Alkyl methylphosphonic acids (RMPAs) and methylphosphonic acid (MPA), both nerve gas hydrolysis products, can be identified by gas chromatography-mass spectrometry (GC-MS). We adopted tert-butyldimethylsilylation (TBDMS) as a derivatization technique for the identification of these products by GC-MS. We found that it was difficult to detect RMPAs and MPA from evidence specimens such as soils and body fluids, and ascertained several causes for this. The first is the interference from the TBDMS derivatization. It was determined that the TBDMS derivatization of RMPAs and MPA was suppressed in the presence of divalent metal cations such as calcium and magnesium ions, as well as some neutral compounds like carbohydrates. A cleanup procedure using a strong anion-exchanger (SAX)-solid phase extraction (SPE) method was optimized to remove the interfering compounds. The second reason that detection was difficult was because of strong matrix adsorption. We elucidated that the aqueous extraction recoveries for all phosphonates were inversely correlated with the phosphate adsorption coefficient in the soil samples. To diminish the adsorption of phosphonates to soils, a method of alkali extraction was adopted. For human serum samples, we adopted acetonitrile or trichloroacetic acid deproteinization which worked to break the binding between the proteins and the phosphonates. The detection yields of RMPAs and MPA from soils and human serum were dramatically increased using these pretreatment procedures combined with extraction and/or SAX-SPE. The method of SAX-SPE was applied to various types of samples, such as seawater, drinks and human urine, and good detection yields of RMPAs and MPA were provided by GC-MS.
Four successive extracts of the whole plant of Enicostemma axillare (E. axillare), were examined for in vitro antioxidant activity using nine different methods. In the 2,2'-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS) method, all the four extracts of E. axillare showed potent antioxidant activity with IC50 values ranging from 13.26 to 24.36 μg/ml. The chloroform extract has shown potent antioxidant activity in H2O2, nitric oxide, and hydroxyl radical using the deoxyribose and lipid peroxidation methods, with IC50 values of 16.99±0.38, 60.66±0.30, 25.06±0.12, and 94.66±2.40 μg/ml, respectively. Potent activity was also observed for the petroleum ether extract with the deoxyribose, p-nitroso dimethyl aniline (p-NDA), and H2O2 methods and for the ethyl acetate extract with the H2O2 and nitric oxide methods. All extracts showed moderate total antioxidant capacity using the phosphomolybdenum method. The activity was not correlated with the total phenol content of the extracts.
The basis for the unique effectiveness of amiodarone treatment on cardiac arrhythmias is incompletely understood. We investigated and compared the effects of amiodarone on K+ channels of hypertrophied ventricular myocytes with normal ventricular myocytes in rats. The pressure overload hypertrophy models of rats were established by partial ligation of ascending aorta for 4 weeks. Ventricular myocytes were exposed to 1, 10 and 50 μmol/l amiodarone, and whole cell patch-clamp technique was used to study the effects of amiodarone on outward currents, such as delayed rectifier outward K+ current (IK), slowly activating delayed rectifier outward K+ current (IKs), transient outward K+ current (Ito) and rectifier K+ current (IK1). Compared with the control group, the current density of IK, IK1 and Ito were all decreased in hypertrophied myocytes group. Hyper-concentration of amiodarone (10 and 50 μmol/l) inhibited Ito of the control group (37.5%±5.8% and 54.3%±5.7%) and the hypertrophied myocytes group (9.3%±2.3% and 22.8%±3.0%), but had effect on IK1 neither the control nor hypertrophied myocytes group. 10 μmol/l amiodarone inhibited IKs 23.3%±6.2% in the control group and 50.1%±9.8% in the hypertrophied myocytes group. Ito and IKs appeared difference affection of amiodarone's action on both groups. IKs in hypertrophy group was higher than that in control, whereas Ito was lower in hypertrophy group. We concluded amiodarone do inhibit Ito and IK in rat normal and hypertrophied cardiomyocytes. Amiodarone application should be treated difference between hypertrophied heart and normal heart.
Malathion residue in wheat kernels is enzymatically degraded into malathion monocarboxylic acids during sample preparation for pesticide residue analysis by the Japanese official method. To investigate whether the hydrolyzing enzyme is identical to carboxylesterase (CE), we compared the effects of various inhibitors against CE activity using p-nitrophenyl acetate as substrate with that against malathion-hydrolyzation activity. Although neither CE nor malathion-hydrolyzation activities were affected by EDTA, they were irreversibly suppressed by several serine esterase inhibitors and reversibly inhibited by cholinesterase inhibitor or sulfhydryl compounds. These inhibitions suggested that characteristics of both enzymes in wheat kernels were close to that of cholinesterase, though the enzymes were not able to be exactly classified by a classification method for mammals. When native polyacrylamide gel electrophoresis (PAGE) with esterase-zymography was performed with the addition of eserine sulfate into the sample and deoxycholic acid into the cathode buffer to prevent aggregation of CE isozymes, three clear bands of the isozymes were observed. These isozymes were confirmed by hydrophobic interaction chromatography (HIC). When malathion was reacted with each isozyme partially purified by chromatography techniques and native PAGE, malathion α-monocarboxylic acid as a major metabolite and malathion β-monocarboxylic acid as a minor metabolite were produced. These results suggested that all isozymes in wheat kernels responsible for the degradation of residual malathion in the kernels, though there are differences among malathion degradability by the isozymes.
The central nervous system (CNS) pharmacological effects of ethylacetate extract (EAS) of Sida tiagii Bhandri collected from northern province (Rajasthan) of India were assessed by elevated plus-maze, pentobarbitone induced sleeping time, spontaneous motor activity, pentylenetetrazole-induced seizure, forced swim test and rotarod tests. The results of the present study demonstrated the CNS depressant potential, i.e., anxiolytic, antiseizure, reduction in spontaneous locomotion and potentiation of pentobarbital-induced hypnosis of the plant under investigation.
Dioxins, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are ubiquitous environmental pollutants. The variety of adverse effects produced by dioxins are a serious problem because they may affect humans and wild animals through the food chain. In this study, we examined the possible protective effects of piperine, which is a major alkaloid in black pepper (Piper nigrum Linn.) and long pepper (Piper longum Linn.), on the toxic effects of TCDD in C57BL/6J mice. The repeated administration of high doses (30 and 45 mg/kg, 14 days, p.o.) of piperine alone produced a weak agonistic effect on the aryl hydrocarbon receptor, which was evaluated based on the increase in hepatic ethoxyresorufin O-deethylase (EROD) activity. No such effect was observed at the lowest dose (15 mg piperine/kg). However, while coadministration (20 mg/kg, 28 days, p.o.) of piperine with TCDD had no effect on TCDD-induced wasting syndrome, it improved the hepatic accumulation of free fatty acids produced by TCDD. In relation to this, the hepatic accumulation of triglycerides by TCDD also tended to be reduced by piperine. Despite the above effects, piperine failed to reduce the increase in hepatic EROD activity and lipid peroxidation produced by TCDD. These results suggest that piperine is a candidate to improve disorders of lipid metabolism produced by dioxins, although the mechanism remains to be clarified.
We examined the effect of oral intake of pure glucosylceramide derived from konjac extract on skin barrier function evaluated by transepidermal water loss (TEWL) in hairless mice with sodium dodecyl sulfate (SDS)-induced skin roughness. The difference of TEWL between SDS-treated site and untreated sites in the pure glucosylceramide-fed group was significantly lower than that in control group on day 14 of ingestion. We investigated interleukin-1α (IL-1α) production in the hairless mouse skin, and it was significantly lower in the glucosylceramide-fed group than that of control animals. This reduced IL-1α production should contribute to improvement of skin barrier function. To investigate the effect of oral intake of glucosylceramide in human, we conducted a randomized double-blind placebo-controlled study including 100 healthy subjects whose TEWL in cheek was relatively high. As a result, cheek TEWL was significantly lower in the test product group as compared with the control group in weeks 8 and 12 of ingestion (p=0.023 and p=0.002 respectively).
There have been studies demonstrating that serum caeruloplasmin acts as an antioxidant in cardiovascular disease. However, several studies have demonstrated that it acts as an independent risk factor in patients with cardiovascular disease. To ascertain the role of caeruloplasmin in normolipidemic acute myocardial infarction patients, we investigated the correlation between the serum caeruloplasmin level and incidence of cardiovascular disease in individuals with normal lipid profiles. The levels of caeruloplasmin were significantly higher in patient sera than in those of controls (p<0.001) and the difference in the lipid profiles was also significant (p<0.001). These results suggest that caeruloplasmin may act as a prooxidant and appears to be a risk factor in this disease.
Many hypertensive patients are continuously prescribed various antihypertensive drugs despite the undesirable side effects such as nausea, dizziness, and vertigo. To decrease the use of cardiovascular drugs for preventing these side effects, alternative pharmacotherapy with supplements such as coenzyme Q10 has been extensively studied. However, the effects of coenzyme Q10 based on clinical trials involving relatively small patient numbers have been varied, and the impact on the cardiovascular system remains to be clarified. Here we report a case of 67-year-old woman with essential hypertension (maximum systolic/diastolic blood pressure: 155/100 mmHg) who had been prescribed candesartan cilexetil as an outpatient for about 5 years. Regardless of the treatment, the symptoms were not relieved, and the systolic and diastolic blood pressures gradually increased with age. However, after week-long supplementation with coenzyme Q10, her diastolic blood pressure returned to normal, and so did her systolic blood pressure after month-long supplementation. Subsequently, she completely ceased taking candesartan. Thus, coenzyme Q10 supplementation may be effective for selected patients with essential hypertension. This should be investigated further in randomized controlled trials.
In the light of a novel mode of action, the antiproliferative effects of green tea catechins were studied with relating to their membrane lipid interactions. Mouse myeloma cells and liposomes consisting of phospholipids and cholesterol were treated with structurally-different catechins of 10 and 100μM for 0.5-48 hr. The induced changes in membrane fluidity were comparatively determined by measuring fluorescence polarization with different probes to characterize the membrane-acting sites. (-)-Epicatechin-3-gallate (ECG) and (-)-epigallocatechin-3-gallate (EGCG) showed the growth-inhibitory effects on tumor cells with the potency increasing in this order, but neither (-)-epicatechin (EC) nor (+)-catechin (C). Simultaneously with inhibiting the cell growth, both catechin gallates rigidified tumor cell membranes by acting on their hydrophilic and hydrophobic regions. The most antiproliferative EGCG predominantly affected the centers of cell membranes and its acting site was deeper with increasing the culture time. Correlating to the comparative effects on tumor cells, EGCG reduced the fluidity of liposomal membranes more intensively than ECG, whereas EC and C were essentially ineffective. The antiproliferative effects of green tea catechins are associated with their structure-dependent interactions with lipid bilayers to modify cell membrane fluidity.
The generation of cyanide ions by the reaction of pharmaceuticals containing nitrogen with sodium hypochlorite was examined using 20 different compounds. The generation of cyanide ions was observed in hexamine (hexamethylenetetramine), losartan potassium, metronidazole, and allopurinol. The largest generation was observed in hexamine. In both hexamine and losartan potassium, the amount of cyanide ion formed increased to a maximum at 45-60 min. The generation of cyanide ions was attributed to the formation of nitroso product by oxidizing action of sodium hypochlorite.
Based on the previous finding of okicamelliaside (OCS), a highly potent anti-degranulation ellagic acid glucoside, in the leaves of Camellia japonica (C. japonica), we evaluated an extract of these leaves and OCS itself for their potential to suppress allergic reactions in vivo. Two conventional animal allergy models were used. In the allergic conjunctivitis model, male S.D. rats were stimulated with anti-ovalbumin (OVA) serum and challenged with OVA/Evans blue mixture. Oral administration of extracts from C. japonica at 1000 mg/kg for 10 days significantly reduced the vascular permeability of conjunctivas. In the second model, male BALB/c mice were stimulated with a Japanese cedar pollen extract and challenged by nasal instillation of the antigen. The sneezing frequency during the 10 min immediately after the challenge tended to decrease by intraperitoneal administration of 0.2 mg/kg of OCS for 24 days. These results suggest that C. japonica extracts (CJE) and OCS prepared from them could be useful to alleviate the symptoms of an immediate-type allergy.
The ethanol extract of Rhodomyrtus tomentosa (Aiton) Hassk. (R. tomentosa) was determined for its antibacterial activity on staphylococci isolated from acne lesions. Antibiotic susceptibility patterns of the isolates were performed. Preliminary screening of the antibacterial property of the extract using disk diffusion method demonstrated pronounced antibacterial activity of the extract on all tested isolates. The average inhibition zones of 64 coagulase-positive and 85 coagulase-negative isolates were 12 mm and 14 mm, respectively. Minimal inhibitory concentration (MIC) was determined by broth microdilution method. The MIC50 and MIC90 ranged from 64-512 μg/ml. Time-kill curves were assessed at 0.5 MIC, MIC, 2 MIC, and 4 MIC by counting viable bacterial cells after time intervals. At 4 MIC, the level of treated staphylococci declined by at least 3 log fold within 6-8 hr. Rhodomyrtone, a pure compound in acylphloroglucinol class, isolated from this plant species was very effective against Staphylococcus aureus (S. aureus) ATCC 25923 with the MIC value at 0.5 μg/ml which is very closed to that of vancomycin. This finding challenges the use of rhodomyrtone as an alternative agent for staphylococcal cutaneous infections.
Menadione, a synthetic vitamin K3, exhibits anti-estrogenic activity on in vitro assay. However, the in vivo anti-estrogenic effects of menadione have not been determined, while correlations between biological effects and structural changes are unclear. Thus, we investigated the in vivo anti-estrogenic activity of menadione under fluorescent light and dark conditions. Suppression of the hepatic estrogen response genes vitellogenin1 (VTG1), VTG2 and estrogen receptor-α (ER-α) was used as an index of anti-estrogenic activity. Male medaka (Oryzias latipes) were treated with nominal concentrations of menadione in the presence or absence of 17β-estradiol (E2), and hepatic VTG1, VTG2 and ER-α mRNA levels were determined by quantitative real-time PCR. In the presence of E2 under dark conditions, expression of hepatic VTG2 and ER-α genes was suppressed by menadione treatment. On the other hand, menadione activity was lost under fluorescent light conditions. These results suggest that menadione has anti-estrogenic activity in vivo, and that this activity is diminished under fluorescent light, probably due to a structural change in menadione.
Glyphosate is an herbicide used for many plants that can be toxic to humans in high doses. Current methods for measuring glyphosate are slow and expensive. Here we describe a fast and simple method for measuring glyphosate in tea beverages by capillary electrophoresis with on-line formation of copper(II)-glyphosate complex. The optimum running conditions were found to be 40 mM acetate buffer (pH 5.0) containing 5 mM CuSO4 with an effective voltage of +15 kV using a sulfonated capillary (FunCap-CE Type S) and direct UV detection at 250 nm. Linearity (r2>0.999) was demonstrated in the range 5-1000 mg/l of glyphosate. Good reproducibilities of peak area (relative standard deviation <1.2%) and migration time (relative standard deviation <0.2%) were obtained. Recovery of glyphosate was between 98 and 100%. With this method, a tea beverage that was mixed with a glyphosate formulation was successfully analyzed.
The affinity for thyroid hormone receptor (TR) of polybromodiphenyl ethers (PBDEs) and hydroxylated PBDEs was examined. 4-Hydroxy-2,2',3,4',5-pentabromodiphenyl ether (4-OH-BDE-90) and 3-hydroxy-2,2',4,4'-tetrabromodiphenyl ether (3-OH-BDE-47) markedly inhibited the binding of triiodothyronine (1×10-10 M) to TR in the concentration range of 1×10-6-1×10-4 M. 2,3,4,5,6-Pentabromophenol (PBP) also showed an inhibitory effect at 1×10-5-1×10-4 M. However, 2,2',3,4,4',5'-hexabromodiphenyl ether (BDE-138), decabromodiphenyl ether (DBDE), 4-methoxy-2,2',3,4',5-pentabromodiphenyl ether (4-MeO-BDE-90), 4'-hydroxy-2,2',4,5'-tetrabromodiphenyl ether (4'-OHBDE-49), 4-hydroxy-2,2',3,4'-tetrabromodiphenyl ether (4-OH-BDE-42), 4'-hydroxy-2,2',4-tribromodiphenyl ether (4'-OH-BDE-17), 3'-hydroxy-2,4-dibromodiphenyl ether (3'-OH-BDE-7), 2,4,6-tribromophenol (TBP) and tetrabromohydroquinone (TBHQ) did not show affinity for TR. In contrast, 4'-OH-BDE-17 and 3'-OH-BDE-7 exhibited estrogenic activity in estrogen-responsive reporter assay using MCF-7 cells at the concentration of 1×10-5 M. However, adjacent bromo substitution of 3- or 4-hydroxylated PBDEs markedly decreased the estrogenic activity. These results suggest that hydroxylated PBDEs act as thyroid hormone-like agents, as well as estrogens, that a 4- or 3-hydroxyl group in PBDEs is essential for thyroid hormonal and estrogenic activities, and that adjacent dibromo substitution favors thyroid hormonal activity, but not estrogenic activity.