Bioactivity-guided isolation of potent anxiolytic compounds from leaves of Citrus paradisi

CONTEXT: Citrus fragrances have been attributed with mood-enhancing properties by aromatherapists. Leaves of this plant have been reported to exert anti-anxiety activity. Till date, no specific phytoconstituent responsible for this has been identified. OBJECTIVE: Isolation of anxiolytic constituent of Citrus paradisi using bioactivity-guided fractionation. MATERIALS AND METHODS: Leaf extracts of four varieties of C. paradisi in petroleum ether, chloroform, methanol and water were evaluated for anti-anxiety activity in mice using elevated plus-maze apparatus. Because of activity in methanol extract, it was used for safety evaluation/acute toxicity studies in animals. Bioactive fraction of methanol extract was subjected to column chromatography and structure of the isolated compound was elucidated by melting point, ultraviolet, infrared, nuclear mass reactor and mass spectroscopy. The isolated constituents were further evaluated for anti-anxiety activity using light/dark model and hole-board model of anxiety. RESULTS: Results showed no mortality at a dose up to 2000 mg/kg body weight that indirectly reflects the safety profile of the leaf extracts. Fractionation of methanol extract led to the isolation of four flavonoids (rutin, quercetin, kaempferol and myricetin). The isolated compounds exhibited significant anxiolytic activity in different animal models. CONCLUSION: The study confirms the presence of four flavonoids responsible for anti-anxiety activity.