*2.6. Antioxidant Activity Assesments*

To evaluate the antioxidant properties of EPSRT7, several tests were conducted using 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH•), hydroxyl radical (•OH), and superoxide anion (O2 −•) as indicators. Ascorbic acid (Vc) was used as the positive control. EPSRT7 was prepared in concentrations ranging from 0.1 to 10 mg/mL. Absorbance measurements were performed using a FLUOstar Omega BMG LABTECH (Aylesbury, UK) spectrophotometer for DPPH (OD525 nm), OH (OD510 nm), and O2 −(OD325 nm).

#### 2.6.1. DPPH Radical Scavenging Activity

Research on the DPPH scavenging activity of EPSRT7 followed the procedure described by Niknezhad et al. [43]. First, 50 μL of EPSRT7 at different concentrations was mixed with 100 μL of DPPH (100 μM DPPH–ethanolic solution). The mixtures were stirred and left to incubate in the dark at 25 ◦C. The absorbance was measured after 30 min.

Formula (3) was used to determine the percentage of radical-scavenging activity for DPPH.

$$\text{DPPH savinging activity [\%]} = \left[ 1 - (\text{A}\_1 - \text{A}\_2)/\text{A}\_0 \right] \times 100\tag{3}$$

where A1 represents the reaction mixture, A2 refers to the reaction mixture without DPPH, and A0 denotes the reaction mixture with DPPH and without EPSRT7.

#### 2.6.2. OH Radical Scavenging Activity

The FeSO4–salicylic acid method described by Sun et al. [44] was used to determine the hydroxyl radical scavenging activity of EPSRT7. The mixtures contained a FeSO4 solution (9 mM, 40 μL), 40 μL of a 9 mM ethanol–salicylic acid solution, EPSRT7 diluted at various concentrations (40 μL), and H2O2 (8.8 mM, 40 μL). The mixtures had then been incubated at 37 ◦C for 30 min before absorbance was measured. Formula (4) was used to determine the percentage of hydroxyl radical scavenging activity.

$$\text{Hydroxyl radical scanning activity} \left[ \% \right] = \left[ 1 - (\text{A}\_1 - \text{A}\_2) / \text{A}\_0 \right] \times 100 \tag{4}$$

where A1 represents the reaction mixture, A2 refers to the reaction mixture without salicylic acid, and A0 denotes the reaction mixture with salicylic acid and without EPSRT7.

#### 2.6.3. O2 − Scavenging Activity

The superoxide scavenging activity of EPSRT7 was assessed as per Balakrishnan et al. [45]. In this method, 0.3 mL of different EPSRT7 concentrations was mixed with 2.6 mL of phosphate buffer (50 mM, pH 8.2) and 90 μL of pyrogallol (3 mM), and dissolved in HCl (10 mM). The absorbance was then monitored at 0 and 10 min. Formula (5) was used to determine the percentage of superoxide scavenging activity:

$$\text{Superoxide scavenging activity } [\%] = 1 - [(\text{A}\_{10}/\text{C}\_{10}) - (\text{A}\_{0}/\text{C}\_{0})] \times 100 \tag{5}$$

where A0 and A10 represent the reaction mixture at 0 and 10 min, respectively; C0 and C10 represent the reaction mixture without pyrogallol at 0 and 10 min, respectively.

#### *2.7. Exopolysaccharide Toxicity Evaluation*

#### 2.7.1. Cell Culture

HeLa cells, which are human epithelial cells derived from cervical carcinoma, were obtained from CLS (Cell Line 76 Service, BW, Eppelheim, Germany) and chosen as the reference cell line to assess the toxicity of EPSRT7. The cells were cultured using Dulbecco's Modified Eagle's Medium (DMEM) and added to 10% fetal bovine serum (FBS), 2 mM L-glutamine, penicillin (100 IU/mL), and streptomycin (100 μg/mL) under a 5% CO2 atmosphere at 37 ◦C [46].

#### 2.7.2. Cytotoxicity Assay

HeLa cells were cultured in a 24-well culture plate at a density of 5 × <sup>10</sup><sup>5</sup> cells per well. Each well was treated with 100 μL of EPSRT7 at various concentrations (0–400 μg/mL) for 24 h. The toxicity of the exopolysaccharide was determined by measuring the reduction in the MTT reagent (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazoliumbromide) to formazan [46,47]. The optical density at a wavelength of 590 nm was recorded using a microplate reader.

The cytotoxicity of EPSRT7 on HeLa cells was assessed using Formula (6):

$$\text{Cell viability} \left[ \% \right] = \left( \text{A}\_1 / \text{A}\_2 \right) \times 100 \tag{6}$$

where A1 refers to cells treated with EPSRT7 and the MTT solution, and A2 refers to cells without any treatment with the MTT solution.

#### *2.8. Evaluation of Antioxidant Ability on HeLa Cells*

#### 2.8.1. Injury Inducement Model

The methodology used to create an injury model for HeLa cells was based on the procedure outlined by Huang-Lin et al. [39]. HeLa cells were seeded at a density of <sup>5</sup> × <sup>10</sup><sup>4</sup> cells per well for 24 h. The medum was then removed and replaced with 100 <sup>μ</sup>L of various concentrations of H2O2 (0.25–2 mM) for 1 h at 37 ◦C under a 5% CO2 atmosphere. Following exposure, the H2O2 solution was removed, and a fresh medium was added to the wells. Cell viability was measured using the MTT method as described in Section 2.7.2.

HeLa cell viability was computed using Formula (7):

$$\text{Cell viability (\%)}=\text{(A}\_1/\text{A}\_2)\times 100\tag{7}$$

where A1 represents the absorbance of HeLa cells treated with H2O2 and the MTT solution, while A2 represents the absorbance of HeLa cells that were not subjected to any treatment with the MTT solution.
