Quassinoids from Malaysian <i>Eurycoma longifolia</i> significantly increased the expression of the melatonin biosynthesis-related enzyme gene (AANAT)

<i>Eurycoma longifolia</i> Jack, also known as Tongkat Ali, is a native plant in the family of Simaroubaceae that grows in Southeast Asian rain forests. <i>E</i>. <i>longifolia</i> has garnered significant attention due to its profound pharmacological properties (antimalarial, anti-pyretic, antiulcer, cytotoxic and aphrodisiac properties). However, the recent report that the intake of Tongkat Ali supplements improve sleep quality in healthy adults has aroused our interest. Three the most characteristic C₂₀-quassinoids (1-3) were isolated from the roots of <i>Eurycoma longifolia</i>, along with eight known secondary metabolites with other structural types (4-11). Their structures were identified by comprehensive analyses of NMR spectroscopy, and HRMS data. The sleep-promoting activity of three quassinoids based on the melatonin (sleep hormone) biosynthesis-related enzyme gene (AANAT) expression test was also evaluated, and the results revealed that quassinoids (1-3) significantly increased AANAT gene expression compared to the control. These results provide new ideas for the development and utilization of the plant for clinical applications of sleep disorders in the future.