Elsevier

Food Chemistry

Volume 135, Issue 3, 1 December 2012, Pages 999-1005
Food Chemistry

Cocoa polyphenols are absorbed in Caco-2 cell model of intestinal epithelium

https://doi.org/10.1016/j.foodchem.2012.05.101Get rights and content

Abstract

Cocoa is an abundant source of polyphenols, mainly flavan-3-ol monomers and polymers. In the literature, there are contradictory data on the absorption limit of procyanidins in humans. In our study, the Caco-2 cell model of intestinal epithelium was used to determine the absorption and secretion of cocoa flavan-3-ols. Three compounds: (+)-catechin, (−)-epicatechin and procyanidin B2 were detected and quantified at the receiver side of Caco-2 monolayer after 2 h transport experiment. The obtained results of apparent permeability coefficient suggest paracellular route of transport of investigated compounds. Additionally, the results suggest that compounds of cocoa powder purified extract are able to affect tight junction functioning.

Highlights

► Cocoa polyphenols are absorbed in Caco-2 cell model of intestinal epithelium. ► Absorption is limited to monomers and dimers. ► Compounds present in cocoa extract affect tight junctions between epithelial cells.

Introduction

The considerable interest in polyphenolic compounds was evoked by a growing body of epidemiological evidence that regular consumption of polyphenol rich foods prevents degenerative diseases like cancer, atherosclerosis and diabetes. Polyphenols exhibit a wide range of biological activities, including antioxidant, anti-cancerogenic, cardioprotective, antimicrobial and neuro-protective activities (Jaganath & Crozier, 2010). All above mentioned attributes are also ascribed to flavan-3-ol and their polymers linked by carbon–carbon bond known as condensed tannins or proanthocyanidins (Aron & Kennedy, 2008). The most common structures of condensed tannins are formed by (+)-catechin and (−)-epicatechin units and called procyanidins (PCs). The PCs with different degree of polymerisation can be found in foods commonly consumed in a balanced diet, including almonds, cranberries, grape, cinnamon, cocoa, peanuts, and wine (Santos-Buelga and Scalbert, 2000, Serrano et al., 2009). Due to their abundance in common foodstuff the estimated daily intake is very high; however, it strictly depends on dietary habits and preferences. The available data report on intake values ranging from 10 to 500 mg/d (Aron and Kennedy, 2008, Gu et al., 2004). Even higher values, exceeding 1 g/d, were reported by Saura-Calixto, Serrano, & Goni (2007) when both extractable and non-extractable tannins were taken into account. Generally, it is believed that bioactive substances have to be absorbed in an active form and distributed to the tissues in order to exhibit physiological activity in the human body. Therefore, the question of bioavailability of PCs is of vital importance. High molecular weight compounds are believed to be poorly absorbed in the intestine, but the route and mechanism of absorption of PC has not been fully elucidated yet. Different hypothesis were proposed, however, it is still not clear if the size of the molecule is the main factor limiting absorption, and if this is the case, the cut-off value was not clearly pointed out. Appeldoorn, Vincken, Gruppen, & Hollman (2009) showed direct absorption of A and B type PC dimers in rats. Shoji et al. (2006) reported on the presence of PC oligomers up to pentamers in rat serum after intragastric injections of high doses of apple procyanidins. Direct absorption of catechin, PC dimer, and trimer in the Caco-2 cell model of small intestine was reported, whereas polymer with average degree of polymerisation 6 was mostly retained at luminal side of the model (Deprez, Mila, Huneau, Tome, & Scalbert, 2001).

Cocoa beans and their products are characterised by high content of PC with highly diversified profile of flavan-3-ols: monomers (catechin and epicatechin) and their polymers up to tetradecamers (Kelm et al., 2006, Wollgast and Anklam, 2000). They were reported to exert vast range of beneficial effects on human well-being. The effect on cardiovascular health was particularly emphasised, but in recent studies also impact on cognition functions was proven (Field et al., 2011, Grassi et al., 2010). The possible link between antioxidant properties of cocoa polyphenols and health has been recently reviewed (Jalil & Ismail, 2008). Phenolic composition of cocoa beans and their products differs. The processing of raw cocoa bean involves many steps, including fermentation, roasting, grinding, alkalising, which affect the polyphenolic compounds composition (Wollgast & Anklam, 2000). However, even the decreased content of polyphenols in processed cocoa remains high comparing to others fruits and their products (Crozier et al., 2011). So far studies aimed to assess intracellular accumulation of cocoa polyphenols in Caco-2 cells were performed (Neilson et al., 2009), but the rate of their transport through intestinal epithelium has not been reported.

The Caco-2 cell model of absorption has been proved to be a good alternative for animal studies and has emerged as one of the standard in vitro tools to predict in vivo intestinal absorption of various substances (Langerholc, Maragkoudakis, Wollgast, Gradisnik, & Cencic, 2011). The Caco-2 permeability results were found to be in good agreement with in situ intestinal perfusion model as well as with in vivo absorption (Zuo, Zhang, Zhou, Chang, & Chow, 2006).

In the present study we intended to isolate flavan-3-ols rich extract from cocoa powder and to test their permeability through Caco-2 monolayers mimicking the human intestinal epithelium in order to specify the degree of polymerisation which is limiting absorption of procyanidins. The approach applied included the estimation of apical to basolateral transport and reversed, as well as the assessment of the effect of pH gradient between mucosal and serosal sides.

Section snippets

Chemicals and standards

All solvents and reagents were of analytical grade or higher. (+)-Catechin, (−)-epicatechin, dimethyl sulfoxide (DMSO), acetic acid, formic acid, Sephadex LH-20, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), magnesium sulphate and Lucifer yellow were obtained from Sigma–Aldrich (Buchs, Switzerland). Acetone, methanol, hexane, and acetonitrile of HPLC grade was acquired from Lab-Scan (Gliwice, Poland). Monosodium phosphate, disodium phosphate, potassium chloride, sodium chloride

Composition of cocoa powder purified extract (CPPE)

RP-HPLC analysis revealed the presence of PC monomers and oligomers up to hexamers in CPPE (Table 1). Four dimeric, three trimeric, two tetrameric, one pentameric as well as one hexameric PC isomers were identified in CPPE. In the case of monomers, isomers were identified on the basis of comparison to retention times of standard compounds. Additionally, quercetin glucosides were detected. Generally, the characterisation of proanthocyanidins rich samples with RP-HPLC method cause many

Discussion

The values of Papp for monomer and dimer from CPPE noted in our study are similar to those reported by Deprez et al. (2001). The Papp values obtained by the authors for monomer, dimer and trimer were not significantly different and ranged within 0.9–2.0 × 10−6 cm/s, similar to the value noted for mannitol, marker of paracellular transport. In the case of procyanidin polymers with DP 6, the same authors reported ten times lower permeability which amounted to 0.1 × 10−6 cm/s. On the other hand, (Zuo et

Acknowledgements

AK is a fellow of the Scientific Exchange Programme founded by Rectors’ Conference of the Swiss Universities (CRUS, Project code: 10.127) and START Programme of the Foundation for Polish Science. Christèle Bellon is acknowledged for providing introduction to cell culture techniques.

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