Stephanoside B modulates metabolic gene expression and lengthens the circadian bmal1 oscillation period in differentiated myotubes revealed by real-time bioluminescence

BACKGROUND: Gymnema (G.) inodorum is a medicinal plant with anti-diabetic, anti-obesity, and anti-inflammatory properties traditionally consumed as tea in Southeast Asia. While bioactive stephanosides and gymnemic acids suppress adipocyte differentiation, their effects on skeletal muscle metabolism and circadian regulation remain unclear. This study investigated the effects of gymnemic acid (GiA-7) and stephanosides B and C in differentiated C2C12 myotubes. METHODS AND RESULTS: Differentiated C2C12 myotubes were treated with GiA-7 and stephanosides B and C, and gene expression was analyzed by quantitative reverse transcription polymerase chain reaction. Stephanoside B selectively and concentration-dependently increased the expression of Pparg and Ppargc1a under basal conditions and after dexamethasone-induced circadian synchronization. It also increased the expression of the core clock genes Nr1d1, Per2, and Cry1, while decreasing Bmal1, consistent with known PPARγ-circadian interactions. To directly monitor circadian rhythmicity, we established a real-time Bmal1-luciferase reporter gene assay in differentiated myotubes. Continuous bioluminescence tracking over several days revealed that stephanoside B specifically lengthened the circadian period, demonstrating an effect on molecular clock function. CONCLUSIONS: Stephanoside B is associated with coordinated changes in metabolic gene expression and circadian parameters in skeletal muscle cells. These findings suggest a potential role of G. inodorum-derived compounds in modulating muscle circadian regulation at the cellular level. This study also provides a practical real-time analytical platform for evaluating dietary compounds that influence skeletal muscle circadian dynamics.