Composition and In Vitro Activity-Cytotoxicity Profiles of Vespa basalis Venom: Combined Proteomic, Metabolomic, and...

Vespa basalis Smith venom (VBsV) causes painful and potentially severe systemic reactions, yet the relationships among its composition, bioactivity, and cytotoxicity remain poorly understood. Proteomic and metabolomic profiling was combined with ultrafiltration and gel filtration to characterize VBsV and obtain >3 kDa, operationally defined 700-3000 Da, and <700 Da fractions. The effect on LPS-induced NO production was evaluated by nitric oxide (NO) release in LPS-stimulated RAW264.7 cells, and cytotoxicity was assessed in tumor and THLE-2 liver cells. Five venom allergen proteins were identified, including two phospholipase A1 isoforms, hyaluronidase A, antigen 5, and venom dipeptidyl peptidase IV. Targeted profiling also detected several neurotransmitters and energy metabolites, including 5-hydroxytryptamine, L-glutamate, histamine, γ-aminobutyric acid, inosine, and adenosine. The 700-3000 Da fraction exhibited the strongest NO-suppressive and tumor-cell-viability effects among the tested fractions. The 700-3000 Da fraction suppressed NO release (IC₅₀, approximately 23 μg/mL) and reduced tumor-cell viability, while minimally affecting THLE-2 cells at 10-80 μg/mL; by contrast, the >3 kDa fraction showed greater THLE-2 cytotoxicity. These findings identify the 700-3000 Da fraction as a priority for direct compositional characterization and isolation of the responsible molecules.