Bioactive peptides derived from food

As interest in the ability of functional foods to impact on human health has grown over the past decade, so has the volume of knowledge detailing the beneficial roles of food-derived bioactive peptides. Bioactive peptides from both plant and animal proteins have been discovered, with to date, by far the most being isolated from milk-based products. A wide range of activities has been described, including antimicrobial and antifungal properties, blood pressure-lowering effects, cholesterol-lowering ability, antithrombotic effects, enhancement of mineral absorption, immunomodulatory effects, and localized effects on the gut. Although there is still considerable research to be performed in the area of food-derived bioactive peptides, it is clear that the generation of bioactive peptides from dietary proteins during the normal digestive process is of importance. Therefore, it will become necessary when determining dietary protein quality to consider the potential effects of latent bioactive peptides that are released during digestion of the protein.     

Bioactive peptides

Peptides with biological activities, released during gastrointestinal digestion or food processing, play an important role in metabolic regulation and modulation, suggesting their potential use as nutraceuticals and functional food ingredients for health promotion and disease risk reduction. Many studies have reported that peptides from various food sources possess bioactivities, including antihypertensive, antioxidant, anticancer, antimicrobial, and opioid activities as well as immunomodulatory and cholesterol-lowering effects. More studies are being performed exploring the sources, bioavailabilities, and possible physiological/functional properties and the mechanisms of action of bioactive peptides. Technological approaches in terms of peptide preparation, purification, and characterization have also been investigated.     

Bioactive molecular sheets from self-assembly of polymerizable peptides

We have demonstrated that polymerizable peptides self-assemble into a unique sheet-like 2D structure in bulk solution that can be covalently fixed to produce 2D molecular objects which were shown to be efficient at delivering cargos into living cells and are nearly nontoxic in contrast to non-polymerized nanostructures.     

Detection of small-molecule enzyme inhibitors with peptides isolated from phage-displayed combinatorial peptide libraries

BACKGROUND: The rapidly expanding list of pharmacologically important targets has highlighted the need for ways to discover new inhibitors that are independent of functional assays. We have utilized peptides to detect inhibitors of protein function. We hypothesized that most peptide ligands identified by phage display would bind to regions of biological interaction in target proteins and that these peptides could be used as sensitive probes for detecting low molecular weight inhibitors that bind to these sites. RESULTS: We selected a broad range of enzymes as targets for phage display and isolated a series of peptides that bound specifically to each target. Peptide ligands for each target contained similar amino acid sequences and competition analysis indicated that they bound one or two sites per target. Of 17 peptides tested, 13 were found to be specific inhibitors of enzyme function. Finally, we used two peptides specific for Haemophilus influenzae tyrosyl-tRNA synthetase to show that a simple binding assay can be used to detect small-molecule inhibitors with potencies in the micromolar to nanomolar range. CONCLUSIONS: Peptidic surrogate ligands identified using phage display are preferentially targeted to a limited number of sites that inhibit enzyme function. These peptides can be utilized in a binding assay as a rapid and sensitive method to detect small-molecule inhibitors of target protein function. The binding assay can be used with a variety of detection systems and is readily adaptable to automation, making this platform ideal for high-throughput screening of compound libraries for drug discovery.     

LocaPep: localization of epitopes on protein surfaces using peptides from phage display libraries

The use of peptides from a phage display library selected by binding to a given antibody is a widespread technique to probe epitopes of antigenic proteins. However, the identification of interaction sites mimicked by these peptides on the antigen surface is a difficult task. LocaPep is a computer program developed to localize epitopes using a new clusters algorithm that focuses on protein surface properties. The program is constructed with the aim of providing a flexible computational tool for predicting the location of epitopes on protein structures. As a first set of testing results, the localization of epitope regions in eight different antigenic proteins for which experimental data on their antibody interactions exist is correctly identified by LocaPep. These results represent a disparate sample of biologically different systems. The program is freely available at http://atenea.montes.upm.es.     

Mass spectrometric sequencing of individual peptides from combinatorial libraries via specific generation of chain-terminated sequences

Combinatorial peptide libraries are a versatile tool for drug discovery. On-bead assays identify reactive peptides by enzyme-catalyzed staining and, usually, sequencing by Edman degradation. Unfortunately, the latter method is expensive and time-consuming and requires free N termini of the peptides. A method of rapid and unambiguous peptide sequencing by utilizing synthesis-implemented generation of termination sequences with subsequent matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometric analysis is introduced here. The required capped sequences are determined and optimized for a specific peptide library by a computer algorithm implemented in the program Biblio. A total of 99.7% of the sequences of a heptapeptide library sample could be decoded utilizing a single bead for each spectrum. To synthesize these libraries, an optimized capping approach has been introduced.     

Diversity of phage-displayed libraries of peptides during panning and amplification

The amplification of phage-displayed libraries is an essential step in the selection of ligands from these libraries. The amplification of libraries, however, decreases their diversity and limits the number of binding clones that a screen can identify. While this decrease might not be a problem for screens against targets with a single binding site (e.g., proteins), it can severely hinder the identification of useful ligands for targets with multiple binding sites (e.g., cells). This review aims to characterize the loss in the diversity of libraries during amplification. Analysis of the peptide sequences obtained in several hundred screens of peptide libraries shows explicitly that there is a significant decrease in library diversity that occurs during the amplification of phage in bacteria. This loss during amplification is not unique to specific libraries: it is observed in many of the phage display systems we have surveyed. The loss in library diversity originates from competition among phage clones in a common pool of bacteria. Based on growth data from the literature and models of phage growth, we show that this competition originates from growth rate differences of only a few percent for different phage clones. We summarize the findings using a simple two-dimensional "phage phase diagram", which describes how the collapse of libraries, due to panning and amplification, leads to the identification of only a subset of the available ligands. This review also highlights techniques that allow elimination of amplification-induced losses of diversity, and how these techniques can be used to improve phage-display selection and enable the identification of novel ligands.     

Bioactive peptides grafted silicone dressings: A simple and specific method

The need for bioactive dressings increases with the population aging and the prevalence of chronic diseases. In contrast, there are very few dressings on the market which are designed to display a chosen bioactivity. In this context, we investigated the surface-functionalization of silicone wound dressing with bioactive peptides. One of the challenges was to avoid multistep grafting reactions involving catalysts, solvents or toxic reagents, which are not suitable for the fabrication of medical devices at an industrial scale. In the other hand, a covalent bonding was necessary to avoid the loss of the biological effect by progressive removal of the peptide in biological fluids generated by the wound. To solve these limitations, we developed a strategy allowing an easy and direct functionalization of silicone. This strategy relies on hybrid silylated bioactive peptides, which chemoselectively react with plasma-activated silicone surfaces. We synthesized three hybrid peptides with wound healing properties, which were grafted on commercially available silicone dressings Cerederm^® and Mepitel^®. Grafted dressings were evaluated in vitro and enabled a quicker scare recovery and extracellular matrix deposition with human dermal fibroblasts. These results were confirmed by in vivo studies showing an enhanced wound-healing of the pig skin. By this simple method, we transformed inert dressing into bioactive dressing which showed properties of wound healing.     

Bioactive Hydrolysates from Chlorella vulgaris: Optimal Process and Bioactive Properties

Microalgae have been described as a source of bioactive compounds, such as peptides. Microalgae are easy to produce, making them a sustainable resource for extracting active ingredients for industrial applications. Several microalgae species have interesting protein content, such as Chlorella vulgaris with around 52.2% of protein, making it promising for peptide hydrolysate production. Therefore, this work focused on the production of water-soluble hydrolysates rich in proteins/peptides from the microalgae C. vulgaris and studied bioactive properties. For that, a design of experiments (DOE) was performed to establish the optimal conditions to produce hydrolysates with higher levels of protein, as well as antioxidant and antihypertensive properties. Four experimental factors were considered (cellulase percentage, protease percentage, hydrolysis temperature, and hydrolysis duration) for three responses (protein content, antioxidant activity, and antihypertensive activity). The optimal conditions determined by the DOE allowed producing a scaled-up hydrolysate with 45% protein, with antioxidant activity, measured by oxygen radical absorbance capacity assay, of 1035 µmol TE/g protein, IC₅₀ for angiotensin-converting enzyme inhibition activity of 286 µg protein/mL, and α-glucosidase inhibition of 31% (30 mg hydrolysate/mL). The obtained hydrolysates can be used as functional ingredients for food and nutraceuticals due to their antioxidant, antihypertensive, and antidiabetic potential. Moreover, the antioxidant potential of the extracts may be relevant for the cosmetic industry, especially in antiaging formulations.     

Bioactive constituents from Michelia champaca

(-)-Anonaine (1), (-)-asimilobine (2), (-)-nuciferine (3), (-)-anolobine (4), (-)-romerine (5), (-)-N-acetylanonaine (6), liriodenine (7), (+)-syringaresinol (8), N-trans-feruloyltyramine (9), N-cis-feruloyltyramine (10), scopoletin (11), 4-acetonyl-3,5-dimethoxy-p-quinol (12), vanillin (13), vanillic acid (14), syringic acid (15), beta-sitosterol (16) and stigmasterol (17) were isolated from branches of Michelia champaca L. In addition, a cell proliferation assay of five of the isolated compounds on human breast and lung cancer cells showed that liriodenine (7) was the strongest inhibitor.     

Bioactive eremophilanolides from Senecio poepigii

A new bioactive eremophilanolide, 1alpha-tigloyloxy-8betaH,10betaH-eremophil-7(11)-en-8alpha,12-olide, was isolated from Senecio poepigii and its structure was elucidated by spectral analysis. 1alpha-Angeloyloxy-8beta-methoxy-10betaH-eremophil-7(11)-en-8alpha,12-olide was also isolated. Antifungal and insect antifeedant properties were evaluated.     

Bioactive diterpenes from Clerodendrum kaichianum

Bioassay-guided isolation studies of the extract of Clerodendrum kaichianum Hsu., a new rearranged abietane diterpene and five known diterpene compounds were isolated by various chromatography methods. Their structures were identified by means of spectroscopic methods, including 1D- and 2D-NMR spectroscopy, as (16R)-12,16-epoxy-11,14,17-trihydroxy-17(15-->16)-abeo-8,11,13-abietatrien-7-one (1), villosin A (2), salvinolone (3), 14-deoxyloleon U (4), 5,6-dehydrosugiol (5), and coleon U (6). Compounds 1, 2, 3, and 5 are reported for the first time for this genus. Compounds 1, 2, and 6 demonstrated potent cytotoxic activities against the HL-60 tumor cell line.     

Combinatorial chemistry: libraries from libraries, the art of the diversity-oriented transformation of resin-bound peptides and chiral polyamides to low molecular weight acyclic and heterocyclic compounds

Combinatorial chemistry has deeply impacted the drug discovery process by accelerating the synthesis and screening of large numbers of compounds having therapeutic and/or diagnostic potential. These techniques offer unique enhancement in the potential identification of new and/or therapeutic candidates. Our efforts over the past 10 years in the design and diversity-oriented synthesis of low molecular weight acyclic and heterocyclic combinatorial libraries derived from amino acids, peptides, and/or peptidomimetics are described. Employing a "toolbox" of various chemical transformations, including alkylation, oxidation, reduction, acylation, and the use of a variety of multifunctional reagents, the "libraries from libraries" concept has enabled the continued development of an ever-expanding, structurally varied series of organic chemical libraries.     

Bioactive stilbene dimers from Gnetum cleistostachyum

Two new stilbene dimers, named bisisorhapontigenin A (1) and cis shegansu B (2), together with gnetuhainin P (3) and gnetulin (4), were isolated from the lianas of Gnetum cleistostachyum C. Y. Cheng. Their structures were elucidated by means of spectroscopic evidences, including UV, IR, MS, 1H NMR, 13C NMR, NOE and 2D NMR, respectively. The pharmacological activities of compounds 1, 2 and 3 also have been tested.     

Characterisation of combinatorial libraries of mucin-2 antigen peptides by high-resolution mass spectrometry

An epitope motif, TX(1)TX(2)T, of mucin-2 glycoprotein was identified by means of a mucin-2-specific monoclonal antibody, mAb 994, raised against a synthetic mucin-derived 15-mer peptide conjugate. For determination of the epitope sequence recognised with highest affinity by mAb 994, a combinatorial approach was applied using the portioning-mixing technique excluding Cys. Antibody binding of libraries was most profound when Gln was at the X(1) position. Analytical characterisation of the TQTX(2)T library was conducted by amino acid analysis and matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) and electrospray ionisation Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometric methods. Control libraries were prepared by mixing 19 individual peptides corresponding to the TQTX(2)T sequence. Thus, mixtures of 6, 10 and 19 pentapeptides were analysed and compared with the combinatorial mixture. MALDI-TOFMS was able to detect only partially the components in the 6- and 10-member mixtures, but failed to characterise a more complex 19-member mixture. In contrast, ESI-FTICRMS resolved all mixtures of higher complexity and provided direct identification at monoisotopic resolution, such as for a peptide library containing 'isobaric' lysine and glutamine (Delta m = 0.0364 Da). The results of this study suggest that ESI-FTICRMS is a powerful tool for characterisation of combinatorial peptide libraries of higher complexity.     

Bioactive constituents from Croton sparsiflorus Morong

Whole plant extracts of Croton sparsiflorus in methanol have shown significant enzyme inhibition and antioxidant activities. Bioassay-guided isolation of chloroform fraction at pH 3 resulted in the identification of crotsparinine (1) and crotsparine (2), while sparsiflorine (3) was purified from the chloroform fraction at pH 9. The structures of the compounds were confirmed through spectral analyses (EI-MS, ¹H and ¹³C NMR). The isolated compounds 1–3 exhibited remarkable enzyme inhibition activity with IC₅₀ values 27.01 ± 1.1, 22.26 ± 1.0 and 18.02 ± 1.3 μM in xanthine oxidase and 48.42 ± 1.5, 48.05 ± 1.4 and 7.42 ± 1.0 μM in acetylcholine esterase assays, respectively. These compounds also showed potent radical scavenging and reducing properties in DPPH and FRAP assays, respectively. The present results suggest the validity of the traditional uses of C. sparsiflorus in rheumatism and gout. Furthermore, the isolated noraporphine alkaloids can be useful in the treatment of neurodegenerative diseases.     

Bioactive phenolic amides from Celtis africana

Nine compounds have been isolated for the first time from Celtis africana, namely trans-N-coumaroyltyramine (1), trans-N-feruloyltyramine (2), trans-N-caffeoyltyramine (3), lauric acid (4), oleic acid (5), palmitic acid (6), lupeol (7), β-sitosterol (8) and oleanolic acid (9), respectively. Their structures have been elucidated by different spectroscopic techniques. The isolated compounds were screened for their antioxidant, anti-inflammatory and acetylcholinestrease enzyme inhibitory activities. Compounds 1-3 showed significant antioxidant and anti-inflammatory activities and weak to moderate acetylcholinestrease enzyme inhibition activity.