In vivo antioxidant activity of mackerel (Scomber japonicus) muscle protein hydrolysate

Abstract

Pacific chub mackerel (Scomber japonicus) is an important fish throughout the world, especially in East Asian countries, including Korea, China, and Japan. Protein hydrolysates from marine sources are commonly used as nutritional supplements, functional ingredients, and flavor enhancers in the food, beverage, and pharmaceutical industries. Antioxidants isolated from fish are relatively easy to prepare, are cost effective, and have no reported side effects. Hence, the present study aimed to investigate the in vivo antioxidant activities of mackerel muscle protein hydrolysate (MMPH) prepared using Protamex. The in vivo bioactivities of MMPH were investigated in alcoholic fatty liver mice (C57BL/6). Serum alanine aminotransferase and aspartate aminotransferase levels were comparable in test and control mice, whereas serum triglyceride and lipid peroxidation levels significantly (p < 0.05; p < 0.001) decreased after administration of MMPH (100-500 mg kg^-1), especially at a concentration of 100 mg kg^-1. A significant (p < 0.05) reduction in xanthine oxidase activity was observed in all groups treated with MMPH (100-500 mg kg^-1), as compared with the control group. Significantly (p < 0.05) higher superoxide dismutase (SOD) activity/protein expression and regulated catalase (CAT) activity/protein expression levels were observed in groups administered MMPH (100-500 mg kg^-1), especially at a concentration of 100 mg kg^-1. These results show that the abundant amino acids of S. japonicus play an important role in the cytosol of the liver cells by directly participating in the expression of xanthine oxidase and the detoxifying SOD and CAT proteins, thereby enhancing antioxidant ability and ultimately, inhibiting lipid peroxidation. This study demonstrated that muscle protein hydrolysate from S. japonicus has strong antioxidant activities.

Keywords: Enzymatic hydrolysis; In vivo antioxidant activity; Protamex; Protein Hydrolysate; SOD and CAT protein expression.

Figure 1. Animal experimental design.

Figure 2. Serum biochemical properties.

Serum (A) ALT; (B) AST; (C) total cholesterol; and (D) triglyceride levelsValues are expressed as mean ± S.E. for groups of eight animals; N: normal; C: control; P: positive control; M100, M250, and M500: mackerel muscle protein hydrolysate at concentrations of 100, 250, and 500 mg kg⁻¹, respectively. n.s: not significant; ∗p < 0.05 vs. control, †p < 0.001 vs. control p: p value of ANOVA and significance between control and test groups using Fisher’s PLSD post hoc test.

Figure 3. Effect of mackerel muscle protein hydrolysate on liver lipid peroxidation levels.

Values are expressed as mean ± S.E. for groups of eight animals; N: normal; C: control; P: positive control; M100, M250, and M500: mackerel muscle protein hydrolysate at concentrations of 100, 250, and 500 mg kg⁻¹, respectively. n.s: not significant; ∗p < 0.05 vs. control p: p value of ANOVA and significance between control and test groups using Fisher’s PLSD post hoc test.

Figure 4. Effect of mackerel muscle protein hydrolysate on aldehyde oxidase and xanthine oxidase activity in liver.

(A) aldehyde oxidase and (B) xanthine oxidase activityValues are expressed as mean ± S.E. for groups of eight animals; N: normal; C: control; P: positive control; M100, M250, and M500: mackerel muscle protein hydrolysate at concentrations of 100, 250, and 500 mg kg⁻¹, respectively. n.s: not significant; ∗p < 0.05 vs. control, †p < 0.001 vs. control p: p value of ANOVA and significance between control and test groups using Fisher’s PLSD post hoc test.

Figure 5. Effect of mackerel muscle protein hydrolysate on SOD and CAT protein levels in liver.

(A) SOD and (B) CAT protein levels; (C) SOD and CAT protein expression measured by western blotValues are expressed as mean  ± S.E. for groups of eight animals; N: normal; C: control; P: positive control; M100, M250, and M500: mackerel muscle protein hydrolysate at concentrations of 100, 250, and 500 mg kg⁻¹, respectively.Protein extracts were separated on 12% SDS-polyacrylamide gels and after transfer to polyvinylidene fluoride membranes, proteins were detected with monoclonal SOD and CAT antibodies (Santa Cruz Biotechnology) and subsequently, visualized with a goat anti-mouse IgG (H+L) HRP-conjugated secondary antibody (Thermo Fisher Scientific). SOD and CAT expression were detected using a West Save Gold western blot detection kit (Abfrontier), and band intensity was measured using a Chemi-Doc XRS system (Bio-Rad). Results were normalized using a β-actin monoclonal antibody (Santa Cruz Biotechnology) as an internal standard. Additionally, experiments were repeated at least three times to minimize errors. n.s: not significant; ∗p < 0.05 vs. control, †p < 0.001 vs. control. p: p value of ANOVA and significance between control and test groups using Fisher’s PLSD post hoc test.