The retinal capillary endothelial cells are connected to each other by tight junctions that play a key role in permeability as the inner blood-retinal barrier (inner BRB). Thus, understanding the inner BRB transport mechanism is an important step towards drug targeting of the retina. Nevertheless, inner BRB transport studies have been very limited in number since it is not easy to use the retinal capillaries, which are very small in size, for in vitro transport studies. Conditionally immortalized rat retinal capillary endothelial cells (TR-iBRB), pericytes (TR-rPCT) and Müller cell lines (TR-MUL) have been established from transgenic rats harboring the temperature-sensitive simian virus 40 large T-antigen gene. These cell lines posses respective cell type markers and maintain certain in vivo functions. Using a combination of newly developed cell lines and in vivo studies, we have elucidated the mechanism whereby vitamin C, L-cystine, and creatine are supplied to the retina. TR-iBRB cells are also able to identify transporters and apply to study regulation of transporters under pathophysiological conditions. Furthermore, these cell lines permit the investigation of cell-to-cell interactions and the expression of inner BRB-specific genes between TR-iBRB and other cell lines.
We developed a system that uses the single-molecule fluorescence detection system MF10S to assess quantitatively the activity of WRN helicase, the product of the causative gene of Werner syndrome that includes premature ageing. Double-strand DNA substrates labeled with the fluorescence dye TAMRA at the 5′ end and with a quencher at the 3′ end of the counter strand were incubated with a single trapper oligonucleotide and Werner helicase, and the resultant single DNA fragments labeled with TAMRA produced by the unwinding of WRN helicase were detected using the MF10S. The results using this system and those using polyacrylamide gel electrophoresis were well correlated. The MF10S system provides a quantitative analysis that is much faster, simpler, and more economical than systems using polyacrylamide gel electrophoresis and radioisotopes, and could be used as a quantitative analysis system for Werner helicase and other DNA helicase activities.
Using a surface plasmon resonance (SPR) system, we investigated the lipid membrane-binding properties of four analogues of the 18-residue linear amphipathic β-sheet cationic antimicrobial peptide (KIGAKI)3-NH2, each of which contains a single isoleucine-to-tryptophan substitution. The results of the SPR study revealed significant differences in the binding characteristics of the peptides depending upon the position of tryptophan residues. These peptides showed higher binding affinity to membranes containing acidic phospholipids than zwitterionic phospholipids. The addition of dimethylsulfoxide to the running buffer was effective in maintaining the solubility of these peptide solutions and obtaining concentration-dependent sensorgrams for the kinetic analysis in this study. The kinetic binding data of SPR correlated closely with both the ability of the peptides to lyse liposomes with the same phospholipid composition and bactericidal activity. The results demonstrate that SPR may be a valuable tool to predict the membrane lytic properties of antimicrobial peptides.
Hemagglutinating activity for glutaraldehyde-fixed, trypsinized human and rabbit erythrocytes was obtained from carp (Ciprinus capio) egg extract. This activity was inhibited by sulfated glycans such as heparin and fucoidan, and salmon testis DNA (stDNA), but not by mono- and oligosaccharides. The active fraction from hydroxyapatite column, HA400, caused aggregation of stDNA at a concentration of 125 μg/ml. HA400-induced aggregation was inhibited by heparin and fucoidan at the concentrations of 25 and 50 μg/ml, respectively. HA400 also bound to short size single- or double-stranded oligonucleotide in dot blot hybridization analysis using 32P-labeled probe. HA400 was further purified by heparin-Sepharose column chromatography, and consequently, active fractions (HS2, HS3 and HS4) were obtained. Hemagglutinating activities of HS2 and HS4 were 16 and 32 times higher than that of HS3, respectively. In contrast, HS3 showed the highest DNA aggregating activity among these fractions, indicating that the activities of DNA aggregation and hemagglutination were separated by heparin-Sepharose column chromatography. HS3-induced stDNA aggregation was inhibited by 0.5 M NaCl or above. Southwestern blot analysis revealed that the DNA aggregating protein (DAP) possessing oligonucleotide-binding capacity was the 18 kDa protein. The N-terminal amino acid sequence of DAP was LKEGECEVCVGFLGR having partial homology with DNA-binding protein HU-α, but showed no homology with nucleohistone proteins. These results indicate that there is a novel, non-histone DAP in carp eggs.
Free radical scavenging and protective actions against chemically induced hepatotoxicity of Crassocephalum crepidioides were investigated. A water extract of C. crepidioides strongly scavenged superoxide anion, hydroxyl radical and also stable radical 1,1-diphenyl-2-picrylhydrazyl. Galactosamine (GalN, 400 mg/kg) and lipopolysaccharide (LPS, 0.5 μg/kg) induced hepatotoxicity of rats as seen by an elevation of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and of lipid peroxidation in liver homogenates was significantly depressed when the herbal extract was given intraperitoneally 1 and 15 h before GalN and LPS treatment. Similarly, carbon tetrachloride (CCl4) induced liver injury as evidenced by an increase in AST and ALT activities in serum was also inhibited by the extract pretreatment. Isochlorogenic acids, quercetin and kaempferol glycosides were identified as active components of C. crepidioides with strong free radical scavenging action. These results demonstrate that C. crepidioides is a potent antioxidant and protective against GalN plus LPS- or CCl4-induced hepatotoxicity.
This study examined the effect of astilbic acid (3β,6β-dihydroxyolean-12-en-27-oic acid), which is a herbal medicine isolated from Astilbe chinensis. Astilbic acid inhibited 5-lipoxygenase (5-LOX)-dependent leukotriene C4 (LTC4) generation in bone marrow-derived mast cells in a concentration-dependent manner with an IC50 value of 2.1 μM. In addition, astilbic acid was tested in a rat passive cutaneous anaphylaxis (PCA) reaction assay by administering 10 to 50 mg/kg i.p. Astilbic acid dose dependently inhibited the PCA reaction, which was activated by anti-dinitrophenyl (DNP) IgE. Furthermore, this compound inhibited mouse acetic acid-induced writhing (47—62% inhibition at 0.4—50 mg/kg) after being administered orally, while aspirin (200 mg/kg) showed 62% inhibition. These results suggest that astilbic acid may be beneficial in regulating various inflammatory processes.
Phellinus linteus (PL) is a fungus mainly found in tropical America, Africa and Asian countries including Korea, Japan and China. PL has been traditionally used for the treatment of arthritis, liver damage and cancer. However, little was known on the biological activity and characterization of Phellinus species in Cambodia. Thus, in the present study, the anti-metastatic mechanism of aqueous extract of Cambodian Phellinus linteus (CPL) was evaluated. Cambodian mushroom was identified as a Phellinus species with 99% homology of Phellinus linteus by DNA sequence analysis and comparison by the National Center for Biotechnology Information. CPL did not exhibit any significant cytotoxicity against B16BL6 cells, invasive melanoma cells at 1 mg/ml. However, CPL inhibited platelet aggregation induced by B16BL6 cells and also disrupted the adhesion to gelatin and invasion of B16BL6 cells in a concentration dependent manner. Similarly, CPL dose-dependently inhibited the pulmonary metastatic colonies in C57BL/6 mice intravenously injected by B16BL6 cells up to 55.5% at a dose of 50 mg/kg compared with untreated control. CPL also down-regulated the expression of urokinase type plasminogen activator (uPA), one of key proteins associated with invasion and metastasis of tumor cells in a concentration dependent fashion, while CPL didn't significantly affect the expression of matrix metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2) by reverse transcriptase–polymerase chain reaction (RT-PCR). Taken together, these findings indicate that Cambodian Phellinus linteus may inhibit metastasis at least partly via regulation of uPA associated with tumor cell induced platelet aggregation (TCIPA) and also suggest a further study for isolation of active ingredients and the involvement of adhesion molecule signaling pathway.
Sodium spirulan (Na-SP) is a sulfated polysaccharide isolated from the blue-green alga Spirulina platensis. Na-SP has anticoagulant and fibrinolytic properties in vitro, including activation of heparin cofactor II, enhancement of vascular endothelial cell fibrinolytic activity and stimulation of endothelial proteoglycan (PG) release. In the present study, we investigated the types of endothelial PGs whose release is stimulated by Na-SP. Na-SP stimulated the PG release in a dose- and time-dependent manner. However, heparin, dextran sulfate and hyaluronan stimulated the release of heparan sulfate PGs rather than chondroitin/dermatan sulfate PGs, whereas the release of both types of PGs was strongly stimulated by Na-SP. Sepharose CL-6B chromatography of [35S]sulfate-labeled PGs showed that PGs were partially released after partial degradation of the core proteins without a change in chain length of the glycosaminoglycan chains after Na-SP treatment. On the other hand, SDS-polyacrylamide gel electrophoresis and Western blot analysis of the PG core proteins indicated that the Na-SP-releasable PGs are both a large heparan sulfate PG, perlecan, and a small chondroitin/dermatan sulfate PG, biglycan, without change in the size of the core proteins. Taken together, these results suggest that Na-SP stimulates the endothelial release of both perlecan and biglycan with the intact structures, possibly by mechanisms different from those of heparin, dextran sulfate and hyaluronan; a part of the PG core proteins may be degraded after Na-SP treatment. Since perlecan and biglycan have antithrombin activities, the present data support the hypothesis that Na-SP may enhance local anticoagulant activity in the liquid phase on the endothelium via stimulation of endothelial PG release.
In this paper we investigated the vascular activity and possible mechanism of Orientin, from bamboo leaves (Phyllostachys nigra), in isolated thoracic aortic rings from New Zealand rabbit. Among the four compounds, studied, only Orientin relaxed phenylephrine-induced contractions with an IC50 value of 2.28 μM in the endothelium intact and with an IC50 value around 7.27 μM in the endothelium removed aortic rings. The vasorelaxant effect of Orientin on endothelium-intact thoracic aortic rings was attenuated by the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester, but not by indomethacin (a cyclooxygenase inhibitor), tetraethylammonium chloride (K+ channels inhibitor) or propranolol (β-receptor inhibitor). Furthermore, Orientin inhibited norepinephrine (NE), CaCl2 and KCl-induced vasoconstriction concentration dependently in a non-competitive manner, and also reduced both the initial fast release and the sustained phases of phenylephrine-induced contractions. Orientin can stimulate NO production from endothelial cells. Orientin also increased cyclic guanosine 3,5-cyclic monophosphate (cGMP) levels without changes in adenosine-3′,5′-cyclic phosphoric acid (cAMP) in rabbit aorta. The results showed that Orientin relaxed thoracic aortic rings by the nitric oxide-cGMP pathway, and in the vascular smooth muscle inhibited the contraction induced by the activation of receptor-operating and voltage-dependent Ca2+ channels. Cyclooxygenase pathway, potassium channels, β-receptors and cAMP pathway, on the other hand, had no apparent roles. The inhibition of both intracellular Ca2+ release and extracellular Ca2+ influx may be one of the main vasorelaxant mechanisms of Orientin.
The acetate (FA), hexanic (FH), hydroalcoholic (FHA) and precipitated hydroalcoholic (FHAppt) fractions from the root of Petiveria alliacea L. were evaluated for antinociceptive effect using the abdominal constriction induced by acetic acid, hot-plate, formalin tests. The open field and rota rod tests were used to evaluate psychomotor function and myorelaxant activity. The fractions were administered intraperitoneally in mice at doses of 100 and 200 mg/kg. Inhibitions of abdominal constrictions were observed with all doses of the fractions, as compared to control. FH and FHAppt, at both doses, reduced the nociception produced by formalin in the 1st (0—5 min) and 2nd (20—25 min) phases, however FHA (100, 200 mg/kg) and FA 200 mg/kg presented significant inhibition on the 1st and 2nd phases, respectively, of this test. A reduction of the locomotor activity was observed in the open field test with all the fractions. These fractions failed to affect the motor coordination in the rota rod test. Results showed that the different fractions of Petiveria alliacea L. have different antinociceptive potentials as demonstrated in the experimental models of nociception in mice, supporting folk medicine use of this plant.
Astaxanthin is a natural antioxidant carotenoid that occurs in a wide variety of living organisms. We investigated, for the first time, antihypertensive effects of astaxanthin (ASX-O) in spontaneously hypertensive rats (SHR). Oral administration of ASX-O for 14 d induced a significant reduction in the arterial blood pressure (BP) in SHR but not in normotensive Wistar Kyoto (WKY) strain. The long-term administration of ASX-O (50 mg/kg) for 5 weeks in stroke prone SHR (SHR-SP) induced a significant reduction in the BP. It also delayed the incidence of stroke in the SHR-SP. To investigate the action mechanism of ASX-O, the effects on PGF2α-induced contractions of rat aorta treated with NG-nitro-l-arginine methyl ester (L-NAME) were studied in vitro. ASX-O (1 to 10 μM) induced vasorelaxation mediated by nitric oxide (NO). The results suggest that the antihypertensive effect of ASX-O may be due to a NO-related mechanism. ASX-O also showed significant neuroprotective effects in ischemic mice, presumably due to its antioxidant potential. Pretreatment of the mice with ASX-O significantly shortened the latency of escaping onto the platform in the Morris water maze learning performance test. In conclusion, these results indicate that astaxanthin can exert beneficial effects in protection against hypertension and stroke and in improving memory in vascular dementia.
We previously reported that Choto-san acts as an antioxidant and cytoprotective agents against H2O2-induced oxidative damage in NG108-15 cells, and the effect is due at least partly to the phenolic compounds. To further investigate the detail mechanisms of this cytoprotection effects of Choto-san and related compounds on enzyme activities of antioxidant systems were examined. Choto-san (5—100 μg/ml) and Chotoko (5—100 mg/ml) stimulated the activity of superoxide dismutase (SOD), catalase and glutathione peroxidase (GPX). These also increased the level of glutathione. Although Choto-san without Chotoko (w/o CKO) did not show the effects on SOD and catalase, GPX activity and glutathion content also, but weakly, stimulated by w/o CKO. The effects of phenolic compounds, epicatechin, caffeic acid and quercetin were also investigated. Epicatechin stimulated catalase, GPX and glutathion content, but not SOD. On the other hand, caffeic acid stimulated SOD activity but had no effects on others. Quercetin stimulated all, although intensities were different among. These results suggest that simultaneous induction of cellular antioxidant defense systems by Choto-sam and its related constituents may be an important mechanisms underlying the protective effects of Choto-san on ischemia-induced neuronal cells injury, and the characteristics of the stimulative effects of phenolic compounds were depend on enzymes.
The anticancer effect of hydroalcoholic extract of Aegle marmelos (AME) was studied in the Ehrlich ascites carcinoma bearing Swiss albino mice. The spatial effect of various AME administration schedules showed that six-day administration increased the survival of tumor bearing mice. The best antineoplastic action of AME was obtained when AME administered through intraperitoneal route than the oral route at equimolar dose. Administration of AME once daily for six consecutive days to the tumor bearing mice caused a dose dependent remission of the tumor at 400 mg/kg body weight, where the greatest antitumor effect was observed and the higher doses showed toxic manifestations. A 24-d lengthening in life span was observed in EAC animals treated with 400 mg/kg AME. This dose of 400 mg/kg was considered as the best dose, where the animals survived up to 43 d post-tumor-cell inoculation as against no survivors in the saline treated control group. The antitumor activity when tested for different schedules for triple administrations, the best effect was observed for 1–2–3, followed by 1–3–5 and 1–5–9 days, respectively. Stage specific evaluation of AME inhibited the increase in body weight gain in animals due to tumor development during early stages only. The AME treatment resulted in a dose dependent elevation in the median survival time (MST) and average survival time (AST) up to 400 mg/kg AME and decline thereafter. The effective dose of 400 mg of AME is 1/6th of the LD50 dose, which increased the MST and AST up to 29 and 27 d, respectively. The acute toxicity study of AME showed that the drug was non-toxic up to a dose of 1750 mg/kg b. wt. The LD10 and LD50 was found to be 2000 and 2250 mg/kg.
The present study was performed to evaluate the intraocular pressure (IOP)-lowering effect of nipradilol in combination with latanoprost on ocular normotensive and hypertensive rabbits. IOP was measured using an applanation pneumatonograph under topical application of 0.4% oxybuprocaine hydrochloride for corneal anesthesia. Ocular hypertension was induced by injection of 0.1 ml hypertonic saline (5% NaCl) into the vitreous body. Saline, nipradilol, latanoprost, sodium nitroprusside (SNP) or indomethacin was then instilled just after 5% NaCl injection. All drugs were instilled in the inferior conjunctival sac, using 50 μl drops. If more than two drugs were used, they were applied 5 min apart. Nipradilol lowered IOP in both ocular normotensive rabbits and ocular hypertensive rabbits, whereas latanoprost did not lower IOP in either. When nipradilol was applied in combination with latanoprost, the reduction in ocular hypertension was significantly enhanced, compared to the effect of nipradilol alone. A significantly potent reduction in ocular hypertension was also observed by the SNP-latanoprost combination. The IOP-lowering effects of SNP in combination with latanoprost were abolished by treatment with indomethacin. These results indicate that the IOP-lowering effect of latanoprost was enhanced when applied in combination with nipradilol or SNP, both of which have nitric oxide (NO)-donating actions. Since both combined effects were abolished by treatment with indomethacin, the mechanisms by which nipradilol combined with latanoprost lowered ocular hypertension may be related, at least in part, to the production of prostaglandins via the NO-donating action of nipradilol.
The effect of radiation on tumor tissue can be optimized by adding radiosensitizing agents, in order to achieve a greater degree of tumor damage than expected from the use of either treatment alone. The ethanolic extract of Aphanamixis polystachya (APE) was tested in Swiss albino mice transplanted with Ehrlich ascites carcinoma (EAC) and exposed to various doses of γ-radiation. EAC mice received 0, 10, 25, 50, 75, 100, 150 or 200 mg/kg body wt APE before exposure to 6 Gy γ-radiation followed by once daily administration for another 8 consecutive days post-irradiation. The optimum radiosensitizing dose was found to be 50 mg/kg APE that was further tested in EAC mice exposed to 0, 1, 2, 4, 6 or 8 Gy hemi body γ-radiation. The best effect of APE and radiation was observed for 6 Gy γ-radiation. The splitting of 50 mg into two equal fractions of 25 mg and administering the split dose with a gap of 8 h on 1, 3, 5, 7 or 9 d of tumor inoculation resulted in an increased survival even when the drug was administered at late stages (day 5) of tumor development. The APE treatment before irradiation elevated lipid peroxidation followed by a reduction in the glutathione contents. Treatment of tumor bearing mice with APE before irradiation further reduced the activities of various antioxidant enzymes like glutathione peroxidase, glutathione-s-transferase, superoxide dismutase and catalase at different post last drug administration (PLDA) times.
The existence of helper-T cell (Th) subsets, types I and II (Th1/Th2), provides a framework for understanding pathological immune responses. We previously reported that a benzoimidazole derivative, M50367, acted directly on naïve Th cells to inhibit their differentiation into Th2 cells. Oral treatment with this compound reduced the Th2 response in vivo and suppressed disease progression in a murine model of atopic asthma. In this study, we investigated the effect of M50367 on 2 other murine disease models, such as atopic dermatitis and intradermal tumor-bearing mice, the pathogenesis of which may be related to the Th2 response. NC/Nga mice treated with a repeated application of picryl chloride developed atopic dermatitis-like skin lesions together with IgE hyper-production. M50367 (30 mg/kg) significantly inhibited the IgE hyper-production without affecting the skin lesions. In C57BL/6 mice bearing intradermal B16F10 melanoma, M50367 (30, 100 mg/kg) significantly inhibited splenomegaly and enhanced spontaneous interferon-γ release from cultured splenocytes in a dose-dependent manner, though its effect on tumor volume was limited. These results suggest that M50367 could reduce the Th2 response (IgE hyper-production) and enhance the Th1 response (splenocytes interferon-γ production) in these models. In contrast to previous results in the asthma model, its immunomodulation did not lead to the suppression of disease progression, indicating that the pathogenesis of these models might not simply depend on Th2 response.
The present study evaluated the effect of fluconazole on the heart, as well as and the toxic interactions between fluconazole and disopyramide in chick embryos. Chick embryos have been widely used in pharmacologic and toxicologic experiments for evaluating drug action. Fertilized eggs of White Leghorns were incubated and investigated. Fluconazole 0.4 mg/egg, 0.8 mg/egg, 1.2 mg/egg alone or disopyramide 0.3 mg/egg alone was injected into the air sac of each fertilized egg. And fluconazole 0.4 mg/egg with disopyramide 0.3 mg/egg was injected into the air sac of each fertilized egg. Electrocardiograms (ECGs) were recorded 0 to 60 min after the drug injection, and heart rate was determined from ECG wave cycles. Changes in heart rate were expressed as mean-percent changes of the drug-treated groups to the matched control. After the administration of fluconazole 0.4 mg/egg alone, the heart rate did not differ compared with that of the controls. However, the heart rate was significantly decreased with the administration of fluconazole 0.8 mg/egg and 1.2 mg/egg. The heart rate was also significantly decreased by the administration of fluconazole 0.4 mg/egg together with disopyramide 0.3 mg/egg. In addition, an arrhythmia was produced by fluconazole and disopyramide. These findings indicate that the interaction between fluconazole and disopyramide has a marked influence on the heart rate in chick embryos.
Uvaretin, isouvaretin and diuvaretin, cytotoxic C-benzylated dihydrochalcones isolated from Uvaria acuminata, displayed growth inhibitory effects against human promyelocytic leukemia HL-60 cells. We examined the mechanism of the cytotoxicity. From the morphological study by staining with Hoechst 33258, the cells treated with C-benzylated dihydrochalcones exhibited significant chromosomal condensation and nuclear degradation. The cell cycle analysis showed the accumulation of cells in the G1 phase and the appearance of a sub-G1 peak. These results suggest apoptotic cell death. Further, from the detection of DNA fragmentation and the activation of caspase-3, the biological hallmarks of apoptosis, these C-benzylated dihydrochalcones appeared to induce apoptosis against HL-60 cells. The cytotoxicity of uvaretin and diuvaretin was stronger than that of isouvaretin, which suggest that the 5′-substitution of the 2-hydroxybenzyl group increased the cytotoxicity.
In connection to developing non-steroidal estrogen analogs, the present study explores the pharmacophore of triphenylacrylonitriles (Fig. 1) for binding affinity to estrogen receptor using Electrotopological State (E-State) indices of constituting atoms. The analysis shows the efficacy of E-State index in developing statistically acceptable model, which defines the electronic environment and topological states of diverse atoms in a molecule. The investigation concluded that electrophilic substitutions at C6 and C18 of the phenyl rings (A and C rings respectively) attached to C2 and C1 of ethylenic moiety, along with presence of hydroxyl substitution at C12 (ring B) and no. of non-hydrogen free terminal atoms of the molecule have influence on the binding affinity to the estrogen receptor.
The herbal extract Yukmijihwang-tang (YMJ) has been widely used for centuries as an anti-aging herbal medicine in Asian countries. Among the various modified prescriptions of YMJ, YMJ derivatives (YMJd) were formulated to enhance memory retention. This study has three goals: 1) to quantitatively evaluate the memory-enhancing effect of YMJd using behavior tasks; 2) to use cDNA micro-array tools to identify candidate genes responsible for enhancing memory; and 3) to statistically evaluate the specific gene expression patterns using Real-time PCR. Memory retention abilities are addressed by the passive avoidance task with SD male rat. The retention time of the YMJd group was significantly delayed (ca. 100%), whereas with Ginkgo biloba and Soya lecithin treatment, this was only delayed 20% and 10%, respectively. The cDNA from the hippocampi of YMJd and rat control groups were applied to an Incyte rat GEM2 cDNA microarray. The microarray results showed that transthyretin and PEP-19 were abundantly expressed in the YMJd treated group. Importantly, PEP-19 is a neuron-specific protein that inhibits apoptotic processes. On the other hand, neuronal genes involved in neuronal death or neurodegeneration, such as pentraxin and spectrin, were abundantly expressed in the control group. The list of differentially expressed genes may provide further insight into the action and mechanism behind the memory-enhancing effect of herbal extracts of YMJd.
As an attempt to identify bioactive natural products with anti-inflammatory activity, we evaluated the effects of the methanol extract of the semen of Xanthium strumarium L. (MEXS) on lipopolysaccharide (LPS)-induced nitric oxide (NO), prostaglandin E2 (PGE2) and tumor necrosis factor-α (TNF-α) production in RAW 264.7 cells. Our data indicate that MEXS is a potent inhibitor of NO, PGE2 and TNF-α production. Consistent with these findings, the expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein and iNOS, COX-2 and TNF-α mRNA were down-regulated in a concentration-dependent manner. Furthermore, MEXS inhibited nuclear factor kappa B (NF-κB) DNA binding activity and the translocation of NF-κB to the nucleus by blocking the degradation of inhibitor of kappa B-α (IκB-α). We further evaluated the anti-inflammatory and anti-nociceptive activities of MEXS in vivo. MEXS (100, 200 mg/kg/d, p.o.) reduced acute paw edema induced by carrageenin in rats, and showed analgesic activities in an acetic acid-induced abdominal constriction test and a hot plate test in mice. Thus, our study suggests that the inhibitions of iNOS, COX-2 expression, and TNF-α release by the methanol extract of the semen of Xanthium strumarium L. are achieved by blocking NF-κB activation, and that this is also responsible for its anti-inflammatory effects.
To search for antiinflammtory 19α-hydroxyursane-type triterpenoids, the MeOH extract of the roots of Rosa rugosa (Rosaceae) was fractionated. The active fraction of the EtOAc extract was hydrolyzed in alkaline solution to give a hydrolyzed fraction. Both extracts showed antiinflammatory/antinociceptive action in acetic acid-induced writhing and hot plate testing and in a carrageenan-induced paw edema model in mice and rats. Repeated chromatography of the EtOAc extract on both silica gel and octadecylsilane columns led to the isolation of kaji-ichigoside F1 (1, euscaphic acid 28-O-glucoside) and rosamultin (2, tormentic acid 28-O-glucoside). The hydrolyzed fraction was also subjected to silica gel column and octadecylsilane column chromatography to produce euscaphic acid (3) and tormentic acid (4). The potencies were observed in the following order: 4>3>2>1. These results suggest that 19α-hydroxyursane-type triterpenoids are responsible for the antiinflammatory/antinociceptive action of R. rugosa roots.
Caulis Sinomenii is the dried plant stems of Sinomenium acutum and Sinomenium acutum var. cinereum and has been used in Chinese medicine for treating rheumatic diseases for over a thousand years. Previous studies have demonstrated that sinomenine is a major active constituent in both plants and can be utilized as an indicator of quality of the medicinal herb Caulis Sinomenii. Currently, S. acutum and S. acutum var. cinereum are growing over a wide geographical range in China, with equally wide variations in growing conditions. The objectives of this research were to determine whether there were difference between the species and varieties, and whether the different growing conditions could result in different quality by determining the content of sinomenine in different samples. A modified HPLC method using a diode array detector (DAD) has been developed for efficiently quantifying sinomenine in the plants. Using this method, fourteen samples of S. acutum var. cinereum and eleven samples of S. acutum from growing regions as well as eighteen herbal samples of Caulis Sinomenii from wholesale herbal markets were evaluated. The results showed that there was no marked difference in the content of sinomenine between the species and varieties collected from growing regions; however, a very large variation was found among the samples collected from different regions. Moreover, the content of sinomenine in the plants of large size (stem diameter>3 cm) was much higher than those of small size (stem diameter<1 cm). This implies that the growing region has greater impact on the quality of Caulis Sinomenii in terms of the content of sinomenine than the species and varieties. The results also showed that the content of sinomenine in commercial Caulis Sinomenii was markedly lower than that in the plants collected directly from growing regions. This suggests that to obtain the herb with higher content of sinomenine and thus ensure greater efficacy, both in clinical applications and in pharmacological investigations, the plant of Caulis Sinomenii with controlled stem size collected directly from growing regions is preferable.
In the theory of traditional Chinese medicine (TCM), eqi (衛気) circulates at the superficial portion of the body to guard against exopathogen. Gyokuheifusan (GHS; 玉屏風散), containing Astragalus Root, Atractylodes Rhizome, and Saposhnikovia Root, is a TCM formula to treat the insufficiency of eqi by invigorating qi and consolidating the superficial resistance. In this study, we evaluated the effect of GHS on murine antibody production against ovalbumin (OVA) used as exopathogen. Balb/c mice were sensitized with OVA and alum via intraperitoneal (i.p.) injection or intranasal (i.n.) infusion daily for 7 d. GHS was orally administered daily at the dose of 10-times amount of human daily dosage from 3 d before the sensitization for 14 d. Fourteen d after the final sensitization, the blood was collected, and the concentrations of OVA-specific or non-specific immunoglobulins were measured. When OVA was sensitized i.p., the concentration of OVA-specific IgG, IgG1, IgG2a and IgA in the sera significantly increased by GHS-treatment. When OVA was sensitized i.n., GHS significantly reduce the concentration of OVA-specific IgG and IgG1 in the sera. Non-specific immunoglobulins were not changed by GHS-treatment. It is suggested that GHS could stimulate immune responses when antigen had already been invaded into the inside of the body, and that GHS might consolidate the resistance of nasal mucosa to protect from the invasion of OVA, then OVA-specific antibodies in sera might be hypocritically suppressed. The present study might provide the experimental evidence for TCM theory.
Twenty-eight 3-hydroxy triterpenoids of taraxastane- (1—7), oleanane- (8—12), ursane- (13—15), lupane- (16,18,19), taraxane- (20), cycloartane- (21—25), tirucallane- (26—28), and dammarane-types (29) isolated from the non-saponifiable lipid fraction of the flower extract of chrysanthemum (Chrysanthemum morifolium) and one lupane-type 3α-hydroxy triterpenoid (17) derived from 16 were tested for their antitubercular activity against Mycobacterium tuberculosis strain H37Rv using the Microplate Alamar Blue Assay (MABA). Fifteen compounds showed a minimum inhibitory concentration (MIC) in the range of 4—64 μg/ml, among which maniladiol (9; MIC 4 μg/ml), 3-epilupeol (17; 4 μg/ml), and 4,5α-epoxyhelianol (27; 6 μg/ml) exhibited the highest activity. Cytotoxicity of compound 17 against Vero cells gave an IC50 value of over 62.5 μg/ml, suggesting some degree of selectivity for M. tuberculosis.
Two known saponins, licorice-saponin H2 and macedonoside A, were isolated from the stolons of Glycyrrhiza lepidota (American licorice) as major saponins. Since licorice-saponin H2 and macedonoside A are minor saponins isolated from the three glycyrrhizin-producing species (i.e. G. glabra, G. uralensis, G. inflata) and the three macedonoside C-producing species (i.e. G. macedonica, G. echinata, G. pallidiflora), respectively, the present study suggests that G. lepidota is an intermediate of both glycyrrhizin-producing and macedonoside C-producing species. The phylogenetic tree constructed from the nucleotide sequences of ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit gene (rbcL) of these seven Glycyrrhiza plants indicated that G. lepidota was separated from the other six Glycyrrhiza species, and this phylogenetic relationship was in accordance with their saponin compositions.
Sho-saiko-to, one of the the most frequently prescribed Kampo medicines, is used to treat chronic hepatitis and has shown confirmed clinical efficacy. The present study was performed with respect to heme metabolism to study the preventive effects of Sho-saiko-to against endotoxemia. Endotoxin was injected intraperitoneally at a dose of 6 mg/kg into Sho-saiko-to (500 mg/kg/d, p.o.)-pretreated rats, and its administration clearly prevented the endotoxin-induced hypoferremia. In rats pretreated with Sho-saiko-to, the activity of hepatic δ-aminolevulinate synthetase and cytochrome P-450 level 18 h after endotoxin injection were significantly increased as compared to rats treated with endotoxin alone. Similarly, Sho-saiko-to significantly depressed the endotoxin-induced increase in heme oxygenase activity in liver microsomes. These findings suggested that the extent of shock syndrome caused by endotoxin may be due, at least in part, to changes in heme metabolic disturbance during endotoxemia. Sho-saiko-to may therefore protect rats against lethality caused by endotoxin through its ability to regulate the heme metabolism in septic shock.
The aim of this study was to determine whether sesamin, a component from Acanthopanax senticosus HARMS (ASH) pharmacologically offers protection against Parkinson's disease (PD) and its related depressive behavior in rats administered rotenone. We also examined how sesamin affected the rotenone-induced loss of tyrosine hydroxylase (TH) or glial cell line-derived neurotrophic factor (GDNF)-positive neurons in the midbrain of rats. Rats were orally administered sesamin (3, 30 mg/kg) once a day for 2 weeks before an intraperitoneal injection of rotenone (2.5 mg/kg). The pole test and catalepsy test were used to evaluate the effects of sesamin administration on bradykinesia and depressive behaviors in the PD model of rats given rotenone for 5 weeks. Those effects were compared with the ASH administrated group (250 mg/kg). Treatment with sesamin for seven weeks resulted in prophylactic effects on rotenone-induced parkinsonian bradykinesia and catalepsy, and the effects were equivalent to ASH effects. Immunohistochemistical analysis using TH or GDNF antibody showed that sesamin provided cytoprotective effects against rotenone-induced loss of DA cells. The results suggest that it may be possible to use the ASH and sesamin for the prevention of nigral degenerative disorders, e.g., PD with depression, caused by exposure to pesticide or environmental neurotoxins in general.
The antiproliferative constituents in the MeOH extract from the aerial parts of Centella asiatica were investigated. Activity-guided fractionation of MeOH extract resulted in the isolation of ursolic acid lactone, ursolic acid, pomolic acid, 2α,3α-dihydroxyurs-12-en-28-oic acid, 3-epimaslinic acid, asiatic acid, corosolic acid, and rosmarinic acid. Antiproliferative activity of the isolated compounds against human gastric adenocarcinoma (MK-1), human uterine carcinoma (HeLa), and murine melanoma (B16F10) cells was estimated.
The ethanol extract of Chinese medicinal ants Polyrhachis lamelliden was evaluated for its analgesic and anti-inflammatory activities in mice. It was shown that the extract significantly inhibited acetic acid-induced writhing response and increased hot-plate pain threshold of mice at doses of 1.5 and 3.0 g crude drug/kg. Meanwhile, the extract significantly inhibited the increase in vascular permeability induced by acetic acid and in ear edema induced by xylene in mice. However, it had no obvious effect on leukocyte migration induced by carboxymethylcellulose sodium (CMC-Na). The ethanol extract suspended in water was partitioned with diethyl ether, ethyl acetate and n-butanol successively to yield four fractions including water fraction. Among these fractions, diethyl ether and ethyl acetate fractions were found to increase hot-plate pain threshold and to inhibit acetic acid-induced writhing response in mice. Water fractions markedly inhibited acetic acid-induced writhing response and reduced the dye leakage to the peritoneal cavity induced by acetic acid and ear edema induced by xylene. These results suggest that P. lamellidens presents remarkable analgesic and anti-inflammatory activity, which supported the traditional use of the medicinal ants in the treatment of various diseases associated with inflammation. The diethyl ether fraction has greater contribution to the overall analgesic activity, whereas the water fraction showed the greatest anti-inflammatory and peripheral analgesic activities.
To characterize the intestinal absorption of digoxin, its transcellular transport and drug interaction activity was investigated using Caco-2 cell monolayers. We examined digoxin transport in the presence and absence of ouabain to determine whether digoxin binding to Na+,K+-ATPase affects its transcellular digoxin transport, and evaluated its influx and efflux clearance by model-dependent pharmacokinetic analysis. Transcellular transport in the basal-to-apical direction was greater than that in the opposite direction. In addition, ouabain decreased the cellular accumulation of digoxin, but it did not alter its transcellular transport profile. The observations for transcellular transport and cellular accumulation in the presence of ouabain were used for the pharmacokinetic analysis, which showed that the efflux clearance of digoxin on the apical side of the monolayer was 15 times greater than that on the basal side. Apical-to-basal transport was increased by carvedilol and pimobendan, and these compounds suppressed the efflux clearance on the apical side and the influx clearance on the basal side. These findings indicate that the intestinal absorption of digoxin is primarily dominated by the efflux process on the luminal side of the intestine, and that carvedilol and pimobendan may vary the rate of intestinal digoxin absorption mainly by inhibiting its exsorptive transport.
We investigated contamination of environmental surfaces by Staphylococcus aureus from April 1 to the end of June in 2002 in the dermatological ward (37 beds) of a university hospital. For surfaces contaminated by high levels of S. aureus, disinfection methods were evaluated. 100—105 colony forming units (cfu) of methicillin-resistant S. aureus (MRSA) or methicillin-sensitive S. aureus (MSSA) were detected on items such as an immersion bathtub (examined area, about 900 cm2), foot washbowl, stretcher for an immersion bath, and chair for the shower. After disinfection, no S. aureus was detected on smooth surfaces such as the immersion bathtub and foot washbowl; however, S. aureus was detected even after disinfection on porous surfaces made of sponge-like materials (polyethylene foam) such as the stretcher for the immersion bath and the shower chair. Scanning electron microscopy of the porous surfaces showed formation of a large amount of coccus and bacillus biofilms on the walls of pores in the multi-pore structure. Material that is porous should not be used in patient care settings because it is not possible to disinfect it properly.
FK228 (FR901228, depsipeptide) is a potent histone deacetylase inhibitor currently in phase II clinical trials for cancer treatment. In the present study, the cytochrome P450 (P450) enzymes responsible for FK228 metabolism in human liver microsomes were investigated. Incubation with human liver microsomes in the presence of an NADPH-generating system revealed that FK228 is metabolized to at least 10 different metabolites. Km and Vmax values for FK228 disappearance were 20.3 μM and 561.9 pmol/min/mg protein, respectively. Further studies were performed at a substrate concentration of 10 μM (half the Km value for FK228 disappearance). FK228 disappearance activities in human liver microsomes from 12 individuals strongly correlated (r2=0.957) with testosterone 6β-hydroxylase activities, a marker enzyme activity of CYP3A4/5, but not with other P450 enzyme-specific activities (CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6, and 4A). Among 14 recombinant heterologously expressed human P450s examined, CYP3A4 exhibited the highest activity of FK228 disappearance. CYP3A5, 1A1, 2B6, and 2C19 showed 16.8%, 5.2%, 1.6%, and 1.3% of the activity of CYP3A4, respectively. Other P450s showed no significant metabolic activity toward FK228. In addition, FK228 disappearance in human liver microsomes was markedly inhibited by ketoconazole, a potent CYP3A4 inhibitor, and an anti-CYP3A4 antibody. These results indicate that the metabolism of FK228 in human liver microsomes is catalyzed mainly by CYP3A enzymes, particularly CYP3A4.
Ritonavir (RTV) is well known as an inhibitor of many drugs that are metabolized by cytochrome P450 (CYP) 3A or fluxed via P-glycoprotein (Pgp), although it is also reported that RTV is a potent inducer for them. In this study, to elucidate these contradictory phenomena, functional changes of CYP3A or Pgp during chronic administration of RTV were examined in rats. After pretreatment with RTV for indicated days (day 3—day 14), rats were used in the experiments. The area under the plasma drug concentration vs. time curve (AUC0—∞) after oral administration of RTV (20 mg/kg) to these rats showed an RTV-treatment period-dependent decrease, and the mean AUC0—∞ of RTV in Day 14 rats decreased significantly by 57% as compared to the control. The AUC0—∞ after intravenous (i.v.) administration of RTV to Day 3 and Day 5 rats increased significantly by 28% and 22%, respectively, while there were no significant changes in the AUC0—∞ in Day 7 and Day 14 rats as compared to the control. As for i.v. administration of erythromycin (EM) or midazolam (MDZ) to RTV-treated rats, the AUC0—∞in Day 3 and Day 5 rats increased significantly as compared to the control, while in Day 7 rats and rifampicin-treated rats, the AUC0—∞ of EM decreased significantly by 82% and 42%, respectively, as compared to the control. For MDZ, there were no significant changes in the AUC0—∞ in Day 7 or Day 14 rats. After i.v. administration of rhodamine123 (Rho123), the excretion clearances from blood circulation to the intestinal lumen and the biliary excretion clearances in Day 14 rats increased markedly by 2.2-fold and 2.6-fold as compared to the control. It has been confirmed that RTV is not only a potent inhibitor but also a potent inducer of CYP3A, and that RTV is a potent inducer of intestinal Pgp. This property of RTV is responsible for regulating the oral bioavailability of drugs that are mediated by CYP3A and Pgp.
The popularity of traditional herbal medicine (THM) being used as complementary medicines or alternative medicines is increasing. On the other hand, the development of multidrug resistance (MDR) remains a major hurdle to successful cancer chemotherapy. Some THMs capable of reversing MDR may contribute to the improvement of clinical outcomes in cancer chemotherapy. Herein, 19 kinds of herb were chosen from the ingredients of major THMs, and their effects on the sensitivity to anticancer drugs of tumor cells were investigated using the human cervical carcinoma HeLa cells. Focusing on the major mechanism for MDR, i.e., MDR1/P-glycoprotein, the effects of herbal extracts on its transport function were also examined using a MDR1 substrate Rhodamine123. Glycyrrhizae Radix, Rhei Rhizoma, Scutellariae Radix, Poria, Zizyphi Fructus, Zingiberis Rhizoma (dry), Coptidis Rhizoma, Ephedrae Herba and Asiasari Radix significantly enhanced the sensitivity to a MDR1 substrate paclitaxel, whereas none of the herbal extracts used had any effect on the sensitivity to 5-fluorouracil, which is not a substrate for MDR1. Rhodamine123 uptake was significantly increased by Rhei Rhizoma, Poria or Ephedrae Herba among nine herbal extracts sensitized to paclitaxel. This suggests that the increase in paclitaxel sensitivity by Glycyrrhizae Radix, Rhei Rhizoma, Poria or Ephedrae Herba was caused, in part, by the inhibition of MDR1 function, and the change in paclitaxel sensitivity by the other herbal extracts was not always dependent on this. Collectively, these findings indicate that the combination of anticancer drugs with some herbal extracts contributes to the enhancement of clinical outcomes in cancer chemotherapy.
Changes in the mRNA expression of hepatic cytochrome P450 (CYP) isoenzymes associated with overdose of fat-free or fat-containing total parenteral nutrition (TPN) were investigated in infant rats. Three-week-old male Sprague–Dawley rats were divided into three groups: group 1 received an oral diet, group 2 received TPN without fat, and group 3 received TPN with 20% of calories from fat (soybean oil emulsion). After TPN administration for 4 d, serum aspartate aminotransferase (AST) levels in group 2 were significantly increased (p<0.01) compared with the other groups. The mRNA expression of hepatic CYP isoenzymes in group 2 decreased to 0.76 to 31% of that in group 1 (p<0.01), but that in group 3 was maintained at 32 to 84% of that in group 1. These results indicate the importance of including fat in TPN regimens to prevent not only hepatic dysfunction but also mRNA down-regulation of liver CYP isoenzymes.
We developed a gene transfer following the administration of naked plasmid DNA (pDNA) to the kidney surface in mice, and found that the luciferase levels produced in the applied kidney were significantly higher than those produced in another kidney. In contrast, stable renal gene expression was not observed in the case of intraperitoneal or intravenous administration of pDNA. The level of gene expression after instillation of pDNA to the kidney surface reached maximum at 12 h and gradually diminished thereafter. The production of luciferase was saturated at 5 μg of pDNA, and was not affected by instillation volume. Furthermore, pDNA uptake from the kidney surface was proved by in situ experiments using a glass-made diffusion cell. We demonstrated a novel unilateral kidney-selective gene transfer following the administration of naked pDNA to the kidney surface in mice.
Acetohexamide (AH) is reduced to its alcohol metabolite by carbonyl reductase. We have previously shown that carbonyl reductase present in the liver microsomes of rats is a male-specific and androgen-dependent enzyme. In the present study, the role of microsomal carbonyl reductase in the pharmacokinetics of AH was examined in male Wistar–Imamichi (WI) and Sprague–Dawley (SD) rats after its intravenous administration. AH was eliminated more slowly from plasma in the WI strain, which lacks most of the microsomal carbonyl reductase, than in the SD strain. Furthermore, several pharmacokinetic parameters were derived from the data for the plasma concentrations of AH. The plasma clearance (CLp) of AH (72.8±11.2 ml/h/kg) in male WI rats was significantly smaller than that (105.5±11.1 ml/h/kg) in male SD rats. Testectomy caused a marked decrease, from 105.5±11.1 to 44.3±11.8 ml/h/kg, in the CLp of AH in male SD rats. These results indicate that microsomal carbonyl reductase plays a critical role in the differential pharmacokinetics of AH in male WI and SD rats.
The effects of sulpiride, an antipsychotic drug, on cytochrome P450 (CYP) activities in human liver microsomes were investigated. Sulpiride at 50 or 500 μM concentration neither inhibited nor stimulated CYP1A2-mediated 7-ethoxyresorufin O-deethylation, CYP2C9-mediated tolbutamide hydroxylation, CYP2C19-mediated S-mephenytoin 4′-hydroxylation, CYP2D6-mediated debrisoquine 4-hydroxylation, CYP2E1-mediated chlorzoxazone 6-hydroxylation, CYP3A4-mediated nifedipine oxidation, or CYP3A4-mediated testosterone 6β-hydroxylation. The free fractions of sulpiride in the incubation mixture estimated by ultracentrifugation were more than 90.5%. These results suggest that sulpiride would not cause clinically significant interactions with other drugs, which are metabolized by CYPs, via the inhibition of metabolism.
We reported previously that fusogenic liposome (FL) introduced antigen protein encapsulated in the liposome directly into the cytoplasm of the antigen presenting cells, and that it induced immune responses. In the present study, we encapsulated TAX38-46, an HTLV-I derived protein and an antigen peptide model, into FL. The ability to induce effective cytotoxic T lymphocytes (CTL) responses in immunized mice was evaluated. Results showed FL could induce CTL response effectively and suggested that FL is a potential peptide vaccine carrier.