The circadian pacemaker of mammals resides in the paired suprachiasmatic nuclei (SCN) and influences a multitude of biological processes, including the sleep-wake rhythm. Clock genes are the genes that control the circadian rhythms in physiology and behavior. Twenty-four hour rhythm has been demonstrated for the function of physiology and the pathophysiology of diseases. The effectiveness and toxicity of many drugs vary depending on dosing time. Such chronopharmacological phenomena are influenced by not only the pharmacodynamics but also the pharmacokinetics of medications. Thus, knowledge of the 24 h rhythm in the risk of disease plus evidence of 24 h rhythm dependencies of drug pharmacokinetics, effects, and safety constitutes the rationale for pharmacotherapy. One approach to increasing the efficiency of pharmacotherapy is the administration of drugs at times at which they are most effective and/or best tolerated. Drugs for several diseases are still given without regard to the time of day. Identification of a rhythmic marker for selecting dosing time will lead to improved progress and diffusion of chronopharmacotherapy. The mechanisms underlying chronopharmacological findings should be clarified from the viewpoint of clock genes. On the other hand, several drugs have an effect on the circadian clock. The knowledge of interactions between the circadian clock and a drug should be very useful in clinical practice. Therefore, the aim of this review is to provide an overview of the dosing time-dependent alterations in therapeutic outcome and safety of drugs. The mechanisms are introduced from the viewpoint of pharmaceutics.
The resorcylic acid lactone hypothemycin has been shown to inactivate protein kinases by binding to a cysteine conserved in 46 protein kinases, including mitogen-activated protein kinase kinase (MEK), extracellular signal-regulated kinase (ERK) and platelet-derived growth factor receptor (PDGFR). We assessed the selectivity of hypothemycin in cellular contexts. Hypothemycin normalized the morphology and inhibited anchorage-independent growth of Ki-ras transformed normal rat kidney (NRK) cells with selectivity and potency comparable to or greater than that of the MEK inhibitor U0126. In Ki-ras-transformed and phorbol 12-myristate 13-acetate (PMA)-treated NRK cells, hypothemycin blocked ERK activation but showed a minimal effect on autophosphorylation of protein kinase D1 (PKD1), another kinase containing the conserved cysteine. Hypothemycin potently inhibited PDGFR autophosphorylation and activation of the MEK-ERK pathway in platelet-derived growth factor (PDGF)-treated NRK cells. However, the phosphoinositide-3-kinase (PI3K) pathway was only modestly attenuated. Hypothemycin also inhibited growth factor- and anchorage-independent growth of human cancer cell lines with a constitutively active MEK-ERK pathway. Although hypothemycin has the potential to inactivate various protein kinases, the results indicate that in intracellular environments, hypothemycin can inhibit the MEK-ERK axis with sufficient selectivity to normalize transformed phenotypes of cells dependent on this pathway.
Timolol, a beta-blocker, has been shown to be an effective ocular hypotensive agent when used alone or with carbonic anhydrase inhibitor on ocular hypertensive or open angle glaucoma patients. The effect of timolol hemihydrate on the CO2 hydration activities of human carbonic anhydrase (HCA) I and II and their reaction mechanisms were investigated. Timolol activates the enzyme activities of HCA I and HCA II. In HCA I and II, the enzyme kinetic results clearly showed that timolol increases the value of Vmax but does not influence the value of Km. The enzyme kinetic method showed that timolol noncompetitively activates HCA I and II activities through the formation of a ternary complex consisting of the enzyme, the substrate, and timolol. These results indicate that timolol binds apart from the narrow cavity of the active site. AutoDocking results showed that timolol binds at the entrance of the active site cavity in a region where the proton shuttle residue, His 64, of HCA I or II, is placed. The enzyme kinetic and AutoDocking results showed that timolol might weakly bind near the proton shuttle residue, His 64, to accelerate the proton transfer rate from His 64 to the buffer components. It is known that efficient activators of carbonic anhydrase possess a bulky aromatic/heterocyclic moiety and a primary/secondary amino group in their molecular structure. Timolol has a heterocyclic moiety and a secondary amino group, which are typical structures in efficient activators of carbonic anhydrase.
The vascular endothelial growth factor (VEGF) receptor 2 (VEGFR-2), also called fetal liver kinase 1 (FLK1) in mice and kinase insert domain receptor (KDR) in humans, is an endothelial cell specific receptor tyrosine kinase that mediates lung cancer angiogenesis. We hypothesized that an active immunotherapy approach targeting FLK1 may inhibit lung cancer growth and metastasis. To test this hypothesis, we evaluated whether immune responses to FLK1 could be elicited in mice by immunization with an orally administered DNA vaccine encoding the extracellular domain (ECD) of FLK1 (pcDNA3.1-FLK1ECD) carried by attenuated Salmonella typhimurium. We found that the vaccine was effective at protective antitumor immunity in Lewis lung carcinoma models in mice by breaking immune tolerance to FLK1 self-antigen. Both FLK1-specific humoral and cellular immune responses against endothelial cells can be induced in mice by immunization with pcDNA3.1-FLK1ECD. Immunization with pcDNA3.1-FLK1ECD resulted in tumor suppression and prolonged survival in mice challenged with Lewis lung carcinomas cells. Experimental pulmonary metastases were strongly inhibited in pcDNA3.1-FLK1ECD immunized mice challenged with Lewis lung carcinoma cells. Thus, we conclude that the plasmid DNA vaccine encoding the extracellular domain of FLK1 could be an important component of FLK1 DNA vaccine to prevent lung carcinoma recurrence and metastasis after surgery.
Chunpoong is one of the most valuable cultivars of Panax ginseng C. A. MEYER, and is widely grown in Korea and China. Insertion/deletion (InDel) markers and single nucleotide polymorphism (SNP) markers are useful tools for marker-assisted selections. The SNP marker for determinate Chunpoong was previously developed from the nad7 gene of mtDNA by Wang et al. (2009) but was effective only on a limited range of cultivars. In this study, we studied the reasons for this limited application and developed new useful markers for application in Chunpoong-breeding programs. The new markers of InDel and SNP were designed in the major latex-like protein (MLP-like) gene which was highly expressed in 4-year-old Chunpoong expressed sequence tags (ESTs). To validate the marker in polymerase chain reaction (PCR), we used an InDel marker for identification of Chunpoong in the 70 Panax samples based on a double-blind test, and the success rate was 100%. For rapid and reliable assay of Chunpoong in numerous samples, we utilized an EvaGreen dye and melting curve method on real-time PCR. Furthermore, we designed an SNP marker that depended on the InDel region for more efficient detection of Chunpoong in real-time PCR. Compared with PCR-based assays, our Chunpoong SNP marker and real-time PCR assay offers a significant savings of time and labor in the analysis of large numbers of Chunpoong samples.
Tribenoside has been used clinically for hemorrhoidal disease associated with coagulation, inflammation, and wounds. However, the pharmacological mechanism of tribenoside activity has never been clear. In this study we examined whether tribenoside affected expression and deposition of laminins that are required for reconstruction of basement membranes (BMs) during wound healing in hemorrhoidal disease. HaCaT cells, which are derived from human epidermis, were treated in growth media supplemented with tribenoside. Reverse transcriptase-polymerase chain reaction (RT-PCR) using primers specific for laminin chains showed that HaCaT cells constitutively expressed laminin α3, α5, β1, β3, γ1, and γ2 chains. Tribenoside treatment of HaCaT cells did not induce expression of other laminin chains. We also quantified the expression of laminin chains in tribenoside-treated cells using real-time PCR. The expression level of laminin α3, β1, β3, γ1, and γ2 chains was not affected. In contrast, the expression of laminin α5 in the tribenoside-treated cells was four times higher than that of control cells. Immunocytochemistry also showed that tribenoside accelerated the focal deposition of laminin-332 (α3, β3, γ2). These results suggest that tribenoside interacts with epidermal cells and regulates the expression and localization of laminins to help reconstruct BMs in wound healing of hemorrhoids.
Dendritic cells (DCs) play an essential role in the immune system. The transition from immature DC (iDCs) to mature DCs (mDCs) requires appropriate stimuli such as pro-inflammatory cytokines. Proteoglycans (PGs) are one of the main components of extracellular matrix, and some types of PGs are known to induce maturation of murine DCs. Recent studies have investigated the potential benefits of PG from nasal cartilage of salmon head (S-PG). This study investigated the effects of S-PG on maturation of human monocyte-derived DCs. iDCs were prepared from human monocytes using the appropriate cytokines and then stimulated by S-PG alone. In another experiment, iDCs were stimulated by a combination of pro-inflammatory cytokines (MIX) plus S-PG. Although the stimulation of S-PG alone did not induce phenotypic maturation from iDCs, CD40 expression on DCs stimulated by S-PG alone was lower than that of iDCs. In contrast, the phenotypic and functional characteristics of DCs stimulated by MIX+S-PG were similar to those of DCs stimulated by MIX alone. As a result, S-PG did not demonstrate a significant effect with regard to maturation of human monocyte-derived DCs.
Phx-3, one of the phenoxazine derivatives, is reported to have inhibitory effect on Mycobacterium species and Chlamydia pneumoniae but not Escherichia coli, Salmonella Typhimurium, Pseudomonas aeruginosa, Staphylococcus aureus, Listeria monocytogenes. The bactericidal activities of Phx-3 against Helicobacter pylori strains have not been assessed. Then, we measured minimum inhibitory concentration of Phx-3 for Helicobacter strains and assessed the morphological and biochemical effects of Phx-3 on H. pylori. In present study, it has shown that H. pylori strains including clarithromycin resistant strain and Helicobacter musterae were killed effectively by the treatment with Phx-3. Furthermore, severe morphological changes such as membrane blebbing and formation of hollows in H. pylori were detected. In addition, induction of heat shock protein 60 was observed. Taken together, Phx-3 has antibacterial activity against Helicobacter pylori.
We recently reported our discovery of small molecule β-1,6-glucan inhibitor named D75-4590 and the anti-Candida activity of its derivatives D11-2040 and D21-6076. In this study, we further evaluated the antifungal profile of D11-2040. It alone strongly inhibited the vegetative growth and/or hyphal development of various Candida species, but no significant activity was observed against Cryptococcus neoformans or any of the filamentous fungi tested. Synergism was detected for C. albicans in the interaction of D11-2040 and caspofungin by the chequerboard method and in that of D11-2040 and fluconazole by the time-kill method. Slight but positive interactions were observed in several combinations for C. neoformans and Aspergillus fumigatus as well. These results suggested that β-1,6-glucan inhibitors have promising potential as single drugs as well as concomitants.
Using the par to rep region of the 24653 bp plasmid pN315, which is present in Staphylococcus aureus strain N315, we constructed three vectors that can be shuttled between Escherichia coli and S. aureus and maintained stably in S. aureus. Due to plasmid incompatibility, the resident plasmid in S. aureus cells can be replaced via transformation with an entering plasmid, which carries a different drug resistance gene. To evaluate the applicability of this plasmid-based approach for identifying genes essential for S. aureus cell growth, the chromosomal mraY gene, which is involved in peptidoglycan biosynthesis, was deleted in cells harboring a resident plasmid with an intact mraY gene. The resultant disruptant was then transformed with an empty vector. Cells with a chromosomal mraY deletion but lacking the plasmid supplying mraY could not be recovered, suggesting that mraY is indispensable for staphylococcal cell growth or viability. In contrast, other two genes were shown to be dispensable by this system. Thus, the pN315-based plasmids appear to be useful for studying genes essential for S. aureus cell growth.
Lysophosphatidic acid (LPA) is a lipid mediator that is known to exhibit chemotactic activity toward a variety of cancer cells. However, its effect on the immune system has not been studied extensively. Another lipid mediator, sphingosine-1-phosphate (S1P), has been shown to influence lymphocyte recirculation by regulating lymphocyte egress from lymphoid organs. In this study, we found that LPA inhibits spontaneous migration of mouse splenic lymphocytes through a chemorepulsive effect. We also demonstrated that LPA inhibits chemokine CCL21-induced lymphocyte migration. This inhibitory effect on CCL21-induced migration was observed for both T and B cells. The involvement of a receptor, LPA1, LPA2 or LPA3, in the inhibition of the CCL21-induced migration was confirmed with a synthetic agonist, oleyl thiophosphate. Considering that the signaling by CCL21 through cognate receptor CCR7 contributes to lymphocyte homing and dendritic cell trafficking to lymph nodes, LPA may play a role as a key regulator of these processes. The inhibitory effect of LPA is in remarkable contrast to the effect of S1P receptor signaling, which is known to potentiate lymphocyte chemotaxis involving CCR7.
Myrsinoic acid B (AMB) is a prenylated-benzoic acid derivative isolated from the Rapanea genus. Recent studies suggest that AMB has antihyperalgesic and antinociceptive properties in different animal models. The present study was designed to investigate the mechanisms involved in antinociception elicited by AMB (60 mg/kg) when administered by intraperitonial route (i.p.) in mice. The antinociceptive response of the compound was characterized by a reduction in contractions of the abdominal muscle, together with stretching of the hind limbs in response to i.p. injection of acetic acid (0.6%, 0.45 ml/mouse). The antinociception caused by AMB in the acetic acid test was significantly attenuated by i.p. treatment of mice with nitric oxide precursor, (L-arginine, 600 mg/kg), α2 and α1-adrenoceptor antagonists (yohimbine, 0.2 mg/kg/prazosin, 0.2 mg/kg), p-chlorophenylalanine (PCPA) an inhibitor of serotonin synthesis (100 mg/kg), 1-(2-methoxyphenyl)-4-(4-phthalimidobutyl)piperazine (NAN 190), a 5-HT1A selective receptor antagonist (0.5 mg/kg) and a non-selective cholinergic antagonist (atropine, 10 mg/kg). Its action was also modulated by the adrenal-gland hormones. In contrast, antinociception was not affected by naloxone (non-selective opioid receptor antagonist, 1.0 mg/kg), phaclofen (2.0 mg/kg) and bicuculline (1.0 mg/kg) GABAB and GABAA receptor antagonists, respectively, ondansetron (0.3 mg/kg) and ketaserin (1.0 mg/kg), (5-HT3 and 5-HT2 receptors, respectively) and haloperidol (0.2 mg/kg), a non-selective dopaminergic receptor. The antinociceptive effects are not related to muscle-relaxant or sedative action. These results indicate that AMB produces antinociception through mechanisms that involve interaction with L-arginine–nitric oxide, the serotonergic and cholinergic systems, as well as interaction with the α-adrenoceptors.
Z-360 is a novel cholecystokinin (CCK)-2/gastrin receptor antagonist that is being developed for the treatment of pancreatic adenocarcinoma in combination with gemcitabine. A previous study shows that the co-administration of Z-360 with gemcitabine significantly prolonged the survival of mice with orthotopically implanted human pancreatic adenocarcinoma cell lines. To clarify the therapeutic effects of Z-360 in combined with gemcitabine, we analyzed gene expression. When gemcitabine was administered, CCK-2/gastrin receptor expression was induced in an orthotropic xenograft model; the result indicating that Z-360 could act on gemcitabine-sensitive cells. Both in vitro and in vivo studies showed that gemcitabine increased the expression of vascular endothelial growth factor A (VEGFA), a prognostic factor for survival in pancreatic cancer, while Z-360 suppressed this induction of VEGFA gene expression. These results help to explain how Z-360 prolongs survival when used in combination with gemcitabine.
The present study was designed to clarify the effects of an ethanol extract of artichoke leaf on acute gastric mucosal injury in rats. Oral administration of artichoke leaf extract dose-dependently prevented absolute ethanol-induced (125—500 mg/kg) or restraint plus water immersion stress-induced gastric mucosal injury (1000—2000 mg/kg). The artichoke leaf extract contains 1% cynaropicrin and 0.8% chlorogenic acid as main components and 70% dextrin as a vehicle. Cynaropicrin at doses of 1/100 of artichoke leaf extract [ethanol-induced mucosal injury: 5 mg/kg, per os (p.o.); stress-induced mucosal injury: 20 mg/kg, p.o.] also prevented gastric mucosal injury in both animal models. However, dextrin and chlorogenic acid at doses contained in the leaf extract were ineffective in both models. When artichoke leaf extract was given orally to normal rats, it (500—2000 mg/kg, p.o.) dose-dependently increased gastric mucus content. In addition, it (125—500 mg/kg, p.o.) dose-dependently prevented the decrease in gastric mucus content by absolute ethanol. When the effects of artichoke leaf extract on basal gastric acid secretion in rats were evaluated, it (500—2000 mg/kg, p.o.) dose-dependently increased the volume of gastric juice in normal rats. However, it was ineffective in decreasing basal gastric acid secretion in normal rats. These results indicate that artichoke leaf extract is effective against acute gastritis and its beneficial effect is due to that of cynaropicrin. The gastric mucus-increasing action of artichoke leaf extract may be, at least in part, related to the anti-gastritic action of the extract.
For the identification of anti-inflammatory ingredients from Forsythiae fructus (FF), we isolated three hydroxyl pentacyclic triterpene acids (HTAs), namely, oleanolic acid, ursolic acid, and betulinic acid, from an ethylacetate fraction of FF, and evaluated the effect of these triterpene acids on asthmatic guinea pigs by measuring specific airway resistance (sRaw) during both immediate-phase response (IAR) and late-phase response (LAR) following ovalbumin challenge using a double-chambered plethysmograph. Evaluation of leukocytes and chemical mediators in bronchoalveolar lavage fluid (BALF), in addition to a histopathological survey, was also performed. Ursolic, oleanolic and betulinic acids dosed at 12.5 mg/kg significantly (p<0.05) decreased sRaw by 46.80%, 46.54% and 44.27% during in IAR, respectively. And ursolic acid (25 mg/kg), and oleanolic and betulinic acids (50 mg/kg) significantly (p<0.05) decreased sRaw by 38.19%, 38.15% and 35.55% in LAR, respectively. Histamine and phospholipase A2 activity in BALF were significantly decreased by HTAs at 12.5 mg/kg, whereas eosinophil peroxide (EPO) activity in BALF and recruitment of eosinophils were significantly decreased by HTAs at 25 mg/kg, as well as improvement of pathological changes. However, betulinic acid at 12.5 mg/kg, and ursolic and oleanolic acids at 25 mg/kg significantly inhibited leukocytes in BALF, especially eosinophils and neutrophils. Three HTAs were found to have dose-dependent anti-asthmatic effects and ursolic acid is the most active, but their activities were less than those of sodium cromoglycate, salbutamol, and dexamethasone. These results indicate HTAs had anti-asthmatic activity by decreasing of sRaw, and eosinophil recruitment and release of inflammatory mediators into the lungs.
Baicalein, an extract from Scutellaria baicalensis, was evaluated for its ability to inhibit the influenza virus in vivo. Oral administration of baicalein to BALB/c mice infected with the influenza A/FM1/1/47(H1N1) virus showed significant effects in preventing death, increasing the mean time to death, inhibiting lung consolidation, and reducing the lung virus titer in a dose-dependent manner. These effects are believed to be due to baicalin, the metabolite of baicalein in the serum. At a concentration of baicalin 2 μg/ml in overlay medium, it showed significant inhibition in the plaque assay, and the mean IC50 value of baicalin was calculated as 1.2 μg/ml in the cytopathic effect assay. Our results showed that baicalein warrants further research as a potential antiinfluenza viral agent.
Z-360, a novel cholecystokinin2 (CCK2) receptor antagonist, has been developed as a therapeutic drug for pancreatic cancer and showed pain relief action in phase Ib/IIa clinical trial. This study was attempted to elucidate the analgesic efficacy of Z-360 in mice. Oral administration of Z-360 (30—300 mg/kg) showed a dose-dependent inhibitory effect on the late phase of nociceptive responses to formalin. YF476, another CCK2 receptor antagonist, was without effects at 1 and 10 mg/kg. In contrast, the CCK1 receptor antagonist devazepide inhibited the nociceptive responses to formalin. In a mouse model of cancer pain, significant anti-allodynic effect of Z-360 was observed after single and repeated oral administration of 100 and 300 mg/kg doses. Anti-allodynic effect was also observed after repeated administration of devazepide. Combined single treatment with morphine and Z-360 caused an increase inhibition of pain-related responses in the pain models produced by formalin and cancer. Although Z-360 has lower affinity for CCK1 receptor than for CCK2 receptor, Z-360 exhibited an inhibitory effect on sulfated CCK-8-induced gallbladder emptying, a CCK1 receptor-mediated effect, at a dose of 100 mg/kg. These results suggest that Z-360 inhibits inflammatory and cancer pain probably through the blockade of CCK1 receptors. Z-360 is expected to become a useful drug for the pancreatic cancer with analgesic effects as well as the prolongation of survival.
Expression levels of cytochrome P450 (CYP) 3A4, CYP3A5 and CYP3A7 mRNAs in placentas and fetal membranes, which were split into amnion and chorion leave attached decidua (chorion/decidua), obtained from pregnant women with normal delivery (5 subjects) and Caesarean section (15 subjects) were determined. These CYP3A mRNAs were also expressed in amnion and chorion/decidua together with placenta, although the expression level of these mRNAs was strikingly different between subjects. The expression level of the CYP3A4 mRNA in the placenta was about 2-fold higher than those in amnion and chorion/decidua. On the other hand, the expression levels of CYP3A5 and CYP3A7 mRNAs were highest in chorion/decidua. The immunologically related protein(s) with CYP3A7 was detected in all tissues examined. Testosterone 6β-hydroxylase activity in homogenate of human placenta, amnion and chorion/decidua were 26.6, 3.7 and 4.6 pmol/h/mg protein, respectively. These results suggest that CYP3As in fetal membranes have the metabolic function to protect the fetus from exposure to drugs.
The present study was conducted to investigate the functional and transcriptional modulation of P-glycoprotein (MDR-1) by several dietary ingredients (piperine, capsaicin, daidzein, genistein, sesamin, curcumin, taurine) in vinblastine-resistant colon carcinoma LS-180 cells (LS-180V cells). The amount of rhodamine 123 accumulated in LS-180V cells was significantly increased by capsaicin, piperine and sesamin, whereas it was significantly reduced by daidzein and genistein which stimulated the efflux of rhodamine 123. These results suggest that the P-glycoprotein-mediated efflux is inhibited by piperine, capsaicin and sesamin and stimulated by daidzein and genistein. The concurrent addition of piperine and capsaicin seemed to inhibit synergistically the P-glycoprotein-mediated efflux. Pretreatment with sesamin for 48 h caused a significant increase in MDR1 mRNA expression without a significant effect on the expression of P-glycoprotein or accumulation of rhodamine 123. Similar pretreatment with other ingredients had little effect on the expression of MDR1 mRNA or P-glycoprotein, suggesting that they do not cause transcriptional modulation of P-glycoprotein. Piperine, genistein and curcumin have been suggested to stimulate P-glycoprotein-mediated efflux without increasing P-glycoprotein expression. In LS-180V cells, significant increases in mRNA levels of multi-drug resistance associated protein 1 (MRP1) or MRP3 were observed on pretreatment with capsaicin, daidzein, piperine and sesamin. In conclusion, our results suggest that P-glycoprotein-mediated efflux is significantly affected by dietary ingredients. Also, capsaicin, daidzein, piperine and sesamin increased significantly the mRNA expression of MRP1 or MRP3. Thus, the present study provides further evidence that repeated exposure to dietary ingredients can cause drug–food interactions by affecting the function and mRNA expression of intestinal transporters such as P-glycoprotein.
In the investigation of chemical constituents in antiosteoporotic active fraction, 90% ethanol fraction of water extract of the rhizomes of Dioscorea spongiosa, twenty-two glycosides were isolated and their structures were elucidated. In order to know the role of each compound played in the fraction and to find the excellent antiosteoporotic constituent, they were subjected to some in vitro assay experiments with cell lines and an animal organ culture system: stimulation on osteoblasts proliferation and mineralization, inhibition on osteoclasts formation and inhibition on bone resorption. Glycosides 1, 3, 4, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18, 19 and 21 showed significant stimulatory activity on proliferation of osteoblasts. Especially, pregnane glycoside 4, showed the strongest stimulatory activity. The stimulatory activity on osteoblasts mineralization of the compounds stimulating the proliferation significantly was examined and compounds 8, 10, 13 and 16 showed stronger stimulation on osteoblasts mineralization. Their inhibitory activity on bone resorption indicated that only glycosides 5, 13, 14 and 21 possessed significant inhibitory activity at high concentration. Glycosides 9, 11, 14, 18 and 19, isolated in large amount, all showed very strong inhibitory activity on formation of osteoclasts at high concentration, which may be induced by cytotoxicity to bone marrow cells and osteoblasts due to the observation under microscope. It implied that their inhibition on bone resorption may origin from their inhibition on formation of osteoclasts.
We analyzed the effects of glycyrrhetinic acid (GA), a licorice compound, on the induction of anoikis-like death and cytoskeletal disruption in the central nervous system (CNS) tumorigenic cells. GA was cytotoxic in time- and dose-dependent manners, and the tumorigenic cells shed floating cells upon the GA treatment and even some of the adherent cells were easily detached from the fibronectin-coated culture dish by gentle shaking and aspiration. Reculture of the detached cells revealed that the longer the duration of GA exposure, the less the number of the proliferatable cells. These results indicate that GA perturbs cell adhesion and induces anoikis-like cell death. Further, GA also induced morphologic changes and disturbed cytoskeletal proteins.
Panax ginseng is known to have anti-diabetic activity, but the active ingredients are not yet fully identified. In this study, we found the inhibitory effect of Rg1 on hepatic glucose production through AMP-activated protein kinase (AMPK) activation in HepG2 cells. Rg1 significantly inhibited hepatic glucose production in a concentration-dependent manner, and this effect was reversed in the presence of compound C, a selective AMPK inhibitor. In addition, Rg1 markedly induced the phosphorylations of liver kinase B1 (LKB1), AMPK and forkhead box class O1 (FoxO1), a key transcription factor for gluconeogenic enzymes, in time- and concentration-dependent manners. Glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) activities were inhibited by 24% and 21%, respectively, when the cells were exposed to 40 μM of Rg1, resulting from phosphorylation of FoxO1 and inhibition of gluconeogenic gene expression. Taken together, our results demonstrated the suppressive effect of Rg1 on hepatic glucose production via LKB1-AMPK-FoxO1 pathway in HepG2 human hepatoma cells.
This study was designed to evaluate the protective effect of kolaviron (KV), a biflavonoid from Garcinia kola seeds, against γ-radiation (5 Gy)-induced oxidative stress in brain of Wistar rats. Vitamin C (VC) served as standard antioxidant. Forty-four rats were divided into 4 groups of 11 animals each. One group was un-irradiated (normal), two groups were treated with KV and VC (250 mg/kg) for 6 weeks prior to and 8 weeks after irradiation, and fourth group was only irradiated. Rats were sacrificed 1 and 8 weeks after irradiation. Cellular alterations were monitored using changes in the levels of malondialdehyde (MDA)—an index of lipid peroxidation, superoxide dismutase (SOD), glutathione-S-transferase (GST), reduced glutathione (GSH), catalase (CAT), alanine and aspartate aminotransferases (ALT and AST), urea and creatinine. MDA levels increased significantly (p<0.05) by 90% and 151% after 1 and 8 weeks of irradiation. Furthermore, levels of GSH and antioxidant enzymes were significantly (p<0.05) decreased in γ-irradiated animals. GSH and GST decreased by 61% and 43% after 1 week, and by 75% and 74%, after 8 weeks of exposure, respectively. γ-Irradiation decreased SOD and CAT levels by 53% and 68%, respectively, and caused significant (p<0.05) increases in serum ALT, AST and urea after 8 weeks of exposure. Treatment with KV and VC significantly decreased the levels of MDA, ALT, AST and urea. The antioxidant indices were significantly ameliorated in KV-treated animals. These data suggest that kolaviron may protect against γ-radiation-induced oxidative stress in brain of exposed rats.
A new β-cyclogeraniol diglycoside (5), along with four known components, cycloartenol (1), p-hydroxybenzoic acid (2), vanilloloside (3), and 5′-O-methyladenosine (4), were first isolated from the n-BuOH fraction of Nelumbo nucifera stamens. The chemical structure of 5 was elucidated as 1-hydroxymethyl-2,6,6-trimethyl-1-cyclohexene 9-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside (nuciferoside) on the basis of chemical and spectroscopic evidence, including 1D, 2D NMR, and MS. The anti-Alzheimer effects of 1—5 were evaluated via the acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) inhibition assays. Compounds 1—3 and 5 showed good and noncompetitive inhibition against AChE with IC50 values of 11.89, 20.07, 4.55, and 3.20 μM and Kivalues of 15.71, 25.44, 7.76, and 5.76 μM, respectively. Compounds 1, 2, and 5 also possessed BChE inhibitory activities with IC50 values of 13.93, 62.29, 205.78, and 83.06 μM, respectively. The selectivity index (SI) values of 1, 2, 3, and 5, calculated from IC50 values of BChE and AChE, were 1.2, 3.1, 45.7, and 26.0. However, all isolated compounds lacked BACE1 inhibition up to 100 μM. Therefore, N. nucifera stamens-derived compounds could potentially exert their primary anti-Alzheimer effects as AChE inhibitors rather than BACE1 inhibitors.
The effects of a silk amino acid (SAA) preparation on the physical stamina and male reproductive function of mice were investigated. Eight-week-old male ICR mice (29—31 g) were orally administered SAA (50, 160 or 500 mg/kg) for 44 d during 30-min daily swimming exercise. The mice were subjected to a weight-loaded (5% of body weight) forced swimming on the 14th, 28th and 42nd day to determine maximum swimming time, and after a 2-d recovery period (treated with SAA without swimming exercise), parameters related to fatigue and reproductive function were analyzed from blood, muscles and reproductive organs. Repeated swimming exercise increased the maximum swimming time to some extent, in spite of a marked reduction in body weight gain, and SAA further enhanced the stamina in a dose-dependent manner. Forced swimming exercises increased blood parameters of tissue injury, but depleted blood glucose and tissue glycogen, which were substantially prevented by SAA treatment. In addition, SAA significantly reduced the muscular thiobarbituric acid-reactive substances and blood corticosterone content increased by forced swimming. Swimming exercise decreased the blood testosterone level, which was recovered by SAA, leading to enhanced sperm counts. These combined results indicate that SAA not only enhances physical stamina by minimizing damage to tissues, including muscles, as well as preventing energy depletion caused by swimming stress, but also improves male reproductive function by increasing testosterone and sperm counts.
In our ongoing project directed toward the discovery of novel treatments for diabetic complications from herbal medicines, sixteen compounds including three caffeoylquinic acids and four flavonoids were isolated from an EtOAc-soluble extract of the stems and leaves of Erigeron annuus. All the isolates were evaluated in vitro for inhibitory activity on the formation of advanced glycation end products (AGEs) and rat lens aldose reductase (RLAR). Of these, 3,5-di-O-caffeoyl-epi-quinic acid (3) exhibited the most potent inhibitory activity in both the AGEs and aldose reductase (AR) assays. Compound 3 markedly reduced AGEs-bovin serum albumin (BSA) cross-linking in a dose-dependent manner. Furthermore, opacity of lenses was significantly prevented when they were treated with 3 in an ex vivo experiment.
We developed a set of molecular markers in Cistanche deserticola Y. C. MA to evaluate the production quality of cultivated C. deserticola individuals. This application utilizes the inter-simple-sequence repeat (ISSR) polymerase chain reaction (PCR) and random amplified polymorphic DNA (RAPD) PCR as molecular markers to determine the echinacoside content in cultivated C. deserticola individuals. The unweighted pair-group method using arithmetic average clustering (UPGMA) confirmed that the combined ISSR and RAPD data could categorize all C. deserticola individuals into three groups according to their respective echinacoside content. The stepwise multiple regression analysis (SMRA) revealed six potential markers associated with echinacoside accumulation in C. deserticola and produced 18 echinacoside-marker prediction models, four of which were successfully used to predict the quality of C. deserticola from Neimenggu. Both clustering and SMRA showed a correlation between the echinacoside content and molecular markers in cultivated C. deserticola. The relative average deviation of prediction (RADP) of the prediction models could be improved by simplifying and adjusting the model. It was found that the RADP value could reach 2.6% after adjustment and the simplified prediction models could successfully predict the quality of cultivated C. deserticola individuals.
To develop a novel sibutramine base-loaded solid dispersion with improved solubility bioavailability, various solid dispersions were prepared with water, hydroxypropylmethyl cellulose (HPMC), poloxamer and citric acid using spray-drying technique. The effect of HPMC, poloxamer and citric acid on the aqueous solubility of sibutramine was investigated. The physicochemical properties of solid dispersion were investigated using scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and X-ray powder diffraction. The dissolution and pharmacokinetics in rats of solid dispersion were evaluated compared to the sibutramine hydrochloride monohydrate-loaded commercial product (Reductil®). The sibutramine base-loaded solid dispersion gave two type forms. Like conventional solid dispersion system, one type appeared as a spherical shape with smooth surface, as the carriers and drug with relatively low melting point were soluble in water and formed it. The other appeared as an irregular form with relatively rough surface. Unlike conventional solid dispersion system, this type changed no crystalline form of drug. Our results suggested that this type was formed by attaching hydrophilic carriers to the surface of drug without crystal change, resulting from changing the hydrophobic drug to hydrophilic form. The sibutramine-loaded solid dispersion at the weight ratio of sibutramine base/HPMC/poloxamer/citric acid of 5/3/3/0.2 gave the maximum drug solubility of about 3 mg/ml. Furthermore, it showed the similar plasma concentration, area under the curve (AUC) and Cmax of parent drug, metabolite I and II to the commercial product, indicating that it might give the similar drug efficacy compared to the sibutramine hydrochloride monohydrate-loaded commercial product in rats. Thus, this solid dispersion system would be useful to deliver poorly water-soluble sibutramine base with enhanced bioavailability.
Fluvoxamine is a selective serotonin reuptake inhibitor widely used in the treatment of depression and other psychiatric diseases. The aim of this study was to assess the clinical impact of cigarette smoking on plasma fluvoxamine concentration in Japanese patients, and evaluate whether the cytochrome P450 (CYP) 1A2 and CYP2D6 genotypes have effects on that concentration. Thirty-two Japanese patients receiving fluvoxamine were enrolled. They were maintained on the same daily dose of fluvoxamine (mean±S.D., 109.4±66.2 mg/d) for at least 4 weeks to obtain the steady-state plasma concentration. The steady-state plasma concentration-to-dose (C/D) ratio of fluvoxamine in patients who smoked (n=6, 11.8±6.5 ng/ml/dose) was significantly lower than that in non-smoker patients (n=26, 22.8±11.2 ng/ml/dose). There was no significant difference for the C/D ratio of fluvoxamine in patients with CYP1A2 −3860G/G, −3860G/A, and −3860A/A between non-smokers and smokers. Among non-smoker patients, the C/D ratios of fluvoxamine in those with one and two mutated alleles of CYP2D6 were 1.6- and 1.4-fold higher, respectively, than those with no mutated alleles, though the differences among those three genotype groups were not significant. Furthermore, stepwise multiple regression analysis revealed that cigarette smoking and daily dose had significant positive correlations with the plasma concentration of fluvoxamine. Our findings suggest that cigarette smoking has a significant impact on the steady-state plasma concentration of fluvoxamine in Japanese patients.
Some lactic acid bacteria (LAB) are known as representative of probiotics. To screen LAB effective to enhance intestinal immunity, in the present study, we developed an accurate and convenient in vitro evaluation system using Peyer's patch cells (PP-cells) isolated from the mice intestine. We observed that the amount of immunoglobulin A (IgA) produced by PP-cells co-cultured with LAB was well correlative to that in PP-cells, intestine and feces isolated from live mice after oral administration of LAB [correlation coefficient (r)=0.888, 0.883, and 0.920, respectively]. In addition, using this in vitro system, we suggest that the IgA level of PP-cells co-culturing with plant-derived LAB might be more enhanced than with animal-derived LAB.
The aim of this investigation was to assess the applicability of lipid bilayer alteration using a combination of isopropyl myristate (IPM) and glyceryl monocaprylate (GEFA-C8) to the enhancement of pentazocine (PTZ) permeation through hairless mouse skin. The skin permeability of PTZ was enhanced by increasing the concentration of GEFA-C8 up to 10% w/w in combination with IPM. Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and small angle X-ray diffraction (SAXD) were carried out to analyze the effects of these enhancers on the biophysical properties of the stratum corneum (SC) of the skin, and on the permeation of PTZ. ATR-FTIR studies revealed that IPM/GEFA-C8 induced higher CH2 stretching frequencies of SC lipids than IPM alone. SAXD showed the disappearance of long lamellar diffraction of SC lipids with IPM/GEFA-C8, resulting in a complete loss of order of the SC lipid bilayers. When 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (DiI), a hydrophobic fluorescence probe, was applied in IPM alone, the amount of DiI which penetrated into the intercellular space of the SC was very low, but this was markedly increased when DiI was applied in IPM/GEFA-C8. These results indicate that the synergistic effects of IPM and GEFA-C8 enhance transdermal permeation of PTZ by disrupting SC lipids.