To investigate the accumulation pattern of metabolites in Glycyrrhiza uralensis by qualitative and quantitative analyses of metabolic constituents in Glycyrrhiza uralensis with different cultivation years, and to search for its differential metabolites.

The separation was performed on an Agilent SB-C₁₈ (100 mm×2.1 mm, 1.8 μm) column with 0.1% formic acid aqueous solution as the mobile phase A and 0.1% formic acid acetonitrile solution as the mobile phase B. The gradient elution was carried out at a flow rate of 0.35 mL·min^-1, and the column temperature was 40 ℃ with an injection volume of 4 μL. The mass spectrometry was performed with positive and negative ions scanning in multiple reaction monitoring mode. The mass spectrometry was performed in multi-response monitoring mode with positive and negative ion scanning. The qualitative and quantitative analyses of the metabolites in Glycyrrhiza uralensis were carried out on the basis of the self-constructed secondary mass spectrometry database, and the multivariate statistical analyses of the metabolites of Glycyrrhiza uralensis with different cultivation were combined with principal component analysis(PCA), orthogonal partial least squares discriminant analysis(OPLS-DA), and cluster heat map analyses.

(1) A total of 1 038 metabolites were detected from the samples of Glycyrrhiza uralensis with different cultivation years, among which 201 differential metabolites existed between annual and biennial Glycyrrhiza uralensis, 125 up-regulated and 76 down-regulated; 223 differential metabolites existed between biennial and three years old Glycyrrhiza uralensis, 64 up-regulated and 159 down-regulated; 185 differential metabolites existed between annual and three years old Glycyrrhiza uralensis, 59 up-regulated and 126 down-regulated. Four metabolites specific to annual Glycyrrhiza uralensis, six to biennial and one to three-year old were found. (2) K-mean cluster analysis was performed on the differential metabolites, and the differential metabolites were classified according to the different accumulation trends, and it was found that most of the metabolites such as flavonoids, phenolic acids, terpenes, lignans, and coumarins peaked in biennial Glycyrrhiza uralensis, and most of the metabolites such as alkaloids, amino acids and their derivatives peaked in annual Glycyrrhiza uralensis, and a part of the flavonoids, phenolic acids and other metabolites reached peaks in three years old Glycyrrhiza uralensis, suggesting that the metabolism of Glycyrrhiza uralensis in the body reached the peaks. peak value, suggesting that there was a certain pattern of metabolite content changes in Glycyrrhiza uralensis. (3) 160 differential metabolites annotated in Kyoto Encyclopedia of Genes and Genomes (KEGG) datebase and flavonoids, amino acids and their derivatives, and organic acids were the differential metabolites that accounted for a relatively large number of them. A total of 79 differential metabolic pathways were enriched among different comparison groups, among which 6 differential metabolic pathways were highly significantly enriched (P<0.01) and 23 significantly enriched (P<0.05), and the distributions of compounds involved in the above pathways were basically the same as before enrichment in comparison of different cultivation year.

The present study elucidate the differences between the metabolic components of Glycyrrhiza uralensis with different cultivation years, and further analyse the metabolic pathways that might cause the differences through the differential metabolites, which can provide a certain reference basis for the determination of the harvesting year of Glycyrrhiza uralensis and the study of the quality formation mechanism.