## 3 Results and Discussion
### Polyphenolic Extract of L. gravelens Has Potential to Induce Apoptosis in Breast Cancer Cells  
According to the profile of compounds with antiproliferative potential identified in the polyphenol extract of _L. gravelens_, as well as the information in the literature regarding the induction of apoptosis of these compounds, we decided to evaluate caspase-9 as the initiator molecule of the intrinsic pathway, the processing of caspase-3 (the effector molecule), and PARP cleavage, one of its main substrates. We detected the pro-caspase form of caspase-9 (47 kDa) and the active form of 37 kDa in the treatments with extract at 24, 48, and 72 h. The same processing of caspase-9 was observed in the reference drug cisplatin, which is an intrinsic apoptosis inducer. According to the activation of the initiator caspase-9 in response to the treatment with _L. gravelens_ extract, we decided to continue with the downstream evaluation of the intrinsic pathway, focusing on caspase-3, and we detected fully processed forms (19/17 kDa) in all three treatments, as well as in cisplatin. The pattern of caspase-3 processing suggests activation; so, to further characterize this pathway, we also evaluated PARP processing. As expected, we observed cleaved PARP (89 kDa) in all treatments except the control under basal conditions (Figure 4). Thus, the data obtained suggest that the polyphenol extract of Mexican oregano _L. gravelens_ promotes the intrinsic pathway of apoptosis in MDA-MB-231 breast cancer cells. The results obtained coincide with those reported by [PERSON] et al. [64] for the treatment with 100 \(\upmu\)g of apigenin for 72 h of BT-47 breast cancer cells, which inhibited cell proliferation up to 90%, inducing apoptosis by upregulation of cleaved caspase-3 and PARP cleavage. In combination with an extract (ethyl acetate and water) of _Morinda citrifolia_, apigenin was also tested on the viability of MDA-MB-231 breast cancer cells. Treatment at 10 \(\upmu\)M of the extract and 10 \(\upmu\)M of apigenin for 96 h reduced cell viability by around 80% through induction of apoptosis, a similar effect to that shown by our extract alone [48]. On the other hand, luteolin at a concentration of 35 \(\upmu\)M combined with lapatinib (a drug used in the treatment of advanced breast cancer) at a concentration of 450 nmol for 72 h inhibited the growth of BT474 cells around 90% by synergistically inducing apoptosis [65]. So, it could be considered a synergistic effect of the polyphenol extract of Mexican oregano _L. gravelens_ with other compounds to promote its antiproliferative effect. In another study, 10 \(\upmu\)M luteolin for 24 h reduced cell proliferation by about 40% in MDA-MB-453 and MCF-7 cells by inducing apoptosis through increased Bax expression, downregulation of Bcl-2, and the promotion of the cleavage of Caspase-3, which coincides with results of our work [47]. In the case of quercetin, it induced apoptosis, through activation of caspase-3 and the mitochondrial-dependent pathway (intrinsic), in MCF-7 and MDA-MB-231 human breast cancer cells, with IC\({}_{50}\) values of 80 and 50 \(\upmu\)m/mL, respectively [66]. Similarly, [PERSON] et al. [67] proved that 100 \(\upmu\)M quercetin for 72 h decreasesBT-474 cell proliferation by around 70%. This inhibition was associated with the activation of the extrinsic apoptotic pathway through the upregulation of cleaved caspase-8 and cleaved caspase-3, as well as the induction of PARP cleavage. On the other hand, the ethanolic extract of _Thymus vulgaris_, rich in naringenin, inhibited the growth of HTB26 breast cancer cells in a dose and time-dependent manner through apoptotic cell death by expression of Bax and caspases 3, 7, 8, and 9 [68]. Therefore, according to these reports, we can attribute the inhibition of antiproliferative activity in MDA-MB-231 breast cancer cells treated with polyphenolic extract of Mexican oregano _Lippia gravelens_, to the possible induction of apoptosis by the presence of these compounds.