Curcumin (C) and resveratrol (R) are two well-known nutraceuticals with strong antioxidant activity that can protect cells from oxidative stress. This study aims to investigate the synergy of CR combinations in protecting human endothelial EAhy926 cells against H₂O₂-induced oxidative stress and its related mechanisms. C and R as individual compounds as well as CR combinations at different ratios were screened for their protective effects against H₂O₂ (2.5mM) induced cell death assessed by cell viability assays. The synergistic interaction was analysed using the combination index model. The effects of optimal CR combinations on caspase-3 activity, ROS level, SOD activity, NAD cellular production, expression of Nrf2 and HO-1, and Nrf2 translocation were determined. CR combinations produced a synergistic protection against that of H₂O₂-induced changes in cell viability, caspase-3 activity, and ROS production. The strongest effect was observed for CR with the ratio of 8:2. Further experiments showed that CR 8:2 exhibited significantly greater effects in increasing Nrf2 translocation and expressions of Nrf2 and HO-1 proteins, as well as SOD activity and total cellular NAD production, than that of C or R alone. The findings demonstrate that combination of C and R produced a strong synergy in activity against H₂O₂-induced oxidative stress in EAhy926 cells. The mechanism of this synergy involves the activation of Nrf2-HO-1 signaling pathway and promotion of antioxidant enzymes. Further studies on CR synergy may help develop a new combination therapy for endothelial dysfunction and other conditions related to oxidative stress.

Background/Objectives: Oxidative and electrophilic stress-response pathways contribute to cellular resilience across various chronic diseases, including neurodegenerative, hepatic, and metabolic disorders. Pharmacological activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is a validated strategy to counteract oxidative damage, as evidenced by the clinical approval of dimethyl fumarate (DMF) and monomethyl fumarate (MMF) for relapsing forms of multiple sclerosis; both electrophilic compounds activate Nrf2 via covalent Keap1 (Kelch-like ECH-associated protein 1) modification. Cynara scolymus L. contains structurally related electrophilic metabolites; however, their contribution to Nrf2-associated signaling remains undefined. This study aimed to identify and characterize the constituents responsible for Nrf2-inducing activity and benchmark their potency against DMF and MMF. Methods: Bioactivity-guided fractionation combining Soxhlet extraction, Nrf2/ARE luciferase reporter screening, semi-preparative HPLC, and spectroscopic identification was employed. Functional validation included extracellular thiol quantification, H₂O₂ cytoprotection assays, and Western blot analysis of heme oxygenase-1 (HO-1). Results: The dichloromethane extract exhibited the highest Nrf2-inducing activity (54.4-fold). Fractionation yielded five guaianolide sesquiterpene lactones (1–5), four of which were active. The α-methylene-γ-lactone moiety was essential for activity. Aguerin B (3) exhibited the highest activity (39.14 ± 11.13-fold), while TBA analysis identified cynaropicrin (2) as the dominant extract-level contributor (62.9% of total activity). Notably, aguerin B (3) and cynaropicrin (2) induced greater reporter activity than DMF and MMF. Downstream pathway induction was confirmed by concentration- and time-dependent HO-1 upregulation, elevated extracellular glutathione and cysteinylglycine levels, and significant protection against H₂O₂-induced cytotoxicity without intrinsic toxicity. Conclusions: Guaianolide sesquiterpene lactones are the primary mediators of Nrf2-associated antioxidant signaling in C. scolymus. Cynaropicrin (2) exhibited stronger in vitro ARE-reporter induction than fumarates, supporting its relevance for further pharmacological investigation.

Metal-free C-H functionalization is a future-oriented approach for sustainability and safety control (eg. due to the limitation of regulatory science on non-predefined impurity) in our future world. In particular, hypervalent iodine chemistry has been frequently used for metal-free redox reaction, C-H functionalization, and cross-coupling based on transition metal-like and benign

Metal-free C-H functionalization is a future-oriented approach for sustainability and safety control (eg. due to the limitation of regulatory science on non-predefined impurity) in our future world. In particular, hypervalent iodine chemistry has been frequently used for metal-free redox reaction, C-H functionalization, and cross-coupling based on transition metal-like and benign

Background/Objectives: Oxidative stress is a well-established driver of neuronal dysfunction and a shared pathological mechanism across neurodegenerative diseases. Pharmacological activation of the Nrf2/ARE pathway is a validated strategy to counteract oxidative damage, as demonstrated by the clinical approval of dimethyl fumarate (DMF) and monomethyl fumarate (MMF) for relapsing forms of multiple sclerosis. These electrophilic compounds activate Nrf2 via covalent Keap1 modification. Cynara scolymus L. structurally related electrophilic metabolites; however, their contribution to Nrf2 activation remains undefined. This study aimed to identify and characterize the constituents responsible for Nrf2 activation and benchmark their potency against DMF and MMF. Methods: Bioactivity-guided fractionation combining Soxhlet extraction, Nrf2/ARE luciferase reporter screening, semi-preparative HPLC, and spectroscopic identification was employed. Functional validation included extracellular thiol quantification, H₂O₂ cytoprotection assays, and western blot analysis of heme oxygenase-1 (HO-1). Results: The dichloromethane extract exhibited the highest Nrf2-inducing activity (54.4 - fold). Fractionation yielded five guaianolide sesquiterpene lactones (1-5), four of which were active. The α-methylene-γ-lactone moiety was essential for activity. Aguerin B (3) exhibited the highest activity (39.14 ± 11.13-fold), while TBA analysis identified cynaropicrin (2) as the dominant extract-level contributor (62.9% of total activity). Notably, aguerin B and cynaropicrin (2) induced greater activation than DMF and MMF in Nrf2/ARE reporter assay. Downstream pathway activation was confirmed by concentration- and time-dependent HO-1 upregulation, elevated extracellular glutathione and CysGly levels, and significant protection against H₂O₂-induced cytotoxicity without intrinsic toxicity. Conclusions: Guaianolide sesquiterpene lactones are the primary mediators of Nrf2 activation in C. scolymus. Cynaropicrin (2) exhibited superior in vitro potency over fumarates, supporting its relevance for further pharmacological investigation.

The nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor is a master regulator of cytoprotective antioxidant gene expression; nevertheless, the ability of electrolyzed water formulations and their metal salt combinations to modulate this pathway remains insufficiently characterized. This study aimed to investigate whether ASEA Redox Signaling Molecules Supplement (ARS) activates the Nrf2 pathway and whether selected metal salts enhance this effect. Nrf2 activation was quantified in AREc32 luciferase reporter cells; cytotoxicity was assessed via Alamar Blue assay. Extracellular glutathione (GSH) and related thiols were quantified by HPLC with fluorescence detection. Western blot analysis of HepG2 cells was performed to assess the expression of Nrf2-regulated cytoprotective proteins, including HO-1 and GCLC. ARS alone induced modest Nrf2 activation (1.78 ± 0.51-fold). Combination treatment with gold(III) chloride trihydrate (HAuCl₄.3H₂O) significantly enhanced activation (7.43 ± 1.15-fold), whereas the salt alone exhibited negligible activity. Zinc and copper salts exhibited moderate activation. Notably, ARS combined with gold(III) chloride trihydrate significantly elevated extracellular GSH (2.46 ± 2.73-fold) and upregulated HO-1 protein expression. Collectively, these data demonstrate that electrolyzed water formulations activate the Nrf2-ARE pathway in vitro, with marked potentiation by specific metal salts, particularly gold chloride trihydrate, implicating synergistic modulation of cellular antioxidant signaling.