The aim of this study was to evaluate the application potential of a recombinant fungal immunomodulatory protein from Ganoderma lucidum (rFIP-glu). First, a recombinant plasmid pPIC9K::FIP-glu-His was transferred into Pichia pastoris for the production of protein. The protein was then to assess its free radical scavenging abilities and the effect on the viability of both human immortalized keratinocytes (HaCaT cells) and mouse B16-F10 melanoma cells (B16 cells) in vitro, followed by the effect on the melanin synthesis of B16 cells. The results of SDS-PAGE and western blot showed that rFIP-glu was successfully expressed. Furtherly, a bioactivity assay in vitro indicated that the scavenging rate of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals reached 84.5% at 6.0 mg/mL (p ≤ 0.0001) of rFIP-glu, showing strong antioxidant activity. Subsequently, a safety evaluation demonstrated that rFIP-glu promoted the proliferation of HaCaT cells, with the cell viability reaching 124.3% at 48 μg/mL (p ≤ 0.01), regarding the cell viability of B16 cells after exposure to rFIP-glu (48 μg/mL) significantly inhibited, to 80.7% (p ≤ 0.01). Besides, rFIP-glu inhibited the melanin synthesis of B16 cells in a dose-dependent manner from 100–1000 μg/mL, and rFIP-glu at 500 μg/mL (p ≤ 0.01) exhibited the highest intracellular melanin amount reduction of 16.8%. Furthermore, a mechanism analysis showed that rFIP-glu inhibited tyrosinase (TYR) activity by up-regulating the expression of the microphthalmia-associated transcription factor (MITF) and down-regulating the gene expression of TYR and tyrosinase-related protein-1 (TYRP-1), thus inhibiting melanin synthesis. The data implied that rFIP-glu had significant antioxidant activity and whitening potency. It should be used as raw materials for cosmeceutical applications.

Ganoderma leucocontextum, a newly identified species from the Tibetan Plateau, has been mainly studied for its polysaccharides and triterpenoids, with no prior reports on fungal immunomodulatory proteins (FIPs). This study explores the biological activity of FIP-gle2, cloned from G. leucocontextum and expressed in Pichia pastoris. The effects and mechanisms of recombinant FIP-gle2 (rFIP-gle2) on cell activity and melanin synthesis in mouse melanoma B16-F10 cells were investigated in vitro. The results showed that the FIP-gle2 gene, with an open reading frame (ORF) of 333 bp, encodes a 111-amino acid polypeptide with a molecular weight of 12.60 kDa and an isoelectric point of 4.48. We achieved a yield of 184.18 mg/L of rFIP-gle2. In vitro functional experiments showed that rFIP-gle2 significantly inhibited the proliferation of B16-F10 melanoma cells and induced apoptosis in a dose-dependent manner, particularly at concentrations above 1 μg/mL. At 3 μg/mL, rFIP-gle2 effectively inhibited tyrosinase activity and reduced melanin content, downregulating microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), and tyrosinase-related proteins (TRP-1 and TRP-2). Furthermore, RNA-seq analysis indicated that differentially expressed genes in treated cells were enriched in the mitogen-activated protein kinase (MAPK) signaling pathway, with Western blotting confirming enhanced phosphorylation of JNK, ERK, and p38 proteins. Thus, P. pastoris is an effective host for rFIP-gle2 production, which shows potential for applications in pharmaceuticals, cosmeceuticals, and food fields.

Aim: This study aimed to analyze the non-volatile chemical components in wild Ophiocordyceps sinensis (O. sinensis) from four distinct production areas in Xizang, and its fermented mycelia, with the goal of chemically evaluating the feasibility of substituting the wild samples with their mycelial preparation, and providing references for their application in daily life. Methods: An untargeted metabolomics approach using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was employed for comprehensive analysis. Results: A total of 89 non-volatile components were identified, mainly covering short peptides, nucleotides/derivatives, glycerophospholipids, glycosides, and amino acids. Multivariate statistical analysis revealed significant regional variation in the content of key metabolites, particularly the short peptide profile. Di- and tri-peptides were confirmed as the dominant bioactive constituents and chemometric markers distinguishing geographical origins. Compared to the wild samples, the fermented mycelia exhibited a statistically significant reduction in both the diversity and relative abundance of these signature short peptides. Conclusions: Wild O. sinensis and its fermented mycelia both contain bioactive compounds, with the latter sometimes surpassing the wild samples in specific components. However, the fermented version doesn’t match the natural samples’ synergistic effects. The wild sample’s efficacy is heavily influenced by its environment and growth conditions. This study provides a basis for using wild O. sinensis or its mycelial products in daily diets or disease prevention.

Ophiocordyceps sinensis (O. sinensis), a precious traditional Chinese medicine (TCM), exhibits remarkable hepatoprotective effects. However, chemical differences between wild-collected and cultivated products remain insufficiently characterized in relation to liver fibrosis. We combined untargeted ultra-high-performance liquid chromatography–Orbitrap high-resolution mass spectrometry with targeted liquid chromatography–tandem mass spectrometry to compare wild type (WT) and artificially cultivated (AC) specimens. The two sample groups showed distinct metabolic profiles, and targeted analysis supported differences in selected nucleosides, purine derivatives, pyroglutamic acid, and ergosterol. Adenosine, cordycepin, and ergosterol were more abundant in AC extracts, whereas hypoxanthine, deoxyguanosine, guanosine, and guanine were more abundant in WT extracts. Network pharmacology associated selected source-related metabolites with 158 fibrosis-related candidate targets and prioritized a kinase-centered network involving epidermal growth factor receptor (EGFR), proto-oncogene tyrosine-protein kinase Src (SRC), protein tyrosine kinase 2 (PTK2), HRas proto-oncogene, GTPase (HRAS), phosphatidylinositol 3-kinase/protein kinase B (PI3K-Akt), and mitogen-activated protein kinase (MAPK) signaling. Molecular docking and replicated molecular dynamics simulations indicated structurally plausible interactions for complexes of uridine diphosphate N-acetylglucosamine (UDP-N-acetylglucosamine) with HRAS and AKT serine/threonine kinase 1 (AKT1), and of ergosterol with SRC. These findings reveal source-associated metabolic differences between the analyzed WT and AC samples and provide mechanistic insights into their potentially divergent roles in liver fibrosis regulation, which warrant further experimental validation.