SYNERGISTIC ANTIOXIDANT AND ANTI-INFLAMMATORY ACTIVITIES OF N-ACETYLCYSTEINE AND GLUTATHIONE EVALUATED THROUGH IN SILICO AND IN VITRO APPROACHES

Objectives: Oxidative stress and inflammation are interconnected pathological mechanisms implicated in the initiation and progression of several chronic disorders. Natural antioxidant molecules, such as N-acetylcysteine (NAC) and glutathione (GSH) possess potential therapeutic benefits; however, their combined effects require further investigation. This study aimed to evaluate the antioxidant and anti-inflammatory activities of NAC and GSH individually and as a 1:2 combination using integrated in silico and in vitro approaches. Methods: The pharmacokinetic and safety profiles of NAC and GSH were predicted using computational absorption, distribution, metabolism, and excretion (ADME) and StopTox-based toxicity analyses. The anti-inflammatory potential was assessed through egg albumin and bovine serum albumin (BSA) protein denaturation assays. Antioxidant activity was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), ferric reducing antioxidant power (FRAP), and hydrogen peroxide (H2O2) scavenging assays. The activities of individual compounds and the NAC: GSH combination was compared with standard antioxidants, including aspirin and ascorbic acid. Results: ADME predictions indicated that NAC exhibited favorable drug-like properties, including low molecular weight, moderate polarity, and low predicted cytochrome P450 interactions, whereas GSH showed higher polarity and reduced membrane permeability. StopTox predictions suggested that both compounds were unlikely to cause acute toxicity via oral, inhalation, or dermal exposure. The NAC: GSH combination demonstrated superior anti-inflammatory activity compared with individual treatments, showing IC50 values of 26.1 μM and 18.31 μM in egg albumin and BSA denaturation assays, respectively. Similarly, the combined formulation showed enhanced antioxidant activity, with lower IC50 values in the DPPH (35.1 μM), ABTS (30.67 μM), and FRAP (28.71 μM) assays than NAC or GSH alone. Although aspirin and ascorbic acid showed the highest overall activity among tested samples, the NAC: GSH combination displayed marked synergistic effects. Conclusion: The combined administration of NAC and GSH enhances antioxidant and anti-inflammatory activities through complementary mechanisms, including NAC-mediated GSH regeneration, GSH-mediated protein stabilization, and efficient reactive oxygen species scavenging. These findings support the potential of NAC: GSH combination therapy as a promising intervention for oxidative stress- and inflammation-associated disorders. Further in vivo investigations are warranted to validate its therapeutic efficacy.