# Antibacterial and Antiproliferative Potential of Green, Roasted, and Spent Coffee Extracts
## 2 Results and Discussion
### Antibacterial Activity
These results coincided with the effect determined in the cell viability assays where, at concentrations with the highest antiproliferative activity, the number of cells per field was reduced, with a greater effect on C33A cells. Notably, morphological changes in cells were correlated with extracts having the highest phenolic content. In A549 cells, the ethanolic extract from SCG diminished up to 35.5% cell proliferation at 12.5 \(\upmu\)g\(\cdot\)mL\({}^{-1}\), while the aqueous extract from SCG and RC only reduced cell viability at the highest concentration evaluated (200 \(\upmu\)g\(\cdot\)mL\({}^{-1}\)); see Figure 2A-C. In C33A cells, aqueous GB or ethanolic SCG extracts seemed to increase cell viability; however, this does not necessarily indicate an increase in cell proliferation. Similar results have been described, as some metabolites contained in GB or SCG coffee extracts can increase enzymatic-mitochondrial activity, specifically a phytochemicals such as kahweol and cafestol, and CGAs can increase and potentiate mitochondrial-reducing enzymes and increased formazan products, which can result in higher percentages of cell viability [39]. Notably, the ethanolic extract from SCG diminished A549 cell viability only at 12.5 \(\upmu\)g\(\cdot\)mL\({}^{-1}\), the lowest concentration evaluated. These results can be explained by the biphasic dose-response or hormesis, with a stimulatory effect at a low concentration, while high concentrations have the opposite effect. The hormesis effect has been widely documented in biological and pharmacological research [40; 41]. It should be noted that we did not evaluate the effects of the coffee extracts on non-transformed or non-cancerous cells to determine their cytotoxicity. However, there are several comparative studies that have shown that the phytochemicals contained in coffee have a high specificity to decrease the cell viability of cancer cells without affecting non-transformed cells. It has been determined that, in fibroblast cell lines (CCD-18 Co), hepatocytes (AML-12) and macrophages (RAW 264.7), the metabolites present in various coffee extracts do not have a cytotoxic effect; these findings suggest their selectivity to inhibit the proliferation and viability of cancer cells [9; 42]. Figure 1: In vitro antiproliferative activity and morphological changes caused by ethanolic (**A**,**B**) and aqueous (**C**,**D**) extracts of GB, RC, and SCG in C33A cells. (**A**) Percentage of viability of C33A cells, treated with different concentrations of ethanolic extracts, to evaluate the effect on cell proliferation. Green bean, GB; roasted coffee, RC; spent coffee grounds, SCG; and paclitaxel as positive control (120 nM) during 24 h. (**B**) Morphological changes caused by coffee ethanolic extracts evaluated at 150 \(\upmu\)g\(\cdot\)mL\({}^{-1}\). Reductions in the number of cells compared with control and intracellular inclusions, such as apoptotic bodies, can be observed. (**C**) Percentage of viability of C33A cells treated with different concentrations of aqueous extracts, to evaluate the effect of the extracts on cell proliferation. (**D**) Morphological changes caused by aqueous coffee extracts evaluated at 150 \(\upmu\)g\(\cdot\)mL\({}^{-1}\). Calibration bar, 50 \(\upmu\)m. Dunnett: *******, \(p\) < 0.0001; ***, \(p\) < 0.001; ***, \(p\) < 0.01; and *, \(p\) < 0.05. Figure 2: In vitro antiproliferative activity and morphological changes caused by ethanolic (**A**,**B**) and aqueous (**C**,**D**) extracts from GB, RC, and SCG in A549 cells. (**A**) Percentage of the viability of A549 cells, treated with different concentrations of ethanolic extracts, to evaluate the effect on cell proliferation.