Wound Healing Mechanisms of Medicago sativa Honey: Antioxidant, Anti-inflammatory, Antibacterial and Phytochemical Characterisation

Background: Honey has long been used in traditional medicine for wound healing. Its therapeutic properties vary
depending on botanical and geographical origin. Aim: To evaluate the physicochemical characteristics, polyphenolic
composition, antioxidant potential, antibacterial activity, and wound healing effects of Palestinian Medicago sativa (alfalfa)
honey. Methods: Polyphenols were profiled by HPLC. Antioxidant capacity was assessed by Total Phenolic Content (TPC)
and DPPH radical scavenging assays. Anti-inflammatory activity was determined by Nitric Oxide (NO) production in LPSactivated
THP-1 macrophages. Wound healing potential was tested via HaCaT keratinocyte proliferation and scratch
migration assays. Antibacterial effects were evaluated against Gram-positive and Gram-negative strains. Results: HPLC
revealed a diverse polyphenolic profile dominated by ellagic acid (23.51 mg/g), pinocembrin, and myricetin, with a TPC of
47.37 mg/g. Antioxidant assays confirmed high phenolic levels (327.5 ± 6.95 mg GAE/100 g) and strong radical scavenging
activity (DPPH IC₅₀: 12.33 ± 0.68 mg/mL). Alfalfa honey significantly reduced NO production in macrophages, enhanced
keratinocyte proliferation at low concentrations, and promoted migration at 1-2 mg/mL, while higher doses showed
cytostatic effects. Antibacterial assays demonstrated broad-spectrum activity, particularly against Bacillus subtilis and
Klebsiella pneumoniae. Conclusion: Palestinian M. sativa honey exhibits potent antioxidant, anti-inflammatory, antibacterial,
and wound healing properties. Its high ellagic acid content, dose-dependent effects on keratinocyte proliferation, and broad
antibacterial activity highlight its potential as a natural therapeutic agent in wound management.
Major Findings: Palestinian alfalfa honey shows exceptional wound-healing potential through strong antioxidant, antiinflammatory,
and antibacterial activities, largely attributed to its high ellagic acid and polyphenolic content.