# Cytoprotective Effect of Acid-Hydrolyzed Sesame Leaves against Oxidative Stress in HepG2 Cells
## 3 Results and Discussion
### Intracellular Antioxidative Activities of Acid-Hydrolyzed Sesame Leaves  
To evaluate the effect of acid hydrolysis in sesame leaves on redox effectors including ROS, MDA, and GSH, and antioxidant enzymes, including GPx, CAT, and GR, sample-treated HepG2 cells were collected after 24 h. As shown in Figure 2, TBHP increased intracellular ROS generation. All extract-treated samples showed markedly decreased ROS generation compared to that in the TBHP-alone group. ASLE treatment decreased ROS generation to almost basal levels. We then measured GSH and MDA levels to gauge the HepG2 cell response to oxidative stress. TBHP significantly decreased GSH levels compared to control cells (Figure 3A). However, all extract-treated samples showed increased GSH levels compared to those treated with TBHP alone. ASLE treatment most strongly preserved GSH levels in HepG2 cells facing oxidative stress. The MDA level was also significantly elevated in TBHP-treated cells relative to control cells (Figure 3B), and verbascoside, SLE, and ASLE significantly abated the TBHP-mediated elevation of MDA levels. Remarkably, treatment with SLE and ASLE reduced MDA levels to near or below control values. To evaluate the effect of sesame leaf acid hydrolys on antioxidant enzyme activity, GPx, CAT, and GR levels in HepG2 cells were evaluated. TBHP treatment decreased antioxidant enzyme activities, and verbascoside, SLE, and ASLE markedly restored GR and GPx activities in TBHP-treated cells (Figure 4A,B). CAT activity was increased only by ASLE (verbascoside and SLE did not increase this activity) (Figure 4C). Excessive intracellular ROS levels indicate an oxidative insult in living cells, and predict damage to lipids, proteins, and DNA [39]. Cellular MDA is a product of lipid peroxidation in cell membranes resulting from reactions between unsaturated fatty acids and radical species [43]. Oxidative stress-induced liver damage leads to reduced levels of glutathione (GSH), which plays a central role in defending cells against oxidative stress and detoxifying their environment [44]. Numerous dietary phytochemicals have been studied and have shown significant effects on antioxidant enzymes under oxidative stress. Figure 2: Mitigating effects of sesame leaf extracts on 0.5 mM _tert_-butyl-hydroperoxide-mediated intracellular generation of reactive oxygen species (ROS) by HepG2 cells. \({}^{\mathrm{a-e}}\) Means with different letters are significantly different through [PERSON]’s multiple range test with significance threshold at \(p<0.05\). B, blank; CON, control; TBHP, _tert_-butyl hydroperoxide; VS, verbascoside (50 \(\mathrm{\SIUnitSymbolMicro M}\)); SLE, sesame leaf extract (50 \(\mathrm{\SIUnitSymbolMicro m}\)/mL); ASLE, acid-treated sesame leaf extract (50 \(\mathrm{\SIUnitSymbolMicro m}\)/mL). Figure 4: Effects of sesame leaves on 0.5 mM _tert_-butyl-hydroperoxide-stimulated GR (**A**), GPx (**B**), and CAT (**C**) activities in HepG2 cells. \({}^{\text{a-c}}\) Means with different letters are significantly different through [PERSON]’s multiple range test with significance threshold at \(p\) < 0.05. GR, glutathione reductase; GPx, glutathione peroxidase; CAT, catalase; CON, control; TBHP, _tert_-butyl hydroperoxide; VS, verbascoside (50 \(\upmu\)M); SLE, sesame leaf extract (50 \(\upmu\)g/mL); ASLE, acid-treated sesame leaf extract (50 \(\upmu\)g/mL). Figure 3: Effects of sesame leaves on 0.5 mM _tert_-butyl-hydroperoxide-stimulated GSH (**A**) and MDA (**B**) levels in HepG2 cells. \({}^{\text{a-d}}\) Means with different letters are significantly different through [PERSON]’s multiple range test with significance threshold at \(p\) < 0.05. GSH, glutathione; MDA, malondialdehyde embryo; CON, control; TBHP, _tert_-butyl hydroperoxide; VS, verbascoside (50 \(\upmu\)M); SLE, sesame leaf extract (50 \(\upmu\)g/mL); ASLE, acid-treated sesame leaf extract (50 \(\upmu\)g/mL).