Comparative Analysis of the Chemical Constituents of Chrysanthemum morifolium with Different Drying Processes Integrating LC/GC-MS-Based, Non-Targeted Metabolomics

Abstract

Chrysanthemum morifolium is a perennial herbaceous plant in the Asteraceae family that is used as a medicine and food owing to its superior pharmacological properties. Irrespective of its application, C. morifolium must be dried before use. Shade drying (YG) and heat drying (HG) are the two drying methods used in most origins. Given the abundance of flavonoids, phenolic acids, and terpenoids, the primary medicinal active constituents of C. morifolium, it is important to determine whether the composition and content of these compounds are altered during the drying processes. To test this, the changes in the chemical composition of C. morifolium flowers after YG and HG using full-spectrum, non-targeted LC/GC-MS-based metabolomics and, subsequently, the three indicator components of C. morifolium-chlorogenic acid, 3,5-dicaffeoylquinic acid, and luteolin-7-O-glucoside-were accurately quantified by HPLC. The results of the non-targeted metabolomics analysis revealed that YG- and HG-processed C. morifolium differed significantly with respect to chemical contents, especially flavonoids, phenolic acids, and terpenoids. The levels of the indicator components and their precursors also differed significantly between the YG and HG treatments. The contents of most of the flavonoids and key phenolic acids, terpenoids, and carbohydrates were higher with YG than with HG pre-treatment. These results revealed the changes in the chemical composition of C. morifolium during the YG and HG processes, thus providing a reference for the further optimization of the production and processing of chrysanthemums.

Keywords: Chrysanthemum morifolium; heat drying (HG); high performance liquid chromatography; non-targeted metabolome; shade drying (YG).

Figure 1

The global metabolite profile of Chrysanthemum morifolium flowers based on gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–mass spectrometry (LC–MS). (A) LC–MS chromatograms in positive mode electrospray ionization (ESI). (B) LC–MS chromatograms in negative mode ESI. (C) GC–MS chromatograms. Blue, shade-drying group (YG); red, heat−drying group (HG). (D) The superclass of 7908 metabolites identified. Different color blocks represent different compound classes.

Figure 2

Multivariate model and its cross-validation. (A,B) Principal Component Analysis (PCA) of the LC/GC–MS data. (Combine both positive and negative ion data to generate) (C,D) Orthogonal Partial Least Squares−Discriminant Analysis (OPLS−DA) of the LC/GC–MS data. (E,F) Response permutation testing of the model predicted by OPLS−DA. R2X (cum): cumulative interpretation rate in the X direction; R2Y (cum): cumulative interpretation rate in the Y direction; Q2 (cum): cumulative forecast rate of the model; R2 and Q2: parameters of the response sequencing test used to measure whether the model was overfitted.

Figure 3

Differentially abundant metabolites between the YG and HG samples. (A) A volcano plot comprising both LC–MS and GC–MS data of the 7908 metabolites identified by metabolomics analysis. VIP > 1 and p < 0.05 served as the criteria for differential metabolite classification. (B) Classification of the 759 differential metabolites. (C) A heat map of top 50 differential metabolites. (VIP: Variable importance in the projection. P: Probability Value. HG: heat drying. YG: Shade drying).

Figure 4

High-performance liquid chromatography (HPLC) analysis of 3 medicinal ingredients in chrysanthemum (boju). (A) HPLC chromatogram for 3 medicinal ingredients in chrysanthemum (boju). (B) Bar charts of the three indicator components of chrysanthemum (boju) (* p < 0.05).

Figure 5

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. (A) The top 20 pathways in terms of −lg (p-value) for the HG samples compared to the YG samples. (B) A KEGG analysis bubble plot of the top 20 enriched pathways. (C) The 10 most significantly (smallest p-value) upregulated and downregulated KEGG metabolic pathways between the HG and YG groups. (D) The chlorogenic acid, 3,5−O−dicaffeoyl−quinic acid, and luteolin−7−O−glucoside synthetic pathways.

Figure 6

A heatmap of the 81 flavonoids in HG− and YG−processed C. morifolium.

Figure 7

Box plots of β-farnesene, mannose, rhamnose, and 1-kestose. (* p < 0.05; ** p < 0.01; *** p < 0.001).