Background
Osteoporosis, a prevalent bone disorder, results in reduced bone mineral density and mass. With minimal side effects, medicinal plant-based natural remedies are increasingly explored for osteoporosis. However, the osteogenic potential of Tagetes erecta L. flower, traditionally used for cardiovascular and renal diseases, has not yet been studies.

Objective
This study investigates the osteogenic effects of the polyphenol-enriched extract from T. erecta L. flowers (TE) and its main components on osteoblast differentiation, with an emphasis on anti-osteoporotic activity.

Methods
The osteogenic activity of TE was assessed in MC3T3-E1 preosteoblast cells, analyzing osteogenic alkaline phosphatase (ALP) activity via a colorimetric assay and mineralization through Alizarin Red S staining over 14 d. Expression levels of osteogenic markers–transcription factor osterix (SP7), runt-related transcription factor 2 (RUNX2), and ALP–were quantified through quantitative reverse transcription-polymerase chain reaction and western blotting. In vivo effects were evaluated using zebrafish larvae for bone formation and anti-osteoporotic properties. Vertebral development was visualized by staining mineralized structures with calcein or Alizarin Red S. Prednisolone (PDS) was administered to zebrafish larvae to model osteoporosis. Furthermore, molecular docking simulations were conducted to assess the binding affinity of TE components to the ATP-binding pocket of glycogen synthase kinase-3β (GSK-3β), and their inhibitory potential on GSK-3β kinase activity were quantified by in vitro kinase assays. Cellular thermal shift assay (CETSA) was performed to monitor direct bindings of TE and its main components to GSK3-3β.

Results
TE promoted vertebral and cranial bone formation in zebrafish larvae, elevating key osteogenic genes, such as sp7, runx2a, runx2b, and alpl. Among TE components, kaempferol and patuletin significantly enhanced vertebral formation, while isorhamnetin showed moderate effects. Patulitrin and quercetagetin did not increased vertebral formation. In MC3T3-E1 cells, TE increased ALP activity, mineralization, and the expression of SP7, RUNX2, and ALP. It also induced GSK-3β phosphorylation at serine 9 and promoted β-catenin nuclear translocation. Inhibition of β-catenin signaling reversed TE-induced osteogenic effects. Molecular docking suggested strong GSK-3β binding by TE components, with patuletin showing notable inhibition GSK-3β activity (half-maximal inhibitory concentration = 379.3 ng/mL) and enhancing vertebral formation. CETSA confirmed that TE and its main components, kaempferol and patuletin, degrades GSK-3β. Additionally, TE alleviated PDS-induced osteoporosis in both cellular and zebrafish models.

Conclusion
: By targeting GSK-3β and activating β-catenin-mediated pathways, TE shows promise as a novel anti-osteoporotic agent. This study highlights the potential of TE for therapeutic use in bone health, warranting further clinical trials to confirm its applicability.