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Gold Standards for Realistic (Poly)phenol Research Pedro Mena*,† and Daniele Del Rio*,†,‡,§ Laboratory of Phytochemicals in Physiology, Department of Food and Drugs, ‡School for Advanced Studies on Food and Nutrition, and §Department of Veterinary Medicine, University of Parma, Via Volturno 39, 43125 Parma, Italy bioavailability and bioactivity of phenolic compounds by applying, as much as possible, realistic principles. Moreover, some notes for future research are provided.
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REALISTIC RESEARCH ON (POLY)PHENOLS Current knowledge on the bioavailability and metabolism of phenolic compounds provides a robust base to achieve key outputs in not only the field of bioavailability but also the bioactivity of (poly)phenolic compounds. In this sense, some recent works have highlighted a series of facts that will likely favor more realistic experimental conditions in the future. For instance, Ottaviani et al.1 emphasized the dynamic, changing profile of circulating metabolites upon consumption of (−)-epicatechin, one of the major flavan-3-ols in the diet. The metabolic profile was dominated by the presence of microbial-derived ring fission products, widely overcoming simple phase II epicatechin metabolites. Moreover, authors reported on significant interspecies differences (human, rat, and mouse) in the metabolism of epicatechin, an insight with important consequences for investigating the mechanisms of action underlying its putative beneficial effects.1 Our research group has recently discovered the existence of three metabolic phenotypes (metabotypes) in the production of microbial metabolites from green tea flavan-3-ols.2 This selective production of colonic metabolites, leading to well-differentiated interindividual differences in the urinary excretion of circulating metabolites, needs to be taken in great consideration, given that flavan-3-ols are the most consumed flavonoids in the human diet.2 Moreover, it should be highlighted that interindividual differences in the colonic metabolism of some phenolic compounds, such as isoflavones, ellagic acid, and lignans, may change along the lifespan, probably as a result of changes in the gut microbiota composition.3 These novel achievements in the understanding of the bioavailability of (poly)phenols, if applied to design future research, will likely contribute to unravel their actual health potential, while taking into account individual differences. However, efforts should be devoted to address the metabolism of phenolic compounds under real-life settings and on the basis of different patterns of consumption. Intervention trials mimicking daily situations of bioactive consumption may require innovative experimental designs not fully aligned with the prevailing bioavailability studies, but they can be pivotal studies explaining what is really in circulation after consumption of phenolic-rich products.4 With respect to the studies carried out to investigate the bioactivity of phenolic compounds, considerations should be made according to the research model used. In vitro studies, if
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INTRODUCTION Research on (poly)phenols, similar to every other scientific discipline, is continuously evolving. As a consequence, there is the urge to use better methodological practices, to meet new requirements. Non-realistic, pharmacological reductionist approaches are usually used to tackle the complexity of (poly)phenol bioavailability and bioactivity, but the scientific community should seek and embrace new concepts to adequately study the effects of phenolic compounds in reallife situations. (Poly)phenols are metabolized and transformed, in humans, into bioavailable molecules, able to impact on different biological processes related to human health. The elucidation of the metabolic fate of phenolics and their bioavailability is a tipping point to fully unravel the molecular forms responsible for the preventive actions of (poly)phenols in the framework of cardiovascular diseases, metabolic syndrome, neurodegenerative disorders, and certain kinds of cancer. However, although much has been reported on this regard, there is still a lot of work to be carried out. Realistic research or experimental realism refers to adhering to physiological conditions when performing scientific research. This should be kept in mind at all research levels from cell studies to human interventions. Dosages, molecules, and experimental designs should all be chosen based on physiological rationales, to make research translatable and really useful. In other words, research should be faithful to real life to yield reliable insights. These statements, although they may look somehow pretentious, should be safeguarded by the whole scientific community, because they fall within the principles of committed, useful, and responsible research. This viewpoint deals with some gold standards to be considered for the application of realistic research on (poly)phenols (Figure 1). It is inspired on recently published works targeting the © 2018 American Chemical Society
Received: June 21, 2018 Published: July 24, 2018 8221
DOI: 10.1021/acs.jafc.8b03249 J. Agric. Food Chem. 2018, 66, 8221−8223
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Journal of Agricultural and Food Chemistry
Figure 1. Overview of some gold standards to be considered when performing realistic research.
carefully designed and critically interpreted, may lead to valuable outcomes, relevant to human health. Regrettably, many cell studies with (poly)phenols did not take into account metabolic transformations, physiological concentrations, or contemporary presence of more than one metabolite. Although these three aspects should be a must in (poly)phenol research, most of the in vitro studies with phenolics test molecules not appearing in vivo or concentrations far from those achievable in the context of a normal diet. The common practice of testing individual compounds also represents an oversimplification of the real scenario, clearly not considering possible interactions. Approaches attempting to tackle these aspects are fortunately growing, as reported for anthocyanin metabolites5 and urolithins.6 Moreover, the use of realistic advanced models, such as three-dimensional (3D) spheroid cultures for colorectal carcinoma cells,6 shear-stressed dynamic systems for vascular endothelial cells, and multi-organ models allowing cross-talk between tissues, should be preferred. Finally, to obtain a full picture of what is really happening in in vitro assays, the metabolic transformations of (poly)phenolic compounds occurring within the test model should be evaluated to assert the real compounds and the potential mechanisms responsible for the biological activity of (poly)phenolic metabolites.7 Animal models should be chosen according to the capability of each species to metabolize phenolic compounds, besides taking into account the comparability of the model for the selected physiological end point. Although it may seem logical, the lack of studies comparing the metabolism of phenolic compounds between humans and animals could make this relevant choice extremely difficult. Other aspects to be considered when designing realistic animal experiments are the amounts of phenolics supplemented to the animals and the route of administration. Dosages used for both individual compounds or food items should be equivalent to doses achievable in the framework of normal food exposure. In addition, the amount of phenolic compounds provided by the chow should always be assessed, to be able to quantify in detail the exact supplemented dose. This is particularly interesting when testing flavan-3-ol and isoflavone metabolites, because many feeds contain plant foods rich in the parent polyphenols. Finally, the way compounds or the food is provided [oral, intravenous, intramuscular, (sub)cutaneous, etc.] should take into account the bioavailability of the investigated phenolics and the physiological plausibility that a certain metabolite
could reach a target tissue/organ. While mechanistic studies allow for a certain degree of deviation from realistic scenarios, conclusions on the physiological effects of a (poly)phenol or a (poly)phenol-rich foodstuff should be drawn using translatable approaches. Human intervention studies obviously represent the best strategy to unravel the biological effects of (poly)phenols under realistic conditions. However, this ideal research scenario should also clearly follow a series of indications of good practice. First of all, any (poly)phenol-rich matrix may contain other bioactive compounds. The co-occurrence of other bioactives may modify both the bioavailability and the biological effect of the studied phenolics, as demonstrated for cocoa flavan-3-ols and methylxanthines on vascular function.8 In this sense, the possible interactions (additive, synergistic, and antagonistic) with other compounds present in the food matrix, in a diet, or even in drugs should be taken into consideration through more holistic approaches. Second, the measure of specific phenolic metabolites in circulation in concomitance with the physiological outcome assessed could greatly increase the scientific relevance of an intervention, because it would directly link a physiological effect to a putatively responsible molecule. This may be easier in acute studies, but it should also be consistent for chronic trials. Once again, then, interindividual differences in the bioavailability and physiological response should be carefully addressed. Although this aspect is currently under the spotlight, with some collaborative initiatives, such as the COST Action POSITIVe (focused on cardiometabolic end points and plant bioactives; https://www6.inra.fr/cost-positive), the number of works addressing this aspect is rather limited. Two good examples of the importance of interindividual variations in the physiological response to phenolic intake have been recently carried out, both reporting a detailed characterization of the differences in the production of colonic metabolites. Hazim et al. demonstrated that soy consumption improves pulse-wave velocity only in men producing equol, while equol consumption by non-equol producers did not lead to any benefit.9 ́ et al. reported that the urolithin metabotype González-Sarrias explains individual differences in both baseline cardiovascular risk and the improvement of some risk biomarkers in overweight−obese individuals upon pomegranate consumption.10 Overall, these examples emphasize the need for comprehensive human trials, considering the bioavailability 8222
DOI: 10.1021/acs.jafc.8b03249 J. Agric. Food Chem. 2018, 66, 8221−8223
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(4) Mena, P.; Tassotti, M.; Martini, D.; Rosi, A.; Brighenti, F.; Del Rio, D. The Pocket-4-Life project, bioavailability and beneficial properties of the bioactive compounds of espresso coffee and cocoabased confectionery containing coffee: Study protocol for a randomized cross-over trial. Trials 2017, 18, 527. (5) Warner, E. F.; Smith, M. J.; Zhang, Q.; Raheem, K. S.; O’Hagan, D.; O’Connell, M. A.; Kay, C. D. Signatures of anthocyanin metabolites identified in humans inhibit biomarkers of vascular inflammation in human endothelial cells. Mol. Nutr. Food Res. 2017, 61, 1700053. (6) Núñez-Sánchez, M. Á .; Karmokar, A.; González-Sarrías, A.; García-Villalba, R.; Tomás-Barberán, F. A.; García-Conesa, M. T.; Brown, K.; Espín, J. C. In vivo relevant mixed urolithins and ellagic acid inhibit phenotypic and molecular colon cancer stem cell features: A new potentiality for ellagitannin metabolites against cancer. Food Chem. Toxicol. 2016, 92, 8−16. (7) Aragonès, G.; Danesi, F.; Del Rio, D.; Mena, P. The importance of studying cell metabolism when testing the bioactivity of phenolic compounds. Trends Food Sci. Technol. 2017, 69, 230−242. (8) Sansone, R.; Ottaviani, J. I.; Rodriguez-Mateos, A.; Heinen, Y.; Noske, D.; Spencer, J. P.; Crozier, A.; Merx, M. W.; Kelm, M.; Schroeter, H.; Heiss, C. Methylxanthines enhance the effects of cocoa flavanols on cardiovascular function: Randomized, double-masked controlled studies. Am. J. Clin. Nutr. 2017, 105, 352−360. (9) Hazim, S.; Curtis, P. J.; Schar, M. Y.; Ostertag, L. M.; Kay, C. D.; Minihane, A. M.; Cassidy, A. Acute benefits of the microbial-derived isoflavone metabolite equol on arterial stiffness in men prospectively recruited according to equol producer phenotype: A double-blind randomized controlled trial. Am. J. Clin. Nutr. 2016, 103, 694−702. (10) Gonzalez-Sarrías, A.; García-Villalba, R.; Romo-Vaquero, M.; Alasalvar, C.; Orem, A.; Zafrilla, P.; Tomás-Barberán, F. A.; Selma, M. V.; Espín, J. C. Clustering according to urolithin metabotype explains the interindividual variability in the improvement of cardiovascular risk biomarkers in overweight-obese individuals consuming pomegranate: A randomized clinical trial. Mol. Nutr. Food Res. 2017, 61, 1600830.
of (poly)phenolic compounds when evaluating their biological effects.
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REMARKS FOR FUTURE RESEARCH Undertaking realistic research may be risky and requires great efforts, costs, and dedication. Nevertheless, sometimes, small shifts toward more physiological and real-life-oriented experimental designs may represent a very good strategy to investigate the complex relationship between (poly)phenolic compounds and health, without the need of unreasonably high human and economic costs. The advances in analytical chemistry and, in particular, mass spectrometry [high resolution, ion mobility, matrix-assisted laser desorption/ ionization (MALDI) imaging, etc.], if applied to the identification and quantification of phenolic metabolites, may favor a better understanding of their health effects. On the other hand, a classical scenario researchers dread is the absence of “positive” results, and this is the reason why there is a diffused tendency to run away from the real-life setting of experimental conditions, by testing, either in vitro or in vivo, overphysiological levels of a certain molecule or an extremely high amount of a specific food item. This is because the expected effect of physiological conditions is rather limited as a result of the essence of realistic nutritional interventions, characterized by the intake of low but sustained amounts of (poly)phenolic compounds. In conclusion, we should not be scared of “negative” results when carrying out well-designed studies but should, as much as possible, pursue what truly happens in real life when consuming (poly)phenols. Fortunately, research performed using realistic, feasible, multidisciplinary approaches may yield cutting-edge, long-lasting, and useful insights. These are benefits that may undeniably boost the importance of (poly)phenol research, being the groundwork for future holistic adventures in the field. A way of moving with the times of scientific thinking.
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AUTHOR INFORMATION
Corresponding Authors
*Telephone: +39-0521-903841. E-mail: pedromiguel.
[email protected]. *Telephone: +39-0521-903830. E-mail: daniele.delrio@unipr. it. ORCID
Pedro Mena: 0000-0003-2150-2977 Daniele Del Rio: 0000-0001-5394-1259 Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Ottaviani, J. I.; Borges, G.; Momma, T. Y.; Spencer, J. P.; Keen, C. L.; Crozier, A.; Schroeter, H. The metabolome of [2-14C](−)epicatechin in humans: Implications for the assessment of efficacy, safety, and mechanisms of action of polyphenolic bioactives. Sci. Rep. 2016, 6, 29034. (2) Mena, P.; Ludwig, I. A.; Tomatis, V. B.; Acharjee, A.; Calani, L.; Rosi, A.; Brighenti, F.; Ray, S.; Griffin, J. L.; Bluck, L. J.; Del Rio, D. Inter-individual variability in the production of flavan-3-ol colonic metabolites: Preliminary elucidation of urinary metabotypes. Eur. J. Nutr. 2018, 1−15. (3) Gaya, P.; Sánchez-Jiménez, A.; Peirotén, Á .; Medina, M.; Landete, J. M. Incomplete metabolism of phytoestrogens by gut microbiota from children under the age of three. Int. J. Food Sci. Nutr. 2018, 69, 334−343. 8223
DOI: 10.1021/acs.jafc.8b03249 J. Agric. Food Chem. 2018, 66, 8221−8223
