Identification and Characterization of Edible Cricket Peptides on Hypertensive and Glycemic In Vitro Inhibition and Their Anti-Inflammatory Activity on RAW 264.7 Macrophage Cells

Abstract

Recent studies continue to demonstrate the potential of edible insects as a protein base to obtain bioactive peptides applicable for functional food development. This study aimed at identifying antihypertensive, anti-glycemic, and anti-inflammatory peptides derived from the in vitro gastrointestinal digests of cricket protein hydrolysates. After sequential fractionation, the protein digest subfraction containing the lowest molecular weight (<0.5 kDa), hydrophobic (C18) and cationic peptides (IEX) was found responsible for the most bioactivity. The cationic peptide fraction significantly reduced (p < 0.05) α-amylase, α-glucosidase, and angiotensin converting enzyme (ACE) activity in vitro, and also inhibited the expression of NF-κB in RAW 264.7 macrophage cells. A total of 28 peptides were identified with mass spectrometry (LC-MS/MS) and de novo sequencing from the potent fraction. Three novel peptides YKPRP, PHGAP, and VGPPQ were chosen for the molecular docking studies. PHGAP and VGPPQ exhibited a higher degree of non-covalent interactions with the enzyme active site residues and binding energies comparable to captopril. Results from this study demonstrate the bioactive potential of edible cricket peptides, especially as ACE inhibitors.

Keywords: ACE inhibition; cationic peptides; cricket protein.

Figure 1

Molecular weight distribution of cricket protein after enzymatic hydrolysis and in vitro digestion. (a) Gel filtration peptide profile of non-hydrolyzed cricket protein, cricket protein hydrolyzed with alcalase, and cricket protein hydrolysates (CPH), after digestion with gastrointestinal enzymes. (b) Gel filtration peptide profile of non-hydrolyzed cricket protein, cricket protein hydrolyzed with alcalase, and CPH, after digestion with gastrointestinal enzymes.

Figure 2

Peptides from (a) CPHD and (b) its cationic peptide fraction inhibits LPS-induced inflammation in RAW 264.7 Cells. Different lowercase letters indicate significant differences between treatments (p < 0.05). Samples were analyzed in quadruplicate (n = 4). Cells were stimulated for 6 h with 500 ng of lipopolysaccharide (LPS) or pre-treated with CPHD (a), cationic fractions, or PBS (NTC = non-treated control). The data are expressed as the Renilla luciferase activity in relative light units (RLU). Data represent the mean ± SD of quadruplicate samples, representative of four independent experiments.

Figure 3

Percentage of macrophage cell viability after exposure to (a) CPHD and (b) its cationic peptide fraction. CPHD = cricket protein hydrolysates after simulated digestion with gastrointestinal enzymes. Cells were incubated with CPHD (100–1000µg/mL), cationic peptide fraction (0.5–10 µg/mL), or DMSO (0.25%), for 12 h, to evaluate cytotoxicity by MTT assay, with peptides. There was no significant (p > 0.05) reduction in viability, as compared to the DMSO-treated cells. Data represent the mean ± SD representative of eight (n = 8) independent experiments.

Figure 4

2D model of the predicted binding mode of (a) YKPRP, (b) PHGAP, (c) VGPPQ, and (d) captopril to the angiotensin-converting enzyme (ACE). Images were obtained with Discovery Studios Visualizer Software and the best scored docking pose is shown.