不同附生基质金钗石斛代谢产物与抗氧化活性比较研究

Comparison of Metabolites and Anti-oxidative Activity of Dendrobium nobile with Different Epiphytic Substrates

目的:研究木制与石制两种附生基质对金钗石斛初生、次生代谢产物与抗氧化活性的影响,筛选二者差异代谢物,为阐述本草所载石斛“唯生石上者胜”的科学内涵提供依据。方法:基于UPLC-ESI-MS/MS广泛靶向代谢组学技术,选择电喷雾离子源在正、负离子模式下进行采集检测,质谱数据经归一化处理后进行主成分分析(PCA)及正交偏最小二乘法-判别分析(OPLS-DA),根据指标fold change≥2、fold change≤0.5和VIP≥1为标准筛选两种附生基质金钗石斛差异代谢物,并对差异代谢物进行京都基因与基因组百科全书(KEGG)分类与富集分析;以2,2’-联氨-双(3-乙基苯井噻唑啉-6-磺酸)二胺盐(ABTS)自由基、2,2-二苯基-1-苦基肼(DPPH)自由基和羟基自由基清除试验与总抗氧化能力(T-AOC)检测试剂盒(FRAP法)微板法,检测不同附生基质金钗石斛抗氧化活性。结果:从木制与石制两种附生基质金钗石斛样品中共鉴定出1031个成分,其中差异成分190个,相对含量上调成分112个,相对含量下调的成分78个,两者相比,石制基质金钗石斛在黄酮、生物碱等含量较高的次生代谢物数量占优。PCA与层次聚类分析(HCA)结果均表明两种附生基质金钗石斛代谢物含量存在明显差异,能够显著区分;KEGG分析结果表明,石制基质金钗石斛在次生代谢产物与氨基酸合成通路发生明显富集;石制基质金钗石斛对DPPH、ABTS自由基清除能力及总抗氧化能力均高于木制基质金钗石斛。结论:基于UPLC-ESI-MS/MS广泛靶向代谢组学技术可准确、全面地鉴定金钗石斛中的代谢产物,附生石制基质金钗石斛代谢产物数量及相对含量显著高于附生木制基质,且抗氧化活性增强,研究结果证实木制与石制两种附生基质对金钗石斛代谢产物与抗氧化活性有显著影响,为探讨本草所载“唯生石上者胜”的科学内涵提供依据。

Objective: To investigate the effects of wood and stone epiphytic substrates on primary and secondary metabolites and the anti-oxidative activity of Dendrobium nobile, and the differential metabolites were screened out to provide references for illustrating the scientific connotation of "the D. nobile grown on the stone has the superior quality" recorded in Collective Commentaries on the Classic of Materia Medica. Methods: Based on the widely targeted metabolomics technology of ultra-performance liquid chromatography-electrospray ionization-mass spectrometry(UPLC-ESI-MS/MS), the ESI ion source was selected for collection and detection in positive and negative ion modes. After the normalization of MS data, principal component analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA) were performed. The differential metabolites of D. nobile with wood and stone epiphytic substrates were screened out according to the indicators fold change≥2, fold change≤0.5, and VIP≥1, and the differential metabolites were classified and analyzed by Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analysis. Diammonium 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonate)(ABTS), 1,1-Diphenyl-2-picrylhydrazyl(DPPH), and hydroxyl radical clearance test and total antioxidant capacity(T-AOC) ferric ion reducing antioxidant power(FRAP) were used to detect the anti-oxidative activities of D. nobile with different epiphytic substrates. Results: A total of 1031 components were identified from the samples of D. nobile with wood and stone epiphytic substrates, including 190 differential components, 112 up-regulated components, and 78 down-regulated components. As compared with D. nobile with wood epiphytic substrates, the number of major secondary metabolites including flavonoids and alkaloids in D. nobile with stone epiphytic substrates was higher. Both PCA and hierarchical cluster analysis(HCA) results showed that the metabolites were significantly different between the D. nobile with wood and stone epiphytic substrates. KEGG analysis showed that D. nobile with stone epiphytic substrates was enriched in the secondary metabolites and the amino acid synthesis pathway. The DPPH and ABTS radical clearing ability and T-AOC of D. nobile with stone epiphytic substrates were significantly higher than those of D. nobile with wood epiphytic substrates. Conclusion: The widely targeted metabolomics technology based on UPLC-ESI-MS/MS accurately and comprehensively identifies the metabolites in D. nobile. As compared with D. nobile with wood epiphytic substrates, the number of metabolites in D. nobile with stone epiphytic substrates is generally higher, and the anti-oxidative activity of D. nobile with stone epiphytic substrates is increased. The results demonstrate the wood and stone epiphytic substrates have significant effects on the metabolites and anti-oxidative activity of D. nobile, which provide references for the scientific connotation of "the D. nobile grown on the stone has the superior quality" recorded in Collective Commentaries on the Classic of Materia Medica.