Liquid chromatography tandem mass spectrometry identification of proanthocyanidins in rat plasma after oral administration of grape seed extract

Abstract

Proanthocyanidin rich plant extracts derived from grape seed extract (GSE), hawthorn and cranberry are on markets for their preventive effects against cardiovascular diseases and uroinfections in woman. However, the importance of these health beneficial effects of these botanicals remains elusive due to incomplete understanding of uptake, metabolism and bioavailability of proanthocyanidins in vivo. In the present study rats were given GSE orally (300 mg/kg, twice a day) and blood and urine were collected over a 24 h period. Monomeric catechins and their methylated metabolites, and proanthocyanidins up to trimers were detected in blood samples treated with GSE using LC-MS/MS operating in the multiple reaction monitoring (MRM) mode. A new tetramethylated metabolite of dimeric proanthocyanidin (m/z 633) in GSE-treated urine was tentatively identified. Using LC-MS/MS, (+)-catechin and (−)-epicatechin were identified in the brain conclusively. These data suggested that GSE catechins cross the blood brain barrier and may be responsible for the neuroprotective effects of GSE.

Introduction

Grape seeds, one of the richest sources of polyphenols, have potent antioxidant activity. Extracts prepared from the grape seeds contain a rich mixture of monomeric flavan-3-ols, phenolic acids and oligomeric proanthocyanidins. There has been a great surge in commercialization of botanical extracts containing proanthocyanidins. In Europe some standardized plant extracts (e.g. cranberry- or hawthorn-extracts) are on the market as conventional drugs which are used because of their content of oligomeric proanthocyanidins for the treatment of recurrent uroinfections in woman and cardiovascular diseases grade I and II, respectively.

Proanthocyanidins are mixtures of dimers and higher oligomers of monomeric flavan-3-ol units linked mainly by a C4–C8 bond and, to a lesser extent, a C4–C6 bond (both are called B-type proanthocyanidins) (Fig. 1) (da Silva et al. 1991; Boukharat 1988; Prieur et al. 1994). The proanthocyanidins (PAC) of hawthorn-extracts (Crataegus spez.) belong to the B-type with only C–C-links between their (epi)catechin monomer units, whereas the major proanthocyanidins of cranberry (Vaccinium macrocarpum) as typical A-type PACs possess also C–O–C-linkages between their monomer units. The latter type only seems to be predestinated for its antihesion and antiuroinfection activity (Howell et al. 1998; Nowack and Schmitt 2008). Emerging evidence has indicated that grape seed proanthocyanidins have cardioprotective effects against reperfusion-induced injury via their ability to decrease, directly or indirectly, free radicals in myocardium (Pataki et al. 2002). It has also been demonstrated that consumption of GSE decreases the incidence of cataracts in the eyes of hereditary cataractous (ICR/f) rats (Yamakoshi et al. 2002). In addition, studies involving grape seed extract (GSE) found that chronic exposure to GSE modulates expression of specific mouse brain proteins that are either linked to Alzheimer's Disease or neuron-degeneration (Deshane et al. 2004).

Given the increasing significance of a potential health beneficial role of GSE proanthocyanidins in human health, there is a need for a fuller understanding of their absorption, metabolism and excretion. (Epi)catechins are absorbed from the intestines in humans and animals, appearing in plasma and urine primarily as glucuronidated, methylated and sulphated metabolites following the ingestion of diet containing monomeric catechins and proanthocyanidis such as chocolate, black and green tea, and red wine (Yang et al. 1998; Rein et al. 2000; Piskula and Terao 1998; Donovan et al. 1999). However, the metabolic fate of proanthocyanidins is still elusive as there is conflicting evidence on the absorption and metabolism of the oligomeric and polymeric flavan-3-ols in humans and animals. For example, Koga et al. (1999) reported the presence of (+)-catechin and (−)-epicatechin and an absence of dimers in the plasma of rats following ingestion of a GSE. However, studies by Holt et al. (2002) demonstrated the presence of dimeric procyanidins in human plasma as early as 30 min after the consumption of a flavanol-rich food such as cocoa. There is evidence in support of absorption of monomeric catechins and proanthocyanidins through the human intestinal caco-2 epithelial cells (Deprez et al. 2001; Faria et al. 2006). Another study by Tsang et al. (2005) reported the absorption and metabolism of catechin and proanthocyanidins up to trimers in urine following the oral intake of GSE. However, this study was inconclusive in establishing the accessibility of catechin in the brain. It is, therefore, critical to understand the metabolic fate of proanthocyanidins in an experimental animal model. In this study, we describe identification of major metabolites of monomeric flavan-3-ols and proanthocyanidins after oral administration of GSE and their accessibility to the brain by LC-MS/MS methods.