*2.9. Cytotoxicity*

The cytotoxicity of different plant parts was evaluated according to a previously described protocol [37,38]. Osteoblast MC3T3-E1 cells were dispensed in a 96-well plate at a density of 2 × 103 cells/mL, then pre-incubated in a CO2 incubator for 24 h. After incubation, the cells were treated with various concentrations of samples (ranging from 0 to200 μg/mL) and incubated for 48 h. The cell culture solution (10 μL) was added to a 96-well plate containing 40 μL PBS. After placing the LDH reagent in each well, the mixture was placed in a dark place for 45 min at 25 ◦C. After the reaction was terminated by mixing 50 μL of stop solution, the cytotoxicity was evaluated by quantifying the plasma membrane damage. LDH is a stable enzyme present in all cell types and is rapidly released into the culture medium when the plasma membrane is damaged. Cell membrane integrity was evaluated by measuring the LDH leakage levels from the cells using the LDH Cytotoxicity Assay Kit (BioVision, Inc., Milpitas, CA, USA). The absorbance value of the samples was measured using a microplate reader (Thermo Fisher Scientific Instrument Co., Ltd., Shanghai, China) at 492 nm.

#### *2.10. ALP Activity*

MC3T3-E1 cells were seeded as described above, and the ALP activity of the media was measured by following the method described by Liu et al. [39]. Initially, MC3T3-E1 cells were dispensed in a 48-well plate at 5 × 104 cells/well and pre-incubated in a CO2 incubator for 24 h. Then, the MC3T3-E1 cells were treated with different sample concentrations (0–20 μg/mL). Subsequently, the culture medium was replaced and supplemented with 100 μL chemiluminescent substrate for alkaline phosphatase (CSPD; Roche, Basel, Switzerland), added to 20 μL total cell lysate, and reacted for 30 min. The amount of protein in the total cell lysate was measured using CSPD (Life Technologies, Carlsbad, CA, USA) according to the Protein Assay Kit (Bio-Rad, Hercules, CA, USA), and the ALP activity value was expressed as the fold change per μg of total protein.
