关键词: 蜂王浆, 代谢组学, 样品提取, 高分辨质谱

Abstract:

【Objective】Royal jelly, a natural product with health-promoting effect, is rich in small-molecule bioactive compounds. The systematic analysis of the metabolite extraction from royal jelly is still lacking. This study performed metabolic profiling of royal jelly with an aim to compare the metabolite extraction efficiency using different solvents, optimize the metabolite extraction protocol, and globally identify the small-molecule compounds in royal jelly. 【Method】Metabolites in royal jelly were extracted separately using six different solvents including 50% and 80% methanol, ethanol, and acetonitrile. They were analyzed by reverse phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC) each combined with Quadrupole-Exactive Orbitrap high resolution mass spectrometry (HRMS). The amount and relative standard deviation (RSD) values of metabolite features among the solvent groups were compared. Principal component analysis (PCA) was conducted on the basis of these metabolite features. The compound identification was based on mass spectrometry databases and was validated with standards. Both the RSD value comparison and clustering heatmap analysis were performed for the identified compounds. Significantly differential metabolites among the solvent groups were screened out by orthogonal partial least squares-discriminant analysis (OPLS-DA), univariate analysis (Student’s t-test) and fold change (FC). 【Result】Highly polar metabolites, including two isomeric sugars (glucose and fructose), and medium and weakly polar metabolites such as lipids were sufficiently separated using the HILIC and PRLC approach, respectively. The combination of the two separation methods enabled comprehensive detection of metabolites with different polarity in royal jelly. A total of 70 high-abundance compounds were identified, including carbohydrates, amino acids, lipids, and vitamins, with the highest difference of 8 340 times in abundance. Among them, 17 compounds were reported for the first time. The lowest number of metabolite features was obtained with acetonitrile, and a worse coverage was obtained with 80% than 50% acetonitrile. For methanol and ethanol, the number of metabolite features was higher at higher solvent concentrations. The RSD values of most metabolite features were lower than 20% in all solvent groups, and the 80% acetonitrile group had the lowest proportion within 10% RSD values compared with other solvents. The RSD values of the 70 identified compounds provided further evidence for the poor repeatability of 80% acetonitrile. The PCA score plots indicated a similar metabolic profiling from the same solvent. Differences in metabolic profiling were observed among the solvents, and the largest difference existed between 80% acetonitrile and the five other solvents. The clustering heatmap showed a lower abundance of medium and weakly polar metabolites obtained from 50% solvents and a lower abundance of highly polar metabolites including, in particular, fructose, glucose, sucrose, lysine, adenosine, choline, phosphorylcholine, and gluconic acid from 80% acetonitrile. 【Conclusion】The combination of RPLC-HRMS and HILIC-HRMS enables comprehensive and accurate detection of small-molecule compounds in royal jelly. 80% methanol or 80% ethanol is an optimal solvent for metabolite extraction from royal jelly.

Key words: royal jelly, metabolomics, sample extraction, high resolution mass spectrometry (HRMS)