Metabolic conversion of dietary flavonoids alters their anti-inflammatory and antioxidant properties

Abstract

The notion that dietary flavonoids exert beneficial health effects in humans is often based on in vitro studies using the glycoside or aglycone forms of these flavonoids. However, flavonoids are extensively metabolized in humans, resulting in the formation of glucuronide, methyl, and sulfate derivatives, which may have different properties than their parent compounds. The goal of this study was to investigate whether different chemical modifications of the same flavonoid molecule affect its biological and antioxidant activities. Hence, we studied the anti-inflammatory effects of several major human metabolites of quercetin and (−)-epigallocatechin-3-O-gallate (EGCG) by assessing their inhibitory effects on tumor necrosis factor α (TNFα)-induced protein expression of cellular adhesion molecules in human aortic endothelial cells (HAEC). HAEC were incubated with 1–30 μM quercetin, 3′- or 4′-O-methyl-quercetin, quercetin-3-O-glucuronide, and quercetin-3′-O-sulfate or 20–100 μM EGCG, 4′′-O-methyl-EGCG, and 4′,4′′-di-O-methyl-EGCG, prior to coincubation with 100 U/ml of TNFα. 3′-O-Methyl-quercetin, 4′-O-methyl-quercetin, and their parent aglycone compound, quercetin, all effectively inhibited expression of intercellular adhesion molecule-1 (ICAM-1) with IC₅₀ values (concentration required for 50% inhibition) of 8.0, 5.0, and 4.4 μM, respectively; E-selectin expression was suppressed to a somewhat lesser but still significant degree by all three compounds, whereas vascular cell adhesion molecule-1 (VCAM-1) was not affected. In contrast, quercetin-3-O-glucuronide (20–100 μM), quercetin-3′-O-sulfate (10–30 μM), and phenolic acid metabolites of quercetin (20–100 μM) did not inhibit adhesion molecule expression. 4′,4′′-Di-O-methyl-EGCG selectively inhibited ICAM-1 expression with an IC₅₀ value of 94 μM, whereas EGCG (20–60 μM) and 4′′-O-methyl-EGCG (20–100 μM) had no effect. The inhibitory effects of 3′-O-methyl-quercetin and 4′,4′′-di-O-methyl-EGCG on adhesion molecule expression were not related either to inhibition of NF-κB activation or to their antioxidant reducing capacity. Our data indicate that flavonoid metabolites have different biological and antioxidant properties than their parent compounds, and suggest that data from in vitro studies using nonmetabolites of flavonoids are of limited relevance in vivo.

Introduction

The health benefits of consuming fruits and vegetables are often attributed, in part, to their high content of polyphenolic compounds [1], [2]. The consumption of polyphenols in a plant-derived diet can be several times higher than the consumption of other phytochemicals and vitamins, including ascorbic acid (vitamin C), α-tocopherol (vitamin E), or carotenoids [3], [4]. Among the polyphenols, flavonoids have attracted considerable attention. Epidemiological studies have found that an increased intake of dietary flavonoids is associated with a decreased risk of chronic diseases, including certain cancers and cardiovascular diseases [5], [6], [7], [8]. Dietary flavonoids have many interesting in vitro properties, including antioxidant and anti-inflammatory effects, which have been postulated to underlie their beneficial health effects [9], [10], [11], [12].

Flavonoids undergo extensive first-pass metabolism, and the chemical forms of flavonoids present in fruits and vegetables, usually glycosides or aglycones, are quite different from their in vivo metabolites. In the intestinal mucosa and the liver, flavonoids are subjected to extensive glucuronidation, methylation, and sulfation [4], [13]. Thus, after intake of flavonoid-rich foods, these flavonoid metabolites are the main forms found in the circulatory system, where they are present for up to 4–6 h (e.g., catechins) [14], [15] or may remain longer, probably as a result of enterohepatic circulation (e.g., quercetin) [16], [17], [18]. As with pharmaceutical drugs and other xenobiotics [19], biotransformation greatly affects the physical and chemical properties of flavonoids, making them more water-soluble and readily excreted in bile and urine.

Therefore, in this study we investigated whether different chemical modifications of the same flavonoid molecule alter its biological activity and antioxidant capacity. To this end, we compared the effects of different derivatives of two representative flavonoids, the flavonol, quercetin, and the flavan-3-ol, (−)-epigallocatechin-3-O-gallate (EGCG), to their respective parent aglycones. Specifically, we studied whether O-methylation of the flavonoid B ring, glucuronidation or sulfation of quercetin, and O-methylation of EGCG affect the antioxidant properties of quercetin and EGCG and their anti-inflammatory effects on tumor necrosis factor α (TNFα)-induced activation of human aortic endothelial cells (HAEC). Endothelial activation was characterized by surface protein expression of cellular adhesion molecules and release of monocyte chemotactic protein-1 (MCP-1), which is known to critically contribute to monocyte recruitment to the vascular wall and, hence, initiation of vascular inflammation and atherosclerotic lesion formation [20], [21]. Our results show that biotransformation of dietary flavonoids can result in loss or gain of biological or antioxidant activity, which cannot be predicted from the chemical nature of the flavonoid.