1. Introduction
Fucoidan is a polysaccharide obtained from brown seaweeds. It is composed of L-fucose, uronic acid, galactose, xylose, and sulfated ester group[1-3]. Fucoidan has been reported to possess numerous biological activities such as antioxidant, anticoagulant, antithrombotic, antiviral, antiproliferative, antifertilizing, and antitumoral activities[4-8]. These activities of fucoidan are mainly due to its sulfur content, structure conformation, and molecular weight[9]. However, pristine fucoidan obtained from seaweed has high molecular weight and low activity, which limited its application, especially in cosmetic, food science, and medication science. To improve its application and biological activities, degradation of polysaccharide has been used[10-12]. A variety of methods, including acid hydrolysis, enzymatic, sonochemistry, and photochemical treatment, can be used to degrade high molecular weight polysaccharide. Chemical treatment has disadvantages as it can cause formation of side products, reduced yields, functionality losses, and pollution. On the other hand, photochemical treatment has been recognized as a green method. Recently, photochemical and oxidation degradation with hydrogen peroxide have been widely used to reduce molecular weights of many compounds[13,14]. Oxidation degradation method is based on hydroxyl radicals which are powerful oxidizing species. Electron beam can increase the yield of hydroxyl radicals by attacking the 1,4-glycosidic bond of polysaccharide.
In this research, fucoidan was degraded in a hydrogen peroxide aqueous solution under irradiation at various doses to determine effects of electron beam and hydrogen peroxide on fucoidan properties such as molecular weight, viscosity, chemical structure, sulfur content, and antioxidant ability. Fucoidan samples were characterized using gel permeation chromatography (GPC), a ubbelohde viscometer, Fourie-transform infrared spectroscopy (FT-IR), a ultra violet viscometer (UV), and an elemental analyzer (EA).
2. Experimental
2.1. Materials
Fucoidan was obtained from Haewon Biotechnology Co., Korea. It was used after sieving through a 200-mesh sieve. Hydrogen peroxide (30 wt%, Samchuk chemical, Korea), ethanol (Dong yang chemical, Korea), and sodium nitrate (Sigma Aldrich, USA) were of reagent grade.
2.2. Degradation of Fucoidan
Fucoidan degradation reaction was carried out in an aqueous solution. First, fucoidan (4 g) was solubilized in deionized water. Different quantities of hydrogen peroxide aqueous solution were then added under stirring. The prepared solution was irradiated in air. Electron beam irradiation was carried out with different doses (5 kGy, 10 kGy, 15 kGy, and 20 kGy) using an e-beam (2.5 MeV) (EB Tech, Daejeon, Korea) at room temperature. After irradiation, treated fucoidan was precipitated in ethanol. Powder sample was then prepared by freezing drying.
2.3. Characterization of degraded fucoidans
The molecular weight of fucoidan was determined with a GPCMALS (WYATT Technology corporation, Detector: Dawn EOS (Light Scattering) + Optilab DSP(RI), softwear: ASTRA, Column: 2XPL aquagel Mixed-OH (7.8X300 mm). Elution was performed at 40 °C with 0.1 N NaNO3 aqueous solution at a flow rate of 1.0 mL/min. Samples were filtrated with 0.45 μm syringe filter membranes before injection.
The solution viscosity of each sample was determined using a ubbelohde viscometer (Kapillar-Viskosimeter NO:525030, Schott Gerate). The inherent viscosity of solution was measured at a concentration of 0.5 g/d L in deionized water at 35 °C.
The number of chain breaks per molecule was calculated according to the following equation (1):
Where N was the number of chain breaks per molecule, Mwo was the molecular weight of the pristine fucoidan, and Mw was the molecular weight of irradiated fucoidan[15].
Polymer degradation process can be described in terms of molecular weight changes due to polymer chain bonds scission. Its efficiency can be estimated based on radiation yield of scission Gs (mol/J) according to the following equation (2):
Where Gs is the radiation yield of scission (mol/J), c is the fucoidan concentration in solution (g/dm3), D is the absorbed dose (Gy), d is the fucoidan density in solution (kg/dm3), Mwo is the molecular weight of the pristine fucoidan, and Mw is the molecular weight of irradiated fucoidan[16].
Sulfur content of each sample was determined with an Elemental Analyzer (Vario-EL Ⅲ, Elemental analyses system GmbH). The chemical structure of fucoidan was analyzed by Fourier transform infrared spectroscopy (Nicolet 6700, Thermo scientific). All fucoidan samples were grounded with KBr powder and compressed into pellets. The number of scans was 32. Scanning was conducted from 4000 cm-1 to 650 cm-1.
2.4. Antioxidant effect of degraded fucoidans
Measurement of antioxidant ability of fucoidan was based on the method described by Zhao[17]. Hydroxyl radicals were generated in a Fenton reaction system and detected by monitoring absorbance at 510 nm. Hydroxyl radicals were generated in 3 ml of reactive solution containing 25 mM FeSO4 and 6 mM hydrogen peroxide. Fucoidan samples to be tested were prepared at different amounts (1~8 mg) in deionized water. These prepared fucoidan solutions were then added to the Fenton reaction solution. After 2 mM sodium salicylate was added to the mixed solution, the mixture was incubated at 37 °C for 1 hr. After incubation, the absorbance was measured at 510 nm. Inhibition of hydroxyl radicals was determined using the following equation (3):
(Ac: absorbance of control group, Ab: absorbance of test group)
3. Results and discussion
3.1. Effects of hydrogen peroxide and e-beam according to molecular weight of fucoidan
Molecular weight of degraded fucoidan is depicted in Figure 1 and 2. When the concentration of hydrogen peroxide was increased under the same irradiation dose, the degree of change in molecular weight was similar. However, with increasing irradiation dose under the same concentration of hydrogen peroxide, the rate of decrease in molecular weight was much higher. Such results indicate that the use of e-beam radiation it is more advantageous for the degradation process of fucoidan in hydrogen peroxide solution than the degradation with hydrogen peroxide alone, consistent with a previous study[14]. The solution viscosity of fucoidan showed a similar pattern, exhibiting the lowest intrinsic viscosity value (0.78) at FC4/HP10-20 kGy (Figure 3).
3.2. Number of chain breaks and radiation yield of scission of fucoidan
The number of chain breaks per molecule was calculated with equation (1). As shown in Figure 4 and Table 1, with increasing irradiation dose and hydrogen peroxide concentration, the number of chain breaks was markedly increased. A synergetic effect between e-beam irradiation and hydrogen peroxide on fucoidan degradation was apparent. It is well-known that hydrogen peroxide is based on the formation of reaction hydroxyl radicals. However, the conversion rate of hydroxyl radicals is rather low when hydrogen peroxide is used alone. According to a previously literature, the formation of hydroxyl radical is enhanced through radiolysis of hydrogen peroxide when ultrasonic and gamma irradiations are present along with hydrogen peroxide[18]. In order to find an optimized degradation condition, the radiation yield of scission of fucoidan was calculated with equation (2). As shown in Figure 5 and Table 2, when the concentration of hydrogen peroxide was 5%, the irradiation dose did not significantly affect the radiation yield of scission value. However, when the concentration of hydrogen peroxide was 10%, the radiation yield of scission value was markedly increased with increasing irradiation dose up to 15 kGy. When hydrogen peroxide concentrations of 5% and 10% were compared, the degradation of fucoidan was much more efficient at 10%. These results showed that the degradation of fucoidan was increased with increasing concentration of hydrogen peroxide and e-beam irradiation dose.
3.3. Elemental analysis of fucoidan
The sulfur content of fucoidan is an interesting factor. According to a previously literature, the high sulfur content in fucoidan is responsible for its various biological activities such as antioxidant capacity and anticancer properties[19]. Results of sulfur content based on elemental analysis of samples are shown in Table 2. With increasing concentration of hydrogen peroxide, sulfur/carbon, hydrogen/carbon, and nitrogen/carbon ratios were slightly reduced, confirming the loss of sulfur and nitrogen during fucoidan degradation. However, the irradiation dose did not have a significantly effect on these ratios.
3.4. Fourier transform infrared spectroscopy (FT-IR) analysis of fucoidan
FT-IR spectra of pristine fucoidan and irradiated fucoidan are shown in Figure 6. FT-IR spectra of irradiated fucoidan were identical to those of pristine fucoidan, including R-O-SO3 at 1315~1220 cm-1 and C-O-S at 850 cm-1. The band at 1315~1220 cm-1 corresponded to sulfate esters. The presence of absorbance at this wavelength range indicates the presence of sulfate in the sample. The band at 850 cm-1 corresponded to sulfate substituents at axial C4 positions. Intensities of absorbance at 1315~1220 cm-1 and 850 cm-1 were almost the same. However, the band at 1725 cm-1 showed an increase in its peak intensity with increasing irradiation dose. This result is consistent with a previously literature on polymer degradation[20]. This peak corresponded to the formation of carbonyl group[20]. These chemical changes arise from the rearrangement of radical to form a stable product after chain scission which has been proposed in the degradation of alginate and pectin[20]. These results suggested that the degradation process of fucoidan by hydrogen peroxide did not significantly change its chemical structure or the content of its sulfate group.
3.5. Hydroxyl radical scavenging assay
It is well-known that hydroxyl radicals are extremely reactive chemical species and can be generated from hydrogen peroxide in the presence of metal ions such as iron[21]. It can react with any biological molecule and their damaging action is the strongest among free hydroxyl radical species. These damages lead to many diseases. So, it is very important to remove or scavenge free hydroxyl radical from our body. The hydroxyl radical scavenging activities of tested samples were used to determine the antioxidant ability of fucoidan samples. As shown in Figure 7, pristine fucoidan and degraded fucoidans had powerful scavenging ability against hydroxyl radical. The scavenging ability was depended on concentration of samples. Moreover, the low molecular weight fucoidan higher antioxidant ability than others. It was reported in previously literature that the antioxidation ability of fucoidan was depended on its molecular weight distribution and molecular weight [22,23].
4. Conclusion
In the present study, fucoidan was irradiated by electron beam in the present of hydrogen peroxide. According to results of the number of chain breaks per molecule, radiation yield of scission, and molecular weight, fucoidan was efficiently degraded in hydrogen peroxide under radiation. The molecular weight of fucoidan was decreased from 99,956 at irradiation dose of 0 kGy to 6,725 at irradiation dose of 20 kGy. When effects of hydrogen peroxide concentration and irradiation dose were compared, it was found that the irradiation dose had a larger effect on the change of molecular weight of fucoidan. These results suggested that the electron beam promoted degradation of fucoidan by increasing the generation of hydroxyl radicals. The number of chain breaks per molecule was increased with increasing dose from 3.064 in H2O2 10 w/v%-5 kGy irradiation to 13.863 in H2O2 10 w/v%-20 kGy irradiation. The radiation yield of scission was slightly increased from 2.428 × 10-4 mol/J in H2O2 10 w/v%-5 kGy irradiation to 2.747 × 10-4 mol/J in H2O2 10 w/v%-20 kGy. However, the radiation yield of scission was markedly increased from 2.194 × 10-4 mol/J in H2O2 5 w/v%-5 kGy irradiation to 2.747 × 10-4 mol/J in H2O2 10 w/v%-20 kGy. These results were considered to be closely related to the above-mentioned efficient decomposition of hydroxyl radicals from hydrogen peroxide. According to FT-IR spectra and elemental analysis, the fucoidan sample did not show a significant change in its chemical structure or the content of sulfate group during the degradation process. The hydroxyl radical scavenging ability was markedly increased with decreasing molecular weight for fucoidan samples: from 19% for pristine fucoidan (Mw: 99,956) to 67.8% for low molecular weight fucoidan (Mw: 6,725).
