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FLAVONOIDS have demonstrated neuroprotective activity. Hesperidin, the major flavonoid in the hydroalcoholic extract of Citrus trifoliata L. fruits was quantified quantitatively using HPLC. The calibration curve obtained by plotting different concentrations of hesperidin standard versus the area under the curve revealed that hesperidin content was up to 30 mg/g. Hesperidin was isolated and identified using 1H and 13C NMR. Self-nano-emulsifying drug delivery system (SNEDDS) was prepared to improve the physical characteristics and optimize the activity of the extract. Monoamine oxidase enzyme (MAO-B) inhibitory effects of the SNEDDS, the extract, and the isolated hesperidin were evaluated. They showed significant decrease in the IC50 up to 129.9008, 252.7341, and 707.7631 ng/ml, respectively, compared with selegiline, with IC50 of 133.8403 ng/ml. The SNEDDS showed the highest activity, whereas the hydroalcoholic extract showed higher activity than the pure hesperidin, which could be attributed to synergistic effect of other flavonoids in extract. Hesperidin molecular docking studies were carried out. The ability of hesperidin to interact with the key amino acids in MAO-B binding site rationalizes this pronouncing activity as proven by its docking pattern and docking score compared with that of the known MOA-B inhibitor, safinamide.