Antioxidant Profiling and Calcium Oxalate Crystal Growth Inhibition by Bioactive Fractions of Madhuca longifolia Stem Bark

Abstract

Background: Urolithiasis is a recurrent urinary tract disorder in which calcium oxalate (CaOx) crystals account for nearly 80% of kidney stones. The search for safer plant-derived antiurolithiatic agents has increased because of the recurrence and adverse effects associated with conventional therapies. The present study aimed to isolate bioactive phytoconstituents from the stem bark of Madhuca longifolia using a bioactivity-guided approach and evaluate their antioxidant and in vitro antiurolithiatic activities.Methodology: Hydroalcoholic extract of Madhuca longifolia stem bark was fractionated using solvents of increasing polarity. The fractions were evaluated for total phenolic content (TPC), total flavonoid content (TFC), DPPH, FRAP, and nitric oxide scavenging activities. The most active ethyl acetate fraction (F3) was purified by silica gel column chromatography, yielding three compounds (ML-1, ML-2, and ML-3), which were characterized by TLC, FTIR, ^1H NMR, ^13C NMR, and ESI-MS. Antiurolithiatic activity was assessed by calcium oxalate crystal nucleation, aggregation, crystal growth, and microscopic crystal morphology assays. Results and Discussion: The ethyl acetate fraction exhibited the highest TPC (84.62 ± 2.15 mg GAE/g) and TFC (58.34 ± 1.27 mg QE/g). It showed potent antioxidant activity with DPPH and nitric oxide scavenging IC₅₀ values of 28.43 ± 0.82 and 35.18 ± 1.06 µg/mL, respectively, and a FRAP value of 812.45 ± 18.36 µmol Fe²⁺/g. Bioactivity-guided purification yielded three purified compounds (ML-1). Fraction F3 demonstrated significant inhibition of calcium oxalate crystal nucleation (82.64 ± 2.31%), aggregation (79.45 ± 1.84%), and crystal growth (75.83 ± 2.07%) at 800 µg/mL, comparable to the standard formulation. Microscopic analysis revealed a marked reduction in crystal number, size, and aggregation following treatment with the active fraction and isolated compounds. Conclusion: The findings suggest that the phenolic-rich fraction of Madhuca longifolia possesses significant antioxidant and antiurolithiatic activities. Bioactivity-guided fractionation successfully enriched the active phytoconstituents, supporting the potential of Madhuca longifolia as a promising source of natural therapeutic agents for the prevention and management of calcium oxalate urolithiasis.