Chocolate from Theobroma cacao
has been investigated as a functional food with potential health-protective and therapeutic activities against several chronic and degenerative diseases. Chocolate products are rich sources of a variety of monomeric, oligomeric, and polymeric flavan-3-ols (1
). Although the profile of flavan-3-ol species present in chocolate can vary greatly, monomers are one of the major forms present (4
). The predominant flavan-3-ol monomers present in chocolate include (−)-epicatechin (EC), (+)-catechin and (−)-catechin (referred to as C, ) (1
). Many of the proposed biological activities associated with chocolate have been linked to the flavan-3-ols, including C and EC. These activities include improved biomarkers of cardiovascular health and function (7
), increased serum and/or plasma antioxidant capacity (17
), and increased insulin sensitivity and decreased insulin resistance (11
Structures of (±)-catechin (C) and (−)-epicatechin (EC), the major monomeric flavan-3-ols present in chocolate.
Continued interest in the potential benefits of cocoa flavan-3-ols has underscored the need to better understand the bioavailability and metabolism of flavan-3-ol monomers in vivo
. The poor absorption of flavan-3-ols (19
) highlights the potential for optimization of bioavailability as an important strategy for maximizing the effects of chocolate flavan-3-ols in vivo
. In order to accomplish this, a more detailed understanding of the effect of product matrix composition on absorption and metabolism of flavan-3-ols from commercial chocolate products is required.
Previous investigations of the overall bioavailability of C and EC from chocolate in human subjects have reported conflicting results regarding the influence of food matrix factors such as milk protein and carbohydrate (sucrose, starch, etc.). Serafini et al.
) reported that the EC area under the plasma pharmacokinetic curve (AUC) was lower for a milk chocolate confection compared to a dark chocolate confection of equal EC content, while consumption of liquid milk with the dark chocolate confection resulted in an intermediate AUC value. However, Schramm et al.
) reported that consumption of a cocoa beverage formulated with milk resulted in a slightly, but not significantly, higher AUC than consumption of a cocoa beverage formulated with water. Additionally, four separate studies found no difference in the overall bioavailability of EC between cocoa beverages formulated with liquid milk versus water (24
). Interestingly, all studies of beverage formulations have shown no difference in overall bioavailability, while the study by Serafini is the only study performed with confections and also the only study to show a pronounced negative effect of milk on overall bioavailability.
Schramm et al.
) also investigated the effect of carbohydrate, and found that co-consumption of cocoa beverages with sucrose or bread resulted in increased plasma AUC and maximal plasma concentrations (CMAX
) of EC after consumption of cocoa with water. However, limited data exist regarding whether carbohydrate levels similarly affect EC bioavailability when chocolate is consumed in confection form. Recently, we investigated the influence of matrix composition and physical form (beverage vs. confection) of the bioavailability of EC from chocolate (28
). We previously found that the overall bioavailability was generally similar between formulations, but that the physical form greatly modulated the serum pharmacokinetic behavior (CMAX
) of EC.
While the impact of matrix formulation on the bioavailability of flavan-3-ol monomers from chocolate has been extensively studied, there is little data regarding the potential modulation of phase-II metabolism and subsequent circulating profiles by milk and/or carbohydrate. Studies of the influence of matrix formulation on phase-II metabolite profiles have predominantly examined urine, with little data on plasma, and therefore do not completely reflect alterations of circulating profiles. Roura et al.
) reported that consumption of a milk-based cocoa beverage by human subjects resulted in increased excretion of EC sulfates and decreased excretion of EC glucuronides in urine, with no effect on total bioavailability, compared to consumption of a water-based cocoa beverage. Mullen et al.
reported that consumption of cocoa beverages resulted in the presence of C/EC sulfates and O-Me sulfates in plasma, and that the presence of milk increased the plasma elimination time and decreased the AUC of the sulfate metabolites relative to water-based beverages (29
). Mullen et al.
also reported that the total urinary excretion of C/EC metabolites (sulfates, O-Me sulfates, and O-glucuronides) was significantly lower from the milk-based beverages than from the water-based beverages from 0–2 h, 2–5 h, and for total (24 h) excretion. However, the studies by Roura et
al. and Mullen et
al. examined beverages, but not solid confections. Other studies in rats and humans have shown that C and EC are extensively metabolized in vivo
, and are present in plasma as 3'-O-Me and 4'-O-Me derivatives, 5-O-β-glucuronides, 3'/4'-O-Me-5-O-β-glucuronides, and a variety of sulfated metabolites (26
). Although the reported quantitative profiles of these metabolites vary greatly, the 5-O-β-glucuronides appear to predominate, while the sulfated metabolites are typically present in low concentrations in the plasma as they are rapidly excreted into the urine.
The present study was designed to use a relevant animal model to further investigate the effects of matrix composition on the bioavailability of flavan-3-ols from cocoa which we previously observed in humans (28
). The objectives of this study were to assess the impact of food matrix composition on the bioavailability and metabolism of C and EC monomers, and to characterize the qualitative and quantitative profiles of circulating native compounds and their predominant phase-II metabolites, following administration of commercially relevant chocolate confections. An understanding of how chocolate matrix factors potentially modulate systemic flavan-3-ol bioavailability and alter the profile of circulating species could potentially lead to the development of formulation and/or dietary strategies designed specifically to optimize the bioavailability and, by extension, the in vivo
activities of monomeric cocoa flavan-3-ols.