The Effect of Simulated In Vitro Digestion on Biological Activity of Viburnum opulus Fruit Juices.

In the present study, an in vitro digestion method has been used to assay the influence of the physiological conditions in the mouth, stomach, and intestine on the stability and activity in different cell models of the main phenolic compounds from Viburnum opulus fresh juice (FJ), phenolic-rich juice (PJ), and the bioavailable fractions (DFJ and DPJ). The data obtained indicate that the V. opulus samples achieved after in vitro digestion had an influence on cellular glucose and lipid metabolism. The bioavailable fraction of both digested juices stimulated glucose uptake and decreased lipid accumulation by L6 myoblasts and HepG2 hepatocytes. Both DFJ and DPJ reduced the secretion of inflammatory cytokines by 3T3-L1 adipocytes: interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Simultaneously, DFJ and DPJ enhanced oxidative stress in MIN6 cells and decreased glucose-stimulated insulin secretion (GSIS). UPLC-MS analysis revealed qualitative and quantitative changes in hydroxycinnamic acids. In particular, the content of chlorogenic acid decreased drastically; its content in the bioavailable fraction was almost 7 times and 30 times lower than in the FJ and PJ, respectively. Our results suggested that although the phenolic compounds of V. opulus juices undergo transformation during digestion, they are still potent antioxidant agents with biological activity.

A new approach for the assessment of the toxicity of polyphenol-rich compounds with the use of high content screening analysis.

The toxicity of in vitro tested compounds is usually evaluated based on AC50 values calculated from dose-response curves. However, there is a large group of compounds for which a standard four-parametric sigmoid curve fitting may be inappropriate for estimating AC50. In the present study, 22 polyphenol-rich compounds were prioritized from the least to the most toxic based on the total area under and over the dose-response curves (AUOC) in relation to baselines. The studied compounds were ranked across three key cell indicators (mitochondrial membrane potential, cell membrane integrity and nuclear size) in a panel of five cell lines (HepG2, Caco-2, A549, HMEC-1, and 3T3), using a high-content screening (HCS) assay. Regarding AUOC score values, naringin (negative control) was the least toxic phenolic compound. Aronox, spent hop extract and kale leaf extract had very low cytotoxicity with regard to mitochondrial membrane potential and cell membrane integrity, as well as nuclear morphology (nuclear area). Kaempferol (positive control) exerted strong cytotoxic effects on the mitochondrial and nuclear compartments. Extracts from buckthorn bark, walnut husk and hollyhock flower were highly cytotoxic with regard to the mitochondrion and cell membrane, but not the nucleus. We propose an alternative algorithm for the screening of a large number of agents and for identifying those with adverse cellular effects at an early stage of drug discovery, using high content screening analysis. This approach should be recommended for series of compounds producing a non-sigmoidal cell response, and for agents with unknown toxicity or mechanisms of action.

The immunomodulatory potential of Leonurus cardiaca extract in relation to endothelial cells and platelets.

The immunomodulatory activity of Leonurus cardiaca L. polyphenol-rich extract (LCE) was tested in vitro on HUVECs to explore its potential therapeutic usefulness in the treatment of inflammatory lesions. The phytochemical composition of LCE, its antioxidant and cytotoxic activity, and the influence of LCE on NO and platelet-activating factor (PAF) secretion by HUVECs and platelet aggregation were all assessed. Total polyphenol contents in LCE reached 137.0 ± 0.8 mg/g, with hydroxycinnamic acid derivatives as the predominant phenolic compounds. LCE expressed antioxidant capacity, which was, however, 13- to 16-fold lower than the antioxidant activity of ascorbic acid. The plant extract was not cytotoxic up to a concentration 4500 µg/ml and did not exhibit proapoptotic activity. LCE significantly increased NO production in HUVECs in a concentration-dependent manner and led to the inhibition of PAF secretion induced by staphylococcal peptidoglycan. The extract used at the concentration of 100 µg/ml significantly reduced platelet aggregation in the presence of arachidonic acid. We provide in vitro data demonstrating the immunomodulatory potential of LCE, which may be beneficial in preventing the development of difficult-to-treat inflammatory lesions within chronically infected tissues.

Effects of Fruit Extracts on Pancreatic Lipase Activity in Lipid Emulsions.

Pancreatic lipase is the most important enzyme in digestion of triglycerides. Reduction of energy intake from dietary fat through inhibition of this enzyme may be a strategy to prevent and treat obesity. In this study, the effect of 31 fruit extracts on pancreatic lipase was investigated using triolein, sunflower oil and rapeseed oil emulsions. Surprisingly, about 30 % of the fruits tested stimulated pancreatic lipase activity in oil emulsions by over 50 %. Only six fruit extracts were found to inhibit pancreatic lipase activity with the IC(50) value varying from 21.11 to 266.48 mg of fruit equivalent/ml of emulsion. Among them, chokeberry demonstrated the highest anti-lipase activity. The inhibitory activity ranks were comparable in all lipid emulsion models and suggest that consumption of chokeberry, red gooseberry and red currant fruits may be a new dietary option for reduction of fat absorption via inhibition of pancreatic lipase.

Antidiabetic effect of polyphenolic extracts from selected edible plants as α-amylase, α -glucosidase and PTP1B inhibitors, and ß pancreatic cells cytoprotective agents - a comparative study.

Type 2 diabetes mellitus, which is usually a result of wrong dietary habits and reduced physical activity, represents 85-95% of all diabetes cases and among other diet related diseases is the major cause of deaths. The disease is characterized mainly by hyperglycemia, which is associated with attenuated insulin sensitivity or beta cells dysfunction caused by multiple stimuli, including oxidative stress and loss of insulin secretion. Since polyphenols possess multiple biological activities and constitute an important part of the human diet, they have recently emerged as critical phytochemicals in type 2 diabetes prevention and treatment. Their hypoglycemic action results from their antioxidative effect involved in recovering of altered antioxidant defenses and restoring insulin secreting machinery in pancreatic cells, or abilities to inhibit the activity of carbohydrates hydrolyzing enzymes (α-amylase and α-glucosidase) or protein tyrosine phosphatase 1B (PTP1B), which is known as the major negative regulator in insulin signaling. This study investigates the total phenolic content (Folin-Ciocalteu and HPLC methods) and antioxidant capacity (ABTS) of 20 polyphenolic extracts obtained from selected edible plants, which were screened in terms of α -amylase, α - glucosidase and protein tyrosine phosphatase 1B inhibitors or protective agents against oxidative stress induced by tertbutylhydroperoxide (t-BOOH) in ßTC3 pancreatic beta cells used as a model target for antidiabetes drugs. The study concludes that Chaenomeles japonica, Oenothera paradoxa and Viburnum opulus may be promising natural sources for active compounds with antidiabetic properties.

Extract from Ribes nigrum leaves in vitro activates nitric oxide synthase (eNOS) and increases CD39 expression in human endothelial cells

The aim of the present study was to evaluate whether blackcurrant leaf extract (BLE) modulates endothelium antithrombotic function, namely increases the expression/activity of ADPase (CD39) and augments the production of nitric oxide in human umbilical vein endothelial cells (HUVEC). It was found that BLE with proanthocyanidins (60 % of the total polyphenol content) increased the CD39-positive endothelial cell fraction (up to 10 % for 2.5 μg/ml, and up to 33 % for 15 μg/ml, p < 0.05 or less) in a concentration-dependent manner, and enhanced endothelial nitric oxide synthase (eNOS) activation (T495 phosphorylation decreased by 31 ± 6 % for 2.5 μg/ml and 48 ± 6 % for 15 μg/ml; S1177 phosphorylation increased by 13 ± 3 % for 2.5 μg/ml and 18 ± 7 % for 15 μg/ml, compared to untreated cells, p < 0.05 or less). Additionally, incubation for 24 or 48 h with BLE at a lower range of polyphenol concentrations, significantly increased cell viability with a maximal effect at 2.5 μg/ml (viability increased by 24.8 ± 1.0 % for 24 h and by 32.5 ± 2.7 % for 48-h time incubation, p < 0.0001). The increased CD39 expression and the increased eNOS activation in HUVEC can be regarded as the beneficial markers of the improvement of antiplatelet action of endothelial cells. Unexpectedly, these assumptions were not confirmed in the experimental model of platelet-endothelial cell interactions. These observations lead to the conclusion that BLE may improve endothelial cell viability at low physiological concentrations without affecting the antiplatelet action of endothelium

Extract from Ribes nigrum leaves in vitro activates nitric oxide synthase (eNOS) and increases CD39 expression in human endothelial cells.

The aim of the present study was to evaluate whether blackcurrant leaf extract (BLE) modulates endothelium antithrombotic function, namely increases the expression/activity of ADPase (CD39) and augments the production of nitric oxide in human umbilical vein endothelial cells (HUVEC). It was found that BLE with proanthocyanidins (60 % of the total polyphenol content) increased the CD39-positive endothelial cell fraction (up to 10 % for 2.5 µg/ml, and up to 33 % for 15 µg/ml, p < 0.05 or less) in a concentration-dependent manner, and enhanced endothelial nitric oxide synthase (eNOS) activation (T495 phosphorylation decreased by 31 ± 6 % for 2.5 µg/ml and 48 ± 6 % for 15 µg/ml; S1177 phosphorylation increased by 13 ± 3 % for 2.5 µg/ml and 18 ± 7 % for 15 µg/ml, compared to untreated cells, p < 0.05 or less). Additionally, incubation for 24 or 48 h with BLE at a lower range of polyphenol concentrations, significantly increased cell viability with a maximal effect at 2.5 µg/ml (viability increased by 24.8 ± 1.0 % for 24 h and by 32.5 ± 2.7 % for 48-h time incubation, p < 0.0001). The increased CD39 expression and the increased eNOS activation in HUVEC can be regarded as the beneficial markers of the improvement of antiplatelet action of endothelial cells. Unexpectedly, these assumptions were not confirmed in the experimental model of platelet-endothelial cell interactions. These observations lead to the conclusion that BLE may improve endothelial cell viability at low physiological concentrations without affecting the antiplatelet action of endothelium.

Leonurus cardiaca L. herb--a derived extract and an ursolic acid as the factors affecting the adhesion capacity of Staphylococcus aureus in the context of infective endocarditis.

The objective was an assessment of the impact of Leonurus cardiaca L. extract (LCE) and ursolic acid (UA) on the adhesive properties of Staphylococus aureus NCTC 8325 strain, expressing virulence factors important in the pathogenesis of infective endocarditis. The adhesion and biofilm formation of bacteria cultured in the presence of subinhibitory concentrations of LCE or UA on the abiotic surface or covered with fibrinogen, fibronectin or collagen, were evaluated. Inhibitory effects of LCE and UA on staphylococcal adherence to both types of surface were demonstrated. This, in the case of UA, resulted in a significant reduction of biofilm formation.

In vitro inhibitory effect on digestive enzymes and antioxidant potential of commonly consumed fruits.

Dietary inhibitors of fats and carbohydrates degrading enzymes can reduce obesity and type 2 diabetes. In this study, we screened crude extracts from 30 commonly consumed fruits to test their in vitro inhibitory effect against key enzymes relevant for obesity (pancreatic lipase) and type 2 diabetes (α-glucosidase and α-amylase), total phenolic content (Folin-Ciocalteu method), and antioxidant capacity (ABTS and FRAP). The IC50 values of the fruits tested varied from 39.91 to >400 mg/mL, from 1.04 to >80 mg/mL, and from 0.72 to 135.07 mg/mL against α-glucosidase, α-amylase, and pancreatic lipase, respectively. Antioxidant capacity ranged from 0.66 to 124.66 µmol of TE/g of fruit and strongly correlated with phenolic content, while the enzyme inhibition was poorly correlated with total phenolic and antioxidant capacity. Among fruits tested, blue honeysuckle and red gooseberry exhibited the highest inhibitory activity with respect to the carbohydrate degrading enzymes, while lingonberry had the strongest anti-lipase activity.

Vaccinium myrtillus leaves and Frangula alnus bark derived extracts as potential antistaphylococcal agents.

Due to constantly increasing antibiotic resistance of pathogens and participation of the biofilms they make in various types of infections, a development of alternative therapeutic strategies becomes an urgent need. Taking advantage of the biological activity of plant-derived compounds can solve this problem. In this study antimicrobial, including those synergistic with classic antibiotics, and cytotoxic properties of newly-obtained extracts from Vaccinium myrtillus leaves (VLE) and Frangula alnus bark (FBE) were evaluated. Both tested extracts exhibited relevant antistaphylococcal activity (MIC range 0.75-1.5 mg/mL) accompanied by a relativly low cytotoxic effect on mammalian cells (BI > 1). Phytochemical analysis of the extracts tested showed a high total content of phenolic compounds with the predominance of hydroxycinnamic acids in VLE and hydroxybenzoic acids and flavanols in FBE. Widely described in the literature antimicrobial properties of phenolics were probably connected with the biological activity of the extracts tested. We also report that the presence of VLE or FBE at sub-MIC concentrations enhances biocidal potential of vancomycin and linezolid. Therefore, we are considering a possibility of an alternative therapy for local infections caused by S. aureus by combining classic antibiotics with plant-derived extracts.

Matrix effects on the stability and antioxidant activity of red cabbage anthocyanins under simulated gastrointestinal digestion.

Red cabbage is, among different vegetables, one of the major sources of anthocyanins. In the present study an in vitro digestion method has been used to assay the influence of the physiological conditions in the stomach and small intestine, as well as faecal microflora on anthocyanins stability in red cabbage and anthocyanin-rich extract. The recovery of anthocyanins during in vitro gastrointestinal digestion was strongly influenced by food matrix. The results showed that other constituents present in cabbage enhanced the stability of anthocyanins during the digestion. The amount of anthocyanins (HPLC method) and antioxidant capacity (ABTS and FRAP assays) strongly decreased after pancreatic-bile digestion in both matrices but total phenolics content (Folin-Ciocalteu assay) in these digestions was higher than in initial samples. Incubation with human faecal microflora caused further decline in anthocyanins content. The results obtained suggest that intact anthocyanins in gastric and products of their decomposition in small and large intestine may be mainly responsible for the antioxidant activity and other physiological effects after consumption of red cabbage.

Influence of the toothpaste with brazilian ethanol extract propolis on the oral cavity health.

Propolis-based therapeutic agents represent this potential for the development of new drugs in dental care. The aim of a clinical-cohort study was to determine the influence of application of toothpaste enriched with Brazilian extract of propolis (EEP) on health status of oral cavity. Laboratory analysis was conducted in order to assess the chemical composition of EEP including total phenolic compounds, the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity, ABTS radical cation scavenging activity, and FRAP assay. Clinical research involved two groups of subjects comprising 32 adult patients, with assessment based on the preliminary evaluation of the state of their marginal periodontium. The investigation of oral health indices API, OHI, and SBI and microbiological examination of oral microflora were also carried out. Results obtained indicated time-dependent microbial action of EEP at 50 mg/L concentration, with antimicrobial activity against Gram-positive bacteria. The total decrease of API, OHI, and SBI mean values was observed. Hygienic preparations with 3% content of Brazilian ethanol extract of green propolis (EEP) efficiently support removal of dental plaque and improve the state of marginal periodontium.