Tumor angiogenesis affords new targets for cancer therapy, since inhibition of angiogenesis suppresses tumor growth by cutting out the supply of oxygen and nutrients. Anti-angiogenic therapy is thought to be free of the severe side effects that are usually seen with cytotoxic anticancer drugs. Furthermore, anti-angiogenic therapy is thought not only to eradicate primary tumor tissues, but also to suppress tumor metastases. However, it is uncertain whether this therapy causes tumor regression because it inhibits only angiogenic events. Recently, a novel anti-angiogenic therapy called anti-neovascular therapy (ANET) has become notable. This therapy inflicts indirect lethal damage on tumor cells by damaging newly formed blood vessels using anti-cancer drugs targeting the angiogenic vasculature, since cytotoxic anti-cancer drugs cause damage to proliferating neovascular endothelial cells as well as tumor cells. Moreover, neovascular endothelial cells would not be expected to acquire drug-resistance. Traditional chemotherapy, which directly targets tumor cells, has potential problems such as low specificity and severe side effects. On the contrary, in ANET, severe side effects may be suppressed, since traditional anti-cancer agents are delivered to the neovessels by DDS technology. Besides the usage of DDS technology, anti-neovascular scheduling of chemotherapy, or metronomic-dosing chemotherapy, has also been attempted in which anti-cancer drugs are administered on a schedule to damage neovessels. In this review, we describe traditional anti-angiogenic therapy and ANET. We also discuss anti-angiogenic cancer photodynamic therapy (PDT), since PDT is clinically applied to treat age-related macular degeneration (AMD), in which uncontrolled angiogenesis occurs.
Immediate early genes (IEG) are rapidly but transiently induced directly by intracellular signaling cascades to alter patterns of gene expression. It has been proposed that histone modifications could be the key to the quick alteration of chromatin structure, as this spread occurs too rapidly to be the consequence of passage of RNA polymerase II. In this review, we will discuss the different modifications on histones and the chromatin remodeling enzymes, allowing the promoter regions of two IEGs, c-fos and c-jun, to be accessed.
To establish a method for quantitative analysis of HM-1 killer toxin (HM-1), two purified mouse monoclonal antibodies, 1F1 and 4A2, and rabbit polyclonal antiserum against HM-1 were prepared. Both monoclonal antibodies were classified as IgG1(κ) subtype, and did not neutralize the killing activity of HM-1. By SPOTs analysis, the epitope of 1F1 was found in the sequence of CDPNTG with a corresponding sequence of 11—16 from N-terminal amino acid residues of HM-1, but the epitope of 4A2 was not determined. Using 4A2 and polyclonal antiserum, the sandwich enzyme-linked immunosorbent assay (ELISA) was applied to establish the quantitative determination of HM-1. The concentration of HM-1 was determined successfully at the range of 2.5—100 ng/ml. But in the case of 1F1, the method was not established. Genes were constructed to apply the system to the measurement of the secreted concentrations of mutant HM-1, and it was evident that the production of mutant toxins varied among HM-1 mutant genes. The findings of this study are unique in determinimg the epitope of monoclonal antibody against HM-1, and in quantifying the HM-1 using the spot analysis and sandwich ELISA methods.
The effects of dantrolene were investigated on carbonic anhydrase (CA) enzyme activities in in vitro human and in in vivo Sprague-Dawley rat erythrocytes. For in vitro study, human carbonic anhydrase-I (HCA-I) and -II (HCA-II) were purified by Sepharose 4B–L-tyrosine-sulfanylamide affinity chromatography, rats were used for in vivo study. In vivo and in vitro CA enzyme activity was determined colorimetrically using the CO2-hydration method of Wilbur and Anderson. Dantrolene (1.64×10−5—6.56×10−5 M) showed in vitro inhibitory effects on HCA-I and HCA-II hydratase activity, when determined using the CO2-hydratase method. 50% inhibitory concentration (IC50) was 4.09×10−5 M for HCA-I and 3.24×10−5 M for HCA-II. Rat erythrocyte CA activity was significantly inhibited by 10 mg/kg dantrolene for up to 3 h (p<0.001) following intraperitoneal administration. In conclusion, Dantrolene inhibited the carbonic anhydrase enzyme activity under in vitro and in vivo conditions.
Lipopolysaccharide (LPS)-stimulated macrophages produce large amounts of nitric oxide (NO) by inducible nitric oxide synthase (iNOS). This is an important mechanism in macrophage-induced septic shock and inflammation. In the present study, we tested a synthetic propenone compound, 1-furan-2-yl-3-pyridin-2-yl-propenone (FPP-3) for its ability to inhibit the production of tumor necrosis factor-α (TNF-α) and an inducible enzyme, iNOS, in the LPS-stimulated murine macrophage-like cell line, RAW264.7. FPP-3 consistently inhibited nitric oxide (NO) and TNF-α production in a dose dependent manner, with IC50 values of 10.0 and 13.1 μM, respectively. Western blotting probed with specific anti-iNOS antibodies showed that the decrease in quantity of the NO product was accompanied by a decrease in the iNOS protein level. In cells transiently transfected with nuclear factor (NF)-κB promoter-luciferase reporter construct, this compound clearly inhibited the LPS-stimulated NF-κB activation. Moreover, this compound inhibited IκB-α degradation in a concentration and time-dependent manner. These results indicate that FPP-3 inhibits NO production via inhibition of degradation of IκB-α through NF-κB activation.
A 2-substituted purine nucleotide analog, 2-hydroxy-2′-deoxyadenosine 5′-triphosphate (2-OH-dATP), was added to a PCR mixture, to examine its mutagenic potential. The 2-OH-dATP enhanced the total mutation frequency. Interestingly, 2-OH-dATP induced both transition and transversion mutations, including A:T→G:C, A:T→C:G and G:C→T:A mutations. In contrast, other 2-substituted purine nucleotide analogs, 2-aminopurine-2′-deoxyriboside 5′-triphosphate and 2-amino-2′-deoxyadenosine 5′-triphosphate, did not affect the total mutation frequency. These results suggest that 2-OH-dATP is useful in random PCR mutagenesis for the in vitro evolution of nucleic acids and proteins, and for analyses of residues in these biomolecules.
We previously demonstrated that the in vitro maturation of mouse immature bone marrow-derived mast cells into a mature connective tissue mast cell-like phenotype is accompanied by a marked induction of N-myc downregulated gene (NDRG) 1, a cytosolic protein with unknown function. Here we show that NDRG1 undergoes phosphorylation in mast cells. Recombinant NDRG1 was phosphorylated by calmodulin kinase-II, protein kinase (PK) A and PKC in vitro. Deletion of the C-terminal tandem repeats of NDRG1 resulted in increased phosphorylation by PKA and PKC, but not by calmodulin kinase-II. Furthermore, NDRG1 was phosphorylated on serine and threonine residues in mast cells, a process that was accelerated transiently following cell activation. Pharmacologic studies using kinase-specific inhibitors demonstrated that this NDRG1 phosphorylation in mast cells depended on calmodulin kinase-II and PKA, but not PKC. Collectively, our results indicate that NDRG1 is a multiphosphorylated protein in mast cells, and that the kinetics of increased NDRG1 phosphorylation parallels signaling events leading to exocytosis.
N-Myc downregulated gene (NDRG) 1 is markedly induced during in vitro maturation of mouse immature bone marrow-derived mast cells (BMMCs) into a mature connective tissue mast cell (CTMC)-like phenotype. However, cellular function of this unique cytosolic protein is currently obscure. In this study, we sought potential NDRG1-binding proteins using yeast two-hybrid analysis and found that NDRG1 is capable of binding to heat-shock cognate protein 70 (Hsc70) both in vitro and in mast cells. The expression of Hsc70 was markedly elevated during the in vitro maturation of BMMCs into CTMC-like cells in accordance with the increased expression of NDRG1. Deletion of the C-terminal hydrophilic tandem repeats from NDRG1 facilitated the interaction with Hsc70 in vitro. Interaction between NDRG1 and Hsc70 was constitutive in mast cells and was not altered following cell activation. Although NDRG1 undergoes phosphorylation (accompanying paper), the binding of NDRG1 to Hsc70 was not affected by this event. Interestingly, the NDRG1-Hsc70 complex transiently appeared in the nuclear fraction of activated mast cells.
The aim of this work was to investigate the effects of Aloe vera leaf pulp and gel extracts on the liver tissue of neonatal streptozotocin (n0STZ)-induced type-II diabetic rats. The diabetic rats were separated into four groups and each group was given the following samples by gavage, daily for 15 d: phosphate buffered saline (PBS; diabetic control), Aloe leaf pulp extract, Aloe leaf gel extract, glibenclamide. Liver tissues were examined histologically. The markers of oxidative stress: glutathione (GSH), non-enzymatic glycosylation (NEG) and lipid peroxidation (LPO), were determined in liver tissue. Biochemical parameters for liver function: serum alkaline phosphatase (ALP), and alanine transaminase (ALP) activities, were evaluated. All parameters were also determined in healthy (non diabetic) rats for comparison. In the diabetic control group, the degenerative changes in liver tissue were remarkable, while in the diabetic groups given Aloe pulp and gel extracts and glibenclamide, the damage to the liver tissue was decreased. The increase of GSH and the decrease of NEG and LPO in liver tissues with the treatment of Aloe gel extract, is consistent with the beneficial effect of Aloe. Serum ALP and ALT activities were also decreased in the groups given Aloe gel extract. It was concluded that Aloe gel extract has a protective effect comparable to glibenclamide against hepatotoxicity produced by diabetes if used in the treatment of type-II diabetes.
Girolline, an antitumor compound isolated from a sponge, has been reported to inhibit the termination step of protein synthesis in vivo. In this study, we found that girolline induced G2/M cell cycle arrest in several tumor cell lines. Immunochemical analysis revealed that polyubiquitinated p53 was accumulated in girolline-treated cells, while other polyubiquitinated cellular proteins were not accumulated, indicating that the effect of girolline is specific for p53. On the other hand, girolline did not inhibit proteasome activity in vitro, and accumulation of polyubiquitinated p53 was scarcely detected in the presence of leptomycin B, an inhibitor of nuclear export. Based on the above findings, we propose that girolline affects the step of recruitment of polyubiquitinated p53 to the proteasome.
Pulmonary fibrosis is a common consequence of numerous pulmonary diseases. The current therapeutic approaches for this condition are unsatisfactory. Feitai, a composite formula consisting of several herbs, is used in China as a folk remedy for treating patients with pulmonary tuberculosis. In this study, we extensively investigate the effects and mechanisms of Feitai on bleomycin (BLM)-induced pulmonary fibrosis in rats. One hundred and twenty male Sprague–Dawley rats were randomly divided into four groups, referred to as the saline–water, saline–Feitai, BLM–water, and BLM–Feitai groups. Following a single instillation of BLM (5 mg/kg) or saline, rats were orally administered Feitai at a dose of 3 g/kg body weight or sterilized distilled water once daily. Rats were killed at 7, 14, or 28 d post-BLM. Inflammatory cell count, protein concentration, and lactate dehydrogenase activity in bronchoalveolar lavage fluid were measured, and myeloperoxidase activity and lipid peroxide content in lung homogenates were analyzed. Treatment with Feitai inhibited lung fibrotic progression induced by BLM, as indicated by the decrease in lung hydroproline content and lung fibrosis score at 28 d post-BLM. This was accompanied by significant amelioration of BLM-induced body weight loss, lung edema, and inflammatory response during the development of lung injury in the acute phase. The results strongly indicate the beneficial effects of Feitai in protecting against BLM-induced pulmonary fibrosis. Furthermore, the inflammatory response and lipid peroxidation were inhibited by Feitai, suggesting that the effect of this formula on BLM-induced lung injury and fibrosis is associated with antiinflammatory and antioxidant properties.
DW-286a is a new class of fluoronaphthyridone antibiotic. Its effect on the central nervous system, general behavior, and cardiovascular, respiratory, and other organ systems were studied. The doses given were 30, 50, 100, 150, 300, and 1000 mg/kg and drugs were administered orally. DW-286a did not show any effects on general behavior, motor coordination, analgesic action, seizure and mortality, blood pressure and heart rate, contractile response of isolated guinea pig ileums, and renal function. On the other hand, DW-286a decreased spontaneous locomotor activity from 120 to 240 min after administration at the doses of 300 and 1000 mg/kg and the respiration rate from 30 to 240 min after the administration of doses up to 100 mg/kg. The sleeping time and body temperature were increased significantly in mice at the dose of 1000 mg/kg. DW-286a increased the charcoal transport significantly at doses of 300 and 1000 mg/kg. DW-286a inhibited gastric acid secretion, reduced the volume of the gastric juice and total acidity, and increased the pH dose dependently. Based on the above results, it was concluded that DW-286a affects spontaneous locomotor activity, respiration and body temperature, gastrointestinal transport, gastric secretory action, and hexobarbital sleeping time at high doses.
Based on a previous report illustrating severe gastrointestinal side effects with ipriflavone, we examined the effects of ipriflavone on cell death in cultured rat gastric epithelial cells compared with other chemicals, including indomethacin. Low concentrations of ipriflavone, indomethacin, dexamethasone, and estradiol all induced cell death in gastric epithelial cells. DNA from cultured cells treated with ipriflavone showed fragmentation by electrophoresis. Also, some of the cultured cells treated with ipriflavone were positively stained by TUNEL. In order to confirm induction of apoptosis by ipriflavone, rescue from ipriflavone-induced cell death with Z-DEVD-FMK (0.02—0.1 mM), which is a caspase 3 inhibitor, or PGE2 (0.01—10 mM), was tested. Only Z-DEVD-FMK was observed to rescue the cells from cell death. Similar results were obtained with indomethacin, dexamethasone and estradiol. These results suggest that ipriflavone, indomethacin, dexamethasone and estradiol induce cell death of cultured rat gastric epithelial cells by apoptosis.
Antioxidant and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities, reducing powers and the amount of total phenolic compounds of aqueous and/or methanolic extracts of some medicinal plants used in Eastern Turkey were studied. These plants are Prangos ferulacea (ÇAŞIR), Sedum sempervivoides (HOROZ LELESI), Malva neglecta (EBEMGÜMECI), Cruciata taurica (SARILIK OTU), Rosa pimpinellifolia (KOYUN GÓZÜ), Galium verum subsp. verum (MADAVUR OTU), Urtica dioica (ISIRGAN). The highest peroxidation inhibitions were shown by aqueous extracts of C. taurica and R. pimpinellifolia (IC50: 0.00022 mg/l and IC50: 23 mg/l, respectively). However, the highest DPPH radical scavenging activity, reducing power and the amount of phenolic compounds were shown by R. pimpinellifolia. The lowest antioxidant properties were shown by aqueous extract of M. neglecta.
The objective of this study was to determine pharmacokinetic differences of acetyl salicylic acid (ASA) and its metabolites: gentisic acid (GA), salicylic acid (SA) and salicyluric acid (SUA) between Otomies and Mesticians healthy subjects. Design. Ten Otomies and 10 Mesticians were included. After a single dose of aspirin given orally (15 mg/kg), blood and urine samples were collected at different times. Results. Pharmacokinetic parameters of salicylates showed significant differences, except distribution volume of SA, and elimination half-life of SUA. Metabolic rates of ASA showed significant differences for all rates between both groups. On the other hand, percentages of dose excreted were more reduced for SA and SUA for the Otomies than for the Mesticians. Conclusion. Results reflect differences in the hydrolysis way i.e. from ASA to SA and aromatic hydroxylation i.e. from SA to GA, which were slower in Otomies subjects, showing a possible pharmacokinetic differences about the capabilities of ASA biotransformation as a consequence of ethnic differences.
The aim of the present study was to determine the antimicrobial and cytotoxic activities of eight novel titanium(III) based coordination complexes [Ti(Pht)2(DL-serine)2, S1], [Ti(Pht)2(glycine)2, S2)], [Ti(Pht)2(cystine)2, S3], [Ti(Pht)2(DL-leucine)2, S4], [Ti(Suc)2(L-leucine)2, S5], [Ti(Suc)2(cystine)2, S6], [Ti(Suc)2(cystein)2, S7] and [Ti(Suc)2(DL-serine)2, S8] against several gram-positive and -negative bacteria, fungi and brine shrimp nauplii. The investigation showed that almost all of the complexes were moderately active against tested bacteria and fungi at high concentration (200 μg/disc) compared with the standard antibiotic, amoxicillin and the antifungal agent, nystatin. In vivo lethality bioassay experiment showed that only S7 and S8 among the complexes had better cytotoxic effect than standard gallic acid. The LC50 values of these two complexes were found to be 1.00 and 1.21 μg/ml, respectively. Thus the results suggest that only two complexes (S7, S8) among the titanium(III) based coordination complexes show the anticancer properties comparable to the standard cytotoxic agent, and further studies of these two complexes may be helpful for their clinical implication.
A large group of flavonoids was investigated for inhibitory effects on sulfo- and glucurono-conjugation of acetaminophen when added to rat cultured hepatocytes and liver subcellular preparations. The flavonoids inhibited the production of both sulfate and glucuronide conjugates in the cultured cells, with potencies that depended on the specific flavonoid. Among the flavonols, quercetin, kaempferol and galangin were much more effective than myricetin and morin. Flavones including luteolin, apigenin and chrysin were as effective as the corresponding three flavonols above. The inhibition of conjugation by other simple flavones such as 3-, 5-, 7- and 3′,4′-OH flavones, and by catechins such as epicatechin and epigallocatechin, was very weak. These data suggest that the presence of both C5 and 7 hydroxyl substitutions on the A-ring in the flavone structure is required for effective inhibitory activity. The effect of flavonoids on sulfo- and glucurono-conjugation was also examined by incubating acetaminophen with isolated liver cytosolic and microsomal preparations, respectively. The active flavonoids in the cells remarkably inhibited the sulfation, but not glucuronidation, in cell-free enzymatic preparations in vitro. The mechanism of inhibition of conjugation by flavonoids in cultured hepatocytes is not likely to depend on the direct inhibition of sulfo- and glucurono-transferase activity by flavonoids.
The pharmacological properties of SWR-0315NA, (E,Z)-[4[[1-[2-[(3-phenoxy-2-hydroxy propyl)]amino]ethyl]-1-propenyl]phenoxy]acetic acid sodium, were compared with those of (−)-isoproterenol. In the radioligand binding studies of [125I]iodocyanopindolol with COS-7 cell membranes that transiently expressed human β-adrenoceptor (β-AR) subtypes, SWR-0315NA exhibited 1-fold and 2-fold greater binding affinities for β3-AR than those for β1- and β2-ARs, respectively. The maximal stimulatory effects of SWR-0315NA on cAMP accumulation in CHO cells expressing all the β-AR subtypes were 79%, 3% and 93% for β1-, β2- and β3-ARs of those produced by (−)-isoproterenol, respectively. SWR-0315NA has 26.3-fold and more than 630-fold greater selectivity for β3-AR than those for β1- and β2-ARs in potency, respectively. These results indicate that although SWR-0315NA has lower binding selectivity towards β-AR subtypes, it is a selective agonist with high intrinsic activity for β3-AR as compared with (−)-isoproterenol.
Oxidative stress caused by an elevation in reactive oxygen species (ROS) plays an important role in Alzheimer's disease and other neurodegenerative diseases. In this study, we examined the neuroprotective effect of danthron (1,8-dihydroxyanthraquinone) against neurotoxicities induced by β-amyloid (25—35), excitotoxins, apoptosis, and oxidative stress in primary cortical cultures. Danthron dose-dependently reduced neuronal injury induced by 30 μM β-amyloid (25—35). Danthron significantly inhibited oxidative injury induced by 100 μM Fe3+ and decreased membrane lipid peroxidation induced by 100 μM Fe3+ as measured by thiobarbituric-acid-reactive substance (TBARS). Danthron (0.5—50 μM) ameliorated the effects of buthionine sulfoximine (BSO, 1 mM), which depletes endogenous glutathione by 10—73%. Danthron also dose-dependently inhibited neuronal injury mediated by nitric oxide (NO) radicals, but failed to inhibit injury due to superoxide radicals (O2−). These results suggest danthron treatment may, in part, reduce neurotoxicity related to β-amyloid protein by both dominant inhibitory effects on membrane lipid peroxidation and glutathione deprivation.
We have investigated the effect of propolis (CB Propolis) on the growth of human histiolytic lymphoma U937 cells. We found that propolis strongly inhibited the growth of the cells and macromolecular synthesis in a dose- and time-dependent manner by apoptosis. Propolis at 0.015—0.5 μl/ml showed antitumor activity with an IC50 of 0.18 μl/ml for 3 d. It also inhibits DNA, RNA and protein synthesis with an IC50 of 0.08, 0.17 and 4.3 μl/ml, respectively. The inhibitory effect on DNA synthesis was partially irreversible. Moreover, an apoptotic DNA ladder and chromatin condensation were observed in the same concentration range in which cell growth was inhibited. The caspase inhibitor, Z-Asp-CH2-DCB, prevented DNA fragmentation. These results suggest that the antitumor activity of propolis occurs through the induction of apoptosis. Propolis may be useful as a cancer chemopreventive and chemotherapeutic agent.
A variety of novel 2-methylthio-3-substituted quinazolin-4-(3H)-ones have been synthesized by reacting (2-methylthio-4-oxo-3H-quinazolin-3-yl)dithiocarbamic acid methyl ester with a variety of amines, the starting material dithiocarbamate was synthesized from methylanthranilate. The title compounds were investigated for analgesic, anti-inflammatory and antibacterial activities. While the test compounds exhibited significant activity, the compounds A1, A2, A3 and A4 shown more potent analgesic activity, and the compound A4 shown more potent anti-inflammatory activity than the reference diclofenac sodium.
A simple and efficient plant propagation system has been developed by asymbiotic germination of seeds in three medicinally important Dendrobium species, namely, Dendrobium tosaense, Dendrobium moniliforme, and Dendrobium linawianum. Plants obtained from natural habitats were grown in the greenhouse. The flowers were hand pollinated. Seeds of the capsules derived after 12 weeks of hand-pollination germinated asymbiotically (50—74%) on half strength Murashige and Skoog's (MS) basal medium with 3% sucrose and solidified with 0.9% Difco agar. Active growth in the germinated seedlings was achieved by re-culturing on full strength MS basal medium supplemented with 8% banana homogenate, 8% potato homogenate, 8% coconut water, 1.5% sucrose and 0.9% Difco agar. Healthy plantlets, transferred to plastic trays containing moss or moss and tree fern, successfully acclimatized (84—100%) in the greenhouse. A marked varied response was observed in the free radical scavenging activity of methanolic extracts of in vitro propagated plants, on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical using a UV spectrophotometer assay. Methanolic extracts were prepared by dissolving the powdered plant material, obtained from six months old in vitro propagated plants, each about 5 g, in boiling methanol. The percentage of scavenging effect of D. tosaense extract was 95.9% at 0.4 mg/ml concentration, whereas D. monoliforme, and D. linawianum extracts scavenged 83.4% and 92.3%, respectively, at a concentration of 0.4 mg/ml. All the extracts scavenged DPPH radical significantly in a concentration dependent manner.
Shimizu and Tsuji established a method of preparing colloidal platinum nanoparticles, whose average size is 2 nm, by ethanol reduction of H2PtCl6 in the absence of protective agents for the particles. Platinum nanoparticles have negative surface potential and are stably suspended from an electric repulsion between them. The platinum nanoparticles reduced hydrogen peroxide and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) below 0.1 ppm. It is necessary to use higher concentration of platinum nanoparticles for the reduction of 2,6-dichlorophenol indophenol (DCIP) than that of hydrogen peroxide and 2,2-diphenyl-1-picrylhydrazyl radical, because reoxidation of DCIPH2 (reduced) by oxygen was not negligible under our experimental conditions. These results indicate that electrons on platinum nanoparticles produced by the method of Shimizu and Tsuji can reduce hydrogen peroxide, DPPH radical or DCIP transferring electrons.
Hairy roots of interspecific hybrid ginseng (Panax ginseng×P. quinquefolium), induced by Agrobacterium rhizogenes ATCC 15834, grew well in B5 liquid media supplemented with 2.5 μM auxins (3-indole butyric acid (IBA), 1-naphtaleneacetic acid (NAA) and 3-indoleacetic acid (IAA)). The hairy roots cultured in B5 liquid medium supplemented with 2.5 μM IBA showed best growth (6.39 g fresh weight per a flask, at week 8). The highest content of the total ginsenosides was 1.63% as dry weight at week 8 when cultured with 2.5 μM NAA. The different auxins affected the numbers and lateral branching roots. Especially, 2.5 μM IBA promoted the lateral root formation (43.7±4.0 roots, at week 8), and 2.5 μM NAA promoted the lateral root growth (45.3±5.6 mm, at week 8). The growth and ginsenosides production of 8-week old hairy roots cultured in B5 liquid media supplemented with IBA and NAA combinations were also investigated. Hairy roots produced higher amounts of ginsenosides in B5 liquid media supplemented with 0.5—1.0 μM IBA and NAA combinations than that cultured in B5 liquid media supplemented with only IBA and NAA. The highest yield of ginsenoside was obtained when cultured with 0.5 μM IBA and 1.0 μM IBA combination (6.38 mg per a flask, at week 8).
Previously, we have determined marker nucleotides on the chloroplast matK gene to identify Rheum palmatum, R. tanguticum and R. officinale used as Rhei Rhizoma officially. In the present study, we further developed a convenient and efficient identification method on the basis of marker nucleotides with Amplification Refractory Mutation System analysis. On the basis of the nucleotide substitutions at positions 367 and 937 among the three species on the matK gene, at each position two kinds of reverse primers with complementary 3′-terminal nucleotides were designed. Upon PCR amplification using three sets of primers and template DNA from each species, one or two fragments (202 bp or/and 770 bp) were detected. As the resultant three fragment profiles were species-specific, the procedure enabled us to classify the botanic origins of 22 drug samples of Rhei Rhizoma.
Sho-seiryu-to (SST) is widely used herbal formula in Japanese traditional medicine (kampo) to treat allergic diseases. Since Japanese physicians frequently prescribe this formula combined with azelastine hydrochloride, one of anti-histamine and anti-allergic medicines, we evaluated the pharmacokinetic interactions between SST and azelastine hydrochloride in rats to obtain the drug information for the prevention from disadvantage or adverse effects by their combined therapy. Oral administration of SST did not influence the plasma concentration profile of azelastine after its intravaneous injection, suggesting that SST would not change the activities of metabolic enzymes such as cytochrome P450s. However, maximum concentration (Cmax) of azelastine after oral administration of azelastine hydrochloride was significantly reduced and mean residence time (MRT) was significantly lengthened when SST was orally administered at 20 times amount of human daily dosage. There was not significant difference in the area under the plasma concentration–time curve (AUC), suggesting that SST might delay the absorption of azelastine without affecting the extent of bioavailability. Since this delay was independent of ephedrine that is a main constituent of SST and that a suppressor for gastric transit, SST might form unsoluble complex with azelastine to reduce its absorption. At the double of human daily dose, SST did not made the absorption of azelastine delay. The possibility that SST reduce the absorption of azelastine hydrochloride could not be denied completely, however, it is suggested that SST would not cause pharmacokinetic interaction with azelastine hydrochloride clinically.
The present study was designed to evaluate the effect of So-Cheong-Ryong-Tang (SCRT, also called Sho-Seiryu-To or Xiao-Qing-Long-Tang) on helper T cell development by monitoring Th1/Th2-specific cytokine secretion patterns in artificially induced Th1 or Th2 polarized conditions. The results demonstrated that Th2 cells were dramatically underpopulated in the Th2-driven condition triggered by SCRT treatment, while the Th1 cells were not altered in the Th1-skewed condition. Furthermore, under Th2-skewed conditions the levels of interleukin-4 were considerably decreased with SCRT treatment. The expression of GATA-3, a transcription factor that plays a pivotal role in Th2 lineage programming, did not change with SCRT treatment, while the expression of another Th2 transcription factor, c-Maf, was dramatically suppressed. These data suggest that SCRT modulates Th2 development by suppressing c-Maf expression. This study implies that the SCRT effect on CD4+ T cells is a key pharmacologic point of effect for treating IgE-mediated allergic asthma. These results also suggest that SCRT might be a useful agent for the correction of Th2-dominant pathologic disorders.
In order to further determine the nature of structure–activity relationship on the cytotoxicities of saponins with 1→2 and 1→3 linkages of disaccharides, we isolated guaianin N, collinsonidin, kalopanaxsaponin A and hederoside D2 as disaccharides, and patrinia glycoside B-II as a trisaccharide, from the n-BuOH extract of Akebia quinata (Lardizabalaceae). Complete acid hydrolysis of the extract afforded oleanolic acid (1) and hederagenin (2). By sulforhodamine B (SRB) assay, kalopanaxsaponin A containing an α-L-rhap-(1→2)-α-L-arap moiety exhibited distinctly higher cytotoxicity (IC50 1.8—2.7 μg/ml) against all of the tested cell lines than the other saponins (IC50, 4—8 μg/ml). These results suggest that the α-L-rhap-(1→2)-α-L-arap moiety has a unique structural significance in terms of its cell biochemistry, compared to those oleanane glycosides with other sugar linkages. On the other hand, kalopanaxsaponin A exhibited a significant inhibitory effect on nitric oxide production by lipopolysaccharide (LPS)-activated macrophage 264.7, whereas other saponins had weaker activities.
Four lavandulyl flavanones, (2S)-2′-methoxykurarinone (1), sophoraflavanone G (2), leachianone A (3), and (−)-kurarinone (4), which are isolated from the roots of Sophora flavescens have been tested for in vitro antimalarial activity against Plasmodium falciparum. Compounds 1—3 showed moderate antimalarial activities with EC50 values of 2.4×10−6, 2.6×10−6, and 2.1×10−6 M, respectively. These compounds did not show selective toxicity against P. falciparum in the toxicity test on mouse mammalian tumor cells, however, it is suggested that the position of methoxyl groups in flavanone skeleton plays an important role on antimalarial activity.
Tumor-bearing mice showed a significant resistance against Candida albicans intravenous infection. Longer survival was observed in groups of mice inoculated with fungal cells 2—3 weeks after tumor transplantation with allogeneic sarcoma 180, syngeneic methylcholanthrene-induced Meth A fibrosarcoma, and MM 46 mammary carcinoma than in non-tumor-bearing mice inoculated only with fungal cells. This effect was not observed when the mice were infected only 1 week after tumor transplantation. A significant decrease in the number of C. albicans cells in the kidneys was observed in mice inoculated with fungal cells 2—3 weeks after tumor transplantation. In the tumor-bearing mice treated with cyclophosphamide (CY), a remarkable decrease in both the number of peripheral blood polymorphonuclear leukocytes (PMNs) and the defense against challenge with C. albicans cells was observed, as compared with the CY-untreated groups (normal and tumor-bearing mice). A marked increase in the calcium concentration in serum and number, candidacidal activity, active oxygen level, and myeloperoxidase activity of PMNs was observed in the 2—3-week tumor-bearing mice. From these results, it is suggested that PMNs, which accumulated in the 2—3-week tumor-bearing mice, play an important role in the protection from C. albicans infection by increasing the number and the types of killing factors.
Administration of a citrus flavonoid hesperidin (HES) to mice before LPS challenge significantly reduced tumor necrosis factor (TNF)-α production in a dose-dependent manner. Treatment of HES 3 h before intraperitoneal (i.p.) infection with 108 CFU Salmonella typhimurium aroA resulted in rescue from lethal shock as similar to LPS-nonresponder mice. Not only bacterial numbers in livers and spleens but also plasma LPS levels significantly decreased by pretreatment with HES. In addition, HES markedly suppressed plasma levels of TNF-α and high mobility group box chromosomal protein 1 (HMGB-1), decreased the number of apoptotic cells in livers and normalized the activated states of blood coagulation factors such as prothrombin time and platelet numbers caused by infection. Pretreatment of LPS with HES suppressed the chromogenic Limulus reaction.
Oxatomide is an antiallergic drug used for the treatment of diseases mediated by type I allergy. Recently, it has been reported that terfenadine and astemizole, which have antiallergic actions similar to those of oxatomide, show side effects on the cardiovascular system, such as QT prolongation, ventricular arrhythmia and cardiac arrest. This might be because concomitant drugs such as itraconazole inhibit cytochrome P450 3A4 (CYP3A4), the enzyme responsible for degradation of terfenadine and astemizole, and thus the blood concentrations of the drugs are abnormally increased. On the other hand, isoforms of P450 involved in the metabolism of oxatomide have not been clarified. Therefore, we attempted to identify these isoforms using microsome preparations of in vitro expression systems derived from a human lymphoblastoid cell line. Oxatomide was metabolized by CYP2D6-Val and CYP3A4, but not by CYP1A2, CYP2C9-Arg, CYP2C9-Cys or CYP2C19. We also examined whether oxatomide showed inhibitory effects on metabolic activity of individual P450 isozymes using model substrates for each isozyme. Oxatomide did not inhibit the metabolism of the model substrates for CYP1A2, CYP2C9-Arg, CYP2C9-Cys and CYP2C19, but inhibited the degradation of those for CYP2D6-Val and CYP3A4. It was found that oxatomide is metabolized by CYP2D6 and CYP3A4 in human liver microsomes, and simultaneously acts as an inhibitor for these isoforms, responsible for the metabolism of the drug itself.
Candida albicans grew in hyphal form in RPMI1640, however, addition of farnesol inhibited the formation. Farnesol did not affect the expression of mRNAs related to cAMP-EFG1 pathways. The mRNAs (HST7 and CPH1) in mitogen activated protein kinase (MAP) cascades were decreased in farnesol-treated cells, but CST20 was not. Furthermore, expression of general amino acid permease 1 (GAP1) was decreased by farnesol. We concluded that farnesol inhibits a MAP kinase cascades, and the suppression is a cause of interruption of hyphae formation.