Melittin- and Apamin-Standardized Bee Venom: UHPLC-ESI-MS Characterization, Antibacterial Activity, and Apoptosis Induction in Human Keratinocytes

Bee venom (BV) is valued therapeutically mainly for its peptides, melittin and apamin. This study determined the dose-dependent biological activity of bee venom standardized to its melittin and apamin content by UHPLC-ESI-MS, as well as that of its principal peptides, melittin and apamin, tested at matched concentrations. Antibacterial activity was assessed as showing the minimum inhibitory and minimum bactericidal concentrations (MIC, MBC) against Staphylococcus aureus (MSSA and MRSA) and Staphylococcus epidermidis (MRSE). Cytotoxicity toward primary human keratinocytes (HEKa) was determined by MTT and LDH assays and the mode of cell death by Annexin V-FITC/propidium iodide staining. The standardized bee venom contained 608.21 µg/mg of melittin and 22.17 µg/mg of apamin. Melittin was the only active constituent (MIC 2.5 to 5 µg/mL, MBC/MIC ≤ 2), whereas apamin was inactive (>10 µg/mL). Bee venom was active only against Staphylococcus epidermidis (MRSE), with both MIC and MBC values equal to 10 µg/mL. At concentrations of 5 and 10 µg/mL, both bee venom and melittin were markedly cytotoxic to keratinocytes. The selectivity index (CC50/MIC) did not exceed 1.6 for melittin and reached 0.2 for bee venom. On a melittin-equivalent basis, whole venom was more cytotoxic than pure melittin (CC50 1.33 versus 3.91 µg/mL), indicating that constituents other than melittin contribute to keratinocyte damage. Melittin is therefore the principal antibacterial constituent, but its narrow selectivity does not support direct topical use. Combination experiments evaluating bee venom or its isolated peptides in combination with antibiotics were not performed here and remain a defined direction for future work. Standardization to melittin content predicts potency but not safety, which has to be assessed on the whole standardized venom.