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  • br in a sealed tube

    2022-08-31

    
    in a sealed tube which fulfilled with nitrogen for 6 h. Subsequently, the residual TFA was removed by adding methanol and evaporating to dryness under stream of nitrogen gas. Then 1.0 mL of 0.3 mol/L NaOH was added to dissolve the residue for derivatization. The sample was added 400 μL of PMP-methanol solution and set at 70 °C for 2 h. After cooled at room temperature, the sample was adjusted to pH 6–7 by adding 400 μL of 0.3 mol/L HCl, then 1, 200 μL of distilled water and an equal volume of chloroform were added and agitated. Subsequently, the chloroform layer was discarded and this step was repeated for an-other two times. The derived sample was filtered through 0.22 μm filter before analysis.
    UV spectra of EPS-CP, EPS-CS and EPS-SS showed that only a single peak in the range of 190–210 nm, respectively. The UV spectra had no detectable peaks at 280 or 260 nm, indicating that all the three EPSs did not have protein or nucleic acid.
    The main structure groups of EPSs were inspected using Fourier transform infrared spectroscopy (FTIR) at room temperature. Briefly, 2 mg of sample was pressed into 20 mg of KBr pellets. The mixture was scanned at the frequency of 400 cm−1–4000 cm−1 at a resolution ratio of 1 cm−1 and the scan number was 32.
    The surface morphology of the EPSs were observed using scanning PD98059 microscopy (SEM, SIGMA 500/VP, ZEISS) at a voltage of 3.0 kV and under 400×, 1000×, 3000× magnification, respectively. Before SEM observation, the sample was coated with a layer of Au and fixed to the SEM stubs with conductive tape.
    2.4. In vitro antioxidant activity assay
    2.4.1. DPPH free radicals scavenging assay
    DPPH radicals scavenging activity was determined according to previous study. Briefly, 2.0 mL of sample (dissolved in distilled water) was added to 2.0 mL of DPPH solution (0.1 μmol/L, dissolved in ethanol) and the absorbance was measured at 517 nm after incubation in dark for 30 min at room temperature [19].
    2.4.2. Hydroxyl radical scavenging assay
    The hydroxyl radical scavenging activity was determined according to Fenton's reaction method [20]. Briefly, the reaction mixture contain-ing 100 μL of 0.435 mM brilliant green, 200 μL of 0.5 mM FeSO4, 150 μL of 3.0% H2O2 and 100 μL of various concentration samples dissolved in dis-tilled water was incubated at 37 °C for 1 h. The mixture was centrifuged at 4696 ×g for 5 min, 200 μL of the supernatant was taken and measured at 624 nm using microplate reader. The hydroxyl radical scavenging activity was evaluated using the following formula: Hydroxyl radical scavenging activity (%) = [(A0 − A1) / (A2 − A1)] × 100. Where A0 is the absorbance of the sample, A1 is the absorbance of blank (absence of sample), A2 is the background absorbance (absence of both sample and Fenton reaction system).
    2.5. Antitumor activity of the EPSs on colon cancer cell lines
    The effects of EPSs on the growth of colon cancer cells were evalu-ated by the analysis of cell viability dispensed on a 96-well culture plate. The cell viability was assayed using cell counting kit-8 (CCK-8) according to the manufacturer's instruction (CK04, DOJINDO). Briefly, HCT8 or HCT116 cells were seeded in 96-well plates at a density of 1 × 104 cells/per well. After treatment with EPSs for 24 h, the medium containing 10% of CCK-8 solution was added in each well and incubated for 1 h at 37 °C followed by monitoring the absorption at 450 nm by microplate reader (Bio-Rad, USA). Experiment was conducted in five replicates.
    Fig. 1. Gel-permeation chromatography (GPC) profiles of EPSs. (a) EPS-SS from Scenedesmus sp., (b) EPS-CP from Chlorella pyrenoidosa, (c) EPS-CS from Chlorococcum sp.
    For the colony formation assay, cells were seeded into 6-well plates at density of 1500 cells per well and cultured with RPMI 1640 medium supplemented with various concentrations of EPSs. The cultures were maintained at 37 °C in a 5% CO2 incubator for one week, and the surviv-ing colonies were fixed with 4% paraformaldehyde (PFA) and stained with 0.1% crystal violet for 20 min. After staining, colonies were washed with PBS buffer and subsequently counted using ImageJ software (1.52a, National Institutes of Health, USA).