Investigating the antioxidant activity of metformin hydrochloride (MtHCl): Theoretical intuitions and experimental evidences

• Validated Approach: Combines DFT and assays to predict and validate MtHCl's antioxidant potential. • Mechanistic Insights: 5N site shows lowest BDE, PA, ETE, indicating high antioxidant efficacy. • Effective Scavenging: DPPH, hydroxyl, and superoxide assays confirm strong free radical neutralization. • Therapeutic Development: Insights optimize MtHCl's formulation for antioxidant-based health therapies. Diabetes mellitus, is characterized by chronic hyperglycemia, which leads to excessive production of reactive oxygen species (ROS) and subsequent oxidative stress. This may further exacerbate associated problems including retinopathy, neuropathy, nephropathy, cardiovascular diseases etc. Therefore, antioxidants are of significant importance in mitigating these complications and improving diabetes management. Metformin hydrochloride (MtHCl), an active pharmaceutical ingredient that contains biguanides is a well-known drug for the management of type 2 diabetes. This study assesses the AO activity of MtHCl using a range of experimental techniques, including the scavenging assays for superoxide (SO), hydroxyl (OH), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. The IC 50 for MtHCl in scavenging radicals of DPPH was 45.68 mg/mL, hydroxyl (OH) was 59.82 mg/mL and superoxide (SO) was 33.5 mg/mL. The effectiveness of a drug in combating oxidative stress depends on its chemical composition, structure-activity relationship, and ability to receive or donate electrons, neutralize free radicals, and regenerate endogenous antioxidants. Quantum computational calculations at DFT/B3LYP/6-311 (d, p) level of theory for predicting and understanding the ability of MtHCl to neutralize free radicals, which are essential in disorders linked to oxidative stress. When comparing the values with ∼3 N and 4 N for radical scavenging (RS) mechanisms, the calculated parameters of MtHCl, such as proton dissociation enthalpy (PDE), electron transfer enthalpy (ETE), proton affinity (PA), ionization potential (IP), and bond dissociation energy (BDE), suggest that donating a H atom is easy from 5 N position. This study highlights the AO activity of MtHCl, suggesting its potential to reduce oxidative stress in diabetes. While direct RS contributes, the primary in vivo benefits likely stem from indirect mechanisms, such as reduced ROS production through improved glycemic control and enhanced endogenous antioxidants, given that therapeutic plasma concentrations (1–2 µg/mL) are lower than the observed IC 50 value.