Ellagic Acid Metabolites Impact on Macrophage Function in Vitro

I Zanotti1, L Mele2, A Piemontese1, F Bernini1, D Del Rio2. 1Dipartimento di Farmacia, Università di Parma, Parma, Italy, 2Dipartimento di Scienze della Nutrizione, Università di Parma, Parma, Italy

Aim: Reduction of the prevalence of atherosclerotic cardiovascular disease through the consumption of polyphenol-enriched diets has been well established in several epidemiological studies (1). Ellagitannins represent a complex subclass of hydrolysable tannins found in berries, walnuts and pomegranate. These compounds are poorly absorbed after oral ingestion and are further metabolized by the colonic microbiota to form a series of catabolites called urolithins, characterized by a common 6H-dibenzo[b,d]pyran-6-one nucleus and a decreasing number of phenolic hydroxyl groups (urolithin D → C → A → B) (2). Whereas several in vitro studies demonstrated anti-atherogenic effects of ellagitannins (3), no data on the activity of these metabolites is currently available.

In the present study we investigated the activity of ellagic acid colonic metabolites urolithin A,B,C,D and B glucoronide on cholesterol efflux, cholesterol uptake, monocyte differentiation into macrophages and cell migration, all processes involved in atherosclerosis.

Methods: Cultured human or murine macrophages were exposed to acetylated LDL 25 µg/ml for 24 hours, in order to promote foam cell formation. Urolithins 1-10µM were incubated for 24h. Cell cholesterol uptake and efflux were evaluated upon cell exposure to human HDL 25µg/ml for 4-8 hours. Cholesterol efflux was quantified by a radioisotope-based assay, whereas cell cholesterol content was measured through a fluorimetric analysis. Cell migration was performed with a Boyden chamber assay, using C5a 10nM as chemotactic factor. THP-1 monocytes differentiation was evaluated by MTT method on adherent cells (4) upon incubation with urolithins for 24-48-72 hours.

Results: None of the compounds analyzed significantly influenced cholesterol exchange between foam cells and HDL. Conversely, all urolithins improved macrophage migration stimulated by C5a from 2 to 15 fold compared to untreated cells (p<0.05 by ANOVA one way). Urolithin C dose and time-dependently inhibited the differentiation of THP-1 monocytes into macrophages, reaching the maximum effect (-45% of adherent cells compared to control; p<0.05 by ANOVA one way) at 72h.

Conclusions: We demonstrated that urolithins, the ellagitannin metabolites that actually appear at cellular level, could exert an antiatherosclerotic activity by affecting macrophage function in vitro. These effects could be, at least in part, explain the antiatherogenic effects following the regular intake of diets enriched in ellagitannins.

(1) Del Rio et al. (2013). Antioxid Redox Sign 18: 1818-1892.

(2) González-Barrio et al. (2010). Journal of Agricultural and Food Chemistry 58: 3933-3939.

(3) Zanotti et al. (2015). Food and Function In press.

(4) Kim et al. (2010). Mediators Inflamm Article ID 529359.