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.

Floccularia luteovirens, an endemic resource of the Tibetan Plateau, possesses significant medicinal and ecological values. However, the understanding of antioxidant capacity and metabolic profiling of F. luteovirens from diverse regions remains elusive due to limited resources. Therefore, to comprehensively comprehend the antioxidant capacity and metabolite diversity of F. luteovirens, we conducted a rounded analysis of its antioxidant capacity from three distinct regions using both untargeted and targeted metabolomics. Determination of antioxidant indices, such as ferric ion-reducing antioxidant power (FRAP), total phenolic content (TPC), and flavonoid content (FC), revealed the robust antioxidant capacity of F. luteovirens. QL F. luteovirens (QLFL) exhibited no significant difference compared to ZD F. luteovirens (ZDFL); however, both were significantly distinct from XH F. luteovirens (XHFL) across multiple indices. Furthermore, a positive correlation was observed between FRAP and flavonoid content. A total of 5782 metabolites were identified and chemically classified. Metabolites of F. luteovirens varied significantly at different regions and eight key differential metabolites were screened. Phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, and cyanoamino acid metabolism were the main different regulatory pathways. Consequently, the disparities in the antioxidant activity of F. luteovirens may primarily be ascribed to the biosynthesis and metabolism of phenylalanine, while vanillic acid could potentially serve as a pivotal metabolite influencing the antioxidative capacity of F. luteovirens by targeted metabolomics. These findings enhance our understanding of the composition of F. luteovirens and provide valuable resources for its comprehensive utilization and targeted development.

To explore the differences in protein quality among classic medicinal entomopathogenic fungi and to evaluate their metabolic adaptability, we analyzed the amino acid composition and proteomic characteristics of Cordyceps sinensis (CS), Cordyceps militaris (CM), and Cordyceps cicadae (CC). Quantitative analysis showed CM contained the highest crude protein and lysine, methionine, threonine, and valine. CS adapted to high-altitude hypoxia and exhibited lower protein but elevated leucine, isoleucine, and histidine contents, which may contribute to membrane stabilization and oxidative stress resistance. CC displayed higher non-essential amino acids such as arginine, proline, and tyrosine, reflecting active nitrogen metabolism. Four-dimensional data-independent acquisition (4D-DIA) proteomics identified 495 differentially expressed proteins (DEPs). Compared with CS, CM and CC displayed upregulated glutamate oxaloacetate transaminases 2 (GOT2), glutamate dehydrogenase (GDH), and argininosuccinate synthase 1 (ASS1) coordinately regulate nitrogen flux through the alanine-aspartate-glutamate metabolic network and urea cycle, supporting metabolic intermediate replenishment for energy metabolism. The upregulation of branched-chain keto acid dehydrogenase E1 subunit alpha (BCKDHA) and acyl-CoA dehydrogenase short/branched chain (ACADSB) in CM and CC facilitated the integration of branched-chain amino acid catabolism with the TCA cycle, explaining species-specific differences in protein content. This study presents the first application of 4D-DIA proteomics to compare CS, CM, and CC, providing insights into quality divergence mechanisms in medicinal fungi.

Ophiocordyceps sinensis, is an entomopathogenic fungus renowned for its medicinal properties, thriving in the frigid and high-altitude regions of the Qinghai–Tibet plateau. Given the limited availability of wild resources and the increasing recognition of their medicinal value, the cultivation of O. sinensis was initiated. However, there is a paucity of research investigating the disparities in their quality. This study evaluated the primary physiological indicators of both wild and cultivated O. sinensis. It also employed proteome and untargeted metabolome approaches to elucidate the differences in quality and underlying mechanisms between the two types. The results revealed that the contents of key representative components, including polysaccharide, crude protein, adenosine, and mannitol, were higher in wild O. sinensis than in cultivated O. sinensis. A total of 499 differentially expressed proteins (DEPs), including 117 up-regulated and 382 down-regulated DEPs, were identified in wild and cultivated O. sinensis. Additionally, 369 up-regulated differentially accumulated metabolites (DAMs) and 737 down-regulated DAMs were also identified. Wild O. sinensis had higher relative levels of lysophospholipid metabolites, while cultivated O. sinensis had higher relative levels of aldehydes and carboxylic acids. Correlation analysis revealed that different habitats altered 47 pathways shared between the proteome and metabolome, including carbohydrate metabolism and energy metabolism. β-glucosidase and α-galactosidase play essential roles in carbohydrate catabolism and may indirectly influence amino acid synthesis through energy metabolic pathways. The differential expression of polyamine oxidase (PAO) could reflect variations in polyamine metabolism and ammonia production between wild and cultivated O. sinensis. These variations may consequently affect nitrogen homeostasis and the biosynthesis of nitrogen-containing compounds, ultimately leading to differences in nutritional quality. In conclusion, these findings offer a novel perspective on the applications of O. sinensis and serve as a reference for the targeted development of cultivated O. sinensis.