*4.4. Chemical Analysis*

The chemical composition of both the PBE and PBP was analyzed using several methods. Official methods of analysis of the Association of Official Analytical Chemists (AOAC) was used to obtain the total polysaccharide content [43]. The polyphenol content was measured accordingly with the method described by Chandler and Dodds (1983) with minor modifications [44]. The sulfate content was evaluated by BaCl2 gelation method [45].

Samples were hydrolyzed in 4M triflouroacetic acid (4 h, 100 ◦C). This was subjected to CarboPac PA1 cartridge column (4.5 × 50 mm) for separation and detected with an ED50 Dionex electrochemical detector (Dionex) concerning monosugar analysis [46].

The attenuated total reflectance Fourier transform infra-red (ATR-FTIR) spectrum was obtained with a Bruker FTIR, Alpha II (Bruker, Karlsruhe, Germany) instrument in the 400–4000 cm<sup>−</sup><sup>1</sup> wavenumber range. Commercial grade fucoidan was analyzed at the same time.

#### *4.5. Radical Scavenging Activity Evaluation via Electron Spin Resonance (ESR) Spectrometer*

Both PBE and PBP were analyzed for its radical scavenging activities. DPPH, alkyl, and hydroxyl radical scavenging activities were evaluated using electron spin resonance spectroscopy (ESR, JES-FA200; JEOL, Tokyo, Japan). The DPPH radical scavenging was assessed via the method defined by Nanjo et al. (1996) [47]. The method, in brief, equal volumes of sample and DPPH was mixed vigorously, transferred to a capillary tube and inserted to the ESR spectrometer for measurement. Alkyl radical scavenging followed the method described by Hiramoto et al. (1993) [48]. The radical was generated via a reaction mixture of AAPH and 4-POBN with tested sample which was incubated in water bath (37 ◦C, 30 min) and subjected to analysis. The method explained by Finkelstein et al. (1980) was used in the evaluation of the hydroxyl radical scavenging potential [49]. This used the Fenton reaction as the basis and mixed sample in phosphate buffer solution (pH 7.4) with equal volumes of 0.3 M DMPO, 10 mM FeSO4, and 10 mM H2O2 (200 μL) for analysis.

## *4.6. Chemical Assay for Hydrogen Peroxide*

A colorimetric assay described by Kim et al. (2014) was implemented in the evaluation of the hydrogen peroxide scavenging [50]. The method in brief; each sample was mixed with 0.1 M phosphate buffer (pH 5.0, 100 μL) in a micro well plate. Hydrogen peroxide (20 μL) was added and was incubated (37 ◦C, 5 min). ABTS (1.25 mM, 30 μL) and peroxidase (1 unit/mL, 30 μL) was added to the above and further incubated at 37 ◦C for 10 min. The absorbance measurements were collected using an ELISA reader at 405 nm.

#### *4.7. Protective E*ff*ects of PBP via In Vitro Methods*
