的关联度。结果：12批金钗石斛醇提物共有18个共有峰，指认了12号峰为石斛酚，各样品相似度为0.911～0.996。金钗石斛S1～S12醇提物均可显著降低细胞培养液中IL-4的含量（P<0.05或P<0.01），金钗石斛S1～S8、S10醇提物均可显著降低细胞培养液中IL-6的含量（P<0.05或P<0.01）。经灰色关联度分析发现，除4、18号峰外，其余共有峰与IL-4含量的关联度均大于0.6，且5、7号峰与IL-4含量的关联度均大于0.8;除9、14、4、3、18号峰外，其余共有峰与IL-6含量的关联度均大于0.6，且1、12、13號峰与IL-6含量的关联度均大于0.8。结论：12批不同产地金钗石斛醇提物的HPLC指纹图谱的相似度较高、所含成分一致性较好;其中1、5、7、12（石斛酚）、13号峰所代表的化学成分可能是金钗石斛潜在的抗炎药效成分。

关键词 金钗石斛;高效液相色谱;指纹图谱;抗炎作用;谱效关系

ABSTRACT   OBJECTIVE： To establish the HPLC fingerprints of ethanol extracts of Dendrobium nobile from different habitats， and to study spectrum effect relationship of its anti-inflammatory effect. METHODS： The ethanol extracts from 12 batches of D. nobile from different habitats （S1-S12） were prepared. The fingerprints of ethanol extracts from 12 batches of D. nobile were established by HPLC and TCM Fingerprint Similarity Evaluation System （version 2012）. Common peaks were identified and the similarity was evaluated. Using the contents of inflammatory factors （IL-4， IL-6） as anti-inflammatory indexes， ELISA was used to investigate the effects of ethanol extracts from 12 batches of D. nobile on RAW264.7 macrophages in inflammatory model induced by lipopolysaccharide. Grey correlation analysis was used to analyze the correlation between common peaks and anti-inflammatory indexes in the fingerprint of ethanol extract from D. nobile. RESULTS： There were 18 common peaks in ethanol extracts from 12 batches of D. nobile， and No. 12 peak was identified as dendrophenol. The similarity of each sample was 0.911-0.996. The content of IL-4 in cell culture medium was significantly reduced by ethanol extracts of D. nobile S1-S12 （P<0.05 or P<0.01）; the content of IL-6 in cell culture was significantly reduced by ethanol extracts of D. nobile S1-S8， S10 （P<0.05 or P<0.01）. Grey correlation analysis found that except for peaks 4 and 18， the correlation degree between the other common peaks and the content of IL-4 was greater than 0.6， and the correlation degree between peaks 5 and 7 and the content of IL-4 was greater than 0.8; except for peaks 9， 14， 4， 3 and 18， the correlation degree between the other common peaks and the content of IL-6 was greater than 0.6， and the correlation degree between peaks 1， 12 and 13 and the content of IL-6 was greater than 0.8. CONCLUSIONS： HPLC fingerprints of ethanol extracts from 12 batches of D. nobile from different habitats have high similarity and good consistency in composition; the chemical constituents represented by peaks 1， 5， 7， 12 （dendrophenol） and 13 may be potential anti-inflammatory components of D. nobile.

KEYWORDS   Dendrobium nobile; HPLC; Fingerprint; Anti-inflammatory activity; Spectrum effect relationship

石斛为兰科植物金钗石斛Dendrobium nobile Lindl、鼓槌石斛D. chrysotoxum Lindl或流苏石斛D. fimbriatum Hook的栽培品及其同属植物近似种的新鲜或干燥茎，其性甘、微寒，具有益胃生津、滋阴清热的功效[1]。现代药理研究表明，石斛具有抗炎、增强免疫力、降血糖、抗衰老、抗肿瘤和保护神经系统等多种药理活性[2]。相关研究结果显示，金钗石斛被较好地应用于慢性咽炎[3]，对炎症因子的分泌和炎症反应的发生具有良好的抑制作用[4-5]。然而，不同产地的金钗石斛因其生长的土壤环境的异质性和微生态差异性[6]，可能在内在性状、化学成分、药理活性等方面表现出一定的差异[7]，而这些方面的差异在一定程度上影响着金钗石斛的临床药效。……