Our study objectives were: To quantitatively determine the effect of radiolabel instability on terminal phase radioactive tissue residues in a multiple dose tissue distribution study. To quantitatively compare tissue residue artifacts (non drug-related radioactivity) from two chemically-distinct radiolabel locations. To conduct a definitive multiple dose tissue distribution study using the better of the two radiolabeled compounds. We compared the excretion and tissue distribution in rats of [¹⁴C]linezolid, radiolabeled in two different locations, after 7 consecutive once daily [¹⁴C] oral doses. The radiolabels were in the acetamide (two carbon) and oxazolidinone (isolated carbon) functional groups. Terminal phase tissue residue and excretion data were compared to data from rats dosed orally with [¹⁴C]sodium acetate. Drug-related radioactivity was excreted rapidly over 24 h. After a single dose, the acetamide and oxazolidinone radiolabel sites both gave 3% of dose as exhaled ¹⁴CO₂. After 7 daily [¹⁴C] oral doses, terminal phase radioactive tissue residues were higher from the acetamide radiolabel, relative to the oxazolidinone radiolabel, and were primarily not drug-related. In the definitive tissue distribution study, low concentrations of drug-related radioactivity in skin and thyroid were observed. We conclude that although small amounts of radiolabel instability do not significantly affect single dose tissue radioactivity C_max and AUC, artifacts arising from radiolabel instability can prolong the apparent terminal phase half life and complicate study data interpretation. When possible, it is always preferable to use a completely stable radiolabel site.

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Pregnenolone sulfate (PREGS) is reported to be present in higher concentration in the brain (more than 5 ng/g tissue in the rat) than in blood and is considered to be a neurosteroid. However, there are some doubts on its brain levels, because they were determined by indirect methods (e.g., GC-MS or radioimmunoassay after solvolysis). In the present study, PREGS in rat brains was determined by an enzyme-linked immunosorbent assay, which did not require solvolysis, after pretreatment with an Oasis HLB cartridge. The absolute recovery rate of PREGS through the pretreatment was 60.8%, and the quantitation limit was 33 pg/g tissue for a 200-mg of brain aliquot. Intra- and inter-assay coefficients of variation were less than 15.1 and 9.2%, respectively. The brain PREGS levels in the control rats (n=10) were less than 0.15 ng/g tissue except for one sample (0.42 ng/g tissue) and were lower than the serum levels (n=5, 0.25—0.47 ng/ml). On the contrary, the brain PREGS levels were sufficiently increased after intrapertioneal injection of 2 mg/kg body of PREGS (n=5, 0.37—1.29 ng/g tissue). These results demonstrate that, in rats, the brain PREGS may be derived from peripheral sources, and its actual levels are much lower than those previously measured by indirect methods.

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Mevalonate pyrophosphate decarboxylase (MPD) is considered to be a cytosolic protein. Recently, other groups reported that MPD is mostly located in the peroxisomes. In this study, we examined whether the expression of MPD in mice depends on the proliferation of peroxisomes, and whether MPD is predominantly located in the peroxisomes or the cytosol of mice. No increase in the protein level of MPD was observed in the crude extract of the livers of mice administered with peroxisome proliferative drugs. The result suggests that the expression of MPD is independent of the proliferation of peroxisomes, and may be maintained via a specific regulatory mechanism, different from the regulation of the expression of peroxisome proliferator-activated receptor alpha. When the subcellular distribution of MPD in mouse melanoma (B16F10) cells was examined by cell fractionation, MPD was detected in the cytosol of B16F10 cells, but not in the peroxisomes. In permeabilized B16F10 cells treated with digitonin, which lack cytosolic enzymes, 80% and 20% of MPD, 75% and 25% of lactate dehydrogenase, or 2% and 98% of catalase, existed in the medium and in the cell, respectively. From these results, it indicated that MPD was predominantly located in the cytosol and did not exist in the peroxisomes of B16F10 cells.

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Ewing's sarcoma (ES) is one of the most malignant bone and soft tissue tumors in childhood. Morphologically, ES belongs to the small round cell tumors (SRCT). ES, peripheral primitive neuroectodermal tumor (PNET), and Askin's tumor are classified as ES family tumors (ESFT) because they share a common chromosomal translocation. The EWS-FLI1 chimeric gene is generated by t (11; 22). Other reciprocal translocations resulting in formation of chimeric genes between EWS and ETS family genes (ERG, ETV1, E1AF, and FEV) are t (21; 22), t (7; 22), t (17; 22), and t (2; 22), respectively. Although it is generally difficult to distinguish ES from SRCT, we could easily and quickly distinguish ES from other SRCT by using reverse transcription polymerase chain reaction (RT-PCR). We looked for specific chimeric genes in 23 tumor samples, including three ES clinical samples. We detected five chimeric genes in the three ES samples. Three chimeric genes, all EWS-FLI1, were detected in one ES sample. Different chimeric genes, EWS-ERG and EWS-ETV1, were detected in the other two ES samples. Moreover, because we could not detect specific chimeric genes in samples from non-ESFT, it may be possible to use this technique to diagnose ESFT and to detect tumor cell contamination before hematopoietic stem cell transplantation.

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The pH dependence for the hydrolysis of β-lactam antibiotics by a metallo-β-lactamase (IMP-1) produced from Serratia marcescens was investigated varying the concentration of Zn(II). The activity of IMP-1 for imipenem was decreased at pH less than pH 5.3 without external addition of Zn(II) ions but was recovered with addition of Zn(II). Varying the concentration of external Zn(II), the molar activity of the enzyme, k_obs, that was defined by the velocity of hydrolysis of imipenem/concentration of IMP-1 was expressed by k_obs=v_init/[E]_T=k_max[Zn]/(K_d+[Zn]) in which K_d stands for the dissociation constant between Zn(II) and IMP-1. The dissociation constants, K_d, vary with pH; K_d=840×10⁻⁶ M at pH 4.3 and K_d=0.19×10⁻⁶ M at pH 6.0. The plot of −log K_d against pH showed a straight line having a slope of 4.0 below pH 5.0, showing the existence of four functional groups which may be protonated upon dissociation of Zn(II) ion(s). The k_cat, K_m, and k_cat/K_m of hydrolysis of imipenem and cephalothin in the presence of sufficient concentration of Zn(NO₃)₂ for saturation of IMP-1 with Zn(II) showed similar dependency to each other on pH between pH 6.0 and 9.0.

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In previous studies in vitro we showed that the quinone fraction (QF) from the heartwood of Auxemma oncocalyx TAUB. presented antiplatelet and antioxidant activities. In the present work, the QF antioxidant property was evaluated in models of CCl₄-induced hepatotoxicity in rats, and prolongation of pentobarbital-induced sleeping time in mice. Our results showed that levels of plasma glutamate-pyruvate-transaminase (GPT), as well as glutamate-oxalate-transaminase (GOT), were increased by the administration of CCl₄. On the other hand, only GPT levels were reduced by the QF treatment. Pentobarbital sleeping time was prolonged by the administration of CCl₄ and reduced by the QF treatment. Moreover, QF did not alter the pentobarbital-induced sleeping time. In conclusion, we showed that QF, represented mainly by oncocalyxone A, has hepatoprotective activity, and this effect is at least in part due to the antioxidant activity of this quinone.

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The metabolic stability of the acyl-CoA: cholesterol O-acyltransferase (ACAT) inhibitor N-(4-benzyloxy-3, 5-dimethoxycinnamoyl)-N′-(2, 4-dimethylphenyl)piperazine (YIC-708-424) and its n-alkoxy derivatives containing an alkyl chain of 3 or 7 to 10 carbons, which exhibited different hypocholesterolemic activities, was investigated in vivo and in vitro in rats. After the oral administration of YIC-708-424 to rats at a dose of 5 mg/kg/d for 7 d, the parent compound was not detected in the blood. On the other hand, when the n-alkoxy derivatives were administered to rats, an increase in the alkyl chain length produced a progressive increase in the blood concentration of the parent compound. Both in the blood of rats administered YIC-708-424 and in the reaction mixture after the incubation of YIC-708-424 with rat hepatic 9000×g supernatants, an inactive major metabolite, N-(4-benzyloxy-3, 5-dimethoxycinnamoyl)-N′-(4-carboxyl-2-methylphenyl)piperazine, was observed. The ratio of the maximum velocity to the apparent Michaelis–Menten constant (V_max/K_m) for the degradation of the n-propyloxy derivative in rat hepatic and intestinal microsomes was almost equivalent to that of YIC-708-424. On the other hand, an increase in the alkyl chain length of n-alkoxy derivatives produced a progressive decrease in V_max/K_m for the degradation of these compounds. Additionally, the in vivo hypocholesterolemic activities of YIC-708-424 and its n-alkoxy derivatives were positively correlated with the blood concentration of the parent compound and were negatively correlated with their V_max/K_m. These results suggest that the metabolic stability of ACAT inhibitors in the liver and intestinal epithelium, which are the major target organs of these compounds, has a strong influence on their pharmacological activities in vivo.

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(−)-Epicatechin-3-O-gallate (ECG), a component of Rhei Rhizoma, is one of the active components of Onpi-to, a herbal medicine composed of five crude drugs (Rhei Rhizome, Glycyrrhizae Radix, Ginseng Radix, Zingiberis Rhizoma and Aconiti Tuber), which has been used in patients with chronic renal failure. Pharmacokinetics of ECG was investigated in male rats employing an HPLC-electrochemical detection method. 1. Following oral administration of ECG, ECG plasma levels revealed curves characterized by peaks at 0.065, 0.063 and 0.085 h corresponding to dosages of 12.5, 25.0 and 50.0 mg/kg at mean concentrations of 49.62, 212.89 and 464.04 ng/ml, respectively. Plasma levels subsequently declined bi-exponentially. ECG demonstrated nonlinear pharmacokinetics in terms of C_max and AUC(0—inf). 2. The absolute bioavailability values (F) were 1.02, 1.47 and 3.30% at doses of 12.5, 25.0, and 50.0 mg/kg, respectively. 3. Following intravenous injection of ECG, plasma levels of ECG decreased with the γ-elimination half-life (t_1/2γ) of 4.03 h. 4. Following oral administration of ECG, urinary levels of ECG were lower than the quantitation limit. Moreover, cumulative excretion of the metabolites, δ-(3,4-dihydroxyphenyl)-γ-valerolactone and δ-(3-methoxy-4-hydroxyphenyl)-γ-valerolactone, was 2.45 and 0.23% of dose, respectively, up to 30 h after dosing.

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Onpi-to, an herbal medicine composed of five crude drugs (Rhei Rhizoma, Glycyrrhizae Radix, Ginseng Radix, Zingiberis Rhizoma and Aconiti Tuber), was administered orally to rats. Onpi-to includes 1.240% of total potential rhein derived from sennoside A, sennoside B, rhein 8-O-glucopyranoside and rhein. Plasma, urinary and biliary levels of rhein were determined by an HPLC-UV method. The plasma levels displayed curves characterized by maximum peaks at 8.3±5.2 min, 8.3±5.2 min and 20.0±21.9 min following dosages of 125, 250 and 500 mg/kg with mean concentrations of 1302.5±926.4, 2973.6±684.3 and 3118.8±1701.2 ng/ml, respectively, followed by a subsequent decline. Area under the concentration–time curve (AUC)(0—48 h) at doses of 125, 250 and 500 mg/kg were 752.3±321.5, 2443.3±554.4 and 4443.2±2641.3 ng·h/ml, respectively. In female rats, rhein plasma levels showed curves which had a maximum peak at 45.0±16.4 min after a dosage of 250 mg/kg with mean concentration of 3058.0±1533.7 ng/ml, followed by a subsequent decline. AUC(0—48 h) was 5537.7±1876.0 ng·h/ml. The cumulative urinary excretion of rhein and of conjugated rhein was 3.14±1.56% and 38.21±18.87% of dose, respectively, 48 h after dosing at 500 mg/kg of Onpi-to in male rats. The cumulative biliary excretion of rhein was 1.34±0.44% of dose 48 h after dosing at 500 mg/kg of Onpi-to in male rats.

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Glucocorticoids are effective drugs for the treatment of allergic skin diseases. In the present study, we observed the effects of prednisolone on the cutaneous reaction and skin barrier function in mice treated with a hapten, 2,4-dinitrofluorobenzene. Repeated hapten application onto the mouse ear resulted in a potent ear swelling with an elevation of specific serum IgE. The ear swelling appeared following the second application of the hapten and peaked at 24 h after each application. Specific serum IgE was detected first after the fourth hapten application. Topical treatment with prednisolone apparently suppressed the swelling, whereas it failed to affect the serum specific IgE level. The hapten application caused an increase in transepidermal water loss, which was potently inhibited by prednisolone, although the water content was not affected. Amounts of triglyceride and cholesterol in the ear skin increased after repeated hapten applications, whereas the relative amount of free fatty acid and ceramide diminished. Prednisolone exhibited an inhibitory effect on the changes in lipid content. Thus prednisolone apparently inhibits the alteration of skin barrier function caused by hapten application as well as the cutaneous reaction.

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We examined the effects of the angiotensin converting enzyme (ACE) inhibitors captopril, enalaprilat, quinapril, and trandolapril, and their active metabolites quinaprilat and trandolaprilat, on hemolysis induced by lysophosphatidylcholine (LPC) in human erythrocytes. LPC induced hemolysis at the concentrations above the critical micelle concentration (4 μM). Propranolol, used as a reference drug, attenuated the 50% hemolysis induced by 6 μM LPC at concentrations ranging from 100 nM to 100 μM. Similarly, quinaprilat (10 μM) and trandolaprilat (10, 100 μM) significantly attenuated the LPC-induced hemolysis, but other ACE inhibitors did not. Since propranolol possesses a membrane stabilizing action correlated with high lipophilicity, it appears that the high lipophilicity of quinaprilat or trandolaprilat is responsible for the protection from the damage induced by LPC. However, quinapril and trandolapril were not effective, although both drugs have higher lipophilicity than quinaprilat and trandolaprilat. Hence, it is suggested that the high lipophilicity alone may not contribute to the protective effects of ACE inhibitors against LPC-induced hemolysis. None of ACE inhibitors attenuated the hypotonic hemolysis (60 mM NaCl), although propranolol did. Furthermore, neither propranolol (100 μM) nor quinaprilat (50 μM) and trandolaprilat (50 μM) affected LPC micelle formation, suggesting that these drugs do not directly bind to LPC. We therefore believe that the protective effects of quinaprilat and trandolaprilat on the LPC-induced hemolysis may be related physicochemically to their highly lipophilic and ACE inhibitory structures, which probably maintain erythrocyte membrane integrity by a mechanism other than ACE inhibition, prevention of LPC micelle formation or protection against osmotic imbalance.

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Chitosan is widely used to treat patients with hypoxia-induced diseases such as ischemia, neuronal death, cerebral stroke, and cerebral infarction. Using the ELISA method, we examined the effect of high molecular weight water-soluble chitosan (WSC) on inflammatory cytokine production in the desferrioxamine (DFX, known to mimic hypoxia)-stimulated human mast cell line HMC-1. DFX significantly increased interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α production compared with the control in a time-dependent manner (p<0.05), but did not affect IL-1α production and mRNA expression. The increase in IL-6, IL-8, and TNF-α levels was significantly inhibited by WSC in a dose-dependent manner with IC₅₀ values of 0.77, 0.88, and 2.5 μg/ml, respectively. The maximal inhibition rate of IL-6, IL-8, and TNF-α production by WSC was 64±9.7%, 80±9.4% and 54±4.5%, respectively. In addition, WSC inhibited DFX-induced activation of nuclear factor (NF)-κB. In conclusion, these results suggest that WSC is an inhibitor of NF-κB under hypoxic conditions, which might explain its beneficial effect in the treatment of hypoxia-induced inflammatory diseases.

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To find new antipruritic herbal medicines for pruritus, we screened the methanol extracts of seven herbal medicines which have been used to treat dermatologic diseases, testing them on mouse models of acute and chronic itch. When administrated perorally (p.o.) at a dose of 200 mg/kg, methanol extracts of Sophora flavescens and Cnidium monnieri, but not the others, significantly inhibited a serotonin (5-HT)-induced itch-related response (scratching) and the spontaneous scratching of NC mice, a mouse model of atopic dermatitis. The inhibitory effect of Sophora flavescens was stronger than that of Cnidium monnieri. The methanol extract from Sophora flavescens (50—200 mg/kg) inhibited 5-HT-induced scratching in a dose-dependent manner, without any effects on the locomotor activity. These results suggest that Sophora flavescens and its constituents widely affect acute and chronic pruritus, and are possible as new antipruritic agents.

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The ability of curcumin, a natural antioxidant isolated from Curcuma longa, to inhibit hydrogen peroxide (H₂O₂)-induced cell damage in NG108-15 cells was examined. When added simultaneously with 500 μM H₂O₂, curcumin (25—100 μM) effectively protected cells from oxidative damage. However, when the cells were pretreated with curcumin (25—100 μM) for 1.5 h before H₂O₂ exposure, curcumin was unable to inhibit H₂O₂-induced cell damage. Instead, it caused a significant concentration-dependent decrease in cell viability after H₂O₂ exposure. This dual action of curcumin suggests that pretreatment with curcumin by itself did not have any significant effect on the viability of the NG108-15 cells, but it sensitized them to oxidative damage induced by H₂O₂ under our experimental conditions. It appears that these events may not relate to the antioxidant and free radical scavenging activities of curcumin.

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Annonaceous acetogenins (ACG) are natural products found in the plant family Annonaceae and which strongly inhibited mitochondrial complex I. The inhibition of NADH oxidase of chicken liver mitochondria by three different structural ACG was studied here, and ACG was shown to have potent inhibitory activities similar to rotenone for NADH oxidase. The IC₅₀ values indicated that bis-adjacent tetrahydrofuran (THF) type squamocin C was more potent than non-adjacent bis-THF type squamostatin B, and the latter was more potent than non-THF type compound 1 in the assay. The roles of structural factors of ACG such as the terminal γ-lactone, the features of other ring moieties and hydroxyl groups, as well as the alkyl chain were simply discussed in this study.

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This study was conducted to develop a new biomaterial to be used for an antioxidative drug. In this study, the hepatoprotective effect of chondroitin sulfate (CS) (100 mg/kg and 200 mg/kg body weight) was investigated at the antioxidative enzyme levels of liver total homogenate and mitochondria fraction. And the carbone tetrachloride (CCl₄)-induced rats were used as hepatotoxic models. The CCl₄ induced rat has been widely used as a hepatotoxic model due to its practicality, convenience and cost effectiveness since the generation of free oxygen radicals by CCl₄ injection was proposed as an important causative agent of hepatotoxicity. Malondialdehyde (MDA) levels were determined as well as the activities of superoxide dismutase (SOD), catalase (CAT), reduced-glutathione (GSH), oxidized-glutathione (GSSG) and glutathione peroxidase (GPx) in the liver. In addition, histopathology of liver tissue was investigated. Liver antioxidative enzyme activity was elevated while MDA concentration was decreased in all CS treated animals. The results demonstrated that CS protected oxidative stress in a dose dependent manner. Moreover, inflammation and cirrhosis in liver tissue of CS treated group were significantly decreased. It gave us an impression that CS might be a radical scavenger.

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We have examined the cytotoxic effect of rhodexin A isolated from the extract of Rhodea japonica on human leukemia K562 cells using a flow cytometer and compared it with that of ouabain. Rhodexin A at 30 nM started to attenuate growth without affecting viability and further increases in the concentration of rhodexin A (100 nM or more) completely inhibited growth with decreasing viability. Rhodexin A at 30—100 nM increased the G₂M population, but decreased the G₀G₁ population, suggesting cell cycle arrest in the G₂M phase. Rhodexin A at 100 nM increased the number of cells with hypodiploid DNA, indicating that rhodexin A induced apoptosis. The potency of rhodexin A to inhibit growth was greater than that of ouabain. The results indicate that rhodexin A exerts a potent inhibitory action on the growth of human leukemia K562 cells by inducing cell cycle arrest and apoptosis. Rhodexin A may also be a candidate for cancer treatment because there have been clinical reports of tumor regression in patients taking cardiac glycosides.

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Transgene expression and skin tumorigenicity were investigated in transgenic TG-AC mice carrying the v-Ha-ras after treatment with benzo[a]pyrene (BP). Animals treated with 40 μg BP (×2/week/mouse) showed 100% tumor response after 25 weeks, as did 40% of the mice treated with 20 μg BP but 10 μg BP did not produce a tumor response. In the case of animals treated with 40 μg BP for 25 weeks, most of the tumors were proven to be carcinomas (80%, 4 out of 5 mice), and all tumors were shown to be positive in terms of transgene expression detected by in situ hybridization. These data suggest that BP was tumorigenic in a dose-dependent manner in TG-AC mice and that TG-AC mice were dependent on transgene expression during BP carcinogenesis.

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Acetophenone derived mono-Mannich bases (Ig1—Ig4), 1-aryl-3-amino-1-propanone hydrochlorides, which are known to have cytotoxicity in Jurkat cells, were synthesized. Then, they were converted to corresponding azine derivatives (D1—D4), N, N′-bis(3-amino-1-aryl-propylidene)hydrazine dihydrochlorides, which are bifunctional agents. The aryl part was replaced by phenyl in Ig1, Ig2, Ig3, D1, D2, and D3, and by p-hydroxyphenyl in Ig4 and D4. The amine part was replaced by dimethylamine in Ig1, D1, Ig4 and D4, by piperidine in Ig2 and D2, and by morpholine in Ig3 and D3. The aim of this study was to investigate whether the modification in chemical structure, converting the mono-Mannich base to a corresponding azine derivative, improves the cytotoxicity. In addition, the effect of the representative compound, D3, N, N′-bis(3-morpholine-4-yl-1-phenylpropylidene)hydrazine dihydrochloride, on cellular glutathione level after 1 h exposure in phosphate buffer at 37 °C was also determined to provide information on a possible mechanism of cytotoxic action. Compounds D2—D4 are reported for the first time in this study. Except for Ig2 and D2, the cytotoxicity of mono-Mannich bases, Ig1, Ig3 and Ig4 and corresponding azine derivatives, D1, D3 and D4 were higher than the reference compound 5-FU. Azine derivatives D1 and D4 had almost equal cytotoxic potency with corresponding mono-Mannich bases Ig1 and Ig4, respectively. On the other hand, azine derivatives D2 and D3, had 1.28 and 1.90-times less cytotoxicity in Jurkat cells compared with the mono-Mannich bases, Ig2 and Ig3, respectively, from which they are derived. Azine derivative D3 dose-dependently decreased the total cellular glutathione level, suggesting that azine derivatives may exert cytotoxicity by thiol alkylation. Azine derivatives with equal or less cytotoxic potency compared to the mono-Mannich bases they are derived from seemed to be less suitable derivatives for the development of new cytotoxic compounds.

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We have previously examined the antiproliferative activity of caffeic acid phenethyl ester (CAPE) and its 20 analogues against six tumor cell lines, and found that CAPE analogues possess selective antiproliferative activity toward the murine colon 26-L5 carcinoma cell line. To extend our study, the effects of CAPE analogues on the metastatic development of murine colon 26-L5 carcinoma cells in the lung were examined. The oral administration of CAPE (5 mg/mice/d) for 7 d after tumor inoculation decreased the tumor weight and the number of tumor nodules in the lung by 50% and 50%, respectively, compared to the control, while CAPE (5 mg/mice/d) administered for 7 d before tumor inoculation showed no significant effect. Besides CAPE, 4-phenylbutyl caffeate, 8-phenyl-7-octenyl caffeate, 2-cyclohexylethyl caffeate and n-octyl caffeate at an oral dose of 2 mg/mice/d caused a 55%, 43%, 55% and 35% reduction of the tumor nodules in their lung metastasis formation, respectively. These results further elaborate the possibility of CAPE and its analogues to become a new class of chemopreventive agents for the treatment of colon cancer metastasis.

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The anti-lipid peroxidation (LP) activity and anti-superoxide formation (SOD) of new N-H and N-substituted indole derivatives were evaluated to determine their antioxidant activity. The results showed that compounds 1, 2, 5, 9 and 12 demonstrate considerable inhibition of lipid peroxidation of mouse liver homogenate. On the other hand, compounds 5 and 12 showed anti-superoxide formation activity at a concentration of 10⁻⁴ M. Consequently, it can be concluded that these compounds exhibit important activity compared with reference compound α-tocopherol (vitamin E).

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Betula platyphylla var. japonica is a rich source of triterpenoid as it contains dammarane type triterpenes in the leaves, and lupane type and oleanane type triterpenes in the bark. Four oxidosqualene cyclase cDNAs (BPX, BPX2, BPW and BPY) were cloned by homology based PCR methods from cell suspension cultures of B. platyphylla var. japonica. Open reading frames consisting of 2274, 2304, 2268 and 2340 bp were ligated into yeast expression plasmid pYES2 under the control of GAL1 promoter and introduced into lanosterol synthase deficient (erg7) Saccharomyces cerevisiae strain GIL77. Analyses of in vitro enzyme activities and/or accumulated products in the transformants demonstrated that they encode cycloartenol synthase (BPX and BPX2), lupeol synthase (BPW) and β-amyrin synthase (BPY) proteins. Phylogenetic tree was constructed for all the known oxidosqualene cyclases (OSCs) including the clones obtained in this study, revealing that OSCs having the same enzyme function form respective branches in the tree even though they derive from different plant species. Intriguing correlation was found between reaction mechanism and molecular evolution of OSCs in higher plants.

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The effects of polygodial isolated from the leaves of Tasmannia lanceolata on necrotizing agents-induced gastric lesions in rats were compared with capsaicin. Polygodial markedly inhibited the gastric mucosal lesions induced by several necrotizing agents, such as ethanol (ED₅₀=0.029 mg/kg, p.o.), 0.6 M HCl (ED₅₀=0.26 mg/kg, p.o.), and aspirin (ED₅₀=0.38 mg/kg, p.o.), and partly inhibited the gastric mucosal lesions induced by indomethacin, but showed no significant effect on acid output in pylorus-ligated rats at doses of 0.05—0.5 mg/kg. The gastroprotection of polygodial was attenuated by pretreatment with indomethacin (10 mg/kg, s.c.), N^G-nitro-L-arginine methyl ester (70 mg/kg, i.p.), N-ethylmaleimide (10 mg/kg, s.c.) and ruthenium red (3.5 mg/kg, s.c.). Polygodial (0.2 mg/kg, p.o.) increased the amount of reduced glutathione in gastric mucosa of ethanol-treated group. These results suggested that endogenous prostaglandins, nitric oxide, sulfhydryl compounds and vanilloid receptor-mediated effects are involved in the protective effect of polygodial.

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A method for semi-micro high-performance liquid chromatography (HPLC) has been established for the simultaneous determination of 3α-hydroxyglycyrrhetic acid and 3-dehydroglycyrrhetic acid together with glycyrrhizin, glycyrrhetic acid and glycyrrhetic acid mono-glucuronide formed by incubation of glycyrrhizin with rat feces. The analysis was accomplished within 25 min with a TSKgel ODS-80TsQA (150×2.0 mm i.d.) column by linear gradient elution using a mobile phase containing aqueous phosphoric acid and acetonitrile at a flow rate of 0.2 ml·min⁻¹, a thermostatic oven at 25 °C, and detection at 254 nm. The detection limits of these compounds were 0.2 pmol per injection (5 μl). The metabolites of glycyrrhizin, by anaerobic or aerobic incubation with rat fecal suspension over 48 h, were determined. Glycyrrhizin was almost completely converted to metabolite glycyrrhetic acid, and metabolites 3α-hydroxyglycyrrhetic acid and 3-dehydroglycyrrhetic acid in negligible amounts in anaerobic conditions. However, the metabolic time courses of 3-dehydroglycyrrhetic acid when incubated in aerobic conditions revealed that it apparently continued increasing during the whole incubation period.

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In the present study, we investigated the anti-oxidant activity of oolong tea in an oxidation model using human low-density lipoprotein (LDL). Oolong tea suppressed the oxidation of LDL induced by 2-2′-azobis 4-methoxy-2,4-dimethyvaleronitrile (V70) in a dose-dependent manner, that is, it prolonged the lag time to 114.3%, 138% and 199.9% as compared with the control group at 0.5 μg/ml, 1.0 μg/ml, and 2.5 μg/ml, respectively. We also determined the scavenging effect of oolong tea on active oxygen radicals using the electron spin resonance (ESR) technique with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trapping agent. The intensity of the ESR signals for the DMPO–OOH adduct formed by the hypoxanthine/xanthine oxidase reaction system with DMPO decreased in the presence of oolong tea. The IC₅₀ of oolong tea was 19.9 μg/ml. These findings suggested that oolong tea has beneficial effects on health related to its anti-oxidative action.

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The antidiabetic properties of prosopis gum alone and as a bioadhesive base for the delivery of metformin are presented. The bioadhesive value of the gum was commensurate with those of Carbopol 974-P and sodium carboxymethyl cellulose (NaCMC). The release of the drug was higher from prosopis gum based bioadhesive formulations than from NaCMC and Carbopol 974-P products. This was shown by the shorter time required to reach t₅₀ (the time required for 50% of the drug to be released) or t₂₀ (time required for 20% of the drug to be released) for the release of metformin. The gum showed moderate antidiabetic properties when used alone. In combination with metformin in a bioadhesive form, the glucose lowering effect was found to be synergistic. The areas under the plasma drug concentration vs. time curves (AUCs) for the bioadhesive combinations were similar to those of the drugs alone in an aqueous system. This shows that the gum did not interfere with absorption of the incorporated drug. However, the areas under the effect vs. time curves (AUECs) were much higher when combined in a bioadhesive form than with the drug alone. The AUCs obtained with NaCMC based bioadhesive formulations were relatively smaller than those of metformin in an aqueous system and the combinations of metformin and prosopis gum.

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We evaluated microbial viability in preparations packaged for single use only which mandate that residual solution be discarded such as albumin and globulin preparations as blood products, preparations containing albumin (such as urokinase and interferon), fat emulsions, and a preparation containing fat emulsions (propofol). In most preparations, Serratia marcescens and Burkholderia cepacia proliferated rapidly at 30 °C. However, in globulin preparations containing 1—2.25% glycine to prevent protein degradation (Gamma-Venin P, Venilon-I, Globulin Injection, and Ahlbulin), no growth of S. marcescens and B. cepacia was detected over 24 h at 30 °C. For globulin preparations containing 1—2.25% glycine, the injunction to “Discard residual solution after the package has been used” in the package inserts can be revised to “It is possible to use residual solution within 24 h after the package has been used with storage in a cool place.”

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The effects of various disinfectants and hot water on planktonic cells and biofilm cells of Burkholderia cepacia were investigated. The survival rate of viable B. cepacia cells in suspension decreased to 0.001% or lower within 15 s of exposure to 0.5% benzalkonium chloride, within 30 s of exposure to 0.5% alkyldiaminoethyl glycine, or within 1 min of exposure to 0.1% alkyldiaminoethyl glycine, and decreased to about 0.1% with 60 min of exposure to 0.1% benzalkonium chloride or 0.5% chlorhexidine gluconate, but did not decrease to 1% or less with 60 min of exposure to 0.1% chlorhexidine gluconate. There were no effects of 0.1% and 0.2% chlorhexidine gluconate and 0.1% benzalkonium chloride against biofilm cells of B. cepacia, and 0.5% chlorhexidine gluconate, 0.5% benzalkonium chloride and 0.1% alkyldiaminoethyl glycine were barely effective against biofilm cells even after 60-min exposure. On the other hand, both planktonic cells and biofilm cells of B. cepacia were eradicated within 15 s by sodium hypochlorite, povidone-iodine, 80% v/v ethanol, and hot water at 65 °C or higher.

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This study was conducted to explore the mechanism of the pharmacokinetic interaction between aspirin (ASP) and indomethacin (IND) using rat erythrocytes (RBCs) and hepatocytes. ASP was hydrolyzed to salicylic acid (SA) in both the RBCs and hepatocytes. Within RBCs, aspirin and/or salicylate (ASP/SA) increased the concentration of IND, accompanied by a constant hydrolysis of IND. In hepatocytes, a low dose of IND was subjected to glucuronidation rather than hydrolysis, and ASP/SA inhibited both the acylglucuronidation of IND and hydrolysis of IND glucuronide. A high dose of IND underwent hydrolysis with about double the glucuronidation, and ASP/SA decreased the ratio of hydrolysis to glucuronidation, accompanied by a loss of ASP, IND and their metabolites from the medium. Collectively, the results provide metabolic insight into the mechanism of drug-drug interaction between ASP/SA and IND in the hepatocytes and RBCs.

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The detachment forces of various polymers are frequently measured to determine their mucoadhesion strength. As the process of mucoadhesion is a consequence of interactions between the mucus layer on mucosa and mucoadhesive polymers, it is greatly dependent on mucus and polymer structure including their charge. It is also known that the glycosaminoglycan layer, which covers the urinary bladder mucosa surface, is highly negatively charged. Therefore, by measuring the zeta potential of polymer dispersions and mucosal homogenates an insight into electrostatic interactions during mucoadhesion can be obtained. In our experiments we chose three polymers, two anionic (polycarbophil, PC; sodium carboxymethyl cellulose, CMCNa) and one cationic (chitosan hydrochloride, CH), for which we expected different zeta potential values and different mucoadhesion strengths. The correlation between the zeta potential and the detachment force was determined. In addition to that, the zeta potential of the scraped surface layer of pig urinary bladders was measured to confirm its negative value. The mucoadhesion strength decreased in the following order: CH>CMCNa=PC. The zeta potentials for all three polymers and for porcine vesical mucosal homogenates were measured in Tyrode solution and two NaCl solutions with different ionic strengths. The lower values of the detachment force correlated well with the more negative zeta potential of the polymer, which might be a consequence of the greater repulsion between negative charges of polymers and glycosaminoglycans.

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The degradation of thirteen flavonoid aglycones—wogonin, diosmetin, hesperetin, baicalein, morin, genistein, daidzein, quercetin, naringenin, luteolin, kaempferol, apigenin and neophellamuretin—were investigated in rabbit, rat and human fecal flora suspensions as well as in artificial intestinal juice, using high performance liquid chromatography. Separation were performed with a Cosmosil 5C₁₈-AR II column by isocratic and gradient elution with 0.1% (v/v) phosphoric acid–acetonitrile as a mobile phase, and detected at 254 nm. The flow rate was 1.0 ml/min. 5,7-Dimethoxycoumarin was used as the internal standard. The result indicated that all flavonoid aglycones except baicalein, diosmetin and quercetin were quite stable in artificial intestinal juice, whereas all were degraded in rabbit, rat and human feces suspension. In rabbit feces, wogonin, diosmetin and hesperetin were less degraded, whereas neophellamuretin, apigenin, kaempferol, luteolin, and naringenin were the most extensively degraded. In rat feces, wogonin and diosmetin were least degraded, whereas kaempferol, quercetin, genistein, luteolin, naringenin and neophellamuretin were extensively degraded. As in human feces, wogonin, daidzein and diosmetin were less degraded, whereas morin, genistein, baicalein, and quercetin were extensively degraded. In conclusion, wogonin and diosmetin were among the less degraded ones for all three feces tested. The presence of a methoxy group on the A or B ring of the flavonoid seems to protect the structure from bacterial degradation.

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SM-11355, cis-[((1R, 2R)-1, 2-cyclohexanediamine-N, N′)bis(myristato)]platinum(II), suspended in Lipiodol (SM-11355/Lipiodol) showed cytotoxic activity in hepatic tumor models in vivo and tumor cell lines in vitro. SM-11355/Lipiodol demonstrated selective retention in tumor tissue in vivo and high accumulation in tumor cells in vitro. This study was aimed to clarify the relation between the cytotoxicity of SM-11355/Lipiodol and intracellular platinum content. The cytotoxic activities were estimated by using WST-1 reagent. Intracellular platinum content and platinum-DNA adduct were estimated following exposure with SM-11355/Lipiodol when methionine was added. Methionine clearly inhibited the cytotoxic activities of SM-11355/Lipiodol. Moreover, intracellular platinum content and platinum-DNA adduct following exposure of SM-11355/Lipiodol decreased with increases in methionine concentration. The characteristic release of SM-11355/Lipiodol was not affected by addition of methionine. The present results suggested that one of platinum compounds exposed to cells following SM-11355/Lipiodol treatment is very similar in cytotoxic mechanism to cisplatin.

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A chitosan (CS)–orotic acid salt (CS-OT) was prepared, and the release of orotic acid (OT) from CS-OT as well as the adsorption of bile acids by CS-OT was investigated in vitro. The amount of OT released from CS-OT was about 2—2.7 μmol/mg CS-OT and this changed depending on the species of CS. CS-OT also adsorbed bile acids and the amount increased incrementally according to the number of amino group contained in CS. Furthermore, CS-OT was given to rats as feed in order to investigate the influence on serum cholesterol levels. A decrease in serum cholesterol levels was observed in the group, which was fed a diet containing CS-OT or CS for 1—2 weeks, but no differences in body weight changes were recognized. Therefore, CS-OT may be applied to treating hyperlipidemia.

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This study was conducted to investigate the chemical component of the hot water (HW) fraction of mycelia of Cordyceps sinensis and its antifatigue and antistress effect against a stimulus in vivo using rats and mice. The growth of mycelia reached a maximum level of 31.6 g/l after 120 h of incubation. The main chemical composition of the HW fraction of mycelia of C. sinensis was found to be carbohydrate (78.9%) with 5% moisture. The swimming endurance capacity of mice orally administered with the HW fraction (150 and 300 mg/kg/d, respectively) was significantly prolonged from 75 to 90 min with a lessening of fatigue. When the HW fraction (150 mg/kg/d) was given to rats for 8 d including a 48 h stress period, the weight changes of the adrenal gland, spleen, thymus, and thyroid, which is an index of stress, were suppressed. The HW fraction also significantly inhibited the increase in total cholesterol and the decrease in alkaline phosphatase levels as biochemical parameters of immobilization stress in rats.

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A study was investigated on the inhibitory effect of 29 drugs that have been reported to induce gynecomastia on the 2-hydroxylation of estradiol (E2) by recombinant P450 CYP3A4 and on the 17-oxidation of E2 by hepatic microsomal type II 17β-hydroxysteroid dehydrogenase (17β-HSD) of human male. The IC₅₀ values were determined for each drug relative to the 2-hydroxylation of E2 (catalytic activity: 1.54 nmol/nmol P450/min), and the inhibition constants (K_i) were determined for 13 drugs of which IC₅₀ values were 100 μM or less. Ketoconazole exhibited the lowest inhibitory concentration, and IC₅₀ and K_i values of 0.007 and 0.01 μM, respectively, were obtained. The IC₅₀ and K_i values for each of the 12 remaining drugs were as follows: cyclosporin A (IC₅₀: 0.064, K_i: 0.30), nicardipine hydrochloride (0.55, 0.29), tacrolimus (0.64, 0.88), mandipine hydrochloride (3.9, 2.6), nisoldipine (10, 3.3), verapamil hydrochloride (10, 20), domperidone (13, 7.2), haloperidol (14, 55), nitrendipine (14, 2.5), chlormadinone acetate (16, 10), flutamide (30, 39) and omeprazole (49, 47). With the exception of cyclosporin A that exhibited a competitive inhibition, the inhibition mechanisms of these drugs were all non-competitive. Next, the percentage inhibition of the above 29 drugs relative to the 17-oxidation of E2 (catalytic activity: 0.47 nmol/mg protein/min) was investigated at the approximate therapeutic concentration (1 μM) and at the non-clinical overdose concentration (100 μM). Although none of the drugs investigated exhibited inhibitory effects at a concentration of 1 μM, spironolactone and ketoconazole at 100 μM demonstrated percentage inhibitions of 96% and 77%, respectively. When the K_i values were determined for these two drugs, the former had a K_i value of 2.4 μM and the latter, 41 μM, and both of their inhibition mechanisms were non-competitive. On the basis of the above results, a total of 14 drugs consisting of the above 13 drugs plus spironolactone were found to inhibit the 2-hydroxylation or 17-oxidation of E2 in the liver, and this is presumed to act as a trigger that causes as increase in the estradiol pool, followed by induction of gynecomastia.

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We focused on dynamic responses to acute heat stress between 34 °C and 38.5 °C. Physiological and neuroendocrinological changes between 34 °C and 38.5 °C were studied in mice. The influence of humid conditions, 85% relative humidity (RH), on these changes was also investigated. Rectal temperatures increased above 34 °C and hematocrit levels increased at 38.5 °C 85% RH for 60 min. Food consumption and body weight gains decreased after a daily 60 min exposure to 34, 37 and 38.5 °C for 2 weeks. The corticosterone and vasopressin levels in the blood, and catecholamine and serotonin metabolite levels in the hypothalamus were not changed at 34 °C, but increased when above 37 °C for 60 min. Above 37 °C, these physiological and neuroendocrinological changes were accelerated by humid conditions. These results indicated that food consumption and body weight gains decreased above 34 °C, and the neuroendocrinological changes, which were accelerated by humid conditions, were induced above 37 °C. In comparison with restraint and water immersion stress, heat stress at 37 °C 85% RH showed a slower increase in serum corticosterone levels, smaller changes in plasma dopamine and dihydroxyphenylacetic acid levels, and, after repeated exposure, larger decreases in food consumption and body weight gains. This study clarified the relationships between temperature and humidity conditions and physiological and neuroendocrinological changes, along with the characteristics of responses in acute heat stress.

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