Project

Development of a combined in-vitro test set-up for screening of bioavailability and cardio-vascular effects of polyphenols

Code
178BW0813
Duration
01 January 2013 → 31 December 2016
Funding
Regional and community funding: IWT/VLAIO
Research disciplines
  • Natural sciences
    • Other chemical sciences
  • Medical and health sciences
    • Nutrition and dietetics
  • Agricultural and food sciences
    • Agricultural animal production
    • Food sciences and (bio)technology
Keywords
polyphenols
 
Project description

The aim of this PhD thesis was to develop an in vitro screening tool to study the bioavailability and health benefits of polyphenols, with a focus on lowered blood pressure. To this aim, (i) protocols were developed to combine in vitro digestion including colonic conversion with intestinal transport and phase I and II metabolism, (ii) the effect of the intestinal matrix and microbial metabolites on intestinal polyphenol transport and metabolism was further studied at the University of Leeds (UK) and (iii) the vasorelaxant activity of a set of polyphenol (phase II) metabolites generated in the intestine or circulating in plasma upon consumption of different classes of polyphenols was assessed and the mechanisms involved were investigated.

The state of the art knowledge about polyphenols in foods, their fate in the human body and their role in the prevention of cardiovascular diseases is discussed in Chapter 1. In this chapter, a concise overview of the bioavailability and bioactivity of two model polyphenols, hesperidin from oranges and ferulic acid from wholegrain cereals and coffee, is presented. We also provide a summary of the prevalence of CVD and the role of polyphenol consumption as a preventive measure for CVD development. Finally, in vitro models to study the different processes involved in cardiovascular health benefits of polyphenols are highlighted.

In Chapter 2, the fate of hesperidin 2S (Cordiart®) in the gastrointestinal system is investigated using the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®). Digestion in the stomach, small intestine and fermentation in the colon resulted in the establishment of the metabolite profile at different sites of the gastro-intestinal tract. The majority of hesperidin 2S metabolism occurred in the in vitro ascending and transverse colon, resulting in the production of hydroisoferulic acid, hydrocaffeic acid, 4-OH phenylacetic acid and hydroferulic acid. To establish the in vitro bioavailable fraction, selected SHIME® samples were applied to Caco-2 cell monolayers as model for the intestinal epithelium, resulting in the transport of dihydroisoferulic acid and dihydroferulic acid-4-O-sulfate, which were considered in vitro bioavailable in our model. Hesperidin 2S treatment also increased the production of butyrate in the descending colon compartment, most likely by stimulation of the butyrate producing cluster Eubacterium rectale/Clostridium coccoides. By combining digestion and transport experiments in Caco-2 cells, the biological relevance of this model of the intestinal epithelium was improved, since these assays are most often performed in standardised cell culture conditions.

Since intestinal metabolites such as SCFA are known regulators of colonocyte differentiation and maturation, the effect of the intestinal matrix or SCFA on hesperetin and ferulic acid transport and phase II metabolism in Caco-2 cells was investigated in Chapter 3. Chronic exposure to intestinal matrix or SCFA altered the transport dynamics and phase II metabolism of hesperetin and ferulic acid and these changes were linked to the expression of MCT1, MCT4 and ABCG2, transporters involved in the transport of phenolics in the intestine. We showed that gut microbial activity profiles could determine polyphenol bioavailability in vitro. These host-microbe interactions provide an interesting target for modifier molecules in support of beneficial health effects of polyphenols.

Finally, the vasorelaxant properties of relevant circulating polyphenol phase II metabolites were studied in isolated mouse arteries (Chapter 4). Hesperetin and ferulic acid-4-O-sulfate caused significant vasorelaxations in this assay and the mechanism of ferulic acid-4-O-sulfate induced vasorelaxations was investigated in more detail. ferulic acid-4-O-sulfate mediated relaxations occurred by direct activation or stimulation of soluble guanylyl cyclase (sGC) or the opening of voltage-dependent potassium (Kv) channels, as indicated by an inhibited response to ferulic acid-4-O-sulfate in the presence of ODQ, a selective sGC inhibitor, in sGC knockout mice and in the presence of 4-aminopyridin, an inhibitor of Kv-channels. These observations were confirmed in anesthetized mice, where ferulic acid-4-O-sulfate decreased mean arterial pressure. For the first time, we demonstrated vasorelaxant properties of a relevant circulating hydroxycinnamic acid phase II metabolite.

In Chapter 5, the info generated in this PhD is integrated to a consice conclusion, providing suggestions for EFSA health claim submission and future perspectives on polyphenol research regarding emerging topics such as inter-individual variability, placebo development, epigenetic modifications, stress and synergies.