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The present study evaluated the impact of different thermal (infrared-drying, hot air-drying and purée pasteurization) and non-thermal (freeze-drying) processing technologies on red-fleshed apple (poly)phenolic compounds. We further investigated the processing effect on the (poly)phenol bioavailability in a crossover postprandial study where three subjects consumed three apple products (freeze-dried snack, hot air-dried snack and pasteurized purée). (Poly)phenolic compounds present in the apple products and their biological metabolites in urine were analyzed using liquid chromatography coupled to mass spectrometry (UPLC-MS/MS). When comparing different processes, infrared-drying caused important losses in most of the apple (poly)phenolics, while hot air-drying and purée pasteurization maintained approximately 83% and 65% of total (poly)phenols compared with the freeze-dried snack, respectively. Anthocyanins in particular were degraded to a higher extent, and hot air-dried apple and pasteurized purée maintained respectively 26% and 9% compared with freeze-dried apple snack. The acute intake showed that pasteurized purée exhibited the highest (poly)phenol bioavailability, followed by hot air-drying and freeze-dried snack, highlighting the impact of processing on (poly)phenols absorption. In conclusion, for obtaining affordable new red-fleshed apple products with enhanced (poly)phenol bioavailability, purée pasteurization and hot air-drying represent viable techniques. However, to obtain a red-fleshed apple snack with high anthocyanin content, freeze-drying is the technique that best preserves them., This study was supported by the Spanish Ministry of Industry, Economy and Competitiveness (MINECO) through the AGL2016-76943-C2-1-R and AGL2016-76943-C2-2-R projects (co-funded by the Agencia Estatal de Investigación (AEI) and the European Regional Development Fund (ERDF)); Iziar A. Ludwig was supported by the Spanish Ministry of Industry, Economy and Competitiveness (Juan de la Cierva, FJCI-2014-20689) and 2017PMF-POST2-19 The Martí I Franqués Research Grants Programme Universitat Rovira i Virgili (Reus, Tarragona, Spain); and Silvia Yuste through a grant by the University of Lleida. In addition, the authors are grateful to NUFRI SAT (Mollerussa, Lleida, Catalonia, Spain) for providing the red-fleshed apples.

The present study aimed to evaluate the metabolism and bioavailability of anthocyanins (ACN) and other phenolics from red-fleshed apple (RFA) and to define the intake biomarkers compared to common white-fleshed apple (WFA). Acute and sustained (6-week) interventions were combined in a randomized, controlled and parallel study with 121 hypercholesterolemic subjects. Another arm consuming ACN-rich infusion from aronia fruit (ARO) provided matched content and profile of ACN. Plasma, urine and faeces samples were analyzed using UPLC-MS/MS. Results showed higher bioavailability of ACN after ARO compared to RFA, showing a clear apple matrix effect. The dihydrochalcone phloretin-2¿ -O-glucuronide was the most discriminant intake biomarker of both apples. The urinary peonidin-3-O-galactoside was a good biomarker after both ARO and RFA intakes, whereas peonidin-O-arabinoside was reported to be specific from ARO. The elucidation of the phenolic metabolism and the selection of intake biomarkers is a promising approach to relate phenolic compounds and human health., This study was supported by the Spanish Ministry of Industry, Economy and Competitiveness through the AGL2016-76943-C2-1-R and AGL2016-76943-C2-2-R projects (co-funded by the European Social Fund, European Union); M.J. Motilva thanks to Consejo Superior de Investigaciones Científicas-CSIC for partial funding throught the “Ayudas incorporacion ´ a escalas científicas CSIC, 2018” (Reference 201870I129). L.R. was Sara Borrell postdoctoral contract and she is a Serra Húnter Fellow. I.A.L. enjoys a post-doctoral contract (2017PMFPOST2-19) from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement and from the Universitat Rovira i Virgili (URV). S.Y. was supported by a pre-doctoral grant from the University of Lleida. The authors are grateful to CALIDAD PASCUAL (Aranda de Duero, Burgos, Spain) for generously providing the Bezoya mineral water to prepare daily Aronia infusion.

The aim of this study was to investigate comprehensively the metabolic pathways and human bioavailability of anthocyanins and other (poly)phenols in an apple matrix, and to elucidate potential intake biomarkers. After the acute intake of a red-fleshed apple freeze-dried snack, plasma and urine were collected and analyzed by UPLC-MS/MS. A total of 37 phase-II and microbial phenolic metabolites were detected in plasma and urine. Among these, phloretin glucuronide, cyanidin-3-O-galactoside (plasma and urine) and peonidin-3-O-galactoside (urine) were the only metabolites detected in all the volunteers and not detected at basal conditions. The maximum urine excretion was detected at 2–4 h, and the main increase in plasma of phloretin glucuronide and cyanidin-3-O-galactoside was observed at 2 h post-intake (61.0 ± 6.82 and 10.3 ± 1.50 nM, respectively). These metabolites could be selected as the best intake biomarkers of red-fleshed apple and might be useful in human intervention studies when studying its health effects., This study was supported by the Spanish Ministry of Industry, Economy and Competitiveness through the AGL2016-76943-C2-1-R and AGL2016-76943-C2-2-R projects (co-funded by the European Social Fund, European Union); I.A.L. enjoys a post-doctoral contract (2017PMF-POST2-19) from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement and from the Universitat Rovira i Virgili (URV). S.Y. was supported by a grant from the University of Lleida. In addition, the authors are grateful to NUFRI SAT (Mollerussa, Lleida, Catalonia, Spain) for providing the red-fleshed apples. A.P. has Torres Quevedo contract (Subprograma Estatal de Incorporación, Plan Estatal de Investigación Científica y Técnica y de Innovación).

BACKGROUND: Barley (Hordeum vulgare L.) is a healthy grain because of its high content of dietary fibre and phenolic compounds. It faces periods of high temperature during grain filling, frequently reducing grain weight. Heat stress may also affect some of the bioactive compounds present in the grain. To produce quality grains that provide nutritional and health benefits, it is important to understand the effect of environmental stresses on the quantity and quality of bioactive compounds. RESULTS: We have studied the effect of post-anthesis thermal stress on barley bioactive compounds and antioxidant capacity under Mediterranean field conditions during two consecutive growing seasons in four barley genotypes. Thermal stress affected grain weight and size and changed the relative composition of bioactive compounds. The relationship between heat stress and grain ⊎-glucans and arabinoxylans content was indirect, as the resulting increases in concentrations were due to the lower grain weight under stress. Conversely, heat stress had a significant direct impact on somephenolic compounds, increasing their concentrations differentially across genotypes, which contributed to an improvement in antioxidant capacity of up to 30%. CONCLUSION: Post-anthesis thermal stress had a significant effect on ⊎-glucans, arabinoxylans, phenolic compound concentration and antioxidant capacity of barley grains. Final grain quality could, at least partially, be controlled in order to increase the bioactive concentrations in the barley grain, by cultivation in growing areas prone to heat stress. Late sowings or late flowering genotypes could also be considered, should a premium be implemented to compensate for lower yields. This work was funded by project AGL 2015–69435-C3-1 from the Spanish Ministry of Economy and Competitiveness. Mariona Martínez-Subirà was supported by a pre-doctoral fellowship (BES-2016-078654/AGL 2015-69435-C3-1).

Berries are rich sources of (poly)phenols which have been associated with the prevention of cardiovascular diseases in animal models and in human clinical trials. Recently, a berry enriched diet was reported to decrease blood pressure and attenuate kidney disease progression on Dahl salt-sensitive rats. However, the relationship between kidney function, metabolism and (poly)phenols was not evaluated. We hypothesize that berries promote metabolic alterations concomitantly with an attenuation of the progression of renal disease. For that, kidney and urinary metabolomic changes induced by the berry enriched diet in hypertensive rats (Dahl salt-sensitive) were analyzed using liquid chromatography (UPLC-MS/MS) and 1H NMR techniques. Moreover, physiological and metabolic parameters, and kidney histopathological data were also collected. The severity of the kidney lesions promoted in Dahl rats by a high salt diet was significantly reduced by berries, namely a decrease in sclerotic glomeruli. In addition, was observed a high urinary excretion of metabolites that are indicators of alterations in glycolysis/gluconeogenesis, citrate cycle, and pyruvate metabolism in the salt induced-hypertensive rats, a metabolic profile counteracted by berries consumption. We also provide novel insights that relates (poly)phenols consumption with alterations in cysteine redox pools. Cysteine contribute to the redox signaling that is normally disrupted during kidney disease onset and progression. Our findings provide a vision about the metabolic responses of hypertensive rats to a (poly)phenol enriched diet, which may contribute to the understanding of the beneficial effects of (poly)phenols in salt-induced hypertension., CB, CNS, CO were funded by Agence Nationale de la Recherche (ANR) (ANR-13-ISV1-0001-01) and Fundação para a Ciência e Tecnologia (FCT) (FCTANR/BEX-BCM/0001/2013). We also acknowledge the Investment for the Future program ANR-11-IDEX-0003-01 within the LABEX ANR-10-LABX-0033 (CB, CO), FCT financial support of AG (SFRH/BD/103155/2014). iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344), which is cofunded by FCT through national funds, and by FEDER under the PT2020 Partnership Agreement is acknowledged. CERMAX - Centro de Ressonância Magnética António Xavier, located within ITQB for the (1H) NMR spectroscopy on an 800 MHz Bruker AvanceII+ funded by FCT project AAC 01/SAICT/2016 and ONEIDA (LISBOA-01-0145-FEDER-016417) which is co-funded by FEEI - “Fundos Europeus Estruturais e de Investimento” from “Programa Operacional Regional Lisboa 2020″ and by national funds from FCT.

The analysis of the fat-soluble vitamins A and E and lipid micronutrients in blood, such as carotenoids, is an important parameter to monitor the micronutrient status in humans. Although the potential of dried blood spot (DBS) cards, the use of this technique for blood sampling and subsequent analysis of these fat-soluble micronutrients has been poorly or not studied. An analytical method based on DBS cards (FTA® DMPK-A) combined with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has been developed and validated for the determination of carotenoids (lutein, zeaxanthin, β-cryptoxanthin and β-carotene), tocopherols (α-tocopherol, γ-tocopherol and δ-tocopherol) and all-trans-retinol in human blood. Under optimum DBS card extraction conditions, the extraction recoveries of the studied compounds were higher than 72%, the sample matrix effect lower than 17%, and the detection limits at hundred nM concentration levels. The developed method was applied to the analysis of human blood, and the concentration ranges obtained fell within the expected ranges previously reported in healthy adults. Moreover, the influence of hematocrit effect was investigated in a range of 25–55% in order to compare the obtained results to those reported in the literature for the analysis of plasma samples. This method represents an improvement over current techniques reported in the literature due to the use of a non-invasive blood collection method, and moreover, this methodology was for the first time 1) validated for the analysis of all-trans-retinol, tocopherols and carotenoids, and 2) applied for the determination of tocopherols in human blood samples., This study was supported by the Spanish Ministry of Industry, Economy and Competitiveness through the AGL2016-76943-C2-1-R and AGL2016-76943-C2-2-R projects (co-funded by the European Social Fund, European Union); M.J. Motilva thanks to Consejo Superior de Investigaciones Científicas-CSIC for partial funding throught the “Ayudas incorporación a escalas científicas CSIC, 2018” (Reference 201870I129). I.A.L. enjoys a post-doctoral contract (2017PMF-POST2-19) from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement and from the Universitat Rovira i Virgili (URV). S.Y. was supported by a pre-doctoral grant from the University of Lleida.

Barley (Hordeum vulgare L.) stands out for its high content on bioactive compounds although it is not frequently found in human food. In this study, a purple hulless barley genotype was used to explore its food potential. β-glucans, arabinoxylans, anthocyanins and other phenolic components were determined in biscuits containing different proportions of whole barley flour and pearling fractions and compared to biscuits prepared with 100% refined (control) and 100% whole wheat flour. Barley biscuits were richer in bioactive compounds, showed higher in-vitro antioxidant capacity and lower estimated glycemic index with slight changes in physical properties. Baking did not affect β-glucans and arabinoxylans while it increased most of the phenolic compounds and antioxidant capacity. Barley anthocyanins were thermally unstable and exhibited high degradation rates but were partially stabilized by tartaric acid. Biscuits baked with 100% flour from purple barley grains fulfill the health claim of “high in fiber”. A single biscuit provides more than 0.75g of β-glucans. Thus, one serving of four biscuits satisfies the 3g of β-glucans per day target to display the label of “reduces blood cholesterol and risk of heart disease”. This work was funded by project AGL 2015-69435-C3-1 from the Spanish Ministry of Economy and Competitiveness. Mariona Martínez-Subirà was supported by a pre-doctoral fellowship (BES-2016-078,654/AGL 2015-69435-C3-1).

Diets rich in (poly)phenols are associated with a reduced reduction in the incidence of cardiovascular disorders. While the absorption and metabolism of (poly)phenols has been described, it is not clear how their metabolic fate is affected under pathological conditions. This study evaluated the metabolic fate of berry (poly)phenols in an in vivo model of hypertension as well as the associated microbiota response. Dahl salt-sensitive rats were fed either a low-salt diet (0.26% NaCl) or a high-salt diet (8% NaCl), with or without a berry mixture (blueberries, blackberries, raspberries, Portuguese crowberry and strawberry tree fruit) for 9 weeks. The saltenriched diet promoted an increase in the urinary excretion of berry (poly)phenol metabolites, while the abundance of these metabolites decreased in faeces, as revealed by UPLC¿MS/MS. Moreover, salt and berries modulated gut microbiota composition as demonstrated by 16S rRNA analysis. Some changes in the microbiota composition were associated with the high-salt diet and revealed an expansion of the families Proteobacteria and Erysipelotrichaceae. However, this effect was mitigated by the dietary supplementation with berries. Alterations in the metabolic fate of (poly)phenols occur in parallel with the modulation of gut microbiota in hypertensive rats. Thus, beneficial effects of (poly)phenols could be related with these interlinked modifications, between metabolites and microbiota environments., C.B., C.N.d.S., C.O. were funded by ANR (ANR-13-ISV1-0001-01) and FCT (FCTANR/BEX-BCM/0001/2013). D.B. was funded by the Austrian Science Fund (FWF P26127-B20) and European Research Council (Starting Grant: FunKeyGut 741623). D.S. and A.F. acknowledge support from Scottish Government: Rural and Environment Science and Analytical Services. We also acknowledge the Investment for the Future program ANR-11-IDEX-0003-01 within the LABEX ANR-10-LABX-0033 (C.B., C.O.), Fundação para a Ciência e Tecnologia financial support of A.G. (SFRH/BD/103155/2014) and C.N.d.S. (IF/01097/2013). iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344), which is cofounded by FCT through national funds, and by FEDER under the PT2020 Partnership Agreement, is acknowledged.

Colorectal cancer (CRC) is the fourth cancer with the most new cases reported in 2018 worldwide. Consumption of fruit and vegetables is a protective factor against the risk of CRC. Beyond this, flavonoids could orchestrate these healthy effects. Apart from containing the typical apple flavonoids, red-fleshed apples also contain anthocyanins, mainly cyanidin-3-O-galactoside (Cy3Gal). Through an azoxymethane rat carcinogenesis model, a study was carried out in order to assess the possible protective effects of apple polyphenols, with special attention to anthocyanins. In addition, apart from negative and positive controls, a group with chemotherapy with 5-fluorouracil (5FU) was included to compare their performance against the output collected from the animal treatments with white-fleshed apple (WF), red-fleshed apple (RF) and Cy3Gal (AE). Although the 5FU group presented the best performance towards aberrant crypt foci (ACF) inhibition (70.1%), rats fed with white-fleshed apples ('Golden Smoothee') were able to achieve 41.3% ACF inhibition, while none of the challenged treatments (WF, RF and AE) suffered mucin depletion in their colonocytes. Expression changes of 17 genes related to CRC were assessed. In detail, the ACF inhibition phenotype detected in 5FU and WF groups could be explained through the expression changes detected in the apoptosis-related genes of Aurka, p53 and Cox2. Moreover, in the apple consumption groups (WF and RF), a reduced protein expression of matrix metalloproteinases with gelatinase activity (MMP-2 and 9) was detected. Overall, our study suggests an effect of apple polyphenols and apple anthocyanin Cy3Gal against colon carcinogenesis, retarding/diminishing the appearance of the precancerous markers studied., FundingThis research was partially supported by the Spanish Ministry of Economy and Competitiveness through the AGL2016-76943-C2-1-R project (cofounded by the State Research Agency and the European Regional Development Fund) and by the corresponding authors. This study was also supported by the Spanish Ministry of Education, Culture and Sport through the “Formación Profesorado Universitario” contract awarded to David BarsCortina (FPU014/02902).

[Scope] The antithrombotic effects of resveratrol (RV) and its derivatives remain unknown. The objective is to evaluate the modulatory effects of RV, its glucoside form, piceid, and its biological metabolites (RV‐3‐O‐β‐d‐glucuronide, RV‐4’‐O‐d‐glucuronide, and RV‐3‐O‐sulfate) on tissue factor (TF). Moreover, the endothelial metabolism of RV is assessed. [Methods and results] Human aortic endothelial cells (HAECs) are incubated with trans‐piceid, trans‐RV, or their biological metabolites and stimulated with tumor necrosis factor‐α (TNF‐α). TF activity, protein levels, and mRNA expression are determined in cell lysates. Moreover, RV conjugation (phase‐II‐metabolism) to its sulfated or glucuronidated metabolites and their deconjugation to their parent compound (free RV) are also assessed in cell lysates and culture media. RV decreased TF activity, protein levels, and mRNA expression, whereas piceid and RV metabolites (RVmet) had no effects. RV‐3‐O‐sulfate was the main metabolite generated in the endothelium, while RVmet are deconjugated to free RV. Isomerization of trans‐RV and its trans‐metabolites to their cis‐forms is observed. [Conclusions] RV exerts antithrombotic effects by modulating TF. RVmet and piceid does not exert this effect. However, the capacity of endothelial cells to deconjugate RVmet to free RV indicates that RVmet function as an endothelial reservoir for RV regeneration, thus, contributing to the antithrombotic effects of RV., This work was supported by the CENIT program from the Spanish Ministry of Science and Innovation (Proyecto CENIT: MET‐DEV‐FUN; 2006–2009) and by the project AGL2016‐76943‐C2 from the Ministerio de Economía, Indústria y Competitividad, the Agencia Estatal de Investigación (AEI), and the European Regional Development Fund (ERDF). Ú.C. has a Pla Estratègic de Recerca i Innovació en Salut (PERIS) post‐doctoral grant (SLT002/16/00239; Catalonia, Spain) from Generalitat de Catalunya. S.F.‐C., Ú.C., and R.S. conceived and designed the experiment.

Beneficial properties attributed to the intake of fruit and red wine have been associated with the presence of significant amounts of anthocyanins. However, their low absorption and consequent accumulation in the gut have generated the suspicion that colonic metabolites of anthocyanins are probably involved in these protective effects. Grape pomace and strawberry extracts, rich in malvidin- and pelargonidin-glucoside, respectively, were fermented in vitro using human feces as microbial inoculum. After 8 h of anaerobic incubation, the anthocyanins were almost completely degraded, whereas their microbial metabolite concentrations were highest at 24 h. Syringic acid and tyrosol were the main metabolites of grape and strawberry extracts, respectively. On the basis of the metabolites detected, metabolic pathways of malvidin- and pelargonidin-glucosides were proposed. Anthocyanin-rich grape and strawberry extracts and their generated metabolites such as hydroxyphenylacetic acid showed apoptotic effects in HT-29 colon cancer cells and may suggest their possible contribution as anticarcinogenic agents.

In this study, a fast and simple blood sampling and sample pre-treatment method based on the use of the dried blood spot (DBS) cards and ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) for the quantification of olive oil phenolic metabolites in human blood was developed and validated. After validation, the method was applied to determine hydroxytyrosol metabolites in human blood samples after the acute intake of an olive oil phenolic extract. Using the FTA DMPK-A DBS card under optimum conditions, with 20µL as the blood solution volume, 100µL of methanol/Milli-Q water (50/50, v/v) as the extraction solvent and 7 disks punched out from the card, the main hydroxytyrosol metabolites (hydroxytyrosol-3-O-sulphate and hydroxytyrosol acetate sulphate) were identified and quantified. The developed methodology allowed detecting and quantifying the generated metabolites at low μM levels. The proposed method is a significant improvement over existing methods to determine phenolic metabolites circulating in blood and plasma samples, thus making blood sampling possible with the volunteer pricking their own finger, and the subsequent storage of the blood in the DBS cards prior to chromatographic analysis.

Arbutus unedo is a small Mediterranean fruit, commonly named strawberry tree, which is a rich source of different sub-classes of phenolic compounds, the more representative being the gallic acid derivatives, including its mono and oligomeric forms esterified with quinic and shikimic acids. In addition, galloyl derivatives, particularly gallotannins, described in A. unedo, are part of a very selective phenolic group, present in a reduced number of plant-products. The aim of the present study is to provide a better understanding of human metabolic pathways of different sub-classes of phenols from the A. unedo fruit after an acute intake by healthy adults. Therefore, the A. unedo phenolic metabolites were studied in whole blood samples (0 to 24 h), urine (24 h) and feces (12 and 24 h). Special focus was placed on the application of dried blood spot (DBS) cards for the sample collection and for the analysis of phenolic metabolites in whole blood samples. The results of the blood analysis revealed two peaks for the maximum concentrations of the main phenolic metabolites. Furthermore, it is appropriate to highlight the application of DBS cards as an efficient and accurate way to collect blood samples in post-prandial bioavailability studies. The analysis of urine (24 h) gave a wide range of phenolic metabolites showing the extensive metabolism that A. unedo phenolic compounds underwent in the human body. The results of the study provide a relevant contribution to the understanding of the in vivo human bioavailability of phenolic compounds, especially galloyl derivatives, a singular phenolic sub-group present in the A. unedo fruit.

Anthocyanins (ACNs) are promising health-enhancing phenolic compounds. We focus on ACN animal tissue bioavailability to provide an evidentiary link between tissue ACNs and their associated health properties. We performed a systematic review of electronic libraries; 279 results were retrieved, and 13 publications met inclusion criteria. Extracted information included animal model employed, administration route, doses, analysis method, and ACN concentration values in tissues. Total ACN concentrations were detected in mice kidney (2.17 × 105 pmol/g), liver (1.73 × 105 pmol/g), heart (3.6 × 103 pmol/g), and lung (1.16 × 105 pmol/g); and in pig brain (6.08 × 103 pmol/g). ACNs showed a predominance of parent ACNs in long-term experiments versus an ACN metabolite predominance in short-term experiments. ACNs detected in animal tissues, such as cyanidin-3-glucoside, suggest it may have an important role in human health. This information could be useful to determine proper ACN-intake biomarkers in biological samples in futures studies. The AppleCOR Project (Subproject AGL2016-76943-C2-2-R and Suproject AGL2016-76943-C2-1-R) has been possible with the support of Ministerio de Economia , Indústria y Competitividad, the Agencia Estatal de Investigación (AEI) and the European Regional Development Fund (ERDF). B.A.S.-R. enjoys a 2017MFP-COFUND-30 predoctoral fellowship contract. This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie Grant Agreement No. 713679 and from the Universitat Rovira i Virgili (URV). Ú.C. has a Pla estratègic de recerca i innovació en salut (PERIS) postdoctoral grant (SLT002/16/00239; Catalunya, Spain) from Generalitat de Catalunya; NFOC-Salut group is a consolidated research group of Generalitat de Catalunya, Spain (2017 SGR 522).

Hydroxytyrosol (HT) and its derivatives represent the minor components of Virgin Olive Oil (VOO) that are of great interest for their pharmacological properties and among the most widely researched natural antioxidant compounds. In this review, the occurrence and metabolic fate of HT and its precursors are presented prior to discussing its beneficial effects on health. Bioavailability studies show that the metabolites detected in plasma depend on the model used (animal or human), the HT source (simple molecule or complex precursors) and the dose administered. However, in all cases HT sulphate appears to be the most ubiquitous metabolite in biofluids and it seems probable that it is responsible to a great extent for HT biological effects. Epidemiological evidence of HT and its derivatives against such lifestyle-associated pathologies as cancer, cardiovascular and neurodegenerative diseases is reviewed together with the newest perspectives on the mechanisms of action based on in-vitro and animal studies. According to the reviewed data, HT and its precursors could have the potential clinical use in cardiovascular diseases; more epidemiological data is needed to demonstrate their neurodegenerative diseases and cancer prevention.

The main objective of this study was to evaluate the impact of the season on the apple phytochemical composition (phenolic compounds, triterpenes, and organic and ascorbic acids). For this proposal, four red-fleshed and five white-fleshed apple varieties from two consecutive seasons (2015 and 2016) were studied. A significant interaction with the season in some compounds was observed. The total phenolic content in the apple flesh from 2015 was higher than that from 2016 probably related with the lower rainfall during the harvest period in 2015 that could have favored hydric stress in the apple trees. The impact of the season on the apple skin was different. The 2016 season was characterized by higher maximum and minimum temperatures resulting in a higher content of flavonols, triterpenes, and organic acids. Anthocyanin concentration in both the flesh and skin of the red-fleshed apples showed no clear relationship to the season, and each variety showed an individual pattern., This study was supported by the Spanish Ministry of Economy and Competitiveness through the AGL2016-76943-C2-1-R project (cofunded by the State Research Agency (AEI) and the European Regional Development Fund ERDF); and by the Spanish Ministry of Education, Culture and Sport through the “Formación Profesorado Universitario” grant awarded to D.B.-C. (FPU014/02902). In addition, L.B. acknowledges support from the MTM2015-69493-R grant from the Spanish Ministry of Economy and Competitiveness.

[Scope] The main findings of the “Virgin Olive Oil and HDL Functionality” (VOHF) study and other related studies on the effect of phenol‐enriched virgin olive oil (VOO) supplementation on cardiovascular disease are integrated in the present work., [Methods and results] VOHF assessed whether VOOs, enriched with their own phenolic compounds (FVOO) or with those from thyme (FVOOT), improve quantity and functionality of HDL. In this randomized, double‐blind, crossover, and controlled trial, 33 hypercholesterolemic subjects received a control VOO (80 mg kg−1), FVOO (500 mg kg−1), and FVOOT (500 mg kg−1; 1:1) for 3 weeks. Both functional VOOs promoted cardioprotective changes, modulating HDL proteome, increasing fat‐soluble antioxidants, improving HDL subclasses distribution, reducing the lipoprotein insulin resistance index, increasing endogenous antioxidant enzymes, protecting DNA from oxidation, ameliorating endothelial function, and increasing fecal microbial metabolic activity. Additional cardioprotective benefits were observed according to phenol source and content in the phenol‐enriched VOOs. These insights support the beneficial effects of OO and PC from different sources., [Conclusion] Novel therapeutic strategies should increase HDL‐cholesterol levels and enhance HDL functionality. The tailoring of phenol‐enriched VOOs is an interesting and useful strategy for enhancing the functional quality of HDL, and thus, it can be used as a complementary tool for the management of hypercholesterolemic individuals., This work was supported by grants: the VOHF Study (AGL2009-13517- C03) and the MEFOPC Project (AGL2012-40144-C03) from the Spanish Ministry of Education and Science. The authors wish to acknowledge the support of the IISPV, the EURECAT-CTNS, and the COS; Reus, Spain. M.C.L.H has predoctoral student grant from the Universitat de Lleida. A.P. has Torres Quevedo contract (Subprograma Estatal de Incorporaci´on, Plan Estatal de Investigaci´on Cient´ıfica y T´ecnica y de Innovaci´on). L.R. and M.F. have Sara Borrell postdoctoral grants (CD14/00275, 2015–2017;CD17/00233, 2018–2021). M.F. was also supported by a joint contract of the ISCIII and Health Department of the Catalan Government (CES12/025; CB06/03/0028). U.C. has a PERIS post-doctoral grant ´ (SLT002/16/00239; Catalunya, Spain). NFOC-Salut group is a consolidated research group of Generalitat de Catalunya, Spain (2017 SGR 522).CIBERDEM and CIBEROBN are initiatives of ISCIII of Spain which is supported by FEDER funds (CB06/03).

[Scope] The application of dried blood spot (DBS) cards for the study in human blood of dietary polyphenol bioavailability has been poorly studied. [Methods and results] An analytical method based on blood sampling with DBS cards combined with LC–MS/MS has been developed and validated. To test the method validation, the phenolic metabolites are determined in human blood and plasma obtained after an acute intake of a red‐fleshed apple snack in ten volunteers. Capillary blood by finger prick is compared to venous blood by venipuncture and whole blood is also compared to their corresponding venous plasma samples. Moreover, the venous plasma results using DBS cards are compared to those obtained by microElution solid phase extraction (µSPE). The main phenolic metabolites detected in blood and plasma samples are phloretin glucuronide, dihydroxyphenylpropionic acid sulphate, (methyl) catechol sulphate, catechol glucuronide, and hydroxyphenyl‐γ‐valerolactone glucuronide. No significant differences are observed between capillary blood, venous blood, and plasma samples using DBS, and neither between plasma samples analyzed by DBS or µSPE. [Conclusions] Finger‐prick blood sampling based on DBS appears to be a suitable alternative to the classic invasive venipuncture for the determination of circulating phenolic metabolites in nutritional postprandial studies, This study was supported by the Spanish Ministry of Industry, Economy and Competitiveness through the AGL2016‐76943‐C2‐1‐R and AGL2016‐76943‐C2‐2‐R projects (co‐funded by the European Social Fund, European Union); by the Spanish Ministry of Industry, Economy and Competitiveness through the I. A. Ludwig postdoctoral research contract (Juan de la Cierva, FJCI‐2014‐20689); and by the University of Lleida through the Silvia Yuste doctoral grant. In addition, the authors are grateful to NUFRI (Mollerussa, Lleida, Catalonia, Spain) for providing the apples. Ú.C. has a PERIS post‐doctoral grant (SLT002/16/00239; Catalunya, Spain). NFOC‐Salut group is a consolidated research group of Generalitat de Catalunya, Spain (2017 SGR 522).

cope The purpose of the study was to evaluate the effect, regarding the metabolic and microbial profile of feces, of diet supplementation of healthy adults with pomegranate juice (PJ). Methods and results Twelve healthy adults were recruited to the study, which consisted of the intake of 200 mL/day of PJ during 4 weeks. Feces were collected before and after the supplementation with PJ. Metabolites (phenolic catabolites, short-chain fatty acids, and fecal steroids) and microbial profile were analyzed at baseline and at 4 weeks. Fecal phenolic metabolites, 3-phenylpropionic acid, catechol, hydroxytyrosol, and urolithin A, showed a significant increase in their concentration after supplementation with PJ. Among fecal steroids, parallel to the significant increase of cholesterol concentration, a significant decrease of coprostanol was observed. Although no significant changes in the microbiota profile were observed, different relationships between initial microbiota and the metabolites produced were found. Catechol showed positive and negative correlation with Oscillospora and Paraprevotella genera, respectively, and 3-phenylpropionic acid was positively correlated with Odoribacter genus. Conclusion Inclusion of PJ in the diet did not significantly alter the gut microbiota composition in healthy adults, but the individual bacterial composition could contribute to the generation of potential health-promoting phenolic metabolites.

Pomegranate fruit contains a wide range of phenolic compounds that have been related to several health benefits. The stability of pomegranate phenols during digestion was tested by the application of in vitro gastrointestinal digestion (GID) to different pomegranate products: juice (PJ), pulp (PP) and peel extract (PE). The resulting non-absorbable fractions were submitted to in vitro colonic fermentation with human faeces to monitor the generation of microbial metabolites. During the duodenal step, we observed a low stability of anthocyanins and flavonoids and an important release of ellagic acid, especially after PE digestion. The poor potential absorption of the studied phenolic compounds led to their high exposure to colonic metabolism. After colonic fermentation, PE appeared to be the best source of microbial substrates leading to a larger generation of gut microbial catabolites in terms of absolute amounts. We suggest that using PE might be a good strategy to enrich food products with potential health benefits in the prevention of chronic diseases.

This study is an exhaustive chemical characterization of the phenolic compounds, triterpenes, and organic and ascorbic acids in red-fleshed apple varieties obtained by different breeding programs and using five traditional and new white-fleshed apple cultivars as reference. To carry out these analyses, solid-liquid extraction (SLE) and ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) were used. The results showed that the red-fleshed apples contained, in either the flesh or peel, higher amounts of phenolic acids (chlorogenic acid), anthocyanins (cyanidin-3-O-galactoside), dihydrochalcones (phloretin xylosyl glucoside), and organic acids (malic acid) but a lower amount of flavan-3-ols than the white-fleshed apples. These quantitative differences could be related to an up-regulation of anthocyanins, dihydrochalcones, and malic acid and a down-regulation of flavan-3-ols (anthocyanin precursors) in both the flesh and peel of the red-fleshed apple varieties. The reported results should be considered preliminary because the complete phytochemical characterization of the red-fleshed apple cultivars will be extended to consecutive harvest seasons.

After the sustained consumption of virgin olive oil (VOO), the unabsorbed native phenols (mainly hydroxytyrosol (HT)) are transformed into its catabolites in the intestine by microbials. The role of these catabolites in preventing colon cancer has not been sufficiently investigated. This work aims to study the antiproliferative and apoptotic activities in colon (Caco-2; HT-29) cancer cell lines of the main catabolites detected in human feces (phenylacetic, phenylpropionic, hydroxyphenylpropionic, and dihydroxyphenylpropionic acids and catechol), after the sustained VOO intake. Additionally, an assessment of the ability of these colonic cells to metabolize the studied compounds was performed. The results showed that HT and phenylacetic and hydroxyphenylpropionic acids produce cell cycle arrest and promote apoptosis. HT-29 cells were more sensitive to phenol treatments than Caco-2. In synthesis, the results of the present study represent a good starting point for understanding the potential apoptotic and antiproliferative effects of VOO phenolic compounds and their colonic metabolites.

Rice bran is an exceptional source of such antioxidant molecules as γ-oryzanol and ferulic acid, but their bioavailability and metabolism within this matrix remain unknown. The aims of this work were to describe the oral bioavailability and metabolic pathways of the ferulic acid-derived phenolic compounds contained in a rice bran enzymatic extract (RBEE), and to determine its effect on NADPH oxidase activity. Wistar rats were administered with RBEE and sacrificed at different times over a period of 24 h to obtain plasma. An additional group was used for collection of urine and faeces over a period of 48 h. The phenolic metabolites were determined by ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS), and plasma pharmacokinetic parameters were calculated. In parallel, aortic rings were incubated in the plasma of rats sacrificed 30 min after RBEE gavage, or in the presence of RBEE, ferulic acid or γ-oryzanol. Endothelin-1-induced superoxide production was recorded by lucigenin-enhanced luminescence. Twenty-five ferulic acid metabolites showing biphasic behaviour were found in the plasma, most of which were found in the urine as well, while in the faeces, colonic metabolism led to simpler phenolic compounds. Superoxide production was abrogated by phenolic compound-enriched plasma and by RBEE and ferulic acid, thus showing the biological potential of RBEE as a nutraceutical ingredient.

Olive oils flavoured with edible herbs have grown in popularity because of their added value and potential health benefits. However, the combined presence of different phytochemicals from olive oil and herbs requires study of their possible interactions during intestinal transport and metabolism. The aim of this study was firstly to evaluate the effect on bioaccessibility of the co-occurring bioactive compounds from olive oil and thyme through an in vitro digestion model of three extracts: olive extract (OE), thyme extract (TE) and a combination of both (OTE). The bioaccessible fractions were exposed to Caco-2 and HepG-2 cell models, as well as to a co-culture of both of these. Results indicated that the bioaccessibility of hydroxytyrosol was enhanced when OTE was digested. After Caco-2 cells exposure, no significant differences were observed in hydroxytyrosol transport, whereas the main flavonoids from thyme seemed to undergo an enhanced basolateral permeation when both phenolic sources where exposed.

Several epidemiological studies throughout the years have suggested that polyphenols from fruits and vegetables promote health and reduce the risk of certain chronic and neurodegenerative diseases. Yet, it has been proved to be extremely difficult to quantitatively establish the benefit afforded by polyphenols, principally due to the limited understanding of the extent of its absorption and metabolic fate. Pharmacokinetics includes the study of the mechanisms of absorption and distribution of an ingested polyphenol, its chemical changes in the body (e.g. by metabolic enzymes), and the effects and routes of excretion of the metabolites. In recent years, there have been major advances in our knowledge of polyphenol absorption and metabolism, and it is apparent that most classes of polyphenols are sufficiently absorbed to have the potential to exert biological effects. The pharmacokinetics of polyphenols includes the same steps as those for orally ingested drugs (LADME) and faces some of the same challenges, including transporters and enzymes. However, unraveling the bioavailability of polyphenols is even more challenging than with drugs, since many other factors, such as the variety in the chemical structure, the food matrix and the gut microbiota, can affect bioavailability of polyphenols during digestion. This review focuses on the most relevant factors that influence polyphenol pharmacokinetics, and also on the most recent technological strategies developed to overcome the poor bioavailability of phenolic compounds and thus increase their potential for greater health benefits.

Two different rapid sample pretreatment strategies, dried spot cards, and microelution solid-phase extraction plates (μSPE), with ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) have been developed and validated for the determination of hydroxytyrosol and its metabolites in spiked human urine samples. Hydroxytyrosol, hydroxytyrosol-3'-O-glucuronide, hydroxytyrosol-4'-O-glucuronide, hydroxytyrosol-3-O-sulphate, and homovanillic alcohol-4'-O-glucuronide were used as the target compounds. Using the FTA DMPK-A dried urine spot card under optimum conditions, with 5 μL of preconcentrated urine volume and 100 μL of methanol/water (50/50, v/v) as the elution solvent, the extraction recovery (%R) of the compounds studied was higher than 80%, and the matrix effect (%ME) was less than 8%. The stability of these cards and punching at the centre or side of the card were also studied, obtaining an excellent stability after 7 days of storage and complete homogeneity across the surface of the dried drop. The different μSPE parameters that affect the efficiency were also studied, and under optimum conditions, the %R and the %ME were higher than 70% and lower than 17%, respectively. The linearity range in dried urine spot cards was 2.5-20 μM for all the metabolites, with the exception of hydroxytyrosol-3-O-sulphate and hydroxytyrosol, which were 0.3-70 μM and 2.5-50 μM respectively. With regards to μSPE, the linearity range was 0.5-5 μM for all the studied compounds, except for hydroxytyrosol-3-O-sulphate, which was 0.08-5 μM. The quantification limits (LOQs) were 0.3-2.5 μM and 0.08-0.5 μM in dried spot cards and in μSPE, respectively. The two developed methods were then applied and compared for determining hydroxytyrosol and its metabolites in human 24 h-urine samples after a sustained consumption (21 days) of a phenol-enriched virgin olive oil. The metabolites identified were hydroxytyrosol in its glucuronide and sulphate forms, homovanillic alcohol in its glucuronide and sulphate forms, homovanillic acid sulphate and hydroxytyrosol acetate sulphate.

Scope—Lipoprotein particle measures performed by nuclear magnetic resonance (NMR), and associated ratios, may be better markers for atherosclerosis risk than conventional lipid measures. The effect of two functional olive oils, one enriched with its polyphenols (FVOO, 500 ppm), and the other (FVOOT) with them (250 ppm) and those of thyme (250 ppm), versus an standard virgin olive oil (VOO), on lipoprotein particle atherogenic ratios and subclasses profiles was assessed. Methods and Results—In a randomized, double-blind, crossover, controlled trial, 33 hypercholesterolemic individuals received 25 mL/day of VOO, FVOO, and FVOOT. Intervention periods were of 3-weeks separated by 2-week washout periods. Lipoprotein particle counts and subclasses were measured by NMR. Polyphenols from olive oil and thyme modified the lipoprotein subclasses profile and decreased the total LDL particle/total HDL particle (HDL-P), small HDL/large HDL, and HDL-cholesterol/HDL-P ratios, and decreased the lipoprotein insulin resistance index (LP-IR) (P

This study investigated and compared the absorption, metabolism, and subsequently, the tissue distribution and excretion of hydroxytyrosol (HT) administered either in its free form or through its naturally occurring esterified precursors, namely oleuropein (OLE) and its aglycone forms known as secoiridoids (SEC). Here, rats were fed a diet supplemented with the equivalent of 5 mg phenol/kg/day for 21 days and the HT metabolites in the gastrointestinal digesta (stomach, small intestine and caecum), plasma, urine and metabolic tissues (liver and kidney) were analysed. Compared to HT and SEC, OLE showed greater stability during digestion, and, consequently, the bioavailability based on the urine excretion of HT metabolites was higher. OLE, as a glycoside molecule, reached the colon unaltered generating more diverse microbial metabolites. In terms of bioavailability, findings suggest that OLE might be the most suitable precursor of HT for incorporation into foods or nutraceutical formulations.

Flavonoids are metabolized by the gut microflora giving rise to a wide range of phenolic acids that may exert systemic effects in the body. Nevertheless, the colonic metabolism pathways and the function of the metabolites formed are poorly studied. In the present study, the individual colonic metabolism of three subclasses of flavonoids (flavonols, flavones and flavanones) and phenolic acids was evaluated. For this, seven standards of flavonoids (quercetin, quercetin–rhamnoside, quercetin–rutinoside, myricetin, luteolin, naringenin and kaempferol–rutinoside) and two phenolic acids (protocatechuic acid and gallic acid) were submitted to an in vitro fermentation model using rat colonic microflora. Simultaneously, a nuts–cocoa cream enriched with these standards of flavonoids was incubated and the colonic metabolism of these compounds was evaluated. The results showed that the greatest number of colonic metabolites came from the fermentation of quercetin and quercetin–rhamnoside, and the maximum concentration of fermentation products was observed after 48 h of fermentation. On the other hand, a smaller number of fermentation products were observed after the colonic fermentation of kaempferol–rutinoside, naringenin, luteolin and myricetin. The phenolic acids were slightly metabolized by the colonic microflora.

Phenolic compounds are one of the main reasons behind the healthy properties of virgin olive oil (VOO). However, their daily intake from VOO is low compared with that obtained from other phenolic sources. Therefore, the intake of VOO enriched with its own phenolic compounds could be of interest to increase the daily dose of these beneficial compounds. To evaluate the effectiveness of enrichment on their bioavailability, the concentration of phenolic compounds and their metabolites in human plasma (0, 60, 120, 240 and 300 min) from thirteen healthy volunteers (seven men and six women, aged 25 and 69 years) was determined after the ingestion of a single dose (30 ml) of either enriched virgin olive oil (EVOO) (961·17 mg/kg oil) or control VOO (288·89 mg/kg oil) in a cross-over study. Compared with VOO, EVOO increased plasma concentration of the phenol metabolites, particularly hydroxytyrosol sulphate and vanillin sulphate (P,0·05). After the consumption of VOO, the maximum concentration of these peaks was reached at 60 min, while EVOO shifted this maximum to 120 min. Despite these differences, the wide variability of results indicates that the absorption and metabolism of olive oil phenols are highly dependent on the individual. The present study was supported by the Spanish Ministry of Education and Science financing the projects AGL2005- 07881-C02-01/ALI and AGL2005-07881-C02-02/ALI; Health Ministry (FIS; PI021307) and CIBERDEM; and the grant received by Manuel Suarez (BES-2006-14136).

Rapid, selective and sensitive methods were developed and validated to determine procyanidins, anthocyanins and alkaloids in different biological tissues, such as liver, brain, the aorta vein and adipose tissue. For this purpose, standards of procyanidins (catechin, epicatechin, and dimer B(2)), anthocyanins (cyanidin-3-glucoside and malvidin-3-glucoside) and alkaloids (theobromine, caffeine and theophylline) were used. The methods included the extraction of homogenized tissues by off-line liquid-solid extraction, and then solid-phase extraction to analyze alkaloids, or microelution solid-phase extraction plate for the analysis of procyanidins and anthocyanins. The eluted extracts were then analyzed by ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry, using a triple quadrupole as the analyzer. The optimum extraction solution was water/methanol/phosphoric acid 4% (94/4.5/1.5, v/v/v). The extraction recoveries were higher than 81% for all the studied compounds in all the tissues, except the anthocyanins, which were between 50 and 65% in the liver and brain. In order to show the applicability of the developed methods, different rat tissues were analyzed to determine the procyanidins, anthocyanins and alkaloids and their generated metabolites. The rats had previously consumed 1g of a grape pomace extract (to analyze procyanidins and anthocyanins) or a cocoa extract (to analyze alkaloids) per kilogram of body weight. Different tissues were extracted 4h after administration of the respective extracts. The analysis of the metabolites revealed a hepatic metabolism of procyanidins. The liver was the tissue which produced a greater accumulation of these metabolites.

Procyanidins are metabolised by endogenous gastrointestinal microflora into several small molecules that may exert systemic effects. However, the metabolic pathways of procyanidins are still largely uncharacterised. The aim of this study was to evaluate the colonic metabolism of procyanidins (catechin, epicatechin, epicatechin gallate, epigallocatechin gallate and dimer B2) and alkaloids (theobromine and caffeine) by an in vitro colonic fermentation model using rat colonic microflora. Simultaneously, a nuts-cocoa cream was incubated and the colonic metabolism of procyanidins and alkaloids was evaluated. Results showed that most of the procyanidins tested were catabolised after 24–48 h of fermentation. Fermentation of the dimer B2 did not produce the same catabolic compounds as epicatechin fermentation and differences were observed between the fermentation of catechin and epicatechin. The alkaloids were not metabolised by the colonic microflora and this fact was verified in vivo. The results showed an intense metabolism of procyanidins and poor metabolism of alkaloids.

Carob flour (CF) and washed carob flour (WCF) (without the water soluble dietary fraction) were submitted to an in-vitro digestion method to evaluate the dietary soluble fibre (DF) and soluble sugars content on the digestibility and bioaccessibility of polyphenols. In addition, combinations of the raw materials (CF and WCF) with hazelnut oil (HO) were proposed in order to evaluate the protection of a vegetable oil rich in polyunsaturated fatty acids (PUFA) on the phenolic compounds submitted to in-vitro digestion conditions. The results showed that the soluble dietary fraction enhanced the stability of the phenolic compounds during the duodenal digestion phase. Similarly, the lipid fraction (rich in PUFA) showed a protective effect on the recovery of the phenolic compounds during duodenal digestion. Disruption of the natural matrix under digestion conditions led to the release of DF and PUFA, which could establish interactions with certain polyphenol compounds enhancing their recovery and stability during digestion.

Natural plant extracts are complex mixtures of molecules that have to be uptaken and metabolized, at least by the intestinal barrier, before they reach their body targets. This paper evaluates the feasibility of using a co-culture system to assay the bioactivity of plant extract on hepatic cells closest to the in vivo situation. Caco-2 cells, mimicking intestinal barrier roles, were grown on inserts on a monolayer of HepG2 hepatocytes for 24 h. The co-culture induced some adaptations, but the Caco-2 cells showed no differences in their abilities to filter and metabolize grape-seed derived extract (GSPE) components. The co-culture clearly reproduced the ability of a procyanidin extract to decrease the triglyceride secretion of hepatocytes, a proven in vivo effect. This system mimics a human physiological system that is useful for assaying the bioactivity of extracts and may be able to be used for developing new functional foods.