The Missing Piece in the Puzzle of Inflammation

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The low molecular weight phenolic metabolites and their concentrations in human circulation

Funding Information: European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme under grant agreement No 804229. iNOVA4Health Research Unit (LISBOA‐01‐0145‐FEDER‐007344), which is cofunded by Fundação para a Ciência e Tecnologia (FCT) / Ministério da Ciéncia e do Ensino Superior, through national funds, and by FEDER under the PT2020 Partnership Agreement, European Research Council (Starting Grant: FunKeyGut 741623). FCT (2020.04630.BD). Funding Information: This work has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme under grant agreement No 804229. iNOVA4Health Research Unit (LISBOA‐01‐0145‐FEDER‐007344), which is cofunded by Fundação para a Ciência e Tecnologia (FCT) / Ministério da Ciência e do Ensino Superior, through national funds, and by FEDER under the PT2020 Partnership Agreement, is acknowledged. D.B. acknowledges funding by the European Research Council (Starting Grant: FunKeyGut 741623). Authors would like to acknowledge FCT for financial support of D.C. (2020.04630.BD). Publisher Copyright: © 2022 The Authors. Comprehensive Reviews in Food Science and Food Safety published by Wiley Periodicals LLC on behalf of Institute of Food Technologists.A large number of epidemiological studies have shown that consumption of fruits, vegetables, and beverages rich in (poly)phenols promote numerous health benefits from cardiovascular to neurological diseases. Evidence on (poly)phenols has been applied mainly to flavonoids, yet the role of phenolic acids has been largely overlooked. Such phenolics present in food combine with those resulting from gut microbiota catabolism of flavonoids and chlorogenic acids and those produced by endogenous pathways, resulting in large concentrations of low molecular weight phenolic metabolites in human circulation. Independently of the origin, in human intervention studies using diets rich in (poly)phenols, a total of 137 low molecular weight phenolic metabolites have been detected and quantified in human circulation with largely unknown biological function. In this review, we will pinpoint two main aspects of the low molecular weight phenolic metabolites: (i) the microbiota responsible for their generation, and (ii) the analysis (quali- and quantitative) in human circulation and their respective pharmacokinetics. In doing so, we aim to drive scientific advances regarding the ubiquitous roles of low molecular weight phenolic metabolites using physiologically relevant concentrations and under (patho)physiologically relevant conditions in humans.publishersversionepub_ahead_of_prin

A Human Pharmacokinetic Study

Funding: This research was supported by the Projects PID2019-103914RB-I00 and PID2019-109369RBI00 from the Ministry of Science and Innovation (MCIN/AEI/10.13039/501100011033, Spain). C.E.I.- A. is the holder of a predoctoral grant FPU18/03961 from MICIN. M.C.L.-H. is a receipt of a Juan de la Cierva Grant IJC2020-044353-I/MCIN/AEI/10.13039/501100011033/EU/PRTR.Exosomes are extracellular vesicles (EVs) that regulate intercellular signaling by transferring small RNAs, proteins, nucleic acids, lipids, and other metabolites to local or distant organs, including the brain, by crossing the blood-brain barrier. However, the transport of (poly)phenols in human EVs has not yet been described. Therefore, we aimed here to explore (i) whether resveratrol and (or) its derived metabolites are found in the cargo of human plasma exosome-containing EVs (E-EVs), (ii) when this incorporation occurs, and (iii) whether resveratrol intake stimulates the release of E-EVs. Thus, in a pharmacokinetic study, healthy volunteers (n = 16) consumed 1 capsule (420 mg resveratrol) in the evening before attending the clinic and one more capsule on the day of the pharmacokinetics. The plasma and the isolated E-EVs were analyzed using UPLC-ESI-QTOF-MS. Of 17 metabolites in the plasma, 9 were identified in the E-EVs, but not free resveratrol. The kinetic profiles of resveratrol metabolites were similar in the plasma and the E-EVs, a higher metabolite concentration being detected in the plasma than in the E-EVs. However, the plasma/E-EVs ratio decreased in the gut microbial metabolites, suggesting their better encapsulation efficiency in E-EVs. In addition, glucuronide conjugates of resveratrol, dihydroresveratrol, and lunularin were incorporated into the E-EVs more efficiently than their corresponding sulfates despite glucuronides reaching lower plasma concentrations. Notably, more E-EVs were detected 10 h after resveratrol consumption. This exploratory study provides the first evidence that (i) resveratrol metabolites are transported by E-EVs, with a preference for glucuronide vs. sulfates, (ii) the gut microbial metabolites concentration and kinetic profiles are closely similar in E-EVs and plasma, and (iii) resveratrol intake elicits E-EVs secretion. Overall, these results open new research avenues on the possible role of E-EVs in (poly)phenol health effects.publishersversionpublishe

(R,S)-Equol 7-β-D-glucuronide, but not other circulating isoflavone metabolites, modulates migration and tubulogenesis in human aortic endothelial cells targeting the VEGF pathway

Funding Information: J. A. G.-B. was supported by a Standard European Marie Curie Individual Fellowship from the European Commission. D. G.-M. was supported by a Miguel Servet contract (AES 2021; CP21/00028) funded by the Institute Carlos III and by “ERDF a way of making Europe”. This work has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie Grant Agreement No. 838991. This work was supported by the Ramón y Cajal grant (RyC2021-032111-I) and by the grants CNS2022-135253 and TED2021-130962B-C22 funded by the MCIN/AEI/10.13039/501100011033 and by the “European Union NextGenerationEU/PRTR” program, grants PID2022-136915NA-I00 and PID2022-136419OB-I00 funded by MCIN/AEI/10.13039/501100011033 and “ERDF A way of making Europe” by the European Union, and the grant 22030/PI/22 funded by the Programa Regional de Fomento de la Investigación Científica y Técnica (Plan de Actuación 2022) de la Fundación Séneca-Agencia de Ciencia y Tecnología de la Región de Murcia, Spain. Publisher Copyright: © 2024 The Royal Society of Chemistry.Current knowledge indicates that the consumption of isoflavone-rich foodstuffs can have a beneficial impact on cardiovascular health. To what extent these isoflavones act as the main actors of that benefit is less clear. Genistein (GEN), daidzein (DAZ), and the DAZ-derived microbial metabolite equol (Eq) exhibit antiangiogenic effects in vitro, but their low bloodstream concentrations make it difficult to rationalize the in vivo effects. Their derived phase-II metabolites (glucuronides and sulfates) are major metabolites found in plasma, but their role as antiangiogenic molecules remains unexplored. We aimed here to first assess the anti-angiogenic activities of the main circulating isoflavone metabolites (glucuronides and sulfates) and compare them with their corresponding free forms at physiological concentrations (0.1-10 μM). The effects of the conjugated vs. free forms on tubulogenesis, cell migration, and VEGF-induced signalling were investigated in primary human aortic endothelial cells (HAECs). While (R,S)-equol 7-β-d-glucuronide (Eq 7-glur) exerted dose-dependent inhibition of tubulogenesis and endothelial migration comparable to that exerted by the free forms (GEN, DAZ, and Eq), the rest of the phase-II conjugates exhibited no significant effects. The underlying molecular mechanisms were independent of the bFGF but related to the modulation of the VEGF pathway. Besides, the observed dissimilar cellular metabolism (conjugation/deconjugation) places the phase-II metabolites as precursors of the free forms; however, the question of whether this metabolism impacts their biological activity requires additional studies. These new insights suggest that isoflavones and their circulating metabolites, including Eq 7-glur, may be involved in cardiovascular health (e.g., targeting angiogenesis).publishersversionepub_ahead_of_prin

Targeting proteases involved in the viral replication of SARS-CoV-2 by sesquiterpene lactones from chicory (Cichorium intybus L.)

Funding Information: This research has been supported by the EU Horizon 2020 research & innovation programme (H2020-NMBP-BIO-2017, project CHIC grant agreement no. 760891). Publisher Copyright: © 2022 The Royal Society of Chemistry.SARS-CoV-2 is a highly transmissible and pathogenic coronavirus causing a respiratory disease that emerged in 2019, leading to a public health emergency situation which continues to date. The treatment options are still very limited and vaccines available are less effective against new variants. SARS-CoV-2 enzymes, namely main protease (Mpro) and papain-like protease (PLpro), play a pivotal role in the viral life cycle, making them a putative drug target. Here, we described for the first time the potential inhibitory activity of chicory extract against both proteases. Besides, we have identified that the four most abundant sesquiterpene lactones in chicory inhibited these proteases, showing an effective binding in the active sites of Mpro and PLpro. This paper provides new insight for further drug development or food-based strategies for the prevention of SARS-CoV-2 by targeting viral proteases.publishersversionepub_ahead_of_prin

Dietary Phenolics against Breast Cancer. A Critical Evidence-Based Review and Future Perspectives

© 2020 by the authors.Breast cancer (BC) is the most common malignancy and the leading cause of cancer-related death in adult women worldwide. Over 85% of BC cases are non-hereditary, caused by modifiable extrinsic factors related to lifestyle, including dietary habits, which play a crucial role in cancer prevention. Although many epidemiological and observational studies have inversely correlated the fruit and vegetable consumption with the BC incidence, the involvement of their phenolic content in this correlation remains contradictory. During decades, wrong approaches that did not consider the bioavailability, metabolism, and breast tissue distribution of dietary phenolics persist behind the large currently existing gap between preclinical and clinical research. In the present review, we provide comprehensive preclinical and clinical evidence according to physiologically relevant in vitro and in vivo studies. Some dietary phenolics such as resveratrol (RSV), quercetin, isoflavones, epigallocatechin gallate (EGCG), lignans, and curcumin are gaining attention for their chemopreventive properties in preclinical research. However, the clinical evidence of dietary phenolics as BC chemopreventive compounds is still inconclusive. Therefore, the only way to validate promising preclinical results is to conduct clinical trials in BC patients. In this regard, future perspectives on dietary phenolics and BC research are also critically discussedThis research was funded by the projects PID2019-103914RB-I00 (MICINN, Spain), 19900/GERM/15 (Fundación Séneca de la Región de Murcia, Spain), and 201870E014 and 201770E081 (CSIC, Spain). J.A.G.B. was supported by a Juan de la Cierva contract (IJCI-2016-27633) from the Ministry of Science, Innovation and Universities (Spain) and a Standard European Marie Curie Fellowship from the European Commission. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 838991.Peer reviewe

Effect of Resistant Dextrin on Intestinal Gas Homeostasis and Microbiota

Previous studies have shown that a resistant dextrin soluble fibre has prebiotic properties with related health benefits on blood glucose management and satiety. Our aim was to demonstrate the effects of continuous administration of resistant dextrin on intestinal gas production, digestive sensations, and gut microbiota metabolism and composition. Healthy subjects (n = 20) were given resistant dextrin (14 g/d NUTRIOSE ®, Roquette Frères, Lestrem, France) for four weeks. Outcomes were measured before, at the beginning, end, and two weeks after administration: anal evacuations of gas during daytime; digestive perception, girth, and gas production in response to a standard meal; sensory and digestive responses to a comfort meal; volume of colonic biomass by magnetic resonance; taxonomy and metabolic functions of fecal microbiota by shotgun sequencing; metabolomics in urine. Dextrin administration produced an initial increase in intestinal gas production and gas-related sensations, followed by a subsequent decrease, which magnified after discontinuation. Dextrin enlarged the volume of colonic biomass, inducing changes in microbial metabolism and composition with an increase in short chain fatty acids-producing species and modulation of bile acids and biotin metabolism. These data indicate that consumption of a soluble fibre induces an adaptative response of gut microbiota towards fermentative pathways with lower gas productio

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

Polifenoles de la dieta frente al cáncer de mama : estudios metabólicos y moleculares en pacientes, animales y modelos celulares.(Estudio Polysen)

El cáncer de mama es el más diagnosticado entre mujeres y supone un 15% de las muertes por cáncer entre la población mundial femenina. Tiene una etiología multifactorial, pero sólo el 10-15% son casos hereditarios. Esto implica que los factores de riesgo modificables son los que se asocian principalmente con su incidencia. Mientras que la obesidad o abuso de alcohol incrementan el riesgo de cáncer de mama, un mayor consumo de alimentos vegetales podría tener posible efecto preventivo. Numerosos estudios preclínicos con compuestos fenólicos muestran multitud de mecanismos de acción protectores frente al cáncer de mama. Sin embargo, la evidencia en humanos es aún escasa y controvertida. Esta Tesis Doctoral incluye una revisión exhaustiva y crítica de los motivos que hay tras esa evidencia aún inconsistente, incluyendo la falta de i) estudios en pacientes con cáncer de mama que relacionen los efectos beneficiosos con los compuestos fenólicos, ii) estudios preclínicos con metodologías adecuadas para extrapolar sus resultados a un contexto clínico, y iii) estudios que evalúen la biodisponibilidad y distribución en tejidos humanos sistémicos. Considerando lo anterior, esta Tesis ha tenido como objetivo principal identificar qué compuestos (formas moleculares y concentraciones) llegan al tejido mamario tumoral (TTM) y sano (TSM) en pacientes con cáncer de mama tras el consumo de una mezcla de extractos vegetales, ricos en fenólicos, representativos de la dieta. Este objetivo serviría para abordar estudios mecanísticos in vitro, más representativos de la situación in vivo, sobre el efecto quimiopreventivo de metabolitos fisiológicamente relevantes en modelos celulares de cáncer de mama. Para abordar estos objetivos se realizó un estudio clínico en pacientes con cáncer de mama, donde se caracterizó el perfil metabólico de diferentes compuestos fenólicos y sus metabolitos en TTM y TSM, plasma y orina de estas pacientes, tras consumir una mezcla de extractos vegetales. Además, se estudió en un modelo animal (ratas Sprague Dawley) la farmacocinética en plasma y tejido mamario de fenólicos y metabolitos derivados, tras el consumo de esta mezcla. El estudio clínico permitió identificar 27 metabolitos fenólicos y 6 metilxantinas en TTM de pacientes con cáncer de mama, siendo la mayoría de las formas moleculares detectadas en TTM, conjugados de Fase-II (glucurónidos y sulfatos). El estudio animal permitió conocer parámetros cinéticos en plasma y tejido mamario de los fenólicos y sus derivados metabólicos, y sirvió para identificar que el ayuno previo a la cirugía en pacientes puede subestimar cualitativa- y cuantitativamente la distribución de metabolitos fenólicos en tejido mamario. Los metabolitos que llegan al TTM no ejercieron efecto antiproliferativo, ni tampoco actividad estrogénica/antiestrogénica, a tiempos cortos de exposición en diferentes modelos celulares de cáncer de mama. Estudios posteriores revelaron que el metabolismo de Fase-II es crítico para dificultar/abolir la actividad biológica de los metabolitos. Sin embargo, resveratrol y sus metabolitos de Fase-II, detectados en TTM humano, sí ejercieron efecto quimiopreventivo a largo plazo, mediante la inducción de senescencia celular (dependiente de la ruta p53/p21) en la línea tumoral MCF-7 (p53-silvestre) pero no en MDA-MB-231 (p53-mutado). Esto último alienta a investigar el efecto quimiopreventivo de otros fenólicos detectados en TTM mediante la inducción de senescencia, lo que sería plausible con un consumo crónico de alimentos ricos en fenólicos. En general, esta Tesis ha contribuido a comprender qué compuestos fenólicos y metabolitos llegan al TTM en pacientes con cáncer de mama tras el consumo de alimentos vegetales, y qué posible papel podrían jugar estos compuestos frente al cáncer. Además, esta Tesis refuerza el concepto de “primero in vivo, luego in vitro”, y establece una posible secuencia de estudios a seguir para caracterizar la actividad anticancerígena de componentes de la dieta en tejidos sistémicos.Breast cancer is the most commonly diagnosed cancer in women worldwide. Overall, this disease accounts for 15% of cancer deaths among the female population. Breast cancer has multifactorial etiology, but only 10-15% of cases are hereditary. This means that the vast majority are caused by modifiable factors. Thus, it has been described that while obesity or alcohol consumption increase the risk of breast cancer, the higher consumption of phenolic-rich plant foods could be related to a possible preventive effect. Although many preclinical studies have reported numerous mechanisms of action of phenolics that would explain their protective role against breast cancer, however, evidence remains still scarce and controversial in humans. This Doctoral Thesis reviews comprehensively and critically the reasons that are behind this inconsistent evidence, including i) the low number of clinical trials that report beneficial effects of phenolics against breast cancer, ii) the big gap between preclinical and clinical research due to the assay of non-physiological conditions, and iii) the lack of studies that evaluate the bioavailability and distribution of phenolics and derived metabolites in systemic human tissues. Considering the above, this Thesis has aimed to identify which compounds (molecular forms and concentrations) can reach malignant (MMT) and normal (NMT) mammary tissues of breast cancer patients after consuming a mixture of phenolic-rich plant extracts, representative of a dietary context. This knowledge could help to design mechanistic studies, illustrative of the in vivo situation, to evaluate the potential chemopreventive effect of physiologically relevant phenolic-derived metabolites using different breast cancer cell models. To address this objective, we conducted a clinical trial with breast cancer patients to characterize the metabolic profile of different dietary phenolics and their derived metabolites in MMT and NMT, plasma, and urine of patients after consuming a mixture of plant extracts. Furthermore, the plasma and tissue pharmacokinetics of phenolics and derived metabolites after consuming the same mixture of plant extracts were evaluated in an animal model (Sprague Dawley rats). The clinical trial allowed identifying 27 phenolic-derived metabolites and 6 methylxanthines in MMT from breast cancer patients. Phenolic-derived Phase-II conjugated metabolites (mainly glucuronides and sulfates) were the main molecular forms detected in MMT and NMT. Furthermore, the animal study revealed the kinetics of phenolics and derived metabolites in plasma and mammary tissues, and determined that fasting before surgery qualitatively and quantitatively was a critical issue to underestimate the distribution of phenolic-derived metabolites in systemic tissues, including mammary tissue. The in vitro assays showed that phenolic-derived metabolites occurring in MMT, even at much higher concentrations, did not exert antiproliferative, neither estrogenic/antiestrogenic, effects in different breast cancer cell models after short-term exposure. Further cell studies demonstrated that Phase-II conjugation was critical to hamper the chemopreventive effects of phenolics and derived metabolites in breast cancer cell models. However, resveratrol and their Phase-II metabolites, as detected in human MMT, induced cellular senescence (dependent on the p53/p21 pathway) in MCF-7 breast cancer cells (wild-type p53), but not in MDA-MB-231 cells (mutant p53), after long-term exposure. These results open new research scenarios to explore possible chemopreventive effects of other relevant phenolic-derived metabolites detected in MMT. These beneficial effects against breast cancer through cellular senescence induction would be plausible with chronic consumption of phenolic-containing foods. Overall, this Thesis has mainly contributed to understanding what phenolics and derived metabolites reach the MMT in breast cancer patients after consuming plant foods, and what possible protective role against this cancer they could play. Besides, this Thesis claims for the concept ‘first in vivo, and then, in vitro’, and establishes a possible sequence of studies to be followed for characterizing the cancer chemopreventive activity of dietary compounds in systemic tissues