Masaru Kunitomo

Differential rapid analysis of ascorbic acid and ascorbic acid 2-sulfate by dinitrophenylhydrazine method

Abstract Ascorbic acid 2-sulfate (AAS) has recently been detected in animals (1,2), and the suggestion has been made that it is involved in some physiological functions (3–5). AAS has been determined by spectrophotometer measurements at 254 nm, ϵ = 17,000, after isolation from animal tissues (1,6,7). This procedure, however, is complicated and time consuming. Baker et al. (8) reported a rapid assay for AAS from biological samples based on a dinitrophenylhydrazine (DNPH) method which is a standard method for ascorbic acid (AA) determination in biological materials. In this method, AA is oxidized to the osazone by incubation with DNPH in a dilute sulfric acid solution. According to Baker et al. (8), AAS reacts with DNPH during a 3-hr incubation at 60°C but not during a 1-hr incubation at 37°C, and the difference in the reading at 540 nm between the two temperatures corresponds to AAS. This report is concerned with a more rapid and specific method with DNPH for differential measurement of AA and AAS, that is, the differential oxidation of AA and AAS with 2,6-dinitrophenolindophenol (2,6-Dye) and KBrO 3 and the determination of the osazone produced with the original method by Roe and Kuether (9).

Antioxidant activity of the extracts from fruiting bodies of cultured Cordyceps sinensis

Cordyceps sinensis is one of the most valued herbs in traditional Chinese medicine. We investigated the antioxidant activities of the cultured fruiting bodies of Cordyceps sinesis. The water and ethanol extracts of Cordyceps sinensis were found to possess a potent antioxidant activity. The scavenging effects of the extracts on superoxide were very weak, but the extracts moderately inhibited malondialdehyde formation via hydroxyl radical induced by SIN‐1, a peroxynitrite generator. Of the extracts examined, the hot water extract (70 °C for 5 min) showed the greatest oxygen free radical scavenging activity. Also, when low‐density lipoprotein (LDL) was incubated with macrophages in the presence of CuCl2 (1 µM), the hot water extract showed a strong inhibitory effect against lipid peroxidation in the medium and consequent accumulation of cholesteryl ester in macrophages. Their activities were comparable to that of authentic Cu/Zn SOD. These results suggest that the extracts of cultured Cordyceps sinensis possess potent antioxidant and anti‐lipid peroxidation activities and inhibit accumulation of cholesteryl ester in macrophages via suppression of LDL oxidation. Copyright

Feeding of Ginkgo biloba extract (GBE) enhances gene expression of hepatic cytochrome P-450 and attenuates the hypotensive effect of nicardipine in rats.

Ginkgo biloba extract (GBE) has been used clinically for improving peripheral vascular diseases in France and Germany and is ingested widely as a herbal medicine in some countries. However, accurate information about its safety as an herbal medicine has not been sufficiently established. To address this issue, we examined the effect of GBE on hepatic drug metabolizing enzymes and their influence on hypotensive drug in rats. Male rats were fed either a control diet or diet containing GBE (0.5% w/w) for 4 weeks. The feeding of a GBE diet did not change the serum transaminase activity, but increased the liver weight and the phospholipid concentration in the liver. In addition, the GBE diet markedly increased the content of cytochrome P-450 (CYP), and the activity of glutathione S-transferase in the liver. Furthermore, the GBE diet markedly induced levels of CYP2B1/2, CYP3A1 and CYP3A2 mRNA in the liver. The levels of CYP1A1, CYP1A2, CYP2E1, CYP2C11 and CYP4A1 were unchanged. The feeding of GBE for 4 weeks significantly reduced the hypotensive effect of nicardipine that was reported to be metabolized by CYP3A2 in rats. These findings suggest that GBE reduces the therapeutic potency of the Ca2+ channel blocker, nicardipine, via enhancement of cytochrome P-450 expression.

Facilitated nitration and oxidation of LDL in cigarette smokers

Background  Cigarette smoking increases the risk of developing atherosclerosis and ischaemic heart disease. Smoking‐induced oxidative stress is considered to favour oxidation of low‐density lipoprotein (LDL) and subsequently promotes the atherogenic process. We investigated whether peroxynitrite, a reaction product of cigarette smoke, is involved in facilitated oxidation of LDL in smokers.

Oxidants in cigarette smoke extract modify low-density lipoprotein in the plasma and facilitate atherogenesis in the aorta of Watanabe heritable hyperlipidemic rabbits.

Epidemiological studies have shown that cigarette smoking is a major cause of atherosclerosis. Oxidants as well as nicotine in cigarette smoke have been implicated in atherogenesis. To clarify the mechanism involved, we examined the chronic effects of nicotine and nicotine-free cigarette smoke extracts (CSE) on oxidative modification of low-density lipoprotein (LDL) in the plasma of Watanabe heritable hyperlipidemic rabbits and atherogenesis in the aorta. CSE was prepared by bubbling the gas phase of smoke (1 ml/three cigarettes) into phosphate buffer saline, and 3 ml of this CSE was injected daily into the ear vein of the rabbit for five months. The rabbits treated with CSE showed an increase in lipid peroxide levels, estimated as thiobarbituric acid reactive substances (TBARS), with a corresponding decrease in vitamin E levels in the plasma. They also showed enhanced oxidative modification of LDL, assessed by anion-exchange HPLC, incorporation into macrophages and measurement of TBARS. These events could be efficiently prevented by administering vitamin E (150 mg/kg/day, p.o.). Nicotine alone (0.5 mg/kg/day, s.c.) led to a temporary increase in the plasma triglyceride level. At the end of the experiment, CSE but not nicotine had caused progression of atherosclerotic lesions together with accumulation of cholesteryl ester in the thoracic aorta, while vitamin E had significantly prevented such atheromatous formation. These results indicate that oxidants in CSE can promote the development of atherosclerosis through oxidative modification of plasma LDL, particularly in hypercholesterolemia, and offer evidence for increased vitamin E utilization in smokers.

Ginkgo biloba extract-induced relaxation of rat aorta is associated with increase in endothelial intracellular calcium level.

Ginkgo biloba extract (GBE) has been used clinically for improving peripheral vascular diseases in France and Germany. In the present study, to clarify the pharmacological properties of vasodilation produced by GBE, we examined the effect of GBE and quercetin, one of the ingredients in GBE, on the thoracic aorta isolated from Wistar rats. GBE produced a dose-dependent relaxation in the aortic ring precontracted with noradrenaline, and the relaxation was abolished by L-N(G)-nitro arginine methyl ester (L-NAME). Quercetin produced a similar relaxation, which was also abolished by L-NAME. We then examined the effects of GBE and quercetin on the intracellular calcium level ([Ca2+]i) of cultured aortic endothelial cells using a fluorescent confocal microscopic imaging system. Both GBE and quercetin produced significant increases in [Ca2+]i in the endothelial cells. The increase in [Ca2+]i by quercetin (10(-6) M) was abolished by removing the extracellular Ca2+, but was not affected by thapsigargin, a calcium pump inhibitor. These findings suggest that a principal ingredient of GBE producing vasodilation is quercetin, which can activate nitric oxide synthesis and release by increasing [Ca2+]i in vascular endothelial cells.

Downregulation of vascular soluble guanylate cyclase induced by high salt intake in spontaneously hypertensive rats

Cyclic guanosine monophosphate (cyclic GMP)‐mediated mechanism plays an important role in vasodilatation and blood pressure regulation. We investigated the effects of high salt intake on the nitric oxide (NO) – cyclic GMP signal transduction pathway regulating relaxation in aortas of spontaneously hypertensive rats (SHR). Four‐week‐old SHR and normotensive Wistar‐Kyoto rats (WKY) received a normal salt diet (0.3% NaCl) or a high salt diet (8% NaCl) for 4 weeks. In aortic rings from SHR, endothelium‐dependent relaxations in response to acetylcholine (ACh), adenosine diphosphate (ADP) and calcium ionophore A23187 were significantly impaired by the high salt intake. The endothelium‐independent relaxations in response to sodium nitroprusside (SNP) and nitroglycerin were also impaired, but that to 8‐bromo‐cyclic GMP remained unchanged. On the other hand, high salt diet had no significant effects on the relaxations of aortic rings from WKY. In aortas from SHR, the release of NO stimulated by ACh was significantly enhanced, whereas the production of cyclic GMP induced by either ACh or SNP was decreased by the high salt intake. Western blot analysis showed that the protein level of endothelial NO synthase (eNOS) was slightly increased, whereas that of soluble guanylate cyclase (sGC) was dramatically reduced by the high salt intake. These results indicate that in SHR, excessive dietary salt can result in downregulation of sGC followed by decreased cyclic GMP production, which leads to impairment of vascular relaxation in responses to NO. It is notable that chronic high salt intake impairs the sGC/cyclic GMP pathway but not the eNOS/NO pathway.

Evaluation of the bitterness of antibiotics using a taste sensor

The bitterness of nine commercial antibiotics (clarithromycin, erythromycin, cefdinil, doxycycline, vancomycin, tetracycline, minocycline, oxytetracycline and bacampicillin) was evaluated in human gustatory sensation tests with nine volunteers. The bitterness of 0.1–0.3 mM solutions (or suspensions in the case of clarithromycin) of the antibiotics was then measured using an artificial multichannel taste sensor. In the sensor measurements, three variables were used to predict estimated bitterness in single and multiple regression analysis and principal component analysis: sensor output as relative value (R), the change of membrane potential caused by adsorption (C) and C/R. Particularly good correlation was obtained between obtained bitterness scores and predicted scores using C from channel 2 of the sensor (r2 = 0.870, P < 0.005) and C/R values for channels 2 and 3 (r2 = 0.947, P < 0.005). The taste sensor was also successful in assessing the bitterness intensity of clarithromycin powder suspensions of various concentrations. Clarithromycin has a low aqueous solubility but is the most bitter of the nine antibiotics. Sensory data from channel 3 of the sensor predicted the bitterness of clarithromycin powder suspensions and their filtered solutions well. Finally, the bitterness intensity of a commercial clarithromycin dry syrup product (Clarith dry syrup, Taisho Pharmaceutical Co. Ltd, Tokyo, Japan) was evaluated in gustatory sensation tests and using the taste sensor. In Clarith dry syrup the drug is coated with aminoalkyl methacrylate polymer using a spray congealing method. The taste sensor results confirmed that the polymer was successful in almost completely masking the bitter taste of the dry syrup product.

Antitumour activity of cordycepin in mice.

1. The antitumour effect of orally administered cordycepin, a component isolated from water extracts of Cordyceps sinensis, was examined in mice inoculated with B16 melanoma (B16‐BL6) cells.

Altered endothelium-dependent responsiveness in the aortas and renal arteries of Otsuka Long–Evans Tokushima Fatty (OLETF) rats, a model of non-insulin-dependent diabetes mellitus

We examined endothelium-dependent relaxation in the aortas and renal arteries of Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of non-insulin-dependent diabetes mellitus, in comparison with non-diabetic Long-Evans Tokushima Otsuka rats as controls. Acetylcholine-induced relaxation in both arteries was attenuated, and the attenuation was restored to the control level by indomethacin. The relaxation was inhibited completely in the aortas, but only partially in renal arteries by N(G)-nitro-L-arginine methyl ester, and the degree of the latter inhibition was greater in OLETF rats than in the controls. The relaxation was inhibited by aminoguanidine in both arteries of OLETF rats but not in the controls. Serum NO(2) plus NO(3) levels significantly increased in OLETF rats. These results suggest that impairment of relaxation in OLETF rat arteries is due to increased release of contractile factors but not decreased release of nitric oxide.