## 4 Discussion  
_Datura innovia_ is a valuable medicinal plant rich in different phytochemicals with a broad medicinal property [25]. In this study, _D. innovia_ was extracted by hydroalcoholic solvent in order to obtain the maximum amount and diversity of biologically active phytochemicals. The results of the present study presented that the _D. innovia_ ethanolic extract exerted a cytotoxic effect on various cancer cells, with high potent activity against LoVo human colon cancer cells. We have also demonstrated that _D. innovia_ ethanolic extract induces a cell cycle arrest at the sub-G1 phase and more precisely an apoptotic cell death. This finding is supported by several pieces of evidence including Annexin V-FITC/PI labeling as well as the activation of the apoptosis signaling molecules. This finding is in line with a prior study that reported a strong antiproliferative effect of _D. innovia_ leaf methanolic extracts against colon (HCT-15) and liver HepG-2 cancer cells [26]. A potent cytotoxic effect against the THP-1 (human leukemia) cell line with a close IC\({}_{50}\) value (4.52 \(\upmu\)g/mL) was also documented for the _D. innovia_ grown in Pakistan [27]. In the same manner, the cytotoxic effect against the MCF-7 cell line was also reported [18] for _D. innovia_ grown in Iran. This variation in IC\({}_{50}\) values may result from different _D. innovia_ geographical sources, growing conditions and various cell types used [28]. National Cancer Institute (NCI, USA) criteria considered a plant crude extract with IC\({}_{50}\) value less than 20 \(\upmu\)g/mL a suitable extract for in vitro cytotoxic activity [29]. Based on this, _D. innovia_ displayed potent cytotoxic activity against LoVo with IC\({}_{50}\) 10 \(\upmu\)g/mL; therefore, LoVo cells were selected to assert the inhibition mechanisms exerted by this extract. The cell cycle distribution of LoVo cells treated with _D. innovia_ was analyzed to explore the growth inhibition mechanism. It was observed that cells treatments with _D. innovia_ accumulated at the sub-G1 cells phase, and this result proposed that LoVo cells undergo apoptosis since the accumulation of the cells in this stage is considered as a biomarker for DNA damage as well as an apoptotic cell death marker [30]. Confirmation of apoptosis induction was then verified through Annexin V-FITC/PI apoptosis detection assay, which is extensively used to discriminate living cells from both early and late apoptosis [31]. It was noted that _D. innovia_ resulted in a strong shift to early and late apoptotic cell populations and no necrotic cells were detected, which emphasizes that _D. innovia_ extract exerted apoptosis cell death mode. The indications of the dual staining of Annexin-V/PI and the accumulation of cells in the sub-G1 phase, as well as nuclear derangement which was observed with Hochest staining after treatment with _D. innovia_ ethanolic extract, clearly indicates that the cells are going in apoptosis. In fact, the induction of the apoptotic signaling pathways to trigger cancer cell death is the main mechanism for most anticancer drugs [32]. Apoptosis is a highly gene regulated process and various gene families, such as the P53 and the Bcl-2 family, are implicated in apoptotic cell death [33]. To support the flow cytometry results, the expression of the genes involved in the apoptosis pathway was analyzed using the qRT-PCR technique. Our results clearly proved that the extract treatments led to a decrease in the antiapoptotic Bcl-2 gene and an increase in the proapoptotic Bax gene, as well as activating caspase genes in the treated groups compared to the control, suggesting that _D. innovia_ acts through a caspase-dependent apoptosis pathway. A similar event was observed in a recent study which reported that _D. innovia_ extract induced apoptosis through increasing the expression of P53, BAX/BCL2 ratio, caspase 9, 3, 6, 7 on human chronic myeloid leukemia cells (K562 cells) [34]. Altogether this study and our study support the apoptosis-inducing effect of _D.