Antioxidant effects of proanthocyanidin-rich natural extracts from grape seed and cupuassu on gastrointestinal mucosa.

BACKGROUND: The gastrointestinal tract (GI) is constantly exposed to reactive species released by the GI tract itself, and those present in food and beverages. Phenolic compounds may help in protecting the GI tract against damage produced by the reactive species. In this paper we have analyzed the effects of a grape seed proanthocyanidin extract (GSPE) on reactive oxygen species (ROS) production in two different intestinal cell types: the absorptive cell line Caco-2 and the enteroendocrine cell line STC-1. RESULTS: We show that GSPE prevents tert-butylhydroperoxide-induced oxidative stress in both cell lines, and that the effects are dose and time dependent. We have also analyzed whether GSPE has any in vivo effect, and found that 25 mg kg(-1) body weight cannot counteract the increase in intestinal ROS induced by the cafeteria diet. However, an acute (1 h) treatment of 1 g GSPE kg(-1) body weight reduced ROS in fasted animals and also decreased ROS induction by food. These effects were found only after a short-term treatment. Furthermore, we have compared the in vitro GSPE effects with those of another proanthocyanidin-rich extract from cupuassu seeds, though it has compounds with different structures. Cupuassu extract also shows antioxidant effects in both cell types, which suggests different mechanisms from those of GSPE. CONCLUSION: Natural proanthocyanidin-rich extracts have an antioxidant effect in the GI tract, acting on absorptive cells and enterohormone-secreting cells, although the effects depend on the dose and period of treatment. © 2015 Society of Chemical Industry.

Gallic acid is an active component for the anticarcinogenic action of grape seed procyanidins in pancreatic cancer cells.

The aim of the present work was to evaluate the effects of a grape seed procyanidin extract (GSPE) on proliferation and apoptosis in the pancreatic adenocarcinoma cell line MIA PaCa-2 and identify the components of the extract with higher activity. The effects of the extract were analyzed on the proliferation and apoptosis processes in MIA PaCa-2 cells, as well as in the levels of the apoptosis markers Bcl-2 and Bax, the mitochondrial membrane potential, and reactive oxygen species levels. Finally, the components of the extract with higher effects were elucidated using enriched fractions of the extract and pure compounds. The results showed that GSPE inhibits cell proliferation and increases apoptosis in MIA PaCa-2 cells, which is primarily mediated by the downregulation of the antiapoptotic protein Bcl-2 and the depolarization of the mitochondrial membrane. GSPE also reduced the formation of reactive oxygen species. The component of the extract that possesses the highest antiproliferative and proapoptotic activity was gallic acid. In conclusion, GSPE acts as anticarcinogenic in MIA PaCa-2 cells, with gallic acid as the major single active constituent of the extract.

Effect of the co-occurring olive oil and thyme extracts on the phenolic bioaccessibility and bioavailability assessed by in vitro digestion and cell models.

Olive oils flavoured with edible herbs have grown in popularity because of their added value and potential health benefits. However, the combined presence of different phytochemicals from olive oil and herbs requires study of their possible interactions during intestinal transport and metabolism. The aim of this study was firstly to evaluate the effect on bioaccessibility of the co-occurring bioactive compounds from olive oil and thyme through an in vitro digestion model of three extracts: olive extract (OE), thyme extract (TE) and a combination of both (OTE). The bioaccessible fractions were exposed to Caco-2 and HepG-2 cell models, as well as to a co-culture of both of these. Results indicated that the bioaccessibility of hydroxytyrosol was enhanced when OTE was digested. After Caco-2 cells exposure, no significant differences were observed in hydroxytyrosol transport, whereas the main flavonoids from thyme seemed to undergo an enhanced basolateral permeation when both phenolic sources where exposed.

Grape seed procyanidin extract reduces the endotoxic effects induced by lipopolysaccharide in rats.

Acute inflammation is a response to injury, infection, tissue damage, or shock. Bacterial lipopolysaccharide (LPS) is an endotoxin implicated in triggering sepsis and septic shock, and LPS promotes the inflammatory response, resulting in the secretion of proinflammatory and anti-inflammatory cytokines such as the interleukins (IL-6, IL-1ß, and IL-10) and tumor necrosis factor-α by the immune cells. Furthermore, nitric oxide and reactive oxygen species levels increase rapidly, which is partially due to the activation of inducible nitric oxide synthase in several tissues in response to inflammatory stimuli. Previous studies have shown that procyanidins, polyphenols present in foods such as apples, grapes, cocoa, and berries, have several beneficial properties against inflammation and oxidative stress using several in vitro and in vivo models. In this study, the anti-inflammatory and antioxidant effects of two physiological doses and two pharmaceutical doses of grape seed procyanidin extract (GSPE) were analyzed using a rat model of septic shock by the intraperitoneal injection of LPS derived from Escherichia coli. The high nutritional (75mg/kg/day) and the high pharmacological doses (200mg/kg/day) of GSPE showed anti-inflammatory effects by decreasing the proinflammatory marker NOx in the plasma, red blood cells, spleen, and liver. Moreover, the high pharmacological dose also downregulated the genes Il-6 and iNos; and the high nutritional dose decreased the glutathione ratio (GSSG/total glutathione), further illustrating the antioxidant capability of GSPE. In conclusion, several doses of GSPE can alleviate acute inflammation triggered by LPS in rats at the systemic and local levels when administered for as few as 15 days before the injection of endotoxin.

Grape seed procyanidins improve ß-cell functionality under lipotoxic conditions due to their lipid-lowering effect.

Procyanidins have positive effects on glucose metabolism in conditions involving slightly disrupted glucose homeostasis, but it is not clear how procyanidins interact with ß-cells. In this work, we evaluate the effects of procyanidins on ß-cell functionality under an insulin-resistance condition. After 13 weeks of cafeteria diet, female Wistar rats were treated with 25 mg of grape seed procyanidin extract (GSPE)/kg of body weight (BW) for 30 days. To determine the possible mechanisms of action of procyanidins, INS-1E cells were separately incubated in high-glucose, high-insulin and high-oleate media to reproduce the conditions the ß-cells were subjected to during the cafeteria diet feeding. In vivo experiments showed that chronic GSPE treatment decreased insulin production, since C-peptide levels and insulin protein levels in plasma were lower than those of cafeteria-fed rats, as were insulin and Pdx1 mRNA levels in the pancreas. GSPE effects observed in vivo were reproduced in INS-1E cells cultured with high oleate for 3 days. GSPE treatment significantly reduces triglyceride content in ß-cells treated with high oleate and in the pancreas of cafeteria-fed rats. Moreover, gene expression analysis of the pancreas of cafeteria-fed rats revealed that procyanidins up-regulated the expression of Cpt1a and down-regulated the expression of lipid synthesis-related genes such as Fasn and Srebf1. Procyanidin treatment counteracted the decrease of AMPK protein levels after cafeteria treatment. Procyanidins cause a lack of triglyceride accumulation in ß-cells. This counteracts its negative effects on insulin production, allowing for healthy levels of insulin production under hyperlipidemic conditions.

Grape seed procyanidin extract modulates proliferation and apoptosis of pancreatic beta-cells.

Grape seed procyanidin extract (GSPE) modulates glucose homeostasis and insulinemia in several animal models. Under pathological conditions, insulin levels are dependent on pancreatic beta-cell functionality, as well as on the beta-cell mass expansion or apoptosis in the pancreas. In this study, we analysed the effects of GSPE on modulating apoptosis and proliferation in beta-cells. We tested the effects of GSPE in the INS-1E pancreatic beta-cell line, either under basal or altered conditions with high glucose, insulin or palmitate levels. GSPE enhanced the pro-apoptotic effect of high glucose and showed clear antiproliferative effects under high glucose, insulin and palmitate conditions. These antiproliferative effects are likely due to high molecular weight compounds contained in the extract. GSPE also modulated pro- and anti-apoptotic markers in the pancreas of rats fed a cafeteria diet, with the effect depending on the dose of GSPE and duration of treatment. Thus, GSPE is able to modulate apoptosis and proliferation of beta-cells under altered, but not basal, conditions.

Procyanidins modulate microRNA expression in pancreatic islets.

Procyanidins modulate glucose metabolism, partly due to its effects on pancreas. Given the role of microRNAs (miRNAs) in the regulation of diabetes and the fact that flavonoids modulate miRNAs in other tissues, we hypothesized that procyanidins might target miRNAs in the pancreas. We investigated the miRNA expression profile in pancreatic islets isolated from rats treated with a daily dose of grape seed procyanidin extract (GSPE) (25 mg/kg of body weight) for 45 days. The miRWalk database identified putative target genes of these miRNAs. We found that GSPE altered significantly the expression of miR-1249, miR-483, miR-30c-1*, and miR-3544. In silico prediction studies suggested that ion transport and response to glucose are among the regulated pathways. As a conclusion, this is the first study showing that procyanidins can also exert their bioactivity on pancreatic islets by modifying the miRNA expression pattern.

Grape Seed Procyanidin Extract Improves Insulin Production but Enhances Bax Protein Expression in Cafeteria-Treated Male Rats.

In a previous study, the administration of a grape seed procyanidin extract (GSPE) in female Wistar rats improved insulin resistance, reduced insulin production, and modulated apoptosis biomarkers in the pancreas. Considering that pharmacokinetic and pharmacodynamic parameters in females are different from these parameters in males, the aim of the present study was to evaluate the effects of GSPE on male Wistar cafeteria-induced obese rats. The results have confirmed that the cafeteria model is a robust model mimicking a prediabetic state, as these rats display insulin resistance, increased insulin synthesis and secretion, and increased apoptosis in the pancreas. In addition, GSPE treatment (25 mg/kg of GSPE for 21 days) in male rats improves insulin resistance and counteracts the cafeteria-induced effects on insulin synthesis. However, the administration of the extract enhances the cafeteria-induced increase in Bax protein levels, suggesting increased apoptosis. This result contradicts previous results from cafeteria-fed female rats, in which GSPE seemed to counteract the increased apoptosis induced by the cafeteria diet.

Identification of PPARgamma partial agonists of natural origin (I): development of a virtual screening procedure and in vitro validation.

BACKGROUND: Although there are successful examples of the discovery of new PPARγ agonists, it has recently been of great interest to identify new PPARγ partial agonists that do not present the adverse side effects caused by PPARγ full agonists. Consequently, the goal of this work was to design, apply and validate a virtual screening workflow to identify novel PPARγ partial agonists among natural products. METHODOLOGY/PRINCIPAL FINDINGS: We have developed a virtual screening procedure based on structure-based pharmacophore construction, protein-ligand docking and electrostatic/shape similarity to discover novel scaffolds of PPARγ partial agonists. From an initial set of 89,165 natural products and natural product derivatives, 135 compounds were identified as potential PPARγ partial agonists with good ADME properties. Ten compounds that represent ten new chemical scaffolds for PPARγ partial agonists were selected for in vitro biological testing, but two of them were not assayed due to solubility problems. Five out of the remaining eight compounds were confirmed as PPARγ partial agonists: they bind to PPARγ, do not or only moderately stimulate the transactivation activity of PPARγ, do not induce adipogenesis of preadipocyte cells and stimulate the insulin-induced glucose uptake of adipocytes. CONCLUSIONS/SIGNIFICANCE: We have demonstrated that our virtual screening protocol was successful in identifying novel scaffolds for PPARγ partial agonists.

Pancreatic islet proteome profile in Zucker fatty rats chronically treated with a grape seed procyanidin extract.

Grape seed procyanidin extract (GSPE) has been reported to modify glucose metabolism and ß-cell functionality through its lipid-lowering effects in a diet-induced obesity model. The objective of the present study was to evaluate the effects of chronically administrated GSPE on the proteomic profile of pancreatic islets from Zucker fatty (ZF) rats. An isobaric tag for relative and absolute quantitation (iTRAQ) experiment was conducted and 31 proteins were found to be differentially expressed in ZF rats treated with GSPE compared to untreated ZF rats. Of these proteins, five subcategories of biological processes emerged: hexose metabolic processes, response to hormone stimulus, apoptosis and cell death, translation and protein folding, and macromolecular complex assembly. Gene expression analysis supported the role of the first three biological processes, concluding that GSPE limits insulin synthesis and secretion and modulates factors involved in apoptosis, but these molecular changes are not sufficient to counteract the genetic background of the Zucker model at a physiological level.

Procyanidins modify insulinemia by affecting insulin production and degradation.

Previous studies from our research group have suggested that procyanidins modify glycemia and insulinemia. The aim of this work was to evaluate the effects of procyanidins on ß-cell functionality in a nonpathological system. Four groups of healthy rats were studied. The animals were given daily acute doses of grape seed procyanidin extract (GSPE) for different time periods and at different daily amounts. A ß-cell line (INS-1E) was treated with 25 mg GSPE/L for 24 h to identify possible mechanisms of action for the procyanidins. In vivo experiments showed that different doses of GSPE affected insulinemia in different ways by modifying ß-cell functionality and/or insulin degradation. The islets isolated from rats that were treated with 25 mg GSPE/kg of body weight for 45 days exhibited a limited response to glucose stimulation. In addition, insulin gene expression, insulin synthesis and expression of genes related to insulin secretion were all down-regulated. In vitro studies revealed that GSPE decreased the ability of ß-cells to secrete insulin in response to glucose. GSPE increased glucose uptake in ß-cells under high-glucose conditions but impaired glucose-induced mitochondrial hyperpolarization, decreased adenosine triphosphate (ATP) synthesis and altered cellular membrane potentials. GSPE also modified Glut2, glucokinase and Ucp2 gene expression as well as altered the expression of hepatic insulin-degrading enzyme (Ide), thereby altering insulin degradation. At some doses, procyanidins changed ß-cell functionality by modifying insulin synthesis, secretion and degradation under nonpathological conditions. Membrane potentials and Ide provide putative targets for procyanidins to induce these effects.

Enhanced anti-inflammatory effect of resveratrol and EPA in treated endotoxin-activated RAW 264.7 macrophages.

Macrophages play an important role in immunogenic challenges by producing reactive oxygen species, NO and proinflammatory cytokines that can aggravate and propagate local inflammation. Multiple mechanisms regulate these inflammatory processes. NF-κB and activator protein 1 pathways are crucial in the expression of proinflammatory genes, such as TNF-α, IL-1 (α or ß) and -6. Some polyphenols, which are present in beverages, vegetables and fruits, and PUFA, which are present in marine oils and fish food, possess anti-inflammatory effects in vivo and in vitro. Our aim in the present study was to assess whether polyphenols and PUFA have synergistic anti-inflammatory effects in murine macrophages in vitro. Inflammation in RAW 264.7 macrophages was induced by lipopolysaccharide at 100 ng/ml. The treatments with molecules were performed by co-incubation for 19 h. A NO production assay by Griess reaction, a phosphoprotein assay by Pathscan ELISA kit and gene expression analysis using the TaqMan® Low-density Array for ninety-one genes related to inflammation, oxidative stress and metabolism were performed to assess the synergistic anti-inflammatory effects of polyphenols, epigallocatechin gallate and resveratrol (Res; 2·5 µg/ml), and the PUFA, DHA and EPA (30 µm). Adding Res+EPA had an enhanced anti-inflammatory effect, in comparison with EPA and Res alone, leading to decreased NO levels; modulating the phospho-stress activated protein kinase/Jun N-terminal kinase (P-SAPK/JNK) level; down-regulating proinflammatory genes, such as IL, chemokines, transcription factors; and up-regulating several antioxidant genes. Therefore, this combination has a stronger anti-inflammatory effect than either of these molecules separately in RAW macrophages.

Additive, antagonistic, and synergistic effects of procyanidins and polyunsaturated fatty acids over inflammation in RAW 264.7 macrophages activated by lipopolysaccharide.

OBJECTIVE: Macrophages play an important role in immunogenic challenges and can aggravate and propagate local inflammation. Nuclear factor-kappa B (NF-κB) and activator protein 1 pathways can regulate these inflammatory processes by modulating expression of proinflammatory genes. Bioactive molecules present in food, such as procyanidins and polyunsaturated fatty acids, possess antiinflammatory effects in vivo and in vitro. Our aim was to assess whether they have synergistic antiinflammatory effects in murine macrophages. METHODS: A nitric oxide production assay, a phosphoprotein assay, and a low-density array for 91-gene expression related to inflammation, oxidative stress, and metabolism were performed to assess the synergistic antiinflammatory effects of dimeric procyanidins (B1, B2, B3, B4) (5 µg/mL), and the polyunsaturated fatty acids, docosahexaenoic acid, and eicosapentaenoic acid (30 µM) coincubated with lipopolysaccharide for 19 h to mimic inflammation in RAW 264.7 macrophages (mouse leukaemic monocyte macrophage cell line). RESULTS: Adding eicosapentaenoic acid plus B3 had synergistic effects leading to decreased nitric oxide levels; the modulation of phosphoprotein levels, such as P-nuclear factor-[kappa] B p65 and P-stress-activated protein kinase/Jun-amino-terminal kinase; the down-regulation of proinflammatory genes, such as interleukins, chemokines, transcription factors; and up-regulation of antioxidant genes. CONCLUSION: This combination has a stronger antiinflammatory effect than either of these molecules separately in RAW macrophages. These results could lead to in vivo studies that may yield novel preventive or palliative nutritional treatments for obesity, atherosclerosis, and cardiovascular diseases.

The effects of a cafeteria diet on insulin production and clearance in rats.

The aim of the present study was to determine the effects of a cafeteria diet on the function and apoptosis of the pancreas, and the activity and expression of the insulin-degrading enzyme (IDE). Female Wistar rats were fed either with a cafeteria diet or a control diet for 17 weeks, and blood and tissues were then collected for analysis. The cafeteria diet-treated rats had higher plasma insulin and C-peptide levels (P<0·05), showing increased insulin secretion by the pancreas. Insulin protein and gene expression levels were higher in the pancreas of obese rats, as was its transcriptional controller, pancreatic duodenal homeobox 1 (P<0·05). Feeding a cafeteria diet down-regulated the gene expression of the anti-apoptotic marker B-cell/lymphoma 2 (BCL2), and up-regulated the protein levels of BCL2-associated X protein, a pro-apoptotic marker (P<0·05). The cafeteria diet caused lipid accumulation in the pancreas and modified the expression of key genes that control lipid metabolism. To assay whether insulin clearance was also modified, we checked the activity of the IDE, one of the enzymes responsible for insulin clearance. We found increased liver IDE activity (P<0·05) in the cafeteria diet-fed animals, which could, in part, be due to an up-regulation of its gene expression. Conversely, IDE gene expression was unmodified in the kidney and adipose tissue; although when the adipose tissue weight was considered, the insulin clearance potential was higher in the cafeteria diet-treated rats. In conclusion, treatment with a cafeteria diet for 17 weeks in rats mimicked a pre-diabetic state, with ectopic lipid accumulation in the pancreas, and increased the IDE-mediated insulin clearance capability.

Development of a coculture system to evaluate the bioactivity of plant extracts on pancreatic ß-cells.

Natural plant extracts are candidates for the development of new functional foods. Most of them are usually complex mixtures of molecules of uncertain bioavailability that are often partially metabolized before they finally reach the target cells IN VIVO. IN VITRO studies of the bioactivity of these extracts suggest that their direct application to some cell cultures might be a long way from becoming a reality. To overcome this limitation, we seeded Caco-2 cells onto culture inserts and after 21 days, cocultured these with INS-1E on the base of the well. After 24 hours of coculture, TEER (transepithelium electrical resistance) measurements indicated no changes in the permeability of the Caco-2 barrier. We also found no changes in either the ability of Caco-2 cells to metabolize the flavan-3-ol component of a grape-seed procyanidin-rich extract, or in the flavanols' ability to pass through the barrier. However, the expression of the Caco-2 SGLT-1 gene increased due to the coculture. GSIS (glucose stimulated insulin secretion) was maintained in the INS-1E cells with higher levels of insulin secretion despite the fact that the insulin gene expression was unmodified by the cocultivation. Furthermore, we found that in some of the assays requiring several medium changes there was a tendency to lose ß-cells. Neutral red assay showed that seeded cells should only be cocultured for a short time to obtain a higher consistency. In conclusion, four hours coculture with Caco-2 cells and INS-1E is a suitable method for checking the bioactivity of natural plant extracts of unknown bioavailability on ß-cells.