In this report, we have investigated the inhibitory action of four organic acids from Radix Isatidis on Escherichia coli growth was investigated at 37 °C by using a microcalorimeter. The four organic acids were: syringic acid, 2-amino-benzoic acid, salicylic acid, benzoic acid. In accordance with thermokinetic model, the pertaining relationships of the drugs, such as growth inhibitory ratio vs. concentration, maximal power-output vs. growth rate constant, growth rate constant vs. concentration, were obtained. Half-inhibitory concentration of the drugs, IC50, was obtained by quantitative analysis. From the view of thermodynamics and molecular structure, the relationship between quantity and effect of the four organic acids has been discussed. The functional groups on phenyl ring had important influence on the antibacterial activities. Our work suggests that microcalorimetry is a fast, simple and more sensitive method that can be easily performed and applied to study the anti-bacterial activities of organic acids from Radix Isatidis on microorganism compared to other biological methods.
Oxidative stress resulting from an imbalance between radical-generating and radical scavenging systems plays an important role in the pathogenesis of pulmonary fibrosis. Epigallocatechin-3-gallate (EGCG), a polyphenol and a major component of green tea, possess a potent antioxidant property. This study was designed to evaluate the potential antioxidative activity of EGCG in the plasma and lungs during bleomycin induced experimental pulmonary fibrosis. Intratracheal administration of bleomycin (6.5 U/kg body weight) to rats resulted in significant reduction of body weight, enzymic antioxidants (superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase) and non-enzymic antioxidants (reduced glutathione, vitamin C, vitamin E and vitamin A). Elevations in lung W/D (wet weight/dry weight) ratio, hydroxyproline content was observed with a synchronized increase in lipid peroxidation markers (thiobarbituric acid reactive substances and hydroperoxides). Intraperitoneal administration of EGCG at a dose of 20 mg/kg body weight significantly improved the body weight, enzymic and non enzymic antioxidants and considerably decreased the W/D ratio, hydroxyproline and lipid peroxidation marker levels. Histological observations also correlated with the biochemical parameters. Thus, this study confirms the beneficial use of EGCG in alleviating the oxidative stress induced during pulmonary fibrosis.
Deoxypodophyllotoxin (DPT), a naturally occurring flavolignan with anti-inflammatory activity, was isolated from Anthriscus sylvestris HOFFM., and we examined its effects on the expression of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-stimulated, murine macrophage-like RAW264.7 cells. Western blot analysis performed with specific anti-iNOS antibodies showed that a decrease in nitric oxide (NO) was accompanied by a decrease in the iNOS protein level. To clarify the mechanistic basis for DPT's ability to inhibit iNOS induction, we examined the effect of DPT on nuclear factor (NF)-κB transcriptional activity and DNA binding activity. DPT potently suppressed both reporter gene activity and DNA binding activity. These findings suggest that DPT in RAW264.7 cells abolished LPS-induced iNOS expression by inhibiting the transcription factor, NF-κB.
Clear cell renal cell carcinoma (CCRCC) is the most common pathological type of renal cell carcinoma and the main cause of renal carcinoma mortality. NDRG2, a new member of the N-Myc downstream-regulated gene (NDRG) family, is a focus for study at present. Up to now, its expression and function in carcinoma remain unclear. The aim of this study was to investigate its expression in CCRCC tissues and several renal carcinoma cell lines. The expression of NDRG2 was evaluated in renal cell carcinoma cell lines, tumor and adjacent non-tumor tissues from same clear cell renal cell carcinoma patients, by using immunohistochemistry, immunofluorescence, RT-PCR and Western blot. By immunohistochemistry and immunofluorescence we found that NDRG2 was predominantly located in the cytoplasm and membrane of renal carcinoma cancer cells, and the positive rate of NDRG2 in renal carcinoma specimens was 30.3% (40/132), which is significantly lower than 91.67% (121/132) in normal renal tissues (p<0.01). The average staining score in normal renal tissues was significantly higher than renal carcinoma (6.12±1.84 versus 2.65±1.23, p<0.01). Moreover, NDRG2 mRNA and protein were down-regulated in 6 fresh CCRCC tissues compared with their adjacent noncancerous tissues and normal tissues. Its expression was also lower in the human CCRCC-derived cell lines A-498 and 786-O than in the human proximal tubular cell lines HK-2 and HKC. These results indicated that NDRG2 might play an important role in the carcinogenesis and development of CCRCC and may function as a tumor suppressor in CCRCC.
Pyrroloquinoline quinone (PQQ) has been shown to play a role as an anti-oxidant in neuronal cells and prevent neuronal cell death in a rodent stroke model. DJ-1, a causative gene product for a familial form of Parkinson's disease, plays a role in anti-oxidative stress function by self-oxidation of DJ-1. In this study, the expression level and oxidation status of DJ-1 were examined in SHSY-5Y cells and primary cultured neurons treated with 6-hydroxydopamine (6-OHDA) or H2O2 in the presence or absence of PQQ. The pI shift of DJ-1 to an acidic point, which was observed in SHSY-5Y cells treated with 6-OHDA, was inhibited by PQQ. TOF-MS analyses showed that while the level of a reduced form of DJ-1, one of the active forms of DJ-1, was decreased in SHSY-5Y cells treated with 6-OHDA or H2O2, PQQ increased the level of the reduced form of DJ-1. These results suggest that PQQ prevents oxidative stress-induced changes in oxidative status of DJ-1. Therefore, the neuroprotective effects of PQQ on oxidative stress-induced neuronal death may be at least in part involved in increased level of an active form of DJ-1.
S-Benzylisothiourea compound A22 induces coccoid forms in Escherichia coli by inhibiting the function of the actin-like cytoskeletal protein, MreB. The minimum inhibitory concentration of A22 and the minimum concentration to induce coccoid forms for various pathogenic bacteria were determined. At 10 μg/ml, A22 induced coccoid forms in Shigella flexneri but did not inhibit the growth. No alteration of coccoid forms in the Gram-positive bacteria and anaerobic bacteria tested were observed following treatment with A22. To study the relationship between pathogenicity and alterations in bacterial shape, the infectious capacity of A22-induced coccoid S. flexneri was examined using CHO-K1 cells. Invasion of the coccoid cells was significantly reduced, however, no changes in adherence were observed. Using a mutant defective in the type III secretion apparatus, which delivers effectors to the host, we examined the secretion of effectors by A22-induced coccoid S. flexneri. The amount of secreted effectors in the coccoid cells was clearly decreased compared to rod-shaped cells. These results showed that the maintenance of rod-shaped cells by MreB in bacteria was essential for the secretion of effectors via the type III secretion system. Therefore, our results suggest that A22 is a useful lead compound for a novel anti-infectious agent without bactericidal activity and MreB is a candidate target site for development of new anti-infectious agents.
We measured the activity and stability of three mannosyltransferases to ascertain the mechanisms of changes in the antigenicity and the mannan structure of Candida albicans cells cultured at high temperature (37 °C) under acidic (pH 2.0) conditions in a liquid medium. The α-1,6-mannosyltransferase (α-1,6-MT) activity of the particulate-insoluble enzyme fractions prepared from C. albicans J-1012 (J-1012) cells cultured at 37 °C was retained compared to those at 27 °C, whereas β-1,2-mannosyltransferase II (β-1,2-MT II) activity was detected in the 27 °C fraction but not in the 37 °C fraction. Similar results were obtained in the fraction prepared from J-1012 cells cultured at pH 2.0. The α-1,6-MT activities of fractions prepared from C. albicans NIH B-792 (B-792) strain cells cultured at 37 °C were retained compared to those at 27 °C, whereas β-1,2-mannosyltransferase VI-6 (β-1,2-MT VI-6) activity was detected in the fraction of B-792 cells cultured at 27 °C but not detected in the 37 °C fraction. We also found that the β-1,2-MT II and β-1,2-MT VI-6 activity of C. albicans cells was more sensitive to both high temperature and low pH compared with α-1,6-MT activity.
We tested the effects of SI000413, a new formula, consisting of Pyrolae herba and Trachelospermi caulis, on type II collagen-induced arthritis (CIA). CIA was induced in DBA/1J mice by immunization with bovine type II collagen (CII) on days 1 and 21. SI000413 was orally administered 3 times per week throughout the experiment and indomethacin was served as a positive control. Clinical scores, the count of arthritic legs, levels of interleukin 6 (IL-6) and anti-CII antibody, and lymphocyte subsets in blood were examined. SI000413 suppressed CIA development in a dose dependent manner and reduced the incidence of arthritic legs in mice. Histological analysis showed administration of SI000413 reduced inflammatory signs and cartilage destruction. Serum levels of IL-6 and anti-CII antibody were significantly decreased in SI000413-treated mice and the percentages of CD4 T cell, CD8 T cell and B cell in blood were restored to normal levels. In conclusion, we demonstrate that SI000413 ameliorates CIA both clinically and histologically and inhibits the production of anti-CII antibody and pro-inflammatory cytokine in the CIA mouse. These findings suggest that SI000413 is a potential new therapeutic herbal formula for the treatment of RA.
Coumarins represent an important class of phenolic compounds with multiple biological activities, including inhibition of lipidic peroxidation and neutrophil-dependent anion superoxide generation, anti-inflammatory and immunosuppressor actions. All of these proprieties are essential for that a drug may be used in the treatment of inflammatory bowel disease. The present study examined intestinal anti-inflammatory activity of coumarin and its derivative, the 4-hydroxycoumarin on experimental ulcerative colitis in rats. This was performed in two different experimental settings, i.e. when the colonic mucosa is intact or when the mucosa is in process of recovery after an initial insult. The results obtained revealed that the coumarin and 4-hydroxycoumarin, at doses of 5 and 25 mg/kg, significantly attenuated the colonic damage induced by trinitrobenzenesulphonic acid (TNBS) in both situations, as evidenced macroscopically, microscopically and biochemically. This effect was related to an improvement in the colonic oxidative status, since coumarin and 4-hydroxycoumarin prevented the glutathione depletion that occurred as a consequence of the colonic inflammation.
This study was aimed to clarify the mechanisms of gastroprotection by centipedic acid (CPA), a natural diterpene from Egletes viscosa LESS. (Asteraceae) using ethanol-induced gastric mucosal damage in mice and gastric secretion in 4-h pylorus-ligated rats as model systems. In mice, intragastrically administered CPA (25, 50, 100 mg/kg) greatly reduced the mucosal lesions induced by 96% ethanol (0.2 ml, p.o.) by 18, 53, and 79%, respectively, whereas N-acetylcysteine (NAC, 300 mg/kg, i.p.), the reference compound produced a 50% inhibition. In 4-h pylorus-ligated rats, CPA (50 mg/kg) applied intraduodenally decreased both gastric secretory volume and total acidity. Similar to NAC, the plant diterpene effectively prevented the ethanol associated decrease in non-proteic sulfhydryls (NP-SH) and the elevated thiobarbituric acid-reactive substances (TBARS) in gastric tissue, suggesting that these compounds exert an antioxidant effect. Pretreatment of mice with indomethacin, the cyclooxygenase inhibitor but not with capsazepine, the transient receptor potential vanilloid-1 (TRPV1)-receptor antagonist greatly suppressed the gastroprotective effect of CPA. Furthermore, CPA gastroprotection was significantly attenuated in mice pretreated with L-NAME or glibenclamide the respective inhibitors of nitric oxide synthase and K+ATP channel activation. These data suggest that CPA affords gastroprotection by different and complementary mechanisms, which include a sparing effect on NP-SH reserve, and roles for endogenous prostaglandins, nitric oxide, and TRPV1-receptor and K+ATP channel activation.
Angiotensin II (Ang II) is an important mediator stimulating liver fibrosis after liver injury. However, it is not known whether Ang II plays a role in liver regeneration. Here, we investigate the effects of Ang II type 1 (AT1) receptor blocker (ARB), angiotensin-converting enzyme inhibitor (ACEI), systemic infusion of Ang II, and genetic deficiency of the AT1a receptor (AT1a-KO) on the hepatic regenerative response to partial hepatectomy (PH) in mice. Administration of ARB (candesartan cilexetil and losartan) or ACEI (enarapril and lisinopril) enhanced 5-bromo-2′-deoxyuridine (BrdU) incorporation into hepatocyte nuclei in remnant liver as well as the restoration of liver weight after PH. Systemic infusion of Ang II (100 ng/kg/min) suppressed the PH-induced BrdU incorporation and the restoration of liver weight. In contrast to Ang II infusion, these hepatic responses to PH were significantly greater in AT1a-KO mice than in wild-type mice. The PH-induced increases in hepatic levels of hepatocyte growth factor (HGF) mRNA and plasma HGF concentrations were greater in candesartan- and enarapril-treated mice or in AT1a-KO mice than in vehicle-treated mice or wild-type mice, respectively, whereas they were less in Ang II-infused mice than in vehicle-infused mice. In contrast to HGF, blockades of the renin-angiotensin system or Ang II infusion produced opposite effects on the PH-induce increases in hepatic transforming growth factor (TGF)-beta 1 mRNA and plasma TGF-beta 1 levels. These studies suggest that Ang II plays a role in the liver regeneration as a suppressor of hepatocyte proliferation via the AT1 receptor-mediated control of growth factor production.
Visceral fat accumulation has been reported as the most important risk factor for the development of various metabolic disorders. In this study, the preventive effects of Bofutsushosan, a Japanese Kampo preparation, on obesity and various metabolic disorders were investigated focusing on visceral fat accumulation using Tsumura, Suzuki, Obese, Diabetes (TSOD) mice, which showed significant accumulation of visceral fat, and developed metabolic disorders including glucose intolerance, hyperlipidemia, hypertension and hyperinsulinemia. At 2 months after initiation of the study, the control TSOD mice developed various metabolic disorders such as marked obesity and visceral fat accumulation, increases in the levels of blood glucose, insulin, total-cholesterol (TC) and triglyceride (TG), and abnormal glucose tolerance, hypertension and peripheral neuropathy as distinct from the control Tsumura, Suzuki, Non-Obesity (TSNO) mice, which do not develop obesity and various metabolic disorders. In the TSOD mice treated with Bofutsushosan, body weight gain and visceral/subcutaneous fat accumulation were significantly suppressed. Biochemical parameters in plasma (glucose, TC, insulin and tumor necrosis factor-α (TNF-α) level) were significantly suppressed, and abnormal glucose tolerance, elevation of blood pressure and peripheral neuropathy accompanying progression of metabolic disorders were also significantly suppressed. On the other hand, in TSNO mice, Bofutsushosan showed no noteworthy impacts on most parameters except for an improvement of the lipid plasma level. The above results suggested that Bofutsushosan could be effective in preventing obesity and various metabolic disorders.
The aim of this study was to evaluate the relationship between the anticholinergic side effects associated with disopyramide (DP) and serum DP or mono-N-dealkyldisopyramide (MND) concentrations and the safety range of DP or MND for prevention of anticholinergic side effects in 141 inpatients. The serum DP and MND concentrations were determined by high-performance liquid chromatography. No correlation was observed between creatinine clearance (Ccr) and the ratio of the serum concentration to the dose (C/D) of DP, but a significant inverse correlation was observed between Ccr and the C/D of MND. It was observed that the ratio of MND concentration to DP concentration in the group, whose Ccr was below 20 ml/min, was higher than that of the other groups. Although no significant difference was observed in the DP concentration between the patients without (Group 1) or with (Group 2) anticholinergic side effects, significant differences were observed in the MND concentration, Ccr, and the ratio of MND/DP. The DP concentrations of both Groups 1 and 2 were distributed from 0.13 to about 5 μg/ml. On the other hand, although the MND concentrations of Group 1 were below about 1 μg/ml, the MND concentrations of Group 2 were above about 1 μg/ml. These results suggest that not only DP concentration but also MND concentration should be monitored in patients whose renal function is decreased to prevent anticholinergic side effects associated with DP, and that when serum MND concentration was over approximately 1 μg/ml, the dose should be decreased or discontinued.
Paroxetine is often coadministered with low-dose aspirin to treat cerebral infarction patients with depression. In order to assess the ulcerogenic risk induced by the coadministration of these drugs, we examined their effects on gastric secretion using the pylorus ligation method and determined the prostaglandin E2 (PGE2) content in the gastric mucosa using enzyme immunoassay (EIA). Paroxetine stimulated the gastric fluid secretion and acid output, dose-dependently, however, it had no effect on the PGE2 content in the gastric mucosa. Furthermore, exogenous serotonin inhibited both the gastric fluid secretion and the acid output. The coadministration of low-dose aspirin and paroxetine significantly enhanced the gastric fluid secretion and acid output, thus resulting in gastric bleeding, but it hardly affected the PGE2 content in the gastric mucosa. The stimulation of gastric secretion by the coadministration of low-dose aspirin and paroxetine is therefore considered to play an important role in the development of gastric injuries.
Rosmarinic acid (RA) is one of major polyphenolic ingredients of Perillae Herba (a leaf of Perilla frutescens), and has an antidepressant-like property in animal models of depression. However, the mechanism(s) underlying this activity are unknown. Recent studies have reported that regulation of hippocampal neurogenesis is associated with the pathogenesis of depression. To elucidate the mode of action of RA-induced antidepressant-like activity, proliferative effect of RA on newborn cells in the dentate gyrus of mouse hippocampus was investigated using immunohistochemical analysis with bromodeoxyuridine (BrdU), a marker of proliferating cells. RA treatment for 7 or 14 d significantly increased in the number of BrdU-positive cells in inverse correlation with significant reductions in immobility in a forced swimming test, an animal model of depression, in a dose-dependent manner. However, locomotor activities were not affected. These results suggest that RA produces an antidepressant-like effect at least in part via the proliferation of newborn cells in the dentate gyrus of the hippocampus.
Salvianolic acid B (Sal B) is one of the major water-soluble compounds isolated from Radix Salviae Miltiorrhizae (Danshen in Chinese) that has been reported to be beneficial to treatment of diabetic complications. However, the mechanisms involved in these effects are not discussed in relation to mesangial proliferation via modulation of NF-κB. To explain this, human mesangial cells were pretreated with or without Sal B (0.1, 1, 10 μM) for 24 h and stimulated with high glucose (30 mM). Then the effects of Sal B on mesangial cells proliferation, extracellular matrix production and the possible mechanisms were evaluated by methylthiazoletetrazolium assay, flow cytometry assay, enzyme-linked immunosorbent assay, gelatin zymography assay and western blot assay. These results indicated that Sal B could inhibit high glucose-induced mesangial cells proliferation and extracellular matrix production in a dose-dependent manner, partially through modulating the cell-cycle progress and MMP-2 and MMP-9 activities via suppressing NF-κB activation, suggesting that Sal B may be a promising agent for treating diabetic nephropathy.
Schisandrin B (Sch B), a dibenzocyclooctadiene derivative isolated from the fruit of Schisandra chinensis, has been shown to enhance mitochondrial antioxidant status in liver, heart and brain tissues in rodents. Whether or not long-term Sch B treatment can protect against oxidative stress-induced cerebral damage remains unclear. In the present study, the effect of long-term Sch B treatment (1—30 mg/kg/d×15) on cerebral ischemia/reperfusion (I/R) injury was examined in rats. Sch B treatment protected against I/R-induced cerebral damage, as evidenced by the significant increase in the percentage of 2,3,5-triphenyl tetrazolium chloride (TTC)-stained tissues in representative brain slices, when compared with the Sch B-untreated and I/R control. The cerebroprotection was associated with an enhancement in cerebral mitochondrial antioxidant status, as assessed by the level/activity of reduced glutathione, α-tocopherol and Mn-superoxide dismutase, as well as the improvement/preservation of mitochondrial structural integrity, as assessed by the extents of malondialdehyde production, Ca2+ loading and cytochrome c release, as well as the sensitivity to Ca2+-induced permeability transition, in control and I/R-challenged rats. In conclusion, long-term Sch B treatment could enhance cerebral mitochondrial antioxidant status as well as improve mitochondrial structural integrity, thereby protecting against I/R injury.
Considering the importance of inflammation and apoptosis in neurodegenerative conditions, the potential suppressive effects of the Rg3, a by-product obtained during the steaming of red ginseng, may indicate that Rg3 could provide a beneficial therapeutic approach to treating or preventing neurodegenerative disease. We investigated the effect of Rg3 on Aβ42-mediated microglial activation and inflammation-mediated neurotoxicity in murine BV-2 microglial and Neuro-2a neuroblastoma cells, respectively. Rg3 effectively reduced inflammatory cytokine expression in Aβ42-treated BV-2, and inhibited the binding of NF-κB p65 to its DNA consensus sequences, and significantly reduced the expression of TNF-α in activated microglia. Pretreatment with Rg3 increased the survival rate of Neuro-2a exposed to TNF-α. These observations suggest that Rg3 reduced neurotoxicity by inhibiting chronic inflammation through the suppression of activated microglia. In addition, the expression of pro-inflammatory cytokines in BV-2 stimulated by Aβ42 was decreased but not eliminated by Rg3 when binding to the macrophage scavenger receptor type A (MSRA) was blocked with fucoidan. This implies that the inflammatory response may not be exclusively triggered via MSRA. More interestingly, iNOS was almost completely inhibited in the presence of Rg3 when MSRA binding was blocked with fucoidan. Moreover, Rg3 increased the expression of MSRA in BV-2 transfected with siRNA targeting MSRA mRNA, and this increased MSRA expression may play a role in the phagocytosis of Aβ42 peptides. Our results indicate that inhibition of the inflammatory repertoire of microglia, neuroprotection, and increased MSRA expression induced by Rg3 may at least partly explain its therapeutic effects in chronic neurodegenerative diseases.
Amiodarone (AMD)-induced pulmonary toxicity (AIPT) is the most life-threatening side-effect of AMD treatment. N-Monodesethylamiodarone (DEA), an active metabolite of AMD, also exhibits cytotoxicity and tends to accumulate in the lung more intensively than AMD. In this study, we characterized the mechanism of DEA accumulation using A549 cells as a model of the alveolar epithelium. Typical ATP-depletion compounds caused an approximately 30% increase in the accumulation of DEA in A549 cells, although these effects were less than those in Caco-2 cells. Triiodothyronine (T3), which exhibited an inhibitory effect on DEA efflux in Caco-2 cells, did not affect the accumulation of DEA in A549 cells. On the other hand, 100 μM AMD caused an approximately 200% increase in DEA content in A549 cells, although AMD accumulation was not affected by 100 μM DEA. Since the reducing effect of AMD on cellular ATP levels and that of FCCP were similar, the mechanism by which DEA accumulation is increased by AMD might be different from the ATP-dependent DEA efflux mechanism. The decrease in cell viability by DEA in the presence of AMD (IC50 value of DEA for A549 cell viability: 25.4±2.4 μM) was more pronounced than that by DEA alone (IC50 value: 11.5±3.0 μM). This further DEA accumulation by AMD might be a factor responsible for the greater accumulation of DEA than that of AMD in the lung in long-term AMD-treated patients.
The microtubule-dependent motor protein Eg5 is essential for the development and function of the mitotic spindle. Now it has become an anti-mitotic drug target in high throughput screening for anticancer dugs in vitro. Here is a protocol for cloning, expression and purification of a human Eg5 that codes for motor and linker domain in Escherichia coli BL21 (DE3) cells. The effects of temperature, pH, metal ions and DMSO on ATPase activity were investigated. A new compound CPUYL064 showed good inhibitory effect against Eg5 (IC50 value, 100 nM). It inhibited the proliferation of human hepatocellular liver carcinoma cell line HepG2 in a dose- and time-dependent manner. CPUYL064 induced a clear G2/M phase arrest and caused the monastral spindle in HepG2 cells. Induction of apoptosis was further confirmed by changes in membrane phospholipids, changes in mitochondrial membrane potential and by detection of DNA fragmentation. These results indicate that CPUYL064 could be developed as a new, potent mitotic arrest compound.
We assessed the effects of different classes of flavonoids on insulin-stimulated 2-deoxy-D-[1-3H]glucose uptake by mouse MC3T3-G2/PA6 cells differentiated into mature adipose cells. Among the flavonoids examined, the flavones, apigenin and luteolin, the flavonols, kaempferol, quercetin and fisetin, an isoflavone, genistein, a flavanonol, silybin, and the flavanols, (−)-epigallocatechin gallate (EGCG) and theaflavins, significantly inhibited insulin-stimulated glucose uptake. Key structural features of flavonoids for inhibition of insulin-stimulated glucose uptake are the B-ring 4′- or 3′,4′-OH group and the C-ring C2–C3 double bond of the flavones and flavonols, the A-ring 5-OH of isoflavones, and the galloyl group of EGCG and theaflavins. Luteolin significantly inhibits insulin-stimulated phosphorylation of insulin receptor-β subunit (IR-β), and apigenin, kaempferol, quercetin and fisetin, also tended to inhibit the IR-β phosphorylation. On the other hand, isoflavones, flavanols or flavanonols did not affect insulin-stimulated IR-β phosphorylation. Apigenin, luteolin, kaempferol, quercetin and fisetin also appeared to inhibit insulin-stimulated activation of Akt, a pivotal downstream effector of phosphatidylinositol 3-kinase (PI3K), and suppressed insulin-dependent translocation of a glucose transporter, (GLUT)4, into the plasma membrane. Although genistein, silybin, EGCG and theaflavins had no effect on the insulin-stimulated activation of Akt, they blocked insulin-dependent GLUT4 translocation. These results provide novel insights into the modulation by flavonoids of insulin's actions, including glucose uptake in adipocytes.
Estrogens, a group of steroid hormones, act primarily by regulating gene expression after binding with estrogen receptor (ER), a nuclear ligand-activated transcription factor, translocates to the nucleus after dimer formation, enhances the gene transcription. Estrogen Receptor Modulators (ERMs) have selective agonist and antagonist effects to different tissues, and the purpose of research on ERMs is to identify new potent and less toxic drug molecules. The present study has been focused on finding the structural requirements of ER ligand, using receptor-independent pharmacophore space modeling studies that can explore 3D structural features and configurations, responsible for the biological activity of structurally diverse compounds. The studies show (R=0.945, RMSD=2.186, Δcost=677.354) the importance of hydrogen bond acceptors in the aromatic rings and a planner hydrophobic region in the molecular architecture along with critical geometrical distance between features are effectively crucial for binding with ER.
Some novel Mannich base isatin derivatives were synthesized by reacting 1-(5-methyl-2-oxoindolin-3-ylidene)-4-(substitutedpyridin-2-yl)thiosemicarbazide with formaldehyde and several secondary amines. Their chemical structure was elucidated by means of spectral (FT-IR, 1H- and 13C-NMR and mass) analysis. Investigation of anti-inflammatory activity of synthesized compounds was done by carrageenan induced paw oedema method using diclofenac sodium as standard drug and analgesic activity was done by acetic acid induced writhing method. The synthesized compounds showed significant anti-inflammatory and analgesic activity.
The symbiosis between Armillaria species and an achlorophylous orchid Gastrodia elata BLUME has been reported. The main species described as a symbiont is Armillaria mellea (VAHL: FR.) KUMMER, known widely as a primary root rot pathogen. Samples collected from the rhizomorphs attached to the tuber of G. elata were separated and analyzed. Molecular analysis based on sequencing of the intergenic spacer 1 (IGS-1) and internal transcribed spacer (ITS) regions of the ribosomal DNA (rDNA) was performed, coupled with restriction fragment length polymorphism (RFLP) of the IGS-1 region. Cultural morphology and features of basidiomata were also used to characterize the isolates. Phylogenetic analysis and morphological data strongly suggested that the fungus present in the tubers of G. elata is Armillaria nabsnona. This is the first report of occurrence of this Armillaria species in association with G. elata.
This study evaluates the protective effect of Juniperus chinensis hot water extract (JCE) against high-fat-diet (HFD)-induced obesity and its molecular mechanisms in the visceral adipose tissue of rats. JCE supplementation significantly lowered body weight gain, visceral fat-pad weights, blood lipid levels, and blood insulin and leptin levels of rats rendered obese by an HFD. Feeding with JCE significantly reversed the HFD-induced down-regulation of the epididymal adipose tissue genes implicated in adipogenesis, such as the peroxisome proliferator-activated receptors γ2 (PPARγ2), adipocyte protein 2 (aP2), sterol regulatory element binding protein 1c (SREBP1c), fatty acid synthase (FAS), and HMG-CoA reductase (HMGR), as well as those involved in uncoupled respiration, such as the uncoupling protein 2 (UCP2) and uncoupling protein 3 (UCP3). Dietary supplementation with JCE also reversed the HFD-induced decreases in the AMP-activated protein kinase (AMPK) and the acetyl-CoA carboxylase 2 (ACC2) expressions at both the mRNA and protein levels and restored the HFD-induced inhibitions in the AMPK and ACC2 phosphorylation, which are related to fatty acid β-oxidation, in the epididymal adipose tissue. This study reports, for the first time, that the JCE can have an anti-obesity effect in a rodent model with HFD-induced obesity through an enhanced gene transcription of the uncoupling protein as well as an elevated AMPK protein expression and phosphorylation in the visceral adipose tissue.
In our previous study, we reported the renoprotective effect of Hachimi-jio-gan, a Chinese traditional prescription consisting of eight medicinal plants, and also reported the effect of Corni Fructus (Cornus officinalis SIEB. et ZUCC.), a component of Hachimi-jio-gan, on diabetic nephropathy using diabetic rats. In this study, we investigated the effects of morroniside isolated from Corni Fructus on renal damage in streptozotocin-treated diabetic rats. Oral administration of morroniside at a dose of 20 or 100 mg/kg body weight/d for 20 d to diabetic rats resulted in significant decreases in increasing serum glucose and urinary protein levels. Moreover, the decreased levels of serum albumin and total protein in diabetic rats were significantly increased by morroniside administration at a dose of 100 mg/kg body weight/d. In addition, morroniside significantly reduced the elevated serum urea nitrogen level and showed a tendency to reduce creatinine clearance. Morroniside also significantly reduced the enhanced levels of serum glycosylated protein, and serum and renal thiobarbituric acid-reactive substances. Protein expressions related to the advanced glycation endproduct (AGE) level and actions, oxidative stress such as Nε-(carboxyethyl)lysine, as well as receptors for AGE and heme oxygenase-1 were increased in diabetic rats, but the levels were also significantly decreased by the administration of morroniside. This suggests that morroniside exhibits protective effects against diabetic renal damage by inhibiting hyperglycemia and oxidative stress. These results indicate that morroniside is one component partly responsible for the protective effects of Corni Fructus and Hachimi-jio-gan against diabetic renal damage.
Naphthoquinones and other compounds with antimycobacterial activity against Mycobacterium tuberculosis have previously been isolated from Euclea species. In this study, several constituents of Euclea natalensis and E. undulata, as well as organic extracts of the leaves, were assessed for efficacy against the zoonotic pathogen, Mycobacterium bovis. Also included in the battery of test organisms were M. bovis BCG and the fast-growing species M. smegmatis and M. fortuitum. The acetone extract of E. natalensis had potent activity against M. bovis (MIC=26 μg/ml). The naphthoquinone 7-methyljuglone was the most active compound, with an MIC as low as 1.55 μg/ml against pathogenic M. bovis. M. bovis BCG was not as susceptible to the test compounds as the pathogenic strain, but similar patterns of activity were observed between all the strains tested. M. smegmatis appeared to be a better predictor of antimycobacterial activity against pathogenic M. bovis (and M. tuberculosis), while MIC values obtained using M. fortuitum correlated well with those of M. bovis BCG.
Agrobacterium-mediated transformation of leaf tissues of Atropa belladonna with an adenosine 5′-diphosphate (ADP)-ribosylation factor gene of carrot, arf-001, was performed employing pBCR82 as an expression vector. This vector co-expresses rol gene cluster together with arf-001, and thus, the transformed host cells were obtained as hairy roots. Two cell lines of the transformed belladonna were established as the liquid cultures of hairy root tissues, and expression of arf-001 and accumulation of its product in the cells were confirmed by RT-PCR and Western blot analyses, respectively. A marked increase in extracellular protein concentrations was observed in the transformed belladonna root cultures as compared with the controls transformed with an empty vector. However, the secretion of the proteins of the transformants was markedly reduced in the presence of a physiological concentration of monensin. These results suggest that over-expression of arf-001 in belladonna results in the enhancement of secretory activity in the transformed cells.
Emphysema, one of chronic obstructive pulmonary disease (COPD), is characterized as destruction of airway wall and small airway inflammation. To assess the kinetics of disease progression in murine model of elastase-induced emphysema, we used micro-computed tomography (CT) compared with morphological changes. Two week after elastase administration, a significant increase in the volume of low-density areas, recognized as −800—−600 Hounsfield units by micro-CT, was observed. Coefficient of correlation between mean linear intercept (Lm) and low-density area examined by CT, was 0.79 (p<0.01). Micro-CT can quantitatively and sequentially detect murine emphysematous changes, offering a practical method to sequentially analyze the therapeutic effects of treatments in a murine model of emphysema.
The aim of this study was to prepare a pentazocine (PTZ) matrix-type transdermal drug delivery system (TDDS) using acrylic pressure-sensitive adhesives. Among the five Duro-Tak® adhesive polymers tested (87-9301, 87-2677, 87-201A, 87-2196, 87-2852), in vitro dissolution studies demonstrated the highest PTZ release flux from the Duro-Tak® 87-9301 matrix. In addition, the effects of permeation enhancers, isopropyl myristate (IPM) and glyceryl monocaprylate (GEFA-C8), and drug content on PTZ skin permeation from prepared patches were evaluated using Franz diffusion cells fitted with hairless mouse skin. IPM and GEFA-C8 were found to produce effective flux of PTZ at a patch concentration of 10% w/w and 5% w/w, respectively. The PTZ flux increased linearly as the loading dose increased up to 30%, whereas no further increase in flux was observed at loading doses of 40% and 50% due to drug crystallization in the matrix. Thus, the highest skin permeation rate (24.2 μg/cm2/h) was achieved when 30% of PTZ was loaded in Duro-Tak® 87-9301 with 10% IPM and 5% GEFA-C8. These results demonstrate the feasibility of a novel narcotic-antagonist analgesic matrix-type TDDS for PTZ.
We previously prepared 2-hydroxypropyl-β-cyclodextrin (HPβCD) solutions containing disulfiram (DSF) and hydroxypropylmethylcellulose (HPMC, DSF solutions), and found the instillation of this DSF solutions delayed lens opacification in ICR/f rats, a recessive-type hereditary cataractous strain. In this study, we determined the corneal penetration mechanism of DSF solutions using human cornea epithelial cell monolayers based on the immortalized human cornea epithelial cell line (HCE-T) developed by Tropainen et al. [Invest. Ophthalmol. Vis. Sci., 42, 2942—2948 (2001)]. The transepithelial electrical resistance (TER) values of HCE-T cells increases from approximately 275 to 388 Ω·cm2 by exposure to an air–liquid interface for 2 weeks. The penetration of DSF into the basolateral chamber was prevented by the increase in TER values. The DSF in solution was converted to diethyldithiocarbamate (DDC) during the penetration experiment using HCE-T cell monolayers, and a close relationship between the penetration coefficient of DDC and aldehyde dehydrogenase (ALDH) 3A1 mRNA expression (y=41.202x+18.587, R=0.9413) was observed. In addition, an anti-ALDH3A1 antibody significantly inhibited the DSF-DDC conversion. These results suggest that DSF in DSF solutions is converted to DDC via catalysis by an ALDH3A1 in the cornea, and then transited from the apical side to the basolateral side.