Mar Larrosa

Resveratrol and Clinical Trials: The Crossroad from In Vitro Studies to Human Evidence

Resveratrol (3,5,4’-trihydroxy-trans-stilbene) is a non-flavonoid polyphenol that may be present in a limited number of food-stuffs such as grapes and red wine. Resveratrol has been reported to exert a plethora of health benefits through many different mechanisms of action. This versatility and presence in the human diet have drawn the worldwide attention of many research groups over the past twenty years, which has resulted in a huge output of in vitro and animal (preclinical) studies. In line with this expectation, many resveratrol-based nutraceuticals are consumed all over the world with questionable clinical/scientific support. In fact, the confirmation of these benefits in humans through randomized clinical trials is still very limited. The vast majority of preclinical studies have been performed using assay conditions with a questionable extrapolation to humans, i.e. too high concentrations with potential safety concerns (adverse effects and drug interactions), short-term exposures, in vitro tests carried out with non-physiological metabolites and/or concentrations, etc. Unfortunately, all these hypothesis-generating studies have contributed to increased the number of ‘potential’ benefits and mechanisms of resveratrol but confirmation in humans is very limited. Therefore, there are many issues that should be addressed to avoid an apparent endless loop in resveratrol research. The so-called ‘Resveratrol Paradox’, i.e., low bioavailability but high bioactivity, is a conundrum not yet solved in which the final responsible actor (if any) for the exerted effects has not yet been unequivocally identified. It is becoming evident that resveratrol exerts cardioprotective benefits through the improvement of inflammatory markers, atherogenic profile, glucose metabolism and endothelial function. However, safety concerns remain unsolved regarding chronic consumption of high RES doses, specially in medicated people. This review will focus on the currently available evidence regarding resveratrol’s effects on humans obtained from randomized clinical trials. In addition, we will provide a critical outlook for further research on this molecule that is evolving from a minor dietary compound to a possible multi-target therapeutic drug.

Anti-inflammatory properties of a pomegranate extract and its metabolite urolithin-A in a colitis rat model and the effect of colon inflammation on phenolic metabolism.

Whether the beneficial effects of pomegranate are due to the ellagitannins or to their microbiota-derived urolithins is not known. Our objectives were to evaluate the effects of pomegranate intake and its main microbiota-derived metabolite urolithin-A (UROA) on colon inflammation and to assess whether UROA is the main anti-inflammatory compound. In addition, the effect of the inflammation on the phenolic metabolism was also explored. Male Fisher rats were fed with 250 mg kg(-1) day(-1) pomegranate extract (PE) or 15 mg kg(-1) day(-1) UROA for 25 days. Dextran sodium sulfate (5%) (DSS) was administered for the five last days and then rats were euthanized. DSS is a well-known model of inflammatory bowel disease. Colon tissue damage, microbiota changes, antioxidant status, prostaglandin E(2) (PGE(2)), nitric oxide production, inducible nitric oxide synthase (iNOS), prostaglandin E synthase (PTGES), gene expression (microarrays and RT-PCR) and polyphenol metabolism (LC-MS-MS) were evaluated. Both PE and UROA decreased inflammation markers (iNOS, cycloxygenase-2, PTGES and PGE(2) in colonic mucosa) and modulated favorably the gut microbiota. The G(1) to S cell cycle pathway was up-regulated in both groups. UROA group showed various down-regulated pathways, including that of the inflammatory response. PE, but not UROA, decreased oxidative stress in plasma and colon mucosa. Only UROA preserved colonic architecture. The normal formation of urolithins in PE-fed rats was prevented during inflammation. Our results suggest that UROA could be the most active anti-inflammatory compound derived from pomegranate ingestion in healthy subjects, whereas in colon inflammation, the effects could be due to the nonmetabolized ellagitannin-related fraction.

Ellagitannins, ellagic acid and vascular health

Hydrolysable tannins are phenolic phytochemicals that show high antioxidant and free-radical scavenging activities. For this reason their potential effects preventing oxidative related diseases, such as cardiovascular diseases, have been largely studied. In vitro studies show that ellagitannins, at concentrations in the range 10-100 μM, show some relevant anti-atherogenic, anti-thrombotic, anti-inflammatory and anti-angiogenic effects, supporting the molecular mechanisms for the vascular health benefits. While there is good evidence supporting the vascular effects in vitro, the evidence on animal models or humans is much scarcer. The in vitro results often do not match the findings in the in vivo studies. This could be explained by the low bioavailability of the antioxidant ellagitannins and ellagic acid. The main ellagitannin metabolites circulating in plasma are ellagic acid microbiota metabolites known as urolithins, and they have lost their free-radical scavenging activity. They are present in plasma as glucuronide or sulphate conjugates, at concentrations in the nM range. Future studies should focus in the bioavailable metabolites, urolithins, and in the form (conjugated with glucuronic acid or sulphate) and concentrations (nM range) in which they are found in plasma. In this review we critically discuss the role of ellagitannins and ellagic acid on vascular health.

One-year supplementation with a grape extract containing resveratrol modulates inflammatory-related microRNAs and cytokines expression in peripheral blood mononuclear cells of type 2 diabetes and hypertensive patients with coronary artery disease

Numerous studies have shown that resveratrol (RES) exerts anti-inflammatory effects but human trials evidencing these effects in vivo are limited. Furthermore, the molecular mechanisms triggered in humans following the oral intake of RES are not yet understood. Therefore, the purpose of this study was to investigate the molecular changes in peripheral blood mononuclear cells (PBMCs) associated to the one-year daily intake of a RES enriched (8 mg) grape extract (GE-RES) in hypertensive male patients with type 2 diabetes mellitus (T2DM). We used microarrays and RT-PCR to analyze expression changes in genes and microRNAs (miRs) involved in the inflammatory response modulated by the consumption of GE-RES in comparison to a placebo and GE lacking RES. We also examined the changes in several serobiochemical variables, inflammatory and fibrinolytic markers. Our results showed that supplementation with GE or GE-RES did not affect body weight, blood pressure, glucose, HbA1c or lipids, beyond the values regulated by gold standard medication in these patients. We did not find either any significant change on serum inflammatory markers except for a significant reduction of ALP and IL-6 levels. The expression of the pro-inflammatory cytokines CCL3, IL-1β and TNF-α was significantly reduced and that of the transcriptional repressor LRRFIP-1 increased in PBMCs from patients taking the GE-RES extract. Also, a group of miRs involved in the regulation of the inflammatory response: miR-21, miR-181b, miR-663, miR-30c2, miR-155 and miR-34a were found to be highly correlated and altered in the group consuming the GE-RES for 12 months. Our results provide preliminary evidence that long-term supplementation with a grape extract containing RES downregulates the expression of key pro-inflammatory cytokines with the involvement of inflammation-related miRs in circulating immune cells of T2DM hypertensive medicated patients and support a beneficial immunomodulatory effect in these patients.

One-year consumption of a grape nutraceutical containing resveratrol improves the inflammatory and fibrinolytic status of patients in primary prevention of cardiovascular disease.

The search for complementary treatments in primary prevention of cardiovascular disease (CVD) is a high-priority challenge. Grape and wine polyphenol resveratrol confers CV benefits, in part by exerting anti-inflammatory effects. However, the evidence in human long-term clinical trials has yet to be established. We aimed to investigate the effects of a dietary resveratrol-rich grape supplement on the inflammatory and fibrinolytic status of subjects at high risk of CVD and treated according to current guidelines for primary prevention of CVD. Seventy-five patients undergoing primary prevention of CVD participated in this triple-blinded, randomized, parallel, dose-response, placebo-controlled, 1-year follow-up trial. Patients, allocated in 3 groups, consumed placebo (maltodextrin), a resveratrol-rich grape supplement (resveratrol 8 mg), or a conventional grape supplement lacking resveratrol, for the first 6 months and a double dose for the next 6 months. In contrast to placebo and conventional grape supplement, the resveratrol-rich grape supplement significantly decreased high-sensitivity C-reactive protein (-26%, p = 0.03), tumor necrosis factor-α (-19.8%, p = 0.01), plasminogen activator inhibitor type 1 (-16.8%, p = 0.03), and interleukin-6/interleukin-10 ratio (-24%, p = 0.04) and increased anti-inflammatory interleukin-10 (19.8%, p = 0.00). Adiponectin (6.5%, p = 0.07) and soluble intercellular adhesion molecule-1 (-5.7%, p = 0.06) tended to increase and decrease, respectively. No adverse effects were observed in any patient. In conclusion, 1-year consumption of a resveratrol-rich grape supplement improved the inflammatory and fibrinolytic status in patients who were on statins for primary prevention of CVD and at high CVD risk (i.e., with diabetes or hypercholesterolemia plus ≥1 other CV risk factor). Our results show for the first time that a dietary intervention with grape resveratrol could complement the gold standard therapy in the primary prevention of CVD.

Biological significance of urolithins, the gut microbial ellagic Acid-derived metabolites: the evidence so far.

The health benefits attributed to pomegranate have been associated with its high content in polyphenols, particularly ellagitannins. This is also the case for other ellagitannin-containing fruits and nuts including strawberry, raspberry, blackberry, walnuts, and muscadine grapes. The bioavailability of ellagitannins and ellagic acid is however very low. These molecules suffer extensive metabolism by the gut microbiota to produce urolithins that are much better absorbed. Urolithins circulate in plasma as glucuronide and sulfate conjugates at concentrations in the range of 0.2–20 μM. It is therefore conceivable that the health effects of ellagitannin-containing products can be associated with these gut-produced urolithins, and thus the evaluation of the biological effects of these metabolites is essential. Recent research, mostly based on in vitro testing, has shown preliminary evidence of the anti-inflammatory, anticarcinogenic, antiglycative, antioxidant, and antimicrobial effects of urolithins, supporting their potential contribution to the health effects attributed to pomegranate and ellagitannin-rich foods. The number of in vivo studies is still limited, but they show preventive effects of urolithins on gut and systemic inflammation that encourage further research. Both in vivo and mechanistic studies are necessary to clarify the health effects of these metabolites. Attention should be paid when designing these mechanistic studies in order to use the physiologically relevant metabolites (urolithins in gut models and their conjugated derivatives in systemic models) at concentrations that can be reached in vivo.

Effect of a low dose of dietary resveratrol on colon microbiota, inflammation and tissue damage in a DSS-induced colitis rat model.

The naturally occurring polyphenol resveratrol has been acknowledged with health-beneficial properties. Most of the studies dealing with its in vivo effects assay huge doses, not representative from a dietary point of view. Our aim was to ascertain whether resveratrol can exert anti-inflammatory activity in vivo at an attainable dietary dose. Rats were fed with 1 mg of resveratrol/kg/day (a human equivalent dose) for 25 days, and in the last 5 days, 5% dextran sulfate sodium (DSS) was administered to induce colitis. Effects on colon tissue damage, gut microbiota, reactive oxygen species, inflammatory markers and nitric oxide production as well as gene expression profile with microarrays were evaluated. Resveratrol increased lactobacilli and bifidobacteria as well as diminished the increase of enterobacteria upon DSS treatment. Resveratrol significantly protected the colonic mucosa architecture, reduced body weight loss, diminished the induced anemia and reduced systemic inflammation markers, colonic mucosa prostaglandin E(2), cycloxygenase-2, prostaglandin E synthase and nitric oxide levels. In addition, the expression of 2,655 genes in distal colon mucosa related to important pathways was varied. These results reinforce the concept of resveratrol as a dietary beneficial compound in intestinal inflammation at doses possibly attainable with resveratrol-enriched nutraceuticals.

NF-κB-dependent anti-inflammatory activity of urolithins, gut microbiota ellagic acid-derived metabolites, in human colonic fibroblasts

Previous studies have reported the anti-inflammatory properties of pomegranate extracts, suggesting that ellagitannins (ET) and ellagic acid (EA) are the main anti-inflammatory compounds. However, both ET and EA are metabolised in vivo by the gut microbiota to yield urolithins (Uro) which can be found in the gut and in systemic bloodstream. The present study was carried out to evaluate the individual effect of EA and their microbiota-derived metabolites Uro on colon fibroblasts upon IL-1beta treatment as an in vitro inflammation model. Uro-A and Uro-B (10 microm) inhibited PGE2 production (85 and 40 %, respectively) after IL-1beta stimulation, whereas EA did not show any effect. Uro-A, but not Uro-B, down-regulated cyclo-oxygenase-2 (COX-2) and microsomal PGE synthase-1 (mPGES-1) mRNA expression and protein levels. Both Uro inhibited NF-kappaB translocation to nucleus. Slight but significant effects were found in the activation of mitogen-activated protein kinase (MAPK) pathways. Uro-A lowered c-Jun N-terminal kinase phosphorylation state, and both Uro inhibited p38 activation. No metabolites derived from Uro or EA were found in the cell media upon incubation of EA or Uro with the cells, and only traces of the compounds were found inside the cells. The present results suggest that Uro, mainly Uro-A, are the main compounds that are responsible for the pomegranate anti-inflammatory properties. The mechanism of action implicated seems to be via the inhibition of activation of NF-kappaB and MAPK, down-regulation of COX-2 and mPGES-1 expressions, and consequently,via the reduction of PGE2 production. Taking into account that Uro did not enter the cells, a competitive binding for IL-1beta membrane receptor cannot be discarded.

Consumption of a grape extract supplement containing resveratrol decreases oxidized LDL and ApoB in patients undergoing primary prevention of cardiovascular disease: A triple-blind, 6-month follow-up, placebo-controlled, randomized trial

SCOPE The cardioprotective role of resveratrol as part of the human diet is not yet clear. Our aim was to investigate the effect of a grape supplement containing 8 mg resveratrol in oxidized LDL (LDLox), apolipoprotein-B (ApoB), and serum lipids on statin-treated patients in primary cardiovascular disease prevention (PCP). METHODS AND RESULTS A triple-blind, randomized, placebo-controlled trial was conducted. Seventy-five patients (three parallel arms) consumed one capsule (350 mg) daily for 6 months containing resveratrol-enriched grape extract (GE-RES, Stilvid®), grape extract (GE, similar polyphenolic content but no resveratrol), or placebo (maltodextrin). After 6 months, no changes were observed in the placebo group and only LDL cholesterol (LDLc) decreased by 2.9% (p = 0.013) in the GE group. In contrast, LDLc (-4.5%, p = 0.04), ApoB (-9.8%, p = 0.014), LDLox (-20%, p = 0.001), and LDLox/ApoB (-12.5%, p = 0.000) decreased in the Stilvid® group, whereas the ratio non-HDLc (total atherogenic cholesterol load)/ApoB increased (8.5%, p = 0.046). No changes were observed in hepatic, thyroid, and renal function. No adverse effects were observed in any of the patients. CONCLUSION This GE-RES reduced atherogenic markers and might exert additional cardioprotection beyond the gold-standard medication in patients from PCP. The presence of resveratrol in the GE was necessary to achieve these effects.

Concentration and Solubility of Flavanones in Orange Beverages Affect Their Bioavailability in Humans

Orange juice is a very rich source of dietary flavanones. The effect of flavanone concentration and solubility of orange beverages on their bioavailability has been studied in a crossover study with 10 healthy volunteers. Five different beverages with different flavanone concentrations were evaluated. Commercial orange juices (29.2-70.3 mg of flavanones/100 mL) were compared with experimental orange beverages in which the flavanone concentration was enhanced (110.2 mg/100 mL). Hesperetin and naringenin glucuronides and sulfates were detected and quantified in plasma and urine. The study shows that the solubility of the flavanones, and particularly that of hesperidin, in the juice is a key factor for the bioavailability as flavanone excretion and the C(max) in plasma correlate well with the soluble flavanone concentration in the juice, whereas it has no correlation with the total flavanone intake. In addition, a large interindividual variation was observed, this being consistent for each individual after the intake of the different beverages, suggesting that flavanone bioavailability is also dependent on the occurrence of specific microbiota that is able to remove the rutinosides from the juice glycosides, which results in aglycones that are then absorbed from the gut.