An ion-pair high-performance liquid chromatographic method for the simultaneous determination of four flavonoids, namely baicalin, wogonoside, baicalein and wogonin, and four berberine-type alkaloids, namely berberine, coptisine, palmatine and jateorrhizine, and glycyrrhizin in Kampo medicines is described. The analysis can be accomplished within 30 min with a Wakosil-II 5C18 HG column by linear gradient elution using a mobile phase containing aqueous phosphoric acid, sodium dodecyl sulfate and acetonitrile at a flow-rate of 1.0 ml·min^-1, a thermostatic oven at 45°C, and detection at 265nm. The method was applied to quantifying these components in three Kampo decoctions : Oren-gedoku-to, San'o-shashin-to and Hange-shashin-to. The decoctions were diluted with 65% methanol at the final stage because a large quantity of precipitate, mainly from baicalin and berberine, was formed. The within-day relative standard deviations were less than 2.02% (n=10). The recoveries of these compounds were 90.3-102%. The detection limits of these compounds were 0.02-1.96 μM per injection (5 μl).

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We determined the endocytosis motif sequence of macrophage scavenger receptors by measuring the internalization of the ligand-bound cell-surface scavenger receptors at the early stage. Mutation experiments have indicated that the 28 amino acids at the N-terminal cytoplasmic domain are important for both efficient internalization and cell-surface expression of the receptor. The substitution of alanine for Val21, Phe23 or Asp24 reduced both the efficiency of internalization and the cell-surface expression of the receptors; in addition, that for Val28 reduced only the internalization efficiency and that for Lys22 reduced only the cell-surface expression of the receptors. Western blot experiments showed that the total levels of expression of the mutant receptors in the cells were approximately the same and all the mutants were identified in the membrane fraction. This suggests that the Val21, Lys22, Phe23 and Asp24 mutants are localized in membranes other than the cell-surface membrane by missorting. All our results indicate that VXFD is the motif sequence essential for not only the coated-pit-mediated endocytosis of macrophage scavenger receptors but also for the trafficking of the receptors to the cell surface.

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The major physiologic effect of thyroid hormone is thought to be initiated by the binding of T3 to the DNA binding thyroid hormone receptor (TR). The aim of this study has been to characterize the organ specific expression of thyroid hormone receptor mRNA, as well as its protein distribution and molecular weight in man. Determination of TRα1, α2, β1 and β2 mRNA molecular size was performed using Northern blot analysis in the human heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. TRα1, α2 and β1 protein expression was characterized by Western blot analysis of human tissues. TRα1 mRNA of 4.9 kb was detected in all 8 tissues analyzed, with varying abundance in the various tissues. TRα2 mRNA was detected in 2 different sizes, with higher intensity at 5.7 and lower intensity at 3.2 kb. There were, however, multiple TRβ1 mRNA of 8, 2 and 1 kb detected. TRβ1 transcripts of 2 kb and 1 kb showed an organ specific pattern of expression. Multiple TRβ2 mRNA of 6.6, 5.2, 2.5 and 2.4 kb were detected, as well as a unique 1 kb transcript, in the heart. TRβ2 transcripts also displayed tissue specific expression. Western blot analysis displayed a single band of 48 kD for TRα1. The abundance of the TRα1 immunoreactive band was highest in the heart, brain, kidney and skeletal muscle, and lowest in the liver, placenta and lung, while no signals were detected in the spleen. The TRα2 specific antibody detected a band of 58 kD in all the tissues analyzed. The relative intensity of the immunoreactive TRα2 band detected was highest in the placenta and lung, with a medium concentration range in skeletal muscle, the heart and kidney. The TRα2 protein concentration was lowest in the spleen, liver and brain. Human TRβ1 protein was detected as 55 and 52 kD bands, as well as a unique band of 45 kD in heart. The 52 kD band was detected in all tissues except the kidney and spleen. The 55 kD band was not detected in the brain or liver. Both the 55 and 52 kD TRβ1 immunoreactive bands were detected in the placenta, lung, heart and skeletal muscle with similar intensity.In conclusion, specific patterns of TR mRNA and protein expression revealed characteristic organ distributions of each subtype. Unique cardiac expression was observed for TRβ₂ mRNA and for TRβ₁ protein.

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We studied the hydroxyl radical (OH·)-generating system in polymorphonuclear leukocytes (PMNs). When phenylalanine was incubated with the α, β and γ fractions prepared from pig polymorphonuclear leukocytes (PMNs) and hydrogen peroxide (H₂O₂), significant levels of formation of m- and o-tyrosine were observed in the α and β fractions, but not in the γ fraction. The amount of tyrosine formation per milligram of protein was greater with the β than with the α fraction. Further, when phenylalanine was incubated with α or β fractions with similar myeloperoxidase (MPO) activities in the presence of H₂O₂, tyrosine formation by the β fraction was also more effective. Using the β fraction in which the MPO activity was destroyed by heat treatment, no significant amount of tyrosine was formed. However, with the heat-treated β fraction and MPO preparations from human neutrophils in the presence of H₂O₂, the amount of tyrosine formation increased with the addition of increasing amounts of heat-treated β fraction. Tyrosine formation by the β fraction in the presence of H₂O₂ was significantly reduced by OH·scavengers. The above results suggest the existence of an OH·-generating system in which MPO and H₂O₂ participate in the granules of PMNs and, especially, in specific granules, there may exist some factors that cause more effective OH·generation.

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Rabbit liver cytosol produced approximately equal amounts of 6α-naloxol and 6β-naloxol from naloxone in the presence of NADPH at pH 7.4, and contained at least four forms of naloxone reductase. The two major forms, NR1 and NR2, which catalyze the stereospecific reduction of naloxone to 6α-naloxol and 6β-naloxol, respectively, were purified to apparent homogeneity by various chromatographic techniques. Both enzymes are monomeric proteins with similar molecular weights of 35000-36000, but NR1 is a basic protein with an isoelectric point (pI) of 9.3, while NR2 is an acidic protein (pI of 5.9). NR1 and NR2 gave the maximal activities at pH 8.0 and 6.1, respectively. NR1 exhibited considerable activity with NADH as well as with NADPH, whereas NR2 showed highly restricted specificity for NADPH. The K_m and V_max values of NR1 and NR2 for naloxone were 1.0 and 0.06 mM, and 76 and 162 munits/mg, respectively. In addition to naloxone, naltrexone and dihydromorphinone served as good substrates for NR2 but were poor substrates for NR1. Both enzymes reduced aliphatic and aromatic aldehydes, cyclic and aromatic ketones, and quinones at higher rates. The two enzymes catalyzed the dehydrogenation of 17β-hydroxysteroids with low K_m values, and NR2 showed an additional 3α-hydroxysteroid dehydrogenase activity. Amino acid sequence data of NR1 (99% of whole protein) and NR2 (66%) showed that both enzymes belong to the aldo-keto reductase (AKR) superfamily and can be classified into the AKR1C subfamily. These findings therefore indicate that they are new members of the AKR superfamily and may be involved physiologically in the steroid metabolism as well as in the detoxification of xenobiotic carbonyl compounds.

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The cytosol of rabbit lung alveolar macrophages contains a high amount of peroxidase, which reduces 5-hydroperoxyeicosatetraenoic acid (5-HPETE) to 5-hydroxyeicosatetraenoic acid (5-HETE) in the presence of glutathione. This peroxidase was purified 69-fold to homogeneity with overall recovery of activity of 18.5%. The molecular mass of the enzyme was approximately 80 kDa by gel filtration, and emerged as a single band at 23.1 kDa under reducing condition by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The amino-terminal sequence of the purified peroxidase was completely identical to the sequence deduced from cellular glutathione peroxidase (cGPx) gene of rabbit liver. No other activity that reduces 5-HPETE to 5-HETE was observed during purification. These results suggest that cGPx plays an important role in metabolism of lipid hydroperoxides, especially HPETE, in lung alveolar macrophages.

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In a mouse macrophage-like cell line, RAW 264.7, increasing the arachidonic acid (AA) content, by culturing cells with AA, caused profound AA release, irrespective of the lipopolysaccharide (LPS)-treatment, while lowering the AA content, by culturing cells with eicosapentaenoic acid (EPA), decreased it compared with the level in non-modified control cells. However, the release of prostaglandin D₂ (PGD₂), which had been generated from AA in response to LPS-treatment, was significantly decreased in both AA- and EPA-treated cells. Furthermore, although the amount of PG endoperoxide synthase-2 (PGHS-2) increased following LPS-treatment in all cases, both AA- and EPA-treatment caused a reduction of PGHS activity in LPS-treated cell lysates. Also, the addition of EPA or preincubation with AA in an in vitro PGHS assay system involving an LPS-treated, un-modified macrophage lysate, resulted in rapid inhibition of PGHS activity. These results suggest that both AA- and EPA-treatment inhibit PGD₂ synthesis by inactivating PGHS-2 without affecting induction of the protein, and that the increase or decrease in AA content following AA- or EPA-treatment correlates simply with the level of AA release but not with that of PGD₂ formation.

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1α, 25-Dihydroxyvitamin D₃ [1α, 25(OH)₂D₃] has been shown to exert both is nuclear vitamin D receptor (nVDR)-mediated genomic actions and membrane vitamin D receptor (mVDR)-mediated nongenomic actions. In this study, the effects of 1α, 25(OH)₂D₃ and its analogues on transmembrane Ca²⁺ influx were examined in the growth phase of rat osteosarcoma ROS17/2.8 cells. Like BAYK8644 (2×10^-5 M), a well-known L-type Ca²⁺ channel agonist, 1α, 25(OH)₂D₃ (10^-8 M) increased transmembrane influx of Ca²⁺ through voltage-dependent Ca²⁺ channels and increased intracellular Ca²⁺ concentration within 2 min of addition to the medium. The 1α, 25(OH)₂D₃-induced Ca²⁺ influx was completely blocked by pre-treatment with nifedipine (2×10^-5 M), an L-type Ca²⁺ channel antagonist. Two vitamin D analogues, 22-oxa-1α, 25(OH)₂D₃ (OCT, 10^-8 M) and 20-epi-22-oxa-24a, 26a, 27a-trihomo-1α, 25(OH)₂D₃ (KH1060, 10^-8 M), which were 3.8 and 3600-fold more active than 1α, 25(OH)₂D₃ in stimulating differentiation on human promyelocytic leukemic HL-60 cells, respectively, also increased intracellular Ca²⁺ concentration, while their Ca²⁺ channeling activities were similar to or significantly weaker than that of 1α, 25(OH)₂D₃. Furthermore, the enhanced transmembrane Ca²⁺ influx induced by 1α, 25(OH)₂D₃ (10^-8 M) or OCT (10^-8 M) was completely blocked by pre-treatment with the respective 1β epimer [1β, 25(OH)₂D₃ and 1β-OCT] at equal concentration. These findings suggest that 1α, 25(OH)₂D₃ and its analogues modulate transmembrane Ca²⁺ influx in osteoblast-like cells by opening L-type Ca²⁺ channels which can recognize 1α-hydroxy analogues as agonists and 1β-hydroxy analogues as antagonists.

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A new antibiotic, korormicin, isolated from a marine bacterium Pseudoalteromonas sp. F-420, was found to strongly inhibit the respiratory chain-linked Na⁺-translocating NADH-quinone reductase (NQR) from the marine Vibrio alginolyticus. Similar to 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), korormicin specifically inhibited the Na⁺-dependent reaction in the NQR complex that is directly coupled to the extrusion of Na⁺ from the cells. Both korormicin and HQNO acted as purely noncompetitive inhibitors with regard to Q-1, and the inhibitor constants were estimated to be 82 pM and 0.3 μM, respectively. Mutual exclusiveness of korormicin and HQNO was analyzed by kinetic methods, which indicated that a part of the binding site of korormicin and HQNO overlapped, preventing a simultaneous binding of the two inhibitors to the NQR complex. The site of Ag⁺ inhibition was the initial reaction of the NQR complex catalyzed by Nqr6 subunit. The time courses of Ag⁺ inhibition and the release of FAD indicate that the Ag⁺-denatured Nqe6 subunit gradually releases FAD.

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The variable regions of heavy- and light-chains of mouse monoclonal antibody 196-14 toward ovarian cancer-associated antigen CA125 were linked with a peptide linker (GSTSGSGKSSEGKG) and a histidine tag was attached at the carboxyl terminal. This single-chain Fv (scFv) with a histidine tag was expressed in Escherichia coli as an inclusion body. The inclusion body was solubilized with guanidium chloride, followed by purification on nickel nitrilotriacetic acid agarose column and refolding into the active form. The scFv thus obtained bound to the Siso cells, which express CA125, and may recognize the same epitope as the parental 196-14 antibody IgG does.

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We investigated the β-adrenergic receptor (AR) agonistic activities in rats and humans, and the anti-obesity and anti-diabetic activities in KK-A^y mice, of a new β₃-AR agonist, SWR-0342SA ((S)-(Z)-[4-[[1-[2-[(2-hydroxy-3-phenoxypropyl)]amino]ethyl]-1-propenyl]phenoxy] acetic acid ethanedioic acid). With regards to its β-AR agonistic activity in rats, SWR-0342SA stimulated the atrial beating rate (β₁-AR activity) and white adipocyte lipolysis (β₃-AR activity), but did not induce uterine muscle relaxation (β₂-AR activity). The β₃-AR agonistic activity of SWR-0342SA was about 20 times stronger than its β₁-AR agonistic activity. Similarly, SWR-0342SA enhanced the accumulation of cAMP in Chinese hamster ovary (CHO) cells expressing human β₁- and β₃-ARs, while having no effect in CHO cells expressing β₂-ARs. Adenylyl cyclase stimulation by SWR-0342SA in CHO cells expressing β₃-ARs was about 35 times higher than that in CHO cells expressing β₁-ARs. With regards to anti-obesity and anti-diabetic activities, SWR-0342SA had no effect on body weight or food intake, but slightly decreased the fat pads weight in KK-A^y mice, an animal model of obesity and non-insulin-dependent diabetes mellitus (NIDDM). On the other hand, SWR-0342SA significantly decreased both blood glucose (to about 46% of control) and serum insulin levels (to about 40% of control) in KK-A^y mice.These results indicated that SWR-0342SA is a selective β₃-AR agonist, and possesses potent anti-diabetic activity, and that the anti-obesity activity is inferior to the anti-diabetic activity.

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The present study was performed to see if first-generation histamine H₁-antagonists are useful sedative-hypnotic drugs. Increases in electroencephalogram (EEG) power spectra of the delta band (0-4Hz) at the frontal cortex and theta band (4-8Hz) at the hippocampus in rats were used as an indexes of sleep. The H₁-antagonists used in this study resulted in a decrease in sleep latency and an increase in sleep duration (slow wave sleep). The rate of REM (rapid eye movement) sleep during slow wave sleep was decreased by H₁-antagonists and brotizolam. The order of potency of H₁-antagonists for the reduction in sleep latency (from greatest to least) was promethazine>chlorpheniramine>diphenhydramine and pyrilamine, and that for the increase in sleep duration was chlorpheniramine>promethazine>diphenhydramine and pyrilamine. Brotizolam was more potent than these H₁-antagonists, with 14-18-fold and 4-14-fold greater effects on sleep latency and duration, respectively. These results clearly show that H₁-antagonists are effective in mild to moderate insomnia as sedative-hypnotic drugs.

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Methylripariochromene A (MRC) was isolated from the leaves of Orthosiphon aristatus (Lamiaceae) and subjected to the examination of several pharmacological actions related to antihypertensive activity. The following four findings were revealed from the present study : 1) MRC caused a continuous decrease in systolic blood pressure and a decrease in heart rate after subcutaneous administration in conscious male SHRSP, 2) MRC exhibited the concentration-dependent suppression of contractions induced by high K⁺, l-phenylephrine or prostaglandin F_2α in endothelium-denuded rat thoracic aorta, 3) MRC showed a marked suppression of contractile force without a significant reduction in the beating rate in isolated bilateral guinea pig atria, and 4) MRC increased urinary volume and the excretion of Na⁺, K⁺ and Cl^- for 3 h after oral administration with a load of saline in fasted rats. These findings indicate that MRC possesses some actions related to a decrease in blood pressure, i.e. vasodilating action, a decrease in cardiac output and diuretic action. Furthermore, it is presumed that the traditional use of this plant in the therapy of hypertension may be partially supported by these actions with MRC.

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The present study was carried out to investigate the reversibility of the action of two nasal absorption enhancers, bile acids and fusidate derivative, on nasal membrane resistance. The nasal mucosa was isolated from rabbit nasal septum and mounted in a Ussing-type chamber to allow the monitoring of the membrane resistance and flux of fluorescein isothiocyanate-labeled dextran (FD10, M.W. 9400). Membrane resistance was reduced by 46% following treatment with 0.5% (w/v) sodium taurodihydrofusidate (STDHF) for 10 min and then gradually returned to the control level after being wash. The resistance was restored to 76% of the control level following a 30 min treatment with 0.5% (w/v) STDHF. However, there was no recovery of resistance following treatment with 0.5% (w/v) STDHF for 120 min or 1% (w/v) STDHF for 10 min. Concurrently, FD10 transport was enhanced while membrane resistance was reduced. Treatment with 0.5% (w/v) sodium deoxycholate (DC) for more than 10 min showed no reversible action and marked FD10 transport enhancement, whereas a 10-30 min treatment with 0.5% (w/v) sodium glycocholate (GC) or sodium taurocholate (TC) resulted in the rapid recovery of membrane resistance without any enhancement of FD10 permeation. STDHF transport across the nasal mucosa was ∼2-fold faster than that of DC, GC, and TC. The accumulation of STDHF in the nasal mucosa was 2-fold lower than that of DC and 1.7-fold higher than that of GC and TC after a 30 min treatment. The rank order of hydrophobicity determined by reverse-phase HPLC was : DC>STDHF>GC>TC. These results suggest that the reduction in membrane resistance and its reversibility appear to be due to a balance between the accumulation and clearance of STDHF.

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To extend the utility of a renal targeting system using carbohydrate derivatives, we investigated the in vivo tissue distribution in rats of arginine-vasopressin (AVP) derivatives modified at the phenolic hydroxy group of tyrosine by linking it to some sugars, namely D-glucose, D-galactose, D-mannose and L-flucose, via an octamethylene group. The glycosyl and mannosyl derivatives of AVP exhibit renal-selective distribution in vivo. In addition, the glucosyl and mannosyl derivatives exhibited specific binding to the kidney microsomal fraction in vitro. Modification with D-glucose D-mannose at the tyrosine side chain is a suitable methodology for renal targeting, as well as at N-terminal amine.

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The aim of the present research is to characterize the leakage of intestinal constituents induced by β-cyclodextrin (β-CyD) derivatives using an in situ perfusion and an in vitro everted sac. The efficacy of 6-O-α-D-glucosyl (G1)- and 6-O-α-D-maltosyl (G2)-β-CyDs as oral carriers was also compared with that of 2-hydroxypropyl-(HP1; average molar degree of substitution, 0.9) and 2, 6-di-O-methyl (DM)-β-CyDs. In the in situ studies, phenol red (PR) penetration and the release profiles of intestinal constituents for G2-β-CyD were fairly close to those for HP1-β-CyD. However, the ability of G2-β-CyD to include cholesterol was greater than that of HP1-β-CyD. To characterize the release of intestinal constituents induced by modified β-CyDs, the capability of including cholesterol was held constant between DM- and branched β-CyDs. The everted sac study showed that the amount of DM-β-CyD transferred to the serosal side was not significantly different from the branched β-CyDs. On the serosal side, the amount of cholesterols released was approximately 3 times higher for DM-β-CyD than for the branched β-CyDs at 60 min. The cumulative amounts of cholesterols for DM-β-CyD increased approximately 6 times at 60 min compared with at 30 min, predominating over the leakage (average 2.6-fold) on the mucosal side. In contrast, the exposure of the branched β-CyDs resulted in an insignificant increase over the period of this experiment.The present study suggests that permeable β-CyD derivatives play an important role in the leakage of intestinal components. G2-β-CyD is preferably recommended as a drug solubilizer in oral formulations as well as HP1-β-CyD, based on the lower release of intestinal constituents.

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Fullerene is known to effectively produce mainly singlet oxygen by absorbing light energy, and therefore may be useful as a virucidal photosensitizer. This study was designed to investigate the virucidal activity of a water-soluble fullerene derivative, which is conjugated with methoxy polyethylene glycol amine to enhance its water solubility. Vesicular stomatitis virus (VSV) was inactivated upon illumination with this water-soluble fullerene, in a concentration- or dose-dependent manner. The titer of VSV was reduced by >5log 10 in the presence of 10 mg/ml (400 μM) fullerene derivative with 120 J/cm² white light irradiation. VSV inactivation was inhibited by oxygen removal or by the addition of sodium azide, a known singlet oxygen scavenger. The substitution of H₂O by D₂O, which is known to prolong the lifetime of singlet oxygen, promoted the virucidal activity. These results indicate that singlet oxygen may play a major role in VSV photoinactivation by the water-soluble fullerene derivative. The concentration needed for virus inactivation is higher than that of other sensitizers such as methylene blue.

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To characterize the promoters of cytochrome P-450cam hydroxylase operon (camDCAB) and the repressor gene (camR), in vitro run-off transcription assays were performed using RNA polymerase (RNAP) holoenzyme reconstituted with the core enzyme and the σ⁷⁰ protein of Pseudomonas aeruginosa. Both the mRNAs of camDCAB and camR were accurately transcribed from the respective promoter by the reconstituted RNAP holoenzyme. Both the transcriptions were repressed by CamR protein and the repressions were suppressed by D-cam-phor, consistent with the regulation in P. putida. These results suggest that the RNA polymerase containing σ⁷⁰ recognizes the promoter of camDCAB as well as that of camR.

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Changes in the level of GSH and γ-Glutamylcysteine Synthetase (γ-GCS) activity with time were determined during regeneration of rat liver after partial hepatectomy. The GSH level and γ-GCS activity increased in regenerating rat liver, reaching a maximum (2.1- and 1.5-fold that in sham-operated controls) on day 2, respectively. Furthermore, γ-GCS mRNA expression increased within 24 h and reached maximum on day 2. These results suggest that the increased GSH level in regenerating rat liver after partial hepatectomy is associated with the transcriptional activation of γ-GCS gene and the sequence elevation in γ-GCS activity.

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Glucose and amino acid metabolism in a human enterocyte-like cell line (Caco-2) cultured in a serum-and hormone-free medium was examined and effects of epidermal growth factor (EGF) on these cells determined. These cells took up glutamine and converted it to alanine and proline. EGF stimulated the metabolism significantly.

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B. cereus can use hemoglobin, heme, and heme-albumin complex as iron sources, but does not use other iron binding proteins such as transferrin and lactoferrin. B. cereus digests heme-protein complexes and elicits heme release from the proteins, but does not digest transferrin and lactoferrin. Dissociation of heme-proteins corresponded to the use of iron sources by B. cereus. This activity was completely inhibited by EDTA and phosphoramidon, and metalloendopeptidase inhibitors, therefore it appeared that the metalloprotease(s) are related to digestion and use of heme-protein complexes by B. cereus. B. cereus could bind released heme to its cells. These findings suggest that B. cereus can dissociate heme from heme-protein complex, and capture the released heme to use as an iron source.

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The physiological significance of the casein kinase II (CK-II)-mediated phosphorylation of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) on its three enzymatic activities [RNA-dependent DNA polymerase (RDDP), DNA-dependent DNA polymerase (DDDP) and ribonuclease H (RNase H)] was investigated in vitro. It was found that (i) the purified recombinant RT (rRT) functioned as an effective phosphate acceptor for CK-II; (ii) the RDDP, DDDP and RNase H activity of rRT was stimulated about 2.8-, 4.1- and 3.9-fold, respectively, after full phosphorylation by CK-II; and (iii) this stimulation was selectively inhibited by potent CK-II inhibitors, such as neocarzinostatin-chromophore (NCS-chrom) and three polyphenol-containing anti-oxidant compounds [quercetin, epigallocatechin gallate (EGCG) and 8-chloro-3', 4', 5, 7-tetrahydroxyisoflavone (8C-3', 4', 5, 7-THI)]. These results suggest that (i) CK-II may be responsible for activation of RT in HIV-1-infected cells; and (ii) the selective inhibition of CK-II-mediated activation of HIV-1 RT by potent CK-II inhibitors may be involved in the mechanism of their anti-HIV-1 effects at the cellular level.

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We investigated whether tumor necrosis factor (TNF) and caspase-3 activity are involved in the induction of hepatocellular apoptosis in D-galactosamine (D-GalN)-induced hepatotoxicity in mice. Acute hepatotoxicity was induced by the intraperitoneal injection of D-GalN into female BALB/c mice. D-GalN (0.75-3.0 g/kg) increased the serum blutamate pyruvate transaminase (s-GPT) activity and the percentage of liver DNA fragmentation, an indicator of hepatotoxicity, after 48 h, in a dose-dependent manner. Furthermore, after D-GalN (3.0 g/kg) administration, increased liver DNA fragmentation was detected biochemically at 24 h, then increased s-GPT activity accompanied by increased liver DNA fragmentation was observed after 48 h. The serum TNF (s-TNF) level and the TNF mRNA expression in the liver after D-GalN (3.0 g/kg, i.p.) administration were examined by an ELISA kit and reverse transcription polymerase chain reaction (RT-PCR), respectively, to investigate the relation between the s-GPT activity and liver DNA fragmentation. The s-TNF level and TNF mRNA expression in the liver after D-GalN (3.0 g/kg) administration were detected earlier than liver DNA fragmentation, then increased with time. However, there was almost no association of caspase-3 activity with the increase in liver DNA fragmentation. Increases in the s-TNF level, TNF mRNA expression and the percentage of DNA fragmentation in the liver and s-GPT activity were inhibited by dexamethasone (Dex; 0.4-2.5 mg/kg, i.p.) in a dose-dependent manner. Based on these findings, it was considered that the intracellular apoptosis signal in D-GalN-induced hepatotoxicity in mice did not depend on caspase-3 activity, and that other signals mediated by TNF may be involved.

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We examined the effect of Dai-kenchu-to (DKCT), a traditional Chinese (Kampo) medicine, on the levels of 3 brain-gut peptides (motilin, gastrin and somatostatin) in plasma from 24 healthy subhects. A single oral administration of DKCT, at a dose of 7.5 g, caused significant increases in plasma motilin levels (about 12 pg/ml) at 60 to 90 min, compared with a placebo-treated group. Transient elevations of gastrin levels were noted after administration of both DKCT (25.9±1.4 pg/ml) and placebo (23.5±1.3 pg/ml). DKCT did not alter the levels (about 5.7 pg/ml) of somatostatin. In conclusion, these results indicate that the action of DKCT closely relates to changes in motilin-immunoreactive substance levels in human plasma.

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To gain insight into the binding manner of androst-4-ene-6, 17-dione (4) and its 5-en-4-one analog 9, good competitive inhibitors of aromatase, in the active site of the enzyme, their 19-oxygenated derivatives were evaluated as inhibitors of human placental aromatase. The 19-hydroxy steroids 6, 7, and 10 and the 19-oxo analogs 8 and 12 were much poorer competitive inhibitors than their corresponding parent 19-methyl steroids. Conversion of the 17-keto inhibitors, 4, 6, and 10 to the 17β-ols 5, 7, and 11, respectively, markedly decreased their affinity to the enzyme. The results suggest that steroids 4 and 9 would bind to the active site in a similar manner to that involved in the binding of the substrate androstenedione (1).

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The known compound bakuchiol (1) was isolated from an extract of Otholobium pubescens (Fabaceae) by bioassay-guided fractionation using the db/db mouse model for tyep 2 diabets. Oral administration of 1 reduced blood glucose levels in a dose-dependent fashion in db/db mice and did not display a hypoglycemic effect when tested in lean mice at 250 mg/kg q.d. Compound 1 was further evaluated in a new rodent model for type 2 diabetes, the fat-fed, streptozotocin (STZ)-treated rat. In this model, an oral dose of 1 at 150 mg/kg q.d. significantly lowered plasma glucose and triglyceride levels.

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A biologically monitored fractionation of methanol extract of the fruit of Evodia rutaecarpa led to the isolation of six quinolone alkaloids, evocarpine (1), 1-methyl-2-[(4Z, 7Z)-4, 7-tridecadienyl]-4(1H)-quinolone (2), 1-methyl-2-[(6Z, 9Z)-6, 9-pentadecadienyl]-4(1H)-quinolone (3), 1-methyl-2-undecyl-4(1H)-quinolone (4), dihydroevocarpine (5), 1-methyl-2-pentadecyl-4(1H)-quinolone (6). They showed potent anti-Helicobacter pylori activity with the minimum inhibitory concentration (MIC) value of 10-20 μg/ml. However, they had no effect on Helicobacter pylori urease activity at the concentration of 300 μg/ml.

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Licorice root traditionally used as an anti-inflammatory drug exhibited an inhibitory effect on lysoPAF (platelet-activating factor) acetyltransferase in vitro : the ether soluble fraction of the crude drug produced a 27.3% inhibition at a concentration 10 μg/ml. From this fraction, licorcidin (1), 1-methoxyphaseollin (2), 6, 8-diprenylgenistein (3) and 1-methoxyphaseollidin (4) were isolated as active components, whose IC₅₀ values were 7.7, 57 19 and 48 μM, respectively. Licoricidin (1) seems to be one of the most potent compounds of plant origin isolated so far.

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Bioassay-guided fractionation of an aqueous extract of Alismatis Rhizoma has furnished two inducible nitric oxide synthase (iNOS) inhibitory compounds, alismol (1) and alisol B monoacetate (2), together with an inactive triterpene, alisol C monoacetate (3). Compounds 1 and 2 inhibited nitric oxide (NO) synthesis in a dose-dependent manner in murine macrophage-like RAW 264.7 cells stimulated with interferon-γ (IFN-γ) plus lipopolysaccharide (LPS). The inhibitory effects of 1 and 2 on NO synthesis were partly due to suppression of iNOS mRNA expression as determined by Northern blotting.

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The extract of the roots of Panax notoginseng (Araliaceae) exhibited a significant anti-tumor-promoting activity on two-stage carcinogenesis of mouse skin tumors induced by 7, 12-dimethylbenz[a]anthracene (DMBA) as an initiator and a mycotoxin, fumonisin B1, as a non-12-O-tetradecanoylphorbol-13-acetate (non-TPA) type promoter. Further, the extract exhibited the anti-tumor-initiating activity on two-stage carcinogenesis of mouse skin tumors induced by a nitric oxide donor, (±)-(E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexenamide (NOR-1) as an intiator and TPA as a promoter.

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