Simple and rapid analysis of aristolochic acid contained in crude drugs and Kampo formulations with solid-phase extraction and HPLC photodiode-array detection

Simple and rapid analysis of aristolochic acid (AA) in crude drugs and Kampo extracts using a solid-phase extraction method and HPLC-PDA analysis was investigated. Extraction of AA from samples was accomplished by adding methanol containing 1% ammonia. The addition of ammonia ionized the AA of acidic substances so that they adhered to an acrylamide copolymer of a strong anion exchange resin (Sep-Pak QMA) coupled to diol silica easily. Furthermore, a mixture of acetonitrile–water–phosphoric acid (75:25:2, v/v) was effective in isolating AA from its carrier. Since almost all interfering peaks originating from contaminants in crude drugs and Kampo extract formulations could be removed, a satisfactory HPLC chromatogram of AA was obtained. A good result was also obtained when Aristolochiaceae and crude drugs containing AA were tested. Particularly in the case of the medicinal parts of Asarum, several interfering peaks and a ghost peak detected near the AA peak were eliminated. The AA contents of two Kampo extract formulations, tokishigyakukagoshuyushokyoto and ryutanshakanto, were calculated by HPLC analysis. The AA content (the sum of AA-I and AA-II) was 1.25–6.13 mg per daily dose. From an additional recovery experiment for Kampo formulations, high recovery rates of AA were obtained. Neither LC/MS nor special instrumentation was necessary. Our results suggest that this simple, quick, and sensitive analytical method to detect AA in crude drugs and Kampo extract formulations would be valuable in safety inspections of AA in crude drugs and their products.     

Effects of angiotensin-converting enzyme inhibition on changes in left ventricular myocardial creatine kinase system after myocardial infarction: their relation to ventricular remodeling and function

We assessed the effects of angiotensin-converting enzyme (ACE) inhibition on changes in the myocardial intracellular creatine kinase (CK) system in relation to left ventricular (LV) remodeling and function in heart failure after myocardial infarction (MI) in rats. We compared the findings at 4 weeks after MI to those at 12 weeks after MI. LV weight and chamber size were significantly increased and percent fractional shortening (%FS) was decreased in untreated MI rats compared with normal control animals both at 4 and 12 weeks after MI. Animals with MI and treated with the ACE inhibitor temocapril showed significantly reduced LV weight and chamber size and increased %FS compared with untreated MI rats at 12 weeks after MI, but not at 4 weeks after MI. At 4 weeks after MI, no significant changes were found in the total creatine and relative distribution of each CK isoenzyme in either the temocapril-treated or untreated animals with MI compared with the normal controls. In contrast, at 12 weeks after MI, untreated MI rats showed significant reductions in the total creatine and mitochondrial and MM-CK fractions and increases in the MB- and BB-CK fractions compared with the controls. The alterations in the mitochondrial and MB-CK fractions were significantly attenuated after 12 weeks of ACE inhibition. Thus, LV myocardial energy metabolism is progressively impaired and its alteration is not related to the magnitude of geometric changes and LV dysfunction after MI. Most of the beneficial effects of ACE inhibition were observed at 12 weeks after MI. Our results may provide an insight into the therapeutic strategy of ACE inhibition in chronic heart failure after MI.     

Genomic characterization of echovirus 6 causing aseptic meningitis in Hokkaido, Japan: a novel cluster in the nonstructural protein coding region of human enterovirus B

We determined four complete nucleotide sequences of echovirus 6 (E6) isolated from an epidemic of aseptic meningitis (AM) in Hokkaido, Japan, in 2011. Phylogenetic analysis of the genes encoding viral capsid protein 1 revealed that the strains were closely related to E6 strains isolated in China in recent years, but they were distantly related to E6 strains isolated from patients with AM in Osaka Prefecture, Japan, in 2011. The genes encoding the viral protease and RNA-dependent RNA polymerase (3CD) were closely related to those of several non-E6 strains of the species Human enterovirus B isolated in China, South Korea, and Australia from 1999 to 2010, resulting in a novel cluster in the phylogenetic tree. These results suggest that the incidence of AM in Japan in 2011 was caused by at least two lineages of E6 strains, and a lineage of the 3CD gene was interspersed among different serotypic strains isolated in Western Pacific countries.     

Retinal neurons of the California ground squirrel,Spermophilus beecheyi: A Golgi study

Although the optic nerve fibers of the cone-dominant ground squirrel retina have been well studied physiologically, the morphological details of the retinal neurons have not. To that end, retinal neurons of the California ground squirrel have been studied in Golgi-impregnated wholemounts. Two types of horizontal cell have been identified: H1 has an axon and axon terminal, whereas H2 is axonless. The dendritic field of H1 cells enlarges in a nonuniform manner with increasing displacement from the central retina. The smallest examples lie centrally in the visual streak, and the largest occur in the superior periphery. Eight types of bipolar cell are distinguished by morphological differences in dendritic branching pattern and field size in the outer plexiform layer, cell body size, and layering within the inner nuclear layer and by the morphology and stratification of axon terminals in the inner plexiform layer. A large bistratified bipolar cell (B8) is introduced here; the other 7 types closely resemble those in the retinas of other sciurid species described by R.W. West (1976, J. Comp. Neurol. 168:355–378; 1978, Vision Res. 18:129–136). The B1 type is proposed as a blue cone bipolar cell. Amacrine cells are classified into 27 cell types. Six of these occur as mirror-image pairs across the inner plexiform layer, the soma of one of each pair being “displaced” to the ganglion cell layer. The best described of these pairs is the very elaborate starburst amacrine cell, A5, which stains regularly in these wholemounted retinas. Changes in dendritic field size of both A5 subtypes with retinal location are quantified. The morphology of three amacrine cell types identified in Spermophilus beecheyi suggests that their possible counterparts in S. mexicanus (West, 1976) were, as displaced amacrine cells, misidentified as ganglion cells. Amacrine cell types that may play roles in the rod pathway, the blue cone pathway, and ganglion cell directional selectivity are discussed. No type of interplexiform cell was observed. Ganglion cells are classified into 19 cell types, 9 of which probably correspond to the ganglion cells described by West (1976) in the Mexican ground squirrel. The bistratified G11 cell is proposed as an ON-OFF directionally selective type. © 1996 Wiley-Liss, Inc.     

Characterization of human cytochrome P450 enzymes involved in the in vitro metabolism of perospirone

In vitro studies were carried out to identify the major contribution of CYP2C8, CYP2D6 and CYP3A4 to the metabolism of perospirone (cis-N-[4-[4-(1,2-benzisothiazol-3-yl)-1-piperazinyl]butyl]cyclohexane-1,2-dicarboximide monohydrochloride dehydrate), a novel antipsychotic agent, using human liver microsomes and expressed P450 isoforms. Quinidine (a specific inhibitor of CYP2D6) did not markedly affect the metabolism of perospirone, whereas quercetin (an inhibitor of CYP2C8) and ketoconazole (an inhibitor of CYP3A4) caused a decrease in the metabolism with human liver microsomes in a concentration dependent fashion. With 10 microM quercetin, the metabolism of perospirone was inhibited by 60.0% and with 1 microM ketoconazole almost complete inhibition was apparent. Anti-CYP2C8 and anti-CYP2D6 antisera did not exert marked effects, whereas anti-CYP3A4 antiserum caused almost complete inhibition. With expressed P450s, K(m) and V(max) values were 1.09 microM and 1.93 pmol/min/pmol P450 for CYP2C8, 1.38 microM and 5.73 pmol/min/pmol P450 for CYP2D6, and 0.245 microM and 61.3 pmol/min/pmol P450 for CYP3A4, respectively. These results indicated that the metabolism of perospirone in human liver was mainly catalysed by CYP3A4, and to a lesser extent CYP2C8 and CYP2D6 were responsible because kinetic data (K(m) and V(max)) of CYP2C8 and CYP2D6 suggested catalytic potential.     

Growth of Bacteroides fragilis in Rabbit Tracheal Organ Culture: Anaerobiosis and Tissue Respiration

Rabbit tracheal explants supporting growth of inoculated Bacteroides fragilis in air were shown to keep low oxygen tension. Treating the explants with sodium azide induced high oxygen tension and arrested reversibly the growth of B. fragilis.