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Antioxidants (Basel)
2021 Jan 14;101:. doi: 10.3390/antiox10010110.
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Potential Antioxidant Properties of Enzymatic Hydrolysates from Stichopus
japonicus against Hydrogen Peroxide-Induced Oxidative Stress.
Lee HG
,
Kim HS
,
Oh JY
,
Lee DS
,
Yang HW
,
Kang MC
,
Kim EA
,
Kang N
,
Kim J
,
Heo SJ
,
Jeon YJ
.
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A comprehensive antioxidant evaluation was performed on enzymatic hydrolysates of Stichopus
japonicus (S. japonicus) using Vero cells and zebrafish models for in vitro and in vivo studies, respectively. S. japonicus was hydrolyzed with food-grade enzymes (alcalase, α-chymotrypsin, flavourzyme, kojizyme, neutrase, papain, pepsin, protamex, and trypsin), and the free radical scavenging activities were screened via electron spin resonance (ESR) spectroscopy. According to the results, the enzymatic hydrolysates contained high protein and relatively low polysaccharide and sulfate contents. Among these hydrolysates, the α-chymotrypsin assisted hydrolysate from S. japonicus (α-chy) showed high yield and protein content, and strong hydroxyl radical scavenging activity. Therefore, α-chy was chosen for further purification. The α-chy was fractionated by ultrafiltration into three ultrafiltration (UF) fractions based on their molecular weight: >10 kDa (α-chy-I), 5-10 kDa (α-chy-II), and <5 kDa (α-chy-III), and we evaluated their antioxidant properties in H2O2 exposed Vero cells. The α-chy and its UF fractions significantly decreased the intracellular reactive oxygen species (ROS) generation and increased cell viability in H2O2 exposed Vero cells. Among them, α-chy-III effectively declined the intracellular ROS levels and increased cell viability and exhibited protection against H2O2 induced apoptotic damage. Furthermore, α-chy-III remarkably attenuated the cell death, intracellular ROS and lipid peroxidation in H2O2 exposed zebrafish embryos. Altogether, our findings demonstrated that α-chy and its α-chy-III from S. japonicus possess strong antioxidant activities that could be utilized as a bioactive ingredient for functional food industries.
Figure 1. Protective effect of SJH against H2O2 induced oxidative stress. Intracellular reactive oxygen species (ROS) scavenging activity (A) and cell viability (B) in H2O2 exposed Vero cells. Experiments were performed in triplicate and data are expressed as mean ± SD; Significant differences identified at * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 as compared to the H2O2 treated group; ####
p < 0.0001 as compared to the control group. Statistical analyses were conducted using Tukey’s post hoc comparison and Duncan’s multiple range test.
Figure 2. Molecular weight distribution and hydrogen peroxide scavenging activity of α-chy and its UF fractions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern (A) and hydrogen peroxide scavenging activity of UF fractions (B). Experiments were performed in triplicate and data are expressed as mean ± SD; Significant difference identified at **** p < 0.0001 as compared to the control group.
Figure 3. α-chy and its UF fractions suppress H2O2 induced oxidative damage in vitro Vero cells. Intracellular ROS scavenging activity (A) and cell viability (B) in H2O2 exposed Vero cells. Experiments were performed in triplicate and data are expressed as mean ± SD; Significant differences identified at * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 as compared to the H2O2 treated group; ####
p < 0.0001 as compared to the control group.
Figure 4. Effects of α-chy-III against H2O2 induced apoptotic cell death, DNA fragmentation and cell cycle regulation in Vero cells. Protective effect of α-chy-III in H2O2 induced apoptotic cell death (A) DNA fragmentation (B) and cell cycle regulation (C). The apoptotic cell death, DNA fragmentation and cell cycle regulation were analyzed via fluorescence microscopy before propidium iodide (PI) and hoechst 33342 staining. Experiments were performed in triplicate and data are expressed as mean ± SD; Significant differences identified at **** p < 0.0001 as compared to the H2O2 treated group; ####
p < 0.0001 as compared to the control group. Statistical analyses were conducted using Tukey’s post hoc comparison and Duncan’s multiple range test.
Figure 5. Effect of α-chy-III on H2O2 induced oxidative stress in survival rate (A), heart rate (B), cell death (C), ROS generation (D), and lipid peroxidation (E) in zebrafish embryos. Levels of fluorescence intensity were calculated using ImageJ software. Experiments were performed in triplicate and data are expressed as mean ± SD; Significant differences identified at * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 as compared to the H2O2 treated group; ##
p < 0.01 and ####
p < 0.0001 as compared to the control group. Statistical analyses were conducted using Tukey’s post hoc comparison and Duncan’s multiple range.
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