Proanthocyanidins and Phenolic Compounds from the Twigs of Salix chaenomeloides and Their Anti-Lipogenic Effects on 3T3-L1 Preadipocytes.

The present study investigated potential bioactive natural products from the EtOH extract of Salix chaenomeloides twigs using column chromatography, leading to the isolation of six compounds (1-6), which were characterized as two proanthocyanidins, procyanidin B2 (1) and procyanidin B1 (2), and four phenolic compounds, 4-hydroxybenzoic acid ß-D-glucosyl ester (3), di-O-methylcrenatin (4), p-coumaric acid glucoside (5), and syringin (6) by the comparison of their NMR spectra with the reported data and high-resolution (HR)-electrospray ionization mass spectroscopy (ESI-MS) analysis. We investigated the potential of six compounds (1-6) to inhibit adipogenesis in 3T3-L1 preadipocytes, which showed that the compounds (1-6) significantly reduced lipid accumulation in 3T3-L1 adipocytes without affecting cell proliferation. Notably, compound 1 demonstrated a remarkable 60% and 90% reduction in lipid levels with 50 and 100 µM treatments, respectively. Oil Red O staining results indicated that compound 1 significantly inhibits the formation of lipid droplets, comparable to the effect of T863, an inhibitor of triglyceride used as a positive control, in adipocytes. Compound 1 had no effect on the regulators PPARγ, C/EBPα, and SREBF1 of adipocyte differentiation in 3T3-L1 preadipocytes, but compound 1 activated the fatty acid oxidation regulator, PPARα, compared to the lipogenic-induced control. It also suppressed fatty acid synthesis by downregulating the expression of fatty acid synthase (FAS). Finally, compound 1 induced the mRNA and protein levels of CPT1A, an initial marker of mitochondrial fatty acid oxidation in 3T3-L1. This finding substantiates the anti-lipogenic and lipolytic effects of procyanidin B2 (1) in 3T3-L1 preadipocytes, emphasizing its pivotal role in modulating obesity-related markers.

Procyanidin B2 alleviates oxidized low-density lipoprotein-induced cell injury, inflammation, monocyte chemotaxis, and oxidative stress by inhibiting the nuclear factor kappa-B pathway in human umbilical vein endothelial cells.

BACKGROUND: Oxidized low-density lipoprotein (ox-LDL) can initiate and affect almost all atherosclerotic events including endothelial dysfunction. In this text, the role and underlying molecular basis of procyanidin B2 (PCB2) with potential anti-oxidant and anti-inflammatory activities in ox-LDL-induced HUVEC injury were examined. METHODS: HUVECs were treated with ox-LDL in the presence or absence of PCB2. Cell viability and apoptotic rate were examined by CCK-8 assay and flow cytometry, respectively. The mRNA and protein levels of genes were tested by RT-qPCR and western blot assays, respectively. Potential downstream targets and pathways of apple procyanidin oligomers were examined by bioinformatics analysis for the GSE9647 dataset. The effect of PCB2 on THP-1 cell migration was examined by recruitment assay. The effect of PCB2 on oxidative stress was assessed by reactive oxygen species (ROS) level, malondialdehyde (MDA) content, and mitochondrial membrane potential (MMP). RESULTS: ox-LDL reduced cell viability, induced cell apoptosis, and facilitated the expression of oxidized low-density lipoprotein receptor 1 (LOX-1), C-C motif chemokine ligand 2 (MCP-1), vascular cell adhesion protein 1 (VCAM-1) in HUVECs. PCB2 alleviated ox-LDL-induced cell injury in HUVECs. Apple procyanidin oligomers triggered the differential expression of 592 genes in HUVECs (|log2fold-change| > 0.58 and adjusted p-value < 0.05). These dysregulated genes might be implicated in apoptosis, endothelial cell proliferation, inflammation, and monocyte chemotaxis. PCB2 inhibited C-X-C motif chemokine ligand 1/8 (CXCL1/8) expression and THP-1 cell recruitment in ox-LDL-stimulated HUVECs. PCB2 inhibited ox-LDL-induced oxidative stress and nuclear factor kappa-B (NF-κB) activation in HUVECs. CONCLUSION: PCB2 weakened ox-LDL-induced cell injury, inflammation, monocyte recruitment, and oxidative stress by inhibiting the NF-κB pathway in HUVECs.

Aromatase inhibitors identified from Saraca asoca to treat infertility in women with polycystic ovary syndrome via in silico and in vivo studies.

Polycystic ovary syndrome (PCOS) is a widely occurring metabolic disorder causing infertility in 70%-80% of the affected women. Saraca asoca, an ancient medicinal herb, has been shown to have therapeutic effects against infertility and hormonal imbalance in women. This study was aimed to identify new aromatase inhibitors from S. asoca as an alternative to the commercially available ones via in silico and in vivo approaches. For this, 10 previously reported flavonoids from S. asoca were chosen and the pharmacodynamic and pharmacokinetic properties were predicted using tools like Autodock Vina, GROMACS, Gaussian and ADMETLab. Of the 10, procyanidin B2 and luteolin showed better interaction with higher binding energy when docked against aromatase (3S79) as compared to the commercial inhibitor letrozole. These two compounds showed higher stability in molecular dynamic simulations performed for 100 ns. Molecular mechanics Poisson-Boltzmann surface analysis indicated that these compounds have binding free energy similar to the commercial inhibitor, highlighting their great affinity for aromatase. Density functional theory analysis revealed that both compounds have a good energy gap, and ADMET prediction exhibited the drug-likeness of the two compounds. A dose-dependent administration of these two compounds on zebrafish revealed that both the compounds, at a lower concentration of 50 µg/ml, significantly reduced the aromatase concentration in the ovarian tissues as compared to the untreated control. Collectively, the in silico and in vivo findings recommend that procyanidin B2 and luteolin could be used as potential aromatase inhibitors for overcoming infertility in PCOS patients with estrogen dominance.Communicated by Ramaswamy H. Sarma.

Phytochemical investigation and antioxidant properties of unripe tomato cultivars (Solanum lycopersicum L.).

Unripe tomatoes are among the main waste produced during tomato cultivation and processing. In this study, unripe tomatoes from seven different Italian cultivars have been investigated to evaluate their nutraceutical potential. Phytochemical investigation allowed shedding light on the identification of seventy-five bioactive compounds. The highest amount of polyphenolic and glycoalkaloids along with the high level of antioxidant activities was found in the Datterini tomatoes variety. The peculiarity of this variety is the high chlorogenic acid content, being ten times higher compared to the other cultivars examined. Moreover, the total α-tomatine amount has been found substantially higher (34.699 ± 1.101 mg/g dry weight) with respect to the other tomato varieties analyzed. Furthermore, the cultivars metabolomic profiles were investigated with the PCA approach. Based on Datterini cultivar's metabolomic profile, its waste-recovery could represent a good option for further added value products in pharmaceutical and nutraceutical areas with a high α-tomatine content.

Exploring separation patterns and mechanisms of proanthocyanidins in grape seeds and pomace with diverse molecular weights, compositions, and structures.

The function of proanthocyanidins (PAs) relies on their structure and requires high-purity PAs. Though Sephadex LH-20 gel permeation chromatography (GPC) is expected to separate PAs based on structure, its usage rules and mechanisms remain unclear. This study delves into the PAs separation patterns on Sephadex LH-20, first confirming the purification mechanisms of PAs with various mean degrees of polymerization (DP) using the adsorption kinetic model. The study found that an increase in the molecular weight or mean DP of PAs results in decreased polarity, reduced hydrogen bonding actions, and intensified hydrophobic effect, causing delayed extraction of PAs on Sephadex LH-20, with galloylated PA as an exception, which was extracted first despite its high DP. Additionally, the principles for separating specific composition, such as monomers, dimers, etc., were evaluated. The study sheds light on enhancing the purification efficiency of PAs, thus advancing the precise separation technology of diverse proanthocyanidins.

Protective effect and mechanism of procyanidin B2 against hypoxic injury of cardiomyocytes.

Background: Cardiomyocyte ischemia and hypoxia are important causes of oxidative stress damage and cardiomyocyte apoptosis in coronary heart disease (CHD). Epidemiological investigation has shown that eating more plant-based foods, such as vegetables and fruits, may significantly decrease the risk of CHD. As natural antioxidants, botanicals have fewer toxic side effects than chemical drugs and have great potential for development. Procyanidin B2 (PB2) is composed of flavan-3-ol and epicatechin and has been reported to have antioxidant and anti-inflammatory effects. However, whether PB2 exerts protective effects on hypoxic cardiomyocytes has remained unclear. This study aimed to explore the protective effect of PB2 against cardiomyocyte hypoxia and to provide new treatment strategies and ideas for myocardial ischemia and hypoxia in CHD. Methods and results: A hypoxic cardiomyocyte model was constructed, and a CCK-8 assay proved that PB2 had a protective effect on cardiomyocytes in a hypoxic environment. DCFH fluorescence staining, DHE staining, and BODIPY lipid oxidation assessment revealed that PB2 reduced the oxidative stress levels of cardiomyocytes under hypoxic conditions. TUNEL staining, Annexin V/PI fluorescence flow cytometry, and Western blot analysis of the expression of the apoptosis marker protein cleaved caspase-3 confirmed that PB2 reduced cardiomyocyte apoptosis under hypoxic conditions. JC-1 staining indicated that PB2 reduced the mitochondrial membrane potential of cardiomyocytes under hypoxia. In addition, transcriptomic analysis proved that the expression of 158 genes in cardiomyocytes was significantly changed after PB2 was added during hypoxia, of which 53 genes were upregulated and 105 genes were downregulated. GO enrichment analysis demonstrated that the activity of cytokines, extracellular matrix proteins and other molecules was changed significantly in the biological process category. KEGG enrichment analysis showed that the IL-17 signaling pathway and JAK-STAT signaling pathway underwent significant changes. We also performed metabolomic analysis and found that the levels of 51 metabolites were significantly changed after the addition of PB2 to cardiomyocytes during hypoxia. Among them, 39 metabolites exhibited increased levels, while 12 metabolites exhibited decreased levels. KEGG enrichment analysis showed that cysteine and methionine metabolism, arginine and proline metabolism and other metabolic pathways underwent remarkable changes. Conclusion: This study proves that PB2 can reduce the oxidative stress and apoptosis of cardiomyocytes during hypoxia to play a protective role. Transcriptomic and metabolomic analyses preliminarily revealed signaling pathways and metabolic pathways that are related to its protective mechanism. These findings lay a foundation for further research on the role of PB2 in the treatment of CHD and provide new ideas and new perspectives for research on PB2 in the treatment of other diseases.

Oral administration of procyanidin B2 3,3"-di-O-gallate ameliorates experimental autoimmune encephalomyelitis through immunosuppressive effects on CD4+ T cells by regulating glycolysis.

Multiple Sclerosis (MS) is a chronic autoimmune disease of the central nervous system caused by the excessive activation of T cells. Procyanidins are polyphenols that exhibit anti-inflammatory activity. Procyanidin B2 (PCB2) gallate [specifically, PCB2 3,3″-di-O-gallate (PCB2DG)] inhibits cytokine production in T cells by suppressing the acceleration of glycolysis. In this study, we determined the effect of PCB2DG on T cell-mediated autoimmune disease in vivo. We examined the immunosuppressive effects of PCB2DG using an experimental autoimmune encephalomyelitis (EAE) model, which is a classic animal model for MS. Our results indicated that the clinical score for EAE symptoms improved significantly following the oral administration of PCB2DG. This effect was associated with the suppression of T cell-mediated cytokines (e.g., IFN-γ, TNF-α, and IL-17) and infiltrating T cells into the spinal cord, which ameliorated spinal cord injury. In addition, spleen cell culture experiments revealed that the increase of T cell-mediated pro-inflammatory cytokines in EAE mice was significantly decreased following PCB2DG treatment. We further analyzed the glycolytic activity of spleen cells to identify the mechanism of the immunosuppressive effects of PCB2DG. The production of lactate and the expression of glycolytic enzymes and transporters were increased following EAE induction, but not in PCB2DG-treated EAE mice. Collectively, our results indicate that a dietary polyphenol, which has a unique structure, improves the onset of EAE symptoms and inhibits the excessive activation of T cells by influencing glycolysis.

Procyanidin B2 alleviates uterine toxicity induced by cadmium exposure in rats: The effect of oxidative stress, inflammation, and gut microbiota.

Environmental exposure to hazardous materials causes enormous socioeconomic problems due to its deleterious impacts on human beings, agriculture and animal husbandry. As an important hazardous material, cadmium can promote uterine oxidative stress and inflammation, leading to reproductive toxicity. Antioxidants have been reported to attenuate the reproductive toxicity associated with cadmium exposure. In this study, we investigated the potential protective effect of procyanidin oligosaccharide B2 (PC-B2) and gut microbiota on uterine toxicity induced by cadmium exposure in rats. The results showed that the expression levels of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were reduced in utero. Proinflammatory cytokines (including tumor necrosis factor-α, interleukin-1ß and interleukin-6), the NLRP3 inflammasome, Caspase-1 and pro-IL-1ß were all involved in inflammatory-mediated uterine injury. PC-B2 prevented CdCl2-induced oxidative stress and inflammation in uterine tissue by increasing antioxidant enzymes and reducing proinflammatory cytokines. Additionally, PC-B2 significantly reduced cadmium deposition in the uterus, possibly through its significant increase in MT1, MT2, and MT3 mRNA expression. Interestingly, PC-B2 protected the uterus from CdCl2 damage by increasing the abundance of intestinal microbiota, promoting beneficial microbiota, and inhibiting harmful microbiota. This study provides novel mechanistic insights into the toxicity of environmental cadmium exposure and indicates that PC-B2 could be used in the prevention of cadmium exposure-induced uterine toxicity.

Procyanidin B2 3,3''-di-O-gallate ameliorates imiquimod-induced skin inflammation by suppressing TLR7 signaling through the inhibition of endosomal acidification in dendritic cells.

The excessive activation of abnormal T helper 17 (Th17) cells and dendritic cells (DCs) in the dermis and epidermis causes severe inflammation of the skin. Toll-like receptor 7 (TLR7)-located in the endosomes of DCs-recognizes nucleic acids from pathogens as well as imiquimod (IMQ), which plays a crucial role in the pathogenesis of skin inflammation. Procyanidin B2 3,3''-di-O-gallate (PCB2DG), a polyphenol, has been reported to suppress the excessive production of proinflammatory cytokines from T cells. The aim of this study was to demonstrate the inhibitory effect of PCB2DG on skin inflammation and TLR7 signaling in DCs. In vivo studies showed that the clinical symptoms of dermatitis were markedly improved by the oral administration of PCB2DG in mouse dermatitis model caused by IMQ application, accompanied by the suppression of excessive cytokine secretion in the inflamed skin and spleen. In vitro, PCB2DG significantly decreased cytokine production in TLR7- or TLR9 ligand-stimulated bone marrow-derived dendritic cells (BMDCs), suggesting that PCB2DG suppresses endosomal toll-like receptors (TLR) signaling in DCs. The activity of endosomal TLRs depends on endosomal acidification, which was significantly inhibited by PCB2DG in BMDCs. The addition of cAMP, an accelerator of endosomal acidification, abrogated the inhibitory effect of cytokine production by PCB2DG. These results provide a new insight into developing functional foods, including PCB2DG, to improve the symptoms of skin inflammation through the suppression of TLR7 signaling in DCs.

Procyanidin B2 regulates the Sirt1/Nrf2 signaling pathway to improve random-pattern skin flap survival.

Random-pattern skin flaps have been widely used in the reconstruction of damaged tissues. Ischemia-reperfusion injury occurring in the distal regions of the flap is a common issue, which often leads to flap necrosis and restricts its clinical applications. Procyanidin B2 (PB2), a naturally occurring flavonoid in large quantities in various fruits, has been demonstrated to exhibit several significant pharmacological properties. However, the effect of PB2 on flap viability is not clearly known. Here, using Western blot analysis, immunohistochemistry, and immunofluorescence staining, we observed that PB2 significantly reduced oxidative stress and inflammation and enhanced angiogenesis. Mechanically, we provided evidence for the first time that the beneficial effects of PB2 occur through the activation of the Sirt1/Nrf2 signaling pathway. Moreover, co-administration of PB2 and EX527, a selective inhibitor of Sirt1, resulted in down-regulation of the expression of Sirt1, Nrf2, and downstream antioxidants. In summary, our study showed that PB2 might be a novel therapeutic strategy for improving the survival of random-pattern skin flaps.

Microwave-Assisted Extraction Optimization and Effect of Drying Temperature on Catechins, Procyanidins and Theobromine in Cocoa Beans.

Cocoa beans (Theobroma cacao L.) are an important source of polyphenols. Nevertheless, the content of these compounds is influenced by post-harvest processes. In this sense, the concentration of polyphenols can decrease by more than 50% during drying. In this study, the process of procyanidins extraction was optimized and the stability of catechins, procyanidins, and theobromine to different drying temperatures was evaluated. First, the effectiveness of methanol, ethanol, acetone, and water as extract solvents was determined. A Box-Behnken design and response surface methodology were used to optimize the Microwave-Assisted Extraction (MAE) process. The ratios of methanol-water, time, and temperature of extraction were selected as independent variables, whereas the concentration of procyanidins was used as a response variable. Concerning the drying, the samples were dried using five temperatures, and a sample freeze-dried was used as a control. The quantitative analyses were carried out by HPLC-DAD-ESI-IT-MS. The optimal MAE conditions were 67 °C, 56 min, and 73% methanol. Regarding the drying, the maximum contents of procyanidins were obtained at 40 °C. To our knowledge, this is the first time that the stability of dimers, trimers, and tetramers of procyanidins on drying temperature was evaluated. In conclusion, drying at 40 °C presented better results than the freeze-drying method.

Procyanidin B2 Attenuates Sepsis-Induced Acute Lung Injury via Regulating Hippo/Rho/PI3K/NF-κB Signaling Pathway.

Acute lung injury (ALI) is a frequent and challenging aspect of sepsis that currently lacks effective treatments. Procyanidin B2 (PB2) has anti-inflammatory and antioxidant properties. The aim of this study was to determine the effectiveness and mechanism of action of PB2 in treating sepsis-induced ALI using animal experiments. A sepsis-induced ALI mouse model was used by administering lipopolysaccharide (LPS) and then evaluating the levels of inflammatory cytokines and lung injury through measurements of cytokine levels using enzyme-linked immunosorbent assay (ELISA), Western blot and real-time PCR, as well as by the examination of relevant signaling pathways. The animal experiments showed that PB2 protected the lungs from injury caused by LPS and reduced the levels of various inflammatory cytokines in both the serum and lung tissue. Western blot analysis showed that PB2 reduced the expression of TLR4/NF-κB and increased the expression of PI3K/Akt, and also inhibited the Hippo and Rho signaling pathways. The results of the study showed that PB2 helps to treat sepsis-induced ALI by controlling cytokine storms and reducing inflammation by altering the expressions of the TLR4/NF-κB, PI3K/Akt, Hippo and Rho signaling pathways. This research provides a foundation for the further investigation of PB2's mechanism and its potential use in treating sepsis.

Mechanism of Procyanidin B2 in the Treatment of Chronic Myeloid Leukemia Based on Integrating Network Pharmacology and Molecular Docking.

OBJECTIVE: To study the pharmacological mechanism of procyanidin B2 (PCB2) on chronic myeloid leukemia (CML) by integrating network pharmacological methods systematically. METHODS: Firstly, the potential target genes of PCB2 were predicted by the pharmacological database and analysis platform (TCMSP and Pharmmapper). Meanwhile, the relevant target genes of CML were collected from GeneCards and DisGene. Pooled data were collected to screen for common target genes. Furthermore, the above intersection genes were imported into the String website to construct a protein-protein interaction (PPI) network, and the Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were further analyzed. Besides, molecular docking was performed to verify the possible binding conformation between PCB2 and candidate targets. Finally, MTT and RT-PCR experiments of K562 cells were performed to verify the above results of network pharmacology. RESULTS: A total of 229 PCB2 target genes were retrieved, among which 186 target genes had interaction with CML. The pharmacological effects of PCB2 on CML were related to some important oncogenes and signaling pathways. The top ten core targets predicted by Network Analysis were as follows: AKT1, EGFR, ESR1, CASP3, SRC, VEGFA, HIF1A, ERBB2, MTOR, and IGF1. Molecular docking studies confirmed that hydrogen bonding was the main interaction force of PCB2 binding targets. According to the molecular docking score, the following three target proteins were most likely to bind to PCB2: VEGFA (-5.5 kcal/mol), SRC (-5.1 kcal/mol), and EGFR (-4.6 kcal/mol). After treatment of PCB2 for 24h, mRNA expression levels of VEGFA and HIF1A decreased significantly in K562 cells. CONCLUSION: Through integrating network pharmacology combined with molecular docking, the study revealed the potential mechanism of PCB2 anti-chronic myeloid leukemia.

The complexes of soybean protein isolate and procyanidin B2 have synergistic hypolipidemic activity at the cellular level by activating the AMPKα pathway.

The alkali method was used to prepare soybean protein isolate (SPI) and procyanidin B2 (PCB2) complexes, and the interaction between SPI and PCB2 was studied using multi-spectroscopic methods. The human hepatoma (HepG2) cell hyperlipidemia model was used to explore whether SPI-PCB2 has the potential for synergistic hypolipidemia. According to the findings, PCB2 was primarily linked to SPI via C-S and C-N bonds, and the addition of PCB2 reduced the α-helix structure content of SPI by 4.1%. At the cellular level, the optimal SPI-PCB2 ratio for lowering blood lipids was 1:1. Compared with the model group, the TG content and TC content in the 1:1 group were reduced by 28.7% and 26.3%, respectively. Western blot analysis revealed that SPI-PCB2 = 1:1 exerted synergistic hypolipidemic activity mainly by activating adenosine monophosphate-activated protein kinase α (AMPKα) phosphorylation, inhibiting 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) and fatty acid synthetase (FAS) protein expression, and upregulating carnitine palmitoyl transferase 1A (CPT1A) protein activity.

Procyanidin B2: A promising multi-functional food-derived pigment for human diseases.

Natural edible pigments play a paramount part in the food industry. Procyanidin B2 (PB2), one of the most representative naturally occurring edible pigments, is usually isolated from the seeds, fruits, and leaves of lots of common plants, such as grapes, Hawthorn, black soybean, as well as blueberry, and functions as a food additive in daily life. Notably, PB2 has numerous bioactivities and possesses the potential to treat/prevent a wide range of human diseases, such as diabetes mellitus, diabetic complications, atherosclerosis, and non-alcoholic fatty liver disease, and the underlying mechanisms were partially elucidated, including mediating signaling pathways like NF-κB, MAPK, PI3K/Akt, apoptotic axis, and Nrf-2/HO-1. This paper presents a review of the natural sources, bioactivities, and the therapeutic/preventive potential of PB2 and the possible mechanisms, with the aim of promoting the development of PB2 as a functional food and providing references for its clinical application in the treatment of diseases.

Investigation of the chemical composition of antibacterial Psidium guajava extract and partitions against foodborne pathogens.

P. guajava was partitioned into aqueous and ethyl acetate fractions and studied for its antibacterial chemical constituents. The minimum inhibitory concentrations of the aqueous and ethyl acetate partitions against Escherichia coli, Salmonella Typhimurium, and Staphylococcus aureus were found to be 0.75, 0.75, 0.15, 0.5, 0.5, and 0.125%, respectively. Using LC-MS-based chemical fingerprinting, auto MS/MS fragmentation and bioactive molecular networking, 18 compounds of interest were detected. The top 10 bioactive compounds and eight additional non-bioactive compounds known to be found in P. guajava are highlighted. We report five compounds to be identified in P. guajava for the first time. Studies have indicated P. guajava to be a plant source of antibacterial compounds that could be useful in the food industry to prevent foodborne illnesses outbreaks, reduce food spoilage, and satisfy consumer demands for less synthetic chemical usage in the food industry.

Procyanidin B2 alleviates oxidative stress-induced nucleus pulposus cells apoptosis through upregulating Nrf2 via PI3K-Akt pathway.

Oxidative stress can lead to nucleus pulposus cell (NPC) apoptosis, which is considered to be one of the main contributors to intervertebral disc degeneration (IVDD). Procyanidin B2 is a natural antioxidant that protects against oxidative stress. However, whether procyanidin B2 protects NPCs from oxidative stress remains unknown. In this study, we demonstrated that procyanidin B2 could reduce tert-butyl hydroperoxide-induced reactive oxygen species in rat NPCs and attenuate rat NPC apoptosis. Further experiments revealed that procyanidin B2 upregulated the expression of both nuclear factor erythroid 2-related factor 2 (Nrf2) and phosphorylation of protein kinase B (Akt). We then used silencing of Nrf2 and LY294002 to silence Nrf2 expression and block the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, respectively, and found that the protective roles of procyanidin B2 in NPCs were inhibited. Therefore, we demonstrated that procyanidin B2 alleviated rat NPC apoptosis induced by oxidative stress by upregulating Nrf2 via activation of the PI3K/Akt signaling pathway. This study provides a potential therapeutic approach for procyanidin B2 in IVDD, which might help in the development of new drugs for IVDD treatment.

Procyanidin B2 3,3″-di-O-gallate suppresses IFN-γ production in murine CD4+ T cells through the regulation of glutamine influx via direct interaction with ASCT2.

Excessive activation of CD4+ T cells increases cytokine production substantially and induces immune-mediated diseases. Procyanidins are polyphenols with anti-inflammatory properties. Procyanidin B2 (PCB2) gallate [specifically, PCB2 3,3''-di-O-gallate (PCB2DG)] inhibits cytokine production through the suppression of glycolysis via mammalian target of rapamycin (mTOR) in T cells. Several amino acids play critical roles in T cell activation, especially glutamine, which is important in mTOR signaling and interferon-γ (IFN-γ) production in CD4+ T cells. However, the mechanisms underlying the effects of PCB2DG, including its interaction partners, have yet to be clarified. In the present study, the mechanisms underlying the inhibitory effect of PCB2DG on IFN-γ through glutamine metabolism regulation were investigated. We found that PCB2DG treatment reduced intracellular glutamine levels in CD4+ T cells, whereas the addition of glutamine abrogated the inhibitory effects of PCB2DG on IFN-γ production. The PCB2DG-induced reduction in intracellular glutamine accumulation led to the upregulated expression of activating transcription factor 4, which was induced by the cytoprotective signaling pathway in the amino acid response. In addition, the mRNA and protein expression levels of alanine serine cysteine transporter 2 (ASCT2), a major glutamine transporter in CD4+ T cells, were not altered by PCB2DG treatment. Further analysis using a target identification strategy revealed that PCB2DG binds to ASCT2, suggesting that PCB2DG interacts directly with this major glutamine transporter to inhibit glutamine influx. Overall, this study indicates that ASCT2 is a novel target protein of a dietary polyphenol and provides new insights into the mechanism underlying the immunomodulatory effects of polyphenols.

Procyanidin B2 Protects TR-iBRB2 Cells Against Hyperglyc emia Stress by Attenuating Oxidative Stress and Inflammasome Activation via Regulation of Redoxosomes/NF-kB Signaling.

BACKGROUND: Microvascular dysfunction is a hallmark of diabetic retinopathy (DR), which may lead to visual impairment and blindness. Procyanidin B2 (PB2) is a subclass of flavonoids and is widely known due to its anti-oxidant and antiinflammatory effects. However, little is known about the effect of PB2 on hyperglycemia stress-induced retinal microvascular dysfunction. OBJECTIVE: The purpose of this study was to investigate the effect of PB2 against hyperglycemia stress in rat retinal capillary endothelial cells (TR-iBRB2) as well as the underpinning mechanism. METHODS: Cell viability was determined using MTT assay. ROS, NOX activity analysis, Western blot analysis, and immunofluorescence analysis were applied in the study. RESULTS: The results showed that PB2 pre-treatment significantly reduced high glucose- induced cytotoxicity in TR-iBRB2 cells by suppressing oxidative stress and inflammasome activation. Mechanistical study revealed that redoxosomes were formed and activated in TR-iBRB2 cells upon hyperglycemia stress, resulting in activation of NF- κB and thus induction of oxidative stress and inflammasomes activation. However, PB2 pre-treatment dose-dependently attenuated the above events, indicating the protective effect of PB2 against hyperglycemia stress was achieved by regulating redoxosomes/ NF-kB signaling. CONCLUSION: Our findings may contribute to the potential clinical use of PB2 in treating DR and suggest redoxosomes/NF-kB signaling may be a potential therapeutic target of this disease.

Coordination of AMPK and YAP by Spatholobi Caulis and Procyanidin B2 Provides Antioxidant Effects In Vitro and In Vivo.

The liver is vulnerable to oxidative attacks from heavy metals, such as iron, as well as some drugs, including acetaminophen. It has been shown that enhanced oxidative stress in the liver leads to excessive ROS production and mitochondrial dysfunction, resulting in organ injury. The beneficial effects of Spatholobi Caulis (SC), a natural herbal medicine, include treating ischemic stroke, inhibiting tumor cell invasion, pro-angiogenic activities, and anti-inflammatory properties. Scientific studies on its effects against hepatotoxic reagents (e.g., iron and acetaminophen), as well as their underlying mechanisms, are insufficient. This study examined the antioxidant effects and mechanisms of SC in vitro and in vivo. In cells, the proinflammatory mediator, arachidonic acid (AA), plus iron, significantly induced an increase in ROS generation, the damage in mitochondrial membrane potential, and the resulting apoptosis, which were markedly blocked by SC. More importantly, SC affected the activation of AMP-activated protein kinase (AMPK)-related proteins, which were vital to regulating oxidative stress in cells. In addition, SC mediated the expression of Yes-associated protein (YAP)-related proteins. Among the active compounds in SC, the procyanidin B2, but not liquiritigenin, daidzein, and genistein, significantly inhibited the cytotoxicity induced by AA + iron, and activated the LKB1-AMPK pathway. In mice, the oral administration of SC alleviated the elevations of ALT and histological changes by the acetaminophen-induced liver injury. These results reveal the potential of SC and a key bioactive component, procyanidin B2, as antioxidant candidates for hepatoprotection.