Comprehensive profiling of bee venom metabolites by multiplatform metabolomics highlighting melittin and cytotoxic assessment against breast cancer cells

This study characterizes Apis mellifera bee venom using an integrated multiplatform approach utilizing 1D NMR, 2D HSQC with 3D views, GC-MS, and LC-MS. The results clearly identified melittin as the main peptide component (28.6% by GC-MS and 48.5% by LC-MS) as the intact peptide in the crude venom. Proton NMR revealed the classic melittin fingerprint: aliphatic peaks (0.8–1.8 ppm) from hydrophobic amino acids, methylene signals (2.3–2.8 ppm) from amines and acids, alpha-protons (3.0-4.3 ppm), and the single tryptophan indole cluster (6.5–8.2 ppm) and perfectly matched the standard. HSQC confirmed the backbone structure, while longer 12-hour runs highlighted fainter lysine/aspartate signals. GC-MS detected melittin at 10.02 min, plus PLA2 (8.9%), glucosidase, norepinephrine (6.3%), and a dozen other venom markers across 3–18 min. LC-MS captured polar components missed by GC, including norepinephrine topping amines at 4 ng/mL, histamine, and acids like malic (3.8 g/mL), all aligning with NMR regions. Together, these components form a highly effective defense mechanism. Bee venom (0.5–10 µg/mL) displayed concentration-dependent reduction in cell viability of MCF-7 cells, as determined by the MTT assay. Further validation of apoptotic cell death was confirmed by trypan blue and DAPI staining that demonstrated morphological changes, nuclear condensation, and fragmentation. This robust NMR-MS toolkit enables reproducible venom metabolite profiling, supporting cross-species comparisons and therapeutic applications in cancer research.