Characterization of Metabolite Landscape Distinguishes Medicinal Fungus Cordyceps sinensis and other Cordyceps by UHPLC-Q Exactive HF-X Untargeted Metabolomics

Abstract

Cordyceps represent a valuable class of medicinal fungi with potential utilization. The overexploitation and resource scarcity of Cordyceps sinensis (CS) have led to the emergence of Cordyceps such as Cordyceps militaris (CM) and Cordyceps cicadae (CC) as substitutes. The medicinal value of CS is often considered superior to other Cordyceps, potentially owing to differences in active ingredients. This study aimed to evaluate the differences in the composition and abundance of the primary and secondary metabolites of CS and its substitutes by untargeted metabolomics. A total of 4671 metabolites from 18 superclasses were detected. CS and its substitutes were rich in amino acids, lipids, organic acids, and their derivatives. We statistically analyzed the metabolites and found a total of 285 differential metabolites (3'-Adenylic acid, O-Adipoylcarnitine, L-Dopachrome, etc.) between CS and CC, CS and CM, and CM and CC, which are potential biomarkers. L-glutamate and glycerophospholipids were differential metabolites. A KEGG enrichment analysis indicated that the tyrosine metabolic pathway and tryptophan metabolism pathway are the most differentially expressed pathways among the three Cordyceps. In contrast, CS was enriched in a higher abundance of most lipid metabolites when compared to CM and CC, which may be an indispensable foundation for the pharmacological functions of CS. In conclusion, systematic, untargeted metabolomics analyses for CS and other Cordyceps have delivered a precious resource for insights into metabolite landscapes and predicted potential components of disease therapeutics.

Keywords: Cordyceps cicadae; Cordyceps militaris; Cordyceps sinensis; differential metabolites; untargeted metabolomics.

Figure 1

(A) Total ion chromatograms (TICs) of the QC samples. (B) TICs of the CS samples. (C) TICs of the CM samples. (D) TICs of the CC samples.

Figure 2

Metabolomics profiles of CS, CM, and CC. (A) Classification of the annotated metabolites in CS, CM, and CC. (B) Principal component analysis (PCA) results show metabolite profile differences between and within groups.

Figure 3

OPLS-DA score plots. (A) CC vs. CM. (B) CS vs. CM. (C) CC vs. CS. Comp1 first predicted principal component explanatory degree, orthogonal comp1 first predicted orthogonal component explanatory degree.

Figure 4

Differential metabolites of three species of Cordyceps. (A) Volcano plot of differential metabolites between CS vs. CC. (B) Volcano plot of differential metabolites between CS vs. CM. (C) Volcano plot of differential metabolites between CM vs. CC. (D) Venn plot of the number of differential metabolites among three comparison groups.

Figure 5

Hierarchical cluster analysis (HCA) revealed the variation in differential metabolites between and within groups. (A) Top 25 HCA of differential metabolites of 3 Cordyceps; (B) comparative analysis of top 25 differential metabolites in multiple groups. The horizontal coordinate indicates the group name and the vertical coordinate indicates the average relative abundance of metabolites in different groups.

Figure 6

(A–C) KEGG enrichment analysis. The horizontal coordinate represents the pathway name, and the vertical coordinate represents the enrichment rate, which represents the ratio of the metabolite number enriched in this pathway to the background number annotated in the pathway. The larger the ratio, the greater the degree of enrichment. The column color gradient indicates the significance of enrichment. The darker the default color, the more significantly enriched the KEGG term, where the p-value or FDR < 0.001 is marked ***, the p-value or FDR < 0.01 is marked **, and the p-value or FDR < 0.05 is marked *. (D–F) KEGG pathway differential-abundance-score maps. The horizontal coordinate represents the DA score and the vertical coordinate represents the KEGG metabolic pathway name. DA score reflects the overall change in all metabolites in the metabolic pathway. A score of 1 indicates that the expression trend of all annotated differential metabolites in the pathway is upregulated; −1 indicates that the expression trend of all annotated differential metabolites in the pathway is downregulated; and the length of the line segment indicates the absolute value of DA score. The size of the dot indicates the number of differential metabolites annotated in the pathway, and the larger the dot, the more differential metabolites in the pathway. The dot distribution on the right side of the central axis and the longer line segment indicate that the overall expression of the pathway tends to be upregulated. The dots are distributed to the left of the central axis, and the longer the line segment, the more inclined the overall expression of the pathway is to downregulate. (A,D) CM vs. CC. (B,E) CS vs. CC. (C,F) CS vs. CM.

Figure 7

The specimens of mature Cordyceps. (A) C. militaris. (B) C. sinensis. (C) C. cicadae.