Materials
Spirulina platensis powder was provided by the Spirulina platensis Research Institute of Beijing Forest University (Beijing, China). Papain (≥ 3 U/mg) was purchased from Aladdin Reagent Company (Shanghai, China). DPPH was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). A T-AOC kit was bought from the Nanjing Built Biological Engineering Research Institute (Nanjing, China). 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) and DCFH-DA were purchased from Sigma-Aldrich Co. (St. Louis, Mo, USA). 3-Amino,4-aminomethyl-2′,7′-difluorescein diacetate (DAFFM-DA) was obtained from Beyotime (Jiangsu, China). The other chemicals were of analytical grade and were commercially available.
Human normal liver cell lines (L02) was purchased at National Infrastructure of Cell Line Resource and approved by Ethics Committee of Peking Union Medical College (0111003). Experiments were implemented in compliance with the Helsinki Declaration.
Extraction of protein and concentration determination
First, Spirulina platensis powder (20 g) was mixed with distilled water (160 mL), frozen using liquid nitrogen, and thawed via the water bath method (25–30 ℃). After repeating these two steps five times, the mixture was subjected to ultrasonication at room temperature and 150 W for 3 min. The sample was centrifuged at 6000 rpm at 4 ℃ for 30 min. The supernatant (protein fraction) was immediately subjected to freeze drying using a vacuum freeze dryer, and the resulting Spirulina platensis protein powder was stored at 4 ℃.
Preparation of Spirulina platensis peptides
The protein concentration was estimated to be 56 ± 0.97%. The hydrolysate was obtained by subjecting the Spirulina platensis protein powder to enzymolysis. Three compounds, ethylene diamine tetra acetic (EDTA), l-cysteine, and NaCl, were added to 30 mL of 2% Spirulina protein liquid, and the final concentrations of the solvents were adjusted to 2.0 mM, 5.0 mM, and 300 mM, respectively. The papain enzymatic hydrolysis conditions included a temperature of 60 ℃ for 9 h at pH 7.0, and the ratio of enzyme and substrate was 1.6%. The resulting hydrolysate was filtered with 30, 10, and 3 kDa MW cutoff ceramic membranes successively in ultrafiltration centrifuge tubes at 6000 rpm at 4 ℃, for 30 min and stored at 4 ℃ after vacuum freeze drying. The antioxidant abilities of the filtered peptides of different molecular weights were individually determined in vitro.
Quantitation of Spirulina platensis peptides
The Spirulina platensis peptides (crude extract protein, 0–3 kDa, 3–10 kDa and 10–30 kDa) concentration was detected by bicinchoninic acid (BCA) assay. The experimental operation steps according to the BCA Protein Assay Kit (Yuangye Biotech CO., Shanghai, China). The absorbance was measured at 562 nm using a microplate reader (PerkinElmer, Shanghai, China).
Antioxidant activity assay
DPPH radical scavenging activity assay
Aliquots of 0.5 mL of three different molecular weights of Spirulina platensis peptides (0–3 kDa, 3–10 kDa and 10–30 kDa) and Vc in ethanol were added to 2.5 mL 0.1 mM DPPH in 100% ethanol. The mixture was shaken and allowed to stand at room temperature in the dark for 30 min, and the absorbance of the resulting solution was measured at 517 nm (AS). Ethanol was used instead of samples in the control experiment (AC), and the blank was prepared as described above but without DPPH (AB). A lower absorbance represented a higher DPPH scavenging activity. The scavenging activity was calculated as \(\left[ {1 - \frac{{{\text{AS}} - {\text{AB}}}}{{{\text{AC}}}}} \right] \times 100\%\).
Hydroxyl radical scavenging activity assay
The reaction mixture containing 2 mL of 2.0 mM FeSO4, 1 mL of 6 mM salicylate in ethanol, 5 mL of samples, and 2 mL of 6.0 mM H2O2 was incubated for 15 min at 37 ℃ in a water bath. After incubation, the absorbance of the resulting solution was measured at 510 nm (AS) using a spectrophotometer. The control was prepared in the same manner except that distilled water was used instead of the samples (AC). The abilities to scavenge hydroxyl radicals were calculated as \(\frac{{{\text{AC}} - {\text{AS}}}}{{{\text{AC}}}} \times 100\% .\)
Total antioxidant capacity assay
The total antioxidant capacity was assayed using the T-AOC kit. The absorbance of solution was measured at 517 nm using a spectrophotometer. The total antioxidant capacities were calculated according to T-AOC kit’s manufacturer.
Determination of cell viability
1 × 104 cells/mL (100 μL per well) L02 were planted into 96-well plates and cultured for 24 h. The cells were maintained in an atmosphere of 5% CO2 at 37 ℃ in DMEM that was supplemented with 10% fetal bovine serum and 0.1% antibiotics (penicillin and streptomycin). When the cells were completely adhered to the wall, they were treated with various concentrations of LMWSPs (0–3 kDa) and the oxidative damaging agent, H2O2 (1000 μg/mL or 2000 μg/mL). After 24 h of incubation, 10 μL of MTT (5 mg/mL) in phosphate buffered saline (PBS) was added to each well, and the plates were placed in the dark. After four hours, the liquids in the upper layer were removed, and 100 μL of DMSO was added to the 96-well plates to dissolve the purple formazan crystals. The L02 cell viability was estimated by reading the absorbance at 492 nm using a microplate reader (PerkinElmer, Shanghai, China). The relative cell viability was defined as the absorbance of treated wells divided by that of the control.
Intracellular reactive oxygen species (ROS) detection
The level of intracellular ROS was detected using the DCFH-DA assay. 1 × 104 cells/mL (100 μL per well) L02 were planted into 96-well plates and cultured for 24 h. When the cells were completely adhered to the wall, they were treated with various concentrations of LMWSPs (0–3 kDa) and the 2000 μg/mL H2O2. The L02 cells were washed mildly twice with PBS and incubated in Krebs’ ringer solution containing 10 μM DCFH-DA at 37 ℃ for 30 min. After three additional washes with PBS, the DCF-labeled L02 cells were observed and scanned under a confocal microscope (FV 500, Olympus, Japan). Digital pictures were analyzed using Image-J software, and the results were used to calculate the integrated optical density (IOD), which increases as the level of intracellular ROS rises.
Intracellular nitric oxide (NO) test
The intracellular nitric oxide level was determined using a fluorescent NO-sensitive dye via the DAFFM DA assay. Shortly after sucking out the cell culture fluid, cells were washed twice with PBS and maintained in the Krebs’ ringer solution supplemented with 10 μM DAFFM DA at 37 ℃ for 20 min. After three additional washes with PBS to remove excess dye, the cells were examined and scanned using a confocal microscope. Digital pictures were processed using Image-J software, and the results were used to calculate the IOD, which increases as the level of intracellular NO rises.
Analysis of antioxidant activities
Determination of the MDA content and SOD activity are often combined with each other, because the level of SOD activity indirectly reflects the ability of the body to clear oxygen free radicals and MDA indirectly reflects the severity of free radical attack on the cells. The MDA and SOD levels in the supernatants were estimated using MDA and SOD assay kits, respectively, from Jiancheng Bioengineering Institute (Nanjing, China). Briefly, the supernatants were collected, and the absorbance was read at 532 nm using a spectrophotometer, and the MDA and SOD contents were calculated as nmol/mL protein and U/mL, respectively.
Statistical analysis
Statistical analyses were compared via one-way analysis of variance (ANOVA), all calculations were performed using IBM SPSS statistics 21.