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.

Genes and elements involved in the regulation of the nervous system and growth affect the development of spinal deformity in Cyprinus carpio.

Spinal deformity is a serious economic and animal welfare problem in intensive fish farming systems, which will be a significant unsolved problem for the fish sector. The aim of this study was to determine the relative expression of genes (Akt1 substrate 1, Calreticulin, Collagen type I alpha 2 chain, Corticotropin-releasing hormone, Chromodomain-Helicase DNA-binding, Growth hormone, Insulin like growth factor 1, Myostatin, Sine oculis-related homeobox 3, Toll-like receptor 2) in different tissues associated with spinal deformity and to determine the macroelement (calcium, magnesium, phosphorus, potassium, sodium, sulfur) and microelement (barium, copper, iron, manganese, strontium, zinc) content of spine in healthy and deformed common carps (Cyprinus carpio) in Hungary. The mRNA levels of the genes were measured in 7 different tissues (abdominal fat, blood, brain, dorsal muscle, genitals, heart, liver) by qRT-PCR. Correlations between gene expression and element content were analyzed by using linear regression and Spearman rank correlation. In a total of 15 cases, we found a statistically significant connection between gene expression in a tissue and the macro- or microelement content of the spine. In these contexts, the genes Akt1 substrate 1 (3), Collagen type I alpha 2 chain (2), Corticotropin-releasing hormone (4), Insulin-like growth factor 1 (4), and Myostatin (2), the tissue's blood (3), brain (6), heart (5), and liver (1), the macroelements sodium (4), magnesium (4), phosphorus (1) and sulfur (2) as well as the microelement iron (4) were involved. We also found statistically significant mRNA level differences between healthy and deformed common carps in tissues that were not directly affected by the deformation. Based on our results, genes regulating the nervous system and growth, elements, and tissues are the most associated components in the phenomenon of spinal deformity. With our study, we wish to give direction to and momentum for the exploration of these complex processes.

Effects of phytonutrient-supplemented diets on the intestinal microbiota of Cyprinus carpio.

In the aquaculture sector, a strategy for the more efficient use of resources and proper disease control is needed to overcome the challenges of meat production worldwide. Modulation of the gastrointestinal tract microbiota is a promising approach for promoting animal health and preventing infection. This feeding experiment was conducted to discover the phytonutrient-induced changes in the gastrointestinal tract microbiota of common carp (Cyprinus carpio). Acclimatized animals aged 7 months (30 weeks) were divided randomly into five experimental groups to investigate the effects of the applied feed additives. The dietary supplements were manufactured from anthocyanin-containing processing wastes from the food industry, specifically the production of Hungarian sour cherry extract, synbiotics from fermented corn, and fermentable oligosaccharides from Hungarian sweet red pepper seeds and carotenoids from Hungarian sweet red pepper pulps, applied at a dose of 1%. The gut contents of the animals were collected at four time points throughout the 6-week study period. To track the compositional and diversity changes in the microbiota of the carp intestinal tract, V3-V4 16S rRNA gene-based metagenomic sequencing was performed. The growth performance of common carp juveniles was not significantly affected by supplementation of the basal diet with plant extracts. Phytonutrients improve the community diversity, increase the Clostridium and Lactobacillus abundances and decrease the abundances of potentially pathogenic and spoilage bacteria, such as Shewanella, Pseudomonas, Acinetobacter and Aeromonas. The phyla Proteobacteria, Tenericutes and Chlamydiae were positively correlated with the body weight, whereas Spirochaetes and Firmicutes exhibited negatively correlations with the body weight. We hypothesize that the application of phytonutrients in aquaculture settings might be a reasonable green approach for easing the usage of antibiotics.

Nutraceuticals Induced Changes in the Broiler Gastrointestinal Tract Microbiota.

Effects of nutraceuticals on the intestinal microbiota are receiving increased attention; however, there are few studies investigating their effects on broiler meat production. The aim of this study was to implement feeding strategies and carry out a comprehensive trial examining the interplay between natural biologically active compounds such as carotenoids, anthocyanins, fermentable oligosaccharides, and synbiotics and the gastrointestinal tract microbiota. Our feeding program was applied to an intensive production system with a flock of 1,080 Ross 308 broilers. Aging induced significant changes through the feeding experiment. Nutraceuticals were shown to modulate broiler intestinal diversity and differentially enriched Lactobacillus, Enterococcus, Campylobacter, and Streptococcus in the core microbiome during the different stages of broiler rearing. Additionally, they did not remarkably affect animal growth performance; nevertheless, a positive correlation was found between body weight and Corynebacteriales and Pseudomonadales Furthermore, a diet high in carotenoid, fermentable oligosaccharide, and anthocyanin contents affected the number of beneficial genera such as Faecalibacterium, Lactobacillus, Blautia, and Ruminococcus With this comprehensive trial, we revealed that nutraceuticals induced modulations in broiler gastrointestinal tract microbiota. We believe that plant-derived immunostimulants, recycled from plant food waste products, can supplement antibiotic-free broiler meat production.IMPORTANCE In this trial, nutraceuticals were manufactured from waste products of food industry processing of Hungarian red sweet pepper and sour cherry and incorporated into the diet of poultry to investigate their effects on broilers' growth and the broiler gastrointestinal tract microbiota. To avoid the generation of food waste products, we believe that this approach can be developed into a sustainable, green approach that can be implemented in commercial antibiotic-free poultry to provide safe and high-quality meat.

Iron and Manganese Retention of Juvenile Zebrafish (Danio rerio) Exposed to Contaminated Dietary Zooplankton (Daphnia pulex)-a Model Experiment.

In present study the effect of iron (Fe) and manganese (Mn) contamination was assessed by modeling a freshwater food web of water, zooplankton (Daphnia pulex), and zebrafish (Danio rerio) under laboratory conditions. Metals were added to the rearing media of D. pulex, and enriched zooplankton was fed to zebrafish in a feeding trial. The elemental analysis of rearing water, zooplankton, and fish revealed significant difference in the treatments compared to the control. In D. pulex the Mn level increased almost in parallel with the dose of supplementation, as well as the Fe level differed statistically. A negative influence of the supplementation on the fish growth was observed: specific growth rate (SGR%) and weight gain (WG) decreased in Fe and Mn containing treatments. The redundancy analysis (RDA) of concentration data showed strong correlation between the rearing water and D. pulex, as well as the prey organism of Fe- and Mn-enriched D. pulex and the predator organism of D. rerio. The bioconcentration factors (BCF) calculated for water to zooplankton further proved the relationship between the Fe and Mn dosage applied in the treatments and measured in D. pulex. Trophic transfer factor (TTF) results also indicate that significant retention of the metals occurred in D. rerio individuals, however, in a much lower extent than in the water to zooplankton stage. Our study suggests that Fe and Mn significantly accumulate in the lower part of the trophic chain and retention is effective through the digestive track of zebrafish, yet no biomagnification occurs. Graphical abstract.

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.

Effect of Carotenoids, Oligosaccharides and Anthocyanins on Growth Performance, Immunological Parameters and Intestinal Morphology in Broiler Chickens Challenged with Escherichia coli Lipopolysaccharide.

This study was conducted to investigate the effect of carotenoid, oligosaccharide and anthocyanin supplementation in broiler diets under Escherichia coli lipopolysaccharide (LPS) challenge. Ross 308 chickens were fed 5 diets: basal diet (control diet), diet supplemented with ß-glucan in 0.05% (positive control) and diets with 0.5% carotenoid-, oligosaccharide- or anthocyanin contents. On the 26th days of age, chickens were challenged intraperitoneally 2 mg LPS per kg of body weight. 12 h after injection, birds were euthanized, then spleen and ileum samples were collected. LPS induced increased relative mRNA expression of splenic (p = 0.0445) and ileal (p = 0.0435) interleukin-1ß (IL-1ß), which was lower in the spleen in carotenoid (p = 0.0114), oligosaccharide (p = 0.0497) and anthocyanin (p = 0.0303)-treated chickens compared to LPS-injected control birds. Dietary supplementation of carotenoids also decreased relative gene expression of splenic interleukin-6 (IL-6) (p = 0.0325). In the ileum, ß-glucan supplementation showed lower relative mRNA expression of toll-like receptor 5 (TLR-5) (p = 0.0387) compared to anthocyanin treatment. Gene expression of both splenic and ileal interferon-α (IFN-α), interferon-γ (IFN-γ), toll-like receptor 4 (TLR-4) and toll-like receptor 5 (TLR-5) were not influenced by dietary supplements. In conclusion, carotenoids, oligosaccharides and anthocyanins could partially mitigate the immune stress caused by LPS challenge. All of the compounds impacted longer villus height (p < 0.0001), villus height:crypt depth ratios were higher after ß-glucan (p < 0.0001) and anthocyanin (p = 0.0063) supplementations and thickened mucosa was observed in ß-glucan (p < 0.0001), oligosaccharide (p < 0.0001) and anthocyanin (p = 0.048) treatments. All of these findings could represent a more effective absorption of nutrients.

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.

Effect of Anthocyanin-Rich Tart Cherry Extract on Inflammatory Mediators and Adipokines Involved in Type 2 Diabetes in a High Fat Diet Induced Obesity Mouse Model.

Male C57BL/6J mice were used to determine the possible therapeutic effects of our previously described tart cherry extract in a chronic obesity mouse model on metabolic parameters, glucose tolerance, inflammatory mediators, and antioxidant capacity. The control group received standard mouse chow, and the high fat control group was switched to a high fat diet and tap water supplemented with 5% sucrose. The high fat + anthocyanin group received the high fat and sucrose diet, but received the anthocyanin-rich tart cherry extract dissolved in their drinking water. After six weeks, an oral glucose tolerance test was performed, and the water-soluble antioxidant capacity (ACW), superoxide dismutase (SOD) activity, and the plasma levels of insulin, C-peptide, leptin, IL-6, MCP-1, adiponectin and resistin were measured. The high fat diet increased body weight, reduced glucose tolerance, and caused an elevation in leptin, IL-6, MCP-1, and resistin levels. Furthermore, antioxidant capacity was decreased with a significant elevation of SOD activity. Anthocyanin treatment failed to reverse the effects of the high fat diet on body weight and glucose tolerance, but significantly reduced the leptin and IL-6 levels. The tart cherry extract also made a significant enhancement in antioxidant capacity and SOD activity. Our results show that chronic anthocyanin intake has a potential to enhance redox status and alleviate inflammation associated with obesity.

Diosgenin and Its Fenugreek Based Biological Matrix Affect Insulin Resistance and Anabolic Hormones in a Rat Based Insulin Resistance Model.

Fenugreek is known since ancient times as a traditional herbal medicine of its multiple beneficial effects. Fenugreek's most studied and employed effect is its hypoglycemic property, but it can also be useful for the treatment of certain thyroid disorders or for the treatment of anorexia. The regulation of glucose homeostasis is a complex mechanism, dependent on the interaction of different types of hormones and neurotransmitters or other compounds. For the study of how diosgenin and fenugreek seeds modify insulin sensitivity, we used a rat insulin resistance model induced by high-fat diet. Diosgenin in three different doses (1mg/bwkg, 10mg/bwkg, and 50 mg/bwkg, respectively) and fenugreek seed (0.2 g/bwkg) were administered orally for 6 weeks. Insulin sensitivity was determined by hyperinsulinemic euglycemic glucose clamp method. Our research group found that although glucose infusion rate was not significantly modified in either group, the increased insulin sensitivity index and high metabolic clearance rate of insulin found in the 1 mg/kg diosgenin and the fenugreek seed treated group suggested an improved peripheral insulin sensitivity. Results from the 10 mg/kg diosgenin group, however, suggest a marked insulin resistance. Fenugreek seed therapy results on the investigated anabolic hormones support the theory that, besides insulin and gastrointestinal peptides, the hypothalamic-hypopituitary axis regulated hormones synchronized action with IGF-1 also play an important role in the maintaining of normal glucose levels. Both diosgenin and fenugreek seeds are capable of interacting with substrates of the above-mentioned regulatory mechanisms, inducing serious hormonal disorders. Moreover, fenugreek seeds showed the ability to reduce the thyroid hormone levels at the periphery and to modify the T4/T3 ratio. It means that in healthy people this effect could be considered a severe side effect; however, in hypothyroidism this effect represents a possibility of alternative natural therapy.

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.

Sour cherry extract inhibits human salivary α-amylase and growth of Streptococcus mutans (a pilot clinical study).

The purpose of this study was to determine whether cherry extract has any effect on salivary α-amylase activity (sAA) or on the level of Streptococcus mutans in human saliva. 70 patients (45 females and 25 males) in three age groups (22 children, 25 young adults, and 23 adults) were examined. All participants completed a questionnaire to obtain information on their oral health behaviour and life style. Clinical examination was performed to record the number of decayed, missing and filled teeth (DMF-T). Saliva samples were collected for the measurement of sAA and the salivary S. mutans level before and after chewing a gum with or without cherry extract. Statistical evaluation of data was performed. S. mutans and the sAA level of unstimulated saliva samples did not depend on either age or gender. The basal sAA value of adult patients was in linear correlation with the dental caries status. Habitual chewing-gum use decreased the resting sAA and the mean of DMF-T. The number of S. mutans cells was significantly lower in the resting saliva of allergic patients. The applied mechanical and gustatory stimuli by chewing gum resulted in higher sAA and S. mutans levels and a slow decrease of values was observed in the control group for the next 30 min. Thereafter, sAA and S. mutans levels decreased earlier in the presence of sour cherry extract than those of control cases. Chewing gum with sour cherry extract may be useful for the prevention of dental caries.

Optimised selenium enrichment of Artemia sp. feed to improve red drum (Sciaenops ocellatus) larvae rearing.

Selenium is an essential microelement for the normal functioning of life processes. Moreover, it is a component of enzymes with antioxidant effects. However, it has the smallest window of any micronutrient between requirement and toxicity. Selenium is a regularly used element in fish feeds; moreover, enriching zooplankton with selenium to rear larvae is also a well-known technology. It is accepted that the most common starter foods of fish larvae, natural rotifers contain the smallest dosage of selenium, but providing selenium enriched Artemia sp. instead could increase survival and growth rate of fish. However, no such references are available for the red drum (Sciaenops ocellatus) larvae. Therefore, in this study, Artemia sp. was enriched with nano-selenium of verified low toxicity and easy availability in 5 treatments (1, 5, 10, 50, 100 mg/l Se), and then, fish larvae were fed with four of these enriched Artemia stocks (1, 5, 10, 50 mg/l Se) and a control group. At the end of the 9-day-long experiment, survival rate (S) and growth parameters (SL, W, K-factor, SGR) of fish larvae were calculated as well as their selenium retention and glutathione peroxidase enzyme activity were analysed. It was revealed that a moderate level of selenium enrichment (~4 mg/kg dry matter) of Artemia sp. positively influences the rearing efficiency (i.e. survival and growth) of fish larvae, but higher dosages of selenium could cause adverse effects.