## 2 Materials and Methods
### Physicochemical Properties of Fucoidan
#### 2.6.3 Thermal Property of Fucoidan  
Thermogravimetric analysis (TGA) tests were performed on a Netzsch DSC 204 with continuous nitrogen flushing to determine the maximum saturation and melting point of pure fucoidan. The temperature estimate was investigated by heating 1.71 mg of the samples at room temperature up to 900 \({}^{\circ}\)C at a heat rate of 20 \({}^{\circ}\)C/min. The thermograms were recorded with increased temperature.  
2.6.4. Fourier Transform Infrared Spectroscopy (FT-IR)  
The sample extracts were combined with 90 parts of dried KBr pellets. The mixture was crushed to form a 3 mm diameter salt disc. Then, the FT-IR spectrophotometer was used to measure the absorbance at 4000-400 cm\({}^{-1}\).  
2.6.5. Nuclear Magnetic Resonance (NMR) Spectroscopy  
A sophisticated spectrophotometer was used to conduct the 1H-NMR studies on extracted and purified fucoidan (400 MHz Bruker model, Bruker, Billerica, MA, USA). In the NMR test tube, the fucoidan sample was combined with NMR quality double distilled water. In the NMR spectrum, the chemical shift of the proton was presented in parts per million (ppm).  
2.6.6. SEM Analysis  
The lyophilized samples were observed under scanning electron microscope (Sophisticated Test & Instrumentation Centre, Cochin University, Cochin, India).  
2.6.7. Atomic Force Microscopy (AFM)  
The AFM examinations of isolated and purified fucoidan samples were carried out by combining the sample solutions with 0.3 M of sodium hydroxide at 200 \(\upmu\)g/mL. The produced fucoidan extract was allowed to equilibrate with sodium hydroxide for 60 min before being neutralized. After alkali treatment, the purified fucoidan sample partly dissociated the helices formation by charging the hydroxyl groups and lowering the H-bonding between the chains on the laminarian backbone. The fucoidan residue solutions were then dialyzed against excess water for three days to eliminate the bulk of NaCl. The chemical solutions were then placed on a 1 cm\({}^{2}\) freshly cleaned glass plate surface through a 0.22 \(\upmu\)m filter. The samples were air-dried at room temperature for 16 h. The microscopic images of the isolated and purified fucoidan samples were taken within 24 h of fucoidan deposition to prevent sample contamination. It should be noted that fucoidan formed comparable patterns with less order when the solutions were not treated with NaOH, dialyzed, and filtered before deposition onto the glass. Atomic force microscopy (AFM; Sri Ramakrishna Engineering, Coimbatore, India) was used to observe the structures of pure fucoidan.  
2.7.1. DPPH Assay  
The antioxidant activity of the isolated and purified fucoidan was tested using the standard technique [38]. Approximately 100 \(\upmu\)L of the extracted sample was added with 3 \(\upmu\)L of DPPH solution per doses. The antioxidant properties of the sample were measured at 517 nm, followed by a 30 min incubation at 30 \({}^{\circ}\)C in the dark. After incubation, the discolorations were detected at 517 nm with a suitable blank. All tests were performed in triplicate to prevent standard error.  
\[\text{Scavenging }\%=\text{A}_{\text{Control}}-\text{A}_{\text{Sample}}/\text{A}_{ \text{control}}\times 100\]  
2.7.2. Hydroxyl Radicals Scavenging Activity  
The hydroxyl radicals scavenging activity of isolated and purified polysaccharides was investigated using [PERSON]'s reaction [39]. The percentage rate obtained from the hydroxyl radical scavenging test was expressed as inhibition rate. The hydroxyl radicals were prepared using 3 mL of Na\({}_{3}\)PO\({}_{4}\) buffer and various polysaccharides. The solutions were incubated for 1 h, and the existence of hydroxyl radical was determined by measuring the absorbance at 510 nm. BHA and L. Ascorbic acid were used as control.