Exposure of rat phenochromocytoma cells (PC12 cells) to aluminum maltolate complex, Al(maltol)3, induced a decrease in intracellular glutathione (GSH) concentration, resulting in a facilitated release of lactate dehydrogenase (LDH) from the cell and an increase in trypan blue-stained cells. Similar phenomena were observed as the cells were treated with L-buthione-[S,R]-sulfoximine (BSO) in the presence of Al(maltol)3. On the other hand, treatment of PC 12 cells with BSO alone in the absence of Al(maltol)3 did not affect the cell viability. Pre-treatment of PC12 cells with N-acetylcysteine (NAC) for 30 min before a 48 h-exposure to Al(maltol)3 effectively protected the cells from Al(maltol)3 toxicity by increasing intracellular GSH concentration. NAC also effectively inhibited reactive oxygen species (ROS) generation induced by treatment of the cells with Al(maltol)3. However, several lipophilic radical scavengers such as α-tocopherol and 3(2)-tert-butyl-4-hydroxyanisole, and an iron chelator, desferrioxamine, did not prevent Al(maltol)3-mediated ROS production or the decrease of cell viability. Based on these results, we discussed the role of intracellular GSH against the onset of aluminum toxicity in the context of ROS production.
Chloroquine has been used as an anti-malarial drug and is known as a lysosomotropic amine as well. The effects of chloroquine on lysosomal integrity in cultured rat hepatocytes were studied by measuring lysosomal enzyme β-glucuronidase (β-G) or lysosomal membrane glycoprotein (lamp-1) in Percoll density gradient fractions, in the cytosolic fraction obtained from cells permeabilized by digitonin or in the cytosolic fraction obtained by conventional cell fractionation. The distribution of β-G on a Percoll density gradient in chloroquine-treated cells was approximately similar to that of a cytosolic protein, mevalonate pyrophosphate decarboxylase, in nontreated cells. Lamp-1 was decreased in the lysosomal fractions on a Percoll density gradient in chloroquine-treated cells, and was increased in the plasma membrane fraction, as compared with the levels in nontreated cells. Furthermore, after cells were cultured in the presence and absence of chloroquine, the proportions of β-G activity in the cytosolic fraction obtained from the digitonin-permeabilized cells were 19% and 4%, while those in the cytosolic fraction obtained by conventional cell fractionation were 54% and 26%, respectively. From these findings, we infer that chloroquine caused the disruption of lysosomes in the living cells, and that lysosomes treated with chloroquine were easily disrupted by homogenization or centrifugation during cell fractionation.
Oxidatively modified low-density lipoprotein (OxLDL) is present in atherosclerotic lesions and has been proposed to play an important role in atherogenesis. Thrombosis is the major mechanism underlying acute complications of atherosclerosis. In the present study, we analyzed the interaction between OxLDL and blood coagulation factors, which are involved in the blood coagulation pathway. We investigated the effect of OxLDL on plasma coagulation by measuring prothrombin time (PT) as a parameter of the extrinsic pathway of blood coagulation and activated partial thromboplastin time (APTT) as a parameter of the intrinsic pathway of blood coagulation following the addition of OxLDL to plasma. OxLDL, but not native LDL, caused prolongation of APTT in a dose- and oxidation time-dependent manner. In addition, the oxidatively modified product of acetylated LDL (AcLDL), but not AcLDL, also caused prolongation of APTT. The inhibition of lysophosphatidylcholine production in OxLDL by phenylmethylsulfonyl fluoride or Pefabloc pretreatment of LDL resulted in a prolongation of APTT, which was equivalent to the effect of OxLDL. Moreover, OxLDL significantly inhibited blood coagulation factor VIII, IX, and XI activity. Furthermore, we demonstrated that recombinant factor VIII binds to OxLDL and that factor VIII associated with OxLDL is detected in the incubation mixture of OxLDL and plasma. These results indicate that the binding of factor VIII to OxLDL affects the intrinsic pathway of the blood coagulation cascade. The present study suggests that the interaction between OxLDL and factor VIII may provide important information on the initiation and progression of atherosclerosis.
Diabetic cardiomyopathy is associated with cardiac hypertrophy and fibrosis. Activation of protein kinase C (PKC) has been implicated in the diabetes-induced cardiovascular complications. PKCβ2 isoform is preferentially found to be activated in the diabetic myocardium. However, the role of PKCβ2 in diabetic cardiomyopathy is not clear. 14-3-3 family members are dimeric phosphoserine-binding proteins that regulate signal transduction, apoptotic and checkpoint control pathways, and have been shown to bind with PKC isozymes and negatively regulate their enzymatic activities. The present study tests whether 14-3-3 protein regulates cardiac hypertrophy and fibrosis in streptozotocin (STZ)-induced diabetic mice, using transgenic mice with cardiac specific over-expression of dominant negative (DN) 14-3-3 protein. In addition, we examined the relationship between 14-3-3 protein and PKCβ2 in the diabetic myocardium. Cardiac myocyte diameter, content of cardiac fibrosis, left ventricular tissue expressions of atrial natriuretic peptide, transforming growth factor β1, collagen III and PKCβ2 were significantly elevated 28 and 56 d after STZ injection in transgenic DN-14-3-3 mice, when compared to their non-transgenic counterparts. These results clearly demonstrate that the functional inactivation of 14-3-3 protein in DN-14-3-3 mice exacerbates diabetes-induced cardiac hypertrophy and fibrosis. The exacerbations of cardiac hypertrophy and fibrosis were significantly and positively correlated with the enhanced expression of PKCβ2 in DN-14-3-3 mice. Our results indicate for the first time that 14-3-3 protein negatively regulates cardiac hypertrophy and fibrosis, possibly through controlling the expression of PKCβ2 in the diabetic myocardium.
We investigated the enzymatic kinetics and interindividual variability of the metabolism of 5-fluorouracil and procainamide by human liver cytosol and/or microsomes. The Km values for the 5-fluorouracil dihydropyrimidine dehydrogenase (DPD) and procainamide N-acetyltransferase activities in pooled liver cytosol, and procainamide hydrolysis in pooled liver microsomes were 3.9, 1670, and 969 μM, respectively, and the intrinsic clearance (Vmax/Km) values for these reactions were 128, 0.192, and 0.0059 μl/min/mg protein, respectively. The cytosolic activities of 5-fluorouracil metabolism and procainamide N-acetylation ranged from 145 to 790 (469±156, mean±S.D., n=22) and <1 to 152 (52±48, n=12) pmol/min/mg protein, respectively, and the DPD activity of 5-fluorouracil was neither gender-related nor age-dependent. Procainamide N-acetylation activities among 12 human cytosol samples were highly correlated with sulfamethazine N-acetylation activities, suggesting that procainamide N-acetylation is catalyzed by N-acetyltransferase-2. These results suggest that the N-acetylation reaction is more important than the hydrolysis in the metabolic pathway of procainamide, and that there are large interindividual differences in the enzyme activities towards the respective metabolic pathways of 5-fluorouracil and procainamide in human liver.
A member (AKR1C19) of the aldo-keto reductase (AKR) superfamily, found by mouse genomic analysis, was shown to be highly expressed in the liver and gastrointestinal tract, but its function remains unknown. In this study, the recombinant AKR1C19 was expressed and purified to homogeneity. The enzyme was a 36-kDa monomer, and reduced α-dicarbonyl compounds such as camphorquinone and isatin using both NADH and NADPH as the coenzymes. Although apparent kinetic constants for the two coenzymes were similar, the NADPH-linked activity was potently inhibited by submillimolar concentrations of NAD+, but the inhibition of the NADH-linked activity was not significant, suggesting that the enzyme exhibits the NADH-linked reductase activity in vivo. AKR1C19 slowly oxidized 3-hydroxyhexobarbital, S-indan-1-ol and cis-benzene dihydrodiol, but was inactive towards steroids, prostaglandins, monosaccharides, and other xenobiotic alcohols. In addition, the enzyme was inhibited only by dicumarol, lithocholic acid and genistein of various compounds tested. Thus, AKR1C19 possesses properties distinct from other members of the AKR superfamily, and may function as a reductase for endogenous isatin and xenobiotic α-dicarbonyl compounds in the liver and gastrointestinal tract.
Physalis peruviana (PP) is a widely used medicinal herb for treating cancer, malaria, asthma, hepatitis, dermatitis and rheumatism. In this study, the hot water extract (HWEPP) and extracts prepared from different concentrations of ethanol (20, 40, 60, 80 and 95% EtOH) from the whole plant were evaluated for antioxidant activities. Results displayed that at 100 μg/ml, the extract prepared from 95% EtOH exhibited the most potent inhibition rate (82.3%) on FeCl2–ascorbic acid induced lipid peroxidation in rat liver homogenate. At concentrations 10—100 μg/ml, this extract also demonstrated the strongest superoxide anion scavenging and inhibitory effect on xanthine oxidase activities. In general, the ethanol extracts revealed a stronger antioxidant activity than α-tocopherol and HWEPP. Compared to α-tocopherol, the IC50 value of 95% EtOH PP extract was lower in thiobarbituric acid test (IC50=23.74 μg/ml vs. 26.71 μg/ml), in cytochrome c test (IC50=10.40 μg/ml vs. 13.39 μg/ml) and in xanthine oxidase inhibition test (IC50=8.97 μg/ml vs. 20.68 μg/ml). The present study concludes that ethanol extracts of PP possess good antioxidant activities, and the highest antioxidant properties were obtained from the 95% EtOH PP.
The current study was designed to determine the effects of a dietary astaxanthin (ASX-O) on vascular reactivity in spontaneously hypertensive rats (SHR), in order to verify its antihypertensive action mechanism. We evaluated contractions induced by phenylephrine (Phe), angiotensin II (Ang II) and the xanthine/xanthine oxidase (Xan/XOD) system, and relaxations induced by sodium nitroprusside (SNP) as well as endothelium-dependent relaxations mediated by acetylcholine (ACh) in thoracic aorta of the SHR, with and without ASX-O intervention. We also investigated the effects of ASX-O on blood rheology using a microchannel array system. In this study, ASX-O showed a significant modulatory effect on nitric oxide (NO)-induced vasorelaxation by the NO-donor SNP (p<0.05). However, it did not show significant effects in restoring the impaired endothelium-dependent relaxation to ACh in the SHR. On the other hand, the constrictive effects by Phe, Ang II and Xan/XOD were ameliorated by ASX-O (p<0.05). ASX-O also demonstrated significant hemorheological effect by decreasing the microchannel transit time of whole blood. In conclusion, the results suggest that ASX-O may act in modulating the blood fluidity in hypertension, and that the antihypertensive effects of ASX-O may be exerted through mechanisms including normalization of the sensitivity of the adrenoceptor sympathetic pathway, particularly [alpha]-adrenoceptors, and by restoration of the vascular tone through attenuation of the Ang II- and reactive oxygen species (ROS)-induced vasoconstriction.
In order to investigate improvement of hepatic tumor detectability by Sonazoid with phase inversion imaging, the contrast effects on the liver of metastatic carcinoma-model rabbits were evaluated by videodensitometry and visual assessment. Correlation between the contrast enhancement of Sonazoid and histopathology was examined using the same animals. Electron microscopy was performed on hepatic tissue from another healthy rabbits to identify the distribution of Sonazoid microbubbles. As a result, all tumors were smaller than 12 mm in diameter, and after intravenous injection of Sonazoid, they were surrounded with a ring of enhanced signal during the early phase (up to 30 s), followed by a clear contrast defect during the delayed phase (after 10 min). Histopathologic observation revealed that the ring-enhancement was caused by neovasculature in the tumor, and the contrast defects corresponded to living and dead parts of tumors, which lack Kupffer cells. Videodensitometric differences between tumor and healthy tissue markedly increased at delayed phase, and visual detectability of tumors was improved when Sonazoid was used. Ultrastructural analysis showed microbubble-like structures in Kupffer cells, which indicated that Sonazoid microbubbles were taken up with these cells. In conclusion, Sonazoid, used with phase inversion imaging, greatly increases the detectability of small hepatic tumors by highlighting neovascularity at early phase and providing clear contrast defects due to absence of Kupffer cells, which take up Sonazoid microbubbles, at delayed phase.
The aim of the present study was to evaluate the effect of the aqueous extract of seeds of two varieties, namely a country and hybrid variety of Momordica charantia (MCSEt1 and MCSEt2) on oxidative stress in plasma and pancreas of streptozotocin (STZ) induced diabetic rats. Oral administration of each of the seed extracts at a dosage of 150 mg/kg body weight for 30 d resulted in a significant reduction in plasma glucose, thiobarbituric acid-reactive substances, lipid-hydroperoxides, alpha-tocopherol and significant improvement in ascorbic acid, reduced glutathione and insulin. The treatment also resulted in a significant reduction in thiobarbituric acid reactive substances, lipid-hydroperoxides, superoxide dismutase, catalase, glutathione peroxidase and significant improvement in reduced glutathione in pancreas of drug treated diabetic rats when compared to the untreated diabetic rats. On the basis of results obtained, it may be concluded that the treatment of Momordica charantia seed varieties may effectively normalize the impaired oxidative stress in streptozotocin induced-diabetes than the glibenclamide treated groups.
We have reported that caspase cascade accompanied by the regulation of Bax/Bcl-2 and MAPK signaling were involved in evodiamine-induced A375-S2 cell death. In this study, pretreatment with interleukin 1 (IL-1) receptor antagonist (IL-1Ra) rescued the cell viability loss and reversed the ratio of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells induced by evodiamine. IL-1Ra was capable of attenuating the expression of Fas-ligand (Fas-L) and the cleavage of procaspas-8 and -3 caused by evodiamine. Subsequently, IL-1Ra reduced evodiamine-induced DNA degradation, p53 activation and up-regulation of Bax/Bcl-2 ratio. However, IL-1Ra attenuated the enhanced phosphorylation level of p38 mitogen-activated protein kinase (p38 MAPK) without affecting extracellular signal-regulated protein kinase (ERK) inactivation induced by evodiamine. In conclusion, IL-1-induced death cascade in melanoma A375-S2 cell might be one of the targets for natural product evodiamine, and increased Fas-L expression via IL-1 mediated pathway stands at the initiation phase, leading to consequent events that culminate in the death of the cells.
We have previously demonstrated the release of endogenous glutamate by activation of DL-α-amino-3-hydroxy-5-methylisoxasole-4-propionate (AMPA) receptors expressed by bone, while there is no information available on the possible functional expression of glutamatergic signaling molecules in cartilage to date. In rat costal chondrocytes cultured for 4 to 28 d, expression of mRNA was seen for several chondral marker genes including sox9, runt-related gene 2/core binding factor α-1 (Runx-2/Cbfa-1), type II collagen and aggrecan, but not for the adipocyte marker gene peroxisome proliferator-activated receptor γ (PPARγ). Expression of mRNA was drastically increased for Runx-2/Cbfa-1 during culturing from 7 to 14 d with a gradual increase thereafter up to 28 d, while a transient increase was seen in mRNA expression for both type-II collagen and sox-9 on 14 d and for aggrecan on 7 d respectively, in chondrocytes cultured for a period up to 28 d. Irrespective of the culture period up to 21 d, marked expression was seen by cultured chondrocytes with mRNA for GluR3 subunit of AMPA receptors, in addition to vesicular glutamate transporter-1 (VGLUT1) required for the condensation and subsequent exocytotic release of glutamate in the glutamatergic neurotransmission in the brain. Cultured rat costal chondrocytes underwent spontaneous release of endogenous glutamate, while an inhibitor of AMPA receptor desensitization significantly prolonged the duration of endogenous glutamate release stimulated by AMPA. These results suggest that endogenous glutamate could be released from intracellular vesicular constituents associated with VGLUT1 through activation of AMPA receptors expressed by cultured rat costal chondrocytes.
Colominic acid (CA) is an α2,8-linked polymer of sialic acid, originally isolated from capsular Escherichia coli K1. Since inhibition of arterial smooth muscle cell hyperplasia is one of the effective strategies to prevent atherosclerosis, we investigated the effect of CA, purified as an α2,8-linked homopolymer of N-acetylneuraminic acid, on the proliferation of bovine aortic smooth muscle cells in culture. The results demonstrate that CA inhibits the proliferation of the cells without nonspecific cell damage. Sulfation did not modify the inhibitory effect of CA. Specifically, the inhibitory effect of sulfated CA was almost equal to that of CA in vascular smooth muscle cell proliferation. On the other hand, it was suggested that the inhibition of the proliferation by CA is in a degree similar to that by heparin but weaker than that by sodium/calcium-spirulans, known sulfated polysaccharides as the potent inhibitor of vascular smooth muscle cells. The present data suggest that CA with or without sulfate groups can be an origin of beneficial agents that prevents atherosclerosis through a moderate inhibition of arterial smooth muscle cell proliferation.
To evaluate the antipsoriatic effect of Chunghyuldan (CHD, Daio-Orengedokuto in Japanese), which exhibited anti-inflammatory and antiischemic actions, the inhibitory activity of CHD metabolized with and without human intestinal microflora was investigated in oxazolone-induced mouse ear dermatitis. The CHD and metabolized CHD (MCHD) at concentrations of 0.1% also potently suppressed mouse ear swelling by 52.7% and 63.2% at 16 d, respectively. The antipsoriatic effect between CHD and MCHD was not significantly different, although that of CHD weakly increased by the metabolism of human intestinal microflora. Both CHD and MCHD also potently reduced the mRNA levels of cyclooxygenase (COX)-2, interferon (IFN)-γ and IL-4 increased in oxazolone-applied mouse ears, but weakly inhibited that of IL-1β and TNF-α. Based on these findings, CHD may improve contact dermatitis or psoriasis by the regulation of COX-2 produced by macrophage cells and IFN-γ and IL-4 produced by Th cells.
The protective effect of panduratin A, isolated from Kaempferia pandurata ROXB. (Zingiberaceae), against tert-butylhydroperoxide (t-BHP)-induced cytotoxicity was investigated in a human hepatoma cell line, HepG2. The tetrazolium dye colorimetric test (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay) was used to monitor cytotoxicity. Lipid peroxidation [malondialdehyde (MDA) formation] and intracellular glutathione level were estimated by fluorometric methods. Intracellular reactive oxygen species (ROS) formation was measured using a fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA). Panduratin A significantly reduced the cell growth inhibition caused by t-BHP. Furthermore, panduratin A ameliorated lipid peroxidation as demonstrated by a reduction in MDA formation, and attenuated glutathione (GSH) depletion in a dose-dependent manner. It was also found that panduratin A reduced intracellular ROS formation caused by t-BHP. These results strongly suggest that panduratin A has significant protective ability against oxidative damage caused by reactive intermediates.
It was found that reactive oxygen species (ROS) were generated in the interactions of nitric oxide (NO) with glutathione (GSH) or cysteine (CySH) under aerobic conditions. When supercoiled DNA was incubated with a mixture of NO/GSH, NO/CySH, NOC-7 (a NO donor)/GSH or NOC-7/CySH under aerobic conditions, DNA single-strand breaks were observed on agarose gel electrophoresis. The strand breaks were inhibited by common ROS scavengers: superoxide dismutase+catalase, the spin trapping agent 5,5-dimethyl-1-pyrroline-N oxide (DMPO), ethanol, and EDTA. The strand breaks were also caused by incubation with a mixture of S-nitrosoglutathione (GSNO) with GSH or CySH, which was inhibited by ROS scavengers. In the reaction of NO/GSH, GSNO rapidly formed and then gradually decreased, and in the reaction of GSNO/GSH, GSNO was gradually decreased. The decrease inf GSNO was accelerated in the presence of superoxide+catalase. Hydroxyl radical was detected in the mixtures of NO with GSH or CySH under aerobic conditions, and thiyl radicals were detected in the mixtures of GSNO with GSH or CySH under anaerobic conditions as examined in electron spin resonance studies using DMPO as a spin trap. The results indicate that the interaction of NO with thiols in the presence of O2 generates ROS that caused DNA single-strand breaks.
The effect of an aryl hydrocarbon receptor (AhR) ligand on the carbonic anhydrase III (CAIII) mRNA level was studied using primary cultured hepatocytes of rats. CAIII gene which is highly suppressible by dioxins in vivo, was also suppressible in primary cultured hepatocytes of rats by an AhR ligand, 3-methylchlanthrene (3MC). The suppression of CAIII by 3MC was observed in a dose-dependent fashion. The suppression was marked at 10 μM MC. It is likely that AhR is involved in the suppression of the CAIII gene. The transcriptional regulation region of rat CAIII gene was cloned by polymerase chain reaction on the basis of the similarity to the mouse and human CAIII genes. A 1.5 kb section upstream of rat CAIII was sequenced and the transcription initiation site of this gene was mapped to 58 bases upstream of the initiation codon. A xenobiotic responsive element (XRE)-like sequence was found at −555 to −549 bp of the transcription initiation site. The location of XRE-like element was conserved between rats and mice those CAIIIs in liver were shown as dioxins-suppressible. Although the roles of the XRE have not been clarified, these results suggest that the AhR ligands could elicit the suppressive effect on hepatic CAIII and the effect on the factors from extrahepatic tissues is not required for the suppression.
A number of novel 2-chloroethylnitrosourea derivatives of Hoechst 33258 were synthesized and examined for cytotoxicity in breast cancer cell cultures and for inhibition of topoisomerases I and II. Evaluation of the cytotoxicity of these compounds employing a MTT assay and inhibition of [3H]thymidine incorporation into DNA in both MDA-MB-231 and MCF-7 breast cancer cells demonstrated that these compounds were more active than Hoechst 33258. The DNA-binding ability of these compounds was evaluated by an ultrafiltration method using calf thymus DNA, poly(dA-dT)2 and poly(dG-dC)2, indicated that these compounds as well as Hoechst 33258 well interact with AT base pair compared with GC pair. Binding studies indicate that these compounds bind more tightly to double-stranded DNA than the parent compound Hoechst 33258. The degree to which these compounds inhibited cell growth breast cancer cells was generally consistent with their relative DNA binding affinity. Mechanistic studies revealed that these compounds act as topoisomerase I (topo I) or topoisomerase II (topo II) inhibitors in plasmid relaxation assays.
A variety of novel 2-butyl-3-substituted quinazolin-4-(3H)-ones have been synthesized by reacting (2-butyl-4-oxo-3H-quinazolin-3-yl)dithiocarbamic acid methyl ester with a variety of amines; the starting material dithiocarbamate was synthesized from anthranilic acid. The title compounds were investigated for analgesic, anti-inflammatory and antibacterial activities. While the test compounds exhibited significant activity, compounds A1, A2, A3 and A4 showed more potent analgesic activity and compound A4 showed more potent anti-inflammatory activity than the reference diclofenac sodium.
Nidularium procerum LINDMAN, a common bromeliaceae from the Brazilian flora, remains poorly studied regarding its chemical and pharmacological properties. We have recently published that N. procerum has potent analgesic and anti-inflammatory activities. In the present work, we have investigated potential mechanisms involved in the anti-inflammatory effects of N. procerum aqueous extract on lipopolysaccharide (LPS)-, platelet activating factor (PAF)- or formyl-methionyl-leucyl-phenylalanine (fMLP)-induced pleurisy models of inflammation. We found that the aqueous extract of N. procerum leaves (leaf aqueous extract; LAE) inhibits the neutrophil migration, production of inflammatory cytokines interleukin-1 and -6 (IL-1 and IL-6) and the generation of prostaglandin E2 (PGE2) in LPS-induced pleural inflammation in mice. Such inhibitory effect of N. procerum on PGE2 generation was tightly correlated to the inhibition of formation of new cytoplasmic lipid bodies within recruited leukocytes. N. procerum also blocked the in vivo neutrophil influx induced by injection of PAF or fMLP into the mouse pleural cavity and directly inhibited PAF-induced neutrophil chemotaxis in vitro. The data obtained in this study indicate that N. procerum LAE exerts its anti-inflammatory effects by interfering with the capacity of the host to respond to injury at different levels. Among the different functions affected by N. procerum LAE, lipid body formation, PGE2 and cytokine production and neutrophil chemotaxis are readily evidenced in relevant surrogate models. The N. procerum bioactive profile makes it an attractive candidate for future development as a drug or phytomedicine.
Ischaemic stroke is a leading cause of death and long-lasting disability. Gastrodia elata blume (GEB) is a Chinese herb that is widely used to treat convulsive disorders, such as epilepsy, and p-hydroxybenzyl alcohol (HBA) is the active ingredient in GEB. The present study was conducted to evaluate the effects of GEB and HBA on the brain damage and transcriptional levels of Protein disulfide isomerase (PDI) and 1-Cys peroxiredoxin (1-Cys Prx) genes known to play a role in antioxidant systems after transient focal ischemia in the rat brain. Focal ischemia was induced in rats by middle cerebral artery occlusion (MCAO). All animals underwent ischemia for 1 h, followed by 24 h of reperfusion. Coronal brain slices were stained with 2,3,5-triphenyltetrazolium chloride or total RNA was extracted for the analysis of gene expression. Histopathologic analysis revealed a significant (p<0.05) decrease in infarct size in the ipsilateral brain with GEB extracts or HBA. Moreover, the levels of PDI and 1-Cys Prx transcription were significantly increased in the GEB extract- or HBA-treated group compared with the untreated group (p<0.05). This study therefore indicated that GEB and HBA provide neuroprotection by preventing brain damage through the increased expression of genes encoding antioxidant proteins after transient focal cerebral ischemia and may be effective as neuroprotective agents at the cellular and molecular levels in the brain.
The MeOH-soluble fraction of the water extract of Catharanthus roseus from Indonesia, having shown potent inhibitory activity on the metabolism mediated by CYP2D6, was subjected to activity-guided isolation to yield two triterpenes, ursolic acid (1) and oleanolic acid (2), and three alkaloids, vindoline (3), ajmalicine (4), and serpentine (5). The isolated compounds were tested for their inhibitory activity on the metabolism mediated by CYP3A4 or CYP2D6 using [N-methyl-14C]erythromycin or [O-methyl-14C]dextromethorphan as a substrate, respectively. Ajmalicine (4) and serpentine (5) showed very potent inhibitory activity against CYP2D6 with IC50 values of 0.0023 and 3.51 μM, respectively. All isolated compounds showed weak or no inhibition against CYP3A4. On time-, concentration-, and NADPH-dependent assay, serpentine (5) appear to be the mechanism-based inhibitor for CYP2D6 enzyme in which the inhibition was irreversible and driven by catalytic process. KI and kinact values for serpentine (5) were 0.148 μM and 0.090 min−1, respectively. On the other hand, ajmalicine (4) showed no time-dependent inhibition or reversible inhibition, and thus appear to be not mechanism-based inhibitor.
Honey bee propolis is rich in cinnamic acid derivatives. Baccharin and drupanin from Brazilian honey bee propolis are cinnamic acid derivatives that contain prenyl moieties. We previously isolated these two compounds and demonstrated that they induce an apoptotic event in several tumor cell lines. In this study, we examined the tumoricidal activity of baccharin and drupanin in mice allografted with sarcoma S-180 and also studied the genotoxic effects on normal splenocytes using the alkaline single cell gel (comet) assay. We found that both baccharin and drupanin effectively suppressed growth of the tumor. Furthermore, these compounds induced a significant genotoxic effect on the tumor cells in comparison with normal splenocytes. Thus, baccharin and drupanin are potent tumor suppressive components of honeybee propolis.
We investigated the changes (increase or decrease in peak intensity) in the expression of plasma proteins in spontaneously diabetic WBN/Kob rats that were with complicated diabetic nephropathy, to determine multiple biomarkers in the plasma of diabetic rats. The present study using surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) demonstrated that six peaks at mass/charge ratios (m/z) of 4678, 4732, 4808, 9058, 9323, and 9465, among approximately 80 peaks per spectrum in the 2000—10000 Da mass range, had increased peak intensities with the development or progression of diabetic nephropathy in plasma of spontaneously diabetic WBN/Kob rats as compared with those of normal Wistar rats. Administration of the Kampo medicine Hachimi-jio-gan was effective at reducing the expression of diabetic nephropathy but not at reducing blood glucose levels. It also improved the increased levels of these plasma proteins. Other biomarker peaks at m/z 5067, 5279, 7598, and 7917 were not affected by Hachimi-jio-gan administration. Further study will be needed to identify these positive biomarkers and to evaluate the relationship between the efficacy and expression patterns of the plasma proteins in greater detail. The expression patterns of proteins and molecular-related ions revealed that several proteins in plasma may be involved in the development and/or progression of diabetic nephropathy in WBN/Kob rats and the efficacy of Hachimi-jio-gan. This study using ProteinChip technology may provide a useful basis in the search for multiple biomarkers in plasma for the diagnosis of disease and therapeutic evaluation of Kampo medicines.
Oxidation of low density lipoprotein (LDL) is thought to be a major factor in the pathophysiology of atherosclerosis. In the present study, we found that coumarins isolated from Weigela subsessilis (Caprifoliaceae) inhibited LDL oxidation mediated by either catalytic copper ions (Cu2+) or free radicals generated with the azo compound 2,2′-azobis-(2-amidinopropane)dihydrochloride (AAPH). Of the coumarins tested, scopoletin (1) and cleomiscosin A (2) increased the lag time of conjugated diene formation and inhibited the generation of thiobarbituric acid reactive substances (TBARS) in a dose-dependent manner. In addition, it was found that compounds 1 and 2 had the capacity to protect the fragmentation of apolipoprotein B-100 (apoB-100). These results suggest that W. subsessilis and its active coumarins, 1 and 2, may have a role to play in preventing the LDL oxidation involved in atherogenesis.
This study reports on the biological properties, antioxidant and antidiabetic, of two varieties of Amaranthus caudatus seeds, Oscar blanco and Victor red. Oil, squalene and phenolic contents were also determined. Seeds of both investigated varieties were found to possess very different levels of squalene (2.2% in Oscar blanco variety and 7.5% in Victor red variety). Although the antioxidant activity of A. caudatus var. Oscar blanco and A. caudatus var. Victor red statistically did not differ significantly from each other (IC50 values of ethyl acetate extracts were 0.50 mg/ml and 0.62 mg/ml, respectively), significant differences were noticed in relation to antidiabetic activity (inhibition of α-amylase, EC 188.8.131.52) of methanolic extracts that showed 50.5% for A. caudatus var. Oscar blanco and 28% for A. caudatus var. Victor red at concentration of 25 μg/ml.
The isolation and characterization of rat lens aldose reductase (RLAR) inhibitors from the fruiting bodies of Ganoderma applanatum were conducted. Among the extracts and fractions from G. applanatum tested, the MeOH extract and EtOAc fraction were found to exhibit potent RLAR inhibition in vitro, their IC50 being 1.7 and 0.8 μg/ml, respectively. From the active EtOAc fraction, seven compounds with diverse structural moieties were isolated and identified as D-mannitol (1), 2-methoxyfatty acids (2), cerebrosides (3), daucosterol (4), 2,5-dihydroxyacetophenone (5), 2,5-dihydroxybenzoic acid (6), and protocatechualdehyde (7). Among them, protocatechualdehyde (7) was found to be the most potent RLAR inhibitor (IC50=0.7 μg/ml), and may be useful for the prevention and/or treatment of diabetic complications.
In order to screen the active constituents of Vaccinium vitis-idaea L., the compounds absorbed into the rat blood after oral administration of ethanol extract of the stems and leaves of V. vitis-idaea (EEV) have been analyzed. Two compounds were detected in the plasma and identified as arbutin and fraxin, which are originally existed in the crude drug. Furthermore, the pharmacological effects of the two compounds involving EEV for curing acute and chronic respiratory tract infection were tested. The results showed that both arbutin and fraxin have the anti-inflammatory, anti-coughing and phlegm-removing effects. Therefore, it was ascertained that the arbutin and fraxin would be the main active constituents of V. vitis-idaea L.
Eleven cDNA clones encoding the open-reading frame of calmodulin and calmodulin-like proteins were isolated from carrot seedlings treated with fungal mycelial walls. These calmodulin genes were mainly expressed in the stems and leaves of carrot, although the transcriptional level was almost negligible in the seeds and root tissues. Based on nucleotide homology, these genes were divided into two classes, class I and class II, and reverse transcription-polymerase chain reaction analyses revealed that the expression level of class II genes transiently increased when carrot seedlings were treated with the mycelial walls. In contrast, the level of class I genes apparently did not show any significant change even after treatment with the fungal components. These results suggest that the defense responses induced in the fungi-infected carrot accompany the increase in the transcriptional levels of a certain calmodulin gene(s).
Comparative analysis of 10 strains of Glycyrrhiza uralensis cultivated in Kyoto, Japan, was undertaken to characterize their variations. Based on the chemical characteristics of their leaves and underground parts, the 10 strains were divided into two chemotypes, the China type and Kazakhstan type. The contents of licoleafol in the leaves of the China type (0—0.03% of dry weight) were lower than those of the Kazakhstan type (0.05—1.16% of dry weight). In addition, a China type-specific unidentified compound was also detected in the leaves of China-type plants. Glycyrrhizin contents in the underground parts of the China type (2.08—5.12% of dry weight) were relatively higher than those of the Kazakhstan type (0.75—2.55% of dry weight). Contents of glycycoumarin, a species-specific flavonoid of G. uralensis, in the underground parts of China-type plants (0.07—0.28% of dry weight) were higher than those of Kazakhstan-type plants (0.01—0.08% of dry weight). These 10 strains were also divided into two genotypes, the GA type and AT type, based on their chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit gene (rbcL) sequences, although there was no correlation between the chemotype and the rbcL genotype.
Seven sterols (1—7) and eight polyisoprenepolyols (8—15), isolated from the non-saponifiable lipid fraction of the dichloromethane extract of an edible mushroom, Hypsizigus marmoreus (Buna-shimeji), were tested for their antitubercular activity against Mycobacterium tuberculosis strain H37Rv using the Microplate Alamar Blue Assay (MABA). Six sterols (2—7) and two polyisoprenepolyols (8, 12) showed a minimum inhibitory concentration (MIC) in the range of 1—51 μg/ml, while the others (1, 9—11, 13—15) were inactive (MIC>128 μg/ml). The seven sterols (1—7) and three polyisoprenepolyols (8, 10, 12) were further evaluated for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Sterols 6 and 7 showed potent inhibitory effects while preserving the high viability of Raji cells.
Phenolic compounds, in general, exhibit antioxidant and antibacterial activities. We studied antimicrobial activity of the phenolic antioxidants, propofol (2,6-diisopropylphenol), tocopherol, eugenol, butylated hydroxyanisole (BHA), and several of their dimer compounds. Dipropofol (dimer of 2,6-diisopropylphenol) showed strong antibacterial activity against gram-positive strains including methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococci (VRE), while propofol and other monomeric and dimeric phenols having methyl or tert-butyl groups showed no remarkable activity. The results indicated that the dimeric structure of 2,6-diisopropylphenol moiety may play an important role in the antibacterial activity.
Isolation of the ethyl acetate soluble fraction from aerial parts of Viola hondoensis W. BECKER et H. BOISSIEU yielded one major isoflavonoid glycoside, tectoridin-4′-O-β-D-glucoside. The structure of the compound was certainly determined by chemical analyses, as well as 1D- and 2D-NMR spectroscopy. The compound exhibited potent inhibitory activity against the expression of matrix metalloproteinase-1 caused by UV-irradiation in cultured human skin fibroblasts.
The goal of this study was to investigate an intranasal cocaine vaccine containing the mucosal adjuvant macrogol-6-glycerol capylocaprate (RhinoVax). Cocaine–KLH conjugate was prepared and administered in two formulations. Ten mice were immunised intranasally using RhinoVax as adjuvant and ten subcutaneously using aluminium hydroxide as an adjuvant. A negative control group (n=10) received unconjugated KLH with RhinoVax intranasally. Specific cocaine antibodies in serum were measured following primary and booster immunisation. Relative antibody responses in serum indicated that the immunisation was successful. Animals were then challenged with cocaine either intranasally or intraperitoneally with subsequent measurement of drug distribution into the serum, brain and olfactory bulb. The cocaine-immunised groups revealed significantly lower cocaine levels in the brain compared to the negative control group. The inhibition of cocaine distribution to the brain in the intranasal immunised group was comparable to that of the subcutaneous immunised group. This was unexpected because the cocaine specific antibody levels in serum were fivefold lower in the intranasal immunised group. However, the presence of mucosal cocaine specific antibodies after intranasal immunisation could play an important role in hindering direct access of cocaine into the brain via the olfactory bulb.
We investigated the intestinal absorption enhancing effect of the saponins from the root bark of Aralia elata (SRBAE) in Caco-2 cell monolayers and rats. SRBAE at concentrations of 0.04% and 0.08% (w/v) decreased the transepithelial electrical resistance (TEER) values and increased the paracellular uptake of chondroitin sulfates (CSs) having different molecular weights (MW 500, 4500, and 18000) in a dose-dependent manner. We also evaluated the cytotoxicity of SRBAE to determine its proper concentration as an absorption enhancer. MTT assay and trypan blue exclusion test indicated that the cytotoxicity of SRBAE at concentrations of 0.04% and 0.08% was negligible. CS (MW 18000) was orally administered with or without SRBAE to rats. The oral administration of SRBAE (250 mg/kg) in 1 h increased the intestinal absorption of CS, by 4.9-fold versus the control (CS alone). Histological examination of the gastrointestinal tissues showed that SRBAE did not cause any damage to tissues. In conclusion, our results suggest that SRBAE acts as an efficient absorption enhancer and makes it easier for hydrophilic molecules to penetrate the intestinal epithelium.
The present study was undertaken to elucidate the stomach- and site-selective delivery of 5-fluorouracil (5-FU) following its application on the gastric serosal surface in rats. An experimental system utilizing a cylindrical diffusion cell attached to the gastric serosal surface was established. To evaluate the gastric distribution of 5-FU, the stomach was separated into the site under the diffusion cell (site 1) and the site not under the diffusion cell (site 2). Furthermore, the mucosal side at site 1 was separated from the serosal side. After intravenous and oral administration of 5-FU, the 5-FU concentrations at sites 1 and 2 until 240 min were similar. After gastric serosal surface application of 5-FU, however, the concentration of 5-FU at site 1 until 240 min was approximately 10-fold higher than that at site 2, and was sustained. Furthermore, the 5-FU concentration on the mucosal side at site 1 and the serosal side at site 1 were comparable after gastric serosal surface application. The blood concentration of 5-FU was low (<4.4 μg/ml) until 240 min after gastric serosal surface application. The maximum blood concentration of 5-FU after gastric serosal surface application was significantly lower than after intravenous administration. Thus, the stomach- and site-selective delivery system following application on the gastric serosal surface could be applied with anticancer drugs for the treatment of gastric cancer.
This study examined the transport of a photosensitizer (hypericin, HY) using the chick chorioallantoic membrane (CAM) as a model of transport after topical administration. The model correlates both the photosensitizer uptake and anti-vasculature effects after photodynamic therapy (PDT). HY formulations were prepared using N-methyl pyrrolidone (NMP) as a solvent and penetration enhancer. Fertilized chicken eggs were disinfected and incubated at 37.4 °C and 60% humidity. Formulations were applied on CAM and incubated for 30 min in the dark. Subsequently, the solutions were removed from the CAM surface and the HY concentration was determined. The CAM was exposed to a fixed light dose of 10 J/cm2 at 50 mW/cm2. The vascular damage induced by the light was quantitatively measured using image-processing techniques. The uptake ratio of HY in 4.8% NMP (HD group) to that of 0.6% NMP (LD group) was found to be 1.96. This ratio is correlated with the vascular damage caused by the PDT effect of HY. The HD treated CAM showed a vessel regression that was 2.37 times higher than that of LD treated CAM. This paper reports the first attempt to develop a quantitative transport study for HY using CAM and to explore the relationship between the vascular regression and amount of drug of uptake. The model has potential for other similar transport studies.
The inhibitory effect of suplatast (ST), an anti-allergic drug, on theophylline (TP) metabolism was investigated in rats in vivo and in vitro. Intravenous injection of aminophylline (AP) at 10 mg/kg of TP equivalent was performed with or without pretreatment by oral administration of 100 mg/kg of ST 2.5 h prior to AP. In the ST-pretreated group, plasma concentration (Cp), the area under Cp–time profile (AUC) and urinary excretion of TP increased significantly, and urinary excretion of TP metabolites, 1,3-dimethyluric acid (DMU) and 1-methyluric acid (1MU) decreased significantly. Metabolic clearance of DMU (CLDMU) and that of 1MU (CL1MU) were remarkably suppressed by ST pretreatment, however, renal clearance (CLr) of TP did not change. To compare the inhibitory effect of ST on TP metabolism with that of its main metabolite (M1) in vivo, a concomitant intravenous injection of AP (10 mg/kg of TP equivalent) with ST or M1 (40 mg/kg of ST equivalent) was carried out. In the M1 group, Cp and AUC of TP increased significantly, and the total body clearance of TP decreased significantly. In contrast, ST did not induce these changes. Then, the inhibitory effect of ST and M1 on TP metabolism in vitro was evaluated using rat-liver microsomes. ST and M1 suppressed DMU formation in a competitively inhibitory manner, and their equilibrium dissociation constants (Ki) were 822 and 731 μM, respectively. In conclusion, inhibition of TP metabolism by ST was demonstrated in vivo and in vitro, and the involvement of M1 and/or other metabolites in this drug interaction was suggested.
Considerable attention has recently been paid to the application of chemokines to cancer immunotherapy because of their chemotactic affinity for a variety of immune cells and because several chemokines are strongly angiostatic. In the present study, the recombinant adenovirus vectors encoding chemokine CCL19 or XCL1 in an E1 cassette (AdRGD-mCCL19 and AdRGD-mXCL1) were developed. The constructed fiber-mutant adenovirus vector, which contained the integrin-targeting Arg-Gly-Asp (RGD) sequence in the fiber knob, notably enhanced the transfection efficiency to OV-HM ovarian carcinoma cells compared to that induced by conventional adenovirus vector. The results of an in vitro chemotaxis assay for chemokine-encoding vector demonstrated that both AdRGD-mCCL19 and AdRGD-mXCL1 could induce the migration of cells expressing specific chemokine receptors. Of the two chemokine-encoding vectors evaluated in vivo, AdRGD-mCCL19 showed significant tumor-suppressive activity in B6C3F1 mice via transduction into OV-HM cells, whereas XCL1 did not exhibit any notable anti-tumor effects, suggesting that CCL19 may be a candidate for cancer immunotherapy.
The stability of spironolactone (SPN) in rat plasma was studied and its degradation was found to be an apparent first-order reaction. The apparent first-order rate constants (kobs) at 37, 23.5 and 0 °C were 3.543±0.261 (h−1, mean±S.D., n=3), 6.278±0.045 (×10−1 h−1), and 7.336±0.843 (×10−2 h−1), respectively. The half-lives were 0.20 h, 1.10 h, and 9.53 h. The degradation rate of SPN in rat plasma was markedly decreased when NaF, an esterase inhibitor, was added to the plasma, and the degradation was catalyzed by esterase in the plasma. These results indicated that not only plasma but also blood and serum samples in rat pharmacokinetic studies should be cooled to 0 °C, the temperature maintained, and treated as soon as possible. In pharmacokinetic studies reported previously, the temperature control of plasma, blood, and serum samples was not described. The pharmacokinetic study in rats after intravenous administration of SPN at 20 mg/kg was performed with strict temperature control of plasma and blood samples. The AUC, MRT, CL and Vdss values (mean±S.E. of 4 rats) for SPN were 4100.8±212.9 ng h/ml, 0.29±0.01 h, 4915.7±248.0 ml/h/kg, and 1435.4±48.4 ml/kg, respectively. The AUC value was much larger than that previously reported. The AUC, MRT, Cmax and Tmax values (mean±S.E. of 4 rats) of canrenone, an active metabolite of SPN, after the administration of SPN were 4196.1±787.5 ng h/ml, 1.99±0.13 h, 1546.3±436.4 ng/ml and 1.0±0.0 h, respectively. This AUC value was almost identical to the value previously reported.
Melanins are pigments of high molecular weight formed by oxidative polymerization of phenolic or indolic compounds. A number of fungi, including Aspergillus nidulans, produce pigments related or identical to melanin, which are located on cell walls or exist as extracellular polymers. The aim of the present study was to assess the antioxidant activity of synthetic melanin and of the pigment extracted from the mycelium and culture medium after growth of the highly melanized strain (MEL1) from A. nidulans. The ability of the melanin pigment to scavenge the oxidants HOCl and H2O2 was evaluated by inhibition of the oxidation of 5-thio-2-nitrobenzoic acid (TNB) using several melanin concentrations. The results showed that the pigment of the MEL1 strain competes with TNB for H2O2 and HOCl, inhibiting TNB oxidation in a concentration-dependent manner. For the HOCl oxidant, this inhibition was comparable to that of synthetic melanin, whose IC50 values were quite close for both pigments. Thus, our results suggest that the melanin from A. nidulans is a potential HOCl scavenger and may be considered a promising material for the cosmetic industry for the formulation of creams that protect the skin against possible oxidative damage.
In the present study we attempted to ascertain whether Bacillus cereus was able to produce catechol-siderophore(s), and whether it was able to utilize transferrin-bound iron. The growth of B. cereus was stimulated in proportion to the iron-saturation level of the transferrin, and catechol-siderophores were produced in inverse proportion to this level. B. cereus was proved to uptake iron from partially iron-saturated transferrin or holotransferrin, without destroying the transferrin by its proteases. The catechol-siderophores from B. cereus were able to sustain and augment its growth on the transferrin-bound iron. These results indicate that B. cereus has the ability to produce catechol-siderophores, and to utilize transferrin-bound iron as an iron source for growth, via the siderophore-mediated iron-uptake system.