Identification of New Cyclocircadins from Cyclocybe erebia and Their Effects on the Circadian Rhythm of Human Osteosarcoma Cells

Circadian rhythms are fundamental regulatory mechanisms for various biological and biochemical functions in diverse organisms. To identify new circadian rhythm modulators, secondary metabolites in the edible mushroom Cyclocybe erebia were examined. Six new diterpenoids, cyclocircadins C–H, were isolated from a mushroom culture. Their structures, including absolute configurations, were determined using HR-ESI-MS, NMR, and quantum chemical calculations. Cyclocircadins are prenylaromadendrane-type diterpenoids with unique fused ring systems consisting of 3-, 5-, and 7-membered rings. Cyclocircadins C–E possess a hydroxy or carbonyl group on the 7-membered ring, whereas cyclocircadins F–H lack these functional groups, and have the double bonds at a different position within the ring. The effects on the circadian rhythm were assessed in human osteosarcoma U2OS cells expressing Emerald Luciferase (ELuc) under the control of the Per2 promoter. Cyclocircadins A, C, D, and F–H delayed the phase of the circadian rhythm, and reduced the amplitude of bioluminescence oscillations. In addition, cyclocircadins A, C, F, and G tended to lengthen the circadian period, whereas cyclocircadin E tended to shorten it. Cyclocircadins A and F–H were more active at lower concentrations than cyclocircadins C–E, indicating that structural variations in the seven-membered ring are critical for their activity.