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OBJECTIVE: This study aims to characterize in vitro D-chiro-inositol intestinal absorption and identify factors able to improve its bioavailability. D-chiro-inositol, one of the natural occurring stereoisomer of myo-inositol, acts as a second messenger in insulin-regulated glucose metabolism in complementary mode with myo-inositol. Because of their insulin-mimetic activities and safety, both myo-inositol and D-chiro-inositol are often employed as supplements in insulin-resistance treatment. MATERIALS AND METHODS: Trans-epithelial passage of D-chiro-inositol was evaluated in the human intestinal Caco-2 cell line differentiated on filter, a widely established in vitro model to study intestinal absorption. D-chiro-inositol transport was assayed in a concentration range corresponding to an estimated in vivo concentration following oral supplementation. α-Lactalbumin peptides, obtained by in vitro simulated gastrointestinal digestion, were tested as possible modulators of the intestinal permeability of D-chiro-inositol. RESULTS: The absorption of this stereoisomer was relatively low and presumably due to passive diffusion, while it was greatly enhanced by the presence of α-Lactalbumin digest. α-Lactalbumin peptides induced an increase in paracellular permeability that was completely reversible, indicating lack of cytotoxicity. This effect involved temporary rearrangement of F-actin apical cytoskeleton and of the tight junction protein ZO-1. CONCLUSIONS: Although further studies are required to identify and characterize the most effective peptides, the ability of α-Lactalbumin digest to act as absorption enhancers may have very interesting and promising applications in the fields of nutritional supplements and pharmacology. [ABSTRACT FROM AUTHOR]

Nowadays, research on plant extracts has attracted increasing interest. The aim of this study was to compare phenolic profile, vitamin C, and carotenoid content, as well as the biological activities of five different rose species, including Rosa canina , R. corymbifera , R. micrantha , R. rubiginosa, and R. rugosa . These species had different morphological characteristics, with R. rugosa showing higher size of flower petals and higher weight of hips. The highest vitamin C content was found in hip extracts of R. rubiginosa and R. rugosa , which also showed the highest carotenoid amount. R. corymbifera showed the highest phenolic content. No significant antimicrobial activity of extracts containing phenolic compounds against different indicator strains could be detected. Cell monolayer integrity was not affected by treatments with the above-mentioned extracts of R. canina , R. micrantha, and R. rugosa at different concentrations for up to 24 h, while those of R. rubiginosa and R. corymbifera affected intestinal permeability at the highest concentration tested. The partial least squares regression analysis generated a predictive model correlating phenolic compounds with cell monolayer integrity, suggesting a relevant role for catechin, quercitrin, and p-coumaric acid. In conclusion, this study highlights how rose hips belonging to different species can have a diverse phenolic profile, differently influencing intestinal monolayer integrity.

In recent years, there has been growing interest in exploring alternative and innovative delivery systems to improve the efficacy of iron supplements, satisfying iron needs and lowering side effects. To address this issue, this study aimed at demonstrating the advantages of Ferro Supremo formulation (composed of encapsulated iron, vitamins, and micronutrients), in terms of capacity to improve iron intestinal absorption, in comparison with standard FeSO 4 . Hence, differentiated Caco-2 cells have been used for assessing the in vitro bioavailability and safety of FS and FeSO 4 . MTT experiments demonstrated that both FS and FeSO 4 are not able to impair the viability of Caco-2 cells. Furthermore, the quantitative and qualitative analysis, conducted by atomic absorption spectrometry and fluorescence determinations, revealed that FS can enter, accumulate in the cytoplasm, and be transported by intestinal cells four times more efficiently than FeSO 4 . Our findings indicate that this formulation can be considered a valuable and efficiently good choice as food supplements for improving iron deficiency.

The remarkable biological activities of oregano essential oils (EOs) have recently prompted a host of studies aimed at exploring their potential innovative applications in the food and pharmaceutical industries. The chemical composition and biological activities of EOs from two Origanum vulgare genotypes, widely cultivated in Sicily and not previously studied for their biological properties, were characterized. Plants of the two genotypes, belonging to the carvacrol (CAR) and thymol (THY) chemotypes and grown in different cultivation environments, were considered for this study. The chemical profiles, including the determination of enantiomeric distribution, of the EOs, obtained by hydrodistillation from dried leaves and flowers, were investigated by GC-MS. Biological activity was evaluated as antimicrobial properties against different pathogen indicator strains, while intestinal barrier integrity, reduction in pathogen adhesion and anti-inflammatory actions were assayed in the intestinal Caco-2 cell line. The chemical profile of the CAR genotype was less complex and characterized by higher levels of the most active compound, i.e., carvacrol, when compared to the THY genotype. The enantiomeric distribution of chiral constituents did not vary across genotypes, while being markedly different from that observed in Origanum vulgare genotypes from other geographical origins. In general, all EOs showed high antimicrobial activity, both in vitro and in a food matrix challenge test. Representative EOs from the two genotypes resulted not altering epithelial monolayer sealing only for concentrations lower than 0.02%, were able to reduce the adhesion of selected pathogens, but did not exert relevant anti-inflammatory effects. These results suggest their potential use as control agents against a wide spectrum of foodborne pathogens.

A preceding paper has shown that a hempseed peptic hydrolysate displays a cholesterol-lowering activity with a statin-like mechanism of action in HepG2 cells and a potential hypoglycemic activity by the inhibition of dipeptidyl peptidase-IV in Caco-2 cells. In the framework of a research aimed at fostering the multifunctional behavior of hempseed peptides, we present here the identification and evaluation of some antioxidant peptides from the same hydrolysate. After evaluation of its diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, a trans-epithelial transport experiment was performed using differentiated Caco-2 cells that permitted the identification of five transported peptides that were synthesized and evaluated by measuring the oxygen radical absorbance capacity (ORAC), the ferric reducing antioxidant power (FRAP), and the 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic) acid (ABTS), and diphenyl-2-picrylhydrazyl radical DPPH assays. The most active peptides, i.e. WVSPLAGRT (H2) and IGFLIIWV (H3), were then tested in cell assays. Both peptides were able to reduce the H 2 O 2 -induced reactive oxygen species (ROS), lipid peroxidation, and nitric oxide (NO) production levels in HepG2 cells, via the modulation of Nrf-2 and iNOS pathways, respectively., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Hamamelis virginiana L. a rich source of both condensed and hydrolyzable tannins, utilized to treat dermatological disorders. Since no experimental and clinical data is available for its use as oral formulation in skin related disorders, the purpose of this study was to investigate the effects of Hamaforton™ ( Hamamelis virginiana extract) metabolites on gene dysregulation induced by ultraviolet A radiation in cultured human dermal fibroblasts. A combination of in vivo and ex vivo experimental designs has been exploited in order to take into account the polyphenol metabolic transformation that occurs in humans. 12 healthy volunteers received either a capsule of Hamaforton™ or a placebo in a randomized, blinded crossover trial. After Hamaforton™ ingestion, the kinetic of appearance of galloyl derivatives was measured in plasma. Then, in the ex vivo experiment, the serum isolated after supplementation was used as a source of Hamaforton™ metabolites to enrich the culture medium of dermal fibroblasts exposed to ultraviolet A radiation. Three different gallic acid metabolites (4-O-methyl gallic acid, 4-O-methyl gallic acid sulphate and trimethyl gallic acid glucuronide) were identified in volunteer plasma. While, ultraviolet A irradiation of dermal fibroblasts affected the expression of extracellular matrix genes, the presence of Hamaforton™ metabolites in the culture media did not affect the expression of most of those genes. However, the activation of the expression of 10 different genes involved in repair processes for the maintenance of skin integrity, suggest that the metabolites can play a role in damage recovery. To our knowledge, this is the first study that demonstrates the bioavailability of Hamaforton™ phenolic compounds, and the effects of its metabolites on cultured dermal fibroblast response to ultraviolet A irradiation., Competing Interests: CB is an employee of Horphag Research. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Natella, Guantario, Ambra, Ranaldi, Intorre, Burki and Canali.)

This work describes an untargeted analytical approach for the screening, identification, and characterization of the trans-epithelial transport of green tea ( Camellia sinensis ) catechin extracts with in vitro inhibitory effect against the SARS-CoV-2 papain-like protease (PLpro) activity. After specific catechin extraction, a chromatographic separation obtained six fractions were carried out. The fractions were assessed in vitro against the PLpro target. Fraction 5 showed the highest inhibitory activity against the SARS-CoV-2 PLpro (IC 50 of 0.125 μg mL -1 ). The untargeted characterization revealed that (-)-epicatechin-3-gallate (ECG) was the most abundant compound in the fraction and the primary molecule absorbed by differentiated Caco-2 cells. Results indicated that fraction 5 was approximately 10 times more active than ECG (IC 50 value equal to 11.62 ± 0.47 μg mL -1 ) to inhibit the PLpro target. Overall, our findings highlight the synergistic effects of the various components of the crude extract compared to isolated ECG.

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.

In the framework of research aimed at promoting the nutraceutical properties of the phenolic extract (BUO) obtained from an extra virgin olive oil of the Frantoio cultivar cultivated in Tuscany (Italy), with a high total phenols content, this study provides a comprehensive characterization of its antioxidant properties, both in vitro by Trolox equivalent antioxidant capacity, oxygen radical absorbance capacity, ferric reducing antioxidant power, and 2,2-diphenyl-1-picrylhydrazyl assays, and at the cellular level in human hepatic HepG2 and human intestinal Caco-2 cells. Notably, in both cell systems, after H 2 O 2 induced oxidative stress, the BUO extract reduced reactive oxygen species, lipid peroxidation, and NO overproduction via modulation of inducible nitric oxide synthase protein levels. In parallel, the intestinal transport of the different phenolic components of the BUO phytocomplex was assayed on differentiated Caco-2 cells, a well-established model of mature enterocytes. The novelty of our study lies in having investigated the antioxidant effects of a complex pool of phenolic compounds in an extra virgin olive oil (EVOO) extract, using either in vitro assays or liver and intestinal cell models, rather than the effects of single phenols, such as hydroxytyrosol or oleuropein. Finally, the selective trans-epithelial transport of some oleuropein derivatives was observed for the first time in differentiated Caco-2 cells.

LTFPGSAED (P7) is a multifunctional hypocholesterolemic and hypoglycemic lupin peptide. While assessing its angiotensin-converting enzyme (ACE) inhibitory activity, it was more effective in intestinal Caco-2 cells (IC 50 of 13.7 μM) than in renal HK-2 cells (IC 50 of 79.6 μM). This discrepancy was explained by the metabolic transformation mediated by intestinal peptidases, which produced two main detected peptides, TFPGSAED and LTFPG. Indeed LTFPG, dynamically generated by intestinal dipeptidyl peptidase IV as well as its parent peptide P7 were linearly absorbed by mature Caco-2 cells. An in silico study demonstrated that the metabolite was a better ligand of the ACE enzyme than P7. These results are in agreement with an in vivo study, previously performed by Aluko et al., which has shown that LTFPG is an effective hypotensive peptide. Our work highlights the dynamic nature of bioactive food peptides that may be modulated by the metabolic activity of intestinal cells.

Zinc deficiency predisposes to a wide spectrum of chronic diseases. The human Zn proteome was predicted to represent about 10% of the total human proteome, reflecting the broad array of metabolic functions in which this micronutrient is known to participate. In the thyroid, Zn was reported to regulate cellular homeostasis, with a yet elusive mechanism. The Fischer Rat Thyroid Cell Line FRTL-5 cell model, derived from a Fischer rat thyroid and displaying a follicular cell phenotype, was used to investigate a possible causal relationship between intracellular Zn levels and thyroid function. A proteomic approach was applied to compare proteins expressed in Zn deficiency, obtained by treating cells with the Zn-specific chelator N , N , N ', N '-tetrakis (2-pyridylmethyl) ethylene-diamine (TPEN), with Zn repleted cells. Quantitative proteomic analysis of whole cell protein extracts was performed using stable isotope dimethyl labelling coupled to nano-ultra performance liquid chromatography-mass spectrometry (UPLC-MS). TPEN treatment led to almost undetectable intracellular Zn, while decreasing thyroglobulin secretion. Subsequent addition of ZnSO₄ fully reversed these phenotypes. Comparative proteomic analysis of Zn depleted/repleted cells identified 108 proteins modulated by either treatment. Biological process enrichment analysis identified functions involved in calcium release and the regulation of translation as the most strongly regulated processes in Zn depleted cells.

Recent investigations have focused on food-derived peptides as novel natural inhibitors of dipeptidyl peptidase IV (DPP-IV), a new target for diabetes. This study aimed to optimize fast, sensitive, and cost-effective DPP-IV assays in situ on human intestinal Caco-2 cells and ex vivo on human serum. Both assays were applied to investigate the inhibitory activity of soy and lupin peptides. The best conditions for in situ DPP-IV activity in Caco-2 cells were obtained using 2-day cells and 50 µM Gly-Pro-AMC. Sitagliptin, used as reference inhibitor, showed a dose-dependent response with a 50% inhibition concentration (IC 50 ) of 0.6 µM. A lower IC 50 (0.2 µM) was obtained for sitagliptin on human serum incubated with the substrate for 24 h. Both assays were applied to assess the activity of Lup1 (LTFPGSAED) and Soy1 (IAVPTGVA) on DPP-IV. Lup1 and Soy1 inhibited DPP-IV in situ, with IC 50 values of of 207.5 and 223.2 µM, respectively, and maintained their inhibitory activity ex vivo on circulating DPP-IV with a slightly lower potency. These assays can be used to characterize the DPP-IV inhibitory activity of food-derived molecules more accurately than in vitro biochemical tests. This combined approach also considers their effects on the circulating form of DPP-IV, correlated to metabolic diseases.

Background: Myo-inositol is a natural molecule with important therapeutic applications and an impaired oral absorption may result in a reduced clinical effect. Aim of this study was to determine if the combined oral administration of α-lactalbumin and myo-inositol in healthy subjects, could increase the plasma level of myo-inositol administered alone. In vitro studies on human differentiated intestinal Caco-2 cells were also conducted to identify the mechanisms involved in myo-inositol absorption., Objective: The in vivo study was conducted on healthy volunteers in two phases. Subjects received a single oral myo-inositol dose. After 7 days washout, the same subjects were administered a single dose of myo-inositol and α-lactalbumin. Cmax, Tmax and AUC for myo-inositol in plasma were calculated from samples collected at different times. Transepithelial myo-inositol passage, with or without addition of digested α-lactalbumin, was measured in vitro in differentiated Caco-2 cells and compared to transepithelial electrical resistance and phenol red passage., Results: The bioavailability of myo-inositol was modified by the concomitant administration of α- lactalbumin. Although peak concentration of myo-inositol at 180 min (Tmax) was similar for both treatments, administration of α-lactalbumin with myo-inositol in a single dose, significantly increased the plasma concentrations of myo-inositol compared to when administered alone. In vitro, myo-inositol absorption in Caco-2 cells was improved in the presence of digested α-lactalbumin, and this change was associated with an increase in tight junction permeability., Conclusion: Better myo-inositol absorption when orally administered with α-lactalbumin can be beneficial in non-responder patients. Preliminary in vitro findings suggest that peptides deriving from α- lactalbumin digestion may modulate tight junction permeability allowing increased absorption of myoinositol., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Chronic intestinal inflammatory disorders, such as Inflammatory Bowel Diseases (IBDs), are characterized by excessive release of proinflammatory mediators, intestinal barrier dysfunction and excessive activation of NF-kB cascade. Previous studies shown that TNF-α plays a central role in intestinal inflammation of IBDs and supported beneficial effects of flavonoids against chronic inflammatory diseases. In this study, we employed an in vitro model of acute intestinal inflammation using intestinal Caco-2 cells exposed to TNF-α. The protective effects of cyanidin-3-glucoside (C3G), an anthocyanin widely distributed in mediterranean diet, were then evaluated. Caco-2 cells exposure to TNF-α activated NF-kB proinflammatory pathway and induced IL6 and COX-2 expression. Cells pretreatment for 24h with C3G (20-40μM) prevented TNF-α-induced changes, and improved intracellular redox status. Our results demonstrated that C3G, also without any kind of stimulus, increased the translocation of the transcription factor Nrf2 into the nucleus so activating antioxidant and detoxifying genes. In conclusion, C3G exhibited protective effects through the inhibition of NF-kB signalling in Caco-2 cells and these beneficial effects appear to be due to its ability to activate cellular protective responses modulated by Nrf2. These data suggest that anthocyanins could contribute, as complementary or preventive approaches, to the management of chronic inflammatory diseases., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Benefits to health from a high consumption of fruits and vegetables are well established and have been attributed to bioactive secondary metabolites present in edible plants. However, the effects of specific health-related phytochemicals within a complex food matrix are difficult to assess. In an attempt to address this problem, we have used elicitation to improve the nutraceutical content of seedlings of Brassica oleracea grown under controlled conditions. Analysis, by LC-MS, of the glucosinolate, isothiocyanate and phenolic compound content of juices obtained from sprouts indicated that elicitation induces an enrichment of several phenolics, particularly of the anthocyanin fraction. To test the biological activity of basal and enriched juices we took advantage of a recently developed in vitro model of inflamed human intestinal epithelium. Both sprouts' juices protected intestinal barrier integrity in Caco-2 cells exposed to tumor necrosis factor α under marginal zinc deprivation, with the enriched juice showing higher protection. Multivariate regression analysis indicated that the extent of rescue from stress-induced epithelial dysfunction correlated with the composition in bioactive molecules of the juices and, in particular, with a group of phenolic compounds, including several anthocyanins, quercetin-3-Glc, cryptochlorogenic, neochlorogenic and cinnamic acids., Competing Interests: The authors declare no conflict of interest.