*3.4. Cell Viability Test*

We used osteoblastic precursor cell lines (MC3T3-E1 cells) derived from *Mus musculus* to induce the expression of osteoblast markers and investigate the effect of *G. littoralis* on bone metabolism. In this study, we attempted to determine the effect of *G. littoralis* extract on in vitro osteogenic induction using MC3T3-E1 cells. We performed a pilot study in which *G. littoralis* extract concentrations varied from 0.5 to 200 μg/mL to determine the optimal concentration. Cell viability studies were performed for different *G. littoralis* parts, including GLSE, GLFE, GLAE, and GLRE extracts. Initially, the purified *G. littoralis* ethanol extracts were re-suspended in 70% ethanol, and various plant extract concentrations were added to the MC3T3-E1 cell culture. The viability of cells varied considerably among the different plant parts used. The viable cell number increased in up to 5 μg/mL of *G. littoralis* treatment. Then, the higher concentrations showed a significant decrease in the cell population in a concentration-dependent manner (Figure 4), indicating *G. littoralis*'s cellular toxicity properties. Lower concentrations of the plant extracts (between 0 and 2 μg/mL) did not significantly affect the cell viability. Among the different extraction samples, GLSE at a concentration of 0.5–10 μg/mL showed 88.5 ± 1.0% to 96.6 ± 3.9% cellular viability, while these values varied in GLFE (90.7 ± 1.2% to 94.5 ± 0.9%), GLAE (85.8 ± 1.6% to 95.4 ± 1.2%), and GLRE (80.0 ± 0.7% to 97.7 ± 0.4%) at the same concentrations (Figure 4). In addition, the lactate dehydrogenase (LDH) cytotoxicity assay proved that plant extracts beyond 5 μg/mL were toxic to the cell lines (Figure 5). We used 5 μg/mL of *G. littoralis* extract in the subsequent experiments to avoid cytotoxicity and promote MC3T3-E1 cell growth.

#### *3.5. ALP Activity*

We evaluated ALP activity to assess how *G. littoralis* extracts affect osteogenic induction (Figure 6). All the sample extracts (GLSE, GLAE, GLRE, and GLFE) significantly increased ALP activity in MC3T3-E1 cells. Comparatively, higher ALP activities were observed at sample concentrations of 0.5 μg/mL. Increasing the treated sample concentration resulted in a decrease in ALP activity. Among the treated samples, GLSE at a concentration of 0.5–20 μg/mL showed higher ALP activity ranging from 158.4 ± 7.9% to 125.9 ± 11.5%, respectively, while the lowest ALP activity was observed in GLFE at a concentration of 0.5–20 μg/mL ranging from 127.4 ± 2.9% to 84.1 ± 3.7%, respectively. The results indicate

that *G. littoralis* extract enhanced the ALP activity required for osteoblast formation and ECM mineralization in MC3T3-E1 cells.

**Figure 4.** Cell viability of extracts from each part of *G. littoralis* in osteoblastic MC3T3-E1 cell line. Each value is the mean ± standard deviation of nine replicate tests. The mean values followed by the same letter are not significantly different based on the DMRT (*p* < 0.05). GLSE: *G. littoralis* leaf, stem extracts, GLFE: *G. littoralis* fruit extracts, GLAE: *G. littoralis* all extracts, GLRE: *G. littoralis* root extracts.

**Figure 5.** Cell cytotoxicity of extracts from each part of *G. littoralis* in osteoblastic MC3T3-E1 cell line. Each value is the mean ± standard deviation of nine replicate tests. Mean values followed by the same letter are not significantly different based on the DMRT (*p* < 0.05). GLSE: *G. littoralis* leaf, stem extracts, GLFE: *G. littoralis* fruit extracts, GLAE: *G. littoralis* all extracts, GLRE: *G. littoralis* root extracts.
