Bioactive peptides as a novel strategy to prevent alcoholic liver injury.

Alcohol intake has become one of the leading risks to human health and wellness, among which acute and/or chronic alcohol-induced liver injury is a leading threaten, with few therapeutic options other than abstinence. In recent years, studies suggested that certain bioactive peptides from food sources could represent natural and safe alternatives for the prevention of alcoholic liver injury. Hence, this chapter focus on the advanced research on bioactive peptides exerting hepatoprotective activity against alcoholic liver injury. The main sources of protein, strategies for the preparation of hepatoprotective hydrolysates and peptides, underlying mechanisms of peptides on hepatoprotection, and possible structure-activity relationship between peptides and hepatoprotective activity were summarized and discussed, aiming to give a systematic insight into the research progress of hepatoprotective peptides. However, more efforts would be needed to give a clearer insight into the underlying mechanisms and structure-activity relationship before using hepatoprotective peptides as functional food ingredients or dietary supplements.

Musculus senhousei peptides alleviated alcoholic liver injury via the gut-liver axis.

Alcoholic liver injury has become a leading threat to human health, with complicated pathogenesis and limited therapeutic options. Our previous study showed that Musculus senhousei peptides (MSPs) exhibit protective potential against early-stage alcoholic liver injury, although the underlying mechanism is not yet clear. In this study, histopathological analysis, mRNA abundance of injury-associated biomarkers, the gut microbiota, and faecal metabolome were evaluated using a mouse model subjected to acute alcohol exposure, aiming to identify the mechanism by which MSP can alleviate alcoholic hepatotoxicity. The results showed that MSP intervention significantly ameliorated symptoms of liver injury (suppressed serum ALT increment, hepatic lipid accumulation, and neutrophil infiltration in liver tissue), and reversed the abnormal mRNA abundance of biomarkers associated with oxidative stress (iNOS), inflammation (TNF-α, IL-1ß, MCP-1, TNF-R1, and TLR4), and apoptosis (Bax and Casp. 3) in the liver. Moreover, MSP improved intestinal barrier function by increasing the expression of tight junction proteins (Claudin-1 and Claudin-3). Further analysis of faecal microbiota and metabolome revealed that MSP promoted the growth of tryptophan-metabolizing bacteria (Clostridiales, Alistipes, and Odoribacter), leading to increased production of indole derivatives (indole-3-lactic acid and N-acetyltryptophan). These results suggested that MSPs may alleviate alcohol-induced liver injury targeting the gut-liver axis, and could be an effective option for the prevention of alcoholic liver injury.

Musculus senhousei as a promising source of bioactive peptides protecting against alcohol-induced liver injury.

Alcohol-induced liver injury has become a leading risk for human health, however, effective strategies for the prevention or treatment are still lacking. Hence, the present study explored the potential of Musculus senhousei as a source of hepatoprotective peptides against alcoholic liver injury using in vitro, in vivo and in silico methods. Results indicated that Musculus senhousei peptides (MSP, extracted by simulated gastrointestinal digestion of cooked mussel) exhibited notable antioxidant (ABTS and DPPH assays) and alcohol dehydrogenase (ADH) stabilizing activity in vitro. The ingestion of MSP markedly alleviated alcohol-induced liver injury in mice, as indicated by the decrease of serum transaminases (AST and ALT). In line with in vitro assays, significantly increased hepatic ADH activity and activated antioxidative defense system (GSH, SOD, GSH-Px and CAT) were observed, whereas the oxidative stress (MDA) was decreased. Peptidomic analysis revealed over 6000 peptides with favorable amino acid compositions, and a total of 20 potentially novel peptides with bioactivity and bioavailability were excavated among 746 of the most influential peptides using an in silico strategy. Peptides (i.e. WLPMKL, WLWLPA, RLC and RCL) were further synthesized and validated in vitro to be bioactive. These findings suggest that Musculus senhousei can be an ideal source of bioactive peptides for the prevention of alcoholic liver injury.

In vitro and in silico analysis of potential antioxidant peptides obtained from chicken hydrolysate produced using Alcalase.

Chicken hydrolysates (CHs) have been reported to protect mice against alcoholic liver injury possibly through oxidative stress reduction. In this study, the antioxidant activity of CHs was studied. Results showed that CHs exhibited significant antioxidant activity (around 600 and 400 µM TEAC/g in ORAC and ABTS assay, respectively) and could resist simulated gastrointestinal digestion. A total of 22 peptides were identified after antioxidant activity-oriented isolation using size-exclusion chromatography and high-performance liquid chromatography. Further in silico analysis and the validation of antioxidant activity revealed that novel peptides (RWGG and YYCQ) exhibited strong antioxidant activity. The most active peptide YYCQ displayed a TEAC value of 3.54 and 4.28 µM TEAC/µM in ORAC and ABTS assay, respectively. These peptides could contribute to reduce oxidative stress and protect against alcohol-induced liver injury. However, further studies understanding the bioactivity of such peptides in vivo are necessary before further applying them as functional food ingredient.

Effect of cooking and in vitro digestion on the peptide profile of chicken breast muscle and antioxidant and alcohol dehydrogenase stabilization activity.

The present study aimed to investigate the effect of cooking and simulated gastrointestinal digestion on the antioxidant and alcohol dehydrogenase (ADH) stabilization activity of peptides extracted from chicken breast muscle. Results showed that cooking would not affect peptide bioactivity, whereas further digestion using gastrointestinal enzymes could lead to significant changes, producing an increase in ORAC (112.5 to 682.0 uM TE/g) and ABTS radical scavenging activities (164.0 to 848.9 uM TE/g), whereas a decrease in DPPH radical scavenging (from 36.1% to 4.4%), ferric-reducing power (OD 700 from 0.50 to 0.15) and ADH stabilization activities (from 44.1% to 20.5%) was observed. The peptidomic analysis resulted in the identification and relative quantitation of 777 peptides from 76 different parent proteins and evidenced that peptides derived from titin and collagen were mainly responsible for the differences detected in the peptide profile. The decrease of DPPH radical scavenging, ferric reducing power, and ADH stabilization activity may result from the release of inactive peptides containing oxidized residues, mainly from collagen, leading to the loss of efficacy of active sequences. The results confirmed the importance of collagen derived peptides on the antioxidant and ADH stabilization activity observed in chicken breast as well as the negative impact of oxidation on the bioactivity of generated peptides after simulated gastrointestinal digestion. Nevertheless, further work would be needed to confirm the peptide sequences responsible for the observed bioactivity.

Chicken breast-derived alcohol dehydrogenase-activating peptides in response to physicochemical changes and digestion simulation: The vital role of hydrophobicity.

Chicken breast muscle hydrolysates (CBMHs) could promisingly activate alcohol dehydrogenase (ADH) and ameliorate alcohol-induced liver injury. The aim of this work was to investigate the stability of CBMHs against physicochemical treatments and gastrointestinal digestion simulation. Results indicated that CBMHs showed good stability towards heating (25-121 °C), pH treatment (pH 2-12) and remained stable in the presence of NaCl (0.01-2 M) and low concentration of metal ions (0.1 mM Zn2+, Ca2+, Fe2+, and Fe3+). Results from in vitro digestion implied a retained activity of CBMHs after gastric tract, but marked decrease (33.42%) after intestinal tract. UPLC-ESI-Q-ToF-MS/MS analysis together with in silico assessments then revealed that the degradation of hydrophobic peptides (i.e., VAPEEHPTLL, YPGIADRM, ADGPLKGIL, and KDLFDPVIQ) during simulated intestinal digestion may be account for the decreased activity. Conformational changes of ADH upon hydrophobic interaction with synthetic peptides were further confirmed by fluorescence quenching study, possibly responsible for the enhanced ADH activity. Hence, CBMHs noticeably showed good stability against physicochemical treatments and digestion simulation, while attempt establishing the structure-activity relationship of peptides is also fundamental before applying CBMHs as functional ingredient.

Chicken breast muscle hydrolysates ameliorate acute alcohol-induced liver injury in mice through alcohol dehydrogenase (ADH) activation and oxidative stress reduction.

In this study, the ameliorative effect of chicken breast muscle hydrolysates (CBMHs) against acute alcohol-induced liver injury was investigated and its probable mechanism was further elucidated. In vitro studies clearly showed that CBMHs are able to activate alcohol metabolic enzymes (i.e. alcohol dehydrogenase, ADH) in an exponential manner. Meanwhile, an in vivo experiment on male NIH mice indicated that the oral administration of CBMHs (150, 300 and 600 mg per kg bw) 30 min prior to acute alcohol ingestion could significantly promote alcohol metabolism as revealed by the reduced duration of the loss of righting reflex (LORR) and the enhanced activity of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in the liver, the latter accelerating alcohol oxidation and therefore the decreased blood alcohol concentration (BAC) level. Pretreatment with CBMHs significantly decreased the elevations of serum aspartate transaminase (AST) and alanine transaminase (ALT) after alcohol administration. CBMHs could also retard lipid peroxidation as revealed by the suppressed malondialdehyde (MDA) level and simultaneously enhance the activities of superoxide dismutase (SOD) in liver tissue. Furthermore, increased histological damage and higher (p < 0.05) hepatic triglyceride (TG) contents in acute alcoholic-diet fed mice were also reduced (p < 0.05) by supplementing with CBMHs. These benefits clearly suggested that CBMHs could be a potential nutraceutical to facilitate alcohol metabolism and prevent or ameliorate early liver injury induced by acute alcohol exposure.

Practical problems when using ABTS assay to assess the radical-scavenging activity of peptides: Importance of controlling reaction pH and time.

Effects of reaction pH and time on the antioxidant behaviors of Tyr, Trp, Cys, and their related peptides (Tyr-Gly, Tyr-Glu, Tyr-Lys, Trp-Gly, Trp-Glu, Trp-Lys, Cys-Gly and Cys-Gly) in ABTS assay were investigated. Results showed that all these amino acids and peptides displayed a biphasic kinetic pattern with a fast initial step and a slow secondary step. The initial reaction rates of Tyr, Trp and their related peptides were strongly dependent on pH, while those of Cys and Cys-containing peptides were unaffected by pH. They failed to reach equilibrium over the short incubation period of 6-10 min typically used in this assay. Longer incubation time was needed for most of the peptides to approach equilibrium at lower pH. The observed biphasic kinetic pattern as well as the high TEAC values for these amino acids and peptides, could be a result of combined antioxidant behaviors of themselves plus the generated reaction products.

Mechanism of the discrepancy in the enzymatic hydrolysis efficiency between defatted peanut flour and peanut protein isolate by Flavorzyme.

Both defatted peanut flour (DPF) and peanut protein isolate (PPI) are widely used to prepare peanut protein hydrolysates. To compare their enzymatic hydrolysis efficiencies, DPF and PPI were hydrolysed by Alcalase, Neutrase, Papain, Protamex and Flavorzyme. Alcalase and Flavorzyme were found to be the most efficient proteases to hydrolyse both DPF and PPI. The efficiency was comparable to each other when using Alcalase, while PPI was hydrolysed less efficiently than DPF when using Flavorzyme. Analysis of changes in the protein solubility, subunit and conformation, and amino acid composition of DPF, PPI and their Flavorzyme hydrolysis residues indicated that the PPI preparation process had minimal effect on it, but peptide aggregation via non-covalent bonding (including hydrophobic interactions and hydrogen bonds) during hydrolysis and/or thermal treatment after hydrolysis were likely responsible for the reduced hydrolysis efficiency of PPI by Flavorzyme.