Bioactive compounds from the seeds of Amomum tsaoko Crevost et Lemaire, a Chinese spice as inhibitors of sphingosine kinases, SPHK1/2

and found that its EtOH extract inhibited sphingosine kinases 1 and 2 (SPHK1/2). Bioactivity-based analysis and chemical investigation of the EtOH extract led to the isolation and identification of four aliphatic alcohols (1-4), five fatty acids (5-9), 12 phenolics (10-21), and four terpenoids (22-25), including four new compounds, an acetylated aliphatic alcohol (2), a fatty acid (5), and two phenolics (10-11). In addition, compound 1 was isolated for the first time from natural sources in this study. The structures of all compounds were elucidated based on spectroscopic analysis, including 1D and/or 2D NMR and HR-ESIMS as well as LC/MS analysis. A recently developed method using competing enantioselective acylation (CEA) coupled with LC/MS analysis was applied for the assignment of absolute configuration of compound 5. The absolute configurations of compounds 10 and 11 were determined using ECD calculations. All of the compounds (1-25) isolated from the active fraction were evaluated for their SPHK1/2 inhibitory effects at the concentration of 10 μM. Aliphatic alcohols 2-4, fatty acids 7 and 9, and phenolic compounds 13-15 and 21 showed inhibition against the activity of SPHK1 up to 20% and aliphatic alcohols 2 and 4, fatty acid 8, and phenolic compounds 10, 11, 18, and 22 showed inhibition against the activity of SPHK2 up to 40% compared with the control. Compound 2 showed the highest potency to inhibit SPHK1 enzymatic activity, by 59.75%, and compound 22 showed the highest potency in inhibiting the activity of SPHK2, by 22.75%, in comparison with the control, where both exhibited higher inhibition compared to those of positive controls. Docking modeling studies were conducted to suggest the binding mode of 2 and 22 in the substrate-binding pocket of SPHK1 and SPHK2, respectively.