*2.1. Chemical Composition*

The celluclast enzyme assisted extract of *P. boryana* (PBE) and ethanol precipitated component PBP were subjected to chemical composition analysis. The results are given in Table 1. The total phenol content of PBE was 1.32 ± 0.17% while PBP exhibited 1.14 ± 0.26%. Sulfated polysaccharide content was higher in the PBP (56.34%). Monosaccharide analysis revealed, high contents of fucose and galactose in PBP compared to PBE.


**Table 1.** Chemical composition of PBE and PBP obtained from *P. boryana.*

The analyzed Fourier transform infrared (FTIR) data are illustrated in Figure 1. Glycosidic bonds are formed among multiple monomer units to form polysaccharides and are represented via the 1025 cm<sup>−</sup><sup>1</sup> (C-O-C stretching vibrations) fingerprint peak. An intense peak at the 1200 cm<sup>−</sup><sup>1</sup> is observed due to the sulfate stretching vibrations (S=O). While the bending sulfate vibrations are represented via the absorptions at 845 cm<sup>−</sup><sup>1</sup> (C-O-S). The moisture content available in the sample was observed via the H-O-H bending vibrations in the 1625 cm<sup>−</sup><sup>1</sup> region [12,15,16]. Results sugges<sup>t</sup> the PBP possesses a close correlation with commercial fucoidan.

**Figure 1.** Chemical characterization of PBP. (**a**) Standard monosaccharide and PBP analyzed by HPAE-PAD spectrum. (**b**) ATR-FTIR spectra of PBP and commercial fucoidan.

#### *2.2. Free Radical and Hydrogen Peroxided Scavenging Activity*

The individual radical scavenging activities corresponding to each sample is expressed in Table 2. Both samples exhibited potent scavenging activities where PBP was more active compared to PBE. Interestingly, H2O2 chemical assay analysis for scavenging also exhibited PBP as the potent, significant scavenger of radicals. Hence, further experiments were planned with the PBP sample.


**Table 2.** Free radical/ROS scavenging activities of PBE and PBP.

All results expressed as means ± SE, based on triplicated trials.

#### *2.3. Protective E*ff*ect of PBP in H2O2 Stimulated Cells*

The PBP exhibited protective effects against the H2O2 induced cells. The cell viability which was declined with the H2O2 treatment was reinstated with the PBP treatment. Similarly, the intracellular ROS level which was increased against the H2O2 stimulation was effectively downregulated with the treatment of PBP. Furthermore, the results exhibited a dose dependent recovery of each indicator (Figure 2).

**Figure 2.** Hydrogen peroxide stimulated Vero cells exhibit oxidative stress. (**a**) Intracellular reactive oxygen species (ROS) scavenging ability of PBP. (**b**) PBPs' potential to protect cells against hydrogen peroxide. Experiments were triplicated and data shown as mean ± SE; \* *p* < 0.05, \*\* *p* < 0.01. (# denotes significance compared to control while \* represents significance compared to H2O2 treated group).

#### *2.4. PBP Protects Cells from H2O2 Induced Apoptosis*

Earlier studies have revealed the effect of H2O2 on DNA damage leading to apoptosis [17]. Hence, the effect was evaluated through nuclear staining methods. This particular study followed the Hoechst 33342 staining method. Viable cells are indicated via homogeneously stained nuclei while fragmented and chromatin condensed nuclei are an indication of apoptotic cells [18]. As indicated in Figure 3, cells that were exposed to H2O2 were associated with increased cell death, indicating higher intensity in the nuclei region. The number of apoptotic bodies was significantly decreased with the PBP treatment, which was indicative of its potential to act as a protective substance against ROS.

**Figure 3.** PBP protects Vero cells against H2O2-induced apoptosis. The apoptotic body formation was observed using Hoechst 33342 staining method under a fluorescence microscope. (**a**) non-treated group, (**b**) H2O2 treated (1 mM) cells, H2O2 stimulated cells treated with PBP (**c**) 25 μg/mL, (**d**) 50 μg/mL, (**e**) 100 μg/mL, (**f**) quantitative representation. The intensity levels were analyzed using ImageJ software. Triplicated experiments were conducted and results are represented as mean ± SE; \* *p* < 0.05, \*\* *p* < 0.01. (# denotes significance compared to control while \* represents significance compared to H2O2 treated group).

#### *2.5. E*ff*ect of PBP on Antioxidant Enzymes and Pathway Proteins*

CAT and SOD are important enzymes in the process of degrading hydrogen peroxide to protect the cells against oxidative damage. It was observed that the particular enzyme protein levels were significantly declined with the H2O2 treatment. The co-treatment of the PBP recovered the enzyme levels dose dependently overcoming the effect of H2O2 (Figure 4a,b). Further, the effect was examined in the Nrf2-Keap1 pathway proteins. The cytoplasm nuclear factor E2-related factor 2 (Nrf2) level was increased while Kelch-like ECH-associated protein 1 (Keap1) exhibited declining intensities. It was observed that PBP encouraged the Nrf2 protein expression and stabilized the Keap1 protein allowing successful translocation of Nrf2 to the nucleus (Figure 4c,d). Collectively, the results obtained suggested the potential of PBP to promote Nrf2 expression and nuclear translocation to induce transcription of antioxidant enzymes such as CAT and SOD.

**Figure 4.** Effect of PBP on the H2O2 induced antioxidant related protein in Vero cells. (**a**) CAT and SOD, (**b**) relevant quantitative data, (**c**) Nrf2 and Keap1 in cytosol western blot, and (**d**) relevant quantitative data. β-actin was used as internal control. Quantification was assisted with the ImageJ software. Results are represented as mean ± SE; \* *p* < 0.05, \*\* *p* < 0.01. (# denotes significance compared to control while \* represents significance compared to H2O2 treated group).

#### *2.6. Potential of PBP to Protect H2O2 Induced Zebrafish in Lipid Peroxidation, ROS Accumulation, and Cell Death*

The survival rate and the heartbeat rate were recorded against the PBP treatment in H2O2 stimulated zebrafish (Figure 5). The ROS production in the zebrafish embryos treated with H2O2 was investigated by 2,7-dichlorofluorescein diacetate (DCF-DA). The results are interpreted as an indication of the fluorescence intensity. The H2O2 treated embryos expressed significantly high fluorescence intensity compared to the non-treated group. PBP pre-treated embryos exhibited a downregulation of fluorescence intensities dose dependently, subduing the effect generated via H2O2. This reflects the gradual decrement of ROS production, which implies PBPs' ability to work as a protective agen<sup>t</sup> (Figure 6a,b). The amount of lipid peroxidation was measured using the diphenyl-1-pyrenylphosphine (DPPP) staining. Similarly, the results indicated a decline in the amount of lipid peroxidation with the PBP treatment, (Figure 6c,d). The cell death which was evaluated through the acridine orange staining exhibited the fluorescent intensities to be declined significantly in H2O2 induced zebrafish embryos with the PBP treatment (Figure 6e,f). The cell death percentage which was 290 in the H2O2 treated group was declined up to 170 with the PBP (100 μg/mL) treatment. These results reveal the prospective of PBP to act as a potent protector against H2O2 stimulated oxidative stress.

**Figure 5.** Embryos pre-treated with H2O2 (5 mM) and followed by PBP treatment (25, 50, 100 μg/mL). (**a**) survival rate, and (**b**) heart beating rate. Experimental procedure followed triplication and data indicated as mean ± SE; \* *p* < 0.05, \*\* *p* < 0.01. (# denotes significance compared to control while \* represents significance compared to H2O2 treated group).

**Figure 6.** in vivo evaluation of antioxidant potential of PBP. (**a**) Hydrogen peroxide induced ROS production with DCF-DA staining, (**c**) Hydrogen peroxide-induced lipid peroxidation production stained with DPPP, (**e**) PBP protects zebrafish embryos against H2O2-induced death, stained with acridine orange and captured under fluorescence microscope. Quantitative results of each analysis are represented in (**b**), (**d**), and (**f**) respectively, measured via ImageJ software. Triplicated experiments were conducted and results are represented as mean ± SE; \* *p* < 0.05, \*\* *p* < 0.01. (# denotes significance compared to control while \* represents significance compared to H2O2 treated group).
