| Summary: | Vitamin D deficiency is endemic worldwide. Although several strategies have been established to enhance vitamin D<sub>3</sub> levels, studies specifically focusing on the inhibition of vitamin D metabolism, which may prolong the availability of active vitamin D in pathological conditions, have been less explored. Studies also suggest that higher doses of vitamin D<sub>3</sub> fail to achieve optimum vitamin D levels. In this context, we focused on the enzyme CYP3A4, which promotes the inactivation of active vitamin D. The current study aimed to decipher the impact of chrysin, a proven CYP3A4 inhibitor, as an intervention and its effects in combination with low-dose vitamin D<sub>3</sub> (40 IU) and bone health in vitamin D deficiency conditions. The in vivo activity of chrysin was evaluated in female Wistar albino rats fed a vitamin-D-deficient diet to attain vitamin D deficiency for 28 days. Chrysin was given alone and in combination with calcium carbonate (CaCO<sub>3</sub>) and/or vitamin D<sub>3</sub>. All therapeutic interventions were assessed for serum 25-hydroxyvitamin D3(25-OH-D<sub>3</sub>) by LC-MS and biochemical, urinary, and bone parameters. Animals treated with chrysin alone and in combination with low-dose vitamin D<sub>3</sub> and/or CaCO<sub>3</sub> showed an eminent rise in serum 25-OH-D<sub>3</sub> levels along with increased serum biochemical parameters. In contrast, a significant decrease in the urinary parameters followed by beneficial effects on bone parameters was noticed in contrast with the vitamin-D-deficient diet group. Our findings revealed that although chrysin alone showed a notable effect on 25-OH-D<sub>3</sub> and osseous tissue, comparatively, it showed an intensified therapeutic effect in combination with vitamin D<sub>3</sub> and CaCO<sub>3</sub><sub>,</sub> which can be employed as a cost-effective option to improve bone health.
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