*2.12. Mineralization Content*

Calcium content was measured in osteoblasts using the method described by Zakłos-Szyda et al. [41]. Osteoblast MC3T3-E1 cells were aliquoted in a 24-well plate at 2 × 10<sup>4</sup> cells/well in a CO2 incubator at 37 ◦C. The cells were treated with plant samples at a concentration of 0–20 μg/mL and placed in an incubator for seven and 14 days to induce mineral deposition. The culture medium was changed at three-day intervals. For harvesting, the MC3T3-E1 cells were washed with PBS twice and fixed with 4% paraformaldehyde for 2 h. The cells were then stained with 40 mM Alizarin Red S (pH 4.5). The stained cells were then rinsed four times with distilled water and observed under an inverted microscope. Calcified nodules appearing bright red were confirmed, and 100 mM cetylpyridinium chloride (Sigma) solution was added to each well to elute the stain. The eluted stain (100 μL) was added to a 96-well microplate. The absorbance of solubilized calcium-bound Alizarin Red S was taken at 570 nm using a spectrophotometer (Eppendorf AG 22331; Hamburg, Germany). Calcium deposition was expressed as the molar equivalent of calcium.
