Laminaria japonica Aresch powder (Xiamen City, Fujian Province, China) was extracted with 80% (v/v) aqueous methanol, then separated by silica column chromatography, a Sephadex LH-20 column, and further purified by reversed-phase HPLC. The following amounts were obtained: Fuco (890 mg), 9'Z-Fuco (28.3 mg), 13Z- and 13'Z-Fuco (59.5 mg) [7]. Fuco and its stereoisomers were analyzed using an HPLC system equipped with photodiode array detector (Shimadzu LC-20AD, Shimadzu Corp., Kyoto, Japan) (Figure 2).

The purities of Fuco, 9'Z-Fuco and 13Z- and 13'Z-Fuco were 99.48%, 98.15%, and 96.72%, respectively. The corresponding structures illustrated in Figure 1 were established by ¹H-NMR, ¹³C-NMR, IR and UV.

Because of DPPH is a stable organic radical, the DPPH oxidation assay is widely used in the quantification of radical-scavenging capacity (RSC) to evaluate the antioxidant activity of biological samples [13,15,16]. The DPPH scavenging activity of three Fuco stereoisomers is presented in Figure 3.

The optical density (OD) of DPPH was found to be linearly dose-dependent (8.6–51.6 μg/mL). The initial concentration of DPPH used to test the scavenging activity was 51.6 μg/mL, which corresponded to an OD of 0.822. As shown in Figure 4, the scavenging activity of Fuco first increased up to 20 min and then remained constant; moreover, it appeared dose-dependent like the scavenging activity of the three others Fuco stereoisomers (Figure 5). With regard to the EC₅₀ values of DPPH radical-scavenging activities were in the order: 13Z- and 13'Z-Fuco > (all-E)-Fuco > 9'Z-Fuco; however, they were all weaker than α-tocopherol (VE) as shown in Figure 6.

The ABTS-radical-scavenging assay is a popular method for indirect determination of a compound’s antioxidant capacity [17,18]. ABTS can be oxidized by peroxy radicals to form a metastable radical that has a strong absorption between 600 nm and 750 nm. When an antioxidant is present and the ABTS radical is scavenged, the absorption decreases. The reaction of ABTS and antioxidants was correlated with reaction time; indeed, ABTS radical-scavenging activity increased rapidly within 10 min and more slowly over 70 min (Figure 7).

As illustrated in Figure 8, the ABTS radical-scavenging activities of the Fuco stereoisomers were dose-dependent in a similar way. Unlike the DPPH scavenging activity trend, 9'-Z-Fuco was a stronger scavenger of ABTS, with an EC₅₀ value of 33.54 μg/mL, than all-E Fuco, while 13-Z and 13'-Z-Fuco had the lowest ABTS scavenging activities (Figure 9).

Excessive free radicals in the body can result in oxidative damage to some organs and cause many diseases [19,20]. Fenton’s reagent can be used to generate hydroxyl radicals and the color of the Fenton system can be used to indicate the radical content. When the radicals are scavenged by antioxidants, the change in color will be proportional to the scavenged radicals. Meanwhile, the scavenging of radicals by carotenoids can be evaluated by chemiluminescence techniques. In the present study, we found that the hydroxyl radicals were inactive when the temperature was lower than 20 °C while their generation improved as the temperature increased. The reaction speed was also related to the temperature. Figure 10 shows that at a temperature of 37 °C to resemble body conditions, the hydroxyl radical-scavenging activity of Fuco increased slowly over the first 5 min, and then rapidly from 5 to 30 min. The activities of the three Fuco stereoisomers were found to be linearly dose-dependent, and were almost equivalent (Figure 11). As regards the EC₅₀ values, in contrast to the case of DPPH and ABTS, Fuco and its stereoisomers presented similar scavenging activities lower than VE (Figure 12).

The 1,2,3-trihydroxybenzene (THDB) autoxidation method was used to produce and detect the superoxide radical activity. It is an indirect method widely used in the initial screening of antioxidants in the food and pharmaceutical industries. The relationship between the autoxidation and time was directly proportional over 180 s (Figure 13) and then the rate slightly diminishes from 180 to 250 s. The autoxidation of THDB stopped when VC was added. A similar dose-dependent trend of the superoxide radical scavenging activities was observed for the three Fuco stereoisomers (Figure 14). Moreover, in term of EC₅₀ values, the order of superoxide radical-scavenging activity was as follows: 13Z- and 13'Z-Fuco > (all-E)-Fuco > 9'Z-Fuco (Figure 15).

1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azinobis-3-ethylbenzothizoline-6-sulphonate (ABTS), α-tocopherol (VE) and 1,10-phenanthroline monohydrate (PHEN) were purchased from Sigma-Aldrich (Shanghai, China). 1,2,3-Trihydroxybenzene (THDB), vitamin C (VC), phosphate buffer saline (PBS), potassium persulfate, ferrous sulfate, aquae hydrogenii dioxide, Tris-HCL solution, methanol, and ethanol were purchased from Sino Pharm Chemical Reagent Co. Ltd. (Shanghai, China). All the other reagents used were of analytical grade. The radical-scavenging activity was determined by an enzyme-labeled instrument (SpectraMa MD Co., Ltd., Sunyvale, CA, USA).

The powder of Laminaria japonica Aresch (10 kg) was extracted with 80% aqueous methanol (50 L) at room temperature for 10 min, and then evaporated under vacuum to a something like a concrete (1.56 L). The Fuco mixture was first isolated from the methanol extract by silica column chromatography using stepwise elution with a petroleum-acetone mixture (20:1 to 1:1), and then further separated using a Sephadex LH-20 (1200 × 20 mm 27–163 μm) column saturated with 100% methanol. Finally, all-E-Fuco, 9'Z-Fuco and 13Z- and 13'Z-Fuco were successively purified by reversed-phase preparative HPLC. The purity of (all-E)-Fuco, 9’Z-Fuco and 13Z- and 13'Z-Fuco was determined on a Shimadzu LC-20AD HPLC (Shimadzu Crop.) equipped with a photodiode array detector. Ten μL of sample was injected onto an ODS C18 column (250 × 4.6 mm, 5 μm), which was equilibrated with 90% aqueous methanol as the mobile phase at a flow rate of 1.0 mL/min with detection at 450 nm.

The radical-scavenging activity of 1,1-diphenyl-2-picrylhydrazyl (DPPH) was measured by the method of Nakkarike [12]. Ten μL of the sample in methanol was mixed with 0.12 mM DPPH in methanol (150 μL) and incubated in the dark at 25 °C for 30 min. Sample and DPPH solutions in methanol were used as control and blank, respectively. The optical density (OD) was measured at 517 nm, and the scavenging activity was calculated as follows: Scavenging (%) = [1 − (A_sample − A_{sample blank})/A_control] × 100%

2,2-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical solution was prepared by mixing ready-to-use ABTS aqueous solution (5 mL, 7 mM) with potassium persulfate aqueous solution (88 μL, 140 mM) at 25 °C for 15 h. Prior to decolorization, the ABTS radical solution was diluted with methanol until its OD decreased to 0.7, which then made it suitable for the ABTS radical-scavenging assay. The discoloration was initiated by mixing ABTS solution (150 μL) with the sample (10 μL) and incubating at 25 °C for 70 min. Sample and DPPH solutions in methanol were used as control and blank, respectively. The OD was measured at 734 nm at the beginning and the end of the incubation period. Finally scavenging activity was calculated as follows: Scavenging (%) = [(A_control − A_sample)/A_control] × 100%

Hydroxyl radical was generated by the Fenton system [13,21]. The A₀ reaction mixture contained PBS solution (60 μL, pH = 7.4), FeSO₄ (60 μL, 1.5 mM), 1,10-phenanthroline monohydrate (60 μL, 1.5 mM), the methanol solution of compound (20 μL) and H₂O₂ (30 μL, 80 mM). The A₁ reaction mixture contained PBS solution (60 μL, pH = 7.4), FeSO₄ (60 μL, 1.5 mM), 1,10-phenanthroline monohydrate (60 μL, 1.5 mM), methanol (20 μL), and H₂O₂ (30 μL, 80 mM). The A₂ reaction mixture contained PBS solution (60 μL, pH = 7.4), FeSO₄ (60 μL, 1.5 mM), 1,10-phenanthroline monohydrate (60 μL, 1.5 mM) and μL methanol (20). The 96-microwell plates were filled with all the reagents except H₂O₂ and incubated at 37 °C for 70 min, then the reaction was started by the automatic addition of H₂O₂. The OD was measured at 536 nm at the start and end of the incubation period. The results were used to calculate the scavenging percent as follows: Scavenging (%) = [(A₀ − A₁)/(A₂ − A₁)] × 100%

Tris-HCl (a total of 150 μL, 50 mM, pH = 8.2) was continuously added to sample (10 μL) and 1,2,3-trihydroxybenzene (THDB, 10 μL, 3 mmol/L). After 3 min, VC (10 μL, 5 mM) was added to stop the reaction (A₀). Solution A₁ consisted of Tris-HCl (150 μL) that was continuously added to a sample (10 μL) containing VC and 1,2,3-trihydroxybenzene. Solution A₂ consisted of Tris-HCl (a total of 150 μL) that was reacted with 1,2,3-trihydroxybenzene (10 μL) for 3 min. Then VC and blank sample solution (10 μL) were added to stop the reaction. Solution A₃ consisted of Tris-HCl (150 μL) that was continuously added to VC and 1,2,3-trihydroxybenzene and blank sample solution (10 μL). The OD was measured at 536 nm at the start and end of the incubation period. The results were used to calculate the scavenging percent as follows: Scavenging (%) = [(A₀ − A₁)/(A₂ − A₃)] × 100%