## 4 Discussion  
HepG2 cell is the most commonly utilized human hepatoma cell line in pharmacologicalized research [22]. HepG2 cells are highly proliferative and successfully cultured in large-scale systems. When grown on a solid surface, HepG2 cells exhibit an epithelial appearance, and their polarization can arise with the formation of bile canaliculi-like structures between adjacent cells [23; 24]. Anti-cancer drugs often suppress cancer cells by apoptotic induction [25; 26]. Apoptotic cells exposed several distinguishing characteristics such as cell shrinkage, the condensation of chromatin, nuclear DNA fragmentation, phosphatidylserine expression, and plasma membrane blebbing [27; 28]. In this study, HepG2 cells maintained epithelial appearance in the low SFA-CHCl3 extract-treated groups. However, morphological changes in HepG2 cells were observed in the high SFA-CHCl3 extract-treated groups (50 ug/mL and 100 ug/mL SFA-CHCl3 extract), concluding the shrinkage of cells, the formation of debris, and the visible loss of contact and rounding of cell shape that are classic morphologies of apoptosis [29]. Moreover, the SFA-CHCl3 extract treatment at IC\({}_{50}\) value also resulted in the morphological changes of nuclear in HepG2 cells at 24, 48, and 72 h. These alterations were demonstrated by the nuclear condensation and fragmented nuclear, which are also unique features during apoptosis [30]. Therefore, SFA-CHCl3 extract treatment induced the morphologic hallmarks of apoptosis, by a series of typical morphological events in the cytoplasm and nuclear in HepG2 cells. These findings suggest that SFA-CHCl3 extract treatment has strong cytotoxicity against HepG2 cells and can limit their proliferation. Cell proliferation is a fundamental process in multi-cellular organisms, and it has been reported to be linked to apoptosis [31]. Uncontrolled proliferation can be associated with a high level of apoptosis. This investigation showed that SFA-CHCl3 extract treatment markedly decreased the proliferation of HepG2 cells in a dose-dependent manner. This reduced proliferation was found to be associated with HepG2 cells from the high SFA-CHCl3 extract-treated groups, which strongly exposed the morphological features of apoptosis. Cell cycle dysregulation is known to be characteristic of tumor cells. Anti-cancer drugs induce apoptosis by stopping the cell cycle progression in the G1, S, or G2/M stages [27; 32; 33]. In this study, flow cytometry demonstrated that the percentage of the G0/G1 phase of HepG2 cells under SFA-CHCl3 extract treatment decreased with the increase in time of the treatment, while the percentage of the G2/M phase increased. This result disclosed that SFA-CHCl3 extract treatment significantly arrested the cell cycle of HepG2 cells at the G2/M phase, leading to the inhibited proliferation of HepG2 cells. Figure 5: The expression of box and bcl-2 gene. (**A**) The transcript expression of box and bcl-2 was performed by real-time RT-PCR, *** indicates significant difference between groups (_p_ < 0.001). (**B**) Western blot was applied to estimate the expression of box and bcl-2. * indicates significant difference between groups (_p_ < 0.05). Phosphatidylserine exposure on the plasma membrane's outer leaflet is a common alteration in apoptotic cells, and it has been considered a unique feature of these cells [34; 35]. This marker can be detected by double-staining with annexin V and PI; flow cytometry analysis revealed a dramatic increase in the apoptotic percentage of HepG2 cells, suggesting that SFA-CHCl3 extract could induce significant apoptosis in HepG2 cells. The Bcl2 family plays a key role in intrinsic mitochondrial apoptosis [36]. Bcl2 interacts with pro-apoptotic proteins such as Bax, limiting pore development and the release of cytochrome c [37]. On the other hand, an increase in Bax expression causes tumor cells to die, resulting in cell death [38]. The present investigation showed that bax up-regulation and bcl-2 down-regulation were determined in HepG2 cells under SFA-CHCl3 extract treatment.