Akio Kawachi

Simultaneous detection of green tea catechins and gallic acid in human serum after ingestion of green tea tablets using ion-pair high-performance liquid chromatography with electrochemical detection

We developed an analytical method for the simultaneous determination of tea catechins and gallic acid (GA) in human serum using ion-pair high-performance liquid chromatography (HPLC) with electrochemical detection. GA was measured to estimate the amount of gallate moiety produced by degradation of gallated catechins ((-)-epicatechin-3-gallate, ECG; (-)-epigallocatechin-3-gallate, EGCG). Ethyl gallate was adopted as an internal standard to correct for the extraction efficiency. To maximize extraction efficiency, a hydrophobic polytetrafluoroethylene (PTFE) filter was selected for pre-treatment prior to separation. HPLC separation was performed using a C18 reversed-phase column with a gradient mobile phase of phosphate buffer (pH 2.5) containing tetrahexylammonium hydrogensulfate as an ion-pair reagent. Using this method, (-)-epicatechin (EC), (-)-epigallocatechin (EGC), ECG, EGCG, ethyl gallate, and GA were detected as single peaks. The resolution values for target analytes were 4.0-13.0 and the mean values of the absolute recoveries of catechins and GA were 77.3-93.9%. The detection limits for catechins and GA in serum were 0.4-3.1ng/mL. The serum catechin levels of eight healthy volunteers after ingestion of a single dose of green tea tablets were measured using this method. The concentration of total catechins (free+conjugated forms) in serum peaked 60min after ingestion. From these results, this method is thought to enable the simultaneous quantification of GA, the hydrolysis product of gallated catechins, and target catechins, and to be sufficiently sensitive for pharmacokinetic studies of catechins following oral administration of green tea.

Effects of Ascorbic Acid on Interactions between Ciprofloxacin and Ferrous Sulphate, Sodium Ferrous Citrate or Ferric Pyrophosphate, in Mice

The absorption of ciprofloxacin has been reported to be impaired by concomitant administration of ferrous sulphate. The effects of sodium ferrous citrate and ferric pyrophosphate, which have been used as extensively as ferrous sulphate, on the absorption of ciprofloxacin were compared with that of ferrous sulphate. The effects of ascorbic acid on the interactions between ciprofloxacin and each iron compound were studied in mice. Mice were treated orally with ciprofloxacin (50 mg kg−1) alone, the iron compound (ferrous sulphate, sodium ferrous citrate or ferric pyrophosphate; 50 mg elemental iron kg−1) alone, ciprofloxacin with each iron compound or ciprofloxacin in combination with each iron compound and ascorbic acid (250 mg kg−1).

Green tea catechin, epigallocatechin‑3‑gallate, attenuates the cell viability of human non‑small‑cell lung cancer A549 cells via reducing Bcl‑xL expression

Clinical and epidemiological studies have indicated that the consumption of green tea has a number of beneficial effects on health. Epigallocatechin-3-gallate (EGCg), the major polyphenolic compound present in green tea, has received much attention as an active ingredient. Among the numerous promising profiles of EGCg, the present study focused on the anticancer effects. Apoptosis induced by EGCg and subsequent cell growth suppression have been demonstrated in a number of cell culture studies. However, the underlying mechanism of apoptotic cell death remains unclear. Thus, the aim of the present study was to identify the major molecule that mediates proapoptotic cell death by EGCg. The effect of EGCg on cell proliferation and the induction of mRNA that modulates apoptotic cell death was evaluated in the A549 human non-small-cell lung cancer cell line. In addition, morphological changes were assessed by microscopy in A549 cells that had been treated with 100 μM EGCg for 24 h. The MTT assay revealed that cell proliferation was significantly reduced by EGCg in a dose-dependent manner (3–100 μM). The mRNA expression level of B-cell lymphoma-extra large (Bcl-xL) was decreased in A549 cells following 24 h incubation with 100 μM EGCg. Therefore, the results indicated that the inhibition of cell proliferation by EGCg may be achieved via suppressing the expression of the cell death-inhibiting gene, Bcl-xL.

Up-regulation of methionine-enkephalin-like immunoreactivity by 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment in the forebrain of the Long-Evans rat.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is considered to be one of the most toxic environmental contaminants, named dioxin. Exposure to TCDD induces a plethora of intoxication symptoms, including anorexia and hypothermia, in several mammals and human. Enkephalin, an endogenous pentapeptide, is an important neuroregulator of autonomic functions, such as food intake and body temperature. In this study, we investigated the effects of TCDD gastric administration on methionine-enkephalin (MEK) immunoreactivity in the brain of the Long-Evans rat, the species strain considered to be the most TCDD-susceptible, using immunohistochemical staining. A single dose of TCDD (dissolved in olive oil, 50 microg/kg) or olive oil alone was administrated to the rats by gavage. Compared with the vehicle-treated rat, a marked increase in the density of MEK immunoreactive cell bodies, fibers and terminals was found 2 weeks after TCDD treatment in the forebrain of the TCDD-treated rat, i.e. the central amygdaloid nucleus, field CA3 of the hippocampus, paraventricular hypothalamic nucleus, medial preoptic nucleus, interstitial nucleus of the posterior limb of the anterior commissure, lateral globus pallidus, ventral pallidum and lateral division of the bed nucleus of the stria terminalis. These results demonstrated for the first time a site-specific increased enkephalinergic activity in certain brain regions of the Long-Evans rat. It is suggested that the increased MEK immunoreactivity may act as a compensatory adaptation for the pathophysiological alterations caused by TCDD exposure.

Role of the vestibular nuclei in endothelin-1-induced barrel rotation in rats

The fourth or lateral ventricular injection of endothelin-1 resulted in a dose-dependent increase in the barrel rotation and produced marked induction of c-Fos-positive cells in the vestibular nuclei. The doses of the former injection were lower and had shorter mean latent periods compared with the later injection. c-Fos expression after endothelin-1 injection was prevented by the pretreatment with the endothelin ET(A) receptor antagonist, cyclo(D-alpha-aspartyl-L-propyl-D-valyl-L-leucyl-D-tryptophyl) (BQ-123), the glutamate NMDA receptor antagonist, dizocilpine maleate (MK-801), or the L-type Ca(2+) channel antagonist, verapamil, in addition to the incidence of the rotational behavior. There was a significant difference in c-Fos expression between the right and left medial vestibular nuclei, and the number of c-Fos-labeled neurons in the medial vestibular nucleus was markedly increased on the opposite side of the rotational direction. These results suggest that the elicitation of the barrel rotation may be mediated by endothelin ET(A) receptors, glutamate NMDA receptors, and L-type Ca(2+) channels. The changes in the receptor and channel systems induced by endothelin-1 injections appeared to exert crucial influences on the vestibular nuclei and then on the maintenance of equilibrium. The direction of the barrel rotation has a deep connection with the imbalance of neuronal activity in the left and right medial vestibular nuclei.

The angiotensin AT1 receptor antagonist, losartan, induces barrel rotation in the rat

Intracerebroventricular injections of [Arg8]vasopressin (500 ng/rat) or endothelin-1 (70 ng/rat) into the right lateral ventricle induced rotation along the long axis of the body (barrel rotation) in rats. Losartan (10-200 microg/rat), an angiotensin AT1 receptor antagonist, also evoked barrel rotation, which was not inhibited by vasopressin and endothelin receptor antagonists. However, barrel rotation was not observed after injections of high doses of another angiotensin II receptor antagonist, [Sar1,Ile8]angiotensin II (100 microg/rat), or after angiotensin II (10 microg/rat). The results indicate that losartan does evoke barrel rotation which may be not mediated via vasopressin and endothelin receptors.

The detection of influenza virus at the community pharmacy to improve the management of local residents with influenza or influenza-like disease

BackgroundAs of 2014, community pharmacies in Japan are approved by the Ministry of Health, Labour and Welfare to measure lipid panel, HbA1c, glucose, ALT, AST and γ-GTP, but not to screen for influenza virus. We provided influenza virus screening tests at a community pharmacy to triage people with symptoms suggestive of influenza. Participants were given appropriate advice on how to prevent the spread of and safeguard against influenza. We subsequently evaluated the effects of community pharmacy-based influenza virus screening and prevention measures.MethodsLocal residents with symptoms suggestive of influenza participated in this study. Influenza virus screening tests using nasal samples were provided to the pharmacy, and we assessed samples for the presence of influenza virus. The study consisted of a preliminary interview, informed consent, and screening test on Day 1, and mail-in survey on Day 14.ResultsA total 52 local residents participated in the study. The number of participants and influenza virus positive results followed the same trend as the influenza epidemic in the study area. Influenza virus was found in 28.8% of samples. There was no significant difference between the appearance ratios of subjective symptoms among influenza-positive and influenza-negative groups. The percentages of participants who were first screened at the pharmacy, and those who were first screened at a clinic and then tested again at the pharmacy, were 71.2% (37/52) and 28.8% (15/52), respectively. In the latter group, 14 of 15 were negative by screening at the clinic, and one was diagnosed with influenza without testing. Subsequently, 46.8% (7/15) of participants tested positive for influenza by pharmacy-based screening. According to the mail-in survey, all influenza-positive (100%, 7/7) and 35.3% (6/17) of influenza-negative participants visited the clinic after being tested at the community pharmacy; test results between the community pharmacy and clinic were consistent. A total 64.7% (11/17) of symptomatic participants who tested negative recovered spontaneously at home.ConclusionsImplementation of influenza virus screening followed by provision of appropriate advice for both influenza-positive and influenza-negative participants at the community pharmacy showed a significant effect on improving the health of the local community.

Green Tea Catechins -Pharmacokinetic Properties and Health BeneficialEffects

In this review, we provide information on the pharmacokinetic properties of green tea catechins and their beneficial health effects. The major catechins in green tea are (-)-epicatechin (EC), its hydroxyl derivative (-)-epigallocatechin (EGC), and their gallic acid esters, (-)-epicatechin-3-gallate (ECg) and (-)-epigallocatechin-3-gallate (EGCg). We developed an analytical method for determination of the presence of green tea catechins in human serum using ion-pair HPLC with electrochemical detection to estimate the pharmacokinetic parameters of target catechins. The Cmaxvalues indicated that catechin absorption was relatively low. One of the gallated catechins, EGCg, had a longer half-life than the non-gallated catechins. Green tea catechins, in particular, have attracted attention as cancer preventive agents in terms of their low toxicity and being readily available to the general population. Several epidemiological studies revealed that green tea consumption reduces cancer incidence. Numerous in vitro cell culture studies have shown that EGCg, which is defined as a major green tea catechin contributing to green tea’s anticancer effects, inhibits cell growth concomitant with induction of apoptosis. We have previously found that the cell death-inhibiting gene, Bcl-xL, was decreased by EGCg. These results support the hypothesis that EGCg regulates cytoplasmic NF-κB and subsequently induction of apoptosis. Green tea consumption may also play a role in preventing other lifestyle-related diseases, such as cardiovascular diseases and stroke, due to its hypocholesterolemic and hypotensive activities. In conclusion, habitual green tea drinking may promote human health by preventing lifestyle-related diseases.