Effect of including a dietary supplement of raisins, a food rich in polyphenols, on cognitive function in healthy older adults; a study protocol for a randomized clinical trial.

BACKGROUND: Polyphenols have been shown to be effective against many chronic diseases, including neurodegenerative diseases. Specifically, the consumption of raisins, being a food rich in polyphenols, has been attributed with neuroprotective benefits. Therefore, our main objective is to evaluate the effect of including 50 g of raisins in the diet daily for 6 months, on the improvement of cognitive performance, cardiovascular risk factors and markers of inflammation in a population of older adults without cognitive impairment. METHODS: Design and intervention: This study will be a randomized controlled clinical trial of two parallel groups. Each subject included in the study will be randomly assigned to one of two study groups: control group (no supplement), intervention group (50 g of raisins daily during 6 months). STUDY POPULATION: The participants will be selected by consecutive sampling in the Primary Care consultations of urban health centers in Salamanca and Zamora (Spain), taking into account the selection criteria. STUDY VARIABLES: Two visits will be made, baseline and at 6 months. Cognitive performance will be evaluated (Mini-Mental State Examination test, Rey Auditory Verbal Learning Test, verbal fluency and montreal cognitive assessment (Moca)). It will also be analyzed the level of physical activity, quality of life, activities of daily living, energy and nutritional composition of the diet, body composition, blood pressure, heart rate, markers of inflammation and other laboratory tests of clinical relevance (glycaemia, total cholesterol, HDL cholesterol, LDL cholesterol and triglycerides). In addition, sociodemographic data, personal and family history, medication use and alcohol and tobacco consumption will be collected. DISCUSSION: In this project, it is intended to contribute to minimize the problems derived from cognitive deterioration in older people. TRIAL REGISTRATION: ClincalTrials.gov Identifier: NCT04966455 Registration date: July 1, 2021.

Wine, Polyphenols, and Mediterranean Diets. What Else Is There to Say?

A considerable amount of literature has been published claiming the cardiovascular benefits of moderate (red) wine drinking, which has been considered a distinguishing trait of the Mediterranean diet. Indeed, red wine contains relevant amounts of polyphenols, for which evidence of their biological activity and positive health effects are abundant; however, it is also well-known that alcohol, even at a low level of intake, may have severe consequences for health. Among others, it is directly related to a number of non-communicable diseases, like liver cirrhosis or diverse types of cancer. The IARC classifies alcohol as a Group 1 carcinogen, causally associated with the development of cancers of the upper digestive tract and liver, and, with sufficient evidence, can be positively associated with colorectum and female breast cancer. In these circumstances, it is tricky, if not irresponsible, to spread any message on the benefits of moderate wine drinking, about which no actual consensus exists. It should be further considered that other hallmarks of the Mediterranean diet are the richness in virgin olive oil, fruits, grains, and vegetables, which are also good sources of polyphenols and other phytochemicals, and lack the risks of wine. All of these aspects are reviewed in this article.

Caffeic and Dihydrocaffeic Acids Promote Longevity and Increase Stress Resistance in Caenorhabditis elegans by Modulating Expression of Stress-Related Genes.

Caffeic and dihydrocaffeic acid are relevant microbial catabolites, being described as products from the degradation of different phenolic compounds i.e., hydroxycinnamoyl derivatives, anthocyanins or flavonols. Furthermore, caffeic acid is found both in free and esterified forms in many fruits and in high concentrations in coffee. These phenolic acids may be responsible for a part of the bioactivity associated with the intake of phenolic compounds. With the aim of progressing in the knowledge of the health effects and mechanisms of action of dietary phenolics, the model nematode Caenorhabditis elegans has been used to evaluate the influence of caffeic and dihydrocaffeic acids on lifespan and the oxidative stress resistance. The involvement of different genes and transcription factors related to longevity and stress resistance in the response to these phenolic acids has also been explored. Caffeic acid (CA, 200 µM) and dihydrocaffeic acid (DHCA, 300 µM) induced an increase in the survival rate of C. elegans under thermal stress. Both compounds also increased the mean and maximum lifespan of the nematode, compared to untreated worms. In general, treatment with these acids led to a reduction in intracellular ROS concentrations, although not always significant. Results of gene expression studies conducted by RT-qPCR showed that the favorable effects of CA and DHCA on oxidative stress and longevity involve the activation of several genes related to insulin/IGF-1 pathway, such as daf-16, daf-18, hsf-1 and sod-3, as well as a sirtuin gene (sir-2.1).

Caernohabditis elegans as a Model Organism to Evaluate the Antioxidant Effects of Phytochemicals.

The nematode Caernohabditis elegans was introduced as a model organism in biological research by Sydney Brenner in the 1970s. Since then, it has been increasingly used for investigating processes such as ageing, oxidative stress, neurodegeneration, or inflammation, for which there is a high degree of homology between C. elegans and human pathways, so that the worm offers promising possibilities to study mechanisms of action and effects of phytochemicals of foods and plants. In this paper, the genes and pathways regulating oxidative stress in C. elegans are discussed, as well as the methodological approaches used for their evaluation in the worm. In particular, the following aspects are reviewed: the use of stress assays, determination of chemical and biochemical markers (e.g., ROS, carbonylated proteins, lipid peroxides or altered DNA), influence on gene expression and the employment of mutant worm strains, either carrying loss-of-function mutations or fluorescent reporters, such as the GFP.

Effects of Quercetin Metabolites on Triglyceride Metabolism of 3T3-L1 Preadipocytes and Mature Adipocytes.

Quercetin (Q) has rapid metabolism, which may make it worthwhile to focus on the potential activity of its metabolites. Our aim was to evaluate the triglyceride-lowering effects of Q metabolites in mature and pre-adipocytes, and to compare them to those induced by Q. 3T3-L1 mature and pre-adipocytes were treated with 0.1, 1 and 10 µM of Q, tamarixetin (TAM), isorhamnetin (ISO), quercetin-3-O-glucuronide (3G), quercetin-3-O-sulfate (3S), as well as with 3S and quercetin-4-O-sulfate (4S) mixture (3S+4S). Triglyceride (TG) content in both cell types, as well as free fatty acid (FFA) and glycerol in the incubation medium of mature adipocytes were measured spectrophotometrically. Gene expression was assessed by RT-PCR. In mature adipocytes, Q decreased TG at 1 and 10 µM, 3S metabolite at 1 and 10 µM, and 3S+4S mixture at 10 µM. 3S treatment modified the glucose uptake, and TG assembling, but not lipolysis or apoptosis. During differentiation, only 10 µM of ISO reduced TG content, as did Q at physiological doses. In conclusion, 3S metabolite but not ISO, 3G, 4S and TAM metabolites can contribute to the in vivo delipidating effect of Q.

Preparation and Characterization of Protocatechuic Acid Sulfates.

Protocatechuic acid (3,4-dihydroxybenzoic acid; PCA) is a phenolic acid present in plants as a secondary metabolite and is also produced in the human organism as a metabolite from the degradation of polyphenols by the intestinal microbiota, particularly of flavonoids. However, PCA, like most polyphenols, is biotransformed in the human body to different conjugates as sulfates, which are found circulating in blood and could be involved in the bioactivity of the original compound. This paper describes a simple process for the preparation of PCA monosulfates with satisfactory yields. Two compounds were obtained that were identified as PCA-3-sulfate and PCA-4-sulfate by mass spectrometry and ¹H and 13C nuclear magnetic resonance using one- and two-dimensional techniques (heteronuclear single-quantum coherence and heteronuclear multiple-bond correlation). Differential MS fragmentation behavior and UV spectra were observed for each compound, which could be used for their identification in samples of unknown composition. The described procedure can be used for the preparation of these polyphenol metabolites in view of their use in in vivo and in vitro studies, as well as standards for their analysis in biological fluids, to contribute to the elucidation of biological effects of dietary polyphenols.

An Integrated View of the Effects of Wine Polyphenols and Their Relevant Metabolites on Gut and Host Health.

Over the last few decades, polyphenols, and flavonoids in particular, have attracted the interest of researchers, as they have been associated with the health-promoting effects derived from diets rich in vegetables and fruits, including moderate wine consumption. Recent scientific evidence suggests that wine polyphenols exert their effects through interactions with the gut microbiota, as they seem to modulate microbiota and, at the same time, are metabolized by intestinal bacteria into specific bioavailable metabolites. Microbial metabolites are better absorbed than their precursors and may be responsible for positive health activities in the digestive system (local effects) and, after being absorbed, in tissues and organs (systemic effects). Differences in gut microbiota composition and functionality among individuals can affect polyphenol activity and, therefore, their health effects. The aim of this review is to integrate the understanding of the metabolism and mechanisms of action of wine polyphenols at both local and systemic levels, underlining their impact on the gut microbiome and the inter-individual variability associated with polyphenols' metabolism and further physiological effects. The advent of promising dietary approaches linked to wine polyphenols beyond the gut microbiota community and metabolism are also discussed.

Chemical characterization and in vitro colonic fermentation of grape pomace extracts.

BACKGROUND: Currently, there is growing interest in extracts derived from winery by-products because of their beneficial health properties, which are associated with the presence of bioactive compounds. In this paper, we have carried out the chemical characterization and in vitro colonic fermentation of four grape pomace (GP) extracts rich in polyphenols and dietary fibre. RESULT: Firstly, phenolic and dietary fibre composition of the GP extracts was determined. The highest individual phenolic concentrations corresponded to gallic and ellagic acids, followed by catechins and flavonols. The non-digestible fibre fraction ranged from 66% to 83% of the GP extracts, which indicated that they mainly contained non-digestible cell wall components. Secondly, when GP extracts were subjected to fermentation by faecal microbiota, a total of 16 bacterial phenolic metabolites were found in the fermented samples, confirming that polyphenols contained in the GP extracts were metabolized to different active metabolites by microbiota. In addition, the GP extracts tended to promote the growth of intestinal microbiota, although it was only significant for the Enterococcus group. CONCLUSION: These findings, together with other information available in the literature, support the high added value of products obtained from winery by-products. © 2016 Society of Chemical Industry.

The flavonoid quercetin induces acute vasodilator effects in healthy volunteers: correlation with beta-glucuronidase activity.

UNLABELLED: Quercetin exerts vasodilator, antiplatelet and antiproliferative effects and reduces blood pressure, oxidative status and end-organ damage in hypertensive humans and animal models. We hypothesized that oral quercetin might induce vasodilator effects in humans and that they might be related to the deconjugation of quercetin-3-O-glucuronide (Q3GA). DESIGN: double blind, randomized, placebo-controlled trial. Fifteen healthy volunteers (26±5 years, 6 female) were given a capsule containing placebo, 200 or 400mg of quercetin in random order in three consecutive weeks. At 2h a dose-dependent increase in Q3GA was observed in plasma (∼0.4 and 1µM for 200 and 400mg, respectively) with minor levels of quercetin and isorhamnetin. No changes were observed in blood pressure. At 5h quercetin induced and increase in brachial arterial diameter that correlated with the product of the levels of Q3GA by the plasma glucuronidase activity. There was an increase in urinary levels of glutathione but there was no increase in nitrites plus nitrates. Quercetin and isorhamnetin also relaxed human umbilical arteries in vitro while Q3GA was without effect. In conclusions, quercetin exerts acute vasodilator effects in vivo in normotensive, normocholesterolemic human subjects. These results are consistent with the effects being due to the deconjugation of the metabolite Q3GA.

Insights into the Neuroprotective Potential of Epicatechin: Effects against Aß-Induced Toxicity in Caenorhabditis elegans.

Medical therapies to avoid the progression of Alzheimer's disease (AD) are limited to date. Certain diets have been associated with a lower incidence of neurodegenerative diseases. In particular, the regular intake of foods rich in polyphenols, such as epicatechin (EC), could help prevent or mitigate AD progression. This work aims to explore the neuroprotective effects of EC using different transgenic strains of Caenorhabditis elegans, which express human Aß1-42 peptides and contribute to elucidating the mechanisms involved in the effects of EC in AD. The performed assays indicate that this flavan-3-ol was able to reduce the signs of ß-amyloid accumulation in C. elegans, improving motility and chemotaxis and increasing survival in transgenic strain peptide producers compared to nematodes not treated with EC. The neuroprotective effects exhibited by EC in C. elegans could be explained by the modulation of inflammation and stress-associated genes, as well as autophagy, microgliosis, and heat shock signaling pathways, involving the regulation of cpr-5, epg-8, ced-7, ZC239.12, and hsp-16 genes. Overall, the results obtained in this study support the protective effects of epicatechin against Aß-induced toxicity.

Characterization and modulation of glucose uptake in a human blood-brain barrier model.

The blood-brain barrier (BBB) plays a key role in limiting and regulating glucose access to glial and neuronal cells. In this work glucose uptake on a human BBB cell model (the hCMEC/D3 cell line) was characterized. The influence of some hormones and diet components on glucose uptake was also studied. ³H-2-deoxy-D-glucose ([³H]-DG) uptake for hCMEC/D3 cells was evaluated in the presence or absence of tested compounds. [³H]-DG uptake was sodium- and energy-independent. [³H]-DG uptake was regulated by Ca²âº and calmodulin but not by MAPK kinase pathways. PKC, PKA and protein tyrosine kinase also seem to be involved in glucose uptake modulation. Progesterone and estrone were found to decrease ³H-DG uptake. Catechin and epicatechin did not have any effect, but their methylated metabolites increased [³H]-DG uptake. Quercetin and myricetin decreased [³H]-DG uptake, and glucuronic acid-conjugated quercetin did not have any effect. These cells expressed GLUT1, GLUT3 and SGLT1 mRNA.

Deglycosylation is a key step in biotransformation and lifespan effects of quercetin-3-O-glucoside in Caenorhabditis elegans.

Due to their purported healthful activities, quercetin and other flavonoids are being increasingly proposed as nutraceuticals. Quercetin occurs in food as glycosides; however, most assays on its activity have been performed with the aglycone, despite glycosylation deeply affects compound bioavailability. In this work, the uptake and lifespan effects of quercetin-3-O-glucoside (Q3Glc) and quercetin have been assessed in Caenorhabditis elegans. Q3Glc was taken up by this nematode in a concentration-dependent manner and rapidly deglycosylated to quercetin, which was accumulated in the worm and partially biotransformed to conjugated metabolites. Significant mean lifespan extension up to 23% compared to controls was observed in wild type worms cultivated in the presence of low concentrations of Q3Glc (10 µM and 25 µM), whereas exposure to greater concentrations of Q3Glc (50-200 µM) caused a reduction in mean and maximum lifespan compared with the control. By contrast, treatment of klo-1 and klo-2 mutant worms lacking ß-glucosidase activity with 200 µM of Q3Glc led to extended mean lifespan (up to 39%), similar to quercetin aglycone at the same concentration levels. In those mutants, Q3Glc was accumulated without important deglycosylation to quercetin was produced. Taken together, these findings indicated that Q3Glc was taken up by the nematode in greater extent than quercetin, and that deglycosylation and subsequent aglycone accumulation in the worm appeared as key points to explain the observed lifespan effects. The obtained results also suggested that facilitated absorption should be more important for the uptake of quercetin derivatives than passive diffusion.

Effects of a Raisin Supplement on Cognitive Performance, Quality of Life, and Functional Activities in Healthy Older Adults-Randomized Clinical Trial.

The objective of this study was to evaluate the effects of consuming 50 g of raisins on cognitive performance, quality of life, and functional activities in healthy older adults. This is a parallel randomized controlled clinical trial, in which 80 subjects over 70 years of age participated. For 6 months, the intervention group (IG; n = 40) consumed 50 g of raisins per day added to their usual diet, whereas the control group (CG; n = 40) received no supplement. All variables were measured at baseline and at 6 months. Cognitive performance assessed with the Montreal Cognitive Assessment (MOCA) test shows a difference of 3.27 points (95% CI 1.59 to 4.96), p ≤ 0.001, favorable to the IG, after the intervention. Among the cognitive performances, an improvement is observed in the IG in orientation, assessed both with the MOCA test 0.49 (95% CI 0.10 to 0.87), p = 0.014, and with the Mini-Mental State Examination (MMSE) test, 0.36 (95% CI 0.02 to 0.70), p = 0.038. In visuospatial/executive capacity and in language, improvements were also observed in the IG, 1.36 (95% CI 0.77 to 1.95), p = 0.001, and 0.54 points (95% CI 0.12 to 0.96), p = 0.014, respectively. Immediate and delayed recall, assessed with the Rey Auditory Verbal Learning Test, improved in the IG. In addition, the IG showed a better quality of life and greater autonomy in instrumental activities of daily living after 6 months. No significant changes were observed in the rest of the variables analyzed. Therefore, the consumption of 50 g of raisins produces a slight improvement in cognitive performance, quality of life, and functional activities in the elderly.

The effects of polyphenols against oxidative stress in Caenorhabditis elegans are determined by coexisting bacteria.

Introduction: Increasing evidence supports the role of gut microbiota in many aspects of human health, including immune, metabolic and neurobehavioral traits. Several studies have focused on how different components of the diet, such as polyphenols, can modulate the composition and function of the gut microbiota leading to health benefits. Methods: The effects on the resistance against thermally induced oxidative stress of C. elegans grown in the presence of flavonoids (quercetin or epicatechin) and fed different probiotic strains, namely Lactobacillus plantarum CLC17, Bifidobacterium longum NCIMB 8809 and Enterococcus faecium CECT 410, were explored. Results: Feeding C. elegans with the assayed bacteria in the absence of flavonoids did not significantly affect body size and fertility of the worms neither improve their resistance against oxidative stress compared to E. coli controls. However, increased resistance to stress was found when C. elegans was cultivated in the presence of both L. plantarum and flavonoids, but not with B. longum or E. faecium. An exploratory study revealed the presence of glycosylated and sulfated metabolites together with the aglycone in worms treated with quercetin and fed any of the different assayed LAB strains. However, in the assays with epicatechin a differential metabolite, tentatively identified as 5-(4'-hydroxyphenyl)-γ-valerolactone 3'-O-glucoside, was detected in the worms fed L. plantarum but not with the other bacteria. Conclusion: The obtained results indicated that the interactions bacteria/polyphenol play a key role in the effects produced in C. elegans regarding resistance against oxidative stress, although those effects cannot be only explained by the ability of bacteria to metabolize polyphenols, but other mechanisms should also be involved.

Effect of Polyphenolic Complements on Cognitive Function in the Elderly: A Systematic Review.

Polyphenols have been shown to be effective against many chronic diseases. These compounds could have a beneficial effect at the cognitive level. The exact mechanism by which they provide positive effects at the cognitive level is not well known, but it is believed that they could counteract neuroinflammation. The objective of this study is to review nutritional interventions that include foods or supplements rich in flavanols, flavonols, or stilbenes to the usual diet on cognitive deterioration in people over 50 years of age. Clinical trials published in PubMed and Web of Science from 1 March 2010 to 1 March 2020 were explored, from which 14 studies were selected. All of them showed some improvement after the intervention. In interventions with flavanols and stilbenes, relevant improvements have been observed both in healthy patients and in patients with established cognitive impairment. Most studies agree that the greatest benefits are found with high doses and longer duration treatments. The changes were fundamentally assessed through cognitive tests, and in some of the studies, through magnetic resonance imaging (fMRI). The type of cognitive test used to assess the effect of the intervention was revealed to be critical. Several studies have also shown improvements in analytical parameters and blood pressure.

Applications of Natural Products in Food.

The term natural products includes any substance produced by living organisms [...].

Distribution of epicatechin metabolites in lymphoid tissues and testes of young rats with a cocoa-enriched diet.

An increasing number of scientific studies support that flavanol-rich foods and beverages such as cocoa can promote human health, and are beneficial agents for the prevention of some diseases. Our previous studies showed that long-term cocoa intake enhances the antioxidant status in lymphoid organs and also modulates lymphocyte functionality in healthy young rats. Cocoa polyphenolic antioxidants seem to be the best candidates for those effects. However, data regarding polyphenol metabolites in tissues after a long-term cocoa intake are scarce. In the present study we mainly focus on the uptake and accumulation of epicatechin metabolites in lymphoid organs, including the thymus, spleen and mesenteric lymphoid nodes, as well as in the liver and testes after a diet rich in cocoa. Ten young weaned Wistar rats were fed randomly with a 10 % (w/w) cocoa diet or a control diet for 3 weeks, corresponding to their infancy and youth. Tissues were treated with a solid-phase extraction and analysed by liquid chromatography-tandem MS. The major compounds recovered in these tissues were glucuronide derivatives of epicatechin and methylepicatechin. The highest concentration of these metabolites was found in the thymus, testicles and liver, followed by lymphatic nodes and spleen. The high amount of epicatechin metabolites found in the thymus supports our previous findings showing its high antioxidant capacity compared with other tissues such as the spleen. Moreover, this is the first time that epicatechin metabolites have been found in high concentrations in the testes, confirming other studies that have suggested the testes as an important site of oxidation.

Assessment of the In Vivo Antioxidant Activity of an Anthocyanin-Rich Bilberry Extract Using the Caenorhabditis elegans Model.

Anthocyanins have been associated with several health benefits, although the responsible mechanisms are not well established yet. In the present study, an anthocyanin-rich extract from bilberry (Vaccinium myrtillus L.) was tested in order to evaluate its capacity to modulate reactive oxygen species (ROS) production and resistance to thermally induced oxidative stress, using the nematode Caenorhabditis elegans as an in vivo model. The assays were carried out with the wild-type N2 strain and the mutant strains daf-16(mu86) I and hsf-1(sy441), which were grown in the presence of two anthocyanin extract concentrations (5 and 10 µg/mL in the culture medium) and further subjected to thermal stress. The treatment with the anthocyanin extract at 5 µg/mL showed protective effects on the accumulation of ROS and increased thermal resistance in C. elegans, both in stressed and non-stressed young and aged worms. However, detrimental effects were observed in nematodes treated with 10 µg/mL, leading to a higher worm mortality rate compared to controls, which was interpreted as a hormetic response. These findings suggested that the effects of the bilberry extract on C. elegans might not rely on its direct antioxidant capacity, but other mechanisms could also be involved. Additional assays were performed in two mutant strains with loss-of-function for DAF-16 (abnormal DAuer Formation factor 16) and HSF-1 (Heat Shock Factor 1) transcription factors, which act downstream of the insulin/insulin like growth factor-1 (IGF-1) signaling pathway. The results indicated that the modulation of these factors could be behind the improvement in the resistance against thermal stress produced by bilberry anthocyanins in young individuals, whereas they do not totally explain the effects produced in worms in the post-reproductive development stage. Further experiments are needed to continue uncovering the mechanisms behind the biological effects of anthocyanins in living organisms, as well as to establish whether they fall within the hormesis concept.

Plant phenolics as functional food ingredients.

Phenolic compounds have attracted much attention in recent times as their dietary intake has been associated with the prevention of some chronic and degenerative diseases that constitute major causes of death and incapacity in developed countries, such as cardiovascular diseases, type II diabetes, some types of cancers or neurodegenerative disorders like Alzheimer's and Parkinson's diseases. Nowadays it is considered that these compounds contribute, at least in part, for the protective effects of fruit and vegetable-rich diets, so that the study of their role in human nutrition has become a central issue in food research. This chapter reviews the current knowledge on the phenolic compounds as food components, namely their occurrence in the diet, bioavailability and metabolism, biological activities and mechanisms of action. Besides, the approaches for their extraction from plant matrices and technological improvements regarding their preparation, stability and bioavailability in order to be used as functional food ingredients are also reviewed, as well as their legal situation regarding the possibility of making "health claims" based on their presence in food and beverages.

The Mechanisms Behind the Biological Activity of Flavonoids.

Flavonoids are phenolic compounds widely distributed in the human diet. Their intake has been associated with a decreased risk of different diseases such as cancer, immune dysfunction or coronary heart disease. However, the knowledge about the mechanisms behind their in vivo activity is limited and still under discussion. For years, their bioactivity was associated with the direct antioxidant and radical scavenging properties of phenolic compounds, but nowadays this assumption is unlikely to explain their putative health effects, or at least to be the only explanation for them. New hypotheses about possible mechanisms have been postulated, including the influence of the interaction of polyphenols and gut microbiota and also the possibility that flavonoids or their metabolites could modify gene expression or act as potential modulators of intracellular signaling cascades. This paper reviews all these topics, from the classical view as antioxidants in the context of the Oxidative Stress theory to the most recent tendencies related with the modulation of redox signaling pathways, modification of gene expression or interactions with the intestinal microbiota. The use of C. elegans as a model organism for the study of the molecular mechanisms involved in biological activity of flavonoids is also discussed.