# Protective Effects Induced by a Hydroalcoholic _Allium sativum_ Extract in Isolated Mouse Heart
## 3 Results and Discussion
### Toxicological and Pharmacological Studies  
The garlic hydroalcoholic extract (1, 10, 50, and 100 \(\upmu\)g/mL) was tested in vitro to evaluate its effects on the viability of H9c2 cells. No significant effect on H9c2 cell proliferation was observed after the addition of the garlic hydroalcoholic extract to the cell medium at various concentrations (1, 10, 50, and 100 \(\upmu\)g/mL) for 24 h and 48 h (Figure 5A,B). Moreover, when the H9c2 cells were treated with H2O2, their viability was significantly decreased, but the garlic hydroalcoholic extract (10, 50, and 100 \(\upmu\)g/mL) was found to be effective in reverting the cytotoxicity at both the experimental times (Figure 6A,B). On the basis of these results, a second set of experiments was conducted, with the aim to investigate the protective effects induced by the garlic hydroalcoholic extract (1, 10 and 100 \(\upmu\)g/mL) on oxidative stress and inflammatory pathways in mouse heart specimens challenged with LPS. Isolated tissues ex vivo, treated with LPS, have been described as a validated experimental model to determine the modulatory activities of plant-derived extracts and drugs on inflammation and oxidative stress [23; 24; 42]. Inflammatory processes and oxidative stress are known to play a pathogenic role in a number of chronic diseases, such as cardiovascular diseases, atherosclerosis, and diabetes [43; 44]. Cardioprotective and anti-atherosclerotic effects, including an improvement in the blood lipid profile, inhibition of cholesterol biosynthesis, regulation of blood pressure, and inhibition of platelet aggregation, have been reported for garlic-based preparations [45; 46]. In particular, the effects of garlic hydroalcoholic extract (1, 10 and 100 \(\upmu\)g/mL) were measured against the increased levels of pro-inflammatory and pro-oxidant mediators, such as PGE2 and 8-iso-PGF2\({}_{\alpha}\). The pro-oxidant mediator 8-iso-PGF2\({}_{\alpha}\) is an isomer of prostaglandan  
Figure 5: MTT assay of H9c2 cells exposed to garlic hydroalcoholic extract (GHE) (1, 10, 50, and 100 \(\upmu\)g/mL) for 24 h (**A**) and 48 h (**B**), in basal conditions. Data are reported as means \(\pm\) SEM. oxygen radical-catalyzed peroxidation of membrane arachidonic acid, which plays a critical role as a stable marker of lipid peroxidation and oxidative stress [47]. We observed that garlic hydroalcoholic extract (1, 10 and 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) decreased LPS-induced 8-iso-\(\mathrm{PGF_{2\alpha}}\) in a dose-independent manner, on mouse heart specimens (Figure 7). Cardioprotective effects, induced by the garlic, were consistently demonstrated on primary cultured cardiac myocytes, fibroblasts and endothelial cells, through the reduction in reactive oxygen species-dependent signaling pathways [48]. In agreement, a garlic hydroalcoholic extract was found to be effective in decreasing the serum oxidative stress  
Figure 6: MTT assay of H9c2 cells exposed to garlic hydroalcoholic extract (GHE) (1, 10, 50, and 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) for 24 h (**A**) and 48 h (**B**), and challenged with 200 \(\mathrm{\SIUnitSymbolMicro m}\) H\({}_{2}\)O\({}_{2}\). Data are reported as means \(\pm\) SEM. ANOVA, \(p\) < 0.01; \(\ast\ast\)\(p\) < 0.01, \(\ast\ast\)\(p\) < 0.001 vs. H\({}_{2}\)O\({}_{2}\) group. Figure 7: Effects of garlic hydroalcoholic extract (GHE) (1, 10, and 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) on 8-iso-\(\mathrm{PGF_{2\alpha}}\) levels in mouse heart specimens. Data are reported as means \(\pm\) SEM. ANOVA, \(p\) < 0.0001; \(\ast\ast\)\(p\) < 0.001 vs. LPS group. index and total oxidative status, nitric oxide and malondialdehydedehyde production, in a rat experimental model of acute inflammation [49]. In addition, garlic extracts exhibited significant antioxidant activity and protective effects against oxidative DNA damage [50], independently of the processing method.