Biological and Pharmaceutical Bulletin
Online ISSN : 1347-5215
Print ISSN : 0918-6158
ISSN-L : 0918-6158
Volume 24 , Issue 8
Showing 1-25 articles out of 25 articles from the selected issue
Analytical Biochemistry
Regular Articles
  • Kazuhiro YOSHIHARA, Reiko KIYONAMI, Yumi SHIMIZU, Masatoshi BEPPU
    2001 Volume 24 Issue 8 Pages 863-866
    Published: 2001
    Released: May 31, 2002
    JOURNALS FREE ACCESS
    Pyrraline is one of the advanced glycation end products formed under non-enzymatic and non-oxidative conditions in vivo. In this study, we developed a novel method for determination of urinary pyrraline using solid-phase extraction as a pretreatment procedure prior to determination by high performance liquid chromatography (HPLC). The OasisTM HLB solid-phase extraction cartridge was used for pretreatment of urine samples without hydrolysis. The chromatogram obtained clearly revealed the peak for urinary pyrraline owing to prior removal of interfering substances in urine samples. The recovery rate of pyrraline was 97.2±3.3% (n=6). The mean excretion level of urinary pyrraline in healthy control (20—77 years old, n=30) was 1.42±0.65 μmol/mmol creatinine, and the daily variation in the excretion level was considered to be insignificant. We propose the above procedure as a simple, rapid, and accurate method for determination of pyrraline levels in urine.
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  • Noriko TAGAWA, Kayoko SAIKI, Yoshiharu KOBAYASHI
    2001 Volume 24 Issue 8 Pages 867-871
    Published: 2001
    Released: May 31, 2002
    JOURNALS FREE ACCESS
    We have developed an enzyme immunoassay (EIA) for serum 16-dehydropregnenolone (3β-hydroxy-5,16-pregnadien-20-one; 16-DHP). The antiserum against 16-DHP-3-hemisuccinate conjugated bovine serum albumin (16-DHP-3HS-BSA) was raised in rabbits. For use as an enzyme labeled antigen, 16-DHP-3HS was conjugated to alkaline phosphatase. The minimal amount of 16-DHP detected was 4pg (0.013 pmol)/assay and the measurable range was from 0.06—60 ng/ml (0.191—191 nmol/l). The intra-assay coefficient of variation (C.V.) was 4.1% (0.73±0.03 ng/ml, mean±S.D., n=6), and inter-assay C.V. was 7.7% (0.13±0.01 ng/ml, n=6). A liner relation was observed between the serum sample dilution and the 16-DHP concentration. For the recovery study, authentic 16-DHP was added to a serum sample (original concentration: 0.10—0.14 ng/ml), and the recovery was found to be 94.4—96.8% (final 16-DHP concentrations calculated: 0.29—16.3 ng/ml). To investigate the reliability of the present EIA, the values from our EIA were compared with those obtained by GC-MS. The 16-DHP concentration could not be measured in serum by GC-MS because of its sensitivity. Therefore, the conjugated steroid, 16-DHPS, was first enzymatically hydrolysed and then the 16-DHP measured by both methods. There was a good correlation between the levels determined by these methods (Pearson’s correlation coefficient: r=0.927, p<0.001, y=0.74x+3.61, n=27). The serum concentrations of 16-DHP in neonates and umbilical vein were 0.53±0.09 ng/ml and 0.88±0.61 ng/ml, respectively. No 16-DHP was detected in serum from normal healthy adults using the present EIA. These results suggest that 16-DHP originates from the fetus and neonate.
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Notes
Biochemistry/Molecular Biology
Regular Articles
  • Jae-Young UM, Hwan-Suck CHUNG, Mi-Sun KIM, Ho-Jeong NA, Hyun-Jeong KWO ...
    2001 Volume 24 Issue 8 Pages 872-875
    Published: 2001
    Released: May 31, 2002
    JOURNALS FREE ACCESS
    In order to develop convenient and reproducible methods for the identification of ginseng drugs at a DNA level, randomly amplified polymorphic DNA (RAPD) and PCR-restriction fragment length polymorphism (PCR-RFLP) analyses were applied within Panax species. To authenticate Panax ginseng among ginseng populations, RAPD analysis was carried out using a 20 mer-random primer. The similarity coefficients among the DNA of ginseng plants analyzed were low, ranging from 0.197 to 0.491. In addition, by using PCR-RFLP analysis, very different fingerprints were obtained within Korean ginseng plants. These results suggest that these methods are able to authenticate the concerned Panax species. Broader application of this approach to authenticate other morphologically similar medicinal materials is rationalized.
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  • Koichi SAKURAI, Mika KATOH, Kimio SOMENO, Yukio FUJIMOTO
    2001 Volume 24 Issue 8 Pages 876-882
    Published: 2001
    Released: May 31, 2002
    JOURNALS FREE ACCESS
    To evaluate the participation of mitochondrial damage, oxygen radicals and cell death in diabetes mellitus, we designed a way to investigate INS-1 cells, rat pancreatic β-cell line, to die by treatment with alloxan which generate reactive oxygen species (ROS). Incubation of INS-1 cells with alloxan for 24 h resulted in a decrease in viability of cells as well as inhibition of glucose-stimulated insulin release; this could be prevented by antioxidants, vitamin E and butylated hydroxyanisol (BHA). The formation of a DNA ladder and the distribution of phosphatidylserine at the external surface of plasma membrane were observed as indicators of apoptosis in the cells treated with alloxan at concentrations below 0.5 mM. The formation of DNA ladder was prevented by vitamin E, BHA and catalase, suggesting that the ROS is involved in the process of apoptosis in INS-1 cells treated with alloxan. Lower levels of intracellular ATP, collapse of mitochondrial membrane potential and release of cytochrome c from mitochondria were also observed in INS-1 cells treated with alloxan, suggesting that alloxan caused the damage of mitochondria in cells and was related to the process of apoptosis. In contrast, rat liver RLC-18 cells treated with alloxan were not observed in the decrease of viability. It follows from the present study that mitochondrial damages by ROS generated from alloxan is linked to apoptosis in INS-1 cells.
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  • Sachiko OKABE, Nobukazu FUJIMOTO, Naoko SUEOKA, Masami SUGANUMA, Hirot ...
    2001 Volume 24 Issue 8 Pages 883-886
    Published: 2001
    Released: May 31, 2002
    JOURNALS FREE ACCESS
    Green tea is the most effective cancer preventive beverage. In the light of this, the mechanisms of action of tea polyphenols were investigated on the molecular levels. We present here the effects of (−)-epigallocatechin gallate (EGCG) on expression of 588 genes in human lung cancer cell line PC-9 cells, using a human cancer cDNA expression array. The levels of gene expression in non-treated control cells, and cells treated with EGCG alone, with the tumor promoter okadaic acid alone, and with EGCG plus okadaic acid, were studied, and their expression levels were classified into down-regulation (under 0.5 fold) and up-regulation (over 2.0 fold) by comparing with the levels of control. Non-treated PC-9 cells expressed 163 genes out of 588, and EGCG-treated cells induced down-regulated expression of 12 genes and up-regulated expression of 4 other genes. From a comparison of gene expression in the cells treated with EGCG and in cells treated with EGCG plus okadaic acid, we found the following genes commonly affected by EGCG: down-regulation of four genes, NF-κB inducing kinase (NIK), death-associated protein kinase 1 (DAPK 1), rhoB and tyrosine-protein kinase (SKY); up-regulation of one gene, retinoic acid receptor α1. Among them, we think down-regulation of NIK gene expression is significant for cancer prevention, based on evidence that inhibition of NF-κB activation is a result of inhibition of NIK/IKK signalling complex.
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  • Wataru TOMISATO, Shinji TSUTSUMI, Tomofusa TSUCHIYA, Tohru MIZUSHIMA
    2001 Volume 24 Issue 8 Pages 887-891
    Published: 2001
    Released: May 31, 2002
    JOURNALS FREE ACCESS
    Gastric mucosal cell death due to various gastric stressors can cause several types of gastric diseases, such as gastric ulcers. In this study, we examined cell death following the short-term treatment of guinea pig gastric mucosal cells in primary culture with various gastric stressors. The short-term treatment of cells with ethanol, hydrogen peroxide or hydrochloric acid caused, in a dose-dependent manner, cell death in the absence of apoptotic DNA fragmentation and chromatin condensation. Cells lost membrane integrity following the treatment with each of these gastric stressors, suggesting that necrosis was induced in gastric mucosal cells by short-term treatment of the cells with gastric stressors. Geranylgeranylacetone, an anti-ulcer drug with heat-shock protein inducing properties, protected gastric mucosal cells from the necrotic cell death caused by each of these gastric stressors. Pretreatment of cells with low concentrations of ethanol (3%), which also induced heat-shock protein, made cells resistant to the necrotic cell death caused by the gastric stressors. These results suggest that heat-shock proteins is involved in the cytoprotective effect of geranylgeranylacetone against necrotic cell death.
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  • Nam-Ho CHOI-MIURA, Kayo OTSUYAMA, Yoshihiro SANO, Kiyomi SAITO, Katsuh ...
    2001 Volume 24 Issue 8 Pages 892-896
    Published: 2001
    Released: May 31, 2002
    JOURNALS FREE ACCESS
    Plasma hyaluronan binding protein (PHBP) is produced only in liver and kidney in mouse. The induction of PHBP mRNA and the conversion of pro PHBP to the active hetero-dimer form were studied after CCl4, D-galactosamine, HgCl2 or turpentine administration and after partial hepatectomy. The results indicated that the administrations of CCl4 and D-galactosamine, which caused hepatic failure, and the partial hepatectomy enhanced the conversion of pro PHBP to the active two-chain form in the plasma. On the other hand, HgCl2 which injured kidney and turpentine which led to inflammation were not involved in the activation of PHBP. The weak induction and suppression of PHBP mRNA were observed in the liver at 3 h and 12 h, respectively, after the CCl4 administration. However, HgCl2 and turpentine did not influence the amount of PHBP mRNA. These results suggested the hepatic injury-specific activation of PHBP in plasma. PHBP may act as an early factor in the cascade for the tissue remodeling in liver following hepatic injury, i.e., PHBP activates urokinase, urokinase activates matrix metalloproteinases (MMPs) and MMPs degrade extracellular matrix for liver regeneration.
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Notes
  • Toshio MAEDA, Rieko SAKITA, Takayuki KAIHATSU, Masao MIWA
    2001 Volume 24 Issue 8 Pages 950-953
    Published: 2001
    Released: May 31, 2002
    JOURNALS FREE ACCESS
    This investigation considered whether defects in insulin secretion and insulin action may result in the development of sustained hyperglycemia induced by refeeding standard chow pellets. Hyperglycemia, sustained for 36 h was induced, in mice that ate standard chow pellets ad libitum after 48 h fasting, but not 24 h fasting. In 48 h-fasted mice, serum insulin levels were remarkably low and the ability of insulin secretion to respond to glucose was decreased, although insulin-stimulated glucose disposal was not impaired. However, hyperinsulinemia was observed after refeeding for 12 h. The 12 h-refed mice had impaired glucose tolerance and were remarkably insulin resistant. These results suggest that hyperglycemia induced by the fasting-refeeding was caused by hyperphagia and the failure of insulin secretion, and maintained the resulting induced insulin resistance.
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  • Takahiro NAKAMURA, Yoshiro SAITO, Norie MURAYAMA, Mayumi SAEKI, Akiko ...
    2001 Volume 24 Issue 8 Pages 954-957
    Published: 2001
    Released: May 31, 2002
    JOURNALS FREE ACCESS
    The members of the cytochrome P450 (CYP) 3A subfamily play an important role in the metabolism of more than 50% of the drugs metabolized by CYPs. Among the CYP3A members, CYP3A5 is known to exhibit polymorphic expression within the human liver. We hypothesized that a single nucleotide polymorphism (SNP) in the 5'-regulatory region of the CYP3A5 gene might be the cause of CYP3A5 polymorphic expression. Due to the existence of “CYP3AP1, ” a highly homologous sequence to the CYP3A5 gene, it was necessary to make specific primers to the CYP3A5 gene. In the present study, we designed a series of oligonucleotide primers for sequencing the proximal promoter region of the CYP3A5 gene in order to search for the putative regulatory single nucleotide polymorphism. We examined 86 established cell lines derived from Japanese individuals as a representation of the Japanese population. However, no SNP was detected in the promoter region of the CYP3A5 gene isolated from the cell lines used, suggesting other causal factors for the observed polymorphism of CYP3A5-dependent drug metabolism.
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  • Yoshinori TSURUSAKI, Masayoshi YAMAGUCHI
    2001 Volume 24 Issue 8 Pages 958-961
    Published: 2001
    Released: May 31, 2002
    JOURNALS FREE ACCESS
    The effect of regucalcin, a regulatory protein of Ca2+ signaling, on guanosine triphosphatase (GTPase) activity in the nuclei of rat liver was investigated. GTPase activity was significantly increased by the addition of CaCl2 (50 μM) in the enzyme reaction mixture. This increase was not seen in the presence of trifluoperazine (25 μM), an antagonist of calmodulin, which could decrease nuclear GTPase activity, suggesting that nuclear endogenous calmodulin is involved in an increase in the enzyme activity related to Ca2+ addition. The presence of regucalcin (0.5 μM) in the enzyme reaction mixture caused a significant decrease in nuclear GTPase activity. The enzyme activity was significantly raised in the presence of anti-regucalcin monoclonal antibody (25 and 50 ng/ml) in the reaction mixture. This increase was completely abolished by the addition of regucalcin (0.5 μM). Also, the effect of regucalcin addition in increasing nuclear GTPase activity was seen in the presence of EGTA (0.1 mM), a chelator of Ca2+. The present study demonstrates that endogenous regucalcin has a suppressive effect on GTPase activity in the nuclei of rat liver.
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Pharmacology
Regular Articles
  • Akira NAITO, Hisashi HASEGAWA, Takashi KURASAWA, Yasuhiro OHTAKE, Hide ...
    2001 Volume 24 Issue 8 Pages 897-901
    Published: 2001
    Released: May 31, 2002
    JOURNALS FREE ACCESS
    The aim of this work was to investigate histopathologically the relationship between the syndrome of inappropriate secretion of antidiuretic hormone (SIADH) and kidney abnormalities and the therapeutic efficacy of VP-343 ((N-[4-[[2S,3aR)-2-hydroxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]qunoxalin-5(1H)-yl]phenyl]-4'-methyl[1,1'-biphenyl]-2-carboxamide], a selective vasopressin V2 receptor antagonist, in an experimental SIADH rat model. In the model, which was prepared by continuously administering 1-desamino-8-D-arginine vasopression (DDAVP), histopathologic abnormalities, such as dilatation of tubules, basophilic changes in tubules, inflammatory cell infiltration, and mineralization were found in the kidney, accompanied by significant increases in the relative weight of the kidney, lung, liver, adrenal gland, and heart. VP-343 was shown to be effective in protecting the kidney from the histopathologic abnormalities and to normalize the relative weight of the kidney and several common pathophysiologic features, such as hyponatremia, hyposmolarity of plasma, hyperosmolarity of urea, and oligurea, as described previously. These results demonstrate the occurrence of histopathologic abnormalities in the kidney and the efficacy of VP-343 in improving abnormalities in the DDAVP-induced SIADH rat model.
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  • Toyofumi KUSAKA, Shizuo YAMADA, Ryohei KIMURA
    2001 Volume 24 Issue 8 Pages 902-905
    Published: 2001
    Released: May 31, 2002
    JOURNALS FREE ACCESS
    The present study was undertaken to characterize simultaneously [3H]nociceptin binding to opioid receptor-like 1 (ORL1) receptors in the rat brain and spinal cord. Specific binding of [3H]nociceptin to crude membranes from the rat brain and spinal cord at 25°C was saturable, reversible and of high affinity, and it also exhibited a pharmacological specificity involving the ORL1 receptor. The Kd and Bmax values for [3H]nociceptin in the spinal cord were significantly lower than those in the brain. At 4°C, there was a significant increase in the dissociation constant (Kd) for [3H]nociceptin in the brain and spinal cord with little change in the maximal number of binding sites (Bmax) compared with that at 25°C. Nociceptin and its analogue, [Phe1Ψ(CH2-NH)-Gly2]nociceptin(1—13)NH2 were found to be potent inhibitors of [3H]nociceptin binding to crude membranes from the brain and spinal cord, while opioid ligands such as naloxone-benzoylhydrazone, naltrindole and nor-binaltorphimine, exhibited an inhibitory effect only at high concentrations. The Ki values for nociceptin, its analogue and opioid ligands in the spinal cord were significantly lower than those in the brain. There were regional variations in the specific [3H]nociceptin binding to crude membranes from the rat brain: a relatively high density of [3H]nociceptin binding in the cerebral cortex, hippocampus, thalamus and midbrain, moderately dense binding in the corpus striatum and pons/medulla oblongata, and the lowest density of binding in the cerebellum. In conclusion, the present study has shown that [3H]nociceptin binds selectively to ORL1 receptors in the rat brain and spinal cord.
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  • Ryoko SAKAMOTO, Maiko OKANO, Hiroko TAKENA, Kenzo OHTSUKI
    2001 Volume 24 Issue 8 Pages 906-911
    Published: 2001
    Released: May 31, 2002
    JOURNALS FREE ACCESS
    The physiological correlation between glycyrrhizin (GL) and high mobility group proteins 1 and 2 (HMG1/2) and the inhibitory effect of GL on their phosphorylation by three protein kinases (CK-I, CK-II and PKC) were investigated biochemically in vitro. It was found that GL binds directly to HMG1/2, because (i) HMG1/2 have a high affinity with a GL-affinity column; and (ii) GL induces the conformational changes in HMG1/2. Both purified HMG1/2 functioned as phosphate acceptors for these two protein kinases (CK-I and PKC), but not phosphorylated by CK-II. Phosphorylation of HMG1/2 by two protein kinases (CK-I and PKC) was completely inhibited by a glycyrrhetinic acid derivative (oGA) at one-tenth the concentration of GL. Also, the DNA-binding abilities of HMG1/2 were reduced by GL in a dose-dependent manner. These results show that the binding of GL to HMG1/2 results in the inhibition of their physiological activities (DNA-binding ability and phosphorylation by PKC or CK-I) in vitro. The GL-induced inhibition of the physiological activities of HMG1/2 may be involved in the anti-inflammatory effect of GL in vivo.
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Notes
  • Takashi HIRANO, Nobuhide KAWASAKI, Hideki MIYATAKA, Toshio SATOH
    2001 Volume 24 Issue 8 Pages 962-963
    Published: 2001
    Released: May 31, 2002
    JOURNALS FREE ACCESS
    The amount of plasma IgE antibody formed and its change over time were investigated by enzyme-linked immunosorbent assay (ELISA) in male and female Sprague-Dawley (SD), Donryu, and Wistar strain rats. IgE antibody formation was initiated by injecting a mixture of 2,4-dinitrophenylated ascaris extract (DNP-As) as antigen and killed Bordetella pertussis as adjuvant into the paws of the animals. The amount of IgE antibody formed was low on day 10 in both male and female SD (40—80 ng/ml) and Donryu (20—40 ng/ml) strain rats, and an increase in the amount was observed on day 20. The peak value of IgE antibody was observed day 10 in Wistar strain rats and was 130 and 200 ng/ml in the male and female rats, respectively. These results suggest that Wistar strain rats produce the most IgE antibody when DNP-As is used as antigen and they can serve as a model for allergic diseases.
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  • Miki NIIKAWA, Takeshi NAKAMURA, Hisamitsu NAGASE
    2001 Volume 24 Issue 8 Pages 964-966
    Published: 2001
    Released: May 31, 2002
    JOURNALS FREE ACCESS
    Chromosome aberrations induced by mitomycin C (MMC) were suppressed by aspirin in a mouse micronucleus test with peripheral blood and bone marrow cells. Aspirin at doses of 0.5, 5, and 50 mg/kg was injected intraperitoneally or per administered orally 0.5, 6, or 24 h after administration of MMC and then peripheral blood and/or bone marrow cells were sampled 48 h after administration of MMC. The suppressive effect of aspirin was more pronounced in the aspirin-treated groups 24 h than 0.5 and 6 h after administration of MMC. In the aspirin-treated group at 24 h, the frequency of polychromatic erythrocytes with micronuclei was decreased by about 60—80% after intraperitoneal injection and by about 40—70% after oral administration. It is suggested that aspirin may directly act on MMC metabolites, but not on MMC itself.
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Pharmacognosy
Regular Articles
  • Hiroaki HAYASHI, Pengyu HUANG, Ara KIRAKOSYAN, Kenichiro INOUE, Noboru ...
    2001 Volume 24 Issue 8 Pages 912-916
    Published: 2001
    Released: May 31, 2002
    JOURNALS FREE ACCESS
    An oxidosqualene cyclase cDNA, termed GgbAS1, was isolated from cultured cells of licorice (Glycyrrhiza glabra) by heterologous hybridization with cDNA of Arabidopsis thaliana LUP1 lupeol synthase. The yeast transformed with an expression vector containing the open reading frame of GgbAS1 produced β-amyrin, indicating that GgbAS1 encodes β-amyrin synthase involved in the glycyrrhizin and soyasaponin biosyntheses in licorice. Northern blot analysis showed that the level of β-amyrin synthase mRNA was drastically changed in the cultured licorice cells, whereas the mRNA level of cycloartenol synthase was relatively constant.
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  • Masaru OGASAWARA, Toshiyuki MATSUBARA, Hideyo SUZUKI
    2001 Volume 24 Issue 8 Pages 917-920
    Published: 2001
    Released: May 31, 2002
    JOURNALS FREE ACCESS
    We have previously reported that evodiamine had a marked inhibitory activity on tumor cell migration in vitro. To extend our study, the effects of evodiamine on invasion, growth, and metastatic development of colon 26-L5 cells were examined here. Evodiamine inhibited the invasion of tumor cells into Matrigel in a concentration-dependent manner, and achieved 70% inhibition at 10 μg/ml. Treatment of tumor cells with evodiamine for 24 h showed little effect on tumor growth at concentrations of less than 10 μg/ml, whereas an over 48-h treatment resulted in a concentration- and time-dependent inhibition. Pretreatment of tumor cells with 10 μg/ml evodiamine before inoculation into mice caused 70% reduction in their lung metastasis formation. When evodiamine at 10 mg/kg was administered into mice from the 6th day after tumor inoculation, the number of tumor nodules in lungs was decreased by 48% as compared to control. The inhibition rate was equivalent to that produced by cisplatin, a potent anti-cancer drug. Evodiamine did not affect the body weight of mice in the experimental period, whereas cisplatin caused serious weight loss. These results suggest that evodiamine may be regarded as a promising agent in tumor metastasis therapy.
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Biopharmacy
Regular Articles
  • Jin Young HUR, Yunjo SOH, Bum-Hoi KIM, Kyungho SUK, Nak Won SOHN, Ho C ...
    2001 Volume 24 Issue 8 Pages 921-924
    Published: 2001
    Released: May 31, 2002
    JOURNALS FREE ACCESS
    Aster scaber T. (Asteraceae) has been used to treat bruises, snakebite, headache, and dizziness in traditional Chinese medicine. In the present study, the neuroprotective effect of four quinic acid derivatives from A. scaber on amyloid Aβ-induced PC12 cell toxicity was investigated. When cells were treated with quinic acid derivatives prior to Aβ, cell toxicity was significantly diminished. Among quinic acid derivatives, (−)4,5-dicaffeoyl quinic acid (1) gave the highest protection against Aβ-induced cell toxicity. In addition, the neurotrophic effects of compounds were evaluated by microscopically monitoring their potency to induce neurite outgrowth in PC12 cells. Four quinic acid derivatives from A. scaber promoted neurite outgrowth in PC12 cells. Interestingly, a novel quinic acid, (−)3,5-dicaffeoyl-muco-quinic acid (2) was more effective than the other compounds in promoting neurite outgrowth. Unlike nerve growth factor, the withdrawal of quinic acids did not result in any significant decrease in cell viability. The results suggest that quinic acid derivatives from A. scaber might potentially be used as a therapeutic agent in Alzheimer disease.
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  • Yoshihiko TAUCHI, Iichi ZUSHIDA, Sumio CHONO, Juichi SATO, Keiji ITO, ...
    2001 Volume 24 Issue 8 Pages 925-929
    Published: 2001
    Released: May 31, 2002
    JOURNALS FREE ACCESS
    In order to confirm the efficacy of dexamethasone palmitate (DP)-low density lipoprotein (LDL) complex on experimental atherosclerosis in vivo, we examined whether DP-LDL complex could be effective for cholesterol ester accumulation in the aorta of atherogenic mice. Nonatherogenic and atherogenic mice were fed with normal and atherogenic diet for 14 weeks, respectively. Dexamethasone (DEX), lipid emulsion containing DP (DP-LE), or DP-LDL complex was intravenously injected once a week from 8 to 14 weeks. Cholesterol levels in serum and aorta in the atherogenic mice were significantly higher than those of nonatherogenic mice. Injection of DP-LDL complex significantly reduced cholesterol ester (CE) accumulation in the aorta of atherogenic mice. The reduction of CE accumulation in aorta treated with DP-LDL complexes was 10 and 100 times more potent than that with DP-LE and DEX, respectively. The radioactivity in the aorta of atherogenic mice treated with 3H-DP-LDL complex was significantly higher than that with 3H-DP-LE and 3H-DEX at 24 h after injection. Even 7 d after injection, a significant amount of radioactivity was present only in the aorta of atherogenic mice treated with DP-LDL complex. This result suggests that DP-LDL complex is selectively delivered to the atherogenic lesions in the aorta of atherogenic mice, and then DP released from the complex inhibits CE accumulation in the aortic intima. Therefore, DP-LDL complex may be a good drug-carrier in drug delivery system for atherosclerosis.
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  • Yuichiro IMAMURA, Kazuteru SHIMIZU, Fumiyoshi YAMASHITA, Kiyoshi YAMAO ...
    2001 Volume 24 Issue 8 Pages 930-934
    Published: 2001
    Released: May 31, 2002
    JOURNALS FREE ACCESS
    The transport characteristics of a selective peripheral H1 receptor antagonist, ebastine, a substrate for cytochrome P450 3A4, and its three major metabolites, i.e., the hydroxy metabolite of ebastine (M-OH), the pharmacologically active metabolite carebastine (Car), and the desbutyrophenone metabolite (des-BP), were studied in cultured human intestinal Caco-2 cells expressing a drug efflux pump, P-glycoprotein (P-gp), on the apical membrane. The polarized transport of [3H]cyclosporin A (CyA), mediated by P-gp in the basolateral to apical direction across the Caco-2 cell monolayers, was affected by the presence of ebastine in a concentration-dependent manner and significant inhibition was observed at high concentrations (>50 μM). M-OH (300 μM) also significantly inhibited whereas Car and des-BP did not. Although no marked polarized transport of [14C]ebastine in a secretory direction was observed in the Caco-2 systems, the flux in the basolateral to apical direction was slightly higher than that in the opposite direction at concentrations less than 30 μM. [14C]Ebastine (2 μM) uptake from the apical side was significantly increased in the presence of an excess of cold CyA, suggesting that the efflux process mediated by P-gp may be involved in the ebastine uptake by Caco-2 cells. Collectively, these results indicate that ebastine (and presumably M-OH) is transported via P-gp in Caco-2 cells, however, the affinity for P-gp is very low. It is unlikely that the secretory transport of ebastine mediated by P-gp will dramatically affect overall intestinal absorption in vivo because efficient passive diffusion of this drug should occur due to its high lipophilicity. However, it may be advantageous for its effecient first-pass metabolism.
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  • Toshiyuki SAKAEDA, Noboru OKAMURA, Shunji NAGATA, Tatsurou YAGAMI, Mas ...
    2001 Volume 24 Issue 8 Pages 935-940
    Published: 2001
    Released: May 31, 2002
    JOURNALS FREE ACCESS
    This study was performed to determine the exclusion criteria that differentiate poorly absorbed drugs from good drug candidates, and to accelerate drug development by exclusion of unnecessary assessment. The molecular and pharmacokinetic properties of 222 commercially available oral drugs were tabulated and their correlations were analyzed. The exclusion criteria obtained were 1) a molecular weight of more than 500, and 2) a ClogP value of more than 5. Exceptions to molecular weight criteria were compounds with a sugar moiety, high atomic weight, and large cyclic structure. It was also suggested that being a substrate for MDR1 (P-glycoprotein) does not always result in poor bioavailability, and that drug development by chemical modification of a seed or lead compound with quantitative structure activity relationship analysis can result in lower bioavailability, higher bound fraction and lower urinary excretion, which would hamper later development processes and might result in considerable drug-drug interaction. The criteria should be adjusted according to the pharmacological profiles of the agents in question and depending on the estimated profit, but ignoring these criteria may result in a significant waste of time and money during drug development.
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Notes
  • Su-Lan HSIU, Chih-Wan TSAO, Yu-Chuan TSAI, Hong-Jing HO, Pei-Dawn Lee ...
    2001 Volume 24 Issue 8 Pages 967-969
    Published: 2001
    Released: May 31, 2002
    JOURNALS FREE ACCESS
    Morin and quercetin are isomeric antioxidant flavonols. High-performance liquid chromatographic methods were developed for the quantitation of morin and quercetin in serum. The method employed a Cosmosil RP-18 column, using acetonitrile/0.2% o-phosphoric acid: 28/72 and 27/73 (v/v) as mobile phases, with ethyl paraben and 6,7-dimethoxycoumarin used as internal standards for morin and quercetin, respectively. Moreover, a strategy to stabilize morin/quercetin released from their glucuronides/sulfates in serum during hydrolysis was established. The present methods are applicable for determining morin, quercetin, and their glucuronides/sulfates in serum.
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  • Jing-Shi ZHANG, Shen-Feng MA, Ayaka SUENAGA, Masaki OTAGIRI
    2001 Volume 24 Issue 8 Pages 970-972
    Published: 2001
    Released: May 31, 2002
    JOURNALS FREE ACCESS
    To assess the stability of a cisplatin (CDDP) complex prepared with chondroitin sulfate A (CSA) relative to protein binding in the circulation and kidney, a trichloroacetic acid (TCA) precipitation method was developed to measure the protein-unbound species of CDDP and the CDDP-CSA complex in plasma and kidney homogenates. The total and unbound drug concentrations were determined up to 3 h following a 2 mg/kg bolus injection of CDDP or CDDP-CSA complex to rats. The stability against plasma binding was evaluated by a determination of the area under concentration-time curve from time 0 to infinite time (AUC(0—∞); the ratio of unbound drug AUC(0—∞) to total drug AUC(0—∞) was employed to estimate the availability of the unbound drug in the circulation. The results showed that a competitive reaction to platinum existed between plasma protein and the CDDP-CSA complex, but the complex accounted for more than 60% of the protein-unbound species for a dosage, compared to 30% obtained by an administration of uncomplexed CDDP. The tissue binding kinetics in kidney for CDDP and the CDDP-CSA complex was investigated by the use of homogenates. The binding rate constants of CDDP and CDDP-CSA in a kidney homogenate were 0.0040 min-1 and 0.0014 min-1, respectively. The results indicate that the CDDP-CSA complex could effectively retard the binding of CDDP to protein in the kidney. These data provide evidence that endogenous protein is able to compete for platinum from the CDDP-CSA complex, but the complex effectively retarded the protein binding reaction with CDDP in plasma and kidney as compared to native CDDP, which has the potential for reducing the accumulation of CDDP in plasma and kidney.
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  • Hyug CHUN, Dong Hoon SHIN, Bum Shik HONG, Hong Yon CHO, Han Chul YANG
    2001 Volume 24 Issue 8 Pages 941-946
    Published: 2001
    Released: May 31, 2002
    JOURNALS FREE ACCESS
    Polysaccharides are involved in biological responses and can activate complement system, which plays an important role in primary host defense mechanisms. We investigated anticomplementary activities from spice plants and selected thyme (Thymus vulgaris L.) as a potent complementary activator. Acidic polysaccharide (TV-3-IIIA-IIa) purified from the hot-water extract of thyme leaves by DEAE-Toyopearl 650C, Butyl-Toyopearl 650M and Sepharose CL-6B column chromatography and preparative HPLC. The purified polysaccharide, TV-3-IIIA-IIa showed potent anticomplementary activity via classical and alternative pathway with the increase proportional to dosage. TV-3-IIIA-IIa seemed to be a homogenous polymer from the results of HPLC and its molecular mass was estimated as 180 kDa. TV-3-IIIA-IIa mainly consisted of galacturonic acid (44.8 mol%), glucuronic acid (16.7 mol%), arabinose (11.1 mol%), rhamnose (9.2 mol%), galactose (8.9 mol%) and small amounts of glucose, xylose, mannose and fucose. By methylation analysis and reactivity to β-glucosyl Yariv reagent, TV-3-IIIA-IIa was assumed to contain small amounts of type II arabinogalactan and large amounts of pectin-like polysaccharides in the structure. Based upon these results, TV-3-IIIA-IIa was suggested to be a complement activator.
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