Quercetins, Chlorogenic Acids and Their Colon Metabolites Inhibit Colon Cancer Cell Proliferation at Physiologically Relevant Concentrations

Several studies have suggested that a phenolic-rich diet may be protective against colon cancer. Most phenolic compounds are not absorbed in the small intestine and reach the colon where they are metabolized by gut microbiota in simple phenolic acids. In this study, the anti-proliferative activity of quercetins, chlorogenic acids, their colon metabolites and mixtures of parent compounds/metabolites was assessed by using two colon cancer cell lines (Caco-2 and SW480) at physiologically relevant concentrations. Chlorogenic acids, quercetin and the metabolite 3-(3 & PRIME;,4 & PRIME;-dihydroxyphenyl)acetic acid exerted remarkable anti-proliferative activity against Caco-2, whereas quercetin derivatives and metabolites were the most active against SW480. Tested compounds arrested the cell cycle at the S phase in both the cell lines. The mixtures of parent compounds/metabolites, which mimic the colon human metabotypes that slowly or rapidly metabolize the parent compounds, similarly inhibited cell growth. SW480 cells metabolized parent phenolic compounds more rapidly and extensively than Caco-2, whereas colon metabolites were more stable. These results suggest that dietary phenolic compounds exert an anti-proliferative effect against human colon cancer cells that can be further sustained by the colon metabolites. Therefore, gut microbiota metabolism of phenolic compounds may be of paramount importance in explaining the protective effect of phenolic-rich foods against colon cancer

Effect of Ripening and In Vitro Digestion on Bioactive Peptides Profile in Ras Cheese and Their Biological Activities

The effect of ripening and in vitro digestion on the biological activities, peptide profiles and release of bioactive peptides in Ras cheese has been investigated. Ras cheese ripening largely influenced the extent of protein hydrolysis. The advancement in ripening resulted in an increase in total peptides (from 0.97 to 2.46 mmol leucine/g in samples at 30 and 180 days of ripening, respectively) and bioactive peptides concentration, especially angiotensin-converting enzyme (ACE)-inhibitory, dipeptidyl-peptidase-IV-(DPP-IV)-inhibitory and antioxidant peptides. In vitro gastro-intestinal digestion further promoted protein hydrolysis and the release of bioactive peptides. Digested Ras cheese at 90 and 180 days of ripening displayed the highest bioactive peptides intensity. The variations in bioactive peptides amount during ripening and in vitro digestion were correlated with the changes in ACE-inhibitory, DPP-IV-inhibitory and antioxidant activities. The highest amounts of VPP and IPP were detected in digested Ras cheese at 90 days of ripening (17.44 and 36.50 mg/kg of cheese, respectively), whereas the highest concentrations of APFPE were found in undigested and digested 180-day ripened Ras cheese (82.09 and 52.01 mg/kg of cheese, respectively). The present investigation underlined potential differences in the biological effect after the ingestion of Ras cheese at different ripening times

Application of a Combined Peptidomics and In Silico Approach for the Identification of Novel Dipeptidyl Peptidase-IV-Inhibitory Peptides in In Vitro Digested Pinto Bean Protein Extract

The conventional approach in bioactive peptides discovery, which includes extensive bioassay-guided fractionation and purification processes, is tedious, time-consuming and not always successful. The recently developed bioinformatics-driven in silico approach is rapid and cost-effective; however, it lacks an actual physiological significance. In this study a new integrated peptidomics and in silico method, which combines the advantages of the conventional and in silico approaches by using the pool of peptides identified in a food hydrolysate as the starting point for subsequent application of selected bioinformatics tools, has been developed. Pinto bean protein extract was in vitro digested and peptides were identified by peptidomics. The pool of obtained peptides was screened by in silico analysis and structure–activity relationship modelling. Three peptides (SIPR, SAPI and FVPH) were selected as potential inhibitors of the dipeptidyl-peptidase-IV (DPP-IV) enzyme by this integrated approach. In vitro bioactivity assay showed that all three peptides were able to inhibit DPP-IV with the tetra-peptide SAPI showing the highest activity (IC50 = 57.7 µmol/L). Indeed, a new possible characteristic of peptides (i.e., the presence of an S residue at the N-terminus) able to inhibit DPP-IV was proposed

Cooking and In Vitro Digestion Modulate the Anti-Diabetic Properties of Red-Skinned Onion and Dark Purple Eggplant Phenolic Compounds

The intake of phenolic-rich foods is an emerging preventive approach for the management of type 2 diabetes, thanks to the ability of these compounds to inhibit some key metabolic enzymes. In this study, the influence of cooking and in vitro digestion on the α-glucosidase, α-amylase, and dipeptidyl-peptidase IV (DPP-IV) inhibitory activity of red-skinned onion (RSO) and dark purple eggplant (DPE) phenolic fractions was assessed. The applied cooking procedures had different influences on the total and individual phenolic compounds gastrointestinal bioaccessibility. DPE in vitro digested phenolic fractions displayed no inhibitory activity versus α-amylase and DPP-IV, whereas the fried DPE sample exhibited moderate inhibitory activity against α-glucosidase. This sample mainly contained hydroxycinnamic acid amides that can be responsible for the observed effect. Contrariwise, raw and cooked in vitro digested RSO phenolic fractions inhibited all three enzymes but with different effectiveness. Fried and raw RSO samples were the most active against them. Statistical analysis pointed out that quercetin mono-hexosides (mainly quercetin-4′-O-hexoside) were responsible for the inhibition of α-glucosidase, whereas quercetin dihexosides (mainly quercetin-3-O-hexoside-4′-O-hexoside) were responsible for the DPP-IV-inhibitory activity of RSO samples. An accurate design of the cooking methods could be essential to maximize the release of individual phenolic compounds and the related bioactivities

Impact of cooking methods of red-skinned onion on metabolic transformation of phenolic compounds and gut microbiota changes

Herein, we investigated the stability and bioaccessibility of phenolics in differently cooked red-skinned onion (RSO) and consequently their impact on the gut microbiota and metabolism of phenolics. In fact, the different processes used to cook vegetables can modify and re-arrange the molecular profiles of bioactive compounds, such as phenolics in phenolic-rich vegetables, such as RSO. Fried and grilled RSO were compared to raw RSO and a blank control and subjected to oro-gastro-intestinal digestion and subsequent colonic fermentation. For upper gut digestion, the INFOGEST protocol was used, and for lower gut fermentation, a short-term batch model, namely, MICODE (multi-unit in vitro colon gut model), was employed. During the process, phenolic compound profile (through high-resolution mass spectrometry) and colon microbiomics (qPCR of 14 core taxa) analyses were performed. According to the results, the degradation driven by the colon microbiota of RSO flavonols resulted in the accumulation of three main metabolites, i.e., 3-(3 '-hydroxyphenyl)propanoic acid, 3-(3 '-hydroxyphenyl)acetic acid and 3-(3 ',4 '-dihydroxyphenyl)acetic acid. Also, colonic fermentation of raw onions resulted in a substantial increase in beneficial taxa, which was larger compared to the heat-treated onions, particularly Lactobacillales and beneficial clostridia. Also, a higher level of inhibition of opportunistic bacteria was seen for the raw onion samples, namely, Clostridium perfringens group and Escherichia coli. Thus, our results showed that RSO, and especially the raw one, is an excellent dietary source of flavonols that are strongly metabolized by gut bacteria and can positively modulate the gut microbiota. Although additional in vivo studies are necessary, this work is one of the first to explore how RSO processed with different cooking methods can differently impact the phenolic metabolism and microbiota composition in the large intestine of humans, fine-tuning the antioxidant nature of foods

An integrated peptidomics and in silico approach to identify novel anti-diabetic peptides in parmigiano-reggiano cheese

Inhibition of key metabolic enzymes linked to type-2-diabetes (T2D) by food-derived compounds is a preventive emerging strategy in the management of T2D. Here, the impact of Parmigiano- Reggiano (PR) cheese peptide fractions, at four different ripening times (12, 18, 24, and 30 months), on the enzymatic activity of α-glucosidase, α-amylase, and dipeptidyl peptidase-IV (DPPIV) as well as on the formation of fluorescent advanced glycation end-products (fAGEs) was assessed. The PR peptide fractions were able to inhibit the selected enzymes and fAGEs formation. The 12-month-ripening PR sample was the most active against the three enzymes and fAGEs. Mass spectrometry analysis enabled the identification of 415 unique peptides, 54.9% of them common to the four PR samples. Forty-nine previously identified bioactive peptides were found, mostly characterized as angiotensin-converting enzyme-inhibitors. The application of an integrated approach that combined peptidomics, in silico analysis, and a structure–activity relationship led to an efficient selection of 6 peptides with potential DPP-IV and α-glucosidase inhibitory activities. Peptide APFPE was identified as a potent novel DPP-IV inhibitor (IC50 = 49.5 ± 0.5 μmol/L). In addition, the well-known anti-hypertensive tripeptide, IPP, was the only one able to inhibit the three digestive enzymes, highlighting its possible new and pivotal role in diabetes management

Resistance of European spring 2-row barley cultivars to Pyrenophora graminea and detection of associated loci

Pyrenophora graminea is the seed-borne pathogen causal agent of barley leaf stripe disease. In this work, we screened a collection of 206 spring two-row barley cultivars from Europe for their resistance to the fungal pathogen. Artificial inoculation with the highly virulent isolate Dg2 revealed a continuous variation for the incidence of infection, with few highly resistant or highly susceptible genotypes. On average, old cultivars showed higher resistance than the more modern ones. Genome- Wide Association Scan was performed by exploiting available molecular data for >4000 SNP markers and revealed a single, highly significant association on the short arm of chromosome 6H, in a genomic position where quantitative trait loci (QTL) for barley resistance to P. graminea were not detected before. Based on the last version of the reference barley genome, genes encoding for proteins with a kinase domain were suggested as candidates for the locus

Cultivable microbial diversity, peptide profiles, and bio-functional properties in Parmigiano Reggiano cheese

Introduction: Lactic acid bacteria (LAB) communities shape the sensorial and functional properties of artisanal hard-cooked and long-ripened cheeses made with raw bovine milk like Parmigiano Reggiano (PR) cheese. While patterns of microbial evolution have been well studied in PR cheese, there is a lack of information about how this microbial diversity affects the metabolic and functional properties of PR cheese. Methods: To fill this information gap, we characterized the cultivable fraction of natural whey starter (NWS) and PR cheeses at different ripening times, both at the species and strain level, and investigated the possible correlation between microbial composition and the evolution of peptide profiles over cheese ripening. Results and discussion: The results showed that NWS was a complex community of several biotypes belonging to a few species, namely, Streptococcus thermophilus, Lactobacillus helveticus, and Lactobacillus delbrueckii subsp. lactis. A new species-specific PCR assay was successful in discriminating the cheese-associated species Lacticaseibacillus casei, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, and Lacticaseibacillus zeae. Based on the resolved patterns of species and biotype distribution, Lcb. paracasei and Lcb. zeae were most frequently isolated after 24 and 30 months of ripening, while the number of biotypes was inversely related to the ripening time. Peptidomics analysis revealed more than 520 peptides in cheese samples. To the best of our knowledge, this is the most comprehensive survey of peptides in PR cheese. Most of them were from β-caseins, which represent the best substrate for LAB cell-envelope proteases. The abundance of peptides from β-casein 38–88 region continuously increased during ripening. Remarkably, this region contains precursors for the anti-hypertensive lactotripeptides VPP and IPP, as well as for β-casomorphins. We found that the ripening time strongly affects bioactive peptide profiles and that the occurrence of Lcb. zeae species is positively linked to the incidence of eight anti-hypertensive peptides. This result highlighted how the presence of specific LAB species is likely a pivotal factor in determining PR functional properties

Identification of phenolic compounds from inflorescences of non-psychoactive Cannabis sativa L. by UHPLC-HRMS and in vitro assessment of the antiproliferative activity against colorectal cancer

Phenolic compounds from Cannabis sativa L. (Cannabaceae family), in particular cannflavins, are known to possess several biological properties. However, their antiproliferative activity, being of great interest from a medicinal chemistry point of view, has not been deeply investigated so far in the literature. In the light of this, the aim of this study was to obtain an enriched fraction of polyphenols (namely PEF) from inflorescences of a non-psychoactive C. sativa (hemp) variety and to evaluate its antiproliferative activity against cancer cells, capitalizing on a new and selective extraction method for hemp polyphenols, followed by preparative flash column chromatography. Untargeted metabolomics, using a new method based on ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS), was applied here for the first time to fully characterize PEF. Then, the main phenolic compounds were quantified by HPLC-UV. The antiproliferative activity of PEF and of the isolated compounds was assessed in vitro for the first time against Caco-2 and SW480 human colon adenocarcinoma cell lines providing promising IC50 values, in comparison with the reference drug used in therapy for this cancer type. Based on these results, PEF can be considered as a new highly potential therapeutic product to be further investigated against colorectal cancer, thanks to the possible synergistic interaction of its compounds