Experimental and In Silico Evaluation of Honey-Derived Phenolic Compounds: Antioxidant Activity, Anti-Inflammatory...

Honey is a natural product rich in phenolic compounds that contribute to its antioxidant and anti-inflammatory properties. The present study aimed to evaluate the biological activities of honey samples collected from different regions of Kazakhstan through experimental assays and computational analyses of major honey-derived phenolic compounds. Ethanolic extracts of seven honey samples were prepared and assessed for extraction yield, total phenolic content, total flavonoid content (TFC), antioxidant activity using the DPPH radical scavenging assay, and anti-inflammatory activity using the albumin denaturation inhibition assay. In addition, five representative honey-derived phenolic compounds, namely fumaric acid, p-hydroxybenzoic acid, p-coumaric acid, trans-2-hydroxycinnamic acid, and chrysin, were subjected to comprehensive in silico analyses including ADME prediction, drug-likeness evaluation, toxicity assessment, and multitarget molecular docking. Extraction yields ranged from 6.8% to 8.7%. The highest total phenolic content was observed in sample B12 (80.1 ± 9.7 mg GAE/g extract), whereas the highest TFC was detected in sample B10 (6.8 ± 0.5 mg quercetin equivalent (QE)/g extract). DPPH radical scavenging activity revealed IC₅₀ values ranging from 2.5 ± 0.1 to 5.6 ± 0.4 mg/mL, with samples B11, B12, and B10 exhibiting the strongest antioxidant activities. In the albumin denaturation assay, all extracts demonstrated concentration-dependent anti-inflammatory activity, with inhibition percentages reaching 33.9% ± 2.0% at 100 mg/mL. The ADME analysis revealed that all investigated compounds exhibited favorable drug-likeness properties and high predicted gastrointestinal (GI) absorption. Chrysin demonstrated the highest lipophilicity and receptor-binding potential, whereas p-coumaric acid and trans-2-hydroxycinnamic acid showed the most balanced pharmacokinetic profiles. Toxicity predictions performed using ProTox-3.0, pkCSM, and admetSAR indicated low risks of hepatotoxicity and nephrotoxicity, with no predicted neurotoxicity, respiratory toxicity, carcinogenicity, or mutagenicity for any of the compounds. Molecular docking analyses against multiple inflammation-related targets, including cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), nuclear factor kappa-B (NF-κB), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and protein kinase B (AKT1), demonstrated strong binding affinities, particularly for trans-2-hydroxycinnamic acid and chrysin. These compounds exhibited favorable interactions with several key mediators of inflammatory signaling, suggesting potential multitarget mechanisms of action. Computational analyses demonstrated favorable drug-likeness characteristics and high predicted GI absorption for all investigated compounds. Toxicity predictions indicated low risks of hepatotoxicity and nephrotoxicity, with no predicted neurotoxicity, respiratory toxicity, carcinogenicity, or mutagenicity. Molecular docking analyses against multiple inflammation-related targets, including COX-2, iNOS, NF-κB, TNF-α, IL-6, and AKT1, identified trans-2-hydroxycinnamic acid and chrysin as the compounds with the strongest binding affinities, suggesting their potential role as multitarget modulators of inflammatory signaling pathways. Overall, the combined experimental and computational findings demonstrate that Kazakhstan honey represents a valuable source of bioactive phenolic compounds with promising antioxidant and anti-inflammatory properties. Among the investigated compounds, trans-2-hydroxycinnamic acid, p-coumaric acid, and chrysin emerged as the most promising candidates for further pharmacological investigation. These findings provide a scientific basis for future studies exploring honey-derived phenolics as potential complementary agents for the management of inflammatory disorders. The integration of experimental bioactivity assays with multitarget computational analyses provides novel insights into the mechanisms through which honey-derived phenolics may contribute to the therapeutic potential of honey.