HPLC Analysis of Polyphenols Derived from Hungarian Aszú from Tokaj Wine Region and Its Effect on Inflammation in an In Vitro Model System of Endothelial Cells.

Many studies have been published in recent years regarding the fact that moderate wine consumption, as a part of a balanced diet can have a beneficial effect on human health. The biologically active components of wine continue to be the subject of intense research today. In this study, the bioactive molecules of Hungarian aszú from the Tokaj wine region were analyzed using high-performance liquid chromatography (HPLC) and investigated in an in vitro model system of endothelial cells induced by bacterial-derived lipopolysaccharide. The HPLC measurements were performed on a reversed phased column with gradient elution. The non-cytotoxic concentration of the active substance was determined based on 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT)-, apoptosis, and necrosis assays. The antioxidant effect of the extract was determined by evaluating its ability to eliminate ROS. The expressions of the interleukin-(IL)1α, IL1-ß, IL-6, and IL-8 pro-inflammatory cytokines and nitric oxide synthase (eNOS) at the mRNA level were evaluated using a quantitative polymerase chain reaction (qPCR). We found that the lipopolysaccharides (LPS)-induced increases in the expressions of the investigated cytokines were significantly suppressed by Hungarian aszú extract, excluding IL-6. In our experimental setup, our treatment had a positive effect on the eNOS expression, which was impaired as a result of the inflammatory manipulation. In our experimental model, the Hungarian aszú extract decreased the LPS-induced increases in the expression of the investigated cytokines and eNOS at the mRNA level, which presumably had a positive effect on the endothelial dysfunction caused by inflammation due to its strong antioxidant and anti-inflammatory effects. Collectively, this research contributes to a more thorough understanding of the bioactive molecules of aszú from the Tokaj wine region.

Comparison of the Modulating Effect of Anthocyanin-Rich Sour Cherry Extract on Occludin and ZO-1 on Caco-2 and HUVEC Cultures.

Increased permeability of the epithelial and endothelial cell layers results in the onset of pathogenic mechanisms. In both cell types, cell-cell connections play a regulatory role in altering membrane permeability. The aim of this study was to investigate the modulating effect of anthocyanin-rich extract (AC) on TJ proteins in inflammatory Caco-2 and HUVEC monolayers. Distribution of Occludin and zonula occludens-1 (ZO-1) were investigated by immunohistochemical staining and the protein levels were measured by flow cytometry. The mRNA expression was determined by quantitative real-time PCR. The transepithelial electrical resistance (TEER) values were measured during a permeability assay on HUVEC cell culture. As a result of inflammatory induction by TNF-α, redistribution of proteins was observed in Caco-2 cell culture, which was reduced by AC treatment. In HUVEC cell culture, the decrease in protein and mRNA expression was more dominant during inflammatory induction, which was compensated for by the AC treatment. Overall, AC positively affected the expression of the examined cell-binding structures forming the membrane on both cell types.

Effect of Anthocyanin-Rich Extract of Sour Cherry for Hyperglycemia-Induced Inflammatory Response and Impaired Endothelium-Dependent Vasodilation.

Diabetes mellitus (DM)-related morbidity and mortality are steadily rising worldwide, affecting about half a billion people worldwide. A significant proportion of diabetic cases are in the elderly, which is concerning given the increasing aging population. Proper nutrition is an important component in the effective management of diabetes in the elderly. A plethora of active substances of plant origin exhibit potency to target the pathogenesis of diabetes mellitus. The nutraceutical and pharmaceutical effects of anthocyanins have been extensively studied. In this study, the effect of Hungarian sour cherry, which is rich in anthocyanins, on hyperglycemia-induced endothelial dysfunction was tested using human umbilical cord vein endothelial cells (HUVECs). HUVECs were maintained under both normoglycemic (5 mM) and hyperglycemic (30 mM) conditions with or without two concentrations (1.50 ng/µL) of anthocyanin-rich sour cherry extract. Hyperglycemia-induced oxidative stress and inflammatory response and damaged vasorelaxation processes were investigated by evaluating the level of reactive oxygen species (ROS) and gene expression of four proinflammatory cytokines, namely, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-1α (IL-1α), as well as the gene expression of nitric oxide synthase (NOS) endothelin-1 (ET-1) and endothelin-converting enzyme-1 (ECE-1). It was found that hyperglycemia-induced oxidative stress was significantly suppressed by anthocyanin-rich sour cherry extract in a concentration-dependent manner. The gene expression of the tested proinflammatory cytokines increased under hyperglycemic conditions but was significantly reduced by both 1 and 50 ng/µL anthocyanin-rich sour cherry extract. Further, although increased ET-1 and ECE-1 expression due to hyperglycemia was reduced by anthocyanin-rich sour cherry extract, NOS expression was increased by the extract. Collectively, these data suggest that anthocyanin-rich sour cherry extract could alleviate hyperglycemia-induced endothelial dysfunction due to its antioxidant, anti-inflammatory, and vasorelaxant effects.

Allithiamine Alleviates Hyperglycaemia-Induced Endothelial Dysfunction.

Diabetes mellitus-related morbidity and mortality is a rapidly growing healthcare problem, globally. Several nutraceuticals exhibit potency to target the pathogenesis of diabetes mellitus. The antidiabetic effects of compounds of garlic have been extensively studied, however, limited data are available on the biological effects of a certain garlic component, allithiamine. In this study, allithiamine was tested using human umbilical cord vein endothelial cells (HUVECs) as a hyperglycaemic model. HUVECs were isolated by enzymatic digestion and characterized by flow cytometric analysis using antibodies against specific marker proteins including CD31, CD45, CD54, and CD106. The non-cytotoxic concentration of allithiamine was determined based on MTT, apoptosis, and necrosis assays. Subsequently, cells were divided into three groups: incubating with M199 medium as the control; or with 30 mMol/L glucose; or with 30 mMol/L glucose plus allithiamine. The effect of allithiamine on the levels of advanced glycation end-products (AGEs), activation of NF-κB, release of pro-inflammatory cytokines including IL-6, IL-8, and TNF-α, and H2O2-induced oxidative stress was investigated. We found that in the hyperglycaemia-induced increase in the level of AGEs, pro-inflammatory changes were significantly suppressed by allithiamine. However, allithiamine could not enhance the activity of transketolase, but it exerts a potent antioxidant effect. Collectively, our data suggest that allithiamine could alleviate the hyperglycaemia-induced endothelial dysfunction due to its potent antioxidant and anti-inflammatory effect by a mechanism unrelated to the transketolase activity.

Anthocyanin-Rich Sour Cherry Extract Attenuates the Lipopolysaccharide-Induced Endothelial Inflammatory Response.

The anthocyanin content of Hungarian sour cherry is remarkable based on our preliminary investigations. Nutraceutical and pharmaceutical effects of anthocyanins have been extensively studied. The objective of this work was to investigate the the effect of purified sour cherry extract using human umbilical cord vein endothelial cells (HUVECs) as the inflammatory model. HUVECs were isolated by enzymatic digestion and characterized by flow cytometry. The optimal concentration range of sour cherry extract was selected based on MTT, apoptosis, and necrosis assays. Cells were divided into three groups, incubating with M199 medium as control, or with lipopolysaccharide (LPS) or with LPS plus anthocyanin extract (ACE). The effect of sour cherry extract on oxidative stress, pro-inflammatory factors, and arachidonic pathway was investigated. An amount of 50 µg/mL ACE (ACE50) was able to increase the level of glutathione and decrease the ROS, thereby improving the unbalanced redox status in inflammation. ACE50 lowered pro-inflammatory cytokine levels including Interleukin-6 (IL-6), regulated on activation, normal T cell expressed and secreted (RANTES), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor alpha (TNF-α). ACE50 affected the arachidonic acid pathway by reducing the LPS-induced enzyme expression (cyclooxygenase-1, cyclooxygenase-2, and prostacyclin synthase). The extract under investigation seems to have a pleiotropic effect including anti-oxidative, anti-inflammatory, hemostatic, and vasoactive effects. Our results indicate that purified sour cherry extract could reduce the LPS-induced inflammatory response, thereby improving endothelial dysfunction.

Determination of Flavonoid and Proanthocyanidin Profile of Hungarian Sour Cherry.

Hungarian sour cherries (SC) are excellent source of anthocyanin (concentrations (100⁻300 mg in 100 g fresh fruit) and melatonin (0.15 mg in 100 g fresh fruit), but other flavonoid derivatives also can be isolated by aqueous alcoholic extraction. We have developed a new process for extracting non-extractable procyanidines bound to the membrane, proteins, and fibers. These compounds were seperated with UHPLC-MS methods, and the structure of individual components were identified on the basis of their mass fragmentation spectra. The antioxidant capacity of soluble and non-soluble antioxidants were measured with ferric reducing antioxidant power (FRAP), 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity (DPPH), trolox equivalent antioxidant capacity (TEAC) assays, and compared to the new measurement methods of water-soluble antioxidant capacity (ACW), lipid-soluble antioxidant capacity (ACL). Furthermore, total phenolic content (TPC) and total procyanidin content (PAC) were determinated. As a result of our investigation, we found that the solvent combination, where in the first step is water⁻ethanol (1:1), then 100% ethanol were suitable for the extraction of the extractable antioxidants. However, the chemiluminescence method that is based on the elimination of the superoxide radical is more accurate than other colorimetric methods which measure antioxidant capacity.