*2.5. SpD Functioned as an Antioxidant in AC16 Cells*

To test the antioxidant ability of SpD, oxidative stress was induced in AC16 cells using H2O2 or cobalt chloride with high glucose for 24 h. The DCF-DA fluorescence showed that SpD treatment reduced ROS in AC16 cells (Figure 7).

**Figure 7.** SpD showed antioxidant activity in AC16 cells treated with H2O2 or cobalt chloride and hyperglycemic stress. (**A**) Reactive oxygen species generation was induced in AC16 cells using 1 mM H2O2 and SpD (0–10 μM) was co-treated for 24 h. \* *p* < 0.05 compared with 1mM H2O2 group without SpD; (**B**) Cobalt chloride (a hypoxia-mimetic agent) and hyperglycemia (33.3 mM glucose in media) were applied to AC16 cells. The cells were incubated with ,7 -dichlorofluorescein diacetate (DCF-DA) (20 μM) for 20 min at 37 ◦C and the intensity of fluorescence was measured at 485 nm. \* *p* < 0.05 compared with no SpD treated group.
