Evaluation of functional attributes and storage stability of novel juice blends from baobab, pineapple, and black-plum fruits.

Several under-utilized tropical fruits have exceptional micronutrients and phytochemical composition with the potential to contribute to the nutrition of people and also enhance food security. This study was carried out to evaluate the quality attributes and storage stability of juice blends of baobab (Adansonia digitata), pineapple (Ananas comosus), and black-plum (Syzygium cumini) fruits for use as functional beverages. Juice blends were analyzed for physicochemical, antioxidant, and sensory properties. Mineral compositions and storage stability of the pasteurized juice blends at 4 °C for 4 weeks were also investigated. Results showed that the vitamin C contents of individual juices synergistically contributed to the high values observed in the blends (317.45-414.51 mg/L). Juice blends of baobab, pineapple, and black-plum fruits are good sources of calcium (57-153 mg/L), magnesium (71-130 mg/L) and antioxidants (ascorbic acid, total polyphenol contents (65-104 mg GAE/100 mL), scavenging ability (105.97-359.71 µmol TE/100 mL), and reducing potential (1376-1829 µMFe2+ L) the consumption of which will promote human health. Blending enhanced the sensory qualities of the individual juices with improved taste and consumer acceptability. The juice blends kept well for two weeks at 4 °C though the color becomes less intense at the end of the storage period. These findings suggest that baobab-fruit pulp, pineapple, and black-plum fruits can be major ingredients in producing a consumer acceptable anti-oxidant rich functional beverage for optimal benefits to consumers.

Nutrients, bioactive compounds, and minerals in the juices of 16 varieties of apple (Malus domestica) harvested in Austria: A four-year study investigating putative correlations with weather conditions during ripening.

This study was conducted to examine putative correlations between weather parameters during April-September and the amounts of nutrients, minerals and bioactive compounds in the juices of 16 apple varieties from four harvest years in Lower Austria. For most sugar-parameters, negative correlations were found with the total precipitation (r between -0.42 and -0.64). Conversely, positive correlations were observed with the mean air temperature (r between 0.32 and 0.66), the global radiation (r between 0.32 and 0.61) and the number of tropical days (r between 0.39 and 0.51). The sum of 14 polyphenols (HPLC quantitation) was positively correlated with the mean air temperature and global radiation (rs 0.44 and 0.42). Negative correlations were observed between the global radiation and potassium, magnesium and calcium contents (correlation coefficients -0.49, -0.68 and -0.69). We conclude that increased temperatures and global radiation can be correlated with enhanced sugar synthesis and polyphenol formation.

Roasted Rye as a Coffee Substitute: Methods for Reducing Acrylamide.

Acrylamide is assumed to be a potential carcinogen, and reference values have therefore been implemented in EU legislation. Thus, the food industry needs to reduce the acrylamide content in consumer products to the lowest possible value. In this study, roasted rye was evaluated for its suitability as a coffee substitution product with respect to its acrylamide content. The influence of process modifiers, free asparagine content, storage, and rye type on the final content of acrylamide was investigated. Changes in carbohydrate composition and brightness caused by the roasting process were described. Sample analysis was conducted via GC-MS and HPLC-CAD. Existing methods were adapted to roasted rye as the sample matrix. CaCl2 and asparaginase treatment as well as pH adjustments prior to roasting did not prove to reduce the acrylamide content. A significantly (* p < 0.027) lower free asparagine content in the raw material resulted in a lower formation of acrylamide in the final product. The acrylamide content significantly decreased (**** p < 0.0001) after 3 (1100 ± 18 µg kg-1) and 6 (490 ± 7 µg kg-1) months of long-term storage. Only samples stored for 6 months (490 ± 7 µg kg-1) met the EU acrylamide content requirements (<500 µg kg-1) for grain-based coffee substitution products.

Guava (Psidium guajava) Fruit Extract Prepared by Supercritical CO2 Extraction Inhibits Intestinal Glucose Resorption in a Double-Blind, Randomized Clinical Study.

Inhibition of intestinal glucose resorption can serve as an effective strategy for the prevention of an increase in blood glucose levels. We have recently shown that various extracts prepared from guava (Psidium guajava) inhibit sodium-dependent glucose cotransporter 1 (SGLT1)- and glucose transporter 2 (GLUT2)-mediated glucose transport in vitro (Caco-2 cells) and in vivo (C57BL/6N mice). However, the efficacy in humans remains to be confirmed. For this purpose, we conducted a parallelized, randomized clinical study with young healthy adults. Thirty-one volunteers performed an oral glucose tolerance test (OGTT) in which the control group received a glucose solution and the intervention group received a glucose solution containing a guava fruit extract prepared by supercritical CO2 extraction. The exact same extract was used for our previous in vitro and in vivo experiments. Blood samples were collected prior to and up to two hours after glucose consumption to quantitate blood glucose and insulin levels. Our results show that, in comparison to the control group, consumption of guava fruit extract resulted in a significantly reduced increase in postprandial glucose response over the basal fasting plasma glucose levels after 30 min (Δ control 2.60 ± 1.09 mmol/L versus Δ intervention 1.96 ± 0.96 mmol/L; p = 0.039) and 90 min (Δ control 0.44 ± 0.74 mmol/L versus Δ intervention -0.18 ± 0.88 mmol/L; p = 0.023). In addition, we observed a slightly reduced, but non-significant insulin secretion (Δ control 353.82 ± 183.31 pmol/L versus Δ intervention 288.43 ± 126.19 pmol/L, p = 0.302). Interestingly, storage time and repeated freeze-thawing operations appeared to negatively influence the efficacy of the applied extract. Several analytical methods (HPLC-MS, GC-MS, and NMR) were applied to identify putative bioactive compounds in the CO2 extract used. We could assign several substances at relevant concentrations including kojic acid (0.33 mg/mL) and 5-hydroxymethylfurfural (2.76 mg/mL). Taken together, this clinical trial and previous in vitro and in vivo experiments confirm the efficacy of our guava fruit extract in inhibiting intestinal glucose resorption, possibly in combination with reduced insulin secretion. Based on these findings, the development of food supplements or functional foods containing this extract appears promising for patients with diabetes and for the prevention of insulin resistance. Trial registration: 415-E/2319/15-2018 (Ethics Commissions of Salzburg).

In Vitro and In Vivo Inhibition of Intestinal Glucose Transport by Guava (Psidium Guajava) Extracts.

SCOPE: Known pharmacological activities of guava (Psidium guajava) include modulation of blood glucose levels. However, mechanistic details remain unclear in many cases. METHODS AND RESULTS: This study investigated the effects of different guava leaf and fruit extracts on intestinal glucose transport in vitro and on postprandial glucose levels in vivo. Substantial dose- and time-dependent glucose transport inhibition (up to 80%) was observed for both guava fruit and leaf extracts, at conceivable physiological concentrations in Caco-2 cells. Using sodium-containing (both glucose transporters, sodium-dependent glucose transporter 1 [SGLT1] and glucose transporter 2 [GLUT2], are active) and sodium-free (only GLUT2 is active) conditions, we show that inhibition of GLUT2 was greater than that of SGLT1. Inhibitory properties of guava extracts also remained stable after digestive juice treatment, indicating a good chemical stability of the active substances. Furthermore, we could unequivocally show that guava extracts significantly reduced blood glucose levels (≈fourfold reduction) in a time-dependent manner in vivo (C57BL/6N mice). Extracts were characterized with respect to their main putative bioactive compounds (polyphenols) using HPLC and LC-MS. CONCLUSION: The data demonstrated that guava leaf and fruit extracts can potentially contribute to the regulation of blood glucose levels.

Sustaining elevated levels of nitrite in the oral cavity through consumption of nitrate-rich beetroot juice in young healthy adults reduces salivary pH.

BACKGROUND: Dietary inorganic nitrate (NO3-) and its reduced forms nitrite (NO2-) and nitric oxide (NO), respectively, are of critical importance for host defense in the oral cavity. High concentrations of salivary nitrate are linked to a lower prevalence of caries due to growth inhibition of cariogenic bacteria. OBJECTIVE: In-vitro studies suggest that the formation of antimicrobial NO results in an increase of the pH preventing erosion of tooth enamel. The purpose of this study was to prove this effect in-vivo. METHODS: In a randomized clinical study with 46 subjects we investigated whether NO3- rich beetroot juice exhibits a protective effect against caries by an increase of salivary pH. RESULTS: Our results show that, in comparison to a placebo group, consumption of beetroot juice that contains 4000 mg/L NO3- results in elevated levels of salivary NO2-, nitrite NO3-, and NO. Furthermore, we determined an increase of the mean pH of saliva from 7.0 to 7.5, confirming the anti-cariogenic effect of the used NO3--rich beetroot juice. CONCLUSIONS: Taken together, we have found that NO3--rich beetroot juice holds potential effects against dental caries by preventing acidification of human saliva. TRIAL REGISTRATION: C-87-15 (Ethics Commissions of Upper Austria).

Biomolecular Characterization of Putative Antidiabetic Herbal Extracts.

Induction of GLUT4 translocation in the absence of insulin is considered a key concept to decrease elevated blood glucose levels in diabetics. Due to the lack of pharmaceuticals that specifically increase the uptake of glucose from the blood circuit, application of natural compounds might be an alternative strategy. However, the effects and mechanisms of action remain unknown for many of those substances. For this study we investigated extracts prepared from seven different plants, which have been reported to exhibit anti-diabetic effects, for their GLUT4 translocation inducing properties. Quantitation of GLUT4 translocation was determined by total internal reflection fluorescence (TIRF) microscopy in insulin sensitive CHO-K1 cells and adipocytes. Two extracts prepared from purslane (Portulaca oleracea) and tindora (Coccinia grandis) were found to induce GLUT4 translocation, accompanied by an increase of intracellular glucose concentrations. Our results indicate that the PI3K pathway is mainly responsible for the respective translocation process. Atomic force microscopy was used to prove complete plasma membrane insertion. Furthermore, this approach suggested a compound mediated distribution of GLUT4 molecules in the plasma membrane similar to insulin stimulated conditions. Utilizing a fluorescent actin marker, TIRF measurements indicated an impact of purslane and tindora on actin remodeling as observed in insulin treated cells. Finally, in-ovo experiments suggested a significant reduction of blood glucose levels under tindora and purslane treated conditions in a living organism. In conclusion, this study confirms the anti-diabetic properties of tindora and purslane, which stimulate GLUT4 translocation in an insulin-like manner.

Differences in pharmacokinetics of apple polyphenols after standardized oral consumption of unprocessed apple juice.

BACKGROUND: Polyphenols are chemical compounds of the secondary plant metabolism. High concentrations can be found in various fruits including apples, berries and grapes. Polyphenols are associated with numerous health beneficial effects including a reduced risk for cardiovascular disease or diabetes. The human body cannot synthesize or store polyphenols and relies on continuous replenishment by daily diet. Unfortunately, knowledge on absorption, metabolization and excretion is still limited. The aim of this study was to determine the pharmacokinetic fate of apple polyphenols in young healthy adults. METHODS: Volunteers consumed 500 mL of an unfiltered apple juice. Blood and urine samples were collected within a time period of ten hours and analyzed for their total phenolic content, concentration of selected individual polyphenolic compounds and antioxidant capacity. RESULTS: Large differences in apple polyphenol pharmacokinetics between single subjects were observed. Those could be divided into subgroups according to fast or slow rates of polyphenol metabolism. Some subjects showed no detectable metabolism within the study time frame at all. An increase in the total phenolic content over time did not correlate with an observed, highly elevated antioxidant capacity (AOC) in the blood plasma after apple juice consumption. The determined increase of the AOC was rather a result of a high fructose content of the apple juice. No differences in renal excretion were detected between female and male subjects. However, relative concentrations were slightly higher in male subjects. CONCLUSIONS: Apple derived polyphenols can be readily detected in human blood and urine after juice consumption. The existence of sub-populations with different pharmacokinetics suggests significant variations in the individual metabolism rates of polyphenolic substances with implications on bioavailability and potential health effects within the body. TRIAL REGISTRATION: O2413 (Ethics Commissions of Upper Austria) and 415-EP/73/233-2013 Salzburg (Ethics Commissions of Salzburg).

Analysis of insulin receptor substrate signaling dynamics on microstructured surfaces.

Insulin receptor substrates (IRS) are phosphorylated by activated insulin/insulin-like growth factor I receptor tyrosine kinases, with this comprising an initial key event for downstream signaling and bioactivities. Despite the structural similarities, increasing evidence shows that IRS family proteins have nonredundant functions. Although the specificity of insulin/insulin-like growth factor signaling and biological responses partly reflects which IRS proteins are dominantly phosphorylated by the receptors, the precise properties of the respective IRS interaction with the receptors remain elusive. In the present study, we utilized a technique that combines micropatterned surfaces and total internal reflection fluorescence microscopy for the quantitative analysis of the interaction between IRS proteins and insulin/insulin-like growth factor in living cells. Our experimental set-up enabled the measurement of equilibrium associations and interaction dynamics of these molecules with high specificity. We revealed that several domains of IRS including pleckstrin homology and phosphotyrosine binding domains critically determine the turnover rate of the receptors. Furthermore, we found significant differences among IRS proteins in the strength and kinetic stability of the interaction with the receptors, suggesting that these interaction properties could account for the diverse functions of IRS. In addition, our analyses using fluorescent recovery after photobleaching revealed that kinases such as c-Jun N-terminal kinase and IκB kinase ß, which phosphorylate serine/threonine residues of IRS and contribute to insulin resistance, altered the interaction kinetics of IRS with insulin receptor. Collectively, our experimental set-up is a valuable system for quantitifying the physiological interaction of IRS with the receptors in insulin/insulin-like growth factor signaling.

Identification of novel insulin mimetic drugs by quantitative total internal reflection fluorescence (TIRF) microscopy.

BACKGROUND AND PURPOSE: Insulin stimulates the transport of glucose in target tissues by triggering the translocation of glucose transporter 4 (GLUT4) to the plasma membrane. Resistance to insulin, the major abnormality in type 2 diabetes, results in a decreased GLUT4 translocation efficiency. Thus, special attention is being paid to search for compounds that are able to enhance this translocation process in the absence of insulin. EXPERIMENTAL APPROACH: Total internal reflection fluorescence (TIRF) microscopy was applied to quantify GLUT4 translocation in highly insulin-sensitive CHO-K1 cells expressing a GLUT4-myc-GFP fusion protein. KEY RESULTS: Using our approach, we demonstrated GLUT4 translocation modulatory properties of selected substances and identified novel potential insulin mimetics. An increase in the TIRF signal was found to correlate with an elevated glucose uptake. Variations in the expression level of the human insulin receptor (hInsR) showed that the insulin mimetics identified stimulate GLUT4 translocation by a mechanism that is independent of the presence of the hInsR. CONCLUSIONS AND IMPLICATIONS: Taken together, the results indicate that TIRF microscopy is an excellent tool for the quantification of GLUT4 translocation and for identifying insulin mimetic drugs.

Quantification and kinetic analysis of Grb2-EGFR interaction on micro-patterned surfaces for the characterization of EGFR-modulating substances.

The identification of the epidermal growth factor receptor (EGFR) as an oncogene has led to the development of several anticancer therapeutics directed against this receptor tyrosine kinase. However, drug resistance and low efficacy remain a severe challenge, and have led to a demand for novel systems for an efficient identification and characterization of new substances. Here we report on a technique which combines micro-patterned surfaces and total internal reflection fluorescence (TIRF) microscopy (µ-patterning assay) for the quantitative analysis of EGFR activity. It does not simply measure the phosphorylation of the receptor, but instead quantifies the interaction of the key signal transmitting protein Grb2 (growth factor receptor-bound protein 2) with the EGFR in a live cell context. It was possible to demonstrate an EGF dependent recruitment of Grb2 to the EGFR, which was significantly inhibited in the presence of clinically tested EGFR inhibitors, including small tyrosine kinase inhibitors and monoclonal antibodies targeting the EGF binding site. Importantly, in addition to its potential use as a screening tool, our experimental setup offers the possibility to provide insight into the molecular mechanisms of bait-prey interaction. Recruitment of the EGFR together with Grb2 to clathrin coated pits (CCPs) was found to be a key feature in our assay. Application of bleaching experiments enabled calculation of the Grb2 exchange rate, which significantly changed upon stimulation or the presence of EGFR activity inhibiting drugs.

Bioanalytical characterization of apple juice from 88 grafted and nongrafted apple varieties grown in Upper Austria.

The compositional characteristics of untreated pure juice prepared from 88 apple varieties grown in the region of Eferding/Upper Austria were determined. Many of the analyzed varieties are noncommercial, old varieties not present in the market. The aim of the study was to quantitate the mineral, phosphate, trace elements, and polyphenolic content in order to identify varieties that are of particular interest for a wider distribution. Great variations among the investigated varieties could be found. This holds especially true for the total polyphenolic content (TPC) ranging from 103.2 to 2,275.6 mg/L. A clear dependence of the antioxidant capacity on the TPC levels was detected. Bioinformatics was employed to find specific interrelationships, such as Mg²âº/Mn²âº and PO4³â»/K⁺, between the analyzed bio- and phytochemical parameters. Furthermore, special attention was drawn on putative effects of grafting on the phytochemical composition of apple varieties. By grafting 27 different apple varieties on two trees grown close to each other, it could be shown that the apple fruits remain their characteristic phytochemical composition. Finally, apple juice prepared from selected varieties was further characterized by additional biochemical analysis including cytotoxicity, epidermal growth factor receptor (EGFR) inhibition, and α-amylase activity tests. Cytotoxicity and inhibition of EGFR activation were found to be dependent on the TPC, while α-amylase activity was reduced by the apple juices independent of the presence of polyphenolic substances. Taken together selected apple varieties investigated within this study might serve as preferable sources for the development of apple-based food with a strong focus on health beneficial effects.

Acetaminophen changes intestinal epithelial cell membrane properties, subsequently affecting absorption processes.

BACKGROUND/AIMS: Acetaminophen (APAP) effects on intestinal barrier properties are less investigated. APAP may lead to a changed bioavailability of a subsequently administered drug or diet in the body. We investigated the influence of APAP on enterocytic cell membrane properties that are able to modify the net intestinal absorption of administered substances across the Caco-2 barrier model. METHODS: The effect of APAP on cytotoxicity was measured by LDH assay, TER value and cell capacitance label-free using impedance monitoring, membrane permeability by FITC-dextrans, and efflux transporter MDR1 activity by Rh123. APAP levels were determined by HPLC analysis. Cell membrane topography and microvilli were investigated using SEM and intestinal alkaline phosphatase (Alpi) and tight junction protein 1 (TJP1) expression by western blot analysis. RESULTS: APAP changed the apical cell surface, reduced the number of microvilli and protein expression of Alpi as a brush border marker and TJP1, increased the membrane integrity and concurrently decreased cell capacitance over time. In addition, APAP decreased the permeability to small molecules and increased the efflux transporter activity, MDR1. CONCLUSION: APAP alters the Caco-2 cell membrane properties by different mechanisms and reduces the permeability to administered substances. These findings may help to optimize therapeutic implications.

Single-molecule reader for high-throughput bioanalysis.

We report here the development of a device for single-molecule imaging on large surface areas. A CCD camera operated in time delay and integration mode is synchronized with the movement of a sample scanning stage, enabling continuous data acquisition. Experiments on single fluorescent lipid molecules in supported lipid bilayers and on stained living cells demonstrate the capabilities of the method. Areas of up to 5 x 5 mm(2) were recorded within 11 min at a pixel size of 129 nm.

Co-localization of CD3 and prion protein in Jurkat lymphocytes after hypothermal stimulation.

While long-term effects of temperature treatment in respect of, e.g., gene-expression and cellular function have already been studied in some detail, nothing is known on the physiological responses of lymphocytes during short-term hypothermal shifts. In this report, we characterized the effects of such a stimulation using the human lymphocyte cell line Jurkat E6.1 and present evidence that warming from 4 to 37 degrees C for only 2 min is sufficient to cause co-localization of CD3, prion protein and the lipid-raft ganglioside GM1 paralleling lymphocyte activation as observed by Ca(2+) mobilization and mitogen-activated protein kinase-phosphorylation.

The physiological functions of prion protein.

Prion proteins are mentioned predominantly as unprecedented infectious pathogens in the context of transmissible spongiform encephalopathies. Since prions are devoid of nucleic acids, disease transmission must be mediated by an entirely novel mechanism. The general accepted theory proposes the conversion of cellular prion protein (PrP(C)) into the pathological isoform solely through conformational changes. This process favors the development of insoluble protein aggregates in the central nervous system typical for prion diseases. However, progress to elucidate the physiological functions of PrP(C) is still slow besides recent indications of a multifaceted network, in which PrP(C) seems to play a fundamental role. Possible contributions of interrupted or disturbed physiological signaling events due to the pathological prion protein isoform are presented in terms of recent findings.