Viewpoint Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX
pubs.acs.org/JAFC
In Vitro Research on Dietary Polyphenols and Health: A Call of Caution and a Guide on How To Proceed
Downloaded via UNIV OF SUSSEX on July 19, 2018 at 17:24:20 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
María Á ngeles Á vila-Gaĺ vez, Antonio Gonzaĺ ez-Sarrías, and Juan Carlos Espín* Laboratory of Food & Health, Research Group on Quality, Safety and Bioactivity of Plant Foods, CEBAS-CSIC, Post Office Box 164, Campus de Espinardo, 30100 Murcia, Spain found in food sources to cell cultures. The limited physiological relevance of this approach is obvious because the direct interaction of specific cells with these matrixes is impossible in most cases. Additionally, in vitro studies have not usually considered the bioavailability of (poly)phenols nor their interaction with the gut microbiota or their tissue disposition.3 To date, only a few in vitro studies have assayed the conjugated phase II metabolites of different dietary (poly)phenols or their gut-microbiota-derived metabolites that are present in the bloodstream and can reach systemic tissues to evaluate their involvement in the beneficial effects attributed to the (poly)phenol intake.4 “First in vivo and then in vitro”; in other words, let us check the in vivo metabolism and effects first, and then let us check the mechanisms of action. Although accurate in vitro−in vivo extrapolation is not possible, following this key rule will partially overcome the critical limitations in the extrapolation of in vitro studies to the physiological context. This approach ur results suggest potential health benef icial effects, although takes into account a cocktail of variables that should be further in vivo studies are needed... Perhaps, this is the considered jointly and could provide reliable information for most repeated conclusion in the in vitro studies dealing with attributing health benefits to dietary (poly)phenols. dietary (poly)phenols in the last few decades. (i) Concentration and metabolic form of (poly)phenols and Because early epidemiological and observational studies exposition time to cells: There is growing knowledge about the suggested that dietary (poly)phenols could play an important bioavailability, metabolism, and tissue disposition of (poly)protective role against chronic−degenerative pathologies phenols, important information that should be used to assay (cardiovascular and neurodegenerative diseases, cancer, and the right concentration and molecular form of (poly)phenols others), a tsunami of in vitro studies filled the scientific in in vitro studies. This is critical to avoid the tempting literature (around 45 000 entries in the Web of Science) pharmacological approach, i.e., to establish conclusions based describing a myriad of “potential” beneficial properties and on the use of very high concentrations but not attainable from mechanisms of action for these compounds (antioxidant, antia dietary point of view. In addition, it is crucial to avoid the inflammatory, anticancer, cardioprotective, neuroprotective, exposure of cells representative of systemic tissues (breast, antiviral, prebiotic, etc.). However, despite such a huge output endothelium, brain, etc.) with physiologically non-relevant of studies, the unequivocal link between the effects of (poly)phenols or derived metabolites, i.e., unconjugated poly(phenols) and/or their metabolites on human health is metabolites that do not reach those tissues. In this regard, still rather weak. In Europe, the European Food Safety the assay of phase II (poly)phenols and/or their microbialAuthority (EFSA) health claims have only been authorized for derived metabolites (mostly also phase II conjugated) that can 1,2 olive oil hydroxytyrosol and cocoa flavanols thus far. reach systemic tissues (sometimes after an intense enterOnly very few in vitro models and for very specific targets ohepatic circulation) must be encouraged. Unfortunately, the mimic in vivo conditions very well. However, the vast majority lack of availability of some of these relevant metabolites is a of in vitro models cannot fully replicate the in vivo situation. serious drawback. In addition, it is becoming evident that With this obvious premise taken as our starting point, the phase II metabolism drastically limits the “potential” beneficial closer the in vitro conditions to the in vivo context, the greater activities shown by their parent (unconjugated) compounds, the relevance of the results obtained. However, despite the which discourages those who prefer to publish always well-known limitations of in vitro studies to mirror a plausible “potential positive effects” instead of those “negative albeit in vivo physiological cell microenvironment, the vast majority physiologically relevant effects”. Therefore, these are perfect of in vitro conclusions are based on unreliable assay conditions. excuses used by some to test non-physiological metabolites How can we change this trend? For decades (and still persisting), a classical error of in vitro studies was the direct exposure of polyphenol-rich products Received: June 27, 2018 (plant or food extracts) as well as single (poly)phenols as
O
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jafc.8b03377 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Viewpoint
Journal of Agricultural and Food Chemistry ORCID
and obtain fantastic results mediated by a plethora of mechanisms (i.e., “potential effects”), regardless of the lack of relevance to human health. (ii) Suitability of cell models: The use of physiologically like assay conditions (molecular form of metabolites and concentrations) should be in line with the cell model assayed, i.e., parent (poly)phenols in stomach cell lines, unconjugated metabolites in colon cell lines, and circulating conjugated metabolites in systemic cell lines (brain, breast, etc.). In addition, although the heterogeneity of the human organism cannot be replicated in vitro, the use of a single cell line should be avoided and, instead, multiple cell lines with different features or different cell types in co-cultures should be assayed. Primary cell cultures are more relevant than commercial cell lines to mimic in vivo conditions. These cells are obtained from a parental animal tissue and have the same genetic background as those cells in the original tissue. This is very important, especially in tumor cells, as a result of the high variability in commercial tumor cell lines regarding mutations and other genetic features. Unfortunately, there are two main drawbacks: (i) the logistics to obtain this type of cell culture are very difficult, and (ii) cells derived from primary cell cultures usually have a very limited lifespan. Organoids [threedimensional (3D) cell cultures] also hold great promise for modeling diseases and predicting the outcome of molecule responses in vitro. These 3D structures are comprised of multiple cell types (cells surrounded by other cells and an extracellular matrix in a 3D fashion), and cells are selforganized and better represent the in vivo situation versus conventional two-dimensional (2D) cell cultures. Unfortunately, the setup of these models is very challenging. (iii) Food matrix effect and the assay of a cocktail of compounds: There is a recurrent endeavor for studying the effects of a single (poly)phenol or derived metabolite without considering the real mixture of compounds present in vivo after the intake of (poly)phenol-containing foods. Thus, the evaluation of physiologically relevant mixtures could provide significant information on the possible synergistic, antagonistic, or additive effects among the relevant compounds. Likewise, the food matrix acquires singular relevance in in vitro studies dealing with the gastrointestinal tract as a result of its critical role on the bioavailability and further bioactivity of (poly)phenols (interactions with other components of the whole complex food matrix, such as proteins, fiber, etc., that usually hamper or even avoid further biological actions). Overall, the present viewpoint tries to counteract the persisting high number of publications dealing with dietary (poly)phenols that describe “potential” biological effects and mechanisms of action but are mostly irrelevant to human health. Responsibility lies with authors but also with reviewers and especially with editors that often prioritize the publication of spectacular results, regardless of the doubtful extrapolation to human health, instead of moderate, borderline effects obtained using a more realistic physiological context. Additional in vitro studies that do not take into consideration the above points will only nourish the speculation of more “potential effects” and will not contribute to the still weak evidence that links dietary (poly)phenols and health.
■
Antonio González-Sarrías: 0000-0002-3407-0678 Juan Carlos Espín: 0000-0002-1068-8692 Funding
This work was funded by Projects AGL2015-64124-R [Ministry of Economy and Competitiveness (MINECO), Spain] and 201770E081 [Spanish National Research Council (CSIC), Spain]. Notes
The authors declare no competing financial interest.
■
REFERENCES
(1) European Food Safety Authority (EFSA).. Scientific Opinion on the substantiation of health claims related to polyphenols in olive and protection of LDL particles from oxidative damage (ID 1333, 1638, 1639, 1696, 2865), maintenance of normal blood HDL-cholesterol concentrations (ID 1639), maintenance of normal blood pressure (ID 3781), “anti-inflammatory properties” (ID 1882), “contributes to the upper respiratory tract health” (ID 3468), “can help to maintain a normal function of gastrointestinal tract” (3779), and “contributes to body defences against external agents” (ID 3467) pursuant to Article 13(1) of Regulation (EC) No 1924/2006. EFSA J. 2011, 9, 2033. (2) European Food Safety Authority (EFSA).. Scientific Opinion on the modification of the authorisation of a health claim related to cocoa flavanols and maintenance of normal endothelium-dependent vasodilation pursuant to Article 13(5) of Regulation (EC) No 1924/ 20061 following a request in accordance with Article 19 of Regulation (EC) No 1924/2006. EFSA J. 2014, 12, 3654. (3) Espín, J. C.; González-Sarrías, A.; Tomás-Barberán, F. A. The gut microbiota: A key factor in the therapeutic effects of (poly)phenols. Biochem. Pharmacol. 2017, 139, 82−93. (4) González-Sarrías, A.; Espín, J. C.; Tomás-Barberán, F. A. Nonextractable polyphenols produce gut microbiota metabolites that persist in circulation and show anti-inflammatory and free radicalscavenging effects. Trends Food Sci. Technol. 2017, 69, 281−288.
AUTHOR INFORMATION
Corresponding Author
*Telephone: +34-968-396200, ext: 6344. Fax: +34-968396213. E-mail:
[email protected]. B
DOI: 10.1021/acs.jafc.8b03377 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
