Antioxidant, Antibacterial Properties of Novel Peptide CP by Enzymatic Hydrolysis of Chromis notata By-Products and Its Efficacy on Atopic Dermatitis

Abstract

This study investigated the antioxidant, antimicrobial, and anti-atopic dermatitis (AD) effects of a novel peptide (CP) derived from a Chromis notata by-product hydrolysate. Alcalase, Flavourzyme, Neutrase, and Protamex enzymes were used to hydrolyze the C. notata by-product protein, and the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical-scavenging activity was measured. Alcalase hydrolysate exhibited the highest ABTS radical-scavenging activity, leading to the selection of Alcalase for further purification. The CHAO-1-I fraction, with the highest ABTS activity, was isolated and further purified, resulting in the identification of the peptide CP with the amino acid sequence Ala-Gln-Val-Met-Lys-Leu-Pro-His-Arg-Met-Gln-His-Ser-Gln-Ser. CP demonstrated antimicrobial activity against Staphylococcus aureus, inhibiting its growth. In a 2,4-dinitrochlorobenzene (DNCB)-induced AD-like skin model in mice, CP significantly alleviated skin lesions, reduced epidermal and dermal thickness, and inhibited mast cell infiltration. Moreover, CP suppressed the elevated levels of interleukin-6 (IL-6) in the plasma of DNCB-induced mice. These findings highlight the potential of CP as a therapeutic agent for AD and suggest a novel application of this C. notata by-product in the fish processing industry.

Keywords: Chromis notata; antimicrobial activity; antioxidant peptide; atopic dermatitis; by-product hydrolysate.

Figure 1

Purification of Alcalase hydrolysate. (A) FPLC pattern of hydrolysate by 3500 Da dialysis membrane, and the ABTS radical-scavenging activities (lower panel) of the fractions. (B) FPLC pattern on GPC column of the CHAO active fraction, and the ABTS radical-scavenging activities (lower panel) of the fractions. (C) Reversed-phase HPLC pattern of the CHAO-1 active fraction, and the ABTS radical-scavenging activities (lower panel) of the fractions. (D) HPLC pattern with a GPC column of the CHAO-1-I active fraction. All values are expressed as mean ± SD. Different letters are significantly different at p < 0.05 according to Duncan’s multiple range test.

Figure 1

Purification of Alcalase hydrolysate. (A) FPLC pattern of hydrolysate by 3500 Da dialysis membrane, and the ABTS radical-scavenging activities (lower panel) of the fractions. (B) FPLC pattern on GPC column of the CHAO active fraction, and the ABTS radical-scavenging activities (lower panel) of the fractions. (C) Reversed-phase HPLC pattern of the CHAO-1 active fraction, and the ABTS radical-scavenging activities (lower panel) of the fractions. (D) HPLC pattern with a GPC column of the CHAO-1-I active fraction. All values are expressed as mean ± SD. Different letters are significantly different at p < 0.05 according to Duncan’s multiple range test.

Figure 2

Structural characterization of C. notata by-product derived from the CP peptide. (A) Chemical structure of the CP peptide. (B) Synthesis results of C. notata by-product-derived CP peptide. (C) An example of a helical wheel diagram illustrated for the CP peptide, showing the net projection (left panel) and the wheel projection (right panel) for the CP peptide. (D) Antiparallel alignment of two helical wheels of the CP peptide.

Figure 2

Structural characterization of C. notata by-product derived from the CP peptide. (A) Chemical structure of the CP peptide. (B) Synthesis results of C. notata by-product-derived CP peptide. (C) An example of a helical wheel diagram illustrated for the CP peptide, showing the net projection (left panel) and the wheel projection (right panel) for the CP peptide. (D) Antiparallel alignment of two helical wheels of the CP peptide.

Figure 3

Antimicrobial activity of novel peptide CP. (A) Agar disk diffusion test; 1 = 1.25 mM CP; 2 = 2.5 mM CP; 3 = 5 mM CP; 4 = 25 ng penicillin. (B) Effect of CP on growth of S. aureus. OD₄₉₀ = optical density at a wavelength of 490 nm.

Figure 4

Effect of CP on body weight and histopathological changes in DNCB-induced AD mouse model. (A) Changes in body weight of DNCB-induced AD mice during the experiment. (B) Clinical severity of inflammatory skin lesions. Photographs were taken on day 28. (C,E,F) The thicknesses of the epidermis and dermis were examined by Masson’s trichrome staining of the skin sections. (D) The infiltration of mast cells in the dermis was examined by toluidine blue staining of the skin section. (G) The mast cells were counted in 3 fields. All results are shown as the mean ± SD (n = 5 per group). ## p < 0.01 compared to the control group. * p < 0.05 and ** p < 0.01, compared to the DNCB group. Normal, untreated group; DNCB, DNCB-sensitized group; CP, 1 mg CP-treated group; Terfenadine, 0.1 mg Terfenadine-treated group; E, epidermal thickness; D, dermal thickness.

Figure 4

Effect of CP on body weight and histopathological changes in DNCB-induced AD mouse model. (A) Changes in body weight of DNCB-induced AD mice during the experiment. (B) Clinical severity of inflammatory skin lesions. Photographs were taken on day 28. (C,E,F) The thicknesses of the epidermis and dermis were examined by Masson’s trichrome staining of the skin sections. (D) The infiltration of mast cells in the dermis was examined by toluidine blue staining of the skin section. (G) The mast cells were counted in 3 fields. All results are shown as the mean ± SD (n = 5 per group). ## p < 0.01 compared to the control group. * p < 0.05 and ** p < 0.01, compared to the DNCB group. Normal, untreated group; DNCB, DNCB-sensitized group; CP, 1 mg CP-treated group; Terfenadine, 0.1 mg Terfenadine-treated group; E, epidermal thickness; D, dermal thickness.

Figure 5

Inhibitory effect of CP on secreted IL-6 cytokines in the DNCB-induced mouse serum. All results are shown as the mean ± SD (n = 5 per group). ## p < 0.01 compared to the control group. ** p < 0.01 compared to the DNCB group. Normal, untreated group; DNCB, DNCB-sensitized group; CP, 1 mg CP-treated group; Terfenadine, 0.1 mg Terfenadine-treated group.

Figure 6

Experimental procedure of the model of DNCB-induced atopic dermatitis. The mice in the Normal group were treated with 9:1 PBS/olive oil. AD was induced by DNCB in BALB/c mice. Following initial sensitization with 1% DNCB, the BALB/c mice were dorsally treated with 0.5% DNCB 3 times a week for 3 weeks. CP and terfenadine preparations of 1 and 0.1 mg in 200 µL of PBS/olive oil (9:1) were applied on the dorsal skin of the mice in the CP and terfenadine groups every day for 21 days. Normal, untreated group; DNCB, DNCB-sensitized group; CP, CP 1 mg treated group; Terfenadine, Terfenadine 0.1 mg treated group.