Albumin extracted from serum by a simple technique using trichloroacetic acid and ethanol has been applied to a fructosamine assay using nitroblue tetrazolium. A fructosamine assay using extracted albumin sample was carried out without interference from low molecular weight substances with reducing activities and other proteins with varying concentrations, half-lives and reducing activities. 1-Deoxy-1-p-toluidino-D-fructose (DTF) was selected as a calibrator for the assay because it is a stable Amadori reaction product with a constant glycation rate. Albumin fructosamine value was calculated using the calibration curve of DTF. The corrected albumin fructosamine value was expressed as the amount of albumin fructosamine per gram of extracted albumin taking into consideration the variation in albumin concentrations in sera from patients. The corrected albumin fructosamine values correlated more closely with the fasting blood glucose levels (r=0.735) than the serum fructosamine values corrected for albumin concentrations (r=0.514) (p<0.05).

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Cytosol treated with guanosine 5′-O-(3-thiotriphosphate) (GTPγS) destroys the dextran-loaded lysosomes (J. Biochem., 123, 637 (1998)). The transfer of the ADP-ribosylation factor (ARF) from the cytosol to the lysosomal membrane is necessary for this lysis to occur. The role of ARF in the biogenesis of the Golgi complex is to generate high-affinity membrane-binding sites for the heterotetrameric adaptor protein complex in Golgi membranes. We have found that ARF also recruits the adaptor protein to lysosomes. The recruitment of protein coats for vesicles is necessary for the GTPγS-stimulated lysis of lysosomes. The GTPγS-induced lysis proceeded via a process similar to that for the assembly of coated proteins to coated vesicles, which serve to transport proteins between intracellular organelles.

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The frequency of generation of multidrug-resistant mutants of Staphylococcus aureus was examined by selection with various antiseptics and fluoroquinolones and the mutated genes were characterized. Multidrug-resistant mutants with mutation of the region between the promoter and structural regions of the norA gene were generated more frequently during selective pressure with antiseptics than during selective pressure with FQs. We also obtained evidence for the existence of at least one other gene related to resistance to antiseptics and fluoroquinolones on the chromosome of S. aureus in addition to norA gene.

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We attempted to identify the exchange mechanism of transferrin–⁶⁷Ga complex to heparan sulfate–⁶⁷Ga complex. The effect of phosphate on the binding ability of ⁶⁷Ga to transferrin and heparan sulfate was studied by the dialysis method. The phosphate inhibited the binding ability of ⁶⁷Ga to transferrin, while the phosphate enhanced the ⁶⁷Ga binding ability to heparan sulfate. The results suggest that the phosphate is involved in the translocation of ⁶⁷Ga from the transferrin to the heparan sulfate.

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Ty2 retrotransposons are the mobile genetic elements of the yeast Saccharomyces cerevisiae. Transcriptional regulation of Ty2-917 requires a complex set of cis acting elements which are located both upstream and downstream of the transcription initiation site. Previously, the glycolysis regulatory protein Gcr1p has been identified as the major transcriptional regulator of Ty2-917. Gcr1p is a DNA binding transcription factor that requires Gcr2p for its functions. In this study, the effect of Gcr2p on the transcriptional regulation of Ty2-917 was analyzed. The result of this study indicates that Ty2-917 transcription decreases 24-fold in gcr2 mutant yeast cells. In addition, Ty2 enhancer element dependent transcriptional activation of a heterolog promoter also decreases at a significant level. These results showed that Gcr2p is essential for the high level transcription of Ty2-917.

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Peroxisome proliferators stimulate hepatocyte growth in rat liver in vivo. Activin A, a homodimer of inhibin β_A, inhibits DNA synthesis in hepatocytes. The inhibitory action of activin A is suppressed by follistatin, an activin-binding protein. In this paper, we investigated whether administration of di-n-butyl phthalate (DBP), a peroxisome proliferator, modifies the production of activin A and follistatin in rat liver by hourly monitoring of inhibin β_A and follistatin mRNA levels by reverse transcriptase polymerase chain reaction analysis. The mRNA levels of the other inhibin β chains (inhibin β_B and β_C) were examined in a similar manner. The inhibin β_A mRNA level decreased to about 30% by 3 h after DBP administration (8.6 mmol/kg body weight), remained low until 12 h, and returned to its original level by 24 h. The follistatin mRNA level increased to about 2 times by 6 h, and returned to its original level by 24 h. The inhibin β_B mRNA had started to increase by 1 h, peaked at 6 h at about 4 times its initial level, and returned to its original level by 12 h. The inhibin β_C mRNA level had doubled by 6 h and it returned to its original level. These results indicate that the growth stimulatory action of peroxisome proliferators may be mediated via the decrease in activin A level and activity and suggest that the increases in follistatin as well as inhibin β_B and β_C chains may play a role in peroxisome proliferator-stimulated hepatocyte growth.

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Rat cytokine-induced neutrophil chemoattractant-3 (CINC-3) has neutrophil chemotactic activity comparable with that of CINC-1 and CINC-2, but induces calcium mobilization more potently than CINC-1 and CINC-2. However, only one CINC receptor, CXCR2, has been found in rat neutrophils. Therefore we attempted to determine the biochemical basis for the differences in neutrophil responses to CINC-1/-2 versus CINC-3. Both chemotactic activity and calcium mobilization induced by CINC-3 were desensitized by a 100-fold excess of CINC-1, which was consistent with our previous results showing that CINC-1 has 70-fold lower affinity to the receptor on rat neutrophils than CINC-3. Desensitization appeares to be reflected by the affinity of the ligands to the receptor. CINC-1- and CINC-3-induced chemotaxis was sensitive to inhibition by pertussis toxin, whereas calcium mobilization induced by CINC-1 and CINC-3 was insensitive. These results suggest that CINCs induce neutrophil chemotaxis and calcium mobilization through distinct G-proteins with different efficiency.

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We constructed a pig 3α/β,20β-hydroxysteroid dehydrogenase (3α/β,20β-HSD) mutant, which lacks 12 carboxyl-terminal amino acids residues. Enzyme activity studies indicated that the deleted amino acids have a role in steroid metabolism and may assist in substrate binding in wild-type 3α/β,20β-HSD. Furthermore, substrate binding likely induces a conformational change allowing the 12 carboxyl-terminal amino acids interact with the steroid substrate [Nakajin S. et al., Biochim. Biophys. Acta, 1550, 175—182 (2001)]. In this paper, we clarified that although pig 3α/β,20β-HSD is potently inhibited by dexamethasone, glycyrrhetinic acid and spironolactone, this inhibition is remarkably attenuated by deleting the 12 carboxyl-terminal residues. The inhibition constant (K_i) of pig 3α/β,20β-HSD for dexamethasone increased 115-fold. These observations also indicate that these amino acid residues interact with steroid substrates or steroid inhibitors and have an important role in substrate or inhibitor binding to the active site.

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The aim of this study was to determine the effects of Hippophae rhamnoides L. extract (HRe-1) and also vitamin E as a positive control on nicotine-induced oxidative stress in rat blood, specifically alterations in erythrocyte malondialdehyde (MDA) level, activities of some erythrocyte antioxidant enzymes, and plasma vitamin E and A levels. The groups were: nicotine (0.5 mg/kg/d, intraperitoneal, i.p.); nicotine+vitamin E (75 mg/kg/d, intragastric, i.g.); nicotine+HRe-1 (1 ml/kg/d, i.g.); and control group (receiving only vehicles). There were 8 rats per group and the supplementation period was 3 weeks. Nicotine-induced increase in erythrocyte MDA level was prevented by both HRe-1 and vitamin E. Nicotine-induced decrease in erythrocyte superoxide dismutase (SOD) activity was prevented by HRe-1, but not vitamin E. HRe-1 increased the erythrocyte glutathione peroxidase (GSH-Px) activity compared with nicotine and the vitamin E groups. Catalase activity was not affected. Vitamin E supplementation increased plasma vitamin E level. Plasma vitamin A level was higher in both vitamin E and HRe-1 supplemented groups compared with nicotine and control groups. The results suggest that HRe-1 extract can be used as a dietary supplement, especially by people who smoke, in order to prevent nicotine-induced oxidative stress.

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The fruit hull of mangosteen, Garcinia mangostana L. has been used as a Thai indigenous medicine for many years. However, its mechanism of action as a medicine has not been elucidated. The present study was undertaken to examine the effects of mangosteen extracts (100% ethanol, 70% ethanol, 40% ethanol and water) on histamine release and prostaglandin E₂ synthesis. We found that the 40% ethanol extract of mangosteen inhibited IgE-mediated histamine release from RBL-2H3 cells with greater potency than the water extract of Rubus suavissimus that has been used as an anti-allergy crude drug in Japan. All extracts of mangosteen potently inhibited A23187-induced prostaglandin E₂ synthesis in C6 rat glioma cells, while the water extract of Rubus suavissimus had no effect. The 40% ethanol extract of mangosteen inhibited the prostaglandin E₂ synthesis in a concentration-dependent manner with relatively lower concentrations than the histamine release. In addition, passive cutaneous anaphylaxis (PCA) reactions in rats were significantly inhibited by this ethanol extract as well as by the water extract of Rubus suavissimus. These results suggest that the 40% ethanol extract of mangosteen has potent inhibitory activities of both histamine release and prostaglandin E₂ synthesis.

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Oldenlandia diffusa (OD) has been used to treat malignant tumors. In this study using mouse peritoneal macrophages, we have examined the mechanism by which OD regulates nitric oxide (NO) production. When OD (1 mg/ml) was used in combination with 10 U/ml of recombinant interferon-γ (rIFN-γ), there was a marked cooperative induction of NO production (36.13±7.12 μM) by the Griess method (nitrite). Treatment of macrophages with rIFN-γ plus OD (1 mg/ml) caused a significant increase in tumor necrosis factor-α (TNF-α) production (4.49±1.43 ng/ml) by enzyme-linked immunosorbent assay. The increased production of NO and TNF-α from rIFN-γ-plus OD-stimulated cells was almost completely inhibited by pretreatment with 100 μM of pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor kappa B (NF-κB). PDTC also inhibited phosphorylation of IκB in rIFN-γ-plus OD-stimulated cells. These findings demonstrate that OD increases the production of NO and TNF-α by rIFN-γ-primed macrophages and suggest that NF-κB plays a critical role in mediating these effects of OD.

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Ethanolic and aqueous extracts of 14 South American medicinal plants were tested for inhibitory activity on human immunodeficiency virus (HIV). Both extracts were relatively non-toxic to human lymphocytic MT-2 cells, but only the aqueous extract of Baccharis trinervis exhibited potent anti-HIV activity in an in vitro MTT assay. To delineate the extract-sensitive phase, some studies of the antiviral properties of the active extract are described in this paper. Based on the results presented here, a separation scheme was devised, which permitted the preliminary fractionation of the extract, with the aim of finding an inhibitor of this virus.

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Quassia amara L., a neotropical forest shrub of the Simaroubaceae family, is widely used in Caribbean folk medicine and in some northern states of Brazil for the treatment of gastric ulcers. This plant is a source of numerous compounds including both β-carbonile and cantin-6 alkaloids as well as, primarily, the bitter compounds known as quassinoids. We analyzed the possible antiulcerogenic activities of four extracts of different polarities: 70% ethanol (70% EtOH), 100% EtOH, 100% dichloromethane (DCM), and 100% hexane (HEX) obtained from Quassia amara bark. All extracts, administered at doses of 5000 mg/kg orally and 1000 mg/kg intraperitoneally, caused neither toxicity or death. In the indomethacin/bethanechol-induced gastric ulcer, 70% EtOH, 100% EtOH, DCM and HEX extracts, 100 mg/kg, p.o., inhibited the gastric ulcer (22.5, 23.4, 50.5, 46.8%, respectively). 70% EtOH, 100% EtOH, DCM, and HEX extracts reduced the gastric injury induced by the hypothermic restraint–stress test in mice (70.7, 80, 60, 82.7%, respectively). In the pylorus ligature of the mouse stomach, following pre-treatment with a single intraduodenal administration of 100 mg/kg of each extract, only 70% EtOH did not change the biochemical parameters of gastric juice. 100% EtOH, DCM and HEX extracts presented decreased gastric juice content, increased pH values and decreased acid output. We also determined the antiulcerogenic activity on HCl–EtOH-induced gastric ulcers in mice at four doses (25, 50, 75, 100 mg/kg, p.o.), then evaluated the possible dose-dependent relation and calculated the ED₅₀ values. Except for 70% EtOH at a dose of 25 mg/kg, the other extracts showed significantly activity (p<0.05). The free mucous amount in the gastric stomach content was also evaluated. All extracts showed significant increases (p<0.05) of free mucous. This effect was abolished when the animals were pre-treated with indomethacin. Prostaglandin synthesis was evaluated by the administration of HEX extracts by the oral route (100 mg/kg). Prostaglandin synthesis was significantly, increased by 52.3% (p<0.05), and this effect was abolished with prior administration of indomethacin. We concluded that Quassia amara is a probable source for a new drug to treat gastric ulcers, and the mechanism of its activity relates to cytoprotective factors, such as mucous and prostaglandins, but there is still the possibility that antisecretory activity is involved in its antiulcerogenic effect.

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The influence of metal ions (Fe²⁺, Cu²⁺, Zn²⁺) on the hepatoprotective activity of epigallocatechin gallate (EGCG) against hepatotoxin-induced cell injury was investigated. Primary cultures of rat hepatocytes were treated with a well-known hepatotoxin, bromobenzene (BB), in the presence of EGCG only or EGCG plus each metal ion. After 24 h, 0.02 mM EGCG did not show protective activity on the cultured hepatocytes. In contrast, the hepatocytes were protected against BB in the presence of 0.02 mM EGCG and 0.02 mM zinc. The addition of only zinc could not protect hepatocytes against BB. These results suggest that the formation of the zinc–EGCG complex is very important in the enhancement of the hepatoprotective activity of EGCG. The complexation of EGCG with zinc was confirmed by UV–VIS absorption spectroscopy.

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Using a progesterone receptor antagonist, onapristone/ZK 98.299, we examined the in-vivo effects of progesterone on the function of uterine cervix during pregnancy. Onapristone was intravenously administered to pregnant rabbits on day 20 post coitum. After 24 h, the antiprogesterone increased the wet weight of the uterine cervix and decreased the DNA concentration in the cervix. In-situ hybridization also indicated that antiprogesterone augmented the expression of matrix metalloproteinase (MMP)-3/stromelysin-1 mRNA in the uterine cervix. These changes are very similar to those observed and reported thus far in ripened and dilated uterine cervix. These results suggest that during pregnancy, progesterone closely participates in the maintenance of the function of uterine cervix by preventing the production of MMPs and thereby destruction of extracellular matrix, and thus add support to the theory that antiprogesterone has the potential to accelerate for the uterine cervical ripening and dilatation.

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The change in plasma level of dipeptide, Val–Tyr (VY), with in vitro angiotensin I-converting enzyme inhibitory activity was investigated after a single oral administration of a VY-drink at doses of 0, 6 or 12 mg given to mild hypertensive subjects. During this protocol for up to 24 h after the intake, patient/subject blood pressure (BP) was measured for a 15 min period at designated times (0, 1, 2, 4, 8, 24 h) with the individual supine. Based on the VY determination, the maximal increment of plasma VY level was observed over the second hour postprandially (12 mg-dose; 2041±148 fmol/ml-plasma). In addition, the plasma VY level increased with the VY dosage. However, no marked BP change was observed with the increase of plasma VY level, suggesting that VY did not exert an acute hypotensive effect. The area under the curve at 12 mg-dose was estimated to be 8644±420 fmol⋅h/ml-plasma, comparable to that in normotensive subjects. This finding suggests that absorption of VY would not be influenced by a complaint of hypertension.

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The abilities of two types of chitosan oligosaccharides, chitosan oligosaccharide I (1-kDa<MW<3-kDa) and chitosan oligosaccharide II (3-kDa<MW<5-kDa), to prevent 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced oxidative stress were examined in ICR mice. Chitosan oligosaccharide I had no effect on TCDD-induced alterations in lipid peroxidation and glutathione S-transferase activity or liver weight change. However, mice treated with chitosan oligosaccharide II were protected from TCDD-induced lipid peroxidation, inhibition of glutathione peroxidase and glutathione S-transferase activities, and losses in body and liver weights. These results suggest that chitosan oligosaccharide might be a potential agent for combating TCDD-induced pathogenesis.

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Mast cells are critical for initiating innate immune and inflammatory responses by releasing a number of pro-inflammatory mediators. The potential immunomodulatory properties of hydrogenated aromatic hydrocarbons have been the subject of extensive investigation, as the immune system is a sensitive target for hydrogenated aromatic hydrocarbon toxicity. In this report, the effects of polychlorinated biphenyl (PCB) on the expression of cyclooxygenase-2 and pro-inflammatory cytokines such as interleukin-1β (IL-1β), IL-6 and tumor necrosis factor (TNF)-α in the human leukemic mast cell line were investigated. TNF-α and IL-1β expressed their respective mRNA in the presence or absence of PCB, while cyclooxygenase-2 (COX-2) and IL-6 mRNA expression were highly induced by PCB after 2 h. Moreover, pre-treatment with the nuclear factor (NF)-κB pathway inhibitor, pyrrolidine dithiocarbamate, suppressed COX-2, TNF-α and IL-1β induction and reduced the IL-6 mRNA levels induced by PCB. The NF-κB activity was determined by electrophoretic mobility shift analysis (EMSA) using an oligonucleotide containing a consensus NF-κB binding sequence. Stimulating the cells with PCB activated NF-κB. However, pre-treating them with a NF-κB pathway inhibitor, pyrrolidine dithiocarbamate, suppressed PCB-induced NF-κB activation. This suggests that PCB induces cycloxoygenase-2 and pro-inflammatory cytokine expression, and that this induction occurs through NF-κB.

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In this study, we evaluated whether taurine treatment has a protective effect on the prooxidant-antioxidant state following chronic ethanol treatment in rats. Rats were given water containing 20% ethanol (v/v) as drinking water for 3 months. Chronic ethanol treatment in drinking water resulted in increased oxidative stress in the liver of rats. Taurine treatment was performed by adding 1% taurine (w/v) to the drinking water plus injection (400 mg/kg body weight) intraperitoneally 3 times/week for 28 d after ethanol cessation in chronically ethanol-treatad rats. This treatment starting after ethanol cessation caused a significant decreases in serum transaminase activities and hepatic total lipid, triglyceride, malondialdehyde, and diene conjugate levels and significant increases in hepatic glutathione, vitamin E, and vitamin C levels, but did not alter the activities of superoxide dismutase, glutathione peroxidase, and glutathione transferase in the liver as compared with chronically ethanol-treated rats. Accordingly, we propose that taurine has a restorative effect on ethanol-induced hepatic damage by decreasing oxidative stres.

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In this study, we investigated the in vitro and ex vivo suppressive effects of Andrographis paniculata on nitric oxide (NO) production in mouse peritoneal macrophages elicited by bacillus Calmette-Guéin (BCG) and stimulated by lipopolysaccharide (LPS). Incubation of BCG-induced macrophages with the methanol extract of A. paniculata reduced LPS stimulated NO production. The diterpene lactones andrographolide and neoandrographolide were isolated as active components from the extract. These compounds suppressed NO production in a concentration-dependent manner in the concentration range from 0.1 to 100 μM and their IC₅₀ values were 7.9 and 35.5 μM. Neoandrographolide also suppressed NO production by 35 and 40% when the macrophages were collected after oral administration of neoandrographolide at doses of 5 and 25 mg/kg/d and LPS stimulated NO production was examined. However, andrographolide did not reduce NO production on oral administration at the same doses. These results indicate that neoandrographolide, which inhibited NO production both in vitro and ex vivo may play an important role in the use of A. paniculata as an anti-inflammatory crude drug.

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Si-Wu-Tang (SWT), a traditional Chinese formula, has been clinically used in the treatment of cutaneous pruritus, chronic inflammation, and other diseases. The present study was carried out to observe the antipruritic and antiinflammatory effects of SWT aqueous extract using compound 48/80 and picryl chloride (PC) models in mice. SWT (500, 1000 mg/kg p.o.) clearly reduced the scratching responses elicited by compound 48/80 in normal mice. At doses of 250 and 500 mg/kg, it inhibited the scratching responses induced by PC in mice actively sensitized with 2,4-dinitrophenol (DNP)-ovalbumin (OVA) plus alum. Furthermore, SWT (250, 500, 1000 mg/kg) significantly inhibited the footpad swelling caused by compound 48/80 in mice. In the biphasic ear skin reactions induced by PC in actively sensitized mice, SWT (250, 500 mg/kg) reduced the immediate—phase reaction, but did not affect the late—phase reaction. In vitro, SWT (50—500 μg/ml) showed a concentration-dependent inhibition of the histamine release induced by compound 48/80 from rat peritoneal mast cells. The crude drugs contained in SWT, Paeoniae Radix (25, 100 μg/ml), Rehmanniae Radix, and Chuanxiong Rhizoma (100 μg/ml), also showed a clear inhibition, but Angelica Radix did not at the concentrations examined. These findings indicate that SWT aqueous extract has antipruritic and antiinflammatory effects in mice. SWT inhibits histamine release from rat mast cells, and Paeoniae Radix probably plays a crucial role in the formula.

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This study was carried out to determine the effects of the 70% ethanolic extract from the anomalous fruits of Gleditsia sinensis LAM. (AFGS) on experimental allergic reactions and inflammation. AFGS (200, 500, 1000 mg/kg, p.o.) dose-dependently inhibited the systemic anaphylactic shock induced by compound 48/80 in mice and cutaneous reactions induced by histamine or serotonin in rats. At doses of 500 and 1000 mg/kg, AFGS showed a clear inhibition on homologous passive cutaneous anaphylaxis in rats. In vitro, AFGS significantly reduced histamine release from rat peritoneal mast cells triggered by compound 48/80 at concentrations of 20 and 50 μg/ml. Moreover, AFGS (500, 1000 mg/kg, p.o.) showed a significant inhibition on the hind paw edema in rats and ear swelling in mice caused by carrageenin and croton oil, respectively. It also clearly inhibited the vascular permeability induced by acetic acid in mice at a dose of 1000 mg/kg. These findings demonstrate that the ethanolic extract from the anomalous fruits of Gleditsia sinensis possesses antiallergic and anti-inflammatory activities, which may be mediated by reducing the release of mediators such as histamine from mast cells and weakening the inflammatory action of these mediators.

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Zingiberis Rhizoma (Shokyo) showed significant ameliorative effect on the BaCl₂-induced delay of gastric emptying in rat. Bioassay-guided fractionation of the aqueous extract of Shokyo resulted in isolation of 6-gingesulfonic acid (1) and shogasulfonic acid A (3). These compounds significantly improved the delay of gastric emptying on both BaCl₂-induced and N^G-nitro-L-arginine (L-NNA)-induced model in rat.

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Crude extracts of Mexican medicinal plants were screened for trypanocidal activity against Trypanosoma cruzi, which is the etiological agent for Chagas' disease, one of the most serious protozoan diseases in Latin America. There were 43 kinds of methanolic and other organic extracts from 39 plants which were examined by the preliminary screening test to see immobilization of epimastigotes of T. cruzi in vitro. Eighteen of them showed activity at the concentration of 2 mg/ml after incubation for 2 h, while 13 showed activity at the concentration of 1 mg/ml after incubation for 48 h. Among them, the MeOH extract of roots of Aristolochia taliscana (Aristolochiaceae), locally known as “Guaco, ” immobilized all the epimastigotes even at lower concentration of 0.5 mg/ml (48 h). In order to identify principal compounds for this activity, the MeOH extract of Guaco was subjected to bioassay-guided fractionation. From the active fractions, four neolignans, eupomatenoid-7 (1), licarin A (2), eupomatenoid-1 (5) and licarin B (6), and two lignans, austrobailignan-7 (3) and fragransin E₁ (4) were isolated. Compounds 1—4 immobilized all the epimastigotes at the minimum concentration of 25—75 μg/ml after incubation for 48 h, while compounds 5 and 6 were inactive. Corresponding concentration of gossypol, berberine chloride and harmine was 280 μg/ml, 300 μg/ml and >500 μg/ml, respectively.

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The antidiabetic activity of Lyophyllum decastes (Tricholomataceae) was investigated in KK-Ay mice, an animal model of genetically type 2 diabetes with hyperinsulinemia. The water extract of Lyophyllum decastes (LD) (500 mg/kg body weight) reduced the blood glucose of KK-Ay mice 7 h after a single oral administration (p<0.05) when compared with control. LD reduced the blood glucose of KK-Ay mice 3 weeks after repeated administration (p<0.05), and also significantly lowered the serum insulin of KK-Ay mice under similar conditions (p<0.01). However, LD did not affect the blood glucose in normal mice. LD tended to decrease of the blood glucose in an insulin tolerance test. In addition, the muscle content of facilitative glucose transporter isoform 4 (GLUT4) protein content in the plasma membrane fraction from muscle significantly increased in the orally LD-treated KK-Ay mice when compared to that of the controls (p<0.01). These results suggest that the antidiabetic activity of LD is derived, at least in part, from a decrease in insulin resistance, due to the increase of GLUT4 protein content in the plasma membrane of the muscle.

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Epidemiological studies have suggested that the consumption of green tea provides protection against stomach cancer. Fractionation of green tea extract, guided by antiproliferative activity against human stomach cancer (MK-1) cells, has resulted in the isolation of six active flavan-3-ols, epicatechin (EC), epigallocatechin (EGC), epigallocatechin gallate (EGCg), gallocatechin (GC), epicatechin gallate (ECg), gallocatechin gallate (GCg), together with inactive glycosides of kaempferol and quercetin. Among the six active flavan-3-ols, EGCg and GCg showed the highest activity, EGC, GC, ECg followed next, and the activity of EC was lowest. These data suggest that the presence of the three adjacent hydroxyl groups (pyrogallol or galloyl group) in the molecule would be a key factor for enhancing the activity. Since reactive oxygen species play an important role in cell death induction, radical scavenging activity was evaluated using the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical. The order of scavenging activity was ECg≥EGCg≥EGC≥GC≥EC. The compounds having a galloyl moiety showed more potent activity. The contribution of the pyrogallol moiety in the B-ring to the scavenging activity seemed to be less than that of the galloyl moiety.

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An antitumor-promoting effect was found in the extracts/ingredients of a plant used as a traditional medicine in mainland China, using the neoplastic transformation assay of mouse epidermal JB6 cell lines. The ethyl acetate soluble fraction of 75 % ethanol extract of the rhizomes of Dioscorea bulbifera L. showed an inhibitory effect against the tumor promotion of JB6 (Cl 22 and Cl 41) cells induced by a promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Further investigation on the constituents of the EtOAc fraction from the rhizomes revealed the chemical structure to be kaempferol-3,5-dimethyl ether (1), caryatin (2), (+)-catechin (3), myricetin (4), quercetin-3-O-galactopyranoside (5), myricetin-3-O-galactopyranoside (6), myricetin-3-O-glucopyranoside (7) and diosbulbin B (8). Constituent antitumor-promoting activities were also examined in the same way. Compounds 1—7, characterized as flavonoids with the two hydroxyl groups at C-7 and C-4′, showed the most potent inhibitory effect, but there seemed to be differences in the inhibitory effect between flavonol aglycones and flavonol glycosides. Compared with (−)-epicatechin, (+)-catechin exhibited much stronger inhibitory activity which suggested that chemical stereo structures of compounds affect the efficiency of inhibition. Compound 8 showed moderate activity. The constituents with antitumor-promoting activity from this plant are reported for the first time.

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The age-related changes in the electrical and physiological properties of the skin were examined in rats at the ages of 5, 10, 21, 90, and 180 d. The resistance of the stratum corneum, the resistance of the viable skin (epidermis and dermis), and the capacitance of the stratum corneum were analyzed from skin impedance data using an equivalent circuit. With development and aging, the resistance of the stratum corneum and the viable skin increased, whereas the capacitance of the stratum corneum decreased. Physiological characteristics such as the thickness of skin strata and the content of lipid and water in the stratum corneum were also measured. The lipid content in the stratum corneum was constant at all ages. The water content in the stratum corneum decreased, and the thickness of skin strata increased with age. Comparison between electrical data and physiological properties suggested that the increase in the resistance of the stratum corneum with aging is primarily caused by the decrease in the water content and that the capacitance of the stratum corneum and the resistance of the viable skin depend on age-related increases in the thickness of skin strata. In conclusion, the age dependency of cutaneous electrical properties may affect the permeation profile of drugs through the skin, and impedance analysis can be used to estimate age-related changes in transdermal drug delivery.

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Oral administration of the perilla leaf extract (PLE) to mice inhibits inflammation, allergic response, and tumor necrosis factor-α production. We also found that PLE suppressed the tumor necrosis factor-α (TNF-α) production in vitro. Using the inhibitory activity of TNF-α production in vitro as the index for isolation, we searched the active constituents from PLE and isolated luteolin, rosmarinic acid and caffeic acid as active components. Among the isolated compounds, only luteolin showed in vivo activity: inhibition of serum tumor necrosis factor-α production, inhibition of arachidonic acid-induced ear edema, inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced ear edema and inhibition of oxazolone-induced allergic edema. These results suggest that luteolin is a genuinely active constituent which is accountable for the oral effects of perilla.

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The purified polysaccharides from Piper nigrum were prepared as follows: a hot water extract of pepper seeds was fractionated by ultrafiltration with a 5-kDa-membrane cartridge. A fraction with 5 kDa or bigger molecules was successively purified by open column chromatography on DEAE-Toyopearl 650C and Bio-gel P-60 with each active fraction, resulting in PN-Ib and PN-IIa, purified anti-complementary polysaccharides. None of the anti-complementary activity of any polysaccharide was changed by pronase digestion or polymyxin B treatment, but they were decreased by periodate oxidation. Analysis of component sugar and molecular mass determination of the anti-complementary polysaccharides indicated that PN-Ib with an average molecular mass of 21 kDa contained 88.5% glucose and other negligible minor monosaccharides, while PN-IIa showed a different monosaccharide composition, which contained a significant proportion of galactose, arabinose, galacturonic acid and rhamnose. The molar ratio of galactose and arabinose of PN-IIa (48 kDa) was 1.93 : 1. PN-I did not react with β-glucosyl Yariv reagent, however, PN-IIa did react, which indicated that PN-IIa might be an arabinogalactan. Based upon these results, the usefulness of purified anti-complementary polysaccharides from Piper nigrum is suggested as a supplement for immune enhancement.

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In order to treat a hyperammonemic patient with adult-onset type-II citrullinemia (CTLN2), sodium phenylacetate powder was prepared from chemical reagent grade phenylacetic acid in Gunma University Hospital. After purification by recrystalization, phenylacetic acid was neutralized with sodium carbonate and dried at 70 °C under reduced pressure. A solution of the prepared powder produced a single peak of m/z=181.0 (M+Na⁺) in electrospray-ionization-MS spectrogram. The content of phenylacetate was 74% of theoretical value, suggesting the existence of water of crystallization. The content of phenylacetate remained constant for 5 months under dark conditions at room temperature. The prepared sodium phenylacetate powder was orally administered to a 16-year-old patient with CTLN2 at a dosage of 12 g/d. The serum ammonia concentration of the patient, who did not show adequate response to intravenous arginine or oral sodium benzoic acid decreased remarkably to less than 100 μg/dl. Sodium phenylacetate powder should be an essential drug for the treatment of hyperammonemia caused by an inborn error of the urea cycle.

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We previously demonstrated the preventive effect of sesamin, a lignan from sesame oil, on the development of several experimental models of hypertension. In the present study, we explored the mechanisms underlying the antihypertensive effect of sesamin using the deoxycorticosterone acetate (DOCA)-salt rat hypertensive model. After a 5-week treatment period, aortic superoxide (O₂⁻) production was measured in the lucigenin chemiluminescence assay. Chemiluminescence signals significantly decreased in sesamin-containing diet-fed DOCA-salt hypertensive rats compared with those in the normal diet-fed DOCA-salt rats, although the signals in sham-operated control animals were not affected by the sesamin feeding. In addition, there was a positive correlation between systolic blood pressure and aortic O₂⁻ production. These findings suggest that sesamin feeding inhibits enhanced vascular O₂⁻ production in DOCA-salt hypertensive rats and that the antioxidative action of sesamin may contribute to its antihypertensive activity.

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