*2.3. Experiment Design*

Cytotoxicity of H2O2 (ranges from 0.2 to 2.0 mM) on Caco-2 cells was tested by a viability assay, and the optimal concentration of H2O2 was chosen for inducing oxidative damage of Caco-2 cells. Cells were treated with 1 mM of H2O2 for 3 h firstly, and then washed with PBS (a pre-warmed at 37 ◦C water bath) one time. Afterward, the cells treated with 100 μg/mL RDE for 24 h were considered the treatment group (T) according to the results. The cells treated with H2O2 and followed with a standard medium [DMEM-Ham's F-12 (1:1) supplemented with 10% FBS and 1% anti-anti] for 24 h were defined as treatment control (TC). The cells without H2O2 and RDE treatment were negative control (NC). In brief, the H2O2 work solution was directly diluted using a medium from 10 M H2O2. The RDE solution was prepared according to the required concentrations using a medium.

#### *2.4. Cell Viability Assay*

After treatments, cell viability was measured using the water-soluble tetrazolium salts (WST-1) Cell Proliferation Reagent (Sigma Aldrich, Roche, Indianapolis, IN, USA) according to the manufacturer's instructions. Briefly, Caco-2 cells were seeded into 96-well plates at a density of 2 × 10<sup>4</sup> cells/well and cultured in a complete medium for about 5 days (90% confluent). After the different treatments (NC, TC, and T), cells were washed one time with PBS and then treated with 100 μL fresh medium containing 10% WTS-1 and incubated for 1.5 h at 37 ◦C in an atmosphere of 5% CO2. The absorbance at 450 nm was measured using a Synergy™ H4 Hybrid Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA). Cell viability was presented as a percentage of control cells.
