Nowadays, notwithstanding their nutritional and technological properties, food bioactive peptides from plant sources garner increasing attention for their ability to impart more than one beneficial effect on human health. Legumes, which stand out thanks to their high protein content, represent valuable sources of bioactive peptides. In this context, this study focused on the characterization of the potential pleotropic activity of two commercially available soybean (SH) and pea (PH) protein hydrolysates, respectively. Since the biological activity of a specific protein hydrolysate is strictly correlated with its chemical composition, the first aim of the study was to identify the compositions of the SH and PH peptides. Peptidomic analysis revealed that most of the identified peptides within both mixtures belong to storage proteins. Interestingly, according to the BIOPEP-UWM database, all the peptides contain more than one active motive with known inhibitory angiotensin converting enzyme (ACE) and dipeptidyl-dipeptidases (DPP)-IV sequences. Indeed, the results indicated that both SH and PH inhibit DPP-IV and ACE activity with a dose-response trend and IC₅₀ values equal to 1.15 ± 0.004 and 1.33 ± 0.004 mg/mL, and 0.33 ± 0.01 and 0.61 ± 0.05 mg/mL, respectively. In addition, both hydrolysates reduced the activity of DPP-IV and ACE enzymes which are expressed on the surface of human intestinal Caco-2 cells. These findings clearly support that notion that SH and PH may represent new ingredients with anti-diabetic and hypotensive effects for the development of innovative multifunctional foods and/or nutraceuticals for the prevention of metabolic syndrome.

P5 (LILPKHSDAD) is a hypocholesterolemic peptide from lupin protein with a multi-target activity, since it inhibits both 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAR) and proprotein convertase subtilisin/kexin type-9 (PCSK9). This work shows that, during epithelial transport experiments, the metabolic transformation mediated by intestinal peptidases produces two main detected peptides, ILPKHSDAD (P5-frag) and LPKHSDAD (P5-met), and that both P5 and P5-met are linearly absorbed by differentiated human intestinal Caco-2 cells. Extensive comparative structural, biochemical, and cellular characterizations of P5-met and the parent peptide P5 demonstrate that both peptides have unique characteristics and share the same mechanisms of action. In fact, they exert an intrinsically multi-target behavior being able to regulate cholesterol metabolism by modulating different pathways. The results of this study also highlight the dynamic nature of bioactive peptides that may be modulated by the biological systems they get in contact with.

The present study aimed at characterizing the possible biological activities of the multifunctional low molecular weight fractions (<3 kDa) peptides isolated from rainbow trout (Oncorhynchus mykiss) obtained by enzymatic hydrolysis. The fish protein hydrolysate (FPH) was tested for its antioxidant property along with its angiotensin converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV) inhibitory activities. In particular, the 2,2-diphenyl-1-picrylhydrazyl (DPPH), the ferric reducing antioxidant power (FRAP), the oxygen radical absorbance capacity (ORAC) assay and the 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assays were carried out for the evaluation of the in vitro antioxidant activity. The cell-free ACE and DPP-IV inhibitory activity assays were also estimated, showing a dose-dependent inhibition. These biological properties were additionally quantified at the cellular level using human intestinal Caco-2 cells. Namely, the antioxidant activity was determined by evaluating the capability of the hydrolysate to reduce the H₂O₂-induced reactive oxygen species (ROS) and lipid peroxidation levels, and the DPP-IV activity assays show a reduction of enzyme activity of up to 27.57 ± 3.7% at 5 mg/mL. The results indicate that Oncorhynchus mykiss-derived peptides may have potential employment as health-promoting ingredients.

Agri-food industry wastes and by-products include highly valuable components that can upgraded, providing low-cost bioactives or used as an alternative protein source. In this context, by-products from olive production and olive oil extraction process, i.e., seeds, can be fostered. In particular, this work was aimed at extracting and characterizing proteins for Olea europaea L. seeds and at producing two protein hydrolysates using alcalase and papain, respectively. Peptidomic analysis were performed, allowing to determine both medium- and short-sized peptides and to identify their potential biological activities. Moreover, an extensive characterization of the antioxidant properties of Olea europaea L. seed hydrolysates was carried out both in vitro by 2,2-diphenyl-1-picrylhydrazyl (DPPH), by ferric reducing antioxidant power (FRAP), and by 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assays, respectively, and at cellular level by measuring the ability of these hydrolysates to significant reduce the H₂O₂-induced reactive oxygen species (ROS) and lipid peroxidation levels in human intestinal Caco-2 cells. The results of the both hydrolysates showed significant antioxidant properties by reducing the free radical scavenging activities up to 65.0 ± 0.1% for the sample hydrolyzed with alcalase and up to 75.7 ± 0.4% for the papain hydrolysates tested at 5 mg/mL, respectively. Moreover, similar values were obtained by the ABTS assays, whereas the FRAP increased up to 13,025.0 ± 241.5% for the alcalase hydrolysates and up to 12,462.5 ± 311.9% for the papain hydrolysates, both tested at 1 mg/mL. According to the in vitro results, both papain and alcalase hydrolysates restore the cellular ROS levels up 130.4 ± 4.24% and 128.5 ± 3.60%, respectively, at 0.1 mg/mL and reduce the lipid peroxidation levels up to 109.2 ± 7.95% and 73.0 ± 7.64%, respectively, at 1.0 mg/mL. In addition, results underlined that the same hydrolysates reduced the activity of dipeptidyl peptidase-IV (DPP-IV) in vitro and at cellular levels up to 42.9 ± 6.5% and 38.7 ± 7.2% at 5.0 mg/mL for alcalase and papain hydrolysates, respectively. Interestingly, they stimulate the release and stability of glucagon-like peptide 1 (GLP-1) hormone through an increase of its levels up to 660.7 ± 21.9 pM and 613.4 ± 39.1 pM for alcalase and papain hydrolysates, respectively. Based on these results, olive seed hydrolysates may represent new ingredients with antioxidant and anti-diabetic properties for the development of nutraceuticals and functional foods for the prevention of metabolic syndrome onset.

Phenylketonuria represents the most prevalent inborn error of amino acid metabolism. In early diagnosed patients adequate and continued dietary treatment results in a good neurologic outcome. However, due to the natural protein and phenylalanine-restricted diet, oxidative stress represents a concern in phenylketonuric patients. Clear evidences suggest that the pathophysiology of PKU is also dependent by mitochondrial impairment and oxidative stress. In this context due to the tight connection between oxidative and inflammatory stress and noncommunicable diseases (NCDs) development, it is reasonable to hypothesize that PKU patients may present a higher risk to develop NCDs during their life. Currently available protein substitutes on the market include free amino acids (L-AAs), prolonged-release protein substitute and formula containing glycomacropeptide (GMP). Our results suggest that free L-AAs significanlty worsens the intestinal hydrogen peroxide (H₂O₂₎ and lipopolysaccharides (LPS)-induced oxidative and inflammatory status in Caco-2 cells, which are significantly restored towards physiological condition by GMP alone and when present in a 1:1 mixture with free L-AAs, providing new preclinical piece of information which can shed a shadow on the mechanism of action of these products on PKU patients and their future management.

Hempseed (Cannabis sativa) protein is an important source of bioactive peptides. H3 (IGFLIIWV), a transepithelial transported intestinal peptide obtained from the hydrolysis of hempseed protein with pepsin, carries out antioxidant and anti-inflammatory activities in HepG2 cells. In this study, the main aim was to assess its hypocholesterolemic effects at a cellular level and the mechanisms behind this health-promoting activity. The results showed that peptide H3 inhibited the 3-hydroxy-3-methylglutaryl co-enzyme A reductase (HMGCoAR) activity in vitro in a dose-dependent manner with an IC₅₀ value of 59 μM. Furthermore, the activation of the sterol regulatory element binding proteins (SREBP)-2 transcription factor, followed by the increase of low-density lipoprotein (LDL) receptor (LDLR) protein levels, was observed in human hepatic HepG2 cells treated with peptide H3 at 25 µM. Meanwhile, peptide H3 regulated the intracellular HMGCoAR activity through the increase of its phosphorylation by the activation of AMP-activated protein kinase (AMPK)-pathways. Consequently, the augmentation of the LDLR localized on the cellular membranes led to the improved ability of HepG2 cells to uptake extracellular LDL with a positive effect on cholesterol levels. Unlike the complete hempseed hydrolysate (HP), peptide H3 can reduce the proprotein convertase subtilisin/kexin 9 (PCSK9) protein levels and its secretion in the extracellular environment via the decrease of hepatic nuclear factor 1-α (HNF1-α). Considering all these evidences, H3 may represent a new bioactive peptide to be used for the development of dietary supplements and/or peptidomimetics for cardiovascular disease (CVD) prevention.