Yoko Kubota

High-performance anion-exchange chromatography of homogeneous D-gluco-oligosaccharides and -polysaccharides (polymerization degree >50) with pulsed amperometric detection

Abstract High-performance anion-exchange chromatography under alkaline conditions with pulsed amperometric detection was applied to the analyses of (1 → 2)-, (1 → 3)-, (1 → 4)- and (1 → 6)-linked homogeneous α- or β- d -gluco-oligosaccharides and -polysaccharides up to a degree of polymerization (DP) of ⩾ 50. Each series of homogeneous d -gluco-oligomers and -polymers showed a linear relationship between log k′ and DP in isocratic elution using 150 mM sodium hydroxide solution containing 100 mM sodium acetate as the eluent. An effective separation of individual members of an homologous series of linear glucans was achieved using gradient elution, accomplished by maintaining the sodium hydroxide concentration at 150 mM and increasing the sodium acetate concentration during the analysis. The detector response per HCOH group in d -gluco-oligomers (DP 2–7) was almost the same.

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.

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.

Determination of cyclic glucans by anion-exchange chromatography with pulsed amperometric detection

Abstract Anion-exchange chromatography with pulsed amperometric detection was applied to the determination of cyclodextrins ( CD s), branched CD s and cyclosophoraoses. These cyclic glucans with degree of polymerization 6–25 were well resolved in each series by using simple isocratic elution with 150 m M sodium hydroxide solution containing 140–200 m M sodium acetate. The separation mode was not only simple anion exchange, but also involved some hydrophobic interactions and, moreover, inclusion interactions also seemed to take part in the retention. The detector response per glucose unit of these cyclic glucans was almost the same, regardless of the molecular weight and linkage form. The limit of determination of the cyclic glucans was 5–10 pmol and the detection limit was 2.5–5 pmol with a signal-to-noise ratio of 3.

Peroxynitrite is Involved in the dysfunction of vasorelaxation in SHR/NDmcr-cp rats, spontaneously hypertensive obese rats.

SHR/NDmcr-cp (SHR-cp) rats display typical symptoms and features of the metabolic syndrome. We previously reported that endothelium-dependent relaxation decreases in the thoracic aortas of SHR-cp rats, despite increased nitric oxide (NO) production from the endothelium. In the present study, to search for the reasons for this contradiction, we investigated whether vascular abnormality could be reduced by treatment of SHR-cp rats with antihypertensive drugs; a calcium channel blocker (amlodipine), an alpha 2 and imidazoline receptor agonist (moxonidine), and an angiotensin II type 1 (AT1) receptor antagonist (telmisartan). Telmisartan but not amlodipine and moxonidine ameliorated the impairment of relaxation in response to acetylcholine and the increased protein expression of endothelium NO synthase in thoracic aortas. All three drugs significantly lowered the blood pressure. Telmisartan decreased the serum levels of lipid peroxide and 8-hydroxy-2′-deoxyguanosine, oxidative stress markers, and also the aortic levels of the protein expression of gp91phox, a component of NADPH oxidase, and 3-nitrotyrosine, a biomarker of peroxynitrite. These findings suggest that NADPH oxidase-derived superoxide, probably produced due to stimulation of AT1 receptors, reacts with NO to form peroxynitrite, and consequently decreases active NO, leading to attenuation of endothelium-dependent relaxation. Angiotensin receptor antagonists may be effective for preventing endothelial dysfunction in metabolic syndrome.

Participation Of Atp In Cell Volume Regulation In The Endothelium After Hypotonic Stress

1. The role of ATP in the regulatory volume decrease (RVD) after hypotonic cell swelling was examined in cultured endothelial cells isolated from the rat caudal artery.

Isolation and characterization of cyclic α-(1→4)-glucans having degrees of polymerization 9–31 and their quantitative analysis by high-performance anion-exchange chromatography with pulsed amperometric detection

Abstract Cyclic α-(1→4)-glucans with degrees of polymerization (DPs) 9–31 were isolated from a mixture of cyclization products formed in the early stage of the action of cyclodextrin glucanotransferase (CGTase) on synthetic amylose, and characterized by matrix-assisted laser desorption ionization time-of-flight MS, 13C-NMR and HPLC of their partial acid hydrolyzates. High-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection for an accurate estimation of cyclic α-(1→4)-glucans was developed using those isolate glucans as quantification standards, and by HPAEC, the time course of the cyclization reaction of CGTase from an alkalophilic Bacillus sp. A2-5a on synthetic amylose was determined.

Pretreatment with Ginkgo biloba extract weakens the hypnosis action of phenobarbital and its plasma concentration in rats

In a previous study, we found that orally administered Ginkgo biloba extract (GBE) induced hepatic cytochrome P450 (CYP) in rats, especially the CYP2B type. This fact suggested that GBE influenced the availability and safety of drugs that were metabolized via CYP2B type enzymes. To confirm this possibility, in this study we examined the effect of feeding a 0.1, 0.5 and 1.0% GBE diet for 2 weeks on the pharmacokinetics and pharmacological action of phenobarbital, which is known to be metabolized by CYP2B in Wistar rats. The feeding of GBE markedly shortened the sleeping time in rats. Furthermore, the maximal phenobarbital plasma concentration (Cmax) and the 24‐h area under the curve (AUC0–24) were decreased in rats fed GBE. These findings indicate that GBE reduces the therapeutic potency of phenobarbital via enhancement of cytochrome P450 expression, and raises the possibility that GBE and drug interactions may occur clinically.

Isolation and characterization of heptakis(2,6-di-O-methyl)cyclomaltoheptaose and over-methylated homologues

Abstract From a mixture of methylation products of cyclomaltoheptaose (β-cyclodextrin, βCD) containing heptakis(2,6-di- O -methyl)-βCD ( 1 ) and its over-methylated homologues, two major components [ 1 and hexakis(2,6-di- O -methyl)-(2,3,6-tri- O -methyl)-βCD ( 2 )] and fourteen minor components were isolated by h.p.l.c. The solubilities and inclusion behaviour of 1 and a commercial DM-βCD (a mixture containing 1 and 2 ) were compared. A 3- O -methyl group had little effect on the inclusion behaviour of 2,6-di- O -methyl-βCD. Fourteen minor over-methylated homologues were characterized.

Analyses of isomeric mono-O-methyl-d-glucoses, d-glucobioses and d-glucose monophosphates by high-performance anion-exchange chromatography with pulsed amperometric detection

Abstract To investigate the contribution of each hydroxyl group of d -glucose to retention on pellicular quaternary amine-bonded resins and to the pulsed amperometric detector response, all sets of isomeric mono-O-methyl- d -glucoses and d -glucobioses and three isomeric d -glucose monophosphates were analysed by high-performance anion-exchange chromatography under alkaline conditions with pulsed amperometric detection. The results showed that the reduction of retention time on mono-O-methylation follows the order 2-OH > 3-OH > 6-OH > /4-OH > -O Although the elution order of 1-, 3- and 6-phosphates was the same as obtained for the corresponding methyl esters, in the case of glucobioses the elution position was somewhat affected by the configurational contribution and hydrophobic interaction. The pulsed amperometric detector response was little affected by the acidity of each hydroxyl group of d -glucose. The suppression of the pulsed amperometric detector response by substitution of hydroxyl group seemed to be due to the inhibitory effect of the substituent group on the vicinal hydroxyl group and, as a rule, the degree of suppression caused by 6-O-substitution was the smallest.