In vitro pharmacological evaluation of the safety and antibacterial efficacy of a Chilean honey hydrogel for veterinary wound applications

Introduction Honey-based biomaterials have gained increasing interest as topical strategies for wound-related applications because of their antimicrobial, antioxidant, and tissue-supportive properties. However, the direct use of raw honey is limited by its viscosity, variability, and lack of standardized delivery systems. This study aimed to perform a preliminary in vitro pharmacological evaluation of a Chilean honey hydrogel (CHH) formulated with honey, chitosan, and gelatin for potential veterinary wound-related applications. Methods Eight Chilean honey samples with varying contributions of Quillay ( Quillaja saponaria Molina) were physicochemical characterized before hydrogel preparation. CHH formulations containing different relative honey proportions were evaluated in NIH/3T3 murine fibroblasts using the resazurin reduction assay as an indirect indicator of cellular metabolic/reducing activity. Nitrite accumulation was assessed using the Griess method as an indirect marker of nitric oxide-mediated activation. Antibacterial activity against clinical isolates of Staphylococcus pseudintermedius was evaluated using agar diffusion and minimum inhibitory concentration assays, and the results were compared with those of raw honey and selected antibiotics. Results All honey samples met basic physicochemical quality criteria. CHH formulations did not reduce fibroblast metabolic activity, whereas CHH-50% and CHH-100% significantly increased resazurin reduction compared with untreated control cells. Raw honey significantly increased nitrite accumulation, whereas CHH formulations did not increase nitrite levels under the experimental conditions tested. In agar diffusion assays, CHH produced larger and more homogeneous inhibition zones than raw honey against S. pseudintermedius , increasing the mean inhibition zone diameter from 10.98 ± 5.00 mm to 16.19 ± 1.82 mm. MIC assays further supported this effect, with CHH showing lower and less variable MIC values than raw honey. The median MIC decreased from 16.92 mg/mL for raw honey to 4.16 mg/mL for CHH, while MIC90 decreased from 104.17 to 10.42 mg/mL. As expected, amoxicillin–clavulanic acid and cefadroxil showed lower MIC values than CHH. Discussion These findings support CHH as a biocompatible honey-derived formulation with improved antibacterial performance compared with raw honey under in vitro conditions. However, this study should be interpreted as a preliminary pharmacological evaluation rather than evidence of wound-healing efficacy. Further hydrogel characterization, mechanistic, bactericidal, canine skin cell, and in vivo studies are required to determine its translational relevance for veterinary wound management.