# Anti-Inflammatory and Antioxidant Effects Induced by
## 3 Results and Discussion
### Toxicological and Pharmacological Studies  
In a previous study of ours, GHE (1-100 ug/mL) was not able to modify cell viability of H9c2 cells (rat cardiomyopathyblasts), in basal conditions, confirming its good biocompatibility [22]. Moreover, the same extract (10-100 ug/mL) was effective in protecting cells from cytotoxicity induced by H\({}_{2}\)O\({}_{2}\) (200 uM) [22]. In the present study, we investigated the effects of GHE and GWE, in the dose range 1-100 ug/mL, on colon cancer SW480 cell line viability, in basal conditions. Compared to the control group, GHE was not able to affect SW480 cell viability (Figure 5). However, GWE (100 ug/mL) significantly suppressed SW480 cell viability, even if the cell viability was not under the biocompatibility limits (70% viability compared to control, respectively) (Figure 5). Figure 5: MTT assay of SW480 cell line treated with garlic hydroalcoholic extract (GHE) (1, 10, and 100 ug/mL), garlic water extract (GWE) (1, 10, and 100 ug/mL), and vehicle (RPMI) for 48 h, in basal conditions. Data are displayed as the means \(\pm\) SEM. ANOVA, \(p<0.001\); \({}^{**}\)\(p<0.01\) vs. vehicle. Accordingly, both GHE and GWE were not able to modify apoptosis of SW480 cell lines following 48 h of treatment in basal conditions (Figure 6). GHE and GWE, in the dose range 1-100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, were then tested to evaluate their potential protective activities on oxidative and inflammation pathways in mouse colon specimens treated with LPS. In particular, GHE, in the tested dose range, was effective in suppressing LPS-induced gene expression of pro-inflammatory markers strongly involved in colon inflammation, including COX-2, TNF-\(\alpha\), NF-\(\kappa\)B, and IL-6 (Figure 7A-E) [50; 51; 52; 53]. In the same experimental paradigm, GWE (1-100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) inhibited LPS-induced TNF-\(\alpha\), NF-\(\kappa\)B, and IL-6 (Figure 7A-E) gene expression. Moreover, the higher concentrations of GWE suppressed COX-2 (Figure 7A) gene expression induced by LPS treatment. On the other hand, GHE and GWE did not modify LPS-induced Nrf2 gene expression (Figure 7E), ruling out a possible role of this mediator in mediating the protective effects exerted by GWE and GHE in mouse colon. Accordingly, [PERSON] and collaborators (2002) [54] showed that, in peripheral blood monocytes, production of various leukocyte pro-inflammatory cytokines, such as IL-6, IL-8 and TNF-\(\alpha\), was reduced by a garlic extract (\(\geq\)10.0 \(\mathrm{\SIUnitSymbolMicro g}\)/mL). On the other hand, it also stimulated IL-10 synthesis in the same experimental model. In this context, the same authors suggested a possible therapeutic use of garlic in the management of inflammatory conditions, including inflammatory bowel disease [54]. Our present findings also agree with those by [PERSON] and collaborators (2013) [55], who observed inhibitory effects induced by fresh and heated raw garlic extracts (FRGE and HRGE) on LPS-induced release of pro-inflammatory cytokines, such as IL-1\(\beta\), TNF-\(\alpha\), and IL-6, in RAW 264.7 macrophages. Moreover, we investigated the effects of the extracts on LPS-induced levels of PGE\({}_{2}\), a pro-inflammatory mediator generated by COX-2, which is strongly involved in the pathophysiology of inflammatory bowel disease [56]. Our present results showed that both GHE (1-100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) and GWE (10-100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) suppressed LPS-induced PGE\({}_{2}\) levels in isolated colon specimens (Figure 8), further supporting the anti-inflammatory effects exerted by these extracts. Figure 6: Apoptosis assay in SW480 cell line treated with garlic hydroalcoholic extract (GHE) (1, 10, and 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL), garlic water extract (GWE) (1, 10, and 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL), and vehicle (RPMI) for 48 h, in basal conditions.