Background: Cissus quadrangularis (family Vitaceae), traditionally known for its osteogenic potential, contains bioactive phytoconstituents that contribute to bone health. However, the seasonal dynamics of these metabolites remain poorly understood.

Methods: Over a three-year period, we investigated seasonal variations in osteogenic metabolites of C. quadrangularis through systematic extraction and fractionation. Pulverized dried plant material was sequentially extracted with ethanol, followed by liquid–liquid partitioning into hexane, chloroform, methanol, water, and finally n-butanol fractions. The n-butanol fraction, known to be enriched in osteogenic phytoconstituents, was prioritized for metabolomic profiling. Extracts were obtained under uniform experimental conditions, with replicate sampling, and analyzed using TLC and comparative metabolite quantification.

Results: Seasonal variation significantly influenced metabolite abundance. Maximum concentrations were observed in the cool and dry months (January–March, November–December), with February showing the highest metabolite yield. A gradual decline was recorded from March to July, followed by a slight increase in August, and subsequent reduction until October. Both ethanolic and n-butanol fractions exhibited comparable seasonal trends, indicating consistent partitioning of osteogenic metabolites. The ratio of ethanol-to-n-butanol extractability further supported differential biosynthesis of metabolites across climatic conditions.

Conclusion: Our findings demonstrate that the osteogenic phytoconstituents of C. quadrangularis exhibit clear seasonal fluctuations, with optimal accumulation during cooler, drier months. This highlights the importance of harvest timing for maximizing bioactive yield and provides new insights into metabolomic variability of osteogenic phytoconstituents in medicinal plants.