UHPLC-MS/MS profiling and antibacterial activity of hydrolysed and native flavonoids from <i>Crocus sativus</i> stamens

The increasing prevalence of multidrug-resistant (MDR) bacteria demands the development of new antibacterial agents derived from natural sources. Crocus sativus stamens, a by-product of saffron production, remain underexplored despite their potential bioactivity. This study aimed to investigate the phytochemical composition and antibacterial activity of hydrolyzed and non-hydrolyzed ethyl acetate fractions from these stamens. Ethyl acetate (EA) fractions were extracted (yield = 8.7 ± 0.3% w/w), hydrolyzed under acidic conditions, and analyzed using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry. Antibacterial activity was assessed against MDR bacterial strains using agar well diffusion and broth microdilution assays. A total of 31 phenolic compounds were identified. Acid hydrolysis led to a 3.2-fold increase in total aglycones based on UHPLC peak areas. The hydrolyzed EA fraction was enriched in quercetin (7.57%) and catechin gallate (15.59%). It exhibited strong antibacterial activity, notably against methicillin-resistant Staphylococcus aureus with a MIC of 6.25 mg mL−1 (4-fold improvement vs. non-hydrolyzed), and inhibition zones of 18.75 ± 0.37 mm (S. aureus) and 18.0 ± 0.5 mm (MRSA). Acid hydrolysis enhances the antibacterial efficacy of C. sativus stamen extracts by increasing aglycone flavonoids, supporting their valorization as sustainable antibacterial agents.