# Enzymatic Degradation of _Gracilariopsis lemaneiformis_
## 2 Results and Discussion
### GDP Reduced ROS Generation in the HFL1 Cells  
ROS is a normal metabolite that plays an important role in cell signaling pathways [43]. However, excessive ROS can lead to oxidative damage in the human body and food system by reacting with biomolecules, such as DNA, lipids, proteins, and sugars, and destroying their structures and functions [44]. Intracellular ROS is an efficient index for oxidative stress evaluation in living cells [45]. Effects of GDPs on intracellular ROS generation in damaged HFL1 cells were investigated using a DCFH-DA fluorescence probe. The green fluorescence intensity of DCFH-DA was proportional to ROS level [46]. In the present study, the level of intracellular ROS in the HFL1 cells increased significantly after 6 h of H2O2 treatment (Figure 4), indicating that H2O2 treatment significantly induced the production of ROS in the HFL1 cells. However, after pretreatment with 200 ug/mL GDP2, GDP3, and GDP4, intracellular ROS levels in the HFL1 cells decreased by 69.42%, 50.37%, and 32.33% compared with that in the negative control cells, respectively, supporting that GDPs effectively scavenged or inhibited ROS production induced by H2O2 in HFL1 cells. GDP Alleviated Lipid Peroxidation Level and Increased the Cellular Antioxidant Enzymes Activities of HFL1 Cells  
Malondialdehydedehyde (MDA), one of the most important products of the lipid peroxidation of unsaturated fatty acids, is a useful tool in characterizing the degree of membrane damage [47]. As shown in Figure 5A-C, the MDA level in the negative control group was significantly higher than that of the normal control cells, indicating that H2O2 treatment caused oxidative damage to HFL1 cells. By contrast, the MDA level decreased by 25.51%, 20.41%, and 23.67% after pretreatment with 200 ug/mL GDP2, GDP3, and GDP4, respectively, suggesting that GDPs alleviated the degree of membrane damage induced by H2O2 in the HFL1 cells. Figure 3: Protective effects of GDP2 **(A)**, GDP3 **(B)**, and GDP4 **(C)** against H2O2-induced injury in the HFL1 cells. Vertical bars represent means \(\pm\) SD (n = 4), and different letters indicate significant difference (\(p<0.05\)). Figure 2: Effects of GDP2 **(A)**, GDP3 **(B)**, GDP4 **(C)** on the cell viability of HFL1 cells. Vertical bars represent means \(\pm\) SD (n = 4), and different letters indicate significant difference (\(p<0.05\)). Enzymatic antioxidant defense is a vital mechanism against ROS-induced oxidative stress in living organisms. Superoxide dismutase (SOD), the first line of defense system against ROS, can catalyze the conversion from O2\({}^{\bullet-}\) into H2O2, which can subsequently be eliminated by peroxididases such as catalase (CAT) and glutathione peroxidase (GSH-Px) while CAT plays a predominant role in decomposing H2O2 into harmless molecules [3]. The increase in antioxidant enzyme activity can reduce the oxidative stress of living cells [48]. In the present study, the activities of antioxidant enzymes (SOD, CAT, and GSH-Px) were measured to evaluating the protective effects of GDPs against H2O2-induced oxidative damage. As shown in Figure 5, SOD, CAT, and GSH-Px activities decreased by more than 50% after exposure to 200 \(\upmu\)M H2O2 compared with those in the normal control group. However, GDPs significantly increased the antioxidant enzyme activities in the HFL1 cells under oxidative stress. SOD activity increased by 141.09%, 123.87%, and 62.79% after pretreatment with 200 \(\upmu\)g/mL GDP2, GDP3, and GDP4, respectively, compared with that in the negative control cells (Figure 5A-C). Likewise, CAT activities in GDP2, GDP3, and GDP4 groups improved by 88.42%, 57.84%, and 52.13%, respectively, and GSH-Px activity increased by 36.66%, 47.00%, and 42.98%, respectively (Figure 5A-C). Similarly, polysaccharides from _S. fusiforme_ can protect HaCaT human keratinocytes from oxidative stress by stimulating SOD and GSH-PX enzyme activities [41]. Moreover, GDP2 had the highest antioxidant activity.