4.1.2. Free Radical Scavenging Activity Analysis

Most in vitro antioxidant activity evaluation uses 2,2-diphenyl-1-picrylhydrazyl (DPPH) to determine the ability of antioxidants for scavenging free radicals [41]. A total of 200 μM DPPH solution dissolved in methanol (80%, HPLC, Sigma-Aldrich, USA) was prepared, then mixed with EGCG solutions at various concentrations from 0.0625 to 1 mg/mL dissolved in H2O (Purelab, ELGA LabWater), respectively. The absorbance was measured at 517 nm using a UV/VIS spectrophotometer (Shimadzu cooperation, Japan). The DPPH scavenging activity of EGCG was calculated using Equations (1).

$$\text{DPPH savinging activity } \left(\%\right) = \left(1 - \frac{\text{absorbrane of DPPH related with cathode in exact}}{\text{absorbrane of DPPH}}\right) \times 100\% \tag{1}$$

#### *4.2. Liposome Preparation*

Solutions for the fabrication of liposome dosage form including 10 mg/mL of L-αphosphatidylcholine (PC), phosphatidylserine (PS), and cholesterol (CH) solutions dissolved in chloroform, 43.6 mM EGCG solution dissolved in 1 mL H2O, and 10 mg/mL α-tocopherol (vitamin E, VE) solution dissolved in ethanol were prepared and then stored in a 4 ◦C refrigerator.

PC, PS, CH, and VE solutions were mixed in the molar ratio listed in Table 1 to a total volume of 1 mL and 3 μL PKH-26 cell linker kit (Sigma-Aldrich) for cellular membrane labeling fluorescent dye was added and mixed homogeneously. The organic solvent was then removed by a rotary evaporator and a vacuum dryer to obtain a layer of translucent lipid film. In preparation for placebo PC- and PS-liposomes, 1 mL H2O was added, and for PC-EGCG-, PS-EGCG-, PC-EGCG-VE-, and PS-EGCG-VE-liposomes, 0.5 mL H2O and 0.5 mL EGCG solution was added to the lipid film. The solution was shaken by a vortex mixer at high speed for 10 min to obtain large unilamellar vesicles. A mini-extruder (Avanti Polar Lipid Inc., Alabama, USA) with two polycarbonate filter membranes with a pore size of 400 and 200 nm was used to squeeze the sample 11 times repeatedly. Thus, the liposome was forced to pass through the filter membrane and self-assembled to obtain liposomes with uniform particle size.
