Gene expression changes by high-polyphenols cocoa powder intake: a randomized crossover clinical study

Abstract

Purpose: To assess the effect of the intake of a single dose of high-polyphenols cocoa on gene expression in peripheral mononuclear cells (PBMCs), and analyze conjugated (-)-epicatechin metabolites in plasma, which may be related with an antioxidant response in healthy human.

Methods: A randomized, controlled, double-blind, cross-over, clinical trial in healthy young adults who consumed a single dose of high-polyphenols cocoa powder and maltodextrins as control, with a one-week washout period. Analysis of circulating metabolites, plasma antioxidant capacity and gene expression changes in PBMCs were performed under fasting conditions and 2-h after treatment using microarray in a subsample. Pathway analysis was conducted using Ingenuity Pathway Analysis (IPA).

Results: Twenty healthy participants (9 F) were included in the study. A significant increase in circulating (-)-epicatechin metabolites was found after cocoa intake in all participants without related changes in antioxidant capacity of plasma. The metabolites profile slightly varied across subjects. Treatments triggered different transcriptional changes in PBMC. A group of 98 genes showed changes in expression after cocoa treatment, while only 18 were modified by control. Differentially expressed genes included inflammatory cytokines and other molecules involved in redox balance. Gene and network analysis after cocoa intake converged in functions annotated as decreased production of reactive oxygen species (p = 9.58E-04), decreased leukocyte activation (p = 4E-03) and calcium mobilization (p = 2.51E-05).

Conclusions: No association was found between conjugated metabolites in plasma and antioxidant capacity. Changes in PBMCs gene expression suggest anti-inflammatory effects.

Keywords: (−)-Epicatechin; Antioxidant; Catechin; Flavanol; Microarray; Molecular; PBMCs; ROS.

Fig. 1

Study design. Double blinded, randomized, placebo-controlled, crossover clinical trial. Participants allocated in Group-1 consumed the treatments in Cocoa-Placebo sequence and participants allocated in Group-2 in Placebo-Cocoa order. Four blood samples were taken from each participant during the study; two samples at baseline state (t₁ and t₃), and two samples 2 h after treatment intake (t₂ and t₄). Tx1 Cocoa, Tx2 Placebo

Fig. 2

Flow diagram of participants during study. Participants allocated in Group-1 consumed treatments in a Cocoa-Placebo sequence and Group-2 in the opposite order. Treatments consisted in high-polyphenols cocoa intake {600 mg polyphenols of which 94 were (−)-epicatechin} and placebo (maltodextrins) pills

Fig. 3

(−)-Epicatechin metabolites profiling after single dose cocoa intake stratified by sex. Ten metabolites were identified and quantified in plasma samples using UHPLC-MS/MS. Only seven metabolites increased their concentration levels after intervention (p < 0.05). Data presented as proportions in all (n = 20; 1474 ± 215 nM), male (n = 11; 1216 ± 295 nM) and female individual (n = 9; 1701 ± 305 nM). 3MES (−)-3′-O-methylepicatechin-4′-sulfate, E3G (−)-epicatechin-3′-O-glucuronide, E3S (−)-epicatechin-3′-O-sulfate; E4G (−)-epicatechin-4′-O-glucuronide, other E4’S (−)-epicatechin-4′-O-sulfate, EC (−)-epicatechin, C4′G (+)-catechin-4′-O-glucuronide

Fig. 4

Flow diagram of RNA samples analyzed by microarray

Fig. 5

Cluster analysis of samples. Dendrogram of unsupervised hierarchical clustering on normalized gene expression values show a good agreement of samples according to their classification. Labels are defined as follows: subject ID {01:06}, treatment {Tx1: cocoa, Tx2: placebo} and condition {B: baseline and 2 h: after 2 h}. Clustering performed with Spearman correlation, Euclidean distance and average linkage. Numbers in red represents AU (approximately unbiased) p values (%). Numbers in green represents BP (bootstrap probability)

Fig. 6

Enrichment analysis of differentially expressed genes after cocoa intake. Enrichment analysis was performed using the software Ingenuity Pathway Analysis (IPA). Three gene expression networks of annotated functions had an activation z-score < − 1.5 or > 1.5; production of ROS (p = 9.58E−4; z-score = − 2.216; activation of leukocytes (p = 4E−3; z-score = − 1.966); and, viral infection (p = 2.72E−2; z score = − 1.667)