The Influence of Maceration and Flavoring on the Composition and Health-Promoting Properties of Roasted Coffee.

Over the years, many methods of refining green beans have been developed, including maceration aimed at enriching the coffee aroma and improving the overall quality. This study aimed to evaluate the influence of different methods of maceration (fruit and wine) and the addition of food flavors to coffee beans on antioxidant activity, caffeine, phenolic and organic acid content, as well as health-promoting properties. This research showed that the use of the maceration in melon and apple fruit pulp (100 g of fruit pulp per 100 g of green coffee, incubated for 24 h, coffee roasting at 230 °C, control trial roasted coffee) ensured the highest polyphenol (hydroxycinnamic acids and their esters-chlorogenic acids) content (in melon pulp-13.56 g/100 g d.b. (dry bean); in apple pulp-13.22 g/100 g d.b., p < 0.05 (one-way ANOVA)) and antioxidant activity. Melon (92.11%, IC50 = 3.80 mg/mL extract) and apple (84.55%, IC50 = 4.14 mg/mL) showed the highest α-amylase (enzyme concentration 10 µmol/mL) inhibition activity (0.5 mg/mL for both fruits). The addition of food flavors reduced the total content of chlorogenic acids to the range of 4.64 to 6.48 g/100 g d.b. and increased the content of acrylamide and 5-HMF, which positively correlated with a low antioxidant potential compared to the macerated samples and the control. Studies have shown that coffee macerated in the pulp of melon and apple fruit, due to its great potential to inhibit α-amylase in vivo, may have a preventive effect on type II diabetes. This study complements the current knowledge on the potential health-promoting properties of coffee flavored using different methods; further research should include more advanced models for testing these health-promoting properties. Statistical analysis was based on the determination of the average values of six measurements and their standard deviation, as well as on the one-way ANOVA (analysis of variation) and the Pearson correlation coefficient, using Statistic 10.0 software. The significance was defined at p ≤ 0.05.

Evaluation of the effect of roasting and digestion on biological activity of compounds of coffee extracts - in vitro assessment of the bioavailability, cytoprotective properties and modulation of inflammatory response.

Coffee is the most consumed beverage in the world. Consumption of phenolic compounds present in coffee protects the body against oxidative stress generation, inflammatory response, and cancer development. The aim of the study was evaluation of biological activity of coffee extracts (obtained from green, as well as light and dark roasted Robusta and Arabica beans) and isolated fractions on human colon adenocarcinoma Caco-2 cells, which are used as a cellular model of intestinal barrier in bioavailability studies. Additionally, impact of coffee phenolics on oxidative stress level and anti-inflammatory activity has been studied with RAW 264.7 macrophages used in immunomodulatory research. It was demonstrated that the coffee constituents protection against oxidative stress, lipotoxicity and secretion of proinflammatory mediators is correlated with the presence of mono- and dichlorogenic acids and roasting process. It was demonstrated that coffee phytochemicals can decrease cells proliferation and bind to topoisomerase IIα being a dietary tool in cancer prevention.

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.

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.

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.

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.

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.

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.

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.

The effects of baking conditions on acrylamide content in shortcrust cookies with added freeze-dried aqueous rosemary extract.

The aim of the present study was to determine the effects of baking air humidity and dough supplementation with freeze-dried aqueous rosemary extract on acrylamide content in shortcrust cookies, as well as on their antioxidant properties and phenolic composition. Shortcrust cookies were baked at 170 °C in dry or humid (90%) air using 0.1, 0.2, or 0.5% of rosemary extract, and were compared to control samples without the extract. Acrylamide concentration in the obtained products ranged from 22.49 to 28.38 µg kg-1. Furthermore, cookies baked in humid air had less acrylamide (by 6% on average) than those baked in dry air, irrespective of extract content. On the other hand, a significant relationship was found between humidity conditions and total phenolic content in the final products. Shortcrust cookies containing 0.5% of rosemary extract and baked in humid air revealed the best antioxidant properties as their total polyphenol content was approx. Three times higher than that in control samples. Furthermore, shortcrust cookies with 0.5% of rosemary extract showed superior DPPH radical scavenging capacity.

The effect on bioactive components and characteristics of chocolate by functionalization with raw cocoa beans.

Chocolate manufacturers often develop their own original blends of cocoa beans, which, after processing, will give an unique sensory properties to their chocolates. In recent years, there has been a trend in the production of an increasing amount of low-processed or unprocessed food. Therefore, we carried out the research in which we obtained and analyzed chocolates with different blends of cocoa liquors prepared from roasted and unroasted cocoa beans of Criollo and Forastero varieties.Studies have shown that viscosity and yield value increased and hardness of chocolates lowered with the increase content of cocoa liquor from unroasted beans. Chocolates with cocoa liquor from roasted beans were characterized by the largest ability to scavenge DPPH radicals. Antioxidant activity determined by the ORAC-FL method increased with increasing polyphenol content in the chocolates. Type of the cocoa liquor used induced minor changes in the polyphenol and fatty acid composition. Asparagine was determined at the highest concentration, followed by alanine, glutamine, and serine. The concentration of acetic acid was the higher the higher the content of unroasted beans. The increased content of cocoa liquor from unroasted beans caused the color of the chocolate to brighten, more red and yellow. All tested chocolates were characterized by good organoleptic properties and microbial stability.

Binding of Red Clover Isoflavones to Actin as A Potential Mechanism of Anti-Metastatic Activity Restricting the Migration of Cancer Cells.

Actin functions are crucial for the ability of the cell to execute dynamic cytoskeleton reorganization and movement. Nutraceuticals that form complexes with actin and reduce its polymerization can be used in cancer therapy to prevent cell migration and metastasis of tumors. The aim of this study was to evaluate the ability of isoflavones to form complexes with actin. Docking simulation and isothermal titration calorimetry were used for this purpose. The formation of complexes by hydrogen bonds, hydrophobic and π-π interactions was demonstrated. Interactions occurred at the ATP binding site, which may limit the rotation of the actin molecule observed during polymerization and also at the site responsible for contacts during polymerization, reducing the ability of the molecule to form filaments. The greatest therapeutic potential was demonstrated by isoflavones occurring in red clover sprouts, i.e., biochanin A and formononetin, being methoxy derivatives of genistein and daidzein.

Influence of diet based on bread supplemented with raw and roasted cocoa bean extracts on physiological indices of laboratory rats.

The aim of the study was to determine the physiological indices of laboratory rats as a response to a diet consisting of a bread supplemented with freeze-dried water extracts of raw and roasted cocoa beans of Forastero variety. The influence of different cocoa extracts on oxidative stress, hyperglycemia, and lipid metabolism was studied during long-term feeding of laboratory rats with bread supplemented with cocoa extracts. During the 4-week experimental feeding samples of rats feces were subjected to analysis and after its completion, biological samples were collected i.e. intestines content, blood, and organs retrieved individually from each rat. The observed changes indicate the biological activity of polyphenol extracts and other components of cocoa beans present in the prepared extracts. The differences in the results obtained for the analyzed parameters of the gastrointestinal tract revealed that the cocoa bean extracts being the subject of this investigation differently affected the physicochemical properties of rats' intestines, including intestinal microflora. The results show the influence of the diet on the activity of both fecal and cecal enzymes. Applied nutrition treatment varied the concentration and total production of volatile fatty acids in the cecum. Furthermore, the diverse composition of the applied diet affected the level of some biochemical indicators of blood serum of rats. Additionally, to a small extent influenced the analyzed indicators of the antioxidative status of rats as characterized by the concentration of substances reacting with thiobarbituric acid in internal organs.

Evaluation of estrogenic activity of red clover (Trifolium pratense L.) sprouts cultivated under different conditions by content of isoflavones, calorimetric study and molecular modelling.

Isoflavones have an affinity for estrogen receptors (ERs) including beneficial affinity for ERß. Widely used soy is a source of poorly absorbed isoflavones glycosides. Red clover contains mostly easily absorbed free aglycones. Red clover sprouts were cultivated under different conditions (white light, UVA or UVB for 12 or 24 h a day at 18 or 25 °C) to maximise the content of isoflavones, especially of high affinity for ERß. The affinity of isoflavones to ERs was evaluated by molecular modelling and isothermal titration calorimetry (ITC). The richest source of isoflavones, especially formononetin were sprouts cultivated for 10 days under continuous white light and at 25 °C (562 mg/100 g of fresh mass). Formononetin and the above sprouts has been shown to have a high affinity for ERß. Red clover sprouts can be considered as a source of phytoestrogens with high biological activity and as a dietary supplement reducing menopausal symptoms.

Phenolic Profile and Antioxidant Potential of Leaves from Selected Cotoneaster Medik. Species.

The antioxidant efficiency of 70% aqueous methanolic extracts from the leaves of twelve selected Cotoneaster Medik. species was evaluated using four complementary in vitro tests based on SET- (single electron transfer) and HAT-type (hydrogen atom transfer) mechanisms (DPPH, FRAP, O2(•-) and H2O2 scavenging assays). The samples exhibited the dose-dependent responses in all assays with activity parameters of EC50 = 18.5-34.5 µg/mL for DPPH; 0.9-3.8 mmol Fe(2+)/g for FRAP; SC50 = 27.7-74.8 µg/mL for O2(•-); and SC50 = 29.0-91.3 µg/mL for H2O2. Significant linear correlations (|r| = 0.76-0.97, p < 0.01) between activity parameters and total contents of phenolics (5.2%-15.4% GAE) and proanthocyanidins (2.1%-15.0% CYE), with weak or no effects for chlorogenic acid isomers (0.69%-2.93%) and total flavonoids (0.28%-1.40%) suggested that among the listed polyphenols, proanthocyanidins are the most important determinants of the tested activity. UHPLC-PDA-ESI-QTOF-MS analyses led to detection of 34 polyphenols, of which 10 B-type procyanidins, 5 caffeoylquinic acids and 14 flavonoids were identified. After cluster analysis of the data matrix, the leaves of Cotoneaster zabelii, C. splendens, C. bullatus, C. divaricatus, C. hjelmqvistii and C. lucidus were selected as the most promising sources of natural antioxidants, exhibiting the highest phenolic levels and antioxidant capacities, and therefore the greatest potential for pharmaceutical applications.