Identification of metabolites of (-)-epicatechin gallate and their metabolic fate in the rat

After intravenous administration of (-)-epicatechin gallate to Wistar male rats, its biliary metabolites were examined. Deconjugated forms of (-)-epicatechin gallate metabolites were prepared by beta-glucuronidase/sulfatase treatment and purified by HPLC. Five compounds were subjected to FAB-MS and NMR analyses. These metabolites were shown to be (-)-epicatechin gallate, 3'-O-methyl-(-)-epicatechin gallate, 4'-O-methyl-(-)-epicatechin gallate, 4' '-O-methyl-(-)-epicatechin gallate, and 3',4' '-di-O-methyl-(-)-epicatechin gallate. After oral administration, five major metabolites excreted in rat urine were purified in their deconjugated forms and their chemical structures identified. They were degradation products from (-)-epicatechin gallate, pyrogallol, 5-(3,4-dihydroxyphenyl)-gamma-valerolactone, 4-hydroxy-5-(3,4-dihydroxyphenyl)valeric acid, 3-(3-hydroxyphenyl)propionic acid, and m-coumaric acid. Time course analysis of the identified (-)-epicatechin gallate metabolites showed that (-)-epicatechin gallate and its conjugate appeared in the plasma with their highest levels 0.5 h after oral administration; their levels rapidly decreased, and then they disappeared by 6 h. The degradation products, mainly in their conjugated forms, emerged at 6 h, peaked at 24 h, and disappeared by 48 h. In urine samples, (-)-epicatechin gallate and its methylated metabolites were hardly detected and the degradation products began to be excreted in the 6-24 h period, peaked in the 24-48 h period, and then began to disappear. The most abundant metabolite in both the plasma and the urine was found to be the conjugated form of pyrogallol. On the basis of these results, a possible metabolic route of (-)-epicatechin gallate orally administered to the rat is proposed.     

Evaluation of the NO scavenging activity of procyanidin in grape seed by use of the TMA-PTIO/NOC 7 ESR system

The nitrogen monoxide (NO) scavenging activity of grape seed extract (GSE) was studied in the TMA-PTIO/NOC 7 system. The procyanidin-rich (>95%) GSE showed strong NO scavenging activity in the system. The activity was found to depend on the condensation rate of cyanidin when synthetic oligomers were tested. Investigation of the NO scavenging activities of other polyphenols (catechin, epicatechin, epigallocatechin, and epigallocatechin gallate) in the TMA-PTIO/NOC 7 system revealed that gallocatechin, epigallocatechin, and epigallocatechin gallate exhibited strong activities. From the results, it was suggested that the high condensation rate of and the gallate ester moiety in procyanidin in GSE may play an important role in the NO scavenging activity. The mechanism of the NO scavenging activity of phenolic compounds such as GSE is speculated to be as follows: NO reacts with phenolic compounds directly to generate phenoxy radicals.     

Synthesis and structure-activity relationships of 1-phenyl-1H-1,2,3-triazoles as selective insect GABA receptor antagonists

To study the interaction of phenylheterocycles with gamma-aminobutyric acid (GABA) receptors, 4- or 5-alkyl(or phenyl)-1-phenyl-1H-1,2,3-triazoles were synthesized and examined for their ability to inhibit the specific binding of [3H]-4'-ethynyl-4-n-propylbicycloorthobenzoate (EBOB), a noncompetitive antagonist, to the housefly and rat GABA receptors, as well as to the beta3 subunit homo-oligomer of the human GABA receptor investigated as a model receptor. 4-Substituted 1-phenyl-1H-1,2,3-triazoles were found to be more potent competitive inhibitors than the 5-substituted regioisomers in the case of all receptors. The 4-tert-butyl or 4-n-propyl analogue of 1-(2,6-dichloro-4-trifluoromethylphenyl)-1H-1,2,3-triazole exhibited the highest level of inhibition of [3H]EBOB binding to all receptors. Most of the synthesized analogues were more active in terms of the inhibition of EBOB binding to the housefly and human beta3 GABA receptors than to the rat receptor. The 4-cyclohexyl analogue showed the highest (185-fold) housefly versus rat receptor selectivity. A three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis demonstrated that both the 4-trifluoromethyl-2,6-dichloro substitution on the phenyl ring and a small, bulky, hydrophobic substituent at the 4-position of the triazole ring played significant roles in conferring high potency in cases involving the housefly and human beta3 receptors. The human beta3 receptor resembled the housefly receptor in terms of their recognition of phenyltriazoles, whereas 3D-QSAR analysis revealed a slight difference between the two receptors in terms of their mechanisms of recognition of the para-substituent on the phenyl moiety. Some of the triazoles synthesized here exhibited insecticidal activity, which was correlated with their ability to inhibit [3H]EBOB binding to the housefly receptor. Thus, 1-phenyl-1H-1,2,3-triazoles with the appropriate substituents exert insecticidal activity by selectively acting at the site for noncompetitive antagonism of insect GABA receptors.     

Cell bioassay for paralytic shellfish poisoning (PSP): comparison with postcolumn derivatization liquid chromatographic analysis and application to the monitoring of PSP in shellfish

We performed a neuroblastoma cell (Neuro2a) culture assay modified slightly from a method reported previously to provide a simple and sensitive evaluation of paralytic shellfish poisoning (PSP) toxicity in shellfish. The cell bioassay was just as sensitive for C-toxins as for gonyautoxins. The sensitivity of our cell bioassay was 4 times that of the current standard mouse bioassay. Using the cell bioassay, we evaluated PSP toxicity in 361 shellfish samples collected from Mikawa Bay and Ise Bay, Aichi Prefecture, Japan, from April 1999-March 2002. The results were compared with those obtained in a postcolumn derivatization liquid chromatographic analysis. PSP toxins were detected in 236/361 samples by both assays, and there was a fairly good correlation (r = 0.9001, n = 236, p < 0.001) between the results from the two assays. We applied this cell bioassay when short-necked clams in the bay turned poisonous in 2001. The chronological changes in PSP toxicity in the short-necked clams were analyzed and compared with those of the cell density of poisonous plankton (Alexandrium tamarense) occurring in the bay. The PSP toxicity in shellfish peaked 2 weeks after the cell density reached a maximum. We recommend using the cell bioassay for routine monitoring of PSP toxicity in shellfish living in natural marine environments.     

Prevalence of intestinal parasites in private-household cats in Japan

The present study is the first national investigation of intestinal parasites in private-household cats in Japan. A total of 942 faecal samples were collected from private-household cats. Giardia species was assessed using an enzyme-linked immunosorbent assay kit and other intestinal parasites were identified microscopically. The overall prevalence of intestinal parasites was 10.1%; two protozoan parasites (Giardia species and Cystoisospora species) and five helminths (Toxocara cati, Toxascaris leonina, Ancylostoma tubaeforme, Taenia species and Spirometra erinacei) were detected. The total prevalence of intestinal parasite infection was significantly higher in cats aged ≤ 6 months old than in cats older than 6 months because of a significantly higher prevalence of Cystoisospora species and T cati. The total infection prevalence was higher among outdoor cats as a result of the significantly higher prevalence of T cati and S erinacei. Sex and faecal condition were not related to intestinal parasite infections. Regional differences were observed in Cystoisospora species and A tubaeforme.     

Relationship between embryo collection results after superovulation treatment of Japanese Black cows and their plasma β-carotene and vitamin concentrations

The objective of this study was to investigate the relationship between the plasma concentrations of vitamin A (VA), vitamin E (VE) and β-carotene (BC) during embryo collection in Japanese Black cows that had undergone superovulation treatment and the embryo collection results. Following superovulation treatment in 116 Japanese Black cows, we collected 1317 embryos by nonsurgical means seven days after artificial insemination. The collected embryos were classified into transferable embryos, unfertilized oocytes and degenerated embryos. After embryo collection, we collected blood samples from the cows and measured the plasma concentrations of VA, VE and BC. The cows were then divided into 2 groups depending on the plasma concentration of VA (L and H: < 80 IU/dl and ≥ 80 IU/dl), VE (L and H: < 150 μg/dl and ≥ 150 μg/dl) and BC (L and H: < 150 μg/dl and ≥ 150 μg/dl). As a result, the number of collected embryos in the H group of VE was significantly (P<0.01) higher than that in L groups. Furthermore, the number of transferable embryos was higher (P<0.05) in all VA, VE and BC H groups than in the L groups. The H group for BC showed a high ratio of transferable embryos compared with the L group (P<0.05). Consequently, the present study suggests that the plasma VE and BC concentrations are positively correlated with the embryo collection results.     

Efficacy of soluble recombinant FliC protein from Salmonella enterica serovar enteritidis as a potential vaccine candidate against homologous challenge in chickens

FliC, the flagellin antigen of Salmonella Enteritidis, was tested as a vaccine candidate for protective effect against a homologous challenge in chickens. After immunization with recombinant FliC (rFliC) or administration of phosphate-buffered saline (PBS) at 56 days old, the chickens were challenged with 10(9) colony-forming units of Salmonella Enteritidis at 76 days old. The vaccinated birds showed significantly decreased bacterial counts in the liver and cecal contents compared to those administered PBS at 7 days postchallenge, but the protection was partial. The replication experiment also showed a similar result. In both experiments, vaccination induced an increased level of serum anti-rFliC IgG, which was also reactive to the native flagella. The intestinal IgA level was slightly higher in the vaccinated birds than in the control. However, neither the proliferative response nor interferon-gamma secretion of splenic cells upon stimulation with rFliC was induced. Therefore, the effect of rFliC as a vaccine is limited, and further improvement is needed.