Green Synthesized TiO₂ Nanoparticles from Calotropis gigantea Leaves for Anti-inflammatory Biomedical Applications

Abstract

Environmentally sustainable synthesis of nanomaterials offers an alternative to conventional preparation methods that may involve toxic chemicals and high energy consumption. This study synthesised titanium dioxide nanoparticles (TiO₂ NPs) using an aqueous extract of Calotropis gigantea leaves as a reducing and stabilising agent and evaluated their preliminary anti-inflammatory and cellular effects. Reaction parameters, including pH, temperature, and extract concentration, were optimised, and the recovered material was calcined at 450°C for 3 h. Anti-inflammatory activity was assessed using human red blood cell (HRBC) membrane stabilisation and bovine serum albumin (BSA) protein denaturation assays, with diclofenac sodium as the reference standard. HRBC membrane protection increased from 11% at 0.6 mg/mL to 82% at 3.0 mg/mL, with a reported IC₅₀ value of 1.65 mg/mL. Inhibition of BSA denaturation increased from 9% at 500 µg/mL to 60% at 2500 µg/mL, with a reported IC₅₀ value of 1643 µg/mL. Diclofenac sodium showed greater activity in both assays. JC-1 staining of HepG2 cells showed a shift from red to green fluorescence after nanoparticle exposure, indicating changes in mitochondrial membrane potential. The findings demonstrate concentration-dependent membrane stabilisation and protein denaturation inhibition, together with measurable mitochondrial-level interaction. These results support further investigation of green-synthesised TiO₂ nanoparticles, although detailed physicochemical characterisation, quantitative cytotoxicity assessment, and additional mechanistic validation are required before biomedical application can be considered.