With the continued progress in genomic drug discovery, the high-throughput production of drug candidates has become possible, and thus today there are a number of candidates that are extremely effective both in cell-free and in cell models. However, a drug delivery system suitable for the high-throughput production has yet to be fully developed. In tissues, the tight junction (TJ) plays a pivotal role as both a barrier to restrict various substances and in intra-tissue maintenance. Claudin, a ca. 23 kDa transmembrane protein with four transmembrane domains, is responsible for the TJ functions. Interestingly, for each of the 24 members of the claudin family, expression profiles and exact barrier functions differ. Therefore, claudin may be a potential target for use as a drug delivery system via a paracellular route. The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) is known to modulate the barrier function of claudin. We found that C-CPE is a potent absorption-enhancer and that this enhancing activity is 400-fold greater than clinically used enhancers. The enhancing activity examined in this study involved an interaction between C-CPE and claudin-4. These findings indicate that claudin might be a novel target for a drug delivery system. In the current review, we describe about background and data on our research about claudin modulator, and we also discuss the possibility of the use of the claudin family in a new approach for developing a drug delivery system.
Development of new delivery systems that deliver the potential drug specifically to the target site in order to meet the therapeutic needs of the patients at the required time and level remains the key challenge in the field of pharmaceutical biotechnology. Developments in this context to achieve desired goal has led to the evolution of the multidisciplinary field nanobiotechnology which involves the combination of two most promising technologies of 21st century—biotechnology and nanotechnology. Nanobiotechnology encompasses a wide array of different techniques to improve the delivery of biotech drugs, and nanoparticles offer the most suitable form whose properties can be tailored by chemical methods. This review highlights the different types of nanoparticulate delivery systems employed for biotech drugs in the field of molecular medicine with a short overlook at its applications and the probable associated drawbacks.
Cystathionine γ-lyase (CSE) is the last key enzyme in the transsulfuration pathway for the biosynthesis of cysteine from methionine in mammals, and catalyzes the hydrolysis of cystathionine into cysteine. Cysteine can be provided through diet; however, several investigators have suggested that infants may require dietary supplements of cysteine because of very low or undetectable CSE activity in their livers. We have previously shown that CSE levels are much lower in the livers and kidneys of fetal and infant mice than in those of adult mice, suggesting that the maternal supply of cysteine is important for the early development of mice. Here we examined changes of CSE expression in the livers and kidneys of dams during gestation and lactation. Hepatic enlargement was observed as early as gestational day 12.5 (G12.5) and thereafter became more prominent, whereas expression of CSE in the livers was found after postpartum day 1 (P1) and reached a peak at P14. The maintenance of lactation was essential for both hepatic enlargement and CSE expression. In contrast, kidneys gained weight only slightly during lactation while CSE expression in kidneys was markedly induced at G15.5 and then gradually declined through to P28. Serum concentrations of homocysteine (the precursor of cystathionine) were significantly lower in G18.5 dams than in virgins or G15.5 dams, suggesting that the expression of CSE in the kidneys contributes to the effective clearance of homocysteine during the late gestational stage.
The antitumoral activity of 13-demethyl or 13-substituted all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9CRA) was tested using the myeloid leukemia cell line HL-60. Cell proliferation, differentiation and apoptosis were evaluated by flow cytometry and DNA fragmentation assay. The ability to bind to human RXRα and to activate either human retinoic acid response element (RARE)-mediated gene expression or rat CRABPII retinoid X response element (RXRα)-mediated gene expression were determined using luciferase reporter plasmids. In terms of the magnitude of the regulatory activity for the proliferation and differentiation of HL-60 cells, the compounds ranked as follows: ATRA>13-ethyl ATRA>13-demethyl ATRA>13-phenylethyl ATRA>13-propyl ATRA>13-butyl ATRA (ATRA analogues) and 9CRA>13-ethyl 9CRA>13-demethyl 9CRA>13-propyl 9CRA>13-phenetyl 9CRA>13-butyl 9CRA (9CRA analogues). Regarding the magnitude of the apoptosis-inducing activity, the order was: 9CRA>13-ethyl 9CRA>13-demethyl 9CRA, with ATRA and its analogues and the other 9CRA analogues virtually inactive. Similar trends were observed in binding affinity for RXRα and transactivation activity toward RARE- or RXRE-mediated gene expression. The results clearly indicate that the presence of a methyl group at C-13 is essential for the antitumoral activity of ATRA and 9CRA, and that bulky substituents exceeding two carbon atoms or the absence of substitution at position 13 significantly reduce the binding affinity for RAR and RXR, leading to a decreased RAR/RARE and/or RXR/RXRE-mediated gene expression.
The inhibitory effect of suramin on the phosphorylation of GST-HBV core fusion protein (GST-Hcore) and two GST-Hcore fusion polypeptides (Hcore157B and Hcore164B) by two α-type cAMP-dependent protein kinases (PKAIα and PKAIIα) was biochemically investigated in vitro. It was found that (i) this phosphorylation was inhibited by suramin at a low concentration (IC50=approx. 10 nM); (ii) a relative high dose of suramin was required to inhibit an autophosphorylation of PKAIIα (IC50=approx. 0.7 μM) and the PKAIIα-mediated phosphorylation of histone H2B (IC50=approx. 0.4 μM); (iii) the PKAIIα-mediated phosphorylation of Hcore157B was more sensitive to suramin than the phosphorylation of Hcore157B by Ca2+-dependent protein kinase (PKC); and (iv) suramin had a high binding affinity for Hcore157B, but not for histone H2B in vitro. These results suggest that suramin selectively inhibits the PKA-mediated phosphorylation of HBV-CP through the direct binding in vitro of suramin to the Arg-rich C-terminal region (containing three potential phosphorylation sites for PKA) on HBV-CP.
Hepatitis C virus (HCV) core protein plays important roles in the pathogeneses of liver steatosis as well as hepatocellular carcinomas due to HCV infection. In this study, we examined de novo fatty acid biosynthesis in hepatic cell line Huh7 cells expressing HCV core protein. The rate of metabolic labeling of cellular fatty acids with [3H]acetate in core-expressing (Uc39-6) cells was ca. 1.5-fold higher than that in non-expressing (Uc321) cells. The enzyme activities responsible for fatty acid biosynthesis were assayed in vitro. Cytosolic acetyl-CoA carboxylase activity in Uc39-6 cells was ca. 1.6-fold higher than that in Uc321 cells. On the other hand, cytosolic fatty acid synthase activity in Uc39-6 cells was only slightly higher than that in Uc321 cells. Immunoblot analysis of acetyl-CoA carboxylase 1 (ACC1), which is a rate-limiting enzyme for fatty acid biosynthesis, revealed a higher expression level of the protein in Uc39-6 cells than in Uc321 cells. The ACC1 mRNA content in Uc39-6 cells was 1.4-fold higher than that in Uc321 cells. These results strongly suggest that enhancement of fatty acid biosynthesis in core-expressing cells is caused by increased expression of fatty acid biosynthetic enzymes, especially ACC1. Up-regulation of de novo fatty acid biosynthesis by HCV core protein may affect cellular lipid metabolism, resulting in neutral lipid accumulation in HCV-infected cells.
Myoblasts respond to growth factor deprivation either by diffentiation into multinucleated myotubes or by undergoing apoptosis. The induction of apoptosis and differentiation in myogenic lineage may use overlapping cellular mechanisms. Here we demonstrate that the expression of the small heat shock protein αB-crystallin as well as MyoD and myogenin is induced during myogenic differentiation in C2C12 cells, and these inductions occur at an early stage in the differentiation in vitro. To investigate the effect of αB-crystallin on myogenic differentiation and apoptosis, C2C12 cells were infected with adenovirus vector bearing full-length αB-crystallin cDNA. Overexpression of αB-crystallin in C2C12 cells suppressed differentiation-induced apoptosis and activation of caspase 3, and also decreased the expression of MyoD and myogenin during myogenic differentiation of C2C12 cells induced by the differentiation medium. Our findings suggest that stress such as growth factor deprivation plays an important role in triggering apoptosis associated with myogenic differentiation and αB-crystallin suppressed the differentiation, apoptosis and caspase 3 activity.
Cytoprotective effect of caffeic acid (3,4-dihydroxy cinnamic acid) on human lung fibroblast (WI-38) cells against hydrogen peroxide induced damage was investigated. Caffeic acid was found to scavenge intracellular reactive oxygen species, and 1,1-diphenyl-2-picrylhydrazyl radical, and thus prevented lipid peroxidation. The caffeic acid protected cell damage of WI-38 cells exposed to hydrogen peroxide (H2O2), via the activation of extracellular signal regulated kinase protein. Caffeic acid increased the activity of catalase and its protein expression. Hence, from the present study, it is suggestive that caffeic acid protects WI-38 cells against H2O2 damage by enhancing the cellular antioxidant activity.
Mammalian colon plays an important role in electrolyte and water balance, and exhibits significant segmental heterogeneity. The different responses to stimulants even exist in different segments of rat distal colon. The present study focus on the segmental heterogeneity of epithelial responses to different stimulants and investigates the possible mechanism by using short circuit current recording technique. Baseline ISC in the segment 4 of distal colon (DC4, 20.8±2.8 μA·cm−2) was significantly lower than that in the segment 1 of distal colon (DC1, 40.5±1.9 μA·cm−2). Basolateral application of indomethacin induced a larger reduction of the baseline ISC in DC4 (−28.2±3.9 μA·cm−2) than that in DC1 (−10.1±3.9 μA·cm−2). Moreover DC4 are more sensitive to foskolin (a cAMP activator, apical side), Ach (basolateral side) and 5-HT (basolateral side) than DC1, which was not affected by pretreatment with amiloride, a blocker of epithelial Na+ channel. Basolateral pretreatment with atropine (muscarinic cholinergic receptor antagonist) for 10 min, Ach-induced ΔISC increases in both DC1 and DC4 were totally blocked. Otherwise, 5-HT4 receptor antagonist GR113808 (basolateral side) and SB-204070 (basolateral side) completely inhibited 5-HT-induced ISC increases in both DC1 and DC4. Taken together, the results suggested that the segmental heterogeneity of epithelial responses to different stimulants exists in rat distal colon. And it is more likely related to the dissimilar distribution of membrane proteins involved in the ion transports within different segments of rat distal colon.
Human ZFHX4 has recently been shown to be a candidate gene for congenital bilateral isolated ptosis. Here, we report molecular cloning of the human ZFHX4 cDNA and genomic organization of this gene. Human ZFHX4 is about 180 kb long, containing 12 exons that encodes a 3599-amino acid protein carrying four homeodomains and 22 zinc fingers. The 11th exon is 3.2 kb in length and encodes all the four homeodomains together with four of the 22 zinc fingers. ZFHX4 is 90% homologous to mouse Zfhx4, 52% to human ATBF1A and 24% to Drosophila ZFH-2. ZFHX4 was mapped to human chromosome 8q13.3-q21.11 by fluorescence in situ hybridization using BAC clone RP11-48D4 as a probe. RT-PCR analysis showed that ZFHX4 transcripts were expressed in adult human brain, liver and muscle. This, together with the finding that Zfhx4 was expressed transiently in differentiating P19 embryonal carcinoma cells and C2C12 myoblasts, suggests that ZFHX4/Zfhx4 is involved in neural and muscle differentiation.
The peroxisomal ATP-binding cassette (ABC) proteins, adrenoleukodystrophy protein (ALDP, ABCD1) and ALD-related protein (ALDRP, ABCD2), were expressed in Spodoptera frugiperda 21 (Sf21) insect cells using a baculovirus-mediated expression system. Immunoelectron microscopy and subcellular fractionation revealed that the overexpressed ALDP was distributed in various subcellular organelles including mitochondria, nucleus and peroxisomes. The ALDP was not extractable with Na2CO3 treatment, suggesting that it integrated into membranes. ATPase activity was detected in the membrane fraction expressing ALDP. The nucleotide-binding capacities of the expressed ALDP were estimated by the binding to ATP- or ADP-agarose. ALDP exhibited an affinity to both ADP and ATP. In contrast, ALDRP exhibited an affinity to ADP but scarcely to ATP. The ALDP in the Sf21 membrane fraction was extracted with n-dodecyl-β-maltoside and successively purified with a chelate column. The nucleotide-binding and ATPase activities of the purified ALDP were, however, not detected. It may be that certain membranous components are required for the activity. We demonstrate for the first time that the peroxisomal ABC proteins can be expressed in Sf21 membranes maintaining their nucleotide-binding abilities and ATPase activities, and the expressed proteins will be of use for further characterization.
Mitochondria are highly dynamic organelles in eukaryotic cells. Although the role of mitochondria in metabolism, ATP production and apoptosis is more widely recognized, alterations in mitochondrial morphology and abundance are also important for cellular functions. Here we investigated mitochondrial dynamics in synchronized HeLa cells in which the major stages of the cell cycle of the observed cells were resolved by staining phosphorylate histones H1 and H3, and showed that mitochondria exist as filamentous network structures throughout the cell cycle progression, changing their morphology, distribution, and abundance. The current results suggest that mitochondrial condensation occurred at prophase is required for the proper progression of mitochondrial division.
A chromene derivative (1) obtained from a brown alga, Sargassum micracanthum, has been proved to be a potent inhibitor of human cytomegalovirus (HCMV). In the present study, we evaluated its mode of action by various experimental assays. Time-of-addition experiments revealed that 1 was active if applied to cells before viral DNA synthesis, indicating that it inhibited early events of virus replication including virus adsorption and penetration, and a step immediately after viral internalization. Virus attachment and penetration studies suggested that one of the targets for anti-HCMV action of 1 was virus adsorption to cells and to a lesser extent, virus internalization was delayed in the presence of the compound. Pretreatment of virus particles with 1 showed that the compound exerted dose-dependent virucidal action. The chromene derivative and ganciclovir (GCV), an anti-HCMV drug, were synergistic inhibitors when used in combination. The synergistic effect could be explained by inhibition of different steps in HCMV replication cycle produced by 1 and GCV.
Recombinant single-chain fragment variable anti-idiotypic antibodies were produced to represent the internal image of HM-1 killer toxin and were used as novel and effective antifungal agents to inhibit in vitro β-1,3-glucan synthase and cell growth. The mechanism of cytocidal activity of anti-idiotypic antibodies was investigated and was compared with the actions of aculeacin A and papulacandin B, the most common antibiotics acting as β-1,3-glucan synthase inhibitors. The degree of inhibition of β-1,3-glucan synthase by both antibodies and antibiotics were examined for yeasts Saccharomyces cerevisiae A451, Cryptococcus albidus NBRC 0612 and Candida albicans IFM 40215. Although the mechanism of actions of the anti-idiotypic antibodies and antibiotics seems identical, the IC50 values for the various yeasts used in this study confirmed that anti-idiotypic antibodies could be used as more effective fungal β-1,3-glucan synthase inhibitors than those of antibiotics.
Candida albicans water soluble fraction (CAWS) is a water-soluble extracellular mannoprotein–beta-glucan complex obtained from the culture supernatant of Candida albicans, which grows in a chemically defined medium. CAWS induced toxic reactions, such as acute anaphylactoid reaction, by intravenous administration and coronary arteritis by intraperitoneal administration. To clarify the structure responsible for these toxic reactions, C. albicans was cultured in pH- and temperature-controlled conditions and prepared with CAWS with or without the beta-1,2-linked mannosyl segment (BM). The structure of CAWS was assessed by immunochemical and spectroscopic methodologies, and we found that CAWS prepared under the natural culture conditions contained only small amounts of BM and CAWS prepared at neutral conditions at 27 °C contained a significantly higher percentage of BM. Both the acute lethal toxicity and coronary arteritis induction was significantly more severe in the absence of BM. Activation of a complement pathway, the lectin pathway, by CAWS was significantly stronger in the absence of BM. These facts strongly suggest that BM linkages in CAWS negatively modulate acute and chronic toxicity of CAWS, and may be strongly related to the lectin pathway of the complement activation.
The inhibitory effects of the Korean red ginseng (steamed root of Panax ginseng C.A. MEYER, family Araliaceae) saponin fraction (KRGS) and its constituents ginsenosides Rg3, Rf, and Rh2 in mouse passive cutaneous anaphylaxis (PCA) and contact dermatitis models were measured. Orally administered KRGS and its genuine ginsenosides potently inhibited the PCA reaction induced by IgE. However, when these ginsenosides were intraperitoneally administered, ginsenoside Rh2 showed the most potent inhibition. The ginsenoside Rh2 also the most potently inhibited the β-hexosaminidase release from RBL-2H3 cells induced by IgE with antigen. KRGS administered topically at a dose of 0.1% suppressed ear swelling in an oxazolone-induced mouse contact dermatitis model by 38.8%. Its constituents ginsenosides Rg3, Rf, and Rh2 at a concentration of 0.05% also potently suppressed mouse ear swelling by 47.5%, 34.8%, and 49.9% at 16 d, respectively. These ginsenosides also significantly reduced mRNA expression levels of cyclooxygenase (COX)-2, interleukin (IL)-1β, tumor necrosis factor-α and interferon-γ induced by oxazolone applied to mouse ears. However, the ginsenosides, except for ginsenoside Rh2, almost did not notably reduce IL-4 levels. The ginsenoside Rh2 also potently inhibited COX-2 and inducible NO synthetase protein expression in liphopolysaccharide-stimulated RAW264.7 cells. Based on these findings, KRGS and its ginsenosides are suggested to improve atopic and contact dermatitis by regulating expression of cytokines.
Nicotiflorin is a single component extracted from traditional Chinese medicine Flos Carthami. In this study, we investigated its neuroprotection in permanent focal cerebral ischemia model in rats, and in an in vitro model of ischemia. At doses of 2.5, 5 and 10 mg/kg, nicotiflorin administered immediately after the onset of ischemia markedly reduced brain infarct volume and neurological deficits. For primarily cultured neurons suffered 2 h hypoxia followed by 24 h reoxygenation, nicotiflorin significantly attenuated cell death and reduced LDH release. Morphological observation also directly confirmed its protective effect on neuron. These results provided strong pharmacological basis for its potential therapeutic role in cerebral ischemic illness.
Previously, we found that human histocytic lymphoma U937 cells possessed high susceptibility to oridonin-induced cell death, but the molecular mechanisms in response to oridonin remain unclear. In this study, U937 cells showed susceptible to apoptosis induced by 27 μM oridonin and an agonistic anti-Fas IgM mAb (CH-11) (500 ng/ml) as a Fas-sensitized positive control. Caspase 8 inhibitor z-IETD, but neither caspase 1 inhibitor Ac-YVAD nor caspase 10 inhibitor z-AEVD, effectively blocked oridonin-induced cell death as well as DNA fragmentation. Western blot analysis showed the up-regulated expression of Fas, FasL, and FADD, and down-regulated expression of procaspase 8, suggesting that Fas/FasL pathway was activated in oridonin-induced cell apoptosis. Further, stimulation of U937 cells with oridonin and CH11 resulted in significant ERK MAPK activation. However, inhibition of ERK by PD98059 reversed oridonin-induced cell death as well as the activation of caspase 8, indicating that ERK-mediated control occured upstream of caspase 8. Simultaneously, ERK activation accounted for the release of cytochrome c, but failed to influence decreased Bcl-2 expression induced by oridonin. Taken together, these results suggest that Fas/FasL signaling pathway-mediated ERK activation sensitized U937 cells to mitochondrial pathway-mediated apoptosis induced by oridonin.
Breviscapine, a traditional Chinese medicine, is extensively used in clinic to treat cardiovascular diseases and cerebrovascular injury. In this study, we demonstrated the effects of breviscapine on vascular dementia (VD) rats, which were mimicked by permanent occlusion of bilateral common carotid arteries. Breviscapine (2 mg/kg for 14 d) improved the performance of learning and memory of VD rats in Morris water maze, decreased the level of lipid peroxidation and free radicals, and attenuated the pathological alterations, such as nuclear shrink, cellular edema and irregular arrangement of pyramidal layer in the hippocampal CA1 area. In vitro experiment, breviscapine (50 μg/l) protected cortical neuron from injury and decreased intracellular calcium overloading induced by H2O2 (10 mM). The results suggest that breviscapine has therapeutic effect on cerebral ischemia and vascular dementia.
We previously reported that a 150 mg or higher daily dose is necessary for treatment of pulmonary aspergillosis with micafungin (MCFG) alone in patients with blood diseases. Since a delay in the treatment of pulmonary aspergillosis has a major influence on patient survival, clarification of the effective blood concentration of MCFG enables rapid treatment. Establishment of an appropriate dose is also useful for reducing the risk of adverse effects, such as MCFG-induced impairment of liver function. Aiming for the rapid and safe treatment of pulmonary aspergillosis, we established new clinical diagnostic criteria of mycosis and MCFG therapeutic effect judgment criteria, and investigated the effective blood concentration of MCFG for mycosis. The blood trough level of MCFG in patients with blood diseases at each clinical improvement rating of pulmonary aspergillosis was 5.23±2.44 μg/ml in markedly improved cases, 4.08±2.63 μg/ml in improved cases, and 3.45±1.63 μg/ml in successfully prevented cases, showing no significant difference among the 3 groups. Based on this finding, it is advisable to target a 5 μg/ml or higher blood trough level of MCFG in establishing the dose for aspergillosis in patients with blood diseases.
Although Polygala tenuifolia WILLD (PT) was classically mentioned as an anti-dementia drug in Chinese and Japanese traditional medicine, basic research showed only enhancement of the cholinergic function. In Alzheimer's disease, neuritic atrophy and synaptic loss occur prior to neuronal death event, and may be the first trigger of the memory impairment. Therefore, we studied effects of Polygala tenuifolia WILLD (PT) on Aβ(25—35)-induced neuronal damage using rat cortical neurons for characterization of activities of PT under Aβ-induced neuronal damage. Treatment with the water extract of PT enhanced axonal length dose-dependently after Aβ(25—35)-induced axonal atrophy. However, dendritic atrophy and synaptic loss induced by Aβ(25—35) were not recovered by treatment with PT extract. In contrast, Aβ(25—35)-induced cell damage was completely inhibited by PT extract. By characterization of PT effects on neuronal morphological plasticity and cell damage, usefulness as well as an insufficiency of PT as an anti-dementia drug was clarified.
Airway hyperresponsiveness (AHR) associated with heightened airway resistance and inflammation is a characteristic feature of bronchial asthma. It has been demonstrated that contraclile responsiveness to endothelin-1 (ET-1) in repeated antigen challenge-induced airway hyperresponsive bronchial preparation was significantly increased. ET-1 is a potent contracting substance for various smooth muscles including airways. In addition to the classical Ca2+-mediated contraction, ET-1 also induced Ca2+ sensitization of contraction. However, it is not clear whether ET-1 stimulation also activates the CPI-17 (PKC-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa) pathway in airway smooth muscles. Therefore, the changes in ET-1-induced activation/phosphorylation of CPI-17 and myosin light chain (MLC) in bronchial smooth muscle of repeatedly antigen-challenged rats were examined. The levels of ET-1-induced phosphorylation of CPI-17 and MLC were increased much more markedly in the AHR group than in the sensitized control animals. It might be suggested that the augmented activation of CPI-17 observed in the hyperresponsive bronchial smooth muscle is responsible for the enhanced agonists-induced contraction of bronchial smooth muscle in AHR rats.
Silibinin, derived from the milk thistle plant, Silybum marianum, has been traditionally used as an antihepatotoxic agent for the treatment of liver disease. Our preliminary study demonstrated that silibinin has protected rat cardiac myocytes against beta-adrenergic agonist isoproterenol-induced injury through resuming mitochondrial function and regulating the expression of SIRT1 and Bcl-2 family members. In this study, we investigate whether silibinin has anti-apoptotic effect on isoproterenol-treated rat cardiac myocytes. DNA damage, detected by the TUNEL and DNA fragmentation assay, was diminished after treatment of silibinin. Results of nitrite and Western blot assays showed that the amount of NO and the expression of iNOS were decreased after treatment with silibinin, while the expression of procaspase-3 and digestion of caspase-3 substrates, the inhibitor of caspase-activated DNase (ICAD) and poly-(ADP-ribose) polymerase (PARP), were increased simultaneously. The DNA damage was reversed by down-regulation of p53 phosphorylation after treatment with silibinin. Result of flowcytometric analysis showed that the cell cycle was not affected, and the expression of cell cycle regulatory protein p21 also had no change. Consequently, silibinin protected cardiac myocytes against isoproterenol-induced DNA damage through caspase pathway and the expression of p53, but independent on regulation of cell cycle.
Berberine is a benzyl tetra isoquinoline alkaloid which is widely used as an antimicrobial and an antidiarrhoeal. As berberine containing plants are virtually used in all forms of traditional medicine, our study aimed to examine the antioxidant activity of berberine using 2,2-diphenyl 1-picrylhydrazyl (DPPH) radical scavenging, nitric oxide scavenging, lipid peroxidation, superoxide scavenging, iron chelating activity and 2,2-azino bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) radical scavenging methods. The IC50 values for all the models were calculated by regression analysis. In all the models tested, berberine showed its ability to scavenge the free radicals in a concentration dependent manner. The present study thereby justifies the therapeutic potential of berberine.
The objectives of this study were to investigate the radical-scavenging activity and protective potential of Sophora flavescens from oxidative damage by the radical generator 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH) in renal epithelial LLC-PK1 cells and to identify the active components using the bioassay-linked fractionation method. The MeOH extract and fractions of CH2Cl2, BuOH, and H2O from S. flavescens showed 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging effects in a dose-dependent manner (p<0.01),whereas only the BuOH and CH2Cl2 fractions showed protective effects against LLC-PK1 cellular damage induced by AAPH in a dose-dependent manner (p<0.01). In particular, the BuOH fraction had the most effective (p<0.05) antioxidative capacity. Employing a bioassay-linked HPLC/MS method, the active constituents from the BuOH fraction of S. flavescens were isolated and characterized as sophoraflavanone G and kurarinone with potent antioxidant effects against the DPPH radical, with IC50 values of 5.26 and 7.73 μg/ml, respectively. Moreover, the compounds dose dependently recovered cell viability decreased by AAPH treatment (p<0.01), suggesting their protective roles against cellular oxidative damage. The results of this study suggest that S. flavescens has excellent antioxidative and kidney-protective potential and that flavonoids from S. flavescens, i.e., sophoraflavanone G and kurarinone, are the active constituents.
Documented ethno-contraceptive use of Tape-vine or Stephania japonica (THUNB.) MIERS., Syn. Stephania hernandifolia (WILLD.) WALP. leaves is evaluated with regards to post-coital pregnancy interceptive activity of its aqueous extract (AE) and an ethnomedicinal formulation (EF) in Wistar rats. EF at 500 and 250 mg/kg doses induced 66.7% and 33.3% post-coital pregnancy interception respectively and the higher dose exhibited significant reduction in number of litters born and also anti-implantation property. In contrast, none of the dose levels of AE interfered in pregnancy but significant anti-implantation property was observed at doses of 2 and 1 g/kg, even as the higher dose produced significant reduction in number of litters born as well. EF at 500 mg/kg also exhibited significant uterotrophic activity and histological changes in uterus. Pair-wise comparison of sex hormone-levels exhibited significant increment in serum estradiol, LH and FSH but decrease in progesterone levels. Assessed blood lipid-carbohydrate profile exhibited substantial decrease in glucose, cholesterol, VLDL and triglyceride contents and significant increase in HDL. It is concluded that EF probably acts as better post-coital pregnancy interceptive agent through restriction of implantation by alteration of gonadal hormone levels and decline in blood-glucose levels that possibly disrupts oxidative energy metabolism in uterus during implantation. High surge in LH and FSH suggests negligible interference in ovulatory mechanism. This preparation also seems to be free of cardiovascular risk factors. HPTLC and HPLC analysis of both EF and AE exhibited marked chemical differences.
In our previous efforts to find new tyrosinase inhibitory materials, we investigated 44 Indonesian medicinal plants belonging to 24 families. Among those plants, the extract of Artocarpus heterophyllus was one of the strongest inhibitors of tyrosinase activity. By activity-guided fractionation of A. heterophyllus wood extract, we isolated artocarpanone, which inhibited both mushroom tyrosinase activity and melanin production in B16 melanoma cells. This compound is a strong candidate as a remedy for hyperpigmentation in human skin.
Melanogenesis inhibitory and free radical scavenging activities of nine cyclic (1—9) and one acyclic diarylheptanoids (10), and two phenolic compounds, (+)-rhododendrol (11) and (+)-catechin (12), isolated from the ethyl acetate-soluble fraction of the MeOH extract of the bark of Acer nikoense MAXIM. (Aceraceae) were examined. Upon evaluation of compounds 1—12 on the melanogenesis in the B16 melanoma cells, two compounds, 2 and 8, exhibited marked inhibitory activity with 55.6% and 46.8% reduction, respectively, of melanin content at 25 μg/ml without inhibition of cell proliferation. In addition, upon an evaluation of eleven compounds, 1—7 and 9—12 against the scavenging activities of free radicals (against the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical), compound 12 (IC50 9.0 μM) followed by compounds 1, 3, 4, and 6 (IC50 40.2—44.0 μM) showed potent scavenging activities.
The protective effect of Hangeshashinto (HST) and its major constituents, baicalin (BA), berberine (BE), saponin fraction of ginseng (GS) and glycyrrhizin (GL) on rat gastric lesion induced by ethanol was examined to clarify its active ingredients and action mechanism. Oral treatment with HST at the doses of 125 and 250 mg/kg suppressed ethanol-induced gastric lesions. The mixture of BA, BE, GL and GS (4M), each of BE, GL and GS at the dosage corresponded to HST (125 mg/kg) also suppressed the ethanol-induced gastric lesion in rats, but BA did not. Treatment of ethanol augmented the activity of myeloperoxidase (MPO) in the stomach, which was significantly suppressed by the administration of HST, BE, GL and GS. These results suggest that the protective effect of HST on ethanol-induced gastric lesion was depended on BE, GL and GS, by, in part, the reduction of MPO activity in stomach.
Fifteen triterpene acids, viz., seven of the β-boswellic acids (ursane-type) (1—7), two of the α-boswellic acids (oleanane-type) (8, 9), two of the lupeolic acids (lupane-type) (10, 11), and four of the tirucallane-type (12—14, 16), and two cembrane-type diterpenes (17, 18), isolated from the MeOH extract of the resin of Boswellia carteri (Burseraceae), together with a triterpene acid 15 (the acetyl derivative of 14), were examined for their inhibitory effects on the induction of Epstein–Barr virus early antigen (EBV-EA) by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells and on activation of (±)-(E)-methyl-2[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexemide (NOR 1), a nitrogen oxide (NO) donor, and cytotoxic activities against three human neuroblastoma cell lines, IMR-32, NB-39, and SK-N-SH in vitro. On evaluation against the EBV-EA activation induced by TPA, seven compounds, 2, 10, 11, and 13—16, showed potent inhibitory effects on EBV-EA induction. Upon evaluation against activation of NOR 1, five compounds, 7, 13, and 14—16, showed potent inhibitory effects. Further, fifteen compounds, 1—7, 9—11, 13—15, 17, and 18, exhibited potent cytotoxic activities with IC50 values of 4.1—82.4 μM against all of the three human neuroblastoma cells tested.
We evaluated inhibitory effects of plastoquinones (1, 2) and chromene derivative (3) from the methanol extract of Sargassum micracanthum on the differentiation of osteoclast progenitors into osteoclast-like cells (OCLs), bone-resorbing activity and the survival of OCLs. When OCL formation was investigated using osteoclast progenitor cells obtained from macrophage-colony stimulating factor (M-CSF)-treated mouse bone marrow, 1—3 inhibited dose-dependently OCL formation at the concentrations of 3—10 μM, the order of inhibitory potency being 1>3>2. In addition, they suppressed dose-dependently the pit formation induced by OCL on dentine slices, the order of inhibitory potency being 1>3>2. The survival of OCLs was inhibited by about 35, 45 and 60% in the presence of 6 μM of 2, 3 and 1, respectively. These results suggest that the inhibitory effects of the three compounds on the differentiation, pit formation and the survival of OCLs might contribute to the suppression of bone resorption.
Mouth washing after inhalation of corticosteroids is effective for prevention of local adverse effects such as hoarseness and oropharyngeal candidiasis. To establish an optimal procedure for such mouth washing, we investigated the removal rates of drug residues remaining on the oropharyngeal mucosa using various mouth washing methods following inhalation. A beclomethasone dipropionate metered dose inhaler (BDP-MDI) (100 μg) and a fluticasone propionate dry powder inhaler (FP-DPI) (100 μg) were used. The effects of different mouth washing methods were evaluated by quantification of drugs in the expectorated rinse solution using an HPLC method. The amounts of BDP recovered in the rinse after gargling and rinsing for 5 s each were 47.1±13.6 μg, while they were 42.9±9.4 μg after rinsing alone for 10 s and 38.7±9.2 μg after gargling alone for 10 s. Under the same conditions, FP amounts were 32.9±7.3 μg, 28.9±2.4 μg, and 27.1±7.9 μg, respectively. In a comparison of washing time, the amounts of BDP recovered were 49.8±9.7 μg after gargling and rinsing for 2 s each, 53.5±10.2 μg after those for 3 s each, and 47.1±13.6 μg after those for 5 s each, while the amounts of FP under the same conditions were 36.4±2.4 μg, 33.3±6.4 μg, and 32.9±7.4 μg, respectively. As for the effect of time lag before mouth washing, the amount of BDP recovered decreased by 65.7% with a lag time of 1 min and by 5.6% after 10 min, while that of FP decreased by 51.1% with a lag time of 1 min and by 7.7% after 10 min. Our results suggest that the amount of drugs removed by mouth washing is significantly associated with the time lag between inhalation and mouth washing. We concluded that immediate gargling and rinsing after inhalation is most useful for the removal of drugs following inhalation of corticosteroids.
To investigate the effect of functional groups in bovine serum albumin (BSA) on its tissue distribution characteristics, tyrosine (Tyr) or tryptophan (Trp) residues of BSA were chemically modified by tetranitromethane (TNM) and 2-hydroxy-5-nitrobenzyl bromide (HNB), respectively. BSA was successfully modified with each reagent depending on the amount of the reagent added to the reaction mixture, and TNM- and HNB-modified BSA derivatives with different degrees of modification were obtained. Circular dichroism measurements showed that slight secondary and large tertiary changes were detectable as the degree of modification increased. After intravenous injection into mice, all synthetic BSA derivatives were eliminated very slowly from the systemic circulation. However, 111In-TNM6.6- and 111In-HNB2.0-BSA, derivatives with a high degree of modification, showed a slightly faster disappearance from the systemic circulation and slightly higher accumulation in the liver than 111In-unmodified BSA. Pharmacokinetic analyses also demonstrated that the modification of Tyr or Trp residues on BSA had only marginal effects on tissue distribution. These results indicate that the Tyr and Trp residues have little effect on the tissue distribution characteristics of serum albumins, and that the specific modification of these residues may be a promising approach to designing sustained drug delivery systems using serum albumins.
The effects of three serotonin-3 (5-HT3) receptor antagonists, azasetron, ondansetron, and ramosetron, on cytochrome P450 (CYP) 1A2-mediated 7-ethoxyresorufin O-deethylation, CYP2C9-mediated tolbutamide hydroxylation, CYP2C19-mediated S-mephenytoin 4′-hydroxylation, CYP2D6-mediated debrisoquine 4-hydroxylation, CYP2E1-mediated chlorzoxazone 6-hydroxylation, CYP3A4-mediated nifedipine oxidation, and CYP3A4-mediated testosterone 6β-hydroxylation activities in human liver microsomes were compared. Azasetron and ramosetron at a concentration of 1 or 10 μM neither inhibited nor stimulated any of the metabolic activities. On the other hand, ondansetron competitively inhibited CYP1A2 and CYP2D6 activities, and the inhibition constants (Ki) were 3.2 and 21.0 μM, respectively, which are much higher than the reported plasma concentrations after clinical intravenous or oral dosing. The free fractions of the three 5-HT3 receptor antagonists in the incubation mixture estimated by ultracentrifugation were more than 68.6%. These results suggest that azasetron, ondansetron, and ramosetron do not cause clinically significant interactions with other drugs that are metabolized by CYPs via the inhibition of metabolism.
We previously observed the enhanced anticancer efficacy of anticancer drugs encapsulated in Ala-Pro-Arg-Pro-Gly-polyethyleneglycol-modified liposome (APRPG-PEG-Lip) in tumor-bearing mice, since APRPG peptide was used as an active targeting tool to angiogenic endothelium. This modality, antineovascular therapy (ANET), aims to eradicate tumor cells indirectly through damaging angiogenic vessels. In the present study, we examined the in vivo trafficking of APRPG-PEG-Lip labeled with [2-18F]2-fluoro-2-deoxy-D-glucose ([2-18F]FDG) by use of positron emission tomography (PET), and observed that the trafficking of this liposome was quite similar to that of non-targeted long-circulating liposome (PEG-Lip). Then, histochemical analysis of intratumoral distribution of both liposomes was performed by use of fluorescence-labeled liposomes. In contrast to in vivo trafficking, intratumoral distribution of both types of liposomes was quite different: APRPG-PEG-Lip was colocalized with angiogenic endothelial cells that were immunohistochemically stained for CD31, although PEG-Lip was localized around the angiogenic vessels. These results strongly suggest that intratumoral distribution of drug carrier is much more important for therapeutic efficacy than the total accumulation of the anticancer drug in the tumor, and that active delivery of anticancer drugs to angiogenic vessels is useful for cancer treatment.
Chitosan is widely used as a dietary weight-loss supplement in Japan. In the present study, we examined the effect of chitosan on the gastrointestinal absorption profiles of the water-insoluble drugs, indomethacin and griseofulvin, and the water-soluble drugs, acetaminophen and cephalexin, after oral administration in rats. Rats received oral administration of chitosan (5 mg/kg or 25 mg/kg) dissolved in 5% acetic acid or vehicle 15 min before oral administration of each drug. Chitosan at a dose of 25 mg/kg, but not 5 mg/kg, significantly decreased the plasma concentrations of indomethacin and griseofulvin after administration as a suspension with a significant delay of the time to reach maximum concentration compared to the corresponding control values (vehicle-pretreated rats). However, pretreatment of chitosan (25 mg/kg) did not change the pharmacokinetics of indomethacin administered as a solution. Further, the same dose of chitosan had no effect on the pharmacokinetics of acetaminophen. The gastrointestinal absorption profile of an amino-β-lactam antibiotic, cephalexin, which is actively absorbed via carrier-mediated transport system, was also unchanged. The present findings at least suggest the possibility that chitosan at high dose reduces the gastrointestinal absorption of water-insoluble drugs such as indomethacin and griseofulvin, but not water-soluble drugs, by diminishing the surfactant-like effect of bile acids.
The effect of glimepiride on metabolism of S-warfarin to 7-hydroxywarfarin was studied using human liver microsomes and recombinant cytochrome P450 2C9 microsomes (CYP2C9.1 and CYP2C9.3), and was compared with the results from the experiments using glibenclamide as an inhibitor. S-Warfarin 7-hydroxylation by recombinant CYP2C9.1 and CYP2C9.3 was inhibited by glimepiride competitively. The apparent Ki value of glimepiride was lower at CYP2C9.3 than at CYP2C9.1. Glimepiride also inhibited 7-hydroxylation of S-warfarin in a competitive manner by microsomes from human liver which showed the genotypes of CYP2C9, as CYP2C9*1/*1 or CYP2C9*1/*3. The apparent Ki value of glimepiride was lower than that of glibenclamide. These results may provide valuable information for optimizing the anticoagulant activity of warfarin when glimepiride is co-administered to patients.
The purpose of the present study was to gain insight into the major factors affecting transfection efficiency with galactosylated lipoplex in HepG2 cells. In this study, lipoplex and galactosylated lipoplex were examined at different charge ratios (− : +): 1.0 : 1.2, 1.0 : 2.3, 1.0 : 3.1, 1.0 : 4.7, and 1.0 : 7.0. The particle size and zeta potential of the both lipoplexes was dependent on the charge ratio. Cellular uptake was evaluated by using [32P]-labeled pCMV-Luc and this showed that the cellular uptake of galactosylated lipoplex was significantly higher than that of lipoplex at a charge ratio ranging from 1.0 : 2.3 to 1.0 : 7.0. As the charge ratio increased in both lipoplexes, the apparent cellular uptake increased. Transfection activity by galactosylated lipoplex was significantly higher than that by lipoplex except at a charge ratio of 1.0 : 7.0. The optimal charge ratio for transfection efficacy was 1.0 : 2.3 and transfection was reduced at higher charge ratios. Both lipoplexes exhibited no significant cytotoxicity at any charge ratio. In conclusion, it is suggested that intracellular trafficking, rather than the degree of uptake and cytotoxicity, is the important process that determines the optimal charge ratio of galactosylated lipoplex in HepG2 cells.
Objective: To obtain potential skin whitening agents from traditional Chinese herbs, we tested changes of melanin content in melanocyte cell lines after treatment with extracts of 90 traditional Chinese herbs. Methods: Mouse melanocyte cell lines were used. Depigmentation activity of the herb extracts were first screened in Mel-Ab cells, and then re-evaluated in melan-a cells and co-culture of melan-a and SP-1 cells. Melanin content and cell viability were the two indications for evaluation. Tyrosinase activity and the expression of melanin synthesis related enzymes in cells treated with the herb extracts were also tested. Results: Nine herb extracts were proved to have depigmentation activity similar to or better than that of arbutin and low cytotoxicity to melanocytes. Two of them were more effective in co-cultured melan-a cells. Most of the effective herb extracts inhibited tyrosinase activity and the expression of tyrosinase. Some of them also inhibited tyrosinase related protein-1 and/or tyrosinase related protein-2 in cultured cells. Conclusions: We have found 9 herb extracts to be promising skin whitening agents. Among them, water extract of Galla Chinensis and ethanol extract of Radix Clematidis exhibited higher depigmentation activity and caused lower tyrosinase activity in cell culture assays and are worthy to be further studied.
The potency of the induction of hepatomegaly, peroxisomal β-oxidation and microsomal 1-acylglycerophoshocholine (1-acyl-GPC) acyltransferase was compared among perfluorinated fatty acids (PFCAs) with 6—9 carbon chain length in the liver of male and female mice. All PFCAs examined induced hepatomegaly and peroxisomal β-oxidation and the potency was in the order of perfluorononanonic acid (PFNA), perfluorooctanoic acid (PFOA), perfluoroheptanoic acid (PFHA) and perfluorohexanoic acid (PFHeA) when compared with the relative doses to induce the two parameters. Microsomal 1-acyl-GPC acyltransferase was induced by PFHA, PFOA and PFNA, as was peroxisomal β-oxidation. No significant sex-related difference was observed in the induction of peroxisomal β-oxidation by any PFCAs examined. PFNA and PFOA accumulated in the liver of both male and female mice in a dose-dependent manner. PFHA accumulated in the liver to a lesser extent; little PFHeA accumulated in the liver. Hepatic concentrations of PFNA, PFOA and PFHA were higher in male mice than those in female mice. One linear regression line was confirmed between the activities of peroxisomal β-oxidation and hepatic concentrations of PFHeA, PFHA, PFOA and PFNA in male mice regardless of their carbon chain lengths, and the activities were saturable at the concentrations over approximately 500 nmol/g liver. Similar linear regression line was obtained between the two parameters in female mice. These results suggest (i) that the longer the perfluoroalkyl chain becomes, the more PFCA accumulates in the liver of both male and female mice, (ii) that the accumulated PFCAs induce hepatomegaly, peroxisomal β-oxidation and microsomal 1-acyl-GPC acyltransferase, and (iii) that the difference observed in the accumulation of PFHA, PFOA and PFNA in the liver between male and female mice is not enough to produce obvious sex-related difference in the induction of peroxisomal β-oxidation.