Stephanoside B Modulates PPARγ-Dependent Metabolic Genes and Lengthens the Circadian Bmal1 Oscillation Period in Differentiated Myotubes Revealed by Real-Time Bioluminescence

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. Here, we investigated the actions of gymnemic acid (GiA-7) and stephanosides B and C in differentiated C2C12 myotubes. Stephanoside B selectively and concentration-dependently upregulated Pparg and Ppargc1a expression under basal conditions and after dexamethasone-induced circadian synchronization. It also enhanced expression of the core clock genes Nr1d1 , Per2 , and Cry1 , while repressing 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 bio-luminescence tracking over several days revealed that stephanoside B specifically lengthened the circadian period, demonstrating modulation of molecular clock function. Collectively, these results indicate that stephanoside B coordinately regulates metabolic gene expression and circadian rhythms in the skeletal muscle. Given the central role of muscle in systemic energy homeostasis and circadian regulation, these findings highlight the chrono-nutritional potential of G. inodorum extracts. Moreover, this study provides one of the first demonstrations of real-time circadian reporter analysis in differentiated myotubes, offering a novel platform to explore dietary compounds that influence muscle-specific metabolic and circadian processes.