Enrichment of Polyglucosylated Isoflavones from Soybean Isoflavone Aglycones Using Optimized Amylosucrase Transglycosylation.

Isoflavones in soybeans are well-known phytoestrogens. Soy isoflavones present in conjugated forms are converted to aglycone forms during processing and storage. Isoflavone aglycones (IFAs) of soybeans in human diets have poor solubility in water, resulting in low bioavailability and bioactivity. Enzyme-mediated glycosylation is an efficient and environmentally friendly way to modify the physicochemical properties of soy IFAs. In this study, we determined the optimal reaction conditions for Deinococcus geothermalis amylosucrase-mediated α-1,4 glycosylation of IFA-rich soybean extract to improve the bioaccessibility of IFAs. The conversion yields of soy IFAs were in decreasing order as follows: genistein > daidzein > glycitein. An enzyme quantity of 5 U and donor:acceptor ratios of 1000:1 (glycitein) and 400:1 (daidzein and genistein) resulted in high conversion yield (average 95.7%). These optimal reaction conditions for transglycosylation can be used to obtain transglycosylated IFA-rich functional ingredients from soybeans.

Agaricus bisporus-derived ß-glucan enter macrophages and adipocytes by CD36 receptor.

ß-glucans are a heterogeneous group of natural polysaccharides. They are ubiquitously found in bacterial or fungal cell walls, cereals, seaweed, and mushrooms. The beneficial role of ß-glucan in tumor, insulin resistance, dyslipidemia, hypertension, and obesity is being continuously documented. Ample evidence showed that ß-glucan could act on several receptors, such as Dectin, complement receptor (CR3), TLR-2, 4, 6 and scavenger. Based on the above, we wanted to explore whether agaricus bisporus-derived ß-glucan acted on these receptors on Raw 264.7 macrophages and 3T3-L1 adipocytes.

Non-starch constituents influence the in vitro digestibility of naked oat (Avena nuda L.) starch.

The present study investigated the effects of proteins, lipids and ß-glucan in naked oat flour (NOF) on the in vitro digestibility of starch. The content of rapidly digested starch (RDS) increased, and the content of resistant starch (RS) decreased in NOF after removing the non-starch constituents. The estimated glycemic index (eGI) of starch in NOF increased after the removal of the non-starch constituents, with a decreasing order of naked oat starch (NOS) > de-ß-glucan flour > de-proteins flour > de-lipids flour > NOF. NOS was found to have an A-type crystalline pattern, but the removal of proteins or ß-glucan rendered NOS a V-type crystalline pattern. The relative crystallinity decreased after removing non-starch constituents. The in vitro digestibility was positively correlated with the short-range molecular order and negatively correlated with the relative crystallinity. These results clearly illustrate the effects of non-starch constituents on the low digestibility of naked oat.

Immune Pharmacodynamic Responses of the Novel Cancer Immunotherapeutic Imprime PGG in Healthy Volunteers.

Imprime PGG (Imprime) is an i.v. administered, yeast ß-1,3/1,6 glucan in clinical development with checkpoint inhibitors. Imprime-mediated innate immune activation requires immune complex formation with naturally occurring IgG anti-ß glucan Abs (ABA). We administered Imprime to healthy human volunteers to assess the necessity of ABA for Imprime-mediated immunopharmacodynamic (IPD) changes. Imprime (4 mg/kg) was administered i.v. in single and multiple infusions. Subsets of subjects were premedicated with antihistamine and corticosteroid. Peripheral blood was measured before, during and after Imprime administration for IPD changes (e.g., ABA, circulating immune complexes, complement activation, complete blood counts, cytokine/chemokine, and gene expression changes). IPD changes were analyzed based on pretreatment serum ABA levels: low-ABA (<20 µg/ml), mid-ABA (≥20-50 µg/ml), and high-ABA (≥50 µg/ml). At the end of infusion, free serum ABA levels decreased, circulating immune complex levels increased, and complement activation was observed. At ∼1-4 h after end of infusion, increased expression of cytokines/chemokines, a 1.5-4-fold increase in neutrophil and monocyte counts and a broad activation of innate immune genes were observed. Low-ABA subjects typically showed minimal IPD changes except when ABA levels rose above 20 µg/ml after repeated Imprime dosing. Mild-to-moderate infusion-related reactions occurred in subjects with ABA ≥20 µg/ml. Premedications alleviated some of the infusion-related reactions, but also inhibited cytokine responses. In conclusion, ABA levels, being critical for Imprime-mediated immune activation may provide a plausible, mechanism-based biomarker to identify patients most likely to respond to Imprime-based anticancer immunotherapy.

Nanoplatform Constructed from a ß-Glucan and Polydeoxyadenylic Acid for Cancer Chemotherapy and Imaging.

A nanoplatform carrying doxorubicin (Dox) for cancer therapy and a dye for imaging was developed based on a natural triple helix ß-glucan (t-LNT) and polydeoxyadenylic acid (poly(dA)). The t-LNT-Dox conjugates were prepared through Schiff-base reaction between the aldehyde group in the oxidized t-LNT and the amino group of Dox, the single chains (s-LNT-Dox) of which interacted with the poly(dA)-dye to form a composite s-LNT-Dox/poly(dA)-dye through hydrogen bonding between s-LNT and poly(dA). t-LNT-Dox was confirmed to acid-responsively release Dox in vitro, showing enhanced cytotoxicity against HeLa cancer cells with time. It was confirmed that Dox and the dye could be simultaneously delivered into HeLa cells or the tumors with a prolonged duration time. Furthermore, LNT-Dox conjugates effectively inhibited tumor growth and decreased adverse effects of the free Dox in vivo. Hence, this work develops a new strategy to fabricate the nanoplatform for therapy and imaging using a natural polysaccharide.

Oral administration of oat beta-glucan preparations of different molecular weight results in regulation of genes connected with immune response in peripheral blood of rats with LPS-induced enteritis.

PURPOSE: Beta-glucans are biologically active polysaccharides having antioxidant, immunomodulatory, and antiinflammatory properties. This study investigated the transcriptomic profile in peripheral blood of rats with LPS-induced enteritis, which were fed a diet supplemented with high- (G1) and low- (G2) molecular-weight oat beta-glucans. METHODS: Two-color rat gene expression microarrays were applied and the analysis was performed using a common reference design to provide easy means of comparing samples from various experimental conditions against one another. Common reference sample was labeled with cyanine 3 (Cy3) and investigated samples from each experimental group: C-G0 (control group fed semi-synthetic diet), LPS-G0 (LPS-challenged group fed semi-synthetic diet), LPS-G1 (LPS-challenged group fed G1 beta-glucan enriched diet), and LPS-G2 (LPS-challenged group fed G2 beta-glucan enriched diet) were labeled with cyanine 5 (Cy5). Each microarray was performed in quadruplicate. Statistical analysis was performed using one-way ANOVA and Tukey's HSD post-hoc test (p < 0.05). A multiple testing correction was performed using Benjamini and Hochberg False Discovery Rate < 5%. A quantitative real-time RT-PCR was performed to verify the expression of chosen transcripts. RESULTS: The microarray analyses revealed differentially expressed transcripts between: the LPS-G0 and the control groups: C-G0 (138 genes), the LPS-G1 and LPS-G0 groups (533 genes), and the LPS-G2 and LPS-G0 groups (97 genes). Several differentially expressed genes in the beta-glucan-supplemented groups encoded proteins belonging to TLR and NLR signaling pathways, as well as prostaglandin synthesis and regulation pathways. Both beta-glucans up-regulated the expression of Atg10, which belongs to the family of autophagy-related genes, suggesting a possible link between autophagy induction and beta-glucan supplementation. CONCLUSION: The changes in gene expression observed in the peripheral blood indicate that oat beta-glucans exerted a protective effect in rats with an induced inflammatory state caused by LPS challenge. The greater number of differentially expressed genes was observed in group supplemented with G1 beta-glucan, pointing at the differences in the mode of action of high- and low-molecular-weight beta-glucans in the organism.

Beta-Glucan based temperature responsive hydrogels for 5-ASA delivery.

A series of temperature responsive hydrogels consisting of (1,3)-(1,6) ß-Glucan and poly (N-isopropyl acrylamide) (PNIPAM) was synthesized by redox polymerization at room temperature. Tetramethylethylenediamine (TEMED) and potassium persulfate (KPS) were used as a redox pair. ß-glucan was methacrylated (MA-ß-Glucan) and used as a biodegradable and bio-compatible cross-linker to prepare ß-glucan-PNIPAM based temperature responsive hydrogels. Swelling behavior of the hydrogels at different temperatures was investigated. The 5-ASA release from the hydrogels was monitored using UV-VIS spectrophotometer at 37 °C. It is notable that, the swelling and release behaviors of the hydrogels significantly change depending on the hydrogel compositions and temperature. Their thermal stability was determined using thermogravimetric analysis (TGA), assuming the extent of intermolecular interaction between PNIPAM and ß-glucan is proportional to thermal stability, which increased with the amount of PNIPAM. Volume phase transition temperature (VPTT) of the hydrogels was precisely determined by derivative differential scanning calorimeter (DDSC). They possessed variable VPTT with the compositions. The presence of ß-glucan in the PNIPAM network brought VPTT closer to the body temperature (from 32.8 °C to 35.5 °C), indicating that the VPTT could be tuned by the hydrogel compositions. Their in-vivo biocompatibility was tested against WS1 human fibroblast cells in phosphate buffer saline (PBS, pH 7.4). It was demonstrated that, using MA-ß-glucan as a cross-linker resulted in more bio-compatible thermo-responsive hydrogels indicating the enhancement of hydrophilic ß-Glucan on the swollen hydrogel surface.

Mechanisms of antimelanoma effect of oat ß-glucan supported by electroporation.

There are still not specified mechanisms how beta-glucan molecules are transported into cells. Supposing, beta-glucan toxicity against tumor cells may be related to the overexpression of the transporter responsible for the transport of glucose molecules in the cells. In this case, glucans - polymers composed of glucose units are much more up-taken by tumor than normal cells. Increased GLUT1 (Glucose Transporter Type 1) expression has been demonstrated earlier in malignant melanomas. GLUT1 expression promotes glucose uptake and cell growth in that cells. Also, in human melanoma tissues a significant correlation between GLUT1 expression and mitotic activity was found. The aim of the study was to verify if oat ß-glucan (OßG) is delivered into cells by GLUT-1 membrane protein. To check it out we blocked GLUT1 transporters by an inhibitor WZB117 and then we investigated cells viability with and without reversible electroporation (EP). The obtained results bring us to elucidate the mechanism of transport of the OßG into the cells is GLUT-1 dependent and moreover can be supported by EP method.

Behavior of new hydroxyapatite/glucan composite in human serum.

Biomaterials for bone tissue regeneration, including polymer-based composites, are typically evaluated in vitro prior to the clinical trials. However, such composites tested in vivo may behave different due to the specific body conditions. For example, some composites implanted into the tissue acidified due to transient postoperative inflammation may unexpectedly swell which delays the wound healing. Such massive swelling in acidic medium was previously observed for new elastic hydroxyapatite (HAp)/ß-glucan biomaterial. However, in further clinical cases concerning the composite implantation in patients without significant inflammation indicators, no side effects were observed. Therefore, it was reasonable to test the effect of human serum of neutral pH (typical for noninflamed tissues) on the composite parameters, in particular volume changes. Thus, this article shows the characterization of physicochemical parameters of the composite after incubation (5 days) in human serum of neutral pH by means of weight and volume measurement, scanning electron microscopy, X-ray diffraction, Fourier-transform infrared spectroscopy, microcomputed tomography, mercury intrusion, and biochemical techniques. Results showed that human serum collected from healthy people caused no uncontrolled changes in weight and volume, porosity and mechanical properties of the composite. Therefore, this suggests the lack of volume change-related side effects of HAp/glucan composite in bone defects treatment if postoperative inflammation is prevented. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2653-2664, 2018.

Design of Salecan-containing semi-IPN hydrogel for amoxicillin delivery.

Salecan is a new linear extracellular ß-glucan. The unique structure and beneficial properties of Salecan makes it an appealing material in biomedical applications. In this work, novel drug devices based on Salecan in a hydrogel matrix of poly(N-(3-dimethylaminopropyl)acrylamide-co-acrylamide) (Salecan/PDA) were fabricated via free radical polymerization for controlled release of amoxicillin. It was demonstrated that amoxicillin was efficiently encapsulated into the developed hydrogels and released in a Salecan dose-dependent and pH-sensitive manner. Furthermore, cell toxicity and adhesion assays confirmed that these drug carriers were biocompatible. Altogether, this study opens a new avenue to fabricate hydrogel devices for controlled delivery of drug.

Fiber supplements and clinically proven health benefits: How to recognize and recommend an effective fiber therapy.

BACKGROUND: Only 5% of adults consume the recommended level of dietary fiber. Fiber supplements appear to be a convenient and concentrated source of fiber, but most do not provide the health benefits associated with dietary fiber. PURPOSE: This review will summarize the physical effects of isolated fibers in small and large intestines, which drive clinically meaningful health benefits. DATA SOURCES: A comprehensive literature review was conducted (Scopus and PubMed) without limits to year of publication (latest date included: October 31, 2016). CONCLUSIONS: The physical effects of fiber in the small intestine drive metabolic health effects (e.g., cholesterol lowering, improved glycemic control), and efficacy is a function of the viscosity of gel-forming fibers (e.g., psyllium, ß-glucan). In the large intestine, fiber can provide a laxative effect if (a) it resists fermentation to remain intact throughout the large intestine, and (b) it increases percentage of water content to soften/bulk stool (e.g., wheat bran and psyllium). IMPLICATIONS FOR PRACTICE: It is important for nurse practitioners to understand the underlying mechanisms that drive specific fiber-related health benefits, and which fiber supplements have rigorous clinical data to support a recommendation. CLINICAL PEARL: For most fiber-related beneficial effects, "Fiber needs to gel to keep your patients well."

Effects of in vitro fermentation of barley ß-glucan and sugar beet pectin using human fecal inocula on cytokine expression by dendritic cells.

SCOPE: This study simulates the fermentation process of barley ß-glucan and sugar beet pectin in the human colon and monitors the degradation products formed. Additionally, immune effects of the degradation products were investigated. METHODS AND RESULTS: Immunostimulatory activity of fermentation digesta was investigated using bone marrow derived dendritic cells (BMDCs) from toll-like receptor 2/4 (TLR2/4) knockout mice, which were unresponsive to microbe-associated molecular patterns. Cytokine responses were elicited to dietary fibers and not to the SCFA and microbiota. The fermentation digesta were analyzed for their SCFA profiles and glycan metabolites over time. During fermentation the amount of insoluble precipitating fibers increased and induced as well as soluble molecules of lower molecular mass greater amounts of cytokines in BMDCs than the parental fiber. Additionally, high amounts of cytokines can be attributed to soluble galactose-rich beet pectin molecules. CONCLUSIONS: The fermentation of the two fibers led to fiber-specific amounts of SCFA, glycosidic metabolites, and different immunomodulatory properties. BMDC from TLR2/4 knockout mice did not respond to the digest microbiota and SCFA, making it a useful approach to study temporal effects of fermentation on the immunomodulatory effects of fibers.

Curdlan in fibers as carriers of tetracycline hydrochloride: Controlled release and antibacterial activity.

Curdlan (CURD) and polyethylene oxide were used to synthesize nanofibers as carriers of hydro soluble tetracycline hydrochloride (TCH). The viscosity, surface tension and conductivity of the precursor multicomponent aqueous solutions were determined and adjusted to produce defect-free fiber webs. Except for a slight increase in diameter, the addition of TCH did not affect the original morphology of the CURD/PEO nanofibers, as determined by FE-SEM imaging. However, the thermal stability of the system was enhanced (TGA and DSC). Moreover, water resistance, as measured with 24-h immersion tests, was observed upon crosslinking with glutaraldehyde. In-vitro activity measurements indicated a sustained and controlled TCH time-release pattern and excellent antibacterial activity against E. coli, as assessed by UV-vis spectroscopy and viable cell counting, respectively. Overall, we propose nanofibers based on CURD as promising platforms for scaffolds for wound dressing and drug delivery.

Two randomized, double-blind, placebo-controlled, dose-escalation phase 1 studies evaluating BTH1677, a 1, 3-1,6 beta glucan pathogen associated molecular pattern, in healthy volunteer subjects.

BACKGROUND: BTH1677 is a beta glucan pathogen associated molecular pattern (PAMP) currently being investigated as a novel cancer therapy. Here, the initial safety and pharmacokinetic (PK) results of BTH1677 in healthy subjects are reported. SUBJECTS AND METHODS: In the Phase 1a single-dosing study, subjects were randomized (3:1 per cohort) to a single intravenous (i.v.) infusion of BTH1677 at 0.5, 1, 2, 4, or 6 mg/kg or placebo, respectively. In the Phase 1b multi-dosing study, subjects were randomized (3:1 per cohort) to 7 daily i.v. infusions of BTH1677 at 1, 2, or 4 mg/kg or placebo, respectively. Safety and PK non-compartmental analyses were performed. RESULTS: Thirty-six subjects (N = 24 Phase 1a; N = 12 Phase 1b) were randomized to treatment. No deaths or serious adverse events occurred in either study. Mild or moderate adverse events (AEs) occurred in 67% of BTH1677-treated subjects in both studies. Treatment-related AEs (occurring in ≥10% of subjects) included dyspnea, flushing, headache, nausea, paraesthesia, and rash in Phase 1a and conjunctivitis and headache in Phase 1b. BTH1677 serum concentration was linear with dose. Clearance, serum elimination half-life (t1/2) and volume of distribution (Vss) were BTH1677 dose-independent. In Phase 1b, area under the curve, t1/2, and Vss values were larger at steady state on days 6-30 versus day 0. CONCLUSIONS: BTH1677 was well tolerated after single doses up to 6 mg/kg and after 7 daily doses up to 4 mg/kg.

Novel orally active inhibitors of ß-1,3-glucan synthesis derived from enfumafungin.

The clinical success of the echinocandins, which can only be administered parentally, has validated ß-1,3-glucan synthase (GS) as an antifungal target. Semi-synthetic modification of enfumafungin, a triterpene glycoside natural product, was performed with the aim of producing a new class of orally active GS inhibitors. Replacement of the C2 acetoxy moiety with various heterocycles did not improve GS or antifungal potency. However, replacement of the C3 glycoside with an aminoether moiety dramatically improved oral pharmacokinetic (PK) properties while maintaining GS and antifungal potency. Installing an aminotetrazole at C2 in conjunction with an N-alkylated aminoether at C3 produced derivatives with significantly improved GS and antifungal potency that exhibited robust oral efficacy in a murine model of disseminated candidiasis.

Effects of orally administered yeast-derived beta-glucans: a review.

Yeast-derived beta-glucans (Y-BG) are considered immunomodulatory compounds suggested to enhance the defense against infections and exert anticarcinogenic effects. Specific preparations have received Generally Recognized as Safe status and acceptance as novel food ingredients by European Food Safety Authority. In human trials, orally administered Y-BG significantly reduced the incidence of upper respiratory tract infections in individuals susceptible to upper respiratory tract infections, whereas significant differences were not seen in healthy individuals. Increased salivary IgA in healthy individuals, increased IL-10 levels in obese subjects, beneficial changes in immunological parameters in allergic patients, and activated monocytes in cancer patients have been reported following Y-BG intake. The studies were conducted with different doses (7.5-1500 mg/day), using different preparations that vary in their primary structure, molecular weight, and solubility. In animal models, oral Y-BG have reduced the incidence of bacterial infections and levels of stress-induced cytokines and enhanced antineoplastic effects of cytotoxic agents. Protective effects toward drug intoxication and ischemia/reperfusion injury have also been reported. In conclusion, additional studies following good clinical practice principles are needed in which well-defined Y-BG preparations are used and immune markers and disease endpoints are assessed. Since optimal dosing may depend on preparation characteristics, dose-response curves might be assessed to find the optimal dose for a specific preparation.

Measurement of barley ß-glucan concentration in the plasma by sandwich ELISA using rat dectin-1.

We used recombinant rat dectin-1 proteins to newly establish sandwich ELISA for determining barley ß-glucan (BßG). The ELISA method had a working range of 15-4,000 µg/L. Plasma BßG was detectable up to 24 h after an intravenous administration of BßG (1 mg/kg of body weight) to rats. This method may be an effective tool for investigating the immune modulatory effects of BßG.

Barley cultivar, kernel composition, and processing affect the glycemic index.

Barley has a low glycemic index (GI), but it is unknown whether its GI is affected by variation in carbohydrate composition in different cultivars and by food processing and food form. To examine the effect of these factors on GI, 9 barley cultivars varying in amylose and ß-glucan content were studied in 3 experiments in separate groups of 10 healthy participants. In Expt. 1, 3 barley cultivars underwent 2 levels of processing: hull removal [whole-grain (WG)] and bran, germ, and crease removal [white pearled (WP)]. GI varied by cultivar (CDC Fibar vs. AC Parkhill, [mean ± SEM]: 26 ± 3 vs. 53 ± 4, respectively; P < 0.05) and pearling (WG vs. WP: 26 ± 4 vs. 35 ± 3, respectively; P < 0.05) with no cultivar × pearling interaction. In Expt. 2, the GI of 7 WG cultivars ranged from 21 ± 4 to 36 ± 8 (P = 0.09). In Expt. 3, WG and WP AC Parkhill and Celebrity cultivars were ground and made into wet pasta. The GI of AC Parkhill pasta (69 ± 3) was similar to that of Celebrity pasta (64 ± 4) but, unlike in Expt. 1, the GI of WP pasta (61 ± 3) was less than that of WG pasta (72 ± 4) (P < 0.05). Pooled data from Expts. 1 and 2 showed that GI was correlated with total fiber (r = -0.75, P = 0.002) but not with measures of starch characteristics. We conclude that the GI of barley is influenced by cultivar, processing, and food form but is not predicted by its content of amylose or other starch characteristics.

Zebrafish (Danio rerio) larvae as a system to test the efficacy of polysaccharides as immunostimulants.

The present study was carried out to examine the use of zebrafish (Danio rerio) as a preliminary screening model for testing the effect of potential immunostimulant substances on the innate immune system. ß-Glucan, a polysaccharide used widely as an immunostimulant, was used as a representative molecule and tested on zebrafish embryos and larvae. The efficacy of the molecule was evaluated by determining the differential expression of some selected genes related to the immune system by RT-qPCR. Larvae from 72 hours post fertilization were found at the optimal developmental stage for assessing the expression of the selected genes. To verify if the ß-glucan entered the larvae and therefore was responsible for the effects produced, the molecule was labeled fluorescently to check its localization by using microscopy. For estimating the effects of ß-glucan on gene expression, zebrafish embryos and larvae were immersed in three different concentrations of ß-glucan (50, 100, and 150 µg/mL) using five different exposure times. A stronger gene induction was observed when longer times of exposure and older larvae were used. The most evident effects of ß-glucan were the overexpression of the genes TNFα, MPO, TRF, and LYZ. Moreover, slight changes in MPO expression were detected using a transgenic line of zebrafish (MPO::GFP), and a temporal increase in resistance against Vibrio anguillarum was found after ß-glucan immersion. The assay used in this study permits the testing potential of immunostimulants in a simple and cost-effective way.

Kinetics of serum ß-D-glucan after Pneumocystis pneumonia treatment in patients with AIDS.

OBJECTIVE: Serum ß-D-glucan has been demonstrated as a reliable, adjunct diagnostic marker for PCP, but its kinetics after PCP treatment are poorly understood. To evaluate the correlation between the levels of ß-D-glucan and the clinical response, we investigated the individual transition of serum ß-D-glucan levels after the initiation of PCP treatment. METHODS: Retrospective study PATIENTS: Seventeen PCP patients with AIDS who were admitted to our hospital were analyzed. RESULTS: All subjects showed the serum ß-D-glucan levels above the cut-off value, and the median level was 224 pg/mL [IQR: 78-597] at the time of PCP diagnosis. There were no correlations between serum ß-D-glucan levels and CRP, LDH, or AaDO(2) at room air. Although there was a downward trend in serum ß-D-glucan level as PCP treatment was initiated, a significant number of subjects showed a marked increase in the serum ß-D-glucan levels despite their evident clinical improvement. CONCLUSION: The serum ß-D-glucan level does not reflect the severity and prognosis of PCP infection, and thus it may not be suitable for monitoring the response to treatment.