Bioactive Peptides from Cartilage Protein Hydrolysate of Spotless Smoothhound and Their Antioxidant Activity In Vitro

Abstract

In the experiment, crude proteins from spotless smoothhound (Mustelus griseus), cartilages were isolated by HCl-Guanidine buffer, and its hydrolysate was prepared using trypsin at pH 8.0, 40 °C with a total enzyme dose of 2.5%. Subsequently, three antioxidant peptides were purified from the hydrolysate using membrane ultrafiltration, anion-exchange chromatography, gel filtration chromatography, and reverse phase high-performance liquid chromatography. The amino acid sequences of isolated peptides were identified as Gly-Ala-Glu-Arg-Pro (MCPE-A); Gly-Glu-Arg-Glu-Ala-Asn-Val-Met (MCPE-B); and Ala-Glu-Val-Gly (MCPE-C) with molecular weights of 528.57, 905.00, and 374.40 Da, respectively, using protein amino acid sequence analyzer and mass spectrum. MCPE-A, MCPE-B and MCPE-C exhibited good scavenging activities on 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH•) (EC₅₀ 3.73, 1.87, and 2.30 mg/mL, respectively), hydroxyl radicals (HO•) (EC₅₀ 0.25, 0.34, and 0.06 mg/mL, respectively), 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radicals (ABTS⁺•) (EC₅₀ 0.10, 0.05, and 0.07 mg/mL, respectively) and superoxide anion radicals ( O 2 - •) (EC₅₀ 0.09, 0.33, and 0.18 mg/mL, respectively). MCPE-B showed similar inhibiting ability on lipid peroxidation with butylated hydroxytoluene (BHT) in a linoleic acid model system. Furthermore, MCPE-A, MCPE-B, and MCPE-C could protect H₂O₂-induced HepG2 cells from oxidative stress by decreasing the content of malonaldehyde (MDA) and increasing the levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione reductase (GSH-Rx). Glu, Gly, Met, and Pro in their sequences and low molecular weight could be attributed to the antioxidant activities of three isolated peptides. These results suggested that GAERP (MCPE-A), GEREANVM (MCPE-B), and AEVG (MCPE-C) from cartilage protein hydrolysate of spotless smoothhound might serve as potential antioxidants and be used in the pharmaceutical and health food industries.

Keywords: antioxidant activity; antioxidant enzyme; cartilage; peptide; protein hydrolysate; spotless smoothhound (Mustelus griseus).

Figure 1

Elution profile of MGCH-I through DEAE-52 cellulose chromatography (A); and 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH•) and hydroxyl radicals (HO•) scavenging activities of MGCH-I and its subfractions at a concentration of 15 mg protein/mL (B). All data are presented as the mean ± SD of triplicate results.

Figure 2

Elution profile of Frac.4 in Sephadex G-15 chromatography (A) and radical scavenging activities of Frac.4 and its fractions at 10 mg protein/mL concentration (B). All data are presented as the mean ± SD of triplicate results.

Figure 3

Elution profile of Frac.4-1 separated by reverse-phase high performance liquid chromatography (RP-HPLC) system on a Zorbax, SB C-18 column (4.6 × 250 mm) from 0 to 40 min.

Figure 4

Mass spectra of Gly-Ala-Glu-Arg-Pro (MCPE-A) (A); Gly-Glu-Arg-Glu-Ala-Asn-Val-Met (MCPE-B) (B); and Ala-Glu-Val-Gly (MCPE-C) (C) from cartilage protein hydrolysate of spotless smoothhound.

Figure 5

DPPH• (A); HO• (B); superoxide anion radicals (${\mathrm{O}}_2^{-}$•) (C); and 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radicals (ABTS⁺•) (D) scavenging activities of MCPE-A, MCPE-B, and MCPE-C from cartilage protein hydrolysate of spotless smoothhound. All data are presented as the mean ± SD of triplicate results.

Figure 6

Lipid peroxidation inhibition assays of MCPE-A, MCPE-B and MCPE-C from cartilage protein hydrolysate of spotless smoothhound. All data are presented as the mean ± SD of triplicate results.

Figure 7

Cytotoxicity of MCPE-A, MCPE-B, and MCPE-C in HepG2 cells at concentrations of 10.0 µg/mL, 25.0 µg/mL, and 50.0 µg/mL, respectively.

Figure 8

Damage effect of H₂O₂ on HepG2 cells (A) and protective effects of MCPE-A, MCPE-B, and MCPE-C on H₂O₂-induced oxidative damage in HepG2 cells at concentrations of 10.0 µg/mL, 25.0 µg/mL, and 50 µg/mL, respectively; (B) All data are presented as the mean ± SD of triplicate results. ^# p < 0.05 vs. the control group. * p < 0.05 vs. the H₂O₂ treated group. ** p < 0.01 vs. the H₂O₂ treated group.

Figure 9

The effects of MCPE-A, MCPE-B and MCPE-C on the levels of dismutase (T-SOD) (A), catalase (CAT) (B), glutathione peroxidase (GSH-Px) (C), glutathione reductase (GSH-Rx) (D), and content of malonaldehyde (MDA) (E) in H₂O₂-induced HepG2 cells. All data are presented as the mean ± SD of triplicate results. ^# p < 0.05 vs. the control group. * p < 0.05 vs. the H₂O₂ treated group.

Figure 9

The effects of MCPE-A, MCPE-B and MCPE-C on the levels of dismutase (T-SOD) (A), catalase (CAT) (B), glutathione peroxidase (GSH-Px) (C), glutathione reductase (GSH-Rx) (D), and content of malonaldehyde (MDA) (E) in H₂O₂-induced HepG2 cells. All data are presented as the mean ± SD of triplicate results. ^# p < 0.05 vs. the control group. * p < 0.05 vs. the H₂O₂ treated group.