# Enzymatic Degradation of _Gracilariopsis lemaneiformis_
## 3 Materials and Methods
### In Vitro Chemical Assays of Antioxidant Activity  
3.6.1. Hydroxyl Radical Scavenging Activity  
The samples were dissolved in distilled water for the preparation of 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL sample solutions. A total of 0.5 mL of each sample solution was mixed with 0.5 mL of 9 mM FeSO\({}_{4}\), 0.5 mL of 8.8 mM H\({}_{2}\)O\({}_{2}\), and 0.5 mL of 9 mM salicylic acid ethanol solution. The reaction solution was incubated at 37 \({}^{\circ}\)C for 30 min and then measured at 510 nm. Distilled water was used as the control. The scavenging capacity of the hydroxyl radical was calculated using the following equation [59]:  
\[\text{Scavenging percentage (\%)}=(1-A_{1}/A_{0})\times 100\% \tag{1}\]  
where \(A_{1}\) is the absorbance of the sample and \(A_{0}\) is the absorbance of the control (distilled water). 3.6.2. DPPH Radical Scavenging Activity  
DPPH radical scavenging activity determination was assayed through the method as described by [PERSON] et al. (2011) [60] with slight modification. Sample solutions of 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL were prepared with distilled water. Then, 1.0 mL of sample solution was mixed with 1.0 mL of 0.2 mM DPPH dissolved in anhydrous ethanol and vortexed. The mixture was incubated at 37 \({}^{\circ}\)C for 30 min in the dark, and absorbance was read at 517 nm. Distilled water was used as the control. The scavenging capacity of DPPH radical was calculated using the following equation:  
\[\text{Scavenging percentage (\%)}=[1-(A_{i}-A_{j})/A_{0}]\times 100\% \tag{2}\]  
where \(A_{i}\) is the absorbance of the sample mixed with the DPPH solution, \(A_{j}\) is the absorbance of the sample mixed with anhydrous ethanol, and \(A_{0}\) is the absorbance of distilled water mixed with the DPPH solution. 3.6.3. Reduction Capacity  
The reducibility of the GLP and degradation products were determined by the potassium ferricanide method described by [PERSON] et al. (2019) [30] with slight modification. A mixture containing 0.5 mL of sample solution, 0.5 mL of PBS (2 M, pH 6.6), and 0.5 mL of 1% potassium ferricanide were prepared and incubated at 50 \({}^{\circ}\)C for 20 min. The reaction was stopped by adding 0.5 mL of 10% TCA after rapid cooling, and then the mixture was centrifuged at 3000 rpm for 10 min. A total of 1.0 mL of the supernatant was mixed with 1.0 mL of distilled water and 0.2 mL of 0.1% FeSO\({}_{4}\) and left for 10 min, and absorbance was measured at 700 nm. 3.6.4. Total Antioxidant Capacity  
For total antioxidant capacity determination, 0.1 mL sample solution was mixed with 3 mL of FRAP working solution (FRAP working solution was prepared with 0.3 M CH\({}_{3}\)CONA buffer solution, 20 mM FeCl\({}_{3}\), and 10 mM TPTZ in a ratio of 10:1:1) and 0.3 mL of distilled water. Absorbance was measured at 593 nm after incubation at 37 \({}^{\circ}\)C for 20 min. An increase in the absorbance of the sample indicated the enhancement of the total antioxidant capacity [61]. 3.7.1. Cell Culture and Cell Viability Assay  
The effects of GDP2, GDP3, and GDP4 on the antioxidant activity of the HFL1 cells treated with H\({}_{2}\)O\({}_{2}\) were investigated. HFL1 cells were cultured in [PERSON]'s modification of [PERSON]'s medium high-glucose medium (containing 10% fetal bovine serum, 100 UI/mL penicillin, and 100 \(\upmu\)g/mL streptomycin) at an atmosphere of 90% relative humidity, 5% CO\({}_{2}\), and 37 \({}^{\circ}\)C. Cell viability was determined with the MTT method.