Daily Ingestion of Eggplant Powder Improves Blood Pressure and Psychological State in Stressed Individuals: A Randomized Placebo-Controlled Study.

Eggplant (Solanum melongena) is a globally popular vegetable and its significant health effect has not been reported in randomized controlled trials. Recently, we reported that eggplant was rich in choline esters, including acetylcholine (ACh), and had an antihypertensive effect in spontaneously hypertensive rats. Here, we evaluated the effects of a continuous intake of eggplant powder on blood pressure (BP), stress, and psychological state (PS) in 100 stressed participants with normal-high BP or grade 1 hypertension in a randomized, double-blind, placebo-controlled, parallel-group comparative study. The participants were randomly assigned to the eggplant or placebo group. Participants in the eggplant group ingested capsules containing eggplant powder (1.2 g/day; 2.3 mg of ACh/day) for 12 weeks, whereas participants in the placebo group ingested placebo capsules. The primary outcome assessed was hospital BP. Secondary outcomes were stress and PS. Eggplant powder intake significantly decreased the hospital diastolic blood pressure (DBP) at week 8 overall and in the normal-high BP group, and the systolic blood pressure (SBP) and DBP at week 12 overall and in the grade 1 hypertension group, compared to those of the placebo group. It also improved negative PSs at week 8 or 12 in the normal-high BP group. This is the first evidence of the BP- and PS-improving effects of eggplant intake in humans. The functional substance responsible for the effects was estimated to be eggplant-derived choline ester, namely ACh.

Biocompatible Polymers Modified with d-Octaarginine as an Absorption Enhancer for Nasal Peptide Delivery.

Peptide and protein drugs, which are categorized as biologics, exhibit poor membrane permeability. This pharmacokinetic disadvantage has largely restricted the development of noninvasive dosage forms of biologics that deliver into systemic circulation. We have been investigating the potential use of cell-penetrating peptide-linked polymers as a novel absorption enhancer to overcome this challenge. Since our previous study revealed that biocompatible poly( N-vinylacetamide- co-acrylic acid) modified with d-octaarginine, a typical cell-penetrating peptide, enhanced in vitro permeation of biomolecules such as plasmid DNA and bovine serum albumin through cell membranes, the present study evaluated whether the polymers enhanced in vivo absorption of biologics applied on the mucosa. Mouse experiments demonstrated that d-octaarginine-linked polymers drastically enhanced nasal absorption of exendin-4, whose injection is clinically used. The mean bioavailability was 20% relative to subcutaneous administration, even though it fell short of 1% when exendin-4 alone was administered nasally. The absorption-enhancing function of the polymers was superior to that of sodium caprate and sodium N-(8-(2-hydroxybenzoyl)amino) caprylate, which have been used for humans as an absorption enhancer. In vitro experiments using several biologics with different characteristics revealed that biologics interacted with d-octaarginine-linked polymers and were taken up into cells when incubated with the polymers. The interaction and cellular uptake were enhanced as molecular weights of the biologics increased; however, their charge-dependent in vitro performance was not clearly observed. The current data suggested that biologics formulated with our polymers became an alternative to their conventional invasive parenteral formulations.

Effects of the Chemical Structures of Oligoarginines Conjugated to Biocompatible Polymers as a Mucosal Adjuvant on Antibody Induction in Nasal Cavities.

We have been investigating the potential of oligoarginine-linked polymers as an adjuvant for mucosal vaccination that induces immunoglobulin G (IgG) in systemic circulation and immunoglobulin A (IgA) secreted on the mucosa. Our latest infection experiments demonstrated that mice immunized nasally with a mixture of inactivated influenza viruses and poly(N-vinylacetamide-co-acrylic acid) (PNVA-co-AA) modified with D-octaarginine were perfectly protected from homologous virus infection. On the contrary, virus infection was observed in mice immunized with the antigen alone. This difference was presumably due to insignificant induction of secreted IgA on the nasal mucosa in the latter mice. Since it was unclear whether the current induction level was sufficient for heterologous virus infection, we evaluated the effects of the chemical structures of oligoarginines conjugated to PNVA-co-AA on induction of intranasal IgA. The number and optical activity of the arginine residues and the degree of modification with oligoarginines in the polymer backbone were listed as a factor that would influence IgA induction. Mouse experiments revealed that maximization of the modification resulted in an increase in adjuvant activities of oligoarginine-linked polymers most effectively. Glycine segments inserted between oligoarginines and the polymer backbone were a prerequisite for the maximization. The highest IgA level was observed when antigens were coadministered with diglycine-D-octaarginine-linked PNVA-co-AA.

Evaluation of a novel fluorescent nanobeacon for targeted imaging of Thomsen-Friedenreich associated colorectal cancer.

The Thomsen-Friedenreich (TF) antigen represents a prognostic biomarker of colorectal carcinoma. Here, using a nanobeacon, the surface of which was fabricated with peanut agglutinin as TF-binding molecules, we demonstrate that the nanobeacon is able to detect TF antigen in frozen and freshly biopsied polyps using fluorescence microscopy. Our results provide important clues about how to detect aberrant colonic tissues in the most timely fashion. Given the versatile application method for this topical nanobeacon, the protocol used in this work is amenable to clinical colonoscopy. Moreover, the prospects of clinical translation of this technology are evident.

Demonstration of d-Octaarginine-Linked Polymers as Promising Adjuvants for Mucosal Vaccination through Influenza Virus Challenge.

Mucosal vaccination is one of the most effective ways to reduce the risk of pandemics as a result of incorrect prediction of epidemic strains of influenza viruses or virus mutation. However, adjuvants and antigen carriers with potent immunostimulatory activities are a prerequisite for significant induction of mucosal immunity because most antigens are poorly immunogenic when solely applied to the mucosa. Our previous studies demonstrated that poly(N-vinylacetamide-co-acrylic acid) bearing d-octaarginine induced the secretion of antigen-specific immunoglobulin A (IgA) on the mucosa when nasally administered with virus antigens and that intranasal IgA reacts to viral strains other than the one used for immunization. Therefore, the present study evaluated capabilities of secreted IgA for protection against virus infection. When mice were inoculated with a mixture of inactivated H1N1 A/Puerto Rico/8/34 influenza viruses and d-octaarginine-linked polymers, antigen-specific secreted IgA was induced on the nasal mucosa. Immunized mice were completely protected from virus infection of the inoculated strain. To the contrary, mice nasally inoculated with inactivated viruses alone were infected with the homologous viruses presumably because of insignificant induction of secreted IgA. Results demonstrated that our polymer would be a promising adjuvant for mucosal vaccination.

Potential of D-Octaarginine-Linked Polymers as an in Vitro Transfection Tool for Biomolecules.

We have been investigating the potential use of cell-penetrating peptide-linked polymers as a novel penetration enhancer. Since previous in vivo studies demonstrated that poly(N-vinylacetamide-co-acrylic acid) bearing D-octaarginine, a typical cell-penetrating peptide, enhanced membrane permeation of biomolecules, its potential as an in vitro transfection tool was evaluated in this study. A plasmid DNA encoding green fluorescent protein (pGFP-C1), ß-galactosidase, and bovine serum albumin (BSA) were used as model biomolecules. Anionic pGFP-C1 interacted electrostatically with cationic d-octaarginine-linked polymers. When the ratio of mass concentration of polymers to that of pGFP-C1 reached 2.5, complexes whose size and zeta potential were approximately 200 nm and 15 mV, respectively, were obtained. GFP expression was observed in cells incubated with complexes prepared under conditions in which the polymer/pDNA concentration ratio exceeded 2.5. The expression level elevated with an increase in the concentration ratio, but physicochemical properties of the complexes remained unchanged. Results suggested that free polymers contributed to pGFP-C1 internalization. Another cell study demonstrated that ß-galactosidase premixed with polymers was taken up into cells in its active tetrameric form. Similar electrostatic interaction-driven complex formation was observed for BSA charged negatively in neutral solution. However, it appeared that the internalization processes of BSA differed from those of pGFP-C1. A mass concentration-dependent increase in internalized BSA was observed, irrespective of the polymer/protein concentration ratio. Due to frail interactions, polymers that were released from the complexes and subsequently immobilized on cell membranes might also contribute to membrane permeation of BSA.

Action of an endo-ß-1,3(4)-glucanase on cellobiosyl unit structure in barley ß-1,3:1,4-glucan.

ß-1,3:1,4-Glucan is a major cell wall component accumulating in endosperm and young tissues in grasses. The mixed linkage glucan is a linear polysaccharide mainly consisting of cellotriosyl and cellotetraosyl units linked through single ß-1,3-glucosidic linkages, but it also contains minor structures such as cellobiosyl units. In this study, we examined the action of an endo-ß-1,3(4)-glucanase from Trichoderma sp. on a minor structure in barley ß-1,3:1,4-glucan. To find the minor structure on which the endo-ß-1,3(4)-glucanase acts, we prepared oligosaccharides from barley ß-1,3:1,4-glucan by endo-ß-1,4-glucanase digestion followed by purification by gel permeation and paper chromatography. The endo-ß-1,3(4)-glucanase appeared to hydrolyze an oligosaccharide with degree of polymerization 5, designated C5-b. Based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF)/ToF-mass spectrometry (MS)/MS analysis, C5-b was identified as ß-Glc-1,3-ß-Glc-1,4-ß-Glc-1,3-ß-Glc-1,4-Glc including a cellobiosyl unit. The results indicate that a type of endo-ß-1,3(4)-glucanase acts on the cellobiosyl units of barley ß-1,3:1,4-glucan in an endo-manner.

[Effect of oral administration of ß-D-glucan from Aureobasidium pullulans ADK-34 on Candida and MRSA infections in immunosuppressed mice].

UNLABELLED: We examined the effect of the oral administration of ß-D-glucan derived from Aureobasidium pullulans ADK-34 (AP-FBG) on Candida albicans or methicillin-resistant Staphylococcus aureus (MRSA) infection in immunosuppressed mice. Mice pretreated with cyclophosphamide (CY) were intraperitoneally administered AP-FBG for 4 days and then infected with 6×10(4) C. albicans cells. In a preliminary experiment, the survival time of the Candida-infected mice treated with AP-FBG was clearly prolonged. Similarly, the effect of the oral administration of AP-FBG was examined. Mice were orally given 2.5% AP-FBG in feed for 42 days from 14 days prior to 2×10(4) C. albicans cells infection. The survival time of mice treated with AP-FBG was significantly prolonged and the viable cell count in the kidneys of the survivors was significantly decreased at 30 days after infection. The effects of the oral administration of AP-FBG on intestinal MRSA infection were also examined. Mice were given 2.5% AP-FBG orally in feed for 30 days before and after oral MRSA infection and treated with CY 12 days after the infection. The number of viable MRSA cells or the IgA production in feces did not significantly change, while AP-FBG administration seemed to relieve temporally the loss of body weight of mice. CONCLUSIONS: These results suggest that oral pre-administration of AP-FBG promoted resistance of CY-treated mice to C. albicans and lessened the weight reduction of CY-mice infected by MRSA.

Antioxidant Activity of ß-Glucan.

ß-Glucans extracted from barley, which mainly contains ß-(1,3-1,4)-D-glucan, are used extensively as supplements and food additives due to their wide biologic activities, including a reduction in blood lipid level. In this study, the antioxidant activity of ß-glucan was examined to assess potential new benefits associated with ß-glucan, because oxidative stress is considered one of the primary causal factors for various diseases and aging. ß-Glucan extracted from barley was found to possess significant antioxidant activity. The amount of antioxidant activity was influenced by different physiologic properties (e.g., structure and molecular size) of ß-glucan, which varied depending on the source and extraction method used. The antioxidant activity of ß-glucan was significantly higher than that of various polymers that are used as food additives. These results indicate that ß-glucan has promise as a polymeric excipient for supplement and food additive with antioxidant and other benefits, which may contribute to enhancing health and beauty.

Barley low molecular weight ß-glucan potently induces maturation of mouse dendritic cells.

BACKGROUND: Accumulating evidence indicates that non-toxic immunostimulants with strong differentiation/maturation-inducing activity for dendritic cells (DCs) might be useful for preventing or even curing cancer. MATERIALS AND METHODS: Mouse bone marrow (BM) cells were cultured in the presence of various glucans and their differentiation/maturation-inducing activities were compared by measuring cytokines secreted in the culture medium. RESULTS: Barley-derived ß-glucan with an average molecular weight of 2 kDa (BBG-Low) remarkably stimulated the formation of mature DCs from immature mouse DCs. The amount of interleukin-6 produced by sequential treatment of BM cells with granulocyte-macrophage colony-stimulating factor and 10 µg/mL of BBG-Low was approximately 30 times higher than that obtained by a similar sequential treatment using barley ß-glucan of 40-70 kDa instead of BBG-Low. CONCLUSION: BBG-Low induces the formation of mature DCs from immature DCs and suggests that BBG-Low will be useful as a potent nontoxic immunostimulator.

Induction of IFN-γ by a highly branched 1,3-ß-d-glucan from Aureobasidium pullulans in mouse-derived splenocytes via dectin-1-independent pathways.

We have previously elucidated the precise structure of a unique type of 1,3-ß-D-glucan, AP-FBG (Aureobasidium pullulans-fermented ß-D-glucan), from the fungus A. pullulans and found that AP-FBG strongly induced the production of various cytokines in DBA/2 mouse-derived splenocytes in vitro. However, the mechanism(s) of action of AP-FBG on in vitro mouse primary cells have not been characterized in detail. Herein, we report that the production of IFN-γ in DBA/2 mouse-derived splenocytes by AP-FBG was not inhibited following treatment with an anti-dectin-1 neutralizing antibody. In addition, AP-FBG not only failed to activate dectin-1-mediated signaling pathways, examined by a reporter gene assay but also failed to bind to dectin-1, a pivotal receptor for 1,3-ß-D-glucan. Taken together, AP-FBG induced cell activation via dectin-1-independent pathways.

Granulocyte macrophage colony-stimulating factor is required for cytokine induction by a highly 6-branched 1,3-ß-D-glucan from Aureobasidium pullulans in mouse-derived splenocytes.

We have previously obtained and elucidated the precise structure of a highly branched 1,3-ß-D-glucan (with 6-monoglucopyranosyl side chains), Aureobasidium pullulans-fermented ß-D-glucan (AP-FBG), from the fungus A. pullulans. However, the mechanism(s) of the effects of AP-FBG on in vitro mouse primary cells have not been analyzed in detail. Herein, we report that the induction of cytokines by AP-FBG was dependent on the existence of a granulocyte macrophage colony-stimulating factor (GM-CSF); this is similar way to be a typical 1,3-ß-D-glucan from Sparassis crispa (SCG), which is a 1,3-ß-D-glucopyranosyl backbone with single 1,6-ß-D-glucopyranosyl side branching units every three residues. In other words, the production of cytokines in DBA/2-mouse-derived splenocytes by AP-FBG was completely hampered by an anti-GM-CSF neutralizing monoclonal antibody. Furthermore, the addition of exogenous GM-CSF to C57BL/6-derived splenocytes, which are less sensitive to AP-FBG, induced the production of cytokines by AP-FBG. Therefore, GM-CSF is indispensable for the induction of cytokines by AP-FBG in mouse-derived splenocytes. This finding has provided a new insight into our understanding of the actions of ß-D-glucan but will also aid in the design and development of more effective ß-D-glucan agents.

The biological activities of (1,3)-(1,6)-beta-d-glucan and porous electrospun PLGA membranes containing beta-glucan in human dermal fibroblasts and adipose tissue-derived stem cells.

In this study, we investigated the possible roles of (1,3)-(1,6)-beta-d-glucan (beta-glucan) and porous electrospun poly-lactide-co-glycolide (PLGA) membranes containing beta-glucan for skin wound healing, especially their effect on adult human dermal fibroblast (aHDF) and adipose tissue-derived stem cell (ADSC) activation, proliferation, migration, collagen gel contraction and biological safety tests of the prepared membrane. This study demonstrated that beta-glucan and porous PLGA membranes containing beta-glucan have enhanced the cellular responses, proliferation and migration, of aHDFs and ADSCs and the result of a collagen gel contraction assay also revealed that collagen gels contract strongly after 4 h post-gelation incubation with beta-glucan. Furthermore, we confirmed that porous PLGA membranes containing beta-glucan are biologically safe for wound healing study. These results indicate that the porous PLGA membranes containing beta-glucan interacted favorably with the membrane and the topical administration of beta-glucan was useful in promoting wound healing. Therefore, our study suggests that beta-glucan and porous PLGA membranes containing beta-glucan may be useful as a material for enhancing wound healing.

A highly branched 1,3-beta-D-glucan extracted from Aureobasidium pullulans induces cytokine production in DBA/2 mouse-derived splenocytes.

We recently elucidated the structure of a highly branched 1,3-beta-D-glucan with 6-monoglucopyranosyl side chains, extracted from Aureobasidium pullulans (AP-FBG). Although the biological effects of beta-D-glucans are known to depend on their structures, the effects of a highly branched 1,3-beta-D-glucan on the production of cytokines by leukocytes in mice have not yet been elucidated. In this study, we found that AP-FBG strongly induced the production of various cytokines, especially Th1 cytokines (e.g., IFN-gamma and IL-12p70) and Th17 cytokines (e.g., IL-17A), but did not induce the production of IL-4, IL-10, and TNF-alpha in DBA/2 mouse-derived splenocytes in vitro.

Barley-derived beta-D-glucan induces immunostimulation via a dectin-1-mediated pathway.

Barley-derived beta-glucan, a linear mixed-linkage beta-glucan composed of 1,3- and 1,4-beta-D-glucopyranose polymers, binds to dectin-1. However, whether it can trigger signal transduction via dectin-1 remains unclear. In this study, we used a reporter gene assay to determine whether barley-derived beta-d-glucan can activate NF-kappaB via dectin-1-mediated signaling when dectin-1 is cotransfected with Syk, CARD9, and Bcl10 in 293T cells. We found that barley-derived beta-D-glucan can activate NF-kappaB leading to cytokine production when dectin-1, Syk, CARD9, and Bcl10 are coexpressed in the cells. We also found that barley-derived beta-D-glucan can induce the phosphorylation of Syk and production of IL-6 in thioglycolate-elicited peritoneal macrophages. These results indicated that the immunostimulatory effects of barley-derived beta-d-glucan might be exerted, at least in part, via dectin-1.

Involvement of branched units at position 6 in the reactivity of a unique variety of beta-D-glucan from Aureobasidium pullulans to antibodies in human sera.

We recently determined the structure of a unique type of 1,3-beta-D-glucan obtained from Aureobasidium pullulans (AP-FBG) and found that it reacted with the antibodies in human sera. The reactivity of AP-FBG to the antibodies was stronger than that of 1,3-beta-D-glucan obtained Grifola frondosa (GRN) but weaker than that of 1,3-beta-D-glucan from Candida albicans (CSBG). Here, we demonstrated that AP-FBG reacted to IgG antibodies, especially those of the subclasses IgG2, IgG1, and IgG3, in human sera. Moreover, the results of competitive enzyme-linked immunosorbent assays (ELISAs) using various glucan competitors showed that these IgGs recognized branched chains at position 6. This is the first study to report that the branched chains at position 6 of beta-D-glucan strongly contribute to its recognition by antibodies in human sera. This high reactivity of AP-FBG to human IgG could be advantageous for the use of this glucan in medicine, e.g., as an immunostimulatory agent.

Structural characterisation and biological activities of a unique type beta-D-glucan obtained from Aureobasidium pullulans.

A beta-D-glucan obtained from Aureobasidium pullulans (AP-FBG) exhibits various biological activities: it exhibits antitumour and antiosteoporotic effects and prevents food allergies. An unambiguous structural characterisation of AP-FBG is still awaited. The biological effects of beta-D-glucan are known to depend on its primary structures, conformation, and molecular weight. Here, we elucidate the primary structure of AP-FBG by NMR spectroscopy, and evaluate its biological activities. Its structure was shown to comprise a mixture of a 1-3-beta-D-glucan backbone with single 1-6-beta-D-glucopyranosyl side-branching units every two residues (major structure) and a 1-3-beta-D-glucan backbone with single 1-6-beta-D-glucopyranosyl side-branching units every three residues (minor structure). Furthermore, this beta-D-glucan exhibited immunostimulatory effects such as the accumulation of immune cells and priming effects against enterobacterium. To our knowledge, 1-3-beta-glucans like AP-FBG with such a high number of 1-6-beta-glucopyranosyl side branching have a unique structure; nevertheless, many 1-3-beta-glucans were isolated from various sources, e.g. fungi, bacteria, and plants.

Binding capacity of a barley beta-D-glucan to the beta-glucan recognition molecule dectin-1.

To clarify whether barley beta-glucans exhibit their biological effects via binding to dectin-1, a pivotal receptor for beta-1,3-glucan, the structure of barley beta-glucan E70-S (BBG-70) was unambiguously investigated by NMR spectroscopy and studied for its binding capacity and specificity to dectin-1 by ELISA. NMR spectroscopy confirmed that BBG-70 contains two different linkage glucans, namely, alpha-glucan and beta-glucan, which are not covalently attached to one another. Beta-glucan within BBG-70 is a linear mixed-linkage beta-glucan composed of 1,3- and 1,4-beta-D-glucopyranose residues but does not contain the continuous 1,3-linkage. Competitive ELISA revealed that highly purified barley beta-glucan E70-S (pBBG-70) inhibits the binding of soluble dectin-1 to sonifilan (SPG), a beta-1,3-glucan, although at a concentration higher than that of SPG and laminarin. It was found that barley beta-glucan can be recognized by dectin-1, implying that barley beta-glucan might, at least in part, exhibit its biological effects via the recognition by dectin-1 of the ligand sugar structure, which may be formed by 1,3-beta- and 1,4-beta-glucosyl linkage.