Dietary Supplementation with Fermented Brassica rapa L. Stimulates Defecation Accompanying Change in Colonic Bacterial Community Structure.

Our previous studies have elucidated that oral administration of Brassica rapa L. extract, known as Nozawana in Japan, alters immune responses and gut microbiota composition, increasing the numbers of butyrate-producing bacteria. Therefore, further investigation would help elucidate the mechanism attributable for the changes and health-promoting effects observed after B rapa L. extract ingestion. To reveal the modulation effects of fermented B. rapa L. on immune function and intestinal bacterial community structure, we conducted an intervention study with healthy volunteers followed by a mouse feeding study. The pilot intervention study was conducted for healthy volunteers aged 40-64 years under the hypothesis that the number of subjects exhibiting any change in gut microbiota in response to fermented B. rapa L. consumption may be limited. In total, 20 volunteers consumed 30 g of fermented B. rapa L. per day for 4 weeks. The fecal bacterial community composition of the volunteers was characterized using terminal-restriction fragment length polymorphism patterning followed by clustering analysis. To evaluate the detailed changes in the immune responses and the gut bacterial composition, assessed by high-throughput sequencing, we fed healthy mice with freeze-dried, fermented B. rapa L. for 2 weeks. The fecal bacterial community composition of the volunteers before the intervention was divided into three clades. Regardless of the clade, the defecation frequency significantly increased during the intervention weeks compared with that before the intervention. However, this clustering detected a specific increase of Prevotella in one cluster (low to zero Prevotella and high occupation of Clostridium at clusters IV and XIVa) post-ingestion. The cytokine production of spleen cells significantly increased due to feeding fermented B. rapa L. to the mice. This supplementary in vivo trial provided comparable results to the volunteer study regarding the effects of ingestion of the material given the compositional change complying with that of dietary fiber, particularly in the increase of genera Prevotella, Lachnospira, and genera in the Ruminococcaceae family, and the increase in daily defecation amount during 2 weeks of administration. We conclude that feeding fermented B. rapa L. may be responsible for the observed modulation in gut microbiota to increase fiber-degrading bacteria and butyrate-producing bacteria which may be relevant to the improvement in bowel function such as defecation frequency.

Soybean-Derived Glycine-Arginine Dipeptide Administration Promotes Neurotrophic Factor Expression in the Mouse Brain.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, plays an important role in cognitive abilities, including memory and learning. We demonstrated that soybean protein hydrolysate (SPH) diet suppresses age-related cognitive decline via the upregulation of BDNF in a mouse model of senescence. Our purpose was to identify novel bioactive peptides in SPH, which enhance BDNF expression. We treated mouse primary astrocytes with SPH as well as with its positively charged chromatographic fraction. Significant increases in the expression of BDNF were observed in the treatment with positively charged fraction of SPH. Among the synthesized peptides, the dipeptide glycine-arginine (GR) increased BDNF expression in vitro, and LC-TOF-MS analysis showed the presence of GR in the SPH. Furthermore, its administration in vivo increased the expression of BDNF in the cerebral cortex and the number of neurons in hippocampus and cerebral cortex. These data indicate that GR might promote neurogenesis by upregulating BDNF levels.

Enhancement of natural killer activity and IFN-γ production in an IL-12-dependent manner by a Brassica rapa L.

Certain food components possess immunomodulatory effects. The aim of this study was to elucidate the mechanism of the immunostimulatory activity of Brassica rapa L. We demonstrated an enhancement of natural killer (NK) activity and interferon (IFN)-γ production in mice that were orally administered an insoluble fraction of B. rapa L. The insoluble fraction of B. rapa L. significantly induced IFN-γ production in mouse spleen cells in an interleukin (IL)-12-dependent manner, and NK1.1+ cells were the main cells responsible for producing IFN-γ. Additionally, the results suggested that the active compounds in the insoluble fraction were recognized by Toll-like receptor (TLR) 2, TLR4, and C-type lectin receptors on dendritic cells, and they activated signaling cascades such as MAPK, NF-κB, and Syk. These findings suggest that B. rapa L. is a potentially promising immuno-improving material, and it might be useful for preventing immunological disorders such as infections and cancers by activating innate immunity.

Relationship of Enhanced Butyrate Production by Colonic Butyrate-Producing Bacteria to Immunomodulatory Effects in Normal Mice Fed an Insoluble Fraction of Brassica rapa L.

This study was performed to determine the effects of feeding a fiber-rich fraction of Brassica vegetables on the immune response through changes in enteric bacteria and short-chain fatty acid (SCFA) production in normal mice. The boiled-water-insoluble fraction of Brassica rapa L. (nozawana), which consists mainly of dietary fiber, was chosen as a test material. A total of 31 male C57BL/6J mice were divided into two groups and housed in a specific-pathogen-free facility. The animals were fed either a control diet or the control diet plus the insoluble B. rapa L. fraction for 2 weeks and sacrificed to determine microbiological and SCFA profiles in lower-gut samples and immunological molecules. rRNA-based quantification indicated that the relative population of Bacteroidetes was markedly lower in the colon samples of the insoluble B. rapa L. fraction-fed group than that in the controls. Populations of the Eubacterium rectale group and Faecalibacterium prausnitzii, both of which are representative butyrate-producing bacteria, doubled after 2 weeks of fraction intake, accompanying a marginal increase in the proportion of colonic butyrate. In addition, feeding with the fraction significantly increased levels of the anti-inflammatory cytokine interleukin-10 (IL-10) and tended to increase splenic regulatory T cell numbers but significantly reduced the population of cells expressing activation markers. We demonstrated that inclusion of the boiled-water-insoluble fraction of B. rapa L. can alter the composition of the gut microbiota to decrease the numbers of Bacteroidetes and to increase the numbers of butyrate-producing bacteria, either of which may be involved in the observed shift in the production of splenic IL-10.

Extracts of Larix Leptolepis effectively augments the generation of tumor antigen-specific cytotoxic T lymphocytes via activation of dendritic cells in TLR-2 and TLR-4-dependent manner.

Type-1 immunity plays a crucial role in host defense against various tumors and infectious diseases. Here, we first demonstrated that extract of Larix Leptolepis (ELL), one of the most popular timbers at Hokkaido area in Japan, strongly activated Type-1 immunity. ELL induced production of Type-1 cytokines such as IL-12 and TNF-α from bone marrow-derived dendritic cells (BMDCs) in TLR2- and TLR4-dependent manner and remarkably up-regulated the expression of MHC and co-stimulatory molecules. In addition, antigen-specific CTLs were significantly augmented by the combined administration of ELL, antigen and BMDCs. Finally, we revealed that combination therapy using ELL, antigen and BMDCs significantly inhibited the growth of established tumor in mouse model. Thus, these findings suggested that ELL would be a novel adjuvant for inducing an activation of Type-1-dependent immunity including activation of BMDCs and induction of tumor-specific CTLs, which is applicable to the therapy of cancer and infectious diseases.

Toll-like receptor-dependent IL-12 production by dendritic cells is required for activation of natural killer cell-mediated Type-1 immunity induced by Chrysanthemum coronarium L.

Type-1 immunity has an essential role for our host defenses against cancer and outer pathogens such as bacteria and virus. We demonstrated here that the edible plant extract of Chrysanthemum coronarium L. (C. coronarium) remarkably activates Type-1 immunity in a Toll-like receptor (TLR)2-, TLR4-, and TLR9-dependent manner. In the present experiments, the extract of C. coronarium significantly induces interferon (IFN)-γ production by mouse spleen cells. In addition, the IFN-γ production by spleen cells was completely blocked by the addition of anti-Interleukin (IL)-12 monoclonal antibodies. We confirmed that NK1.1(+) natural killer (NK) cells, NKT cells, and CD11c(+) dendritic cells (DC) were immediately activated after the stimulation with the extract of C. coronarium and the IFN-γ production was abolished in NK1.1(+) cell-depleted spleen cells. The stimulation with the extract of C. coronarium caused DC maturation involving with up-regulations of surface expression levels of MHC class I, MHC class II, CD40, and CD86 as well as induction of IL-12 production. The IFN-γ production induced by the extract was significantly reduced in the spleen cells depleted CD11c(+) cells. Furthermore, the IFN-γ production after the stimulation was strongly reduced in TLR4- and partially in TLR2- and TLR9-deficient spleen cells. Thus, we demonstrated the cellular mechanism for the activation of Type-1 immunity via NK cells, NKT cells, and DC by the extract of C. coronarium. These findings strongly suggest that C. coronarium would be a promising immuno-improving adjuvant, which might be useful for prevention of infectious, cancer, and allergic diseases through the activation of Type-1 immunity.

The extract of Japanese soybean, Kurosengoku activates the production of IL-12 and IFN-γ by DC or NK1.1(+) cells in a TLR4- and TLR2-dependent manner.

During the search for immuno-improving foods, we found that a variety of the Japanese soybean, Glycine max cv. Kurosengoku (Kurosengoku), which activated Type-1 immunity in a Toll-like receptor (TLR)4- and TLR2-dependent manner. Namely, the extract of Kurosengoku first caused production of IL-12 from DC and sequentially induced IFN-γ production by NK1.1(+) NK cells and NKT cells. The IFN-γ production was significantly blocked by neutralizing mAb against IL-12 or TLR4- and TLR2-deficient condition, indicating that TLR4- and TLR2-dependent activation of DC to produce IL-12 was essential for the production of IFN-γ from spleen cells by Kurosengoku. Moreover, the extract of Kurosengoku also enhanced production of IFN-γ from human PBMC by co-stimulation with anti-CD3 mAb in a TLR2- and TLR4-dependent manner. Thus, our findings strongly suggest that Kurosengoku might a novel immuno-improving food, which would be a useful tool for preventing the tip of immune balance in developed countries.