# Antibacterial and Antiproliferative Potential of Green, Roasted, and Spent Coffee Extracts
## 2 Results and Discussion
### Antibacterial Activity
Green bean coffee, GB; roasted coffee, RC; spent coffee grounds, SCG; paclitaxel as a positive control (120 nM) during 24 h. (**B**) Morphological changes caused by coffee ethanolic extracts evaluated at 200 \(\upmu\)g-mL\({}^{-1}\) in GB and RC; the last photograph presents the cells exposed to SCG at 12.5 \(\upmu\)g-mL\({}^{-1}\), a reduction in the number of cells compared with control and intracellular inclusions, such as apoptotic bodies, can be observed. (**C**) Percentage of viability of A549 cells treated with different concentrations of aqueous extracts, to evaluate the effect on cell proliferation. (**D**) Morphological changes caused by aqueous coffee extracts evaluated at 200 \(\upmu\)g-mL\({}^{-1}\). Calibration bar, 50 \(\upmu\)m. Dunnett: ***, \(p<0.0001\); ***, \(p<0.001\); ***, \(p<0.01\); and *, \(p<0.05\). To assess whether the extracts increased the number of apoptotic cells or caused cell-cycle arrest in C33A and A549, which could explain the reduction in cell viability, we performed a flow cytometry analysis. We only evaluated the concentrations and extracts in which we observed a notable effect on the reduction of cell viability; as such, the aqueous extracts were not evaluated. In C33A cells, we evaluated ethanolic extracts at 25, 75, 125, and 200 \(\upmu\)g\(\cdot\)mL\({}^{-1}\) concentrations, with a differential effect on the increase in the number of apoptotic cells between 13.5% and 15.7%. The highest percentage of apoptotic cells was observed with the GB ethanolic extract at 12.5 \(\upmu\)g\(\cdot\)mL\({}^{-1}\). In the analysis of cell-cycle changes, the ethanolic RC extract at 125 \(\upmu\)g\(\cdot\)mL\({}^{-1}\) decreased the number of cells in the G1/S phase by 12.7%, promoting cell-cycle arrest (see Figure 3A,B; Table 3). Figure 3: Effect of ethanolic coffee extracts on apoptosis and cell-arrest cycle in C33A and A549 cells. (**A**) Representative result of Annexin V assay to detect apoptosis, by the addition of 125 \(\upmu\)g\(\cdot\)mL\({}^{-1}\) of the SCG ethanolic extract, compared with control. (**B**) An example of the analysis of cell-cycle phases by flow cytometry; with the addition of 125 \(\upmu\)g\(\cdot\)mL\({}^{-1}\), the SCG ethanolic extract caused cell-cycle arrest. Different concentrations of ethanolic and aqueous extracts from GB and SCG were evaluated in A549 cells. (**C**) Representative result of Annexin V assay to detect apoptosis, with induction of apoptosis by the addition of 12.5 \(\upmu\)g\(\cdot\)mL\({}^{-1}\) of the GB ethanolic extract compared with control. (**D**) An example of the analysis of cell-cycle phases by flow cytometry, with the addition of 12.5 \(\upmu\)g\(\cdot\)mL\({}^{-1}\) of the ethanolic extract. In A549 cells, the ethanolic extracts from GB, RC, and SCG were evaluated at 12.5, 150, and 200 \(\mathrm{\SIUnitSymbolMicro mL^{-1}}\). The ethanolic extract from GB increased apoptotic cells by 8.7%. Furthermore, the aqueous extract from SCG exhibited the highest percentage of increase in apoptotic cells (10.3%). Regarding changes in the cell cycle, the ethanolic extracts from SCG and GB reduced G1/S-phase proliferative cells by 15.1% and 15.3%, respectively, in comparison with the control (see Figure 3C,D; Table 3). The antiproliferative and proapoptotic effects of the ethanolic and aqueous extracts of GB, RC, and SCG can be attributed to coffee metabolites, such as caffeine, caffeic acid, trigonelline, chlorogenic acids, and diterpenes (e.g., cafestol and kahweol), as well as some molecular mechanisms that have been described in several cell lines [43; 44; 45; 46; 47]. However, coffee roasting and other processes can change the profile of the bioactive compounds and, therefore, can affect its bioactivity or physiological effects [48; 49]. In this regard, few studies have been focused on extracts from SCG, and analysis of the compound profile in SCG has indicated a high content of melanoidins, chlorogenic acids, caffeic acid and, at a lesser concentration, caffeine [25; 50]. [PERSON] et al.