Dimeric Procyanidin B2 Research Articles

Procyanidin B2 induces porcine skeletal slow-twitch myofiber gene expression by AMP-activated protein kinase signaling pathway

In this study, our aim is to investigate the effect of dimer procyanidin B2 [epicatechin-(4β-8)-epicatechin] (PB2) on porcine skeletal myofiber gene expression in vitro. Our data showed PB2 promoted the protein expression of slow myosin heavy chain (MyHC) in porcine myotubes, concomitant with the increases in mRNA levels of MyHC I, MyHC IIa and Tnni1. We also found PB2 activated AMPK signaling in porcine myotubes. NRF1 and CaMKKβ that are two important upstream factors of AMPK, and Sirt1 and PGC-1α that are two major downstream factors of AMPK, were also up-regulated by PB2. The mechanism study showed the effect of PB2 on slow-twitch myofiber gene expression was abolished by AMPK inhibitor compound C or by AMPKα1 siRNA. Together, we found PB2 induced porcine skeletal slow-twitch myofiber gene expression by AMPK signaling pathway.

Effects of catechins on cellular energy supply in the heart

Main cocoa catechins (-)-epicatechin and its derived dimer procyanidin B2 are effective antioxidants capable to protect cardiovascular system from reactive oxygen species (ROS) damage although their detailed mechanism of action has not been fully clarified yet. Mitochondria are not only the central players in the regulation of cell energy homeostasis but also the main sites of ROS generation as byproducts of oxidative phosphorylation. The essential role of mitochondria in oxidative stress imply that they could be also the potential targets of catechins in the cell. In this work we evaluated the direct effects of (-)-epicatechin and procyanidin B2 on the functions of cardiac mitochondria. Our results showed that (-)-epicatechin starting from 0.2 mg/mL in a concentration-dependent manner significantly increased the substrate-driven respiration rate of isolated rat heart mitochondria with all tested substrates - pyruvate and malate, palmitoyl-L-carnitine and succinate. Procyanidin B2 from 0.7 ng/mL to 3.5 ng/mL also increased the substrate-driven respiration rate of isolated rat heart mitochondria, the highest effect was observed with succinate, then with pyruvate and malate, and the lowest – with palmitoyl-L-carnitine as substrates. Thus, (-)-epicatechin and procyanidin B2 stimulated substrate-driven mitochondrial respiration and phosphorylation at lower concentrations, whereas they inhibited the respiratory chain at higher concentrations. As the substrate-driven mitochondrial respiration rate mainly depends on the passive proton flux through the mitochondrial inner membrane, our results show that both (-)-epicatechin and procyanidin B2 in a concentration-dependent manner could uncouple oxidative phosphorylation in cardiac mitochondria. Furthermore, (-)-epicatechin-reduced the release of apoptosis marker - cytochrome c from mitochondria supporting the evidence of membrane stabilizing properties of this flavonol. Since catechins are weak acids of hydrophobic character, they could directly transfer protons through the inner mitochondrial membrane. As the mild mitochondrial uncoupling is implicated to be a highly effective in vivo antioxidant strategy, catechins could serve as potential compounds for further studies on the development of novel cardioprotective agents. Furthermore, the catechin-rich diet could help to improve cardiac mitochondrial functions thus ddecreasing the risk of cardiovascular diseases.

Procyanidin B2 Promotes Skeletal Slow-Twitch Myofiber Gene Expression through the AMPK Signaling Pathway in C2C12 Myotubes.

Dimer procyanidin B2 [epicatechin-(4β-8)-epicatechin] (PB2) has attracted a lot of interest in nutrition and medicine because of its significant health-promoting abilities. However, the function of PB2 on different types of skeletal myofiber is still unclear. Here, we have found that PB2 significantly increased protein expression of the slow myosin heavy chain (MyHC) and decreased fast MyHC protein in C2C12 myotubes, accompanied by upregulation of mRNA expression of MyHC I, MyHC IIa, and Tnni1 and downregulation of MyHC IIx and MyHC IIb. We have also found that PB2 enhanced the activities of malate dehydrogenase and succinic dehydrogenase and reduced lactate dehydrogenase activity. PB2 promoted phosphorylation of AMPK and significantly increased mRNA expression of AMPKα1. The upstream factors of AMPK, such as phospho-LKB1, NRF1, and CaMKKβ, and the downstream factors of AMPK, including Sirt1 and PGC-1α, were also increased by PB2. Specific suppression of AMPK signaling by AMPKα1 siRNA or by AMPK inhibitor compound C significantly attenuated the PB2-induced upregulation of phospho-AMPK, PGC-1α, and slow MyHC and downregulation of fast MyHC. Our findings suggested that PB2 promotes skeletal slow-twitch myofiber gene expression through the AMPK signaling pathway in C2C12 myotubes.

Monomeric catechin and dimeric procyanidin B2 against human norovirus surrogates and their physicochemical interactions

Plant polyphenols have shown antiviral activity against several human pathogens, but their physicochemical interactions are not well-understood. The objectives of this study were to compare the antiviral activity between monomeric catechin and dimeric procyanidin B2 (PB2) using cultivable human norovirus surrogates (feline calicivirus (FCV-F9) and murine norovirus (MNV-1)) and to understand their potential antiviral mechanism using virus-like particles (VLPs) and the P domain of human norovirus GII (HNoV GII.4). Surrogate viruses at 5 log PFU/mL were treated with 0.5–5 mg/mL monomeric catechin monohydrate, PB2 or phosphate buffered saline (PBS, pH 7.2; control) at 37 °C over 24 h. Infectivity was determined using plaque assays and data from triplicate experiments were statistically analyzed. PB2 at 0.5 mg/mL and 1 mg/mL reduced FCV-F9 to undetectable levels after 3 h and MNV-1 by 0.21 and 1.23 log PFU after 24 h, respectively. Monomeric catechins at 1 mg/mL reduced FCV-F9 to undetectable levels after 6 h and MNV-1 titers to undetectable levels after 24 h. In addition, PB2 was shown to directly bind the P domain, the main capsid structure of HNoVs in the ratio of 1:1 through spontaneous interactions. Electrostatic interactions played a dominant role between PB2 and the P domain. PB2 significantly altered tertiary but not secondary structures of VLPs. Transmission electron microscopy demonstrated that PB2 aggregated VLPs, further indicating interactions between them. These findings indicate that PB2 causes structural changes of the P domain of VLPs, mainly through direct interaction leading to HNoV inactivation.

Preparative separation of cacao bean procyanidins by high-speed counter-current chromatography.

In this work, an efficient method for preparative separation of procyanidins from raw cacao bean extract by high-speed counter-current chromatography (HSCCC) was developed. Under the optimized solvent system of n-hexane-ethyl acetate-water (1:50:50, v/v/v) with a combination of head-tail and tail-head elution modes, various procyanidins fractions with different polymerization degrees were successfully separated. UPLC, QTOF-MS and 1H NMR analysis verified that these fractions contained monomer up to pentamer respectively. Dimeric procyanidin B2 (purity>86%) could be isolated by HSCCC in a single run. Other individual procyanidins in these fractions could be further isolated and purified by preparative HPLC. The developed HSCCC together with preparative HPLC techniques appeared to be a useful tool for large preparation of different procyanidins from cacao beans. Furthermore, by antioxidant activity assays, it was proved that both fractions and individual procyanidins possessed greater antioxidant activities compared to standard trolox. The antioxidant activities of procyanidins increase as the increase of their polymerization degree.

Direct effects of (-)-epicatechin and procyanidin B2 on the respiration of rat heart mitochondria.

Flavonol (−)-epicatechin and its derived dimer procyanidin B2, present in high amounts in cocoa products, have been shown to exert beneficial effects on the heart and cardiovascular system; however, their mechanism of action has not been fully elucidated. We studied effects of (−)-epicatechin and procyanidin B2 on the oxidative phosphorylation of isolated rat heart mitochondria. (−)-Epicatechin and procyanidin B2 had stimulating effect (up to 30% compared to control) on substrate-driven (State 2) mitochondrial respiration. Their effect was dependent on the respiratory substrates used. (−)-Epicatechin at higher concentrations (from 0.27 µg/mL) significantly decreased (up to 15%) substrate- and ADP-driven (State 3) mitochondrial respiration in case of pyruvate and malate oxidation only. Procyanidin B2 (0.7–17.9 ng/mL) inhibited State 3 respiration rate up to 19%, the most profound effect being expressed with succinate as the substrate. (−)-Epicatechin at concentrations of 0.23 µg/mL and 0.46 µg/mL prevented loss of the cytochrome c from mitochondria when substrate was succinate, supporting the evidence of membrane stabilizing properties of this flavonol. Thus, both (−)-epicatechin and procyanidin B2 directly influenced mitochondrial functions and the observed effects could help to explain cardiometabolic risk reduction ascribed to the consumption of modest amounts of cocoa products.

Chemical constituents from Gouania longipetala and Glyphaea brevis

Five compounds were isolated altogether from the two medicinal plants. Glycerol monotricosanoate (1), palmarumycin BG1 (2) and de-O-methyllasiodiplodin (3) were isolated from Gouania longipetala. In addition, epicatechin (4) and its dimer procyanidin B2 (5) were isolated from the stem bark of Glyphaea brevis. Their structures were elucidated by using spectroscopic experiments. They exhibited radical scavenging and moderate antibacterial effects.

Preparation of procyanidin B2 from apple pomace and its inhibitory effect on the expression of cyclooxygenase-2 in lipopolysaccharide-treated RAW264.7 macrophages

Dimeric procyanidin B2 (PB2) is one of phenolic compounds in apple pomace, an agro-industrial byproduct in apple juice processing. This work focused on purification of PB2 from apple pomace using sephadex column chromatography and its potential effect on lipopolysaccharide (LPS)-induced inflammation using RAW264.7 macrophages. PB2 with the purity of 72.28 ± 1.85% was successfully afforded using resin and gel column chromatographic technique. Anti-inflammatory tests suggested that the expression of cyclooxygenase-2 (COX-2) in LPS-induced murine RAW264.7 macrophages was suppressed in a PB2 concentration-dependent manner. PB2 at no less than 50 ?g·mL -1 could significantly suppress inflammation in the LPS-induced cells. Moreover, this suppressive effect was not correlated with PB2 pretreating. However, the COX-2 expression was not reduced in LPS pretreatment way followed by PB2 exposure, which suggested that PB2 has no repairing function. The results showed that high pure PB2 prepared from apple pomace has a remarkable anti-inflammatory property.

Comparison of TLC and HPLC methods used for analysis of (−)-epicatechin and its dimer procyanidin B2 in chocolate

TLC test solutions of baking chocolate (Standard Reference Material (SRM) 2384, National Institute of Standards and Technology, USA) were prepared by extraction of 0.0200 g pulverized defatted chocolate (the amount [%] of fat was determined separately) with 2 mL 70% acetone (aq.) followed by centrifugation and filtration (0.45 μm PVDF filter; Millipore, USA). Liquid–liquid partitioning (1 mL TLC test solution, 1 mL 20% aqueous sodium chloride, and 1.5 mL ethyl acetate) was needed to obtain sufficiently pure test solutions for the HPLC analysis.

Polyphenol Contents and Antioxidant Activities of Extracts from Flowers of Two Crataegus azarolus L. Varieties

Phenolic contents of the ethyl acetate extracts prepared from floral buds and opened flowers harvested on Crataegus azarolus trees native in two localities were performed. The antioxidant activity was measured by DPPH' (2,2-diphenyl-picrylhydrazyl), ABTS+ (2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radicals scavenging using spectrophotometric method. The C. azarolus var. aronia (Willd.) Batt., producing yellow fruits, was richer in total phenols (1638.7 +/- 89.9 mg acid gallic/100 g dry weight) according to C. azarolus var. eu-azarolus Maire (1415.5 +/- 23.8 mg acid gallic/100 g dry weight), producing red ones. Ethyl acetate extract from opened flowers has less content in total phenols, proanthocyanidins and flavonoids compared to this from floral buds. Floral buds from the two C. azarolus varieties occurring in Siliana-Djebel Serdj showed the highest radical scavenging activities (2431.8 +/- 32.7 and 2267.7 +/- 22.7 micromol Trolox/100 g dry weight). Hawthorn from Tunisia contains eight antioxidants of phenolic type (chlorogenic acid, hyperoside, rutin, spiraeoside, isoquercitrin, quercetin, (-)-epicatechin and the dimer procyanidin B2). These compounds identified specially in floral bud extracts presented a strong radical-scavenging activity.

Dimeric procyanidin B2 inhibits constitutively active NF-κB in Hodgkin's lymphoma cells independently of the presence of IκB mutations

Due to long-term toxicity of current Hodgkin's lymphoma (HL) treatment, the present challenge is to find new therapies that specifically target deregulated signaling cascades, including NF-κB, which are involved in Hodgkin (H) and Reed-Sternberg (RS) cell proliferation and resistance to apoptosis. We previously presented evidence that dimeric procyanidin B2 (B2) can interact with NF-κB proteins inhibiting the binding of NF-κB to DNA. Herein, we investigated if B2, acting at a late event in NF-κB signaling cascade, could be effective in inhibiting NF-κB in H–RS cells with different mechanisms of constitutive NF-κB activation. B2 caused a concentration-dependent inhibition of NF-κB-DNA binding to a similar extent (41–48% inhibition at 25μM B2) in all the tested H–RS cell lines (L-428, KM-H2, L-540, L-1236 and HDML-2). This was associated with the inhibition of NF-κB-driven gene expression, including cytokines (IL-6, TNFα and RANTES) and anti-apoptotic proteins (Bcl-xL, Bcl-2, XIAP and cFLIP). The finding of similar amounts of RelA and p50 proteins in the nucleus, but decreased NF-κB-DNA binding, even in those H–RS cells characterized by mutations in the inhibitory IκB proteins, supports that B2 acts by preventing the binding of NF-κB to DNA. B2 did not inhibit AP-1 and STAT3 constitutive activation in H–RS cells, indicating that the moderate effects of B2 on cell viability are due to the complex signaling aberrations in HL. Thus, several signaling pathways should be targeted when designing therapeutics for HL. In this regard, the capacity of B2 to inhibit NF-κB could be valuable in a multi-drug approach.

Mono- O-methylated flavanols and other flavonoids as inhibitors of endothelial NADPH oxidase

The dietary flavan-3-ol (−)-epicatechin improves the bioactivity of nitric oxide in arterial vessels in vivo. Moreover, it effectively protects cultured vascular endothelial cells from signs of oxidative stress and elevates intracellular nitric oxide in vitro. We addressed the effects of (−)-epicatechin, its metabolic conversion products and structurally related compounds on NADPH oxidase activity in intact human umbilical vein endothelial cells (HUVEC) and in cell lysates. (−)-Epicatechin proved to be an O 2 −-scavenger but did not inhibit NADPH oxidase activity, whereas the converse pattern was observed for the metabolites 3′- and 4′- O-methyl epicatechin. The dimer procyanidin B2 and (−)-epicatechin glucuronide were O 2 −-scavengers and inhibited NADPH oxidase. Analysis of structure–activity relations with 45 compounds suggests an apocynin-like mode of NADPH oxidase inhibition. Notably, HUVEC converted (−)-epicatechin to NADPH oxidase-inhibitory methyl ethers. These data identify endothelial NADPH oxidase as candidate target of dietary flavonoids and particularly of their metabolites.

High‐performance liquid chromatography/electrospray ionization mass spectrometric characterization of new products formed by the reaction between flavanols and malvidin 3‐glucoside in the presence of acetaldehyde

Monomer and polymer flavan-3-ols and anthocyanins are the two major groups of phenolic compounds in red wine and one of the most important reactions during red wine ageing is the condensation reaction, either direct or indirect, between these two classes of compounds. In this work, the reaction of (+)-catechin, (-)-epicatechin and dimer procyanidin B2 with malvidin 3-glucoside, in the presence of acetaldehyde, was carried out in wine model solutions at pH 1.7 and 3.2. Identification of the reaction products was carried out by high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) and MS(n) analysis. Various reaction products were detected. The structures of three condensation products - malvidin-3-glucoside-ethyl-(epi)catechin-1''-hydroxyethyl, malvidin-3-glucoside-ethyl-dimer-vinyl and malvidin-3-glucoside-ethyl-dimer-1''-hydroxyethyl - were identified in a model solution for the first time. Moreover, in the reaction solution containing dimer B2 at pH 3.2, monomer epicatechin, trimer and tetramer procyanidins were also detected indicating that, under these conditions, dimer procyanidins can either be hydrolyzed to monomers or polymerized to higher oligomer or polymers.

Anti-HIV activity of flavonoids and proanthocyanidins from Crataegus sinaica

In addition to quercetin, hyperoside, rutin, vitexin, 2″-O-rhamnosylvitexin, (4″'-O-acetyl)-2″-O-rhamno-sylvitexin, epicatechin, procyanidin B-5, proanthocyanidin A-2, procyanidin B-2 and procyanidin C-1, which were reported before in Crataegus sinaica (Rosaceae), three more flavonoids were isolated from the same plant, i.e. (+)-taxifolin, 3-O-β-arabinopyranosyl-(+)-taxifolin and 3-0-β-xylopyranosyl-(+)-taxifolin. The in vitro anti-HIV activity of some selected flavonoids and proanthocyanidins was evaluated. The dimeric procyanidin B2 and proanthocyanidin A2, and especially the trimeric procyanidin C1 showed some in vitro anti-HIV activity.