基于“初生-次生代谢产物”互作途径的大果沙棘和中华沙棘差异性研究

Difference between Hippophae rhamnoides vat.sp and Hippophae rhamnoides based on“primary-secondary metabolite”interaction pathway

目的分析大果沙棘Hippophae rhamnoides vat.sp L.与中华沙棘H.rhamnoides初生/次生代谢物的差异,并对其生物合成途径变化进行分析。方法采用超高效液相色谱-四级杆-飞行时间质谱(UPLC-Q-TOF-MS/MS)法对2个种属的沙棘进行初生及次生代谢产物表征,基于二水平析因-星点设计法(plackett burman-central composite design,PB-CCD)设计对沙棘UPLC-DAD指纹图谱进行研究,优化液相方法,指认共有峰;以偏最小二乘-判别分析(partial least squares discriminant analysis,PLS-DA)、聚类分析及京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)通路富集分析对大果沙棘、中华沙棘进行差异初生代谢物筛选及相关代谢通路富集;进一步采用质谱多反应检测(multiple reaction monitoring,MRM)对筛选得到具有差异的初生代谢物和次生代谢物进行定量分析;最后,对2个品种沙棘的差异性成分进行分析并对其进行生物合成途径的关联和阐释。结果基于成分表征和PLS-DA初步鉴定出中华、大果沙棘含量差异较大的成分有11个,其中包括2个氨基酸类初生代谢物和9个黄酮类次生代谢物。结论建立的方法能鉴定出中华沙棘、大果沙棘中的差异性成分。分析结果显示,总黄酮在中华沙棘中含量较高,说明中华沙棘在药用功效上可能强于大果沙棘;其次异鼠李素在大果沙棘中含量较高,说明其对临床的质控和应用可能不太适用。

Objective To analyze the differences of primary/secondary metabolites of Hippophae rhamnoides vat.sp and H.rhamnoides,and analyze the changes of their biosynthetic pathways.Methods Firstly,the ultra performance liquid chromatography-quadrupole-time of flight-mass spectrometry(UPLC-Q-TOF-MS/MS)technology was utilized to characterize the primary and secondary metabolites of two species of Hippophae Fructus.Then,based on the plackett burman-central composite design(PB-CCD),the UPLC-DAD fingerprint of Hippophae Fructus was studied,the liquid phase method was optimized,and the common peaks were identified.Next,partial least squares discriminant analysis(PLS-DA),cluster analysis and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis were conducted for the differential primary metabolites screening and related metabolic pathways enrichment of H.rhamnoides vat.sp and H.rhamnoides.At the same time,multiple reaction monitoring(MRM)was used to quantitatively analyze the primary and secondary metabolites with differences.Finally,the differential components of the two species of Hippophae Fructus were analyzed and their biosynthetic pathways were correlated and explained.Results Based on component characterization and PLS-DA,a total of 11 components with large differences in the content of two species of Hippophae Fructus were preliminarily identified,including two amino acid primary metabolites and nine flavonoid secondary metabolites.Conclusion The method established in this study can identify the different components in two species of Hippophae Fructus.The results showed that the content of total flavonoids was higher in Hippophae Fructus,indicating that Hippophae Fructus may have stronger medicinal efficacy than H.rhamnoides vat.sp L.;secondly,the determination result of isorhamnetin was higher in Hippophae rhamnoides vat.sp L.,indicating that it may not be suitable for clinical quality control and application.