Ken-ichiro Minato

Anti-tumor polysaccharide from the mycelium of liquid-cultured Agaricus blazei mill

Anti‐tumor active polysaccharide against Sarcoma 180 was isolated by DEAE‐Sepharose CL‐6B and Sepharose 4B column chromatography from the hot‐water soluble fraction of the mycelium of liquid‐cultured Agaricus blazei mill. This polysaccharide did not react with antibodies of anti‐tumor polysaccharides such as lentinan, gliforan, and FIII‐2‐b which is one of anti‐tumor polysaccharides from Agaricus blazei. Moreover, the analyses of 13C‐NMR and GC‐MS suggested that this polysaccharide was preliminarily glucomannan with a main chain of β‐1,2‐linked D‐mannopyranosyl residues and β‐D‐glucopyranosyl‐3‐O‐β‐D‐glucopyranosyl residues as a side chain. This polysaccharide was completely different from the anti‐tumor polysaccharide from fruiting body of Agaricus blazei, β‐1,6‐glucan.

Lemon flavonoid, eriocitrin, suppresses exercise-induced oxidative damage in rat liver

To examine the preventive effect of the lemon flavonoid, eriocitrin (eriodictyol 7-O-rutinoside), on oxidative stress during acute exercise in vivo, levels of N( epsilon )- (hexanoyl)lysine, HEL; o,o-dityrosine, DT; and nitrotyrosine, NT, as oxidative stress markers, were determined by ELISA in livers of trained rats in addition to thiobarbituric acid-reactive substance (TBARS). Eriocitrin administration prior to exercise significantly suppressed the increases in TBARS caused by lipid peroxidation during acute exercise. The contents of HEL, DT, and NT in rat liver increased dramatically by exercise without eriocitrin administration. However, these increases were significantly suppressed by eriocitrin administration before exercise. Moreover, in this study, to clarify whether eriocitrin influences glutathione metabolite system that is considered to be important for a defense against the damage by oxidative stress, the levels of glutathione in rat liver were determined during exercise. The level of reduced glutathione after exercise was maintained by administration of eriocitrin. The increase in the concentration of oxidized glutathione caused by exercise was significantly suppressed by eriocitrin. This result suggested that eriocitrin might play an important role in the control of the change in glutathione redox status in rat liver during exercise. These findings showed that eriocitrin was effective in the prevention of oxidative damages caused by acute exercise-induced oxidative stress.

Fucogalactan isolated from Sarcodon aspratus elicits release of tumor necrosis factor-α and nitric oxide from murine macrophages

Eight species of mushrooms were evaluated for mitogenic activity by the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method using spleen cells of C3H/HeN female mice. The hot water-soluble (HWS) fraction extracted from Sarcodon aspratus showed the highest activity. The mitogen in Sa. aspratus was isolated by Sepharose 6B and DEAE-Sepharose CL-6B column chromatography. Preliminary structural analyses indicated that the mitogen was a fucogalactan. Fucogalactan elicited the release of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in macrophages of mice in vitro. TNF-alpha production induced with 50 microg/ml of fucogalactan was significantly higher than that induced by lentinan (500 microg/ml) by approximately 4.3-fold. Also, fucogalactan showed dose dependence at concentrations from 5 to 500 microg/ml in NO production. Thus, fucogalactan does elicit the release of cytokines such as TNF-alpha and NO.

New potent antioxidative hydroxyflavanones produced with Aspergillus saitoi from flavanone glycoside in citrus fruit.

Potent antioxidative hydroxyflavanones were produced with Aspergillus saitoi from hesperidin or naringin, which are flavanone glycosides in citrus fruit with weak antioxidative activity. The hydroxyflavanone produced from hesperidin was identified as 8-hydroxyhesperetin (8-HHE), a novel substance, and those from naringin were identified as carthamidin (6-hydroxynaringenin) and isocarthamidin (8-hydroxynaringenin) by FAB-MS, 1H-NMR and 13C-NMR analyses. The antioxidative activity of these hydroxyflavanones was examined by using the free radical-scavenging system of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and the methyl linoleate oxidation system. The hydroxyflavanones (8-HHE, carthamidin, and isocarthamidin) exhibited stronger activity than the flavanone glycosides (hesperidin or naringin) and their aglycones (hesperetin or naringenin). The activity of 8-HHE and isocarthamidin was comparable to that of α-tocopherol, and that of carthamidin was weaker than that of isocarthamidin. The hydroxyflavanones, which were hydroxylated on A ring of flavanone by Aspergillus saitoi, were obtained as potent antioxidants.

Suppressive effect of polysaccharides from the edible and medicinal mushrooms, Lentinus edodes and Agaricus blazei, on the expression of cytochrome P450s in mice.

To investigate the effects of lentinan from Lentinas edodes and polysaccharides from Agaricus blazei (ABPS) on the expression of cytochrome P450s (CYPs), lentinan (10 mg/kg/day) or ABPS (200 mg/kg/day) was administered to female BALB/c mice four times every other day by intraperitoneal injection. Lentinan and ABPS suppressed both the constitutive and 3-methylcholanthrene-induced CYP1A expression and ethoxyresorufin-O-deethylation activity in the liver.

Preparation and specificity of antibodies to an anti-tumor β-glucan, lentinan

Antibodies against β‐glucan, lentinan from “Shiitake” (Lentinus edodes), were raised in the rabbit by subcutaneous immunization. Our antibodies reacted significantly with lentinan by inhibition assay of ELISA. The antibodies did not recognize the other polysaccharides such as amylose, dextran, laminarin and galactan. It was proved that lentinan contents in mushroom could be measured by ELISA with the anti‐lentinan antisera. Its contents were 3.5 mg/g fresh weight in Lentinus edodes. However, lentinan was not contained in Agaricus brazei, Agaricus bisporus and Ramaria bitrytis.

3-Amino-1,4-dimethyl-5H-pyrido(4,3-b)indole (Trp-P-1) induces caspase-dependent apoptosis in mononuclear cells

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), one of the tryptophan pyrolysates, is a dietary carcinogen and is formed in cooked meat and fish in our daily diet. Trp-P-1 will affect the cells in the blood circulation system before it causes carcinogenicity in target organs such as the liver. In this study, the cytotoxicity of Trp-P-1 was investigated in mononuclear cells (MNCs) from blood. Trp-P-1 (10-15 microM) decreased cell viability and induced apoptosis characterized both by morphological changes and by DNA fragmentation 4 h after treatment. DNA fragmentation was also observed following treatment at 1 nM after 24 h in culture. This result suggested that apoptosis would occur in the body following unexpected intake of foods containing Trp-P-1. To determine the mechanism of apoptosis, we investigated the activation of the caspase cascade in MNCs. Trp-P-1 (10-15 microM) activated the caspase cascade, i.e. the activity of caspase-3, -6, -7, -8 and -9 increased dose-dependently using peptide substrates, the active forms of caspase-3, -8 and -9 were detected by immunoblotting, and cleavage of poly(ADP-ribose) polymerase and protein kinase C-delta as the intracellular substrates for caspases was observed. A peptide inhibitor of caspase-8 completely suppressed activation of all other caspases, while an inhibitor of caspase-9 did not. These results indicated that caspase-8 may act as an apical caspase in the Trp-P-1-activated cascade.

Mushrooms: Immunomodulating Activity and Role in Health Promotion

Lentinula edodes, Grifola frondosa, Pleurotus cornucopiae, Pholiota nameko, and many other kinds of edible mushrooms have shown their potent and unique immunomodulating activities. It has mainly been recognized that their activities result from the action of their polysaccharides or polysaccharide–protein complexes. The polysaccharides appear to stimulate the human immune response and to be effective in preventing immunodeficiency diseases via activation of cytokine networks. In this chapter, these properties of the immunomodulating polysaccharides from L. edodes, G. frondosa, P. nameko, P. cornucopiae, and others are described.

A Proinflammatory Effect of the β-Glucan from Pleurotus cornucopiae Mushroom on Macrophage Action

PCPS from P. citrinopileatus mushroom extract is a β-1,6-glucan possessing a proinflammatory effect on innate immune cells. The PCPS stimulated THP-1 macrophages to secrete significant levels of TNF. Moreover, the mRNA expressions of TNF and IL-1β were significantly enhanced by PCPS treatment. However, the PCPS did not induce to express both IL-12 and IL-10 mRNA in the macrophages. Next, the P. cornucopiae extract (containing mainly PCPS) treatment against mice showed significant increases in TNF and IL-1β mRNA expressions in the peritoneal macrophages of them. In this study, the expression levels of IFNγ mRNA in the spleen were almost the same between the extract- (PCPS-) treated group and control group. However, the expression of IL-4 mRNA showed a lower level in the extract-treated group than that in the control. Our results suggested that the PCPS could induce proinflammatory action in the immune response. In addition, the proinflammatory effect of the PCPS on THP-1 was enhanced by 5′-GMP-Na, while it was reduced by vitamin D2. These two compounds are majorly contained in the P. citrinopileatus mushroom. Therefore, these results suggested that the P. citrinopileatus mushroom might contain other immune regulative compounds, such as vitamin D2, as well as PCPS.

Hexanoyl-Lysine as an Oxidative-Injured Marker – Application of Development of Functional Food

We could proposed that N(ε)-(hexanoy)lysine, HEL, become a useful biomarker for detection of oxidative stress damage occurred by exhaustive exercise. We examined the preventive effect of flavonoid compound, eriocitrin, against exercise-induced oxidative damage in rat liver. Eriocitrin administration prior to exercise significantly suppressed the increases in thiobarbituric acid-reactive substance caused by lipid peroxidation during exhaustive exercise. The increase in the contents of HEL in rat liver was also abolished by eriocitrin administration. The concentration of oxidized glutathione was significantly increased by exercise, but the eriocitrin administration suppressed this increase. These results suggested that eriocitrin administration prior to exercise prevented oxidative damages caused by exhaustive exercise-induced oxidative stress. Therefore, it was suggested that HEL could be a good biomarker for oxidative stress, especially at earlier stage when oxidative damage was occurred by lipid peroxidation than a stage of harmful aldehyde formation. Moreover, it was suggested that eriocitrin metabolites, eriodictyol and 3, 4 - dihydroxyhydrocinnamic, might scavenge free radicals and reactive oxygen species, resulting in suppression of lipid peroxidation and reactive proteins with radicals to form HEL. These findings implied that eriocitrin might be useful as an anti-oxidative compound to protect oxidative stress damages.