In the 21st century, emerging genomic technologies (i.e., bioinformatics, functional genomics, and pharmacogenomics) are shifting the paradigm of drug discovery research and improving the strategy of medical care for patients. In order to realize the personalized medicine, it is critically important to understand molecular mechanisms underlying inter-individual differences in the drug response, namely, pharmacological effect vs. side effect. Evidence is now accumulating to strongly suggest that drug transporters are one of the determinant factors governing the pharmacokinetic profile of drugs. Effort has been made to identify genetic variation in drug transporter genes. In particular, genetic variations of the human ABCB1 (P-glycoprotein/MDR1) gene have been most extensively studied. Hitherto more than fifty single nucleotide polymorphisms (SNPs) and insertion/deletion polymorphisms in the ABCB1 gene have been reported. However, at the present time, information is still limited with respect to the actual effect of those genetic polymorphisms on the function of ABCB1. In this context, we have undertaken functional analyses of ABCB1 polymorphisms. To quantify the impact of genetic polymorphisms on the substrate specificity of ABCB1, we have developed a high-speed screening system and a new structure–activity relationship (SAR) analysis method. This review addresses functional aspects of the genetic polymorphism of ABCB1 and provides the standard method to evaluate the effect of polymorphisms on the function.

View full abstract

In brain ischemia, cell destructive necrosis occurs in the core, which in turn links to cell death expansion in the vicinity. Apoptosis, on the other hand, occurs in the surroundings of the core, called the penumbra, several days later. As cells showing apoptosis disappear by microglial phagocytosis in the brain, cell death induced by ischemic stress should eventually be terminated. Thus, the authors propose the hypothesis that the cell death mode switch in the event of brain ischemia is an in vivo self-protective mechanism. The authors attempt to overview the current understanding of the molecular mechanisms of necrosis and apoptosis in relation to the ATP hypothesis, and also introduce novel mechanisms for an in vitro cell death mode switch.

View full abstract

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a unique antioxidant enzyme that markedly reduces lipid hydroperoxide generated in biomembranes. Overexpression of mitochondrial PHGPx potentially suppresses the release of cytochrome c (cyt. c) from mitochondria and apoptosis. The hydroperoxide level in mitochondria was elevated in 2-deoxyglucose (2DG)-induced apoptosis, but not in apoptosis-resistant cells in which mitochondrial PHGPx was overexpressed. From studies of the overexpression of PHGPx, the generation of hydrogen peroxide and lipid hydroperoxide in mitochondria might be important triggers of apoptosis. In particular lipid hydroperoxide could be involved in the initiation of cyt. c liberation from mitochondria in 2DG-induced apoptosis since lipid hydroperoxide is a primary substrate for PHGPx. The release of cyt. c from mitochondria is an important proapoptotic signal in the mitochondrial death pathway. Several reports demonstrated the reactive oxgen species could be involved in cyt. c liberation, although its mechanism is still unknown. Cardiolipin (CL), which exclusively locates in the innermembrane of mitochondria, shows strong affinity for cyt. c is required for the adenine nucleotide translocator (ANT) that cotnrols the opening and closing of the permeability transition pore. Association of cyt. c with CL is lost upon peroxidation. CL hydroperoxide (CLOOH), in contrast to CL, does not bind to cyt. c. Furthermore, CLOOH can open the permeability transion pore by the inactivation of ANT. These previous results suggest that mitochondrial PHGPx inhibits the release of cyt. c from mitochondria by the scavenging CLOOH and could prevent apoptosis.

View full abstract

This review presents recent findings with regard to the cellular and molecular mechanisms of neuronal apoptosis induced by cerebral ischemia/hypoxia. The protection of neuronal death by hypoxia-induced proteins in the endoplasmic reticulum (ER) is also reviewed. The excess amount of nitric oxide (NO) generated by inducible NO synthase (iNOS) up-regulated in response to ischemic stress causes neuronal apoptosis through following processes; 1) reduction in mitochondrial membrane potential, 2) release of cytochrome c from mitochondria, and 3) activation of caspase-9 and -3, although low concentrations of NO protect against neuronal death. In contrast, hypoxia induces expression of several proteins such as protein disulfide isomerase (PDI), ubiquilin and HRD1 in the endoplasmic reticulum (ER). PDI and ubiquilin are involved in the protection against neuronal apoptosis probably by interacting with each other and enhancing the effects of PDI as a molecular chaperon. HRD1 is also up-regulated by hypoxia in the ER and induces protection against hypoxia-induced neuronal apoptosis by activating the protein degradation system. The present review hopefully gives pertinent suggestions for further studies on the development of novel prophylactic/therapeutics for neuronal apoptosis-related diseases.

View full abstract

Neurons in the central nervous system (CNS) are vulnerable to radical stress caused by reactive oxygen species, including nitric oxide (NO). Those radicals play crucial roles in glutamate neurotoxicity associated with ischemic brain injury and a wide range of neurodegenerative disorders. In our previous studies, we have shown evidence suggesting that glutamate neurotoxicity is regulated by certain endogenous substances such as neurotrophins, nicotinic acetylcholine, prostanoids and vitamins. Based on those findings, we have used the term ‘neuroprotective factor’ for endogenous substances possessing protective activity against glutamate neurotoxicity, and have further searched for a candidate with unique structure. We isolated a novel neuroprotective substance named ‘serofendic acid’ derived from fetal calf serum. The compound exhibited potent protective action against neurotoxicity induced by glutamate and by an NO donor without inhibiting glutamate receptors. Electron spin resonance analysis demonstrated that serofendic acid had no direct scavenging activity on NO, but was capable of inhibiting the generation of a hydroxyl radical, a presumed ‘executor’ radical in the nitric oxide-mediated neurotoxic cascade. The chemical structure was determined by mass spectrometry and nuclear magnetic resonance spectroscopy, and was confirmed by synthesis. The structure was unique among known endogenous substances because the compound was a sulfur-containing atisane type diterpenoid. The discovery of serofendic acid may provide a new scope for the investigation of low-molecular weight bioactive factors promoting the survival of CNS neurons.

View full abstract

A novel caspase-3-specific inhibitory peptide and an agonistic peptide that binds to the Fas molecule were discovered using our computer screening strategy called the amino acid complement wave (ACW) method, which is based on the complementarities of interacting amino acids between comprehensive testing peptides and a target protein surface pocket. The precise binding configurations of the designed peptides on the three-dimensional (3D) structure of the target protein and the prediction of binding affinities (ΔG) are determined by the molecular docking program. A designed novel tetrapeptide inhibitor of caspase-3, Ac-DNLD-CHO, was revealed to have potent and specific inhibitory activity. When a designed Fas ligand mimic peptide (Fas reactive peptide-4, FRAP-4) was multimerized by carboxyl terminal linkages of polylysine branches (MAP), the octamer (FRAP-4)₈-MAP effectively induced apoptosis of human ovarian cancer cell line NOS4 cells. Thus the ACW method for structure-based design of optimized small peptides can be used to further develop small peptidomimetic and nonpeptidic organic forms into a new generation of effective pharmaceuticals.

View full abstract

This paper reports a sensitive and specific enzyme-linked immunosorbent assay for screening of furanocoumarin derivatives as cytochrome P450 3A4 inhibitors in citrus fruits. Anti-6′,7′-dihydroxybergamottin antibody was obtained by immunizing rabbits with 6′,7′-dihydroxybergamottin conjugated with bovine serum albumin using the N-succinimidyl ester method. An enzyme marker was similarly prepared by coupling 6′,7′-dihydroxybergamottin with β-D-galactosidase. The enzyme-linked immunosorbent assay is capable of detecting as little as 800 pg/ml of 6′,7′-dihydroxybergamottin and 4 ng/ml of bergamottin. Cross-reactivity data showed that the antibody well recognizes both the furanocoumarin and 6,7-dihydroxy-3,7-dimethyloct-2-enyloxy moieties of the 6′,7′-dihydroxybergamottin, and is thus specific to the structure of furanocoumarin derivatives containing geranyloxy side chain as the cytochrome P450 3A4 inhibitors in grapefruit juice. The antibody was, therefore, used for screening a large number of citrus fruits for furanocoumarin derivatives such as 6′,7′-dihydroxybergamottin. Fifteen citrus fruits were examined and significant reactivity was observed in 8 of these: red pummelo, sweetie, melogold, banpeiyu pummelo, hassaku, sour orange, lime and natsudaidai. This enzyme-linked immunosorbent assay may be a powerful tool for screening for furanocoumarin derivatives as cytochrome P450 3A4 inhibitors in grapefruit juice.

View full abstract

Feed containing β-carotene was administered orally to BALB/c mice immunized intraperitoneally with ovalbumin (OVA) for approximately 1 month. The titers of OVA-specific IgE, OVA-specific IgG1 and OVA-specific IgG2a in the mouse sera were determined. The OVA-specific IgE titer and OVA-specific IgG1 titer by mice fed β-carotene were significantly inhibited. On the other hand, the OVA-specific IgG2a titer in mice fed β-carotene was significantly greater than those of control mice. The OVA-specific IgE suppression of β-carotene feeding was dose-dependent. We also examined the effect of fed β-carotene on active systemic anaphylaxis. Feeding β-carotene to mice immunized with OVA inhibited the immediate reduction of the body temperature induced by antigen stimulation. Furthermore, the increase in serum histamine in the mice fed β-carotene under active systemic anaphylaxis was lower than in controls. We then examined the pattern of cytokine production by spleen cells from mice followed by restimulation with OVA in vitro. The spleen cells from the mice fed β-carotene produced more IFN-γ, IL-12 and IL-2 than those from the control group. In contrast, the spleen cells from the mice fed β-carotene produced less IL-4, IL-5, IL-6, IL-10 than those from the control group. Furthermore, analysis of IFN-γ mRNA levels of the splenocytes using the real-time quantitative RT-PCR technique revealed higher levels in the splenocytes from the mice fed β-carotene. These findings suggest that feeding β-carotene improves the helper T cell (T_H)1–T_H2 balance, inhibiting specific IgE and IgG1 production and antigen-induced anaphylactic response.

View full abstract

RAW 264.7 macrophage-like cells are known to release prostaglandins (PGs), mainly PGD₂ to the culture medium after lipopolysaccharide (LPS)-treatment. This release was inhibited by non-steroidal anti-inflammatory drugs (NSAIDs), which are known to inhibit prostaglandin H₂ synthase (PGHS) activity. In this study, we examined the effect of removal of NSAID after induction of PGHS with LPS, on the release of PGs, which has not been studied well. Re-incubation of RAW 264.7 cells after treatment of both LPS and NSAIDs resulted in enhanced release of PGD₂ compared with the cells pretreated with LPS alone. Besides, PGHS activity was detectable in these cell homogenates and the amount of PGHS-2 protein showed similar changes to PGD₂ release. However, addition of NSAIDs again in the re-incubation period almost completely inhibited the PGD₂ release but increased the amount of PGHS-2 protein to the higher levels. Various types of NSAIDs used in this study showed similar effects on the changes in PGD₂ release and PGHS-2 protein amounts, except those on PGHS activity in cell homogenates; while indomethacin, aspirin, and NS-398 inhibited it, but nimesulide and acetaminophen did not. These results seem to suggest an importance for the caution that the enhanced induction of PGHS-2 protein and the concomitant release of PGs release would occur after removal of the NSAID not only from the medium in in vitro experiments but also from therapeutic prescription.

View full abstract

All-trans retinoic acid (ATRA) differentiates HL-60 cells into granulocyte-like cells and cellular proliferation is repressed markedly along with the morphological and physiological changes specific for cellular differentiation. To elucidate the implication of cyclin-dependent kinase (CDK) inhibitors during differentiation, we examined the expression of CDK inhibitors during the differentiation of HL-60 cells. The expression of p21 and p27 among the CDK inhibitors we examined increased during the differentiation induced with ATRA. Then, we established stable transformants of HL-60 cells expressing antisense RNA for p21 and p27 and examined the ability of these cells to differentiate into granulocyte-like cells. The extents of fully differentiated HL-60 cells transfected with genes for antisense RNA of p21 and p27 were only 53% and 60%, respectively, whereas 90% of the parental HL-60 cells differentiated by the ATRA treatment. These results suggest that increased expression of CDK inhibitors, p21 and p27, is necessary for the differentiation of HL-60 cells induced with ATRA.

View full abstract

The selective induction of apoptosis of gambogic acid (GA) on MGC-803 cells and its probable molecular mechanism were studied. GA greatly inhibited (24, 48, 72 h) the growth of MGC-803 cells (by MTT); the IC₅₀ value was 0.96 μg/ml at 48 h. Meanwhile, no influence was observed on body weight, number of WBC (white blood cells) in blood or karyote in marrow of rats after GA was injected intravenously. We conclude that GA does not affect normal cells, but that it can induce apoptosis in tumor cells selectively and there were marked morphological changes. A great quantity of apoptotic cells and increasing G₂/M phase cells were observed by flow cytometry, and a significant percentage of early apoptotic cells were observed by Annexin-V/PI double staining assay. The increase of bax gene and the decrease of bc1-2 gene expressions were detected by immunohistochemistry. Activation of bax and suppression of bc1-2 may contribute to the apoptosis mechanism.

View full abstract

We studied the protective effects of Echinacea purpurea against radiation by evaluating changes in the peripheral blood cell count and peripheral blood antioxidant activity. E. purpurea administration had a suppressive effect on radiation-induced leukopenia, especially on lymphocytes and monocytes, and resulted in a faster recovery of blood cell counts. Mouse peripheral blood antioxidant activity was increased by E. purpurea, and a relationship between the suppressive effect on radiation-induced leukopenia and the antioxidant effect was suggested. Furthermore, we reviewed the evidence of augmentation of found in this study humoral immunity. The effecst of immune activation by E. purpurea were investigated by measuring total immunoglobulin (IgG, IgM). The radioprotective effects of immune activation by E. purpurea were investigated by measuring T lymphocyte subsets in the peripheral blood of mice following whole-body irradiation. E. purpurea activates macrophages to stimulate IFN-γ production in association with the secondary activation of T lymphocytes, resulting in a decrease in IgG and IgM production. Cytokines released from macrophages in mouse peripheral blood after E. purpurea administration activated helper T cells to proliferate. In addition, it is reported that activated macrophages in association with the secondary T lymphocyte activation increases IFN-γ production and stimulates proliferation of cytotoxic T cells and suppressor T cells. We think that CD 4 and CD 8 subsets were more immunologically enhanced by E. purpurea than helper T cells and suppressor T cell these results reflect activation. In addition, we think that these results reflect cell-mediated immune responses.

View full abstract

Qing Nao Yi Zhi Fang (QNYZ), a Chinese medicine, has been developed as a drug for the prevention and treatment of vascular dementia. We examined the effects of QNYZ-treated serum and QNYZ on the proliferation of human arterial smooth muscle cells (SMCs) and endothelial cells (ECs). SMCs did not grow in a medium containing 10% QNYZ serum. QNYZ also completely inhibited the proliferation of SMCs at higher concentrations (500—1000 μg/ml), although no detectable inhibitory activity on the SMC proliferation was observed at lower concentrations (100—200 μg/ml). In contrast, QNYZ (250—500 μg/ml) significantly stimulated the proliferation of ECs and prostacyclin production in ECs. These results suggest that QNYZ may have suppressive effects on the development of intimal thickening in atherosclerosis and after arterial wall injury.

View full abstract

The present study was conducted to determine whether iridoid total glycoside from Cornus officinalis was effective in regulating expression of transforming growth factor beta 1 (TGF-β1) and preventing overdeposition of extracellular matrix (ECM) in a diabetes state. An experimental rat model of diabetic nephropathy (DN) was successfully induced by one intraperitoneal injection of streptozotocin at a dose of 60 mg·kg⁻¹ and maintained for 12 weeks. All rats had free access to standard chow and water. Four groups: normal control, diabetic control, diabetic rats with aminoguanidine treatment and diabetic rats with iridoid total glycoside treatment were used in this experiment. All treatments were administered by intragastric gavage (ig). At the end of the experiment, serum was collected for ELISA determination of TGF-β1 protein level; renal cortex was dissected for reverse transcription polymerase chain reaction (RT-PCR) analysis of its mRNA expression; and immunohistochemistry was introduced to observe ECM deposition. A significantly higher level of protein and mRNA expression of TGF-β1, and also overdeposition of fibronectin and laminin was found in diabetic rats. Both iridoid total glycoside and aminoguanidine were effective in decreasing serum protein level and glomerular mRNA expression of TGF-β1, and in preventing renal overdeposition of fibronectin and laminin. This study suggests that iridoid total glycoside is a beneficial agent for prevention and therapy of DN.

View full abstract

To evaluate the pharmacological characteristics of the new ethacrynic acid (ECA) derivative SA9000, we examined its ocular hypotensive effects in cats and cynomolgus monkeys, its corneal toxicity in rabbits, and its binding affinities for forty-three receptors, ion channels, and second messenger systems. A 20 μl injection into the anterior chamber of eye (intracameral injection) of 0.1 mM SA9000 significantly reduced intraocular pressure (IOP) 3.8 mmHg in cats. A 10 μl intracameral injection of 1 mM SA9000 significantly reduced IOP 7 mmHg in living monkeys without evidence of in vivo (or in vitro) toxicity. The ocular hypotensive effect of SA9000 in monkeys was greater than that of ECA. The morphology of corneal endothelial and epithelial cells in rabbit eyes after intracameral injection of SA9000 was observed using electron microphotography. SA9000 at 2 mM did not induce any abnormalities, indicating that it has no corneal toxicity at a concentration higher than the minimum needed for an ocular hypotensive effect (1 mM). SA9000 at 0.01 mM showed negligible binding affinity for, or inhibition of, forty-three different receptors, ion channel proteins, and second messenger systems. These findings indicate that SA9000 has the potential to be both effective and safe as an ocular hypotensive drug, although the mechanism of action remains unclear.

View full abstract

Two sesquiterpenoids, mansonone E (ME) and mansonone F (MF) were first isolated from the dried root bark of Ulmus pumila (shironire in Japanese), and their antiproliferative activities on human tumor cells were evaluated in vitro. ME had more potent cytotoxic effects on four tumor cell lines, human cervical cancer HeLa, human malignant melanoma A375-S2, human breast cancer MCF-7, and human histiocytic lymphoma U937, than those of MF. The results showed that ME induced oligonucleosomal fragmentation of DNA in HeLa cells and activated caspase-3, followed by the degradation of the inhibitor of caspase-activated DNase, decreased the expression of anti-apoptotic mitochondrial proteins Bcl-2 and Bcl-_XL, and increased that of proapoptotic Bax.

View full abstract

Silymarin, a plant flavonoid from milk thistle (Silybum marianum [L.] GAERTNER) was first evaluated for its protective effect against UV irradiation-induced apoptosis in human malignant melanoma cells (A375-S2 cells). Treatment with silymarin 500 μM for 12 h significantly inhibited UV irradiation (2.4 J/cm², 5 min)-induced apoptosis in A375-S2 cells. Activities of caspase-9 and caspase-3 in UV-irradiated A375-S2 cells were effectively reduced by silymarin in a dose-dependent manner, while the expression of the inhibitor of caspase-activated DNase (ICAD), protein expression of Bcl-x_L (Bcl-2 family member), and the activity of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) were increased simultaneously. It is suggested that the inhibitory effect of silymarin is exerted by blockage of the caspase/ICAD pathway after increased expression of Bcl-x_L protein and activation of the ERK/MAPK pathway.

View full abstract

Increasing evidence in both experimental and clinical studies suggests that oxidative stress plays a major role in the pathogenesis of diabetes mellitus type 1 and type 2. Xanthine oxidase (XO) has been proposed as one of the sources of free radical formation in diabetes. We therefore investigated the preventive effects of Laminaria japonica aqueous extract (LJE) on alterations in the activity of hepatic XO and oxidative stress in the streptozotocin-induced experimental diabetes. We found that lipid peroxide levels and xanthine oxidase activity were increased, whereas glutathione (GSH), GSH reductase and GSH peroxidase were decreased in the liver of streptozotocin-induced diabetic rats. Pretreatment with LJE of 100 mg/kg orally for 5 d significantly reduced blood glucose levels and hepatic lipid peroxidation in the diabetic rats. In addition, the content of glutathione was restored to the control level by LJE pretreatment. Furthermore, LJE significantly suppressed the increased activity of XO and type conversion of the xanthine dehydrogenase to XO in diabetic rat liver. The results suggest that Laminaria japonica would be of great value in preventing hyperglycemia in diabetes mellitus as a dietary supplement possibly, through its antioxidant activity.

View full abstract

We evaluated the effects of pre-germinated brown rice (hatsuga genmai, PGR) on learning and memory and compared them with those of polished rice or cornstarch. In mice that were fed pellets of polished rice or PGR for two weeks, the learning ability in the Morris water maze test was significantly enhanced compared with mice that were fed cornstarch pellets. In the Y-maze test, the intake of food pellets for two weeks failed to affect spontaneous alternation behavior. β-Amyloid_25—35 (Aβ_25—35: 3 nmol/mouse, i.c.v.) protein impaired spontaneous alternation behavior in mice that were fed pellets of cornstarch or polished rice. In contrast, PGR pellets prevented the Aβ_25—35-induced impairment of spontaneous alternation behavior. These results suggest that polished rice and PGR have facilitating effects on spatial learning. In particular, it is surmised that PGR may prevent Alzheimer's disease associated with Aβ.

View full abstract

Wood creosote, a mixture of simple phenolic compounds, has long been used as an herbal antidiarrheal medicine. Previous studies have shown that wood creosote has antimotility activity on the gastrointestinal (GI) tract, although its mechanism of action is not completely understood. The in vitro efficacy of wood creosote on calcium mobilization in guinea pig colonic smooth muscle was evaluated using a digital video camera system mounted on an inverted fluorescence microscope. The effects of wood creosote on spontaneous periodic increases in the free cytosolic calcium concentration ([Ca²⁺]_i), acetylcholine (ACh)-enhanced periodic increases in [Ca²⁺]_i, and tetrodotoxin- or nifedipine-resistant spontaneous periodic increases in [Ca²⁺]_i were evaluated. Wood creosote decreased the amplitude of spontaneous (IC₅₀=21 μg/ml) and ACh-enhanced (IC₅₀=40 μg/ml) periodic increases in [Ca²⁺]_i in guinea pig colonic smooth muscle. Wood creosote also decreased the amplitude of both tetrodotoxin- and nifedipine-resistant spontaneous periodic increases in [Ca²⁺]_i. These results suggest that antimotility activity through inhibition of Ca²⁺ mobilization in the GI tract is at least partially responsible for the antidiarrheal activity of wood creosote. Wood creosote may exert its antimotility effect, at least in part, on network regions of interstitial cells of Cajal, which act as pacemaker cells and mediators of neurotransmission in the GI tract.

View full abstract

Neurolathyrism is a human motoneuron disease caused by the overconsumption of grass pea (Lathyrus sativus) that contains a toxic non-protein amino acid, 3-N-oxalyl-L-2,3-diaminopropanoic acid (L-β-ODAP). The preventive activities of various glutamatergic agents from acute neuronal death caused by L-β-ODAP were studied using rat primary cortical neuron/glia culture. Nearly 80% of the rat primary cortical neurons were killed by 300 μM L-β-ODAP within 24 h. Though antagonists acting on the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor prevented most of the toxicity, antagonists acting on group I metabotropic glutamatergic receptors (mGluRs), including (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA), (S)-α-methyl-4-carboxyphenylglycine (MCPG), and 2-methyl-6-(2-phenylethenyl)pyridine (SIB1893) partially and significantly prevented neuronal death due to L-β-ODAP. These antagonists, within limited concentrations, did not have any inhibitory effects on the currents through AMPA receptors expressed in Xenopus oocytes. L-β-ODAP itself did not induce the currents through group I mGluRs expressed in Xenopus oocytes. These results suggest that the neurotoxicity induced by L-β-ODAP is partially mediated by the activation of group I mGluRs by an indirect mechanisms.

View full abstract

Diosgenyl saponins are the most abundant steroid saponins, and exert a large variety of biological functions. In a previous report, we showed that dioscin was able to induce cytotoxicity and apoptosis in human myeloblast leukemia HL-60 cells. This study further investigated the action mechanisms underlying this effect. The activation of caspase-9 and -3, but not caspase-8, together with the down-regulation of anti-apoptotic Bcl-2 protein, demonstrated that the apoptotic signaling triggered by dioscin was mediated through the intrinsic mitochondria-dependent pathway. We also investigated its anti-proliferative effect on human chronic myelogenous leukemia K562 cells. Flow cytometry analysis showed that dioscin treatment induced the accumulation of cells in the G₂/M phase. Cytomorphology with DAPI and Wright-Giemsa staining demonstrated the enlargement of cell volume and multinucleation in the treated cells. Subsequent apoptosis was delineated with phosphatidylserine externalization and DNA hypodiploidy. Trillin was one of the hydrolysates of dioscin. We demonstrated that it could induce multinucleation in HL-60, K562 and human promyelocytic leukemia NB₄ cells, suggesting its extensive mitotic-arresting effects. As the diosgenyl sapogenin, diosgenin was also shown to be able to induce multinucleation and apoptosis in K562 cells in a similar manner to dioscin. These findings suggest that diosgenyl saponins have the properties to induce mitotic arrest and apoptosis, suggesting that they may be a new kind of antimitotic agent.

View full abstract

Schizandra chinensis fruit has long been used for the treatment of cardiovascular symptoms associated especially with menopausal symptoms in Korea. To provide a scientific rationale for such uses, we have investigated the vasorelaxant effects of Schizandra chinensis fruit on the vasomotor tone of the rat thoracic aorta in an organ bath. The crude extracts of Schizandra chinensis fruit (SC-Ex) elicited a transient relaxing response in the endothelium-intact rat aorta contracted with norepinephrine. This relaxant effect was abolished by removal of the endothelium, and also by pretreatment with nitric oxide synthase inhibitor. We then examined whether this vasodilatory effect occurs through estrogen receptor by reporter assays. SC-Ex activated the estrogen-responsive luciferase gene in COS cells transiently transfected with estrogen receptor and reporter plasmids. The activation was maintained in the butanol-soluble fraction and further increased in the successively fractionated C₁₈ cartridge-adsorbed fraction (SC-ADF). Reporter gene activation by SC-ADF was inhibited by the specific estrogen receptor antagonist ICI 182,780, indicating that the effect is estrogen receptor dependent. However, SC-ADF failed to activate the androgen receptor in COS cells transfected with the corresponding receptor and reporter plasmids. These data show that extracts of Schizandra chinensis fruit act as a weak phytoestrogen.

View full abstract

Rhinacanthus nasutus (L.) KURZ (Acanthaceae) is a shrub widely distributed in South China and India. In this study, the antiproliferative activity of the ethanol extract of root and aqueous extract of leaves of R. nasutus, and the supposed active moiety rhinacanthin C was assessed in vitro using the human cervical carcinoma cell line HeLa, its MDR1-overexpressing subline Hvr100-6, human prostate carcinoma PC-3 cells and human bladder carcinoma T24 cells. Rhinacanthin C was chemically synthesized and its content in the R. nasutus extracts was determined by HPLC with a photodiode array detector. The antiproliferative activity of the R. nasutus extracts was also assessed in vivo using sarcoma 180-bearing mice. It was suggested that 1) the in vitro antiproliferative activity of rhinacanthin C was comparable with or slightly weaker than that of 5-FU, 2) rhinacanthin C showed antiproliferative activity for MDR1-overexpressing Hvr100-6 cells, similarly to parent HeLa cells, 3) the in vitro antiproliferative activity of the ethanol extract of root R. nasutus was due to rhinacanthin C, whereas that of the aqueous extract of leaves of R. nasutus was due to constituents other than rhinacanthin C, and 4) both of the R. nasutus extracts showed in vivo antiproliferative activity after oral administration once daily for 14 d.

View full abstract

In this study, cycloheximide (CHX) and VP-16 alone and in combination (C&V) have been used to strongly trigger apoptosis in U937 cells. The presence of CHX markedly prevented VP-16-induced apoptosis, suggesting that in this process de novo protein synthesis is required. But interestingly, C&V had shown more similarities with CHX but not VP-16 alone, including the effects on cell cycle distribution and induction of apoptosis, which occurred more quickly and was steadily enhanced by increasing concentrations of CHX or by N-α-tosyl-L-lysyl-chloromethyl ketone (TLCK), a serine protease inhibitor. These results indicate that CHX, not VP-16, is indeed the dominant inducer of U937 apoptosis, when they are coadministered. In particular, VP-16 even promoted CHX-induced apoptosis, but did not alter its selection of cell types. In T-cells resistant to CHX (Molt-4), we have detected no apoptotic response to their combination. These findings may well explain why the inhibitory effects of CHX on apoptosis induced by the same stimuli are usually different according to the cell type used, and also suggest that CHX may have the potential to lower side effects and drug resistance of cancer therapy.

View full abstract

Ameliorating effects of methanol extracts of Basidiomycetes against in vitro and in vivo model of Alzheimer's disease were investigated. The extracts of Cordyceps ophioglossoides and Hypocrea citrina var. citrina prevented the β-amyloid_(25—35) (Aβ_(25—35))-induced cell death in SK-N-SH neuronal cells. However, in rat model of Alzheimer's disease, 30-d intraperitoneal administration with only the extract of Cordyceps ophioglossoides significantly prevented spatial memory loss by intracranial injection of Aβ_(25—35), which was assessed in water maze task. Interestingly, the scavenging activity of the reactive oxygen species (ROS) generated in Aβ_(25—35)-treated cells was also found in the extract of Cordyceps ophioglossoides, but not in the extract of Hypocrea citrina var. citrina. These results suggest that the extract of Cordyceps ophioglossoides may protect the Aβ-induced neuronal cell death and memory loss through free radical scavenging activity. These results further suggest that Cordyceps ophioglossoides mycelium may be valuable for the protection from Alzheimer's dementia.

View full abstract

We investigated the gene expression of β₁-adrenergic receptor (β₁AR) and stimulatory G-protein Gsα, important signal transduction elements for regulating heart rate and contractility, in ventricle after chronic treatment with isoproterenol (ISO) in rat. Rats were treated with ISO (4 mg/kg, intraperitoneal) twice a day for 4 d. Ventricle weight of the heart and ventricle weight/body weight ratio were increased by 23% and 25% compared with control, respectively. Positive inotropic responses to ISO in left atrial muscle preparations isolated from ISO-treated rats were markedly decreased. Northern blot hybridization showed that the mRNA transcript of β₁AR was significantly decreased in ventricle of ISO-treated rats, whereas Gsα mRNA level was unchanged. Present results demonstrate that the gene expression of myocardial β₁AR, but not Gsα, was decreased in rat myocardium of ISO-induced cardiac hypertrophy, and suggesting that decrease in the gene expression of β₁AR may be one of the mechanisms responsible for the diminished cardiac function.

View full abstract

Effect of Mao-Bushi-Saishin-to (Ma-Huang-Fu-Zi-Xi-Xin-Tang: MBS) on prostaglandin E₂ (PGE₂) production was investigated using C6 rat glioma cells. Mao or Saishin inhibited histamine-induced PGE₂ production while MBS slightly decreased and Bushi increased it. MBS and Mao inhibited and Bushi enhanced A23187-induced PGE₂ production while Saishin had no effect. Concomitantly, Mao inhibted, but Bushi fascilitated, histamine- and A23187-induced phosphorylation of extracellular signal-regulated kinase (ERK)1/2. Treatment of MBS, Mao and also Saishin increased cAMP content. From these results, MBS inhibit PGE₂ production in C6 cells, mainly due to Mao but also due to Saishin at least in part, and the counteraction of Bushi. The former effect is mediated by formation of cAMP and resulting inhibition of ERK1/2-phosphorylation.

View full abstract

Little is known about the development of infectious diseases during exposure to endocrine disrupting chemicals (EDCs), although several studies have reported on the effect of EDCs on the immune function of the human body. To assess the effect of EDCs on the development of infectious disease, we investigated the effect of eighteen possible EDCs on mouse macrophage production of tumor necrosis factor-α (TNF-α) and nitric oxide (NO) in response to bacterial endotoxin in vitro and ex vivo. Of chemicals we examined, simazine, nitrofen, and benzyl butyl phthalate inhibited lipopolysaccharide (LPS)-induced TNF-α production by mouse macrophage cell line RAW 264 in vitro. Carbaryl, alachlor, nonylphenol, octylphenol, tributyltin, and triphenyltin inhibited LPS-induced NO production in vitro, whereas 2,4-dichlorophenoxy acetic acid and bisphenol A enhanced its production. Zineb and alachlor, on the other hand, enhanced LPS-induced TNF-α production by mouse peritoneal macrophages ex vivo, while alachlor inhibited LPS/interferon-γ-induced NO production ex vivo. These results indicate that some EDCs exert modulatory activity on endotoxin-induced macrophage activation either positively or negatively, suggesting that these compounds may affect the development of infectious diseases. This is the first report that systematically compared the effect of EDCs on LPS action.

View full abstract

We studied the cognitive improving and cerebral protective constituents in the roots of Polygala tenuifolia WILLDENOW, a well-known Chinese traditional medicine prescribed for amnesia, neurasthenia, palpitation, noctural emission and insomnia. Tenuifoliside B (1), which is one of the acylated oligosaccharides in the roots of P. tenuifolia, showed the cerebral protective effect on potassium cyanide (KCN)-induced anoxia in mice, widely used as an animal model for cerebrovascular disease, and also had an ameliorative effect on the scopolamine-induced impairment of performance in passive avoidance task in rats. Compound 1 significantly enhanced oxotremorine-induced tremors in mice, suggesting that it ameliorated the scopolamine-induced impairment of passive avoidance response by enhancing the cholinergic system. These findings show that compound 1 has cognitive improving and cerebral protective effects.

View full abstract

The cultured cells and intact plants of Glycyrrhiza glabra (Fabaceae) produce betulinic acid and oleanane-type triterpene saponins (soyasaponins and glycyrrhizin). To elucidate the regulation of triterpenoid biosynthesis in G. glabra, the cDNA of lupeol synthase, an oxidosqualene cyclase (OSC) responsible for betulinic acid biosynthesis, was cloned, and expression patterns of lupeol synthase and two additional OSCs, β-amyrin synthase and cycloartenol synthase, were compared. The mRNA expression levels of lupeol synthase and β-amyrin synthase were consistent with the accumulation of betulinic acid and oleanane-type triterpene saponins, respectively. The transcript of lupeol synthase was highly expressed in the cultured cells and root nodules. The transcript of β-amyrin synthase, an OSC responsible for oleanane-type triterpene biosynthesis, was highly expressed in the cultured cells, root nodules and germinating seeds, where soyasaponin accumulates, and in the thickened roots where glycyrrhizin accumulates. In the cultured cells, the addition of methyl jasmonate up-regulated β-amyrin synthase mRNA and soyasaponin biosynthesis, but down-regulated lupeol synthase mRNA. Furthermore, the addition of gibberellin A₃ down-regulated β-amyrin synthase mRNA but not lupeol synthase mRNA in the cultured cells. The mRNA levels of cycloartenol synthase, an additional OSC responsible for sterol biosynthesis, in the intact plant and cultured cells were relatively constant in these experiments.

View full abstract

In the present study, we investigated the antioxidative effect of oolong tea in vitro and in vivo using the oxygen radical absorbance capacity (ORAC) assay. An oolong tea extract, catechin and related compounds suppressed the oxidation of fluorescence induced by AAPH in a dose-dependent manner, that is, they prolonged the antioxidant time in vitro. Oral administration of the oolong tea extract to mice treated with restraint stress increased ORAC activity in plasma as compared with a stress control group. The extract also increased plasma vitamin C levels, and there was a good relationship between ORAC activity and the vitamin C level in plasma. The elevation of plasma ORAC and vitamin C level may have been related to the stress-relieving effect of oolong tea. These effects are probably due to the antioxidative properties of the tea. Thus, these findings suggested that oolong tea has beneficial effects on health related to its antioxidative action.

View full abstract

Tectoridin isolated from the flowers of Pueraria thunbergiana (Leguminosae) are metabolized to tectorigenin by human intestinal microflora. When tectoridin was orally administered to rats, tectorigenin, but not tectoridin, was detected in urine after β-glucuronidase hydrolysis. The main metabolite tectorigenin potently inhibited the passive cutaneous anaphylaxis reaction and inhibited in vitro the release of β-hexosaminidase from RBL-2H3 cells induced by IgE. These results suggest that tectoridin is a prodrug, which can be transformed into the active agent tectorigenin by human intestinal bacteria and can be a candidate for antiallergic agent.

View full abstract

The effect of corosolic acid (CA) on blood glucose was studied in KK-Ay mice, an animal model of type 2 diabetes. CA (10 mg/kg) reduced the blood glucose (p<0.05) of KK-Ay mice 4 h after single oral administration when compared with the control group. However, CA did not change the plasma insulin. The muscle facilitative glucose transporter isoform 4 (GLUT4) translocation from low-density microsomal membrane to plasma membrane was significantly increased in the orally CA-treated mice when compared with that of the controls (p<0.05). These results suggest that the hypoglycemic effect of CA is derived, at least in part, from an increase in GLUT4 translocation in muscle. Therefore, it may be that CA has beneficial effects on hyperglycemia in type 2 diabetes.

View full abstract

Yomogin is an active compound isolated from Artemisia princep, a traditional Oriental medicinal herb, which has been shown to inhibit tumor cell proliferation. In this study, we investigated the effects of yomogin on the cytotoxicity, induction of apoptosis, and putative pathways of its actions in human promyelocytic leukemia cells. Yomogin-treated HL-60 cells displayed several features of apoptosis, including DNA fragmentation, formation of DNA ladders in agarose gel electrophoresis, and externalization of annexin-V targeted phosphatidylserine residues. We observed that yomogin caused activation of caspase-8, caspase-9, and caspase-3. A general caspase inhibitor (z-VAD-fmk), caspase-8 inhibitor (z-IETD-fmk) and caspase-3 inhibitor (z-DEVD-fmk), almost completely suppressed the yomogin-induced DNA fragmentation. We further demonstrated that yomogin induced Bid cleavage, mitochondrial translocation of Bax from the cytosol, and cytochrome c release from mitochondria in a caspase-8-dependent manner. Taken together, our data indicate that yomogin is a potent inducer of apoptosis and facilitates its activity via caspase-8 activation, Bid cleavage, Bax translocation to mitochondria, and subsequent release of cytochrome c into the cytoplasm, providing a potential mechanism for the anticancer activity of yomogin.

View full abstract

The inhibitory effects of 40 extracts prepared from 38 traditional Turkish folk medicines on human aldose reductase (h-AR) and hematological activity were investigated. Seven plants containing 5 species of Cistus genus exhibited a potent inhibition of h-AR. Ferulago amani (root) inhibited the platelet aggregation induced by sodium arachidonate, while C. laurifolius (fruit) was found to possess strong inhibition in the blood coagulation assay. An AcOEt extract derived from the leaf of C. laurifolius was purified to isolate three known flavonoids. The activity of one, quercetin-3-O-methyl ether, was found to be as potent as that of eparlestat, which is known to be a remedy for treating complications associated with diabetes.

View full abstract

An extract of the roots of Angelica dahurica BENTH. et HOOK. (Umbelliferae) showed potential tyrosinase inhibition against mushroom tyrosinase. Employing a bioassay-linked HPLC method, followed by semipreparative HPLC, the active principle was isolated and characterized as 9-hydroxy-4-methoxypsoralen.

View full abstract

Activity-guided fractionation based on topoisomerase I inhibitory activity lead to the isolation of ten lignans (1—10) from the methylene chloride extract of the bark of Machilus thunbergii SIEB. et ZUCC. (Lauraceae). These were identified as machilin A (1), erythro-austrobailignan-6 (2), meso-monomethyl dihydroguaiaretic acid (3), meso-dihydroguaiaretic acid (4), galbacin (5), machilin F (6), nectandrin A (7) nectandrin B (8), (−)-acuminatin (9) and (7S,8S)-7-(4-hydroxy-3-methoxyphenyl)-1′-formyl-3′-methoxy-8-methyldihydrobenzofuran (10) by spectral evidence. In DNA topoisomerase I and II assays in vitro at a concentration of 100 μM, 4 showed the most potent inhibitory activity, 93.6 and 82.1% inhibition, respectively, and 8 showed 79.1 and 34.3% inhibition, respectively. All of these compounds exhibited weak or no cytotoxicities against either the human colon carcinoma cell line (HT-29) or the human breast carcinoma cell line (MCF-7).

View full abstract

The effects of γ-oryzanol, a drug mainly used for the treatment of hyperlipidaemia, on several cytochrome P450 (CYP) specific reactions in human liver microsomes were investigated to predict drug interactions with γ-oryzanol in vivo from in vitro data. The following eight CYP catalytic reactions were used in this study: CYP1A1/2-mediated 7-ethoxyresorufin O-deethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated 7-benzyloxyresorufin O-debenzylation, CYP2C8/9-mediated tolbutamide methylhydroxylation, CYP2C19-mediated S-mephenytoin 4′-hydroxylation, CYP2D6-mediated bufuralol 1′-hydroxylation, CYP2E1-mediated chlorzoxazone 6-hydroxylation, and CYP3A4-mediated testosterone 6β-hydroxylation. γ-Oryzanol had little inhibitory effects on CYP activities, indicating that this compound would not be expected to cause clinically significant interactions with other CYP-metabolized drugs at expected therapeutic concentrations.

View full abstract

The tissue distribution after an intravenous dose of micafungin (1 mg/kg), a new echinocandin-like lipopeptide antifungal agent, to male rats was investigated. Micafungin in plasma disappeared biexponentially with a terminal half-life of 5.03 h. Micafungin concentrations in liver, kidney, and lung at the first sampling time (5 min) after dosing were 1.15, 1.64, and 2.58-fold higher than the plasma concentration, and the AUC_0—∞ were 1.61, 3.42, and 2.89-fold higher than that for plasma. The terminal half-lives for these tissues were 5.14, 4.87, and 5.31 h, respectively, which were comparable to those for plasma. These results suggest that micafungin distributes rapidly and moderately into tissues such as the liver, kidney, and lungs, and that the concentrations in tissues decreased in parallel with the unchanged drug in plasma.

View full abstract

Bupleuri Radix (Chai-hu in Chinese and Saiko in Japanese) is one of the most important traditional Chinese crude drugs for treating hepatitis malaria and intermittent fever. B. kaoi is one of the Bupleurum spp. families locally found in Taiwan. The effects of saponin-enriched fraction (SEF) from Bupleurum Kaoi in human non-small cell lung cancer A549 cells were investigated in this study. An enhancement in Fas and its two forms of ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), was responsible for the apoptotic effect induced by SEF. Taken together, our study suggests that the activity of the Fas/Fas ligand apoptotic system may participate in the antiproliferative activity of SEF in A549 cells.

View full abstract

Amlodipine, a cardiovascular drug, exhibited remarkable antibacterial action in vitro against 504 bacterial strains belonging to both Gram positive and Gram negative genera, as well as in vivo against a mouse-virulent bacterium. Based on such findings, the present study was undertaken to determine whether the efficacy of this non-antibiotic drug could be enhanced in the presence of any antibiotic. Twelve bacterial strains, sensitive to amlodipine as well as to 6 antibiotics, viz., benzyl penicillin, streptomycin, chloramphenicol, tetracycline, erythromycin and ciprofloxacin were chosen. Disc diffusion test with amlodipine and streptomycin revealed marked synergism between the combination, compared with their individual effects. The synergism was found to be statistically significant (p<0.01). To assess the degree of synergy, the checkerboard analysis was performed. The fractional inhibitory concentration (FIC) index of this combination turned out to be 0.24, which confirmed synergism. This antibiotic–non-antibiotic pair was then administered to mice, challenged with S. typhimurium to determine whether this was effective in vivo. Statistical analysis of the mouse protection tests suggested that the combination was highly synergistic (p<0.001), according to Student's t-test. This synergistic drug combination may help us in enhancing the scope of prolonged antibiotic therapy in various types of infections, and might open a new therapeutic approach to combat drug resistance in bacterial diseases.

View full abstract

Monoacetyldiglycerides purified from deer antler, and identical synthetic compounds, have been shown to stimulate hematopoiesis. In the present study, we synthesized eight monoacetyldiglycerides, one of which, 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (3), was structurally identical to one of the naturally occurring monoacetyldiglycerides and showed the most potent stimulation of hematopoiesis by colony forming unit in culture (CFUc) assay, having a stimulation index (SI) of 1.54±0.23 at a concentration of 1.0 μg/ml. Moreover, 1.0 μg/ml 3 showed potent growth-stimulation activities on megakaryocyte culture, long term culture of Lin⁻Sca-1⁺ cells with irradiated MS-5 stromal cells (SI, 1.69±0.16), and on the number of cobblestone colonies (SI, 10.4±0.25). In a murine model, 3, at concentrations of 0.5, 5 and 50 mg/kg/d, i.p. and p.o., effectively stimulated hematopoiesis in vivo 7 d after syngenic bone marrow transplantation of irradiated C57BL/6 mice, when assayed by the colony forming units in spleen (CFUs) assay. These data suggest that monoacetyldiglycerides may have significant clinical potential for the acceleration of hematopoiesis.

View full abstract