Common Phenolic Metabolites of Flavonoids, but Not Their Unmetabolized Precursors, Reduce the Secretion of Vascular Cellular Adhesion Molecules by Human Endothelial Cells

Abstract

Background: Flavonoids have been implicated in the prevention of cardiovascular disease; however, their mechanisms of action have yet to be elucidated, possibly because most previous in vitro studies have used supraphysiological concentrations of unmetabolized flavonoids, overlooking their more bioavailable phenolic metabolites.

Objective: We aimed to explore the effects of phenolic metabolites and their precursor flavonoids at physiologically achievable concentrations, in isolation and combination, on soluble vascular cellular adhesion molecule-1 (sVCAM-1).

Method: Fourteen phenolic acid metabolites and 6 flavonoids were screened at 1 μM for their relative effects on sVCAM-1 secretion by human umbilical vein endothelial cells stimulated with tumor necrosis factor alpha (TNF-α). The active metabolites were further studied for their response at different concentrations (0.01 μM-100 μM), structure-activity relationships, and effect on vascular cellular adhesion molecule (VCAM)-1 mRNA expression. In addition, the additive activity of the metabolites and flavonoids was investigated by screening 25 unique mixtures at cumulative equimolar concentrations of 1 μM.

Results: Of the 20 compounds screened at 1 μM, inhibition of sVCAM-1 secretion was elicited by 4 phenolic metabolites, of which protocatechuic acid (PCA) was the most active (-17.2%, P = 0.05). Investigations into their responses at different concentrations showed that PCA significantly reduced sVCAM-1 15.2-36.5% between 1 and 100 μM, protocatechuic acid-3-sulfate and isovanillic acid reduced sVCAM-1 levels 12.2-54.7% between 10 and 100 μM, and protocatechuic acid-4-sulfate and isovanillic acid-3-glucuronide reduced sVCAM-1 secretion 27.6% and 42.8%, respectively, only at 100 μM. PCA demonstrated the strongest protein response and was therefore explored for its effect on VCAM-1 mRNA, where 78.4% inhibition was observed only after treatment with 100 μM PCA. Mixtures of the metabolites showed no activity toward sVCAM-1, suggesting no additive activity at 1 μM.

Conclusions: The present findings suggest that metabolism of flavonoids increases their vascular efficacy, resulting in a diversity of structures of varying bioactivity in human endothelial cells.

Keywords: VCAM-1; endothelial; inflammation; metabolism; phase II conjugate; polyphenol.

FIGURE 1

Combination treatments used in this study. Shaded boxes represent inclusion of respective compounds in equimolar concentrations to a cumulative concentration of 1 μM; for example, a combination comprising 4 constituents would require 0.25 μM of each to yield a final concentration of 1 μM. PCA, protocatechuic acid.

FIGURE 2

Effect of 1 μM flavonoids and phenolic acid metabolites on TNF-α–stimulated sVCAM-1 protein secretion by HUVECs. Flavonoids (A), anthocyanin glucosides (B), unconjugated phenolic acids (C), and conjugated phenolic acids (D) are shown. Data were normalized to a TNF-α control, and columns represent the mean ± SD (n = 3 independent measures). Labeled means without a common letter differ, P ≤ 0.05 (ANOVA with post hoc LSD). *Different from DMSO, P ≤ 0.05 (t test). BA4G, benzoic acid-4-glucuronide; BA4S, benzoic acid-4-sulfate; C3G, cyanidin-3-glucoside; EPI, (-) epicatechin; HES, hesperetin; HUVEC, human umbilical vein endothelial cell; IVA, isovanillic acid; IVA3G, IVA-3-glucuronide; IVA3S, IVA-3-sulfate; LSD, least square difference; NAR, naringenin; PCA, protocatechuic acid; PCA3G, PCA-3-glucuronide; PCA3S, PCA-3-sulfate; PCA4G, PCA-4-glucuronide; PCA4S, PCA-4-sulfate; P3G, peonidin-3-glucoside; QUE, quercetin; sVCAM-1, soluble vascular cellular adhesion molecule 1; VA, vanillic acid; VA4G, VA-4-glucuronide; VA4S, VA-4-sulfate; 4HBA, 4-hydroxybenzoic acid.

FIGURE 3

Effect of 1 μM mixtures of flavonoids and phenolic acid metabolites on TNF-α stimulated sVCAM-1 protein secretion by HUVECs. Flavonoid mixtures (A), phenolic acid mixtures (B), and conjugated and unconjugated phenolic metabolite mixtures (C) are shown. Data were normalized to a TNF-α control, and columns represent the mean ± SD (n = 3 independent measures). *Different from DMSO, P ≤ 0.05 (t test). Where unequal variance was identified (B and C), group differences were established via Kruskal-Wallis nonparametric ANOVA. BA4G, benzoic acid-4-glucuronide; BA4S, benzoic acid-4-sulfate; C3G, cyanidin-3-glucoside; EPI, (-) epicatechin; HES, hesperetin; HUVEC, human umbilical vein endothelial cell; IVA3G, isovanillic acid-3-glucuronide; IVA3S, isovanillic acid-3-sulfate; NAR, naringenin; PCA, protocatechuic acid; PCA3G, PCA-3-glucuronide; PCA3S, PCA-3-sulfate; PCA4G, PCA-4-glucuronide; PCA4S, PCA-4-sulfate; P3G, peonidin-3-glucoside; QUE, quercetin; sVCAM-1, soluble vascular cellular adhesion molecule 1; VA, vanillic acid; VA4G, VA-4-glucuronide; VA4S, VA-4-sulfate; 4HBA, 4-hydroxybenzoic acid.

FIGURE 4

Effect of concentration of phenolic acid metabolites on TNF-α–stimulated sVCAM-1 protein secretion by HUVECs. PCA (A), PCA3S (B), PCA4S (C), IVA (D), and IVA3G (E) are shown. Data were normalized to a TNF-α control, and columns represent the mean ± SD (n = 3 independent measures). Labeled means without a common letter differ, P ≤ 0.05 (ANOVA with post hoc LSD). *Different from DMSO, P ≤ 0.05 (t test). HUVEC, human umbilical vein endothelial cell; IVA, isovanillic acid; IVA3G, IVA-3-glucuronide; LSD, least square difference; PCA, protocatechuic acid; PCA3S, PCA-3-sulfate; PCA4S, PCA-4-sulfate; sVCAM-1, soluble vascular cellular adhesion molecule 1.

FIGURE 5

Effect of concentration of PCA on TNF-α–stimulated VCAM-1 mRNA expression in HUVECs. Data were normalized to a TNF-α control, and columns represent the mean ± SD (n = 3 independent measures). Labeled means without a common letter differ, P ≤ 0.05 (ANOVA with post hoc LSD). *Different from DMSO, P ≤ 0.05 (t test). HUVEC, human umbilical vein endothelial cell; LSD, least square difference; PCA, protocatechuic acid; VCAM, vascular cellular adhesion molecule.