Antioxidant characterization of gamma-irradiated medical-grade honey and its hormetic effect on ROS in rat chondrocytes

Abstract

Introduction
Medical-grade honey (MGH), produced by gamma irradiation, concentrates polyphenols and flavonoids with antioxidant activity. Chondrocyte oxidative stress contributes to cartilage degradation and osteoarthritis, for which no treatment halts progression. Characterizing the antioxidant properties of MGH and its effect on chondrocytes could guide its use as a joint-protective agent.

Objective
To characterize the antioxidant capacity and flavonoid profile of medical-grade honey generated by gamma irradiation, and to evaluate its antioxidant effect on rat chondrocytes under oxidative stress.

Methods
MGH was generated by gamma irradiation (⁶⁰Co, 0–20 kGy). Total phenolic content, total flavonoid content, total antioxidant capacity, FRAP, ABTS, and DPPH were quantified as a function of irradiation dose. Flavonoids were identified and quantified by HPLC with a UV-DAD detector. The effect on ROS was assessed in primary rat chondrocytes by flow cytometry (CM-H2DCFDA) after exposure to tert-butyl hydroperoxide (tBHP, 50 µM) with or without MGH (0.12–4 mg/mL). Viability was assessed in rat chondrocytes by calcein-AM/EthD-1 and alamarBlue. Data were analyzed with the Kruskal-Wallis test and Dunn pairwise comparisons with Benjamini-Hochberg adjustment.

Results
Gamma irradiation produced a dose-dependent, significant increase in phenolic content (+38 %; from 51.2 ± 5.4 to 70.5 ± 5.0 mg GAE/100 g; p<0.001), flavonoid content (+42 %; from 18.9 ± 1.4 to 26.8 ± 2.2 mg QE/100 g; p<0.001), and total antioxidant capacity (+33 %; p<0.001). Consistently, the antioxidant capacities measured by FRAP, ABTS, and DPPH were significantly higher in irradiated (20 kGy) than in non-irradiated honey (+27 %, +68 %, and +67 %, respectively; p<0.001), whereas total protein content remained unchanged (p=0.199), indicating that the antioxidant effect of MGH is attributable to polyphenols rather than proteins. HPLC-UV/DAD analysis identified and quantified phenolic acids and flavonoids including gallic acid, caffeic acid, naringenin, quercetin, baicalin, eriodictyol, and apigenin, several of which increased after irradiation. Viability was preserved in rat chondrocytes up to 4 mg/mL. tBHP raised ROS about 1.7-fold over basal; MGH modulated this response with a hormetic (biphasic) behavior. The net antioxidant effect, normalized to basal levels, was reached at 4 mg/mL (ns vs basal), whereas intermediate doses maintained a stimulatory response without compromising viability.

Conclusion
Gamma irradiation enhances the antioxidant capacity and polyphenol content of MGM, which is well tolerated by rat chondrocytes and modulates oxidative stress-induced ROS with a hormetic profile, with a net antioxidant effect at high doses. These findings support its characterization as an antioxidant source with chondroprotective potential and provide a basis for evaluating its efficacy in osteoarthritis models, to preserve joint function and prevent musculoskeletal disability.