Effects of In Vitro Digestion of Polyphenols from Coffee on Binding Parameters to Human Topoisomerase II α.

Type II topoisomerase (TOPII) is an enzyme that influences the topology of DNA. DNA breaks generated by TOPII may result in mutagenic or cytotoxic changes in cancer cells. In this study, we characterized interactions of TOPIIα with coffee extracts and individual chlorogenic acids (CHAs) from the extracts by performing isothermal titration calorimetry (ITC) and molecular docking (MD) simulations. The study showed that the highest affinity to TOPIIα was found in green coffee (ΔG = -38.23 kJ/mol) and monochlorogenic acids fraction of coffee extracts (ΔG = -35.80 kJ/mol), resulting from the high content of polyphenols, such as CHAs, which can bind to the enzyme in the active site. Coffee extracts and their fractions maintained a high affinity for TOPIIα after simulated digestion in the presence of probiotic bacteria. It can be concluded that coffee may be a potential TOPIIα inhibitor considered as a functional food for cancer prevention.

Effect of Inhibiting Butyrylcholinesterase Activity Using Fractionated Coffee Extracts Digested In Vitro in Gastrointestinal Tract: Docking Simulation and Calorimetric and Studies.

Butyrylcholinesterase (BChE) is a major enzyme from the alpha-glycoprotein family that catalyzes the hydrolysis of neurotransmitter acetylcholine (ACh), lowering the concentration of ACh in the nervous system, which could cause aggravation of Alzheimer's disease (AD). In select pathological conditions, it is beneficial to reduce the activity of this enzyme. The aim of this study was to evaluate the degree of BChE inhibition by coffee extracts fractionated into mono- and diesters of caffeic acid/caffeine, digested in vitro in the gastrointestinal tract. The bioactive compounds from coffee showed high affinity for BchE, -30.23--15.28 kJ/mol, and was the highest for the caffeine fraction from the green Arabica extract. The isolated fractions were highly effective in inhibiting BChE activity at all in vitro digestion phases. It has been shown that the fractionation of coffee extracts could be potentially used to obtain high prophylactic or even therapeutic effectiveness against AD.

Reply to Zarguan et al. Comment on "Budryn et al. Hydroxybenzoic Acids as Acetylcholinesterase Inhibitors: Calorimetric and Docking Simulation Studies. Nutrients 2022, 14, 2476".

We thank Prof. Zarguan's group for their comment [...].

Evaluation of Activity of Sesquiterpene Lactones and Chicory Extracts as Acetylcholinesterase Inhibitors Assayed in Calorimetric and Docking Simulation Studies.

The aim of the study was to explain the effects of sesquiterpene lactones (SLs) from chicory (Cichorium intybus L.) root extracts as inhibitors of acetylcholinesterase (AChE) at the molecular level and to determine the inhibition of AChE activity by specific SLs (lactucin and lactucopicrin) and different chicory extracts. The obtained SLs-rich extracts were purified by the countercurrent partition chromatography (CPC) technique. AChE inhibitors were analyzed using two models: isothermal titration calorimetry (ITC) and docking simulation. The results of ITC analysis of the enzyme and the ligands' complexation showed strong interactions of SLs as well as extracts from chicory with AChE. In a test of enzyme activity inhibition after introducing acetylcholine into the model system with SL, a stronger ability to inhibit the hydrolysis of the neurotransmitter was observed for lactucopicrin, which is one of the dominant SLs in chicory. The inhibition of enzyme activity was more efficient in the case of extracts, containing different enzyme ligands, exhibiting complementary patterns of binding the AChE active site. The study showed the high potential of using chicory to decrease the symptoms of Alzheimer's disease.

Hydroxybenzoic Acids as Acetylcholinesterase Inhibitors: Calorimetric and Docking Simulation Studies.

One of the symptoms of Alzheimer's disease (AD) is low acetylcholine level due to high acetylcholinesterase (AChE) activity. For this reason, AChE inhibitors are used in the treatment of AD, the prolonged use of which may cause a cholinergic crisis. There is a need to search for safe natural AChE inhibitors. The study analyzed 16 hydroxybenzoic acids using calorimetry and docking simulation as AChE inhibitors. All tested compounds were shown to inhibit the hydrolysis of ACh. The best properties were shown by methyl syringinate, which acted as competitive inhibitor at a catalytic site. The tested compounds also interacted with the anionic or peripheral binding site known to block ß-amyloid plaques formation. The activity of the tested hydroxybenzoic acids IC50 ranged from 5.50 to 34.19 µmol/µmol of AChE, and the binding constant Ka from 20.53 to 253.16 L/mol, which proves their reversible, non-toxic effect, and activity at physiological concentrations.

Bioaccessibility of coffee bean hydroxycinnamic acids during in vitro digestion influenced by the degree of roasting and activity of intestinal probiotic bacteria, and their activity in Caco-2 and HT29 cells.

In coffee beans, especially roasted, a significant part of hydroxycinnamic acid (HCAs) and their esters chlorogenic acids (CHAs) is attached to melanoidins through both covalent and non-covalent bonds. Bound and, to a greater extent, unbound HCAs, including those released from the polymerized material during digestion, can be pivotal in preventing of many chronic civilization diseases. The aim of the study was to determine the amount of free CHAs and those released from coffee extracts during in vitro digestion in various sections of the gastrointestinal tract, in the presence and absence of probiotic bacteria. The concentration of free CHAs was the lowest in the stomach and achieved the highest levels in the large intestine. Probiotic bacteria caused significant release of CHAs, and in the colon their concentration was the highest. The studies with Caco-2 and HT-29 cell lines showed that digested coffee extracts had cytoprotective potential against tert-BOOH induced oxidative stress.

Evaluation of the thermal stability of bioactive compounds in coffee beans and their fractions modified in the roasting process.

Coffee is used as flavor or health-promoting additive in thermally processed food. In this study, ground coffee and freeze-dried coffee extracts were evaluated in terms of their thermal stabilities, and for the first time heat resistance of fractions (mono-, dichlorogenic acids and caffeine) with different roasting levels was evaluated. It observed that the degradation of green coffee bean ingredients began at 150 °C, and for the re-heated light and dark roasted, in the range of 171-188 °C. The lyophilized extracts were more stable and their degradation began around 160 °C. However, with the re-treatment (cooking, baking, frying) of the coffee extract fractions, the degradation of the monochlorogenic acids commenced at 114 °C, while for dichlorogenics at 108 °C and caffeine at 146 °C. Monochlorogenic acids in Robusta coffee showed high antioxidant activity (55-70%) and the highest content of fiber (13-17%). Coffee could be used to fortify food.

Chlorogenic Acid Protects against Advanced Alcoholic Steatohepatitis in Rats via Modulation of Redox Homeostasis, Inflammation, and Lipogenesis.

The aim of this study was to evaluate the therapeutic effects of chlorogenic acid (CGA) in rats with advanced alcoholic steatohepatitis. The rats were fed on a high-fat diet and gavaged with ethanol (4 g/kg) for 8 weeks. The livers of ethanol-treated rats showed steatosis; necrosis and mononuclear infiltration; and significant upregulation of the mRNA expression of the prooxidant (Cyp2e1, iNos), lipogenic (Srebp1, Acc), proinflammatory (Tlr4, Nf-κb, TnfA, Il-1B, and Il-6), and profibrogenic (TgfB, Col1, VegfA) genes. Simultaneously, a downregulation of level of Sod and Nrf2 was observed, which was accompanied by increased serum transaminase, TnfA, and serum and liver triglycerides levels. CGA administration (40 and 80 mg/kg, 8 weeks) to ethanol-fed group reduced the liver expression levels of Cyp2e1 and iNos, whereas it markedly enhanced the expression of Sod, Nrf2, and Ho-1. CGA at both doses downregulated the expressions of lipogenic, proinflammatory, and profibrogenic genes, while the expression of Tlr4 was lowered only after the higher dose of CGA. The higher dose of CGA efficiently prevented the progression of alcohol-induced steatosis and reduced inflammation through regulation of the expression of genes encoding the proteins involved in the Tlr4/Nf-κB signaling pathway and fibrosis. The study revealed hepatoprotective and anti-inflammatory effects of CGA through the regulation of expression of genes encoding Cyp2e1/Nrf2 involved in oxidative stress modulation. These results demonstrate CGA as a therapeutic candidate for the prevention and treatment of alcoholic steatohepatitis.

Effect of Fermented Soymilk-Honey from Different Probiotics on Osteocalcin Level in Menopausal Women.

Osteoporosis has been discovered to be a risk factor for menopausal women. Although synbiotics (probiotics and prebiotics) are found in fermented soymilk-honey made using local probiotics, their effect on osteocalcin levels is still unknown. Therefore, this study's objective was to determine the influence of fermented soymilk-honey from different probiotics on osteocalcin levels. A 90-day pre-post quasi-experimental study with a control design was conducted on 54 postmenopausal women divided into three intervention groups namely, the soymilk (SM) group, the soymilk-honey fermented with Lactobacillus casei subsp. casei R-68 (SMH Lc) group, and the soymilk-honey fermented with Lactobacillus plantarum 1 R 1.3.2 (SMH Lp) group. Participants consumed 100 mL of soymilk (SM) or fermented soymilk with honey (SMH Lc or SMH Lp) for 90 days. At the beginning and end of the study, the blood serum osteocalcin level was measured and subjects' health status was assessed, such as cholesterol total, random blood glucose, and uric acid levels. Our results presented that in the SMH Lp group, 90 days supplementation of soy-honey milk fermented with Lactobacillus plantarum 1 R 1.3.2 significantly reduced the level of blood serum osteocalcin. Based on these results it is justified to perform more detailed studies on the effect of fermented soy-honey milk on bone health.

The Interaction of Heptakis (2,6-di-O-Methyl)-ß-cyclodextrin with Mianserin Hydrochloride and Its Influence on the Drug Toxicity.

One tetracyclic antidepressant, mianserin hydrochloride (MIA), has quite significant side effects on a patients' health. Cyclodextrins, which are most commonly used to reduce the undesirable features of contained drugs within their hydrophobic interior, also have the potential to alter the toxic behavior of the drug. The present paper contains investigations and the characteristics of interaction mechanisms for MIA and the heptakis (2,6-di-O-methyl)-ß-cyclodextrin (DM-ß-CD) system, and evaluated the effects of the complexation on MIA cytotoxicity. In order to assess whether there was an interaction between MIA and DM-ß-CD molecules, isothermal titration calorimetry (ITC) have been chosen. Electrospray ionization mass spectrometry (ESI-MS) helped to establish the complex stoichiometry, and circular dichroism spectroscopy was used to describe the process of complex formation. In order to make a wider interpretative perspective, the molecular docking results have been performed. The viability of Chinese hamster cells were investigated in the presence of DM-ß-CD and its complexes with MIA in order to estimate the cytotoxicity of the drug and the conjugate with the chosen cyclodextrin. The viability of B14 cells treated with MIA+DM-ß-CD is lower (the toxicity is higher) than with MIA alone, and no protective effects have been observed for complexes of MIA with DM-ß-CD in any ratio.

Phenolics-Rich Extracts of Dietary Plants as Regulators of Fructose Uptake in Caco-2 Cells via GLUT5 Involvement.

The latest data link the chronic consumption of large amounts of fructose present in food with the generation of hypertension and disturbances in carbohydrate and lipid metabolism, which promote the development of obesity, non-alcoholic fatty liver disease, insulin resistance, and type 2 diabetes. This effect is possible after fructose is absorbed by the small intestine cells and, to a lesser extent, by hepatocytes. Fructose transport is dependent on proteins from the family of glucose transporters (GLUTs), among which GLUT5 selectively absorbs fructose from the intestine. In this study, we examined the effect of four phenolic-rich extracts obtained from A. graveolens, B. juncea, and M. chamomilla on fructose uptake by Caco-2 cells. Extracts from B. juncea and M. chamomilla most effectively reduced fluorescent fructose analogue (NBDF) accumulation in Caco-2, as well as downregulated GLUT5 protein levels. These preparations were able to decrease the mRNA level of genes encoding transcription factors regulating GLUT5 expression-thioredoxin-interacting protein (TXNIP) and carbohydrate-responsive element-binding protein (ChREBP). Active extracts contained large amounts of apigenin and flavonols. The molecular docking simulation suggested that some of identified phenolic constituents can play an important role in the inhibition of GLUT5-mediated fructose transport.

Mitochondrial O-GlcNAc Transferase Interacts with and Modifies Many Proteins and Its Up-Regulation Affects Mitochondrial Function and Cellular Energy Homeostasis.

O-GlcNAcylation is a cell glucose sensor. The addition of O-GlcNAc moieties to target protein is catalyzed by the O-Linked N-acetylglucosamine transferase (OGT). OGT is encoded by a single gene that yields differentially spliced OGT isoforms. One of them is targeted to mitochondria (mOGT). Although the impact of O-GlcNAcylation on cancer cells biology is well documented, mOGT's role remains poorly investigated. We performed studies using breast cancer cells with up-regulated mOGT or its catalytic inactive mutant to identify proteins specifically modified by mOGT. Proteomic approaches included isolation of mOGT protein partners and O-GlcNAcylated proteins from mitochondria-enriched fraction followed by their analysis by mass spectrometry. Moreover, we analyzed the impact of mOGT dysregulation on mitochondrial activity and cellular metabolism using a variety of biochemical assays. We found that mitochondrial OGT expression is glucose-dependent. Elevated mOGT expression affected the mitochondrial transmembrane potential and increased intramitochondrial ROS generation. Moreover, mOGT up-regulation caused a decrease in cellular ATP level. We identified many mitochondrial proteins as mOGT substrates. Most of these proteins are localized in the mitochondrial matrix and the inner mitochondrial membrane and participate in mitochondrial respiration, fatty acid metabolism, transport, translation, apoptosis, and mtDNA processes. Our findings suggest that mOGT interacts with and modifies many mitochondrial proteins, and its dysregulation affects cellular bioenergetics and mitochondria function.

Evaluation of the inhibition of monoamine oxidase A by bioactive coffee compounds protecting serotonin degradation.

Monoamine oxidase A (MAO-A) is a major enzyme responsible for the deamination of neurotransmitters such as serotonin (5-HT) in the central nervous system. The decrease in 5-HT levels is accompanied by disorders at the affective and somatic levels, leading to depression and disorders of the satiety center. The aim of this study was to evaluate the degree of MAO-A inhibition by chlorogenic acids, as well as green, light-, and dark-roasted coffee extracts and bioactive compounds from beans of the species Coffea canephora and Coffea arabica. Data for analysis was obtained using isothermal titration calorimetry and molecular docking. The results showed that caffeine and ferulic acid, as well as green Robusta coffee, demonstrated the greatest inhibition of MAO-A activity, which may increase the bioavailability of serotonin. We believe that green coffee shows potential antidepressant activity by inhibiting MAO-A, and may be used for treating depression and potentially, type 2 diabetes.

Novel Biodegradable Starch Film for Food Packaging with Antimicrobial Chicory Root Extract and Phytic Acid as a Cross-Linking Agent.

The aim of the study was to obtain and evaluate the properties of biodegradable starch film with the addition of phytic acid (0.05%) as a cross-linking agent and chicory root extract (1-5%) as an antimicrobial agent. To prepare biodegradable film, extracts from chicory root obtained with water or methanol were used. The content of bioactive compounds (sesquiterpene lactones and total polyphenols) was evaluated in chicory extracts. The antibacterial activity of the extracts was tested against Gram-negative bacteria (Pseudomonas fluorescens, Escherichia coli) and Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus) using the microculture method. The extracts acted as bacteriostatic agents, decreasing the growth rate (µmax), and extending the lag phase (tlag). The most sensitive bacterium in terms of film bacteriostatic activity was P. fluorescens; all extracts, irrespective of the solvent used, decreased its µmax value. S. aureus was the least sensitive. The obtained films were tested for their properties as food packaging (color, thickness, permeability, mechanical strength). Phytic acid improved the tensile strength and barrier properties of the films. The antimicrobial activity of the films was studied by the disk diffusion method against Gram-negative (P. fluorescens, E. coli) and Gram-positive (B. subtilis, S. aureus) bacteria, as well as fungi (Candida albicans, Aspergillus niger). The growth-inhibiting activity of each obtained film was observed for all tested microorganisms, and the most beneficial effect was observed for films with the 5% level of added extracts obtained with water. The growth-inhibiting activity for fungi, in particular for the yeast C. albicans, was low.

Cicer arietinum L. Sprouts' Influence on Mineralization of Saos-2 and Migration of MCF-7 Cells.

In the present study, we investigated the biological activity of four extracts obtained from Cicer arietinum L. sprouts. The fermentation of the sprouts with Lactobacillus casei and their incubation with ß-glucosidase elevated the concentrations of isoflavonoids, especially coumestrol, formononetin and biochanin A. To study the biological activity of C. arietinum, the human osteosarcoma Saos-2 and human breast cancer MCF-7 cell lines were used. The extracts obtained from fermented sprouts exhibited the strongest ability to decrease intracellular oxidative stress in both types of cells. They augmented mineralization and alkaline phosphatase activity in Saos-2 cells, as well as diminished the secretion of interleukin-6 and tumor necrosis factor α. Simultaneously, the extracts, at the same doses, inhibited the migration of MCF-7 cells. On the other hand, elevated concentrations of C. arietinum induced apoptosis in estrogen-dependent MCF-7 cells, while lower doses stimulated cell proliferation. These results are important for carefully considering the use of fermented C. arietinum sprouts as a dietary supplement component for the prevention of osteoporosis.

Effects of Raw and Roasted Cocoa Bean Extracts Supplementation on Intestinal Enzyme Activity, Biochemical Parameters, and Antioxidant Status in Rats Fed a High-Fat Diet.

The aim of the study was to analyze the influence of diet containing the polyphenol-rich material on intestinal enzyme activity, oxidative stress markers, lipid metabolism and antioxidant status of laboratory rats. The animals were fed high-fat diet supplemented with freeze-dried water extracts of raw and roasted cocoa beans of Forastero variety. The observed changes indicated the biological activity of polyphenols and other components of the prepared cocoa beans extracts (CBEs). The presence of raw and roasted CBEs in the diets diversified the activity of the enzymes of the cecal microflora of rats. Both CBEs beneficially affect the antioxidant status of the serum, even in relation to the control standard group. The experimental cocoa bean preparations showed no significant effect on the mass of rats' liver, heart, and kidneys, but varied some parameters of the antioxidant status of their organisms. The raw CBE in rats fed with the high-fat diet shows a high ability to inhibit lipid peroxidation in heart and more effectively increases hepatic reduced glutathione (GSH) concentrations compared to the roasted CBE, which did not show any significant effect. Moreover, supplementation with both CBEs significantly affects the volatile fatty acids concentration in the rats' cecum. Results of this study contribute to the evidence that dietary supplementation with raw and roasted CBEs can exert health-promoting effects, however further studies are necessary.

The Effects of Trifolium pratense L. Sprouts' Phenolic Compounds on Cell Growth and Migration of MDA-MB-231, MCF-7 and HUVEC Cells.

Uncontrolled growth and migration and invasion abilities are common for cancer cells in malignant tumors with low therapeutic effectiveness and high mortality and morbidity. Estrogen receptor ß (ERß), as a member of the nuclear receptor superfamily, shows potent tumor suppressive activities in many cancers. Phytoestrogens' structural resemblance to 17 ß-estradiol allows their binding to ERß isoform predominantly, and therefore, expression of genes connected with elevated proliferation, motility and invasiveness of cancer cells may be downregulated. Among polyphenolic compounds with phytoestrogenic activity, there are isoflavones from Trifolium pratense L. (red clover) sprouts, containing high amounts of formononetin and biochanin A and their glycosides. To determine the source of the most biologically active isoflavones, we obtained four extracts from sprouts before and after their lactic fermentation and/or ß-glucosidase treatment. Our previous results of ITC (isothermal titration calorimetry) modelling and a docking simulation showed clover isoflavones' affinity to ERß binding, which may downregulate cancer cell proliferation and migration. Thus, the biological activity of T. pratense sprouts' extracts was checked under in vitro conditions against highly invasive human breast cancer cell line MDA-MB-231 and non-invasive human breast cancer cell line MCF-7 cells. To compare extracts' activities acquired for cancer cells with those activities against normal cells, as a third model we choose human umbilical vein endothelial cells (HUVEC), which, due to their migration abilities, are involved in blood vessel formation. Extracts obtained from fermented sprouts at IC0 dosages were able to inhibit migration of breast cancer cells through their influence on intracellular ROS generation; membrane stiffening; adhesion; regulation of MMP-9, N-cadherin and E-cadherin at transcriptional level; or VEGF secretion. Simultaneously, isolated phenolics revealed no toxicity against normal HUVEC cells. In the manuscript, we proposed a preliminary mechanism accounting for the in vitro activity of Trifolium pratense L. isoflavones. In this manner, T. pratense sprouts, especially after their lactic fermentation, can be considered a potent source of biological active phytoestrogens and a dietary supplement with anti-cancer and anti-invasion properties.

Evaluation of Isoflavones as Bone Resorption Inhibitors upon Interactions with Receptor Activator of Nuclear Factor-κB Ligand (RANKL).

Receptor activator of nuclear factor-κB ligand (RANKL) is a cytokine responsible for bone resorption. It binds its receptor RANK, which activates osteoporosis. High levels of osteoprotegerin (OPG) competitively binding RANKL limit formation of ligand-receptor complexes and enable bone mass maintenance. The new approach to prevent osteoporosis is searching for therapeutics that can bind RANKL and support OPG function. The aim of the study was to verify the hypothesis that isoflavones can form complexes with RANKL limiting binding of the cytokine to its receptor. Interactions of five isoflavones with RANKL were investigated by isothermal titration calorimetry (ITC), by in silico docking simulation and on Saos-2 cells. Daidzein and biochanin A showed the highest affinity for RANKL. Among studied isoflavones coumestrol, formononetin and biochanin A showed the highest potential for Saos-2 mineralization and were able to regulate the expression of RANKL and OPG at the mRNA levels, as well as osteogenic differentiation markers: alkaline phosphatase (ALP), collagen type 1, and Runt-related transcription factor 2 (Runx2). Comparison of the osteogenic activities of isoflavones showed that the use of physicochemical techniques such as ITC or in silico docking are good tools for the initial selection of substances showing a specific bioactivity.

Bioavailability and metabolism of selected cocoa bioactive compounds: A comprehensive review.

Cocoa beans and their co-products are a rich source of beneficial compounds for health promotion, including polyphenols and methylxanthines. Knowledge of bioavailability and in vivo bioactivity of these phytochemicals is crucial to understand their role and function in human health. Therefore, many studies concerning bioavailability and bioactivity of cocoa bioactive compound have been done in both in vivo animal models and in humans. This critical review comprehensively summarizes the existing knowledge about the bioavailability and the major metabolic pathways of selected cocoa bioactive compounds (i.e. monomeric flavan-3-ols, procyanidins, anthocyanins, flavonols, phenolic acids, N-phenylpropenoyl-L-amino acids, stilbenes, and methylxanthines). The compiled results indicated that many of these compounds undergo extensive metabolism prior to absorption. Different factors have been suggested to influence the bioavailability of polyphenols and methylxanthines among them the role of gut microbiota, structure of these compounds, food matrix and occurrence of other substances were the most often considered. Aforementioned factors decided about the site where these bioactive compounds are digested and absorbed from the alimentary tract, as well as the pathway by which they are metabolized. These factors also determine of the type of transport through the intestine barrier (passive, involving specific enzymes or mediated by specific transporters) and their metabolic path and profile.

Influence of Diet Enriched with Cocoa Bean Extracts on Physiological Indices of Laboratory Rats.

Cocoa bean is a rich source of polyphenols, mainly flavonoids which have a wide range of biological properties. The aim of the study was to determine the physiological indices of laboratory rats as a response to diets containing water extracts of raw or roasted cocoa beans of Forastero variety, as well as purified monomeric flavan-3-ols fraction isolated from them. The influence of these extracts on selected parameters was studied during 4 weeks feeding. The samples of rats feces were collected throughout the experiment and after its completion, biological samples (intestines content, blood, and organs) were retrieved individually from each rat and subjected to analyses. The observed changes in the gastrointestinal tract functioning indices and metabolism indicators, determined throughout the study and after its completion, confirm to some extent the biological activity of polyphenol extracts of cocoa beans. The differences in the results obtained for the analyzed parameters of the gastrointestinal tract revealed that the cocoa bean extracts differently affected the physicochemical properties of rats' intestines. The results indicate the beneficial effects of the applied nutrition treatment on the activity of cecal enzymes and the content of volatile fatty acids in the gut.