Characterization and pharmacokinetic evaluation of microcomposite particles of alpha lipoic acid/hydrogenated colza oil obtained in supercritical carbon dioxide.

The work reported here is an extension of our previous findings in which supercritical composite particles (SCP) of alpha lipoic acid (ALA) masked with hydrogenated colza oil (HCO) named as ALA/HCO/SCP were obtained by the modified particles from gas-saturated solutions (PGSS) process in supercritical carbon dioxide in order to obscure the unpleasant taste and odor of ALA. The masking effect on ALA/HCO/SCP was compared with the widely used mechano-chemically masked formulation of ALA and HCO named as MC-50F. In the present study, ALA/HCO/SCP particles were found to have a significant improvement in regard to bitterness, numbness, and smell compared to ALA bulk powders suggesting they were well coated. The pharmacokinetic parameters for ALA/HCO/SCP and ALA bulk powder gave similar values but were significantly different from those of MC-50F. The amount of ALA absorbed into the body, in the administered ALA/HCO/SCP, was comparable to that absorbed by ALA bulk powder, whereas about half portion of ALA of the MC-50F was not absorbed, because the ALA/HCO/SCP particles were small enough and the particles of MC-50F were relatively large and had smaller specific surface area. Therefore, this study suggested a newly masked candidate may offer functional particles with maintained efficacy.

Enhancement and regulation effect of myrcene on antibody response in immunization with ovalbumin and Ag85B in mice.

BACKGROUND: MF59, which is an adjuvant belonging to C30 member of the terpene family, is a T helper type-2 (Th2)-biased immune enhancer. Our previous studies showed that pyriproxyfen, a member of the terpene family with fewer carbon atoms (C20) than MF59, enhanced active T helper type-1 (Th1)-biased immune responses. OBJECTIVE: This study was performed to investigate the enhancement of antigen-specific immune responses by myrcene, a member of the terpene family with fewer carbon atoms (C10) than pyriproxyfen. METHOD: Ovalbumin (OVA) was used as an antigen to determine the effects of myrcene on the immune response. The IgG subtypes and cytokines induced by immunization of OVA with or without myrcene were monitored. Thereafter, we determined the effects of myrcene in the immune response against Ag85B, which is a dominant protective antigen for tuberculosis. RESULTS: The results showed that 0.8 mg/dose of myrcene enhanced antigen-specific total IgG immune response to OVA. Direct mixing of the antigen with myrcene was required for the enhancement of antibody production. Myrcene increased OVA-specific IgG2a titer, suggesting induction of Th1-immune response. The level of Th1 cytokines, IFN-γ was increased at 8 weeks after immunization, although IL-13 was also increased at the same time point. However, finally myrcene was found to increase Ag85B-specific total IgG titers at 5 weeks and specific IgG2a titer was increased at both 5 and 8 weeks. The results suggested that myrcene could enhance Th1 immune response. CONCLUSIONS: Myrcene enhanced specific immune responses against OVA and Ag85B. This study suggested the tendency of the enhancement of Th1 immune response by myrcene.

Ultrasonically enhanced extraction of luteolin and apigenin from the leaves of Perilla frutescens (L.) Britt. using liquid carbon dioxide and ethanol.

The present study reports on the ultrasonic enhancement of the liquid carbon dioxide (CO2) extraction of luteolin and apigenin from the leaves of Perilla frutescens (L.) Britt., to which ethanol is added as a cosolvent. The purpose of this research is also to investigate the effects of the particle size, temperature, pressure, irradiation power, irradiation time, and ethanol content in the liquid CO2 solution on the extraction yield using single-factor experiments. We qualitatively and quantitatively analyzed the yields in the extract using HPLC (high-performance liquid chromatography). The liquid CO2 mixed with ethanol was used at temperatures of 5, 20 and 25 °C with extraction pressures from 8 to 14 MPa. The yields of luteolin and apigenin in the extraction were clearly enhanced by the ultrasound irradiation, but the selectivity of the extract was not changed. The yields of luteolin and apigenin in the extract were also significantly improved by adjusting the operating temperature, the irradiation time, and the ethanol content in the liquid CO2 solution, but no change in the selectivity of the extract was observed.

Gas-saturated solution process to obtain microcomposite particles of alpha lipoic acid/hydrogenated colza oil in supercritical carbon dioxide.

Alpha lipoic acid (ALA), an active substance in anti-aging products and dietary supplements, need to be masked with an edible polymer to obscure its unpleasant taste. However, the high viscosity of the ALA molecules prevents them from forming microcomposites with masking materials even in supercritical carbon dioxide (scCO2). Therefore, the purpose of this study was to investigate and develop a novel production method for microcomposite particles for ALA in hydrogenated colza oil (HCO). Microcomposite particles of ALA/HCO were prepared by using a novel gas-saturated solution (PGSS) process in which the solid-dispersion method is used along with stepwise temperature control (PGSS-STC). Its high viscosity prevents the formation of microcomposites in the conventional PGSS process even under strong agitation. Here, we disperse the solid particles of ALA and HCO in scCO2 at low temperatures and change the temperature stepwise in order to mix the melted ALA and HCO in scCO2. As a result, a homogeneous dispersion of the droplets of ALA in melted HCO saturated with CO2 is obtained at high temperatures. After the rapid expansion of the saturated solution through a nozzle, microcomposite particles of ALA/HCO several micrometers in diameter are obtained.

Expression and secretion of cholera toxin B subunit in lactobacilli.

Lactic acid bacteria (LAB) are used in various fields, including in food and medical supplies. There has been a great deal of research into vaccine development using LAB as carriers due to their "generally recognized as safe" status. Cholera is an infectious disease that causes diarrhea due to cholera toxin (CT) produced by Vibrio cholerae. The pentameric cholera toxin B (CTB) subunit has no toxicity, and is used as an antigen in cholera vaccines and as a delivery molecule in vaccines to various diseases. In this study, we generated recombinant LAB expressing and secreting CTB. Here, we first report that CTB expressed and secreted from LAB bound to GM1 ganglioside. The secreted CTB was purified, and its immunogenicity was determined by intranasal administration into mice. The results of the present study suggested that it may be useful as the basis of a new oral cholera vaccine combining LAB and CTB.

Pyriproxyfen enhances the immunoglobulin G immune response in mice.

Pyriproxyfen is a juvenile hormone mimic of vital importance for insect development with little risk to humans. This study was performed to investigate whether large doses of pyriproxyfen affect the immune response in mammals. Mice were immunized thrice with ovalbumin in 5% ethanol, with or without pyriproxyfen or alum. Large doses of pyriproxyfen (9 or 15 mM) significantly enhanced specific total IgG immune response. This enhancement was no longer present 24 hr after treatment with pyriproxyfen. These results suggest that pyriproxyfen is a safe chemical. Moreover, pyriproxyfen induced higher titers of IgG2a and enhanced tumor necrosis factor-alpha and gamma-interferon responses whereas alum induced IgG1 with enhanced interleukin-4 and -10. These observations indicate that the mechanism of immune enhancement by pyriproxyfen may differ from that of alum.

Differences in TLR9-dependent inhibitory effects of H(2)O(2)-induced IL-8 secretion and NF-kappa B/I kappa B-alpha system activation by genomic DNA from five Lactobacillus species.

Lactic acid bacteria (LAB) show anti-inflammatory effects, and their genomic DNA was identified as one of the anti-inflammatory components. Despite the differences in anti-inflammatory effects between live LAB dependent not only on genus but also species, this effect has not been compared at the genomic DNA level. We compared the anti-inflammatory effects of the genomic DNA from five Lactobacillus species-Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus gasseri, Lactobacillus plantarum, and Lactobacillus reuteri-using Caco-2 cells. To evaluate anti-inflammatory effects, decreases in H(2)O(2)-induced IL-8 secretion and inhibition of H(2)O(2)-induced NF-κB/IκB-α system activation were examined. All LAB genomic DNAs dose-dependently decreased H(2)O(2)-induced IL-8 secretion and inhibited H(2)O(2)-induced NF-κB/IκB-α system activation. Comparison of these effects between Lactobacillus species showed that the anti-inflammatory effects of L. acidophilus genomic DNA are lower than those of the other species. Furthermore, suppression of Toll-like receptor 9 (TLR9), a specific receptor of bacterial DNA, expression by RNAi abolished the decrease of H(2)O(2)-induced IL-8 secretion and inhibition of H(2)O(2)-induced NF-κB/IκB-α system activation by LAB genomic DNA. Our results demonstrated that the anti-inflammatory effects of genomic DNA differ between Lactobacillus species and TLR9 is one of the major pathways responsible for the anti-inflammatory effect of LAB genomic DNA.

Trace metal zinc stimulates secretion of antimicrobial peptide LL-37 from Caco-2 cells through ERK and p38 MAP kinase.

Infectious diseases, especially, diarrhoea, are responsible for high mortality rates in developing countries. Zinc supplementation shows beneficial effects against such diseases, but the mechanism of action is poorly understood. Here, we examined whether zinc supplementation can improve mucosal innate immunity through induction of antimicrobial peptide secretion from intestinal epithelial cells. Zinc was found to induce secretion of the antimicrobial peptide LL-37 from Caco-2 cell in a dose (0.63±0.09ng/mL and 0.54±0.06ng/mL at 20µM and 50µM respectively) and time dependent manner. LL-37 secretion increased immediately (1h) after exposure to 20µM Zn (0.29±0.04ng/mL), which continued up to 48h of exposure (0.58±0.05ng/mL). Zinc induces the phosphorylation of ERK and p38 MAP kinase and regulates LL-37 secretion through these MAP kinases. Zinc supplementation may have beneficial effects on mucosal innate immunity via secretion of LL-37.