It has been reported that the copper (Cu) content of hepatocytes increases in chronic liver diseases and small hepatocellular carcinoma (HCC). In cells, Cu exists mainly as Cu-metallothionein (MT) or Cu, zinc (Zn)-superoxide dismutase (SOD). In this study, we investigated the biochemical state of Cu in the hepatocytes of patients with HCC using high-performance liquid chromatography (HPLC). The subjects of present study were 23 patients with HCC who underwent liver resection. The cancerous tissue and non-cancerous hepatic parenchyma with chronic disease were analyzed. In addition, as a normal control, hepatic tissue was collected at autopsy from 13 patients with no liver disease. Each sample was diluted with buffer, chilled, homogenized, and centrifuged. The supernatant was fractionated using HPLC. The metal contents of each fraction were measured using a desktop-type inductively coupled plasma (ICP) emission spectrochemical analyzer. HPLC analysis showed that MT existed mainly as Zn-MT in the normal hepatic tissue. The case of Cu,Zn-MT was significantly greater than Zn-MT in the non-cancerous, but diseased hepatic parenchyma than in the normal hepatic tissue (p<0.01). In comparison with non-cancerous hepatic parenchyma, the Cu-MT in the cancerous section was significantly greater than the Cu,Zn-MT (p<0.01). The Cu content for MT was significantly higher in small HCC (<40 mm) (p<0.01), and the absence of Cu or Zn in the MT fraction was significantly more frequent in the large HCC (≥40 mm) (p<0.01). The Cu and Zn content for SOD in the samples showed no significant difference. Increase in the Cu content in the cancerous hepatic tissue were, thought to be reflecting changes in the distribution of Cu in the MT fraction of hepatic tissues.

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The age dependence of ketone body levels (KBLs) and oxidative damages in selenium-deficient (SeD) and normal rats were compared. The feeding SeD diets gave ketogenesis and higher KBLs especially in younger rats. However, KBLs in SeD rats seemed to decrease with their age. Feeding 0.1 mg/kg Se in water with SeD diet did not affect the KBLs in young (8 week old) rats, whereas the addition of Se reduced the KBLs in older (20 week old) rats. Blood KBLs showed some correlations with tissue damage. TBARSs showed no correlations with the tissue damages and KBLs when the values were compared between the same age, while better correlation was obtained between urinary KBLs of 6—20 week old normal rats and the liver TBARSs of 4—16 week old normal rats. The oxidative injury might induce liver damage with some delay. SeD rat kidney TBARS levels normalized by protein had some correlations with BUN and blood KBL. Kidney may be sensitive to the oxidative stresses and/or injuries. Tissue damages of SeD rats decreased with age. In contrast, oxidative injuries might be gradually accumulated in normal rat tissue. Oxidative stress can be visible by gradual accumulation of small damages during the aging, while large stress in young rats can be buffered and masked. The aging based accumulation of oxidative injuries might also be correlated with KBLs, while it might not give notable tissue damages.

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We developed a simple HPLC method for the simultaneous quantitative determination of seven HIV protease inhibitors: amprenavir (APV), atazanavir (ATV), indinavir (IDV), lopinavir (LPV), nelfinavir (NFV), ritonavir (RTV), saquinavir (SQV), and a nonnucleoside reverse transcription inhibitor, efavirenz (EFV). This method involves a rapid liquid–liquid drug extraction from plasma, the use of an isocratic elution on a reversed-phase C₁₈ column, and an ultraviolet detection at a single wavelength (205 nm). The mobile phase consisted of 39% 50 mM phosphate buffer (pH 5.9), 22% methanol and 39% acetonitrile. Forty-eight samples could be measured in one day since the runtime of one sample is 30 min. The assay has been validated over a concentration range of 0.05 to 12.20 μg/ml for APV, 0.09 to 12.05 μg/ml for ATV, 0.05 to 12.01 μg/ml for IDV, 0.12 to 12.36 μg/ml for LPV, 0.18 to 12.20 μg/ml for NFV, 0.12 to 12.33 μg/ml for RTV, 0.12 to 12.06 μg/ml for SQV, and 0.05 to 12.17 μg/ml for EFV. Calibration curves were linear in the described concentration ranges. The average accuracy ranged from 97.2 to 106.8%. Both the interday and intraday coefficients of variation for all drugs tested were less than 8.5%. This method provides a simple, accurate, and precise method for the therapeutic drug monitoring of the seven protease inhibitors and EFV in clinical routine use.

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Acetaminophen (APAP) is widely used for the treatment of pain and fever. Although it is safe at therapeutic doses, APAP is toxic at higher doses and can cause severe damage to the liver. To clarify the mechanism of APAP-related liver damage, we attempted the identification of the differential gene expression in response to APAP treatment in hepatic HepG2 cells. In the present study, we used the technique of suppression subtractive hybridization (SSH) for the identification of the differentially expressed genes between untreated and treated cells and identified 14 candidate genes showing increased expression in response to APAP treatment. RT-PCR and real-time RT-PCR analysis confirmed that the expression of two genes was increased within 24 h following APAP treatment. Among them, only lysyl hydroxylase 2 expression was increased in a time- and dose-dependent manner. Furthermore, the expression of lysyl hydroxylase 2 was shown to be increased in the livers of APAP-treated mice compared to untreated controls. The increased expression of lysyl hydroxylase 2 was also observed when the cells were exposed to other hepatotoxins, ethanol and isoniazid. Since lysyl hydroxylase 2 is known to be a key enzyme of liver fibrosis, the increased expression of lysyl hydroxylase 2 may be involved in hepatotoxins-related liver fibrosis.

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Peripheral inflammation is accompanied by neurobehavioral alterations such as depression of feeding, exploratory and sexual behaviors. Our previous investigation reported that dietary enrichment with n-3 polyunsaturated fatty acids (PUFA) attenuated the depression of food-motivated behavior and social exploration, but not endocrinological and metabolic disturbances in the mice with systemic inflammation induced by lipopolysaccharide (LPS). We here demonstrate that dietary n-3 PUFA also attenuate the reduction of food-motivated behavior during zymosan-induced peritonitis in mice without influencing plasma leakage into peritoneum and writhing response. Our results suggest that the common mechanism is involved in the attenuation of behavioral depression during systemic and local inflammation by dietary n-3 PUFA.

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This study evaluated the protective effect of Puerariae radix against the oxidative stress induced by hydrogen peroxide (H₂O₂) and streptozotocin in vitro and in vivo, respectively. The ethanol extract scavenged intracellular reactive oxygen species (ROS), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, and prevented lipid peroxidation. This radical scavenging activity of the ethanol extract protected the cell viability of Chinese hamster lung fibroblast (V79-4) cells exposed to H₂O₂. Furthermore, this extract reduced the formation of apoptotic cells induced by H₂O₂, which was demonstrated by the decreased number of sub G₁ hypo-diploid cells and apoptotic cell body formation. The extract increased the activities of the cellular antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT). Administration of the extract to the streptozotocin induced diabetic rats decreased the blood glucose levels. The diabetic rats showed low activities of superoxide dismutase and catalase in the liver, and the ethanol extract increased the CAT activity. The increased level of lipid peroxidation in the diabetic rats reverted to near normal levels after being treated with the extract. This study showed that Puerariae radix was effective in the amelioration of diabetes, which may be a consequence of its antioxidant potential.

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Dihydropyrazines (DHPs), which are derived from sugars, inhibit the growth of Escherichia coli. Addition of copper(II) ion (Cu²⁺) accelerates the effect of DHPs, resulting in cell death. Investigation of the lethal effect in several DNA repair-deficient or detoxifying enzyme-deficient mutant strains and radical scavengers suggested that the cytotoxic and genotoxic damage was caused by radical species (hydroxyl, superoxide anion, and carbon-centered radicals) generated from reaction of DHPs with Cu²⁺.

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Vibrio vulnificus extracellular protease (VvpE) is believed to destroy its hemolysin (VvhA) in the late growth phase, without obvious experimental evidence. So, we attempted to elucidate the mechanism. The hemolytic activity steeply increased with the expression of the VvhA in the early growth phase, and then abruptly declined with the expression of VvpE in the late growth phase. However, the VvhA activity also abruptly declined in a VvpE-deficient mutant. In Western blot, the degradation of VvhA was not observed; instead, the oligomerization of VvhA increased with the concomitant loss of hemolytic activity. These results evidently indicate that the inactivation of VvhA is due to the novel oligomerization of VvhA by unknown mechanism, but not to the destruction of VvhA by VvpE, so that the routine functional assay measuring hemolytic activity cannot reflect the actual production of VvhA.

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Cardiac toxicity is a major adverse effect caused by doxorubicin (DOX) therapy. Many recent studies have shown that DOX toxicity involves generation of reactive oxygen species (ROS). Although protection or alleviation of DOX toxicity can be achieved by administration of antioxidant vitamins such as ascorbic acid and vitamin E, their cardioprotective effect remains controversial. Thus alternative naturally occurring antioxidants may potentially be candidates for antioxidant therapy. In this study, we investigated the antioxidative and cytoprotective effects of Phyllanthus urinaria (PU) against DOX toxicity using H9c2 cardiac myoblasts. The total antioxidant capacity of PU (1 mg/ml) was 5306.75±461.62 FRAP value (μM). DOX IC₅₀ values were used to evaluate the cytoprotective effects of PU ethanolic extract (1 or 10 μg/ml) in comparison with those of ascorbic acid (VIT C, 100 μM) and N-acetylcysteine (NAC, 100 μM). PU treatments (1 or 10 μg/ml) dose dependently caused rightward DOX IC₅₀ shifts of 2.8- and 8.5-fold, respectively while treatments with VIT C and NAC increased DOX IC₅₀ by 3.3- and 4.2-fold, respectively. Additionally, lipid peroxidation and caspase-3 activity were parameters used to evaluate cytoprotective effect. All antioxidants completely inhibited cellular lipid peroxidation and caspase-3 activation induced by DOX (1 μM). Endogenous antioxidant defense such as total glutathione (tGSH), catalase and superoxide dismutase (SOD) activity was also modulated by the antioxidants. PU treatment alone dose dependently increased tGSH, and this effect was retained in the presence of DOX. Similar effect was observed in the assessment of catalase and SOD enzyme activity. The nuclear factor κB (NFκB) transcription factor assay demonstrated that all antioxidants significantly inhibited DOX-induced NFκB activation. Our results suggest that PU protection against DOX cardiotoxicity was mediated through multiple pathways and this plant may serve as an alternative source of antioxidants for prevention of DOX cardiotoxicity.

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Streptozotocin (STZ)-induced diabetic rats were divided into mild diabetic (MD) and severe diabetic (SD) on the basis of fasting blood glucose (FBG) levels. Diabetes was confirmed here by intravenous glucose tolerance test (GTT), biochemical assay of glycogen content in liver and skeletal muscle, glucose-6-phosphatase activity in liver, and serum insulin levels. Hyperlipidemia developed in these experimental diabetic rats was assessed by quantification of total cholesterol (TC), high-density lipoprotein cholesterol (HDLc), low-density lipoprotein cholesterol (LDLc) and triglyceride (TG) in serum. Aqueous extract of seed of Tamarindus indica was given to MD and SD rats at the dose of 80 mg and 120 mg/0.5 ml distilled water/100 g body weight/d respectively for 14 d. Significant attenuation of hyperglycemia was indicated by measuring FBG, glycogen level and glucose-6-phosphatase activity along with monitoring of intravenous GTT and serum insulin level. Similarly, correction of hyperlipidemia in diabetic rats after this extract supplementation was confirmed by significant reduction in the levels of above-mentioned hyperlipidemic indicators. Intravenous GTT was performed that highlights the antidiabetic action of this extract is not due to its effect on the intestinal rate of glucose absorption but may be due to modulation of intracellular glucose utilization in target organs. This study focus the efficacy of this extract for the management of experimental diabetes in rat model which may shed some light on the scientific basis of ancient herbal therapy in this line using this seed.

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Gamisanghyulyunbueum (GSHYBE) has been used clinically to treat skin related disease in South Korea. We investigated GSHYBE-mediated changes in downstream T cell signal transduction. To determine the mechanism of inhibition, we have studied many of the major pathways in phytohemagglutinin (PHA)-activated T cell. We show that among the mitogen-activated protein kinase family activation of phosphorylation of extra cellular signal-regulated kinase 1/2 (ERK1/2, p44/42) and p38, but not c-jun NH₂-terminal kinase is inhibited. In activated MOLT-4 cells, the nuclear localization of nuclear factor of activated T cells (NFATc) was blocked by GSHYBE (1 mg/ml). Also, degradation of inhibitor κB-α and transactivation by nuclear factor-κB (NF-κB)/Rel A were impaired by GSHYBE (1 mg/ml). Furthermore, interlukin (IL)-2, IL-4 and Interferen (IFN)-γ secretion by PHA activated MOLT-4 cells and peripheral blood mononuclear cells (PBMC) were significantly diminishes following GSHYBE treatment (1 mg/ml). Also, oral administration of GSHYBE inhibited IL-2 secretion in skin allergic reaction. In conclusion, our data indicate that GSHYBE treatment of T cells inhibits ERK1/2 and p38 activation and nuclear translocation of NFATc, NF-κB, resulting in diminished secretion of IL-2.

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The inhibition of the proliferation rate of the immortalized human cell line ECV 304 after oxidant damage by oxygen radicals generated in a hypoxanthin–xanthine oxidase system and the protection provided by various propolis extracts was determined. Best inhibition was demonstrated by 60—80% ethanolic extracts (IC₅₀ of approximately 2 μg dry weight/ml) and by the ethyl acetate extract (IC₅₀ of 6.9 μg dry weight/ml). The beneficial effect of polar extracts was quite weak. Human neutrophil elastase activity was inhibited distinctively by ethanolic (60 to 96%) and ethyl acetate extracts (IC₅₀ of approximately 2 μg dry weight/ml). Both activities seem to be responsible for the anti-inflammatory effect of the extract.

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Activation of the volume-regulated anion channels (VRAC) is considered to be involved in arrhythmia, but it has not yet been fully elucidated because of the lack of its high affinitive and selective compounds. A newly synthesized compound, YM-198313 (sodium 4-({[2-(methylthio)benzyl]amino}-5-[(1-phenylethyl)thio]isothiazol-3-olate), strongly inhibited VRAC in HeLa cells with an IC₅₀ of 3.03±0.05 μM. However, YM-198313 weakly affected both the Ca²⁺-activated Cl⁻ channels in HTC cells and the cAMP-activated Cl⁻ channels in T84 cells, demonstrating that this compound is selective for VRAC among Cl⁻ channels. At 10 μM, YM-198313 almost completely (100±7.8%) inhibited the VRAC current in guinea pig atrial myocytes. However, at the same concentration, YM-198313 showed little inhibitory effect on the cardiac cation currents in ventricular myocytes. We believe that YM-198313 is a potent and selective VRAC inhibitor, therefore, it should be use to clarify the role VRAC plays in arrhythmia.

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Retinoid X receptor (RXR) forms heterodimers with peroxisome proliferator-activated receptors (PPARs, with subtypes of α, δ and γ), and the heterodimers can be activated by either an RXR or a PPAR subtype-specific ligand. Based on the chemical structure of the RXR natural ligand, 9-cis-retinoic acid (9-cis-RA), we designed and synthesized a retinoid-like compound, CS018. In vitro characterizations by cell-based reporter gene assays indicated that CS018 activated RXR homodimers and the heterodimers of RXR with PPARs, but not with farnesoid X-activated receptor (FXR) and liver X-activated receptor (LXR). Furthermore, RT-PCR results showed that CS018 induced the expression of the PPARγ target genes, CD36 and lipoprotein lipase (LPL). In vivo studies on the diabetic db/db mice demonstrated that CS018 dramatically lowered the animal blood glucose levels. CS018 thus may represent a new retinoid-like compound that activates RXR/PPARs and has potential therapeutic applications in type 2 diabetes and other metabolic diseases.

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Monocarboxylate transporter (MCT), which cotransport L-lactic acid and protons across cell membranes, are important for regulation of muscle pH. However, it has not been demonstrated in detail whether MCT isoform contribute to the transport of L-lactic acid in skeletal muscle. The aim of this study was to characterize L-lactic acid transport using an human rhabdomyosarcoma (RD) cell line as a model of human skeletal muscle. mRNAs of MCT 1, 2 and 4 were found to be expressed in RD cells. The [¹⁴C] L-lactic acid uptake was concentration-dependent with a K_m of 1.19 mM. This K_m value was comparable to its K_m values for MCT1 or MCT2. MCT1 mRNA was found to be present markedly greater than that MCT2. Therefore, MCT1 most probably acts on L-lactic acid uptake at RD cells. [¹⁴C] L-Lactic acid efflux in RD cells was inhibited by α-cyano-4-hydroxycinnamate (CHC) but not by butyric acid, a substrate of MCT1. Accordingly, MCT2 or MCT4 is responsible for L-lactic acid efflux by RD cells. MCT4 mRNA was found to be present significantly greater than that MCT2. We conclude that MCT1 is responsible for L-lactic acid uptake and L-lactic acid efflux is mediated by MCT4 in RD cells.

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We studied the cytotoxic effect of various anticancer agents on lymphoblastoid cell lines transformed by Epstein–Barr virus. Post-immortal N0005 (post-N0005) is an immortalized cell line derived from pre-immortal N0005 (pre-N0005) accompanied by increased telomerase activity, short-telomere, abnormal karyotypes, mutation of p53 gene, down regulation of p16/Rb and the ability to grow in soft agar medium. Compared with pre-N0005 cells, post-N0005 cells were significantly (p<0.001 by the Student t test) more resistant to the killing activity of seven DNA-modifying agents: camptothecin, etoposide, bleomycin, fluorouracil, thioguanine, melphalan and actinomycin D. However, both pre-N0005 and post-N0005 cells showed similar levels of cytotoxicity against four DNA-non-modifying agents: colchicine, paclitaxel, vincristine and methotrexate. DNA-modifying and DNA-non-modifying agents are distinguished by their different sensitivities with pre-N0005 and post-N0005. Based on these results, we propose that pre-N0005 and post-N0005 cell lines be used as a new method to assess and screen anticancer agents.

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A new beverage made of red wine vinegar and grape juice (Budo-no-megumi^TM) was developed for people who wish to take effective amount of both polyphenols and vinegar. Since the beverage was recently demonstrated to exert hypotensive effect in rats, we analyzed its underlying mechanisms in this study. Sprague-Dawley rats were anesthetized with pentobarbital, and the blood pressure and lead II ECG were continuously monitored (n=6). The effects of recommended volume of the beverage (3 ml/kg, p.o.) on the renin-angiotensin system were assessed in vivo. At the basal control state, the increase in the mean blood pressure induced by the angiotensin I (1 μg/kg, i.v.) and norepinephrine (0.3—3 μg/kg, i.v.) were +57±2 and +36±8 mmHg, respectively. Sixty minutes after the administration of the beverage, the angiotensin I-induced pressor response decreased to +45±7 mmHg at 60 min (p<0.05), whereas no significant change was detected in the norepinephrine-induced pressor response. In another parallel series of the experiment using Sprague-Dawley rats (n=6), the serum angiotensin-converting enzyme activity was 39.4±1.2 IU/l at basal control state, which was slightly but significantly decreased to 37.0±1.4 IU/l at 60 min after the administration of the beverage (p<0.01). These results suggest that previously described hypotensive action of the beverage may be partly induced by the inhibition of angiotensin-converting enzyme.

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Inhibitory effects of docosahexaenoic acid (DHA) on the muscle contractions induced by U46619, a thromboxane A₂ (TXA₂) mimetic, and phenylephrine were compared in guinea-pig aorta. In de-endothelialized guinea-pig aortic ring preparations, DHA at 10 μM strongly inhibited a sustained contraction produced by U46619 (3—100 nM) whereas it did not exhibit an appreciable effect on phenylephrine (3—10 μM)-induced contraction. The present findings indicate that DHA inhibits more selectively TXA₂ receptor (TP receptor)-mediated vascular contraction than α-adrenoceptor-mediated response. Selective inhibition by DHA of TP receptor-mediated contraction of blood vessels seems underlie in part the mechanisms by which this polyunsaturated fatty acid exerts its circulatory-protective effects.

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The adrenal steroid, dehydroepiandrosterone (DHEA) acts as a peroxisome proliferator in the rodents. The present study examined the effects on cellular antioxidants ubiquinone and catalase in the liver of DHEA-treated rats. When administered to male F-344 rats for 8 weeks, DHEA produced a significant increase in hepatic ubiquinone-9 and lipid peroxide levels while no change was observed after 2 weeks. Activity of catalase, in contrast, followed an inverse pattern, being significantly induced at 2 weeks with a return to normal levels after 8 weeks. A marked reduction of ubiquinone-10 in DHEA-treated rat livers was only observed after 2 weeks. These findings indicate the potentials of high dose DHEA to modulate ubiquinone in rat hepatic tissue.

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A hydroalcoholic (50%) extract of Emblica officinalis (fruit) (EO-50) reduced the severity of hepatic fibrosis induced by carbon tetrachloride (CCl₄) and thioacetamide (TAA). Improved liver function was observed by measuring the levels of aspartate aminotransaminase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and bilirubin in serum. Hepatic parameters monitored were the levels of glutathione (GSH), lipid peroxidation (LPO) and hydroxyproline and the activities of catalase, glutathione peroxidase (GPx), Na⁺,K⁺-ATPase and cytochrome P450 (CYP 450 2E1) (aniline hydroxylation). The results suggested that EO-50 effectively reversed profibrogenic events possibly due to its promising antioxidative activity.

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Erythromycin, an antibiotic agent, is known to be a motilin receptor agonist. Motilin is a peptide hormone that regulates gastric motility. One of the gastrointestinal motility regulatory factors has been assumed to be the induction of changes in the levels of peptides (gastrin, somatostatin and motilin) in plasma. We have elucidated the effects of erythromycin by examining changes in the plasma levels of gastroinitestinal peptides. In this study, we investigated the effects of erythromycin on the plasma levels of gastrointestinal peptides (somatostatin, motilin, and gastrin) in healthy volunteers and patients with delayed gastric emptying (DGE). After a single oral administration, erythromycin caused a significant increase in plasma gastrin-like immunoreactive substance (IS) levels at 60 min. But the agent did not alter the levels of somatostatin- and motilin-IS. DGE is the most frequent postoperative complication after pylorus-preserving pancreatoduodenectomy. Molitin is assumed to be one important factor that influences DGE. We also examined the effects of erythromycin on the plasma motilin-IS levels of postoperative patients. The plasma motilin-IS levels were increased after 1 week of oral administration of erythromycin compared with preadministration. These results suggest that the pharmacologic effects of erythromycin in promoting gastric emptying are closely related to changes in plasma motilin-IS levels.

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The aim of this study is to determine the anti-inflammatory activity of a new sphingosine derivative (1) and cembrenoid diterpene (lobohedleolide) (2) isolated from the soft corals of Sinularia crassa and Lobophytum species respectively, collected on the coasts of Andaman and Nicobar Islands. The anti-inflammatory activity was evaluated using carrageenin-induced rat hind paw edema model for acute inflammation and cotton pellet granuloma model for chronic inflammation. Indomethacin was used as a standard drug in this study. Both the sphingosine derivative (1) and the cembrenoid diterpene (2) produced the maximum effect at a dose of 10 mg/kg and this is comparable to that of indomethacin (2 mg/kg, p<0.001). The observed anti-inflammatory activity is almost identical in both the types of experimental inflammation.

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Six polyphenolic acid esters were synthesized and their antioxidative properties were evaluated in three model systems [2,2′-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay, 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH)-induced lipid peroxidation system, and the dye-bleaching assay of peroxynitrite radical]. Among these compounds, we found that compounds 4 [3,4-dihydroxy-benzoic acid-(2-phenoxyethyl ester)], and 5 [3,4-dihydroxy-cinnamic acid-(2-phenoxyethyl ester)] provided comparable activity to caffeic acid phenethyl ester (CAPE) in the DPPH model. Compound 3 [2,5-dihydroxy-benzoic acid-(2-phenoxyethyl ester)], was found to be more active than CAPE in the AAPH system, it also displayed about 2-fold greater activity than CAPE in the peroxynitrite radical model. These results suggest that these phenolic acid ester derivatives, with their potent anti-oxidant activities, may have useful applications as antioxidants.

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Starting from 6-hydroxy-3,4-dihydro-1H-quinoline-2-one, a series of 1-substituted-7-benzyloxy-4,5-dihydro-[1,2,4]triazolo[4,3-a]quinolines was synthesized and their structures were characterized using IR, ¹H-NMR, MS, and elemental analysis techniques. Anticonvulsant activity was evaluated in the maximal electroshock (MES) test, subcutaneous pentylenetetrazol (scMet) test, and rotarod neurotoxicity test. The most active compound was 7-benzyloxy-4,5-dihydro-[1,2,4]triazolo[4,3-a]quinoline 4a. Its ED₅₀ in the MES and scMet tests was 17.3 and 24 mg·kg⁻¹, respectively. The safest compound was 4g, 1-phenyl-7-benzyloxy-4,5-dihydro-[1,2,4]triazolo[4,3-a]quinoline, with TD₅₀ and protective index (PI) (PI=TD₅₀/ED₅₀) values of greater than 300 mg·kg⁻¹ and 13, respectively. The PI value of compound 4g was better than that of most marketed drugs. Structure–activity relationships are also described in this paper.

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A total of 44 variously halogenated derivatives of aniline, phenol, and thiophenol were subjected to analysis of their inhibitory effect on human cytochrome P450 (CYP) 2E1 to investigate the structure–activity relationships in halogenated phenyl derivatives. The activity of human CYP2E1 of the microsomes from baculovirus-transfected insect cells expressing recombinant human CYP2E1 was determined by measuring quinoline 3-hydroxylation, which was detectable by fluorescence monitoring (Ex=355 nm and Em=460 nm). Diethyldithiocarbamate (DDTC), a specific inhibitor of CYP2E1, potently inhibited quinoline 3-hydroxylation (IC₅₀=8.9 μM). The effects of halogen-substitution in 32 aniline derivatives on the CYP2E1 inhibition can be summarized as follows: more enhancement by chlorine- and bromine-substitution than by fluorine-substitution, more enhancement by para- and metha-halogen-substitution than by ortho-halogen-substitution, and more enhancement by dihalogen-substitution than by mono- and trihalogen-substitution except for trifluorine-substitution. The greatest enhancement of the inhibitory activity was observed in 3,4-dichloroaniline (IC₅₀=8.0 μM) and 3,5-dichloroaniline (IC₅₀=9.2 μM), and their inhibitory activities were very close to that of DDTC. All of the dichlorophenols and dichlorothiophenols were compared with dichloroanilines for CYP2E1 inhibition. Although dichlorothiophenols showed similar or more potent inhibitory activities than dichloroanilines, dichlorophenols showed less inhibitory activities. 3,4-Dichlorothiophenol and 3,5-dichlorothiophenol showed very potent inhibition and their IC₅₀ values were 5.3 and 5.2 μM, respectively. These results suggest that 3,4- and 3,5-dichlorophenyl derivatives may be useful as potent CYP2E1 inhibitors.

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We evaluated the antipyretic, analgesic, and muscle relaxant activities of Pueraria isoflavonoids and their metabolites in mice. The glycosides daidzin and genistin significantly reduced fever induced by lipopolysaccharide (LPS). Their metabolites, daidzein and p-ethylphenol, also significantly reduced fever induced by LPS. In addition, daidzin, daidzein, dihydrodaidzein, and p-ethylphenol showed analgesic activity as assessed by the acetic acid-induced writhing test. Furthermore, equol and p-ethylphenol showed muscle relaxant activity in the rotarod and horizontal wire test. These results suggest that these compounds play a major role in the therapeutic activity of Pueraria isoflavonoids.

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Melanogenesis stimulation activities of seven ethanolic extracts obtained from Umbelliferae plants used as Chinese crude drugs, namely the roots of Angelica dahurica BENTH. et HOOK., A. biserrata SHEN et YUAN, Notopterygium incisum TING, Heracleum lanatum MICHX., and H. candicans WALL., and the fruits of Cinidium monnieri (L.) CUSSON and C. formosanum YABE, were examined by using cultured murine B16 melanoma cells. Among them, the extract (5, 25 μg/ml) of H. lanatum showed a potent stimulatory effect on melanogenesis with significant enhancement of cell proliferation in a dose-dependent manner. The melanogenesis stimulatory effects of sixteen coumarins (1—16) isolated from the seven Umbelliferae crude drugs were also examined. Among them, linear-furocoumarins [psoralen (1), xanthotoxin (2), bergapten (3), and isopimpinellin (4)] and angular-furocoumarin [sphondin (13)] exhibited potent melanogenesis stimulation activity. From the view point of structure–activity relationships, it may be assumed that a linear-furocoumarin ring having a hydrogen and/or methoxyl group at 5 and 8 positions such as 1, 2, 3 and 4 was preferable for the melanogenesis stimulation activity. The introduction of a prenyl group into the furocoumarin ring was disadvantageous. Coumarin derivatives having a simple coumarin ring were inactive.

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To provide some pharmacological evidence for its clinical use in inflammatory diseases, anti-inflammatory effect of the aqueous extract from Radix Ophiopogon japonicus (ROJ-ext), a traditional Chinese herb, was examined in mouse and rat models. ROJ-ext significantly inhibited xylene-induced ear swelling and carrageenan-induced paw edema in mice when given orally at doses of 25 and 50 mg/kg. Moreover, ROJ-ext also remarkably suppressed carrageenan-induced pleural leukocyte migration in rats and zymosan A-evoked peritoneal total leukocyte and neutrophil migration in mice, while had no obvious effect on pleural prostaglandin E₂ level. Furthermore, two active compounds were isolated from ROJ-ext and identified as ruscogenin and ophiopogonin D. As the results, ROJ-ext, ruscogenin and ophiopogonin D dose-dependently reduced phorbol-12-myristate-13-acetate (PMA)-induced adhesion of HL-60 cells to ECV304 cells, with IC₅₀ of 42.85 μg/ml, 7.76 nmol/l and 1.38 nmol/l, respectively. However, they showed no inhibitory effect on PMA-induced cyclooxygense-2 (COX-2) mRNA expression in ECV304 cells. Ruscogenin and ophiopogonin D also notably decreased zymosan A-induced peritoneal leukocyte migration, in comparison with ROJ-ext. These results demonstrate that ROJ-ext presents remarkable anti-inflammatory activity and ruscogenin and ophiopogonin D are two of its active components, which supported its traditional use in the treatment of various diseases associated with inflammation.

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Chronic inhibition of nitric oxide (NO) synthesis by administration of high dose of N^G-nitro-L-arginine methylester (L-NAME) induces vascular inflammation and subsequent atherosclerosis. We aimed to investigate whether the methanol extract of Sorbus commixta cortex (MSC) is able to prevent inflammatory process in a rat model of L-NAME-induced atherosclerosis. Chronic treatment with low or high doses of MSC prevented the L-NAME-induced increase in monocyte chemoattractant protein-1 (MCP-1) and nuclear factor-κB (NF-κB) p65 expressions as well as adhesion molecules including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and E-selectin in aorta. In addition, increased endothelin-1 (ET-1) and angiotensin converting enzyme (ACE) expressions and decreased endothelial cell NO synthase (ecNOS) expression in aorta from L-NAME treated group was reversed by treatment with MSC. From the histological examination, aortic segment from the L-NAME-treated rats revealed a thickening of intima and media, which was ameliorated by treatment with MSC. In conclusion, our results indicate that MSC can prevent atherosclerosis by inhibiting vascular over-expressions of vasoactive materials, pro-inflammatory transcription factor, and adhesion molecules and by augmenting ecNOS in chronic L-NAME-treated rat model.

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Ultraviolet-B (UVB) irradiation has been demonstrated to produce reactive oxygen species (ROS) in the cells and skin, which induces the synthesis of matrix metalloproteinases (MMPs), causing skin photoaging. Using the human skin fibroblast HS68 cell line in the present study, we investigated the photoprotective effects of aucubin from Eucommia ulmoides. Pretreatment with aucubin significantly inhibited the production of MMP-1 by 57% when compared to the UVB-irradiated cells. Additionally, the senescence-associated β-galactosidase (SA β-gal) activity was markedly decreased in the presence of aucubin, which indicates it as an antiphoto-induced aging compound. As the effect of aucubin was determined against ROS, the inhibited ROS formation and malondialdehyde (MDA) levels, and the increased cell viability and glutathione (GSH) level were observed with aucubin under UVB irradiation. Based upon these results, it was suggested that aucubin might play an important role in the cellular defense mechanism against UV radiation-induced photoaging. An understanding of the antioxidant properties of aucubin could, in part, act to elucidate its protective mechanism on the human skin photoaging.

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Crude extracts of Mexican medicinal plants were screened for trypanocidal activity against Trypanosoma cruzi, which is the etiological agent for Chagas' disease, one of the most serious protozoan diseases in Latin America. There were 71 kinds of methanolic and other organic extracts from 65 plants, which were newly examined by a preliminary screening test to observe immobilization of epimastigotes and trypomastigotes of T. cruzi in vitro. The MeOH extract of seeds of Persea americana (avocado) showed moderate activity against epimastigotes. In order to identify the principal compounds for the activity, the MeOH extract was subjected to bioassay-guided fractionation. From the active fractions, six 1,2,4-trihydroxyheptadecane derivatives and two 1,2,4-trihydroxynonadecane derivatives including a new one were isolated. These compounds showed moderate activity against epimastigotes and trypomastigotes.

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Although substances, which can increase the paracellular permeability of intestinal mucosa, could be very helpful for increasing the bioavailability of hydrophilic drugs, they are not used therapeutically due to the possibilities of acute or long-term toxicity (intestinal inflammations due to penetration of bacterial fragments into subepithelial spaces). In this paper the abilities of a calcium chelator EDTA and clodronate (a first generation bisphosphonate) to increase the paracellular permeability were assessed using rat jejunum in side-by-side diffusion chambers while the viability of the tissue was monitored by transepithelial potential difference. Although clodronate is less potent than EDTA in depleting calcium from the intestinal tissue, it significantly increased the paracellular permeability of viable rat jejunum “in vitro” when tested at 15 mM and higher concentrations (the highest therapeutic dose dissolved in 250 ml gives a 22 mM solution of clodronate). This effect was reversible under “high-calcium” conditions. Since clodronate therapy does not have any long-term consequences it was concluded that a safe, transient increase of small intestinal permeability is possible. However, the acute gastrointestinal undesired effects, which can develop during the therapy with high doses of clodronate, might also occur after oral applications of paracellular permeability enhancers. Namely, 30 mM and higher concentrations of clodronate caused a loss of the tissue viability in all rat jejunal segments tested in “in vitro” conditions. A similar effect was observed with much lower concentrations of EDTA.

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We have previously shown that repeated administration of nonsteroidal anti-inflammatory drugs (NSAIDs) to mice treated with β-glucan, a biological response modifier, induced severe lethality. The lethality would be strongly related to the translocation of enterobacterial flora to the peritoneal cavity and disruption of the cytokine network. Reports suggest that nitric oxide (NO) can have an effective or detrimental role in septic shock. In the present study, we examined the effect of NO, an inflammatory mediator, on β-glucan/indomethacin (IND)- induced septic shock by inhibiting its synthesis with N^G-nitro-L-arginine methyl ester (L-NAME), a nonselective NO synthase (NOS) inhibitor. Nitrite concentration was used as an indicator of NO generation. Mortality in β-glucan/IND-treated mice was increased by administering L-NAME. Numbers of bacteria in various organs of mice treated with β-glucan/IND rose significantly within a couple of days of the administration of L-NAME. Additionally, TNF-α, IL-1β, and IL-6 concentrations were enhanced in peritoneal exuded cells in culture. These results suggest a significant loss of the bactericidal activity of macrophages on the administration of a NOS inhibitor which enhanced the rate of enterobacterial invasion to the peritoneal cavity, resulting in systemic inflammatory response syndrome. The production of NO, therefore, provides a protective effect in β-glucan/IND-induced sepsis.

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A liposomal formulation of UCN-01 was studied to prevent binding of drug to human α₁-acid glycoprotein (hAGP). The release of drug from liposomes added to various media was investigated by monitoring the concentration of UCN-01 in different fractions. Protein bound UCN-01 was separated from liposomal UCN-01 and free UCN-01 by gel chromatography and the drug content in the fractions was measured by high-performance liquid chromatography. Also, the blood levels of hAGP bound drug and drug retained in liposomes were assessed after intravenous administration to rats of UCN-01 liposomes together with hAGP. In media containing hAGP, but not rat AGP, UCN-01 was released from liposomes. When UCN-01 liposomes were mixed with rat plasma plus hAGP, the UCN-01 in the liposomes was only gradually released so that some drug remained in the liposomes, and therefore not bound to hAGP, for up to 24 h. After the mixture of liposomal UCN-01 and hAGP was injected into rats, some UCN-01 was retained in liposomes for several hours. Encapsulation of UCN-01 into liposomes is an effective method of preventing binding of UCN-01 to hAGP.

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Changes in albumin and antioxidant enzyme mRNA expression in infant rat liver following administration of total parenteral nutrition (TPN) with/without soybean oil emulsion were studied. Infant rats were divided into three groups: group 1=oral diet, group 2=TPN without fat, and group 3=TPN with 20% of calories from soybean oil emulsion. The period of TPN administration was 4 d. Serum aspartate aminotransferase and alanine aminotransferase levels were higher in group 2 than in the other groups, with similar levels seen in the other groups. Albumin, Cu, Zn-superoxide dismutase, and glutaredoxin 1 mRNA expression levels were lower in group 2 than in the other groups, with similar levels seen in the other groups. Catalase mRNA expression was higher in group 1 than in the other groups, with the lowest level seen in group 2. Soybean oil emulsion should be included in TPN regimens to prevent down-regulation of albumin and antioxidant enzyme mRNA expression.

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Bacillus polyfermenticus SCD, commonly referred to as Bispan strain, is used as a host in bioindustry and has been shown to have several human health benefits. In a recent in vitro study, we discovered that B. polyfermenticus SCD exerts cholesterol-lowering and antioxidant effects. Here, we evaluate the effects of B. polyfermenticus SCD on the lipid and antioxidant metabolisms of hypercholesterolemic rats. Twenty male Sprague–Dawley rats were divided into two groups after a 1-week adaptation period and were fed for 6 weeks on either a high fat-high cholesterol diet, or a high fat-high cholesterol diet supplemented with B. polyfermenticus SCD (3.1×10⁶ cfu/d). B. polyfermenticus SCD significantly reduced plasma low-density-lipoprotein cholesterol, hepatic total cholesterol, and triglycerides, while increasing the fecal excretion rates of total cholesterol and triglycerides. In addition, B. polyfermenticus SCD might reduce the risk of atherosclerosis, as the ratio of high-density-lipoprotein cholesterol to total cholesterol was significantly higher than in the control group. B. polyfermenticus SCD led to an increase in total radical trapping antioxidant potential (TRAP) and a decrease in conjugated dienes in plasma. The erythrocytic glutathione peroxidase (GSH-Px) activity in the B. polyfermenticus group was significantly lower than that in the control group. Plasma TRAP levels exhibited a highly significant negative correlation with hepatic total cholesterol and a marginally significant negative correlation with total plasma cholesterol, while a significant positive correlation was detected between fecal total cholesterol and plasma TRAP. These results suggest that B. polyfermenticus SCD exerts significant health benefits through the modulation of physiologic functions including a variety of atherogenic lipid profiles and antioxidants in hypercholesterolemia.

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Morphinone, identified in the bile of guinea pigs and rats given morphine, is a reactive electrophile and has the ability to bind to glutathione (GSH) and tissue macromolecules, leading to GSH depletion and cell damage. We previously demonstrated that the livers of various animal species are capable of forming morphinone from morphine. In this study, we examined whether the human liver can produce morphinone from morphine. HPLC analysis revealed that the incubation of morphine with the 9000×g supernatant of human liver in the presence of NAD(P) and 2-mercaptoethanol (ME) gave a peak corresponding to the synthetic morphinone-ME adduct (MO-ME), which is readily formed by a nonenzymatic reaction of morphinone with ME. The reaction product was isolated and was unambiguously identified as MO-ME using FAB-MS and NMR analyses in comparison with synthetic MO-ME. The conversion of morphine to morphinone required NAD(P), and NAD was a preferred cofactor over NADP. All the 9000×g supernatants from six human livers could produce morphinone at different rates, ranging from 30 to 120 nmol/g liver/30 min with NAD at pH 7.4. The enzyme activity responsible for the formation of morphinone from morphine was mainly localized in the microsomes. The microsomal enzyme activity was inhibited by steroids, lithocholic acid and indomethacin. Among these compounds, steroids with a 17β-hydroxyl group almost completely depressed morphinone formation. In conclusion, the metabolic pathway of morphine to morphinone, a toxic metabolite, in human was shown for the first time in in vitro experiments.

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Candida albicans NIH A-207 (serotype A) and NIH B-792 (serotype B) strains secreted one acid proteinases (AP) each in a yeast carbon-based medium supplemented with bovine serum albumin (BSA) as the sole nitrogen source. Isolation of AP from the culture filtrates was achieved by dialysis, followed by DEAE-Sepharose and Biogel P-100 column chromatographies. It was found that both enzymes from the two strains had very similar properties when examined. The molecular weights and isoelectric points were found to be 43 kDa and pH 4.0, respectively. The amino acid components and first 12 N-terminal amino acid sequences were virtually identical in both enzymes. The optimum pH of the enzymes was 3.5—4.0. The enzymes were heat-labile, and decreases in their activities were found above 37 °C. The AP activities were completely inhibited by the addition of pepstatin. No other inhibitor among those tested had any effect. The enzymes degraded all proteins examined, especially host defense factors such as immunoglobulin G and the granulocyte colony stimulating factor. The enzymes also caused similar degrees of enhancement of vascular permeability when they were injected into the dorsal skin of guinea pigs.

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Transfusion-associated graft-versus-host disease (TA-GVHD) is a fatal complication of blood transfusion resulting from the contamination of blood products by leukocytes. In order to prevent this disease, gamma or X-ray irradiation of blood components,which can inactivate leukocytes, is currently used. However, the minimal doses needed to destroy lymphocytes promote the leakage of potassium from red blood cells (RBCs), which can induce other side effects, such as hyperpotassemia and cardiac arrest. The reactive oxygen species (ROS) generated by the irradiation of aqueous solutions may accelerate the leakage through oxidation of the RBC membrane. Here we studied the effect of dipyridamole, Trolox, human plasma or mannitol on the leakage of potassium from RBCs following irradiation. RBC preparations (hematocrit; 30%) containing antioxidants were irradiated at 30 Gy and stored at 4 °C for 7 d. The leakage of potassium from the RBCs caused by the irradiation was significantly suppressed by dipyridamole (more than 50 μM), Trolox (more than 5 mM) or human plasma (50%). Mannitol (80 mM) is used to inhibit hemolysis as a constituent of MAP solution, which is a solution used for the storage of RBC products in Japan. Here it was clarified that the leakage of potassium from not only irradiated but also non-irradiated RBCs was unexpectedly promoted by mannitol. The amount of mannitol in MAP solution may have to be reconsidered. The osmotic pressure of the RBC preparation increased in a manner dependent on the concentration of mannitol. The elevated osmotic pressure may promote the leakage. In conclusion, although antioxidants have the potential to suppress the leakage of potassium ascribed to the irradiation, the extent of the supression (10—20%) by dipyridamole (DPM), Trolox or human plasma seems insufficient for the clinical use of these agents as an additive for MAP solution.

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