Putative Effect of Spirulina Extract on Enzyme Activities Participating in Lipid and Carbohydrate Digestion Processes.

Excessive calorie intake is generally accepted as a primary cause of metabolic syndrome, and therefore a well-balanced diet and moderate exercise can be expected to be the most effective measures to avoid the disorder of energy utilization and storage. Furthermore, as any other way to improve the disorder of energy balance, it may be effective to delay and lower the digestion and/or absorption of energy sources, lipids, and carbohydrates. As a primary screening of effective substances to delay and lower the digestion and absorption processes among natural materials, the protein-deprived extract was prepared from blue-green algae Spirulina platensis, and the effect of this extract on lipase and α-glucosidase activities was examined. The extract was shown to inhibit lipase activity but not α-glucosidase activity, thus proposing the possibility that the extract prevented the postprandial elevation of blood triglyceride (TG) levels as a result of reducing the digestion and absorption of lipids in the intestinal tract. Therefore, it seems possible to speculate that nonprotein components of Spirulina may be able to effectively improve the disorder of energy balance as a consequence of suppressing the excessive intake of calories by reducing the absorption of lipids in patients with metabolic syndrome.

Lotus Root Extract Stimulates BDNF Gene Expression Through Potential Mechanism Depending on HO-1 Activity in C6 Glioma Cells.

Polyphenolic compounds have been suggested to be involved in the preservation of neural function via the production of neurotrophic factors in the brain. The nonedible joint part of lotus root (a rhizome of Nelumbo nucifera) has been reported to contain large amounts of polyphenolic compounds and, therefore, is expected to improve neural function by stimulating the production of brain-derived neurotrophic factor (BDNF) in glial cells. The effect of the aqueous extract prepared from the joint part of lotus root on BDNF gene expression was examined in C6 glioma cells as an in vitro model. This extract was shown to increase BDNF messenger ribonucleic acid (mRNA) levels to the elevation of HO-1 mRNA levels in the glioma cells, but failed to cause the elevation of BDNF mRNA levels in the cells pretreated with a HO-1 antisense oligodeoxynucleotide (ODN) or an HO-1 enzyme inhibitor zinc protoporphyrin (ZnPP). These findings strongly suggest that the aqueous extract prepared from the nonedible joint part of lotus root might be able to stimulate BDNF gene expression by enhancing HO-1 activity in the glioma cells, proposing the possibility that the joint part of lotus root might potentially improve neural function through the stimulation of BDNF production in glial cells.

Suppressive effect of Okara on intestinal lipid digestion and absorption in mice ingesting high-fat diet.

Soymilk residue Okara is paid attention as a low-calorie foodstuff effective for the amelioration of obesity, and expected to have the potential ability to reduce calorie intake by suppressing the digestion and absorption of high-calorie nutrients in the intestinal tract. Then, the direct effect of Okara extract on lipase activity was examined, and this extract was shown to inhibit the enzyme activity. On the other hand, the spray-dried powder of Okara extract was suspended in a drinking water and given to mice fed with a high-fat diet. Consequently, Okara suspension was shown to reduce triglyceride (TG) levels in the blood, thus suggesting that Okara ingestion could suppress the digestion and absorption of lipids through the inhibition of lipase activity in the intestinal tract. Therefore, Okara is speculated to have the potential ability to prevent the excess intake of calories, thereby being effective and useful for the amelioration of metabolic syndrome.

Soy Pulp Extract Inhibits Angiotensin I-Converting Enzyme (ACE) Activity In Vitro: Evidence for Its Potential Hypertension-Improving Action.

Soy pulp, called "okara" in Japanese, is known as a by-product of the production of bean curd (tofu), and expected to contain a variety of biologically active substances derived from soybean. However, the biological activities of okara ingredients have not yet been fully understood, and the effectiveness of okara as a functional food seems necessary to be further evaluated. Then the effect of okara extract on angiotensin I-converting enzyme (ACE) activity was examined in vitro, and the extract was shown to cause the inhibition of ACE activity in a manner depending on its concentration. Kinetic analysis indicated that this enzyme inhibition was accompanied by an increase in the Km value without any change in Vmax. Further studies suggested that putative inhibitory substances contained in the extract might be heat stable and dialyzable, and recovered mostly in the peptide fraction obtained by a spin-column separation and a high performance liquid chromatography (HPLC) fractionation. Therefore, the extract was speculated to contain small-size peptides responsible for the inhibitory effect of okara extract on ACE activity, and could be expected to improve the hypertensive conditions by reducing the production of hypertensive peptide.

Fermented Brown Rice Extract Stimulates BDNF Gene Transcription in C6 Glioma Cells: Possible Connection with HO-1 Expression.

Fermented brown rice with Aspergillus oryzae, designated as FBRA, is known to be commercially available dietary fiber-rich food, which is appreciated as prebiotics to improve intestinal microflora, and also shown to contain various biologically active substances including polyphenolic compounds. On the other hand, polyphenolic compounds have been suggested to stimulate the expression of brain-derived neurotrophic factor (BDNF) gene in connection with the expression of heme oxidase-1 (HO-1) gene in glial cells, thus resulting in the augmentation of BDNF production in the brain, thereby being anticipated to have a putative effect on the brain function. Then, the effect of FBRA extract on HO-1 and BDNF messenger ribonucleic acid (mRNA) levels in C6 glioma cells was examined, and the extract was shown to stimulate both HO-1 and BDNF gene transcription in the glioma cells. Further studies showed that the stimulatory effect of FBRA extract on BDNF gene transcription was almost completely suppressed by silencing HO-1 gene expression with an HO-1 antisense oligodeoxynucleotide and also inhibiting HO-1 activity with an inhibitor zinc protoporphyrin, thus suggesting that FBRA might have a potential ability to induce BDNF gene expression through HO-1 activity in glial cells.

Spirulina non-protein components induce BDNF gene transcription via HO-1 activity in C6 glioma cells.

Blue-green algae are known to contain biologically active proteins and non-protein substances and considered as useful materials for manufacturing the nutritional supplements. Particularly, Spirulina has been reported to contain a variety of antioxidants, such as flavonoids, carotenoids, and vitamin C, thereby exerting their protective effects against the oxidative damage to the cells. In addition to their antioxidant actions, polyphenolic compounds have been speculated to cause the protection of neuronal cells and the recovery of neurologic function in the brain through the production of brain-derived neurotrophic factor (BDNF) in glial cells. Then, the protein-deprived extract was prepared by removing the most part of protein components from aqueous extract of Spirulina platensis, and the effect of this extract on BDNF gene transcription was examined in C6 glioma cells. Consequently, the protein-deprived extract was shown to cause the elevation of BDNF mRNA levels following the expression of heme oxygenase-1 (HO-1) in the glioma cells. Therefore, the non-protein components of S. platensis are considered to stimulate BDNF gene transcription through the HO-1 induction in glial cells, thus proposing a potential ability of the algae to indirectly modulate the brain function through the glial cell activity.

Polyamines cause elevation of steroid 5α-reductase mRNA levels by suppressing mRNA degradation in C6 glioma cells.

Polyamines are widely distributed in living organisms, and considered to play a potential role in various cellular processes. The effects of polyamines on gene expression as well as cell proliferation have been suggested to be closely associated with the physiological and pathological functions. However, it seems necessary to investigate their potential roles in the regulation of cellular metabolism and functions. Previously, glial cells have been suggested to be involved in the protection and preservation of neuronal functions, probably through the production of neurotrophic factors in the brain. On the other hand, neuroactive 5α-reduced steroids promote glial cell differentiation, resulting in enhancement of their ability to produce brain-derived neurotrophic factor (BDNF). Based on these findings, polyamines are assumed to stimulate the expression of the gene encoding steroid 5α-reductase (5α-R), which can induce the production of neuroactive 5α-reduced steroids in glial cells. The effects of polyamines on 5α-R mRNA levels in C6 glioma cells were examined as a model experiment. In consequence, spermine (SPM) and spermidine (SPD), but not putrescine (PUT), have been shown to elevate 5α-R mRNA levels without activating the 5α-R promoter. Furthermore, SPM increased 5α-R mRNA levels under the conditions in which the mRNA biosynthesis was inhibited. Therefore, it can be speculated that polyamines increase 5α-R mRNA levels as a consequence of suppressing the degradation of mRNA.

Trichostatin A enhances glutamate transporter GLT-1 mRNA levels in C6 glioma cells via neurosteroid-mediated cell differentiation.

The neurotoxic effects of excitatory amino acids (EAAs) are suggested to be connected with the chronic loss of neuronal cells, thereby being responsible for the age-related neurodegenerative diseases. Therefore, it seems conceivable that the excitatory amino acid transporters may contribute to the protection of neuronal cells against the excitotoxic damage by facilitating the removal of EAAs from the brain tissue. On the other hand, previous studies have suggested that glial cell differentiation may be involved in the protection and recovery of neural function probably through the elevation of BDNF gene expression in the brain. Based on these findings, histone deacetylase (HDAC) inhibitors are assumed to induce glutamate transporter-1 (GLT-1) gene expression probably through the promotion of glial cell differentiation. Then, we examined the effects of HDAC inhibitors on GLT-1 mRNA levels in rat C6 glioma cells and found that trichostatin A can induce GLT-1 gene transcription following steroid 5α-reductase and GFAP gene expression. Therefore, it seems conceivable that glial cell differentiation may play a potential role in the removal of EAAs probably through the expression of GLT-1, thereby being involved in the protection of neuronal cells against the chronic excitotoxic insults in the brain.

Palytoxin causes nonoxidative necrotic damage to PC12 cells in culture.

Palytoxin (PTX) is a potent marine toxin that causies serious damage to various tissues and organs. It has been reported to affect the transport of cations across the plasma membranes, which is commonly recognized as being the principal mechanism of its highly toxic action on mammals, including humans. However, although some marine toxins have been shown to cause toxic effects on the nervous system by interfering with the transmission of nerve impulses, the effect of PTX on neuronal cells has not yet been fully elucidated. Therefore, the toxic action of PTX on PC12 cells was examined as an in vitro model experiment to elucidate the neurotoxic properties of this toxin, and PTX was shown to reduce the viability of PC12 cells in a concentration-dependent manner. The cytotoxic action of PTX was not significantly altered by the presence of the antioxidant N-acetylcysteine and reduced-form glutathione in the cultures. Fluorescence staining of the cells and the electrophoretic analysis of genomic DNA showed that PTX failed to cause chromatin condensation and DNA fragmentation within the cells. On the other hand, the exposure to PTX caused positive staining of the cytoplasmic space of the cells with propidium iodide and the release of lactate dehydrogenase into the culture medium. Based on these observations, PTX is considered to cause cell death as a consequence of disrupting the plasma membranes, thus causing nonoxidative necrotic damage to PC12 cells.

Extract of fermented brown rice induces apoptosis of human colorectal tumor cells by activating mitochondrial pathway.

Brown rice fermented with Aspergillus oryzae, designated as FBRA, is a dietary fiber-rich food, and fully appreciated as one of the prebiotics, which are generally considered to be beneficial to the health of the body, because of stimulating the growth and/or the activity of bacteria in the digestive system. To assess the effectiveness of FBRA as a functional food, the direct effect of FBRA extract on human colorectal tumor cells was examined. The exposure of HCT116 cells to FBRA extract reduced their viabilities in a concentration-dependent manner, and the reduction of the cell viability might be attributed to the induction of apoptosis probably through the oxidative damage to the cells. Further studies showed that FBRA extract caused a significant elevation of Bax protein and a slight reduction of Bcl2 protein levels, and furthermore caused the activation of caspase-3 in the cells. Thus, it seems reasonable to conclude that FBRA extract can exert oxidative damage to the cells, resulting in apoptotic cell death by activating the mitochondrial pathway in human colorectal tumor cells. Therefore, daily intake of FBRA can be expected to be beneficial for preventing carcinogenesis and/or suppressing tumor growth in the digestive tract.

In vitro cytotoxic effect of ethanol extract prepared from sporophyll of Undaria pinnatifida on human colorectal cancer cells.

Brown seaweed Undaria pinnatifida (Harvey) Suringar is popular as a foodstuff, and used for medical care in East Asian countries. The major components of this seaweed are shown to benefit hypertension and hyperlipidemia, and considered to reduce the risks of infarction and ischemic diseases. Furthermore, the intake of dietary fiber of seaweeds is considered to prevent the production and proliferation of cancer in the gastrointestinal tract. The direct effect of an ethanol extract prepared from Undaria pinnatifida sporophyll (mekabu) on HCT116 human colorectal cancer cells was examined, and the mekabu extract was shown to induce the non-oxidative apoptotic damage to the cells, thus resulting in the reduction of their viabilities in a concentration-dependent manner. Moreover, the cytotoxic effects of carcinostatic drugs, such as 5-fluorouracil (5-FU) and irinotecan (CPT-11), were observed only in the medium containing sera, while the mekabu extract could effectively reduce the cell viabilities even in the serum-free medium. These findings suggest that the mekabu extract may contain a potential active substance inducing the non-oxidative apoptotic cell death probably through a mechanism different from those of 5-FU and CPT-11, and hence mekabu is possibly useful as an auxiliary drug to the chemotherapy of colorectal cancer.

[Toxicity and toxin profile of the dinoflagellate Alexandrium tamiyavanichii and toxic mussels in Harima-Nada of Seto Inland Sea, Japan].

From October to November 2004, the paralytic shellfish poison (PSP)-producing dinoflagellate Alexandrium tamiyavanichii was observed at Harima-Nada, Seto Inland Sea at a maximum cell density of 4,960 cells/L. The wild cells of the dinoflagellate collected from the same seawaters, and cultured cells derived from them showed toxicity scores of 6.25-15.4 x 10(-4) and 2.7-3.5 x 10(-4) MU/cell, respectively, both of which were much higher than those of previously reported strains. PSP of the wild cells was mainly composed of gonyautoxin (GTX) 5 (40.6-52.4 mol%) and GTX4 (15.6-24.8 mol%), showing a unique composition that was greatly different from those of the previously reported strains, or of the cultured cells, whose main toxin component was GTX3 (average 37.6 mol%). The mussel Mytilus galloprovincialis collected from the same area in the same period accumulated a relatively high level of PSP (13-28 MU/g), suggesting a risk that A. tamiyavanichii may induce high-level PSP contamination of bivalves even at a cell density as low as around 5,000 cells/L.

[Toxicity and toxin profiles of xanthid crabs collected around Nakanoshima in the Tokara Islands, Japan].

A total of 36 specimens of 5 xanthid crab species, Zosimus aeneus (n=16), Xanthias lividus (n=4), Leptodius sanguineus (n=3), Daira perlata (n=10) and Eriphia sebana (n=3), were collected around Nakanoshima Island, which is located at the northeastern part of the Tokara Islands, Kagoshima Prefecture, Japan in May and July 2000, and their toxicity was determined by mouse bioassay. Nine of 16 Z. aeneus specimens and all of 4 X. lividus specimens showed lethal potency to mice (2.1-11 MU/g, 2.8-8.6 MU/g, respectively), whereas all the other species were non-toxic (less than 2.0 MU/g). LC/MS analyses indicated that the toxin of the Z. aeneus specimens was mainly composed of tetrodotoxin (41% of total toxicity), and 11-oxotetrodotoxin contributed to the remaining toxicity. The toxin of the X. lividus specimens, however, was apparently not tetrodotoxin. In HPLC-FLD analyses, no paralytic shellfish poison component was detected in either of the two toxic species.

Occurrence of the polyamines caldopentamine and homocaldopentamine in axenic cultures of the red tide flagellates Chattonella antiqua and Heterosigma akashiwo (Raphidophyceae).

The polyamines caldopentamine and homocaldopentamine were detected in axenic strains of Chattonella antiqua and Heterosigma akashiwo (Raphidophyceae), respectively, as well as spermidine, the most abundant polyamine in both phytoplankton species. Trace amounts of putrescine, diaminopropane and norspermine were also detected in both species. Spermine was detected only from C. antiqua. These long linear polyamines are characteristic components of thermophilic bacteria. The detection from two species of Raphidophyceae indicates that the occurrence of long linear polyamines is not restricted to thermophilic microorganisms.

A new analytical method for gonyautoxins based on postcolumn HPLC.

A new ion-pairing high-performance liquid chromatography (HPLC) method on a C30 column with a volatile mobile phase was developed to separate the gonyautoxin group (GTXs) from contaminants, allowing the utilization of liquid chromatography/mass spectrometry (LC/MS) with higher performance. A mobile phase consisting of 5 mmol/L heptafluorobutyric acid and 2% acetonitrile in 10 mmol/L ammonium acetate was adopted for separation of GTXs because the C30 column strongly retains GTXs under acidic conditions. The newly adopted method could efficiently separate GTXs from contaminants, especially in the toxic short-necked clam, whereas the routine HPLC so far used has poor resolution to separate GTXs from unknown interfering substances. In our method, GTXs were eluted in the order of GTX5, GTX3, GTX4, GTX2 and GTX1 from the C30 column, and were successfully determined by sonic spray ionization mass spectrometry (SSI-MS) with high sensitivity. This method is characterized by the combination of HPLC using a fluorescence detection system for PSP, and SSI-MS for measurement of the mass number.

First paralytic shellfish poison (PSP) infestation of bivalves due to toxic dinoflagellate Alexandrium tamiyavanichii, in the southeast coasts of the Seto Inland Sea, Japan.

The mussel Mytilus edulis and the cultured ark shell Anadara broughtonii in the southeast coasts of the Seto Inland Sea were contaminated with paralytic shellfish poison (PSP) following the appearance of the dinoflagellate Alexandrium tamiyavanichii in early December 1999. A. tamiyavanichii plankton collected around the Straits of Naruto on December 3, 1999 showed PSP toxicity, of which 83 mol% was accounted for by GTX2, GTX3 and GTX4. Its specific toxicity was 112.5 fmol/cell, and one MU was equivalent to 7,200 cells. Toxicity values at the beginning of toxification were 4.7 MU/g for the ark shell and 7.3 MU/g for the mussel. In the former, the value remained at almost 4 MU/g, resulting in prohibition of marketing for about two months. In the latter, it sharply decreased to less than 4 MU/g. These bivalves collected during the toxification period were dissected into five tissues, mantle, adductor muscle, hepatopancreas, gills and "others", and submitted to high-performance liquid chromatography (HPLC). The cultured ark shell accumulated GTX2, GTX3 and STX as major components and GTX1, GTX4, GTX5, neoSTX, dcSTX and PX1-3 (C1-C3) as minor ones. The amount of GTX3 decreased with time, while STX tended to increase. At the early stage of PSP toxification, toxins were accumulated in the gills and "others", most of which were quickly detoxified. On the other hand, PSP of the toxified mussel consisted of GTX4 as a main component, and GTX1, GTX2, GTX3, GTX5, STX and PX1-2 (C1-C2) as minor ones. Its toxin composition pattern was similar to that of the ingested causative plankton. Its total toxin decreased soon after disappearance of the dinoflagellate. During the decrease of toxicity, PSP tended to be retained in the hepatopancreas, resulting in accumulation of 50 mol% of total toxin.