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Abstract Janus Kinases (JAKs) have emerged as an important drug target for the treatment of a number of immune disorders due to the central role that they play in cytokine signalling. 4 isoforms of JAKs exist in mammalian cells and the ideal isoform profile of a JAK inhibitor has been the subject of much debate. JAK3 has been proposed as an ideal target due to its expression being largely restricted to the immune system and its requirement for signalling by cytokine receptors using the common γ-chain. Unlike other JAKs, JAK3 possesses a cysteine in its ATP binding pocket and this has allowed the design of isoform selective covalent JAK3 inhibitors targeting this residue. We report here that mutating this cysteine to serine does not prevent JAK3 catalytic activity but does greatly increase the IC50 for covalent JAK3 inhibitors. Mice with a Cys905Ser knockin mutation in the endogenous JAK3 gene are viable and show no apparent welfare issues. Cells from these mice show normal STAT phosphorylation in response to JAK3 dependent cytokines but are resistant to the effects of covalent JAK3 inhibitors. These mice therefore provide a chemical-genetic model to study JAK3 function.

Estrogen receptor α (ERα) is the key transcriptional driver in a large proportion of breast cancers. We report that APOBEC3B (A3B) is required for regulation of gene expression by ER and acts by causing C-to-U deamination at ER binding regions. We show that these C-to-U changes lead to the generation of DNA strand breaks through activation of base excision repair (BER) and to repair by non-homologous end-joining (NHEJ) pathways. We provide evidence that transient cytidine deamination by A3B aids chromatin modification and remodelling at the regulatory regions of ER target genes that promotes their expression. A3B expression is associated with poor patient survival in ER+ breast cancer, reinforcing the physiological significance of A3B for ER action.

TP53 is conventionally thought to prevent cancer formation and progression to metastasis, while mutant TP53 has transforming activities. However, in the clinic, TP53 mutation status does not accurately predict cancer progression. Here we report, based on clinical analysis corroborated with experimental data, that the p53 isoform Δ133p53β promotes cancer cell invasion, regardless of TP53 mutation status. Δ133p53β increases risk of cancer recurrence and death in breast cancer patients. Furthermore Δ133p53β is critical to define invasiveness in a panel of breast and colon cell lines, expressing WT or mutant TP53. Endogenous mutant Δ133p53β depletion prevents invasiveness without affecting mutant full-length p53 protein expression. Mechanistically WT and mutant Δ133p53β induces EMT. Our findings provide explanations to 2 long-lasting and important clinical conundrums: how WT TP53 can promote cancer cell invasion and reciprocally why mutant TP53 gene does not systematically induce cancer progression.

miR-132 and miR-212 are two closely related miRNAs encoded in the same intron of a small non-coding gene, which have been suggested to play roles in both immune and neuronal function. We describe here the generation and initial characterisation of a miR-132/212 double knockout mouse. These mice were viable and fertile with no overt adverse phenotype. Analysis of innate immune responses, including TLR-induced cytokine production and IFNβ induction in response to viral infection of primary fibroblasts did not reveal any phenotype in the knockouts. In contrast, the loss of miR-132 and miR-212, while not overtly affecting neuronal morphology, did affect synaptic function. In both hippocampal and neocortical slices miR-132/212 knockout reduced basal synaptic transmission, without affecting paired-pulse facilitation. Hippocampal long-term potentiation (LTP) induced by tetanic stimulation was not affected by miR-132/212 deletion, whilst theta burst LTP was enhanced. In contrast, neocortical theta burst-induced LTP was inhibited by loss of miR-132/212. Together these results indicate that miR-132 and/or miR-212 play a significant role in synaptic function, possibly by regulating the number of postsynaptic AMPA receptors under basal conditions and during activity-dependent synaptic plasticity.

MiRNAs can regulate gene expression through versatile mechanisms that result in increased or decreased expression of the targeted mRNA and it could effect the expression of thousands of protein in a particular cell. An increasing body of evidence suggest that miRNAs action can be modulated by proteins that bind to the same 3'UTRs that are targeted by miRNAs, suggesting that other factors apart from miRNAs and their target sites determine miRNA-modulation of gene expression. We applied an affinity purification protocol using biotinylated let-7 miRNA inhibitor to isolate proteins that are involved in let-7 mediated gene regulation that resulted in an affinity purification of Polypyrimidine Tract Binding protein (PTB). Here we show that PTB interacts with miRNAs and human Argonaute 2 (hAgo2) through RNA as well as identified potential mammalian cellular targets that are co-regulated by PTB and hAgo2. In addition, using genetic approach, we have demonstrated that PTB genetically interacts with Caenorhabditis elegans let-7 indicating a conserved role for PTB in miRNA-mediated gene regulation.

Abstract Shotgun metabarcoding was conducted to examine the microbiota in a total of 48 samples from 12 critically ill patients, analyzing samples from both the oropharynx and rectum. We aimed to compare their postadmission microbiota, characterized as moderately dysbiotic, with the severely dysbiotic antemortem microbiota associated with patients’ deaths. We found that, compared with postadmission samples, patient antemortem swab samples presented moderate but not significantly decreased diversity indices. The antemortem oropharyngeal samples presented an increase in biofilm-forming bacteria, including Streptococcus oralis, methicillin-resistant Staphylococcus aureus (MRSA), and Enterococcus faecalis. Although the septic shock rate was 67%, no significant differences were detected in the potential pathogen ratios when the microbiota was analyzed. A notable strain-sharing rate between the oropharynx and intestine was noted. By comparing postadmission and antemortem samples, microbial biomarkers of severe dysbiosis were pinpointed through the analysis of differentially abundant and uniquely emerging species in both oropharyngeal and rectal swabs. Demonstrating strong interconnectivity along the oral-intestinal axis, these biomarkers could serve as indicators of the progression of dysbiosis. Furthermore, the microbial networks of the oropharyngeal microbiota in deceased patients presented the lowest modularity, suggesting a vulnerable community structure. Our data also highlight the critical importance of introducing treatments aimed at enhancing the resilience of the oral cavity microbiome, thereby contributing to better patient outcomes.

A comprehensive study of sorghum bran and flour was performed to explore the secondary metabolite profiles of differently coloured genotypes and to evaluate the variability in the antioxidant properties based on differences in polarity and solubility. This research included one red variety and one white variety. Among the samples, the red variety contained significantly greater amounts of secondary metabolites than did the white variety, with total polyphenol contents of 808.04 ± 63.89 mg.100 g-1 and 81.56 ± 3.87 mg.100 g-1, respectively. High-molecular-weight condensed tannin-type flavonoid extracts with high antioxidant activity were obtained by using relatively low-polarity acetone-water solvents, which was reflected by the measured antioxidant values. Among the methods used, the electron-donating Trolox equivalent antioxidant assay provided the highest antioxidant capacity, with values ranging from 118.5 to 182.6 μmol g−1 in the case of the red variety, in accordance with the electron donor properties of condensed tannins. Key secondary metabolites were identified using MS techniques and quantified using HPLC. Catechin and procyanidin B1 were found in the red variety at concentrations of 3.20 and 96.11 mg.100 g-1, respectively, while the concentrations in the white variety were under the limit of detection. All four tocopherols were found in sorghum, with the red variety containing a higher amount than the white variety, but the vitamin B complex concentrations were higher in the white variety. Overall, the red sorghum variety proved to be a better source of secondary metabolites with potential health benefits and could be used as a nutrient-rich food source.

A Hungarian survey of Tokaj-Mád vineyards was conducted. Shotgun metabarcoding was applied to decipher the microbial-terroir. The results of 60 soil samples showed that there were three dominant fungal phyla, Ascomycota 66.36% ± 15.26%, Basidiomycota 18.78% ± 14.90%, Mucoromycota 11.89% ± 8.99%, representing 97% of operational taxonomic units (OTUs). Mutual interactions between microbiota diversity and soil physicochemical parameters were revealed. Principal component analysis showed descriptive clustering patterns of microbial taxonomy and resistance gene profiles in the case of the four historic vineyards (Szent Tamás, Király, Betsek, Nyúlászó). Linear discriminant analysis effect size was performed, revealing pronounced shifts in community taxonomy based on soil physicochemical properties. Twelve clades exhibited the most significant shifts (LDA > 4.0), including the phyla Verrucomicrobia, Bacteroidetes, Chloroflexi, and Rokubacteria, the classes Acidobacteria, Deltaproteobacteria, Gemmatimonadetes, and Betaproteobacteria, the order Sphingomonadales, Hypomicrobiales, as well as the family Sphingomonadaceae and the genus Sphingomonas. Three out of the four historic vineyards exhibited the highest occurrences of the bacterial genus Bradyrhizobium, known for its positive influence on plant development and physiology through the secretion of steroid phytohormones. During ripening, the taxonomical composition of the soil fungal microbiota clustered into distinct groups depending on altitude, differences that were not reflected in bacteriomes. Network analyses were performed to unravel changes in fungal interactiomes when comparing postveraison and preharvest samples. In addition to the arbuscular mycorrhiza Glomeraceae, the families Mycosphaerellacae and Rhyzopodaceae and the class Agaricomycetes were found to have important roles in maintaining soil microbial community resilience. Functional metagenomics showed that the soil Na content stimulated several of the microbiota-related agrobiogeochemical cycles, such as nitrogen and sulphur metabolism; steroid, bisphenol, toluene, dioxin and atrazine degradation and the synthesis of folate.

Background: Our previous studies demonstrated that sour cherry anthocyanins (AC) reduce the salivary count of Streptococcus mutans and inhibit salivary amylase activity within 30 minutes after chewing AC gum. AC gum and changing toothbrushes after scaling reduced the Gram-negative species in the unstimulated salivary microbiota. The present study examined the effect of AC gums on salivary factors, including changes in microbiome. Methods: The study was conducted over three weeks with two groups; young adults (18–30) and adults (30–45). Ten participants changed their toothbrushes, while the other 10 participants did not change after the control period. After scaling, all participants received three doses of AC gum daily. The salivary mRNA and protein levels of cytokines, mucins, melatonin, and the microbiota of unstimulated and stimulated saliva were determined by polymerase chain reaction, enzyme-linked immunosorbent assay, and 16S rRNA gene sequencing. Results: Significantly higher levels of tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), mucin5B (MUC5B), mucin7 (MUC7), and melatonin were detected in stimulated saliva. Correlation analysis of these factors with the microbiota showed positive correlations with the genera Lachnospiraceae, Eikenella, Saccharibacteria_(TM7), Streptococcus, Prevotella, and Haemophilus. Conclusions: AC chewing gum has a beneficial effect on the composition of the oral microbiome, and toothbrush replacement leads to changes in the levels of salivary pro-inflammatory cytokines.

ABCG2 is an exporter-type ABC protein that can expel numerous chemically unrelated xeno- and endobiotics from cells. When expressed in tumor cells or tumor stem cells, ABCG2 confers multidrug resistance, contributing to the failure of chemotherapy. Molecular details orchestrating substrate translocation and ATP hydrolysis remain elusive. Here, we present methods to concomitantly investigate substrate and nucleotide binding by ABCG2 in cells. Using the conformation-sensitive antibody 5D3, we show that the switch from the inward-facing (IF) to the outward-facing (OF) conformation of ABCG2 is induced by nucleotide binding. IF-OF transition is facilitated by substrates, and hindered by the inhibitor Ko143. Direct measurements of 5D3 and substrate binding to ABCG2 indicate that the high-to-low affinity switch of the drug binding site coincides with the transition from the IF to the OF conformation. Low substrate binding persists in the post-hydrolysis state, supporting that dissociation of the ATP hydrolysis products is required to reset the high substrate affinity IF conformation of ABCG2.

Melatonin is an ancient hormone whose physiological effects have been extensively studied in animals and human. We now know that it also plays a prominent role in the growth and development of plants. In our present experiment, the relationship between endogenous melatonin and the antioxidant system was investigated in potato plant grown in vitro. Changes in redox homeostasis under ultrasound stress were examined. The concentration of small molecule antioxidants and enzymes of the three-level antioxidant pathway was measured. ELISA method was used to determine the melatonin levels in plant tissues at each growth stage (0 h, 24 h, 48 h, 1 week, and 4 weeks after subculturing the explants) both in control and ultrasound-treated plants. Ultrasound stress activated the three-level defense system and decreased the endogenous melatonin levels. Melatonin was able to provide protection against membrane damage caused by drastic ultrasound treatment. Melatonin at the heart of the redox network is a key component regulating various biochemical, cellular, and physiological responses. It has a dual role, as it is able to act both as a growth regulator and an antioxidant. A close relationship was evidenced between the plant hormone indole-3-acetic acid and melatonin and ascorbic acid.

Sorghum bicolor (L.) Moench is an increasingly important crop grown in many countries as a food source due to its excellent nutritional value, drought and pest resistance, and gluten-free properties. In this study, the bioactive profiles and antioxidant potentials of brans of six sorghum varieties were evaluated using spectrophotometric methods. The effects of weather and environmental conditions and different nitrogen nutrition were also evaluated. The bran of red varieties contained a higher amount of polyphenols and tannins and exhibited higher antioxidant capacities than the bran of white varieties, with the exception of one red genotype. The highest total polyphenol contents were measured in samples from two red varieties (Zádor, Alföldi1) with 1084.52 ± 57.92 mg 100 g−1 GAE and 1802.51 ± 121.13 mg 100 g−1 GAE values, respectively, while condensed tannin content varied between 0.50 mg g−1 and 47.79 mg g−1 in sorghum brans. Red varieties showed higher antioxidant activities/capacities with 70–281 µmol TE g−1 and 71–145 µmol TE g−1 for DPPH and TEAC. Correlation analysis showed a strong interaction between DPPH, TEAC, and the amounts of polyphenols and tannins, but not with FRAP values. In conclusion, red-colored varieties are a good source of polyphenols, but seed color alone is not enough to determine the nutritional value of a genotype, and the environmental conditions greatly affect the bioactive profile of sorghum.

The sour cherry contains anthocyanins, which have bactericide action against some oral bacteria (Klebsiella pneumoniae, Pseudomonas aeruginosa). Sour cherry also has antibiofilm action against Streptococcus mutans, Candida albicans, and Fusobacterium nucleatum. Our earlier research proved that chewing sour cherry anthocyanin gum significantly reduces the amount of human salivary alpha-amylase and Streptococcus mutans levels. The microbiota of a toothbrush affects oral health and regular toothbrush change is recommended. A total of 20 healthy participants were selected for the study. We analysed saliva samples with 16S rRNA sequencing to investigate the effect of 2 weeks (daily three times, after main meals) of chewing sour cherry anthocyanin gum—supplemented by toothbrush change in half of our case–control study cohort—after scaling on human oral microbiota. A more stable and diverse microbiome could be observed after scaling by the anthocyanin gum. Significant differences between groups (NBR: not toothbrush changing; BR: toothbrush changing) were evaluated by log2 proportion analysis of the most abundant family and genera. The analysis showed that lower level of some Gram-negative anaerobic (Prevotella melaninogenica, Porphyromonas pasteri, Fusobacterium nucleatum subsp. vincentii) and Gram-positive (Rothia mucilaginosa) bacteria could be observed in the case group (BR), accompanied by build-up of health-associated Streptococcal network connections.

ABSTRACT 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.

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.

P-glycoprotein (Pgp, ABCB1) is a member of one of the largest families of active transporter proteins called ABC transporters. Thanks to its expression in tissues with barrier functions and its broad substrate spectrum, it is an important determinant of the absorption, metabolism and excretion of many drugs. Pgp and/or some other drug transporting ABC proteins (e.g., ABCG2, MRP1) are overexpressed in nearly all cancers and cancer stem cells by which cancer cells become resistant against many drugs. Thus, Pgp inhibition might be a strategy for fighting against drug-resistant cancer cells. Previous studies have shown that certain polyphenols interact with human Pgp. We tested the effect of 15 polyphenols of sour cherry origin on the basal and verapamil-stimulated ATPase activity of Pgp, calcein-AM and daunorubicin transport as well as on the conformation of Pgp using the conformation sensitive UIC2 mAb. We found that quercetin, quercetin-3-glucoside, narcissoside and ellagic acid inhibited the ATPase activity of Pgp and increased the accumulation of calcein and daunorubicin by Pgp-positive cells. Cyanidin-3O-sophoroside, catechin, naringenin, kuromanin and caffeic acid increased the ATPase activity of Pgp, while they had only a weaker effect on the intracellular accumulation of fluorescent Pgp substrates. Several tested polyphenols including epicatechin, trans-ferulic acid, oenin, malvin and chlorogenic acid were ineffective in all assays applied. Interestingly, catechin and epicatechin behave differently, although they are stereoisomers. We also investigated the effect of quercetin, naringenin and ellagic acid added in combination with verapamil on the transport activity of Pgp. In these experiments, we found that the transport inhibitory effect of the tested polyphenols and verapamil was additive or synergistic. Generally, our data demonstrate diverse interactions of the tested polyphenols with Pgp. Our results also call attention to the potential risks of drug–drug interactions (DDIs) associated with the consumption of dietary polyphenols concurrently with chemotherapy treatment involving Pgp substrate/inhibitor drugs.

Neurotrophins are growth factors that are important in neuronal development and survival as well as synapse formation and plasticity. Many of the effects of neurotrophins are mediated by changes in protein expression as a result of altered transcription or translation. To determine whether neurotrophins regulate the production of microRNAs (miRNAs), small RNA species that modulate protein translation or mRNA stability, we used deep sequencing to identify BDNF (brain-derived neurotrophic factor)-induced miRNAs in cultured primary cortical mouse neurons. This revealed that the miR-212/132 cluster contained the miRNAs most responsive to BDNF treatment. This cluster was found to produce four miRNAs: miR-132, miR-132*, miR-212 and miR-212*. Using specific inhibitors, mouse models and promoter analysis we have shown that the regulation of the transcription of the miR-212/132 miRNA cluster and the miRNAs derived from it are regulated by the ERK1/2 (extracellular-signalregulated kinase 1/2) pathway, via both MSK (mitogen and stress-activated kinase)-dependent and -independent mechanisms. [ABSTRACT FROM AUTHOR]

The small intestine is the longest part of the chicken (Gallus gallus) gastrointestinal system that is specialized for nutrient absorption. It is known that decrease in intestinal villi area or height in early age can cause a reduction in essential nutrient intake, which may lead to delayed growth and consequently poorer performance of broiler chickens. The small intestinal absorptive surface is known to be affected by various factors, among others things the nutritional state. In our experiment, we aimed to investigate the possible protein expression alterations that lie behind the villus area and height decrease caused by feed deprivation. A total of 24 chickens were divided into three groups, namely ad libitum fed, fasted for 24 h, fasted for 24 h then refed for 2 h. The morphometric parameters were also measured in the duodenum, jejunum and ileum tissue sections using image analysis. Differential proteome analyses from jejunum samples were performed using two-dimensional difference gel electrophoresis followed by tryptic digestion and protein identification by matrix-assisted laser desorption/ionization mass spectrometry. Overall 541 protein spots were detected after 2D. Among them, eleven showed 1.5-fold or higher significant difference in expression and were successfully identified. In response to 24 h fasting, the expression of nine proteins was higher and that of two proteins was lower compared to the ad libitum fed group. The functions of the differentially expressed proteins indicate that the 24 h fasting mainly affects the expression of structural proteins, and proteins involved in lipid transport, general stress response, and intestinal defense.

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 β-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-1β (IL-1β), 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, β-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 β-glucan (p < 0.0001) and anthocyanin (p = 0.0063) supplementations and thickened mucosa was observed in β-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.

A natural fat-soluble thiamine derivative, namely N-[(4-amino-2-methylpyrimidin-5-yl)methyl]-N-[(2E)-5-hydroxy-3-(prop-2-en-1-yldisulfanyl)pent-2-en-2-yl]formamide (allithiamine) has been identified only in garlic (Allium sativum) until now. Hungarian red sweet pepper (Capsicum annuum) was found as a new source of allithiamine. Extraction procedure and analytical method were developed for the isolation of allithiamine and a chemical synthesis of the compound was also developed. First solid-liquid extraction was performed with 96 % ethanol to isolate allithiamine from pepper seeds. Thereafter, solid phase extraction was applied from ethanolic extract using C18 cartridge to concentrate and purify samples for further analysis. The structure of the synthesized and the isolated compounds was verified by reverse phase HPLC, HPLC-MS, MALD-TOF MS and NMR. Furthermore, effect of allithiamine was investigated on streptozotocin-induced diabetic mice with neuropathy. The results show that neuropathic pain sensation is improved by allithiamine treatment similarly to benfothiamine.

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.

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.

Among diabetes patients, ophthalmological complications are very frequent. High blood glucose and (consequential) ischemia-reperfusion (I/R) injury contribute significantly to the severity of retinopathies. Diabetic retinopathy is among the leading causes of blindness. Our study demonstrates the effect of sour cherry seed extract (SCSE) on blood glucose and function of the retina with electroretinography (ERG) in a diabetic setting with or without ischemia-reperfusion (I/R) injury in Zucker Diabetic Fatty (ZDF) rats. Our results prove that the SCSE has a retinoprotective effect in diabetic rats: according to ERG measurements, SCSE treatment mitigated the retinal function-damaging effect of diabetes, and proved to be protective in the diabetic eye against ischemia-reperfusion injuries of the retina. Outcomes suggest that the protective effects of SCSE may occur through several pathways, including HO-1 dependent mechanisms. The observation that SCSE treatment decreases blood glucose is also novel. These findings offer the possibility for development of novel therapeutic strategies utilizing this emerging functional food, in particular in the prevention of conditions resulting from high blood glucose or I/R injury, such as deterioration of retinal microcirculation.