Susana González-Manzano

Vascular deconjugation of quercetin glucuronide: The flavonoid paradox revealed?

SCOPE The dietary flavonoid quercetin exerts protective cardiovascular effects. Because quercetin is rapidly metabolized into less active or inactive glucuronidated metabolites and the plasma concentrations of free quercetin are very low, a huge amount of scientific data generated along decades with the unconjugated compounds in vitro has been questioned. We aimed to determine whether glucuronidated quercetin can deconjugate in situ and whether deconjugation leads to a biological effect. METHODS AND RESULTS Quercetin and quercetin-3-O-glucuronide (Q3GA) were perfused through the isolated rat mesenteric vascular bed. Quercetin was rapidly metabolized in the mesentery. In contrast, the decay of Q3GA was slower and was accompanied by a progressive increase of quercetin in the perfusate and in the tissue over 6 h, which was prevented by the β-glucuronidase inhibitor saccharolactone. Incubation of mesenteric arterial rings mounted in a wire myograph with Q3GA for ≥1 h resulted in a significant inhibition of the contractile response which was also prevented by saccharolactone. Moreover, the intravenous administration of Q3GA resulted in a slow onset and sustained blood pressure lowering effect, demonstrating for the first time that Q3GA has effects in vivo. CONCLUSION We propose that Q3GA behaves as a quercetin carrier in plasma, which deconjugates in situ releasing the aglycone which is the final effector.

Flavonoid metabolites transport across a human BBB model

This study aimed to evaluate the transmembrane transport of different flavonoids (flavan-3-ols, anthocyanins and flavonols) and some of their metabolites (methylated and conjugated with glucuronic acid) across hCMEC/D3 cells (a blood-brain barrier (BBB) model). Further metabolism of the tested compounds was assayed and their transport modulated in an attempt to elucidate the mechanisms behind this process. The transport across hCMEC/D3 cells was monitored in basolateral media at 1, 3 and 18 h by HPLC-DAD/MS. All the flavonoids and their metabolites were transported across hCMEC/D3 cells in a time-dependent manner. In general, the metabolites showed higher transport efficiency than the native flavonoid. No further biotransformation of the metabolites was found as consequence of cellular metabolism. Anthocyanins and their metabolites crossed this BBB cell model in a lipophilicity-dependent way. Quercetin transport was influenced by phosphatase modulators, suggesting a phosphorylation/dephosphorylation regulation mechanism. Overall, this work suggests that flavonoids are capable of crossing the BBB and reaching the central nervous system.

Extraction and isolation of phenolic compounds.

Phenolic compounds constitute a major class of plant secondary metabolites that are widely distributed in the plant kingdom and show a large structural diversity. These compounds occur as aglycones or glycosides, as monomers or constituting highly polymerized structures, or as free or matrix-bound compounds. Furthermore, they are not uniformly distributed in the plant and their stability varies significantly. This greatly complicates their extraction and isolation processes, which means that a single standardized procedure cannot be recommended for all phenolics and/or plant materials; procedures have to be optimized depending on the nature of the sample and the target analytes, and also on the object of the study. In this chapter, the main techniques for sample preparation, and extraction and isolation of phenolic compounds have been reviewed-from classical solvent extraction procedures to more modern approaches, such as the use of molecularly imprinted polymers or counter-current chromatography.

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.

Study of Zalema Grape Pomace: Phenolic Composition and Biological Effects in Caenorhabditis elegans

The phenolic composition of the extractable fraction of Zalema grape pomace has been analyzed by HPLC-DAD-MS and consisted of mainly flavanols and flavonols (122.75 and 23.11 mg/100 g dry pomace, respectively). The antioxidant activity has been determined by in vitro FRAP, ABTS, and ORAC assays (11.7, 34.9, and 63.6 mmol of Trolox equivalents (TE) per 100 g of dry pomace, respectively) and in vivo using the model organism Caenorhabditis elegans . Cultivation of C. elegans in media containing 100 μg/mL dry pomace extract increased the survival of worms submitted to thermally induced oxidative stress, whereas a decrease in the rate of worm survival was found for 300 μg/mL extract. Interestingly, the levels of reactive oxygen species (ROS) were significantly decreased in stressed worms treated with the pomace extract at the two concentration levels. Further studies are required to explain this unexpected behavior, as well as to determine the compounds and mechanisms involved in the observed effects.

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.

Different cardiovascular protective effects of quercetin administered orally or intraperitoneally in spontaneously hypertensive rats

We tested whether the administration procedure of quercetin affects its metabolite profile and antihypertensive activity. Spontaneously hypertensive rats (SHR) were randomly assigned to four experimental treatments: (1) 1 mL of 1% methylcellulose by oral gavage and 2% DMSO i.p. (control group); (2) 10 mg kg⁻¹ quercetin by oral gavage once daily and 2% DMSO i.p.; (3) 10 mg kg⁻¹ quercetin by oral gavage divided in two daily doses (5 + 5 at 12 h intervals) and 2% DMSO i.p.; (4) 1 mL of 1% methylcellulose by oral gavage and 10 mg kg⁻¹ quercetin i.p. injection. Rats were treated daily for 5 weeks. Single dose and two daily doses, in a long-term oral treatment were equally efficient, both restoring the impaired aortic endothelium-dependent vasodilatation and reducing mesenteric contractile response to phenylephrine, systolic blood pressure, heart rate, and heart and kidney hypertrophy. Attenuation of vascular NADPH oxidase-driven O₂⁻ production was also found in orally treated rats. Intraperitoneal administration reduced, to lesser extent than oral administration, the increased systolic blood pressure, being without effect to the endothelial dysfunction and vascular oxidative stress. In contrast, greater levels of metabolites were quantified following intraperitoneal compared to oral administration at any time point, except for higher plasma methylated quercetin aglycone in oral as compared to intraperitoneal administration at 2 but not at 8 h. In conclusion, oral quercetin was superior to intraperitoneal administration for the protection from cardiovascular complications in SHR. No differences were found between the oral administration as a single daily dose or divided into two daily doses.

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.

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 β-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.

Detailed phenolic composition of white grape by-products by RRLC/MS and measurement of the antioxidant activity

The development and validation of a rapid method of RRLC has been carried out to determine the phenolic composition of winemaking by-products (pomaces, seeds, skins and stems). Thirty-one phenolic compounds belonging to three groups (flavanols, flavonols and phenolic acids) have been identified by use of standards and mass spectrometric detection, and quantified by using the corresponding external standard calibration plot, in a 16-min run. The validation was realized calculating the repeatability, the reproducibility and the limits of detection (LOD) and quantification (LOQ), from standards solutions. The limits of detection and quantification were in the range of 0.16-1.09 and 0.52-3.63 mg/L, respectively, and good repeatability (R.S.D. values <1.5%) and reproducibility (R.S.D. values <5.5%) were found. Results confirmed that the method is effective and suitable for determination of phenolic compounds in winemaking by-products. Seeds, skins, stems and pomaces exhibited a different qualitative and quantitative phenolic profile and different antioxidant activities.