Bovine pancreatic ribonuclease (RNase) A, which consists of 124 amino acids, was synthesized in a conventional manner by assembling relatively small 30 peptide fragments of established purity, followed by applying a new deprotecting procedure with trifluoromethanesulfonic acid-thioanisole. After formation of disulfide bond by a glutathionemediated air-oxidation and subsequent two step purifications by affinity chromatography and ion-exchange chromatography, a protein with the full enzymatic activity of RNase A was obtained. The synthetic protein was subsequently crystallized from aqueous ethanol according to Kunitz. A totally synthetic enzyme with full RNase activity was thus obtained in a crystalline form for the first time.
In the previous paper, the transacylation between substituted phenyl acylates and substituted benzamides in the molten state at high temperature was described. The transacetylation was examined in inert organic solvents under mild conditions, and the reaction order and the substituent effect were discussed according to kinetic data and Hammett's plots. It was found that the substituted benzamides were acetylated with pseudo first-order kinetics and the Hammett's plots were straight lines of slope (p)+1.19 (substituted phenyl acetates) and -0.68 (substituted benzamides).
The degradation of 5β-pregnane-3α, 20β-diol disulfate (2) by refluxing with 3N hydrochloric acid was investigated. In addition to the main product, 17α-methyl-D-homo-5β-androstane-3α, 17aβ-diol (5), various kinds of minor degradation products were detected by gas chromatography and their structures were determined by gas chromatography-mass spectrometry as : 17α-methyl-17aβ-chloro-D-homo-5β-androstan-3α-ol (6), 17α-ethyl-17β-methyl-5β-androst-13-en-3α-ol (7), 5β-pregn-20-en-3α-ol (8), E-isomer (9) of 5β-pregn-17 (20)-en-3α-ol, 5β-pregn-3-en-20β-ol (13), 5β-pregn-2-en-20β-ol (14), and pregnanediol (3). The D-homoannulation of 2 to give 5 and 6 was considered to occur mainly by a concerted mechanism, while stepwise mechanism involving C20-carbonium ion was assigned to the formation of the minor products. Stereochemistry of the degradation of 2 by hot acid is discussed.
Twenty one kinds of 8-acylamino-5-(2-hydroxy-3-substituted aminopropoxy)-3, 4-dihydrocarbostyril derivatives were synthesized and their antagonistic activities on isoproterenol were examined. Several compounds having 3, 4-dimethoxyphenethyl or 4-carbamoylphenoxyethyl moiety in 3-substituted amino group were found to have less antagonistic activity on isoproterenol than those with tert-butyl or isopropyl moiety in 3-substituted amino group, but showed relative higher selectivity for β1-adrenoreceptors in the anesthetized dog.
A diarylheptanoid glycoside named myricanol glucoside (3), C27H36O10·1/2 H2O, mp 220-223°C, [α]D-53.7°, was isolated from the stem bark of Myrica rubra in addition to myricanol (1) and myricanone (2). On the basis of chemical and spectral evidence the glucoside (3) was elucidated to be myricanol 5-β-D-glucopyranoside. This is the first example as a glucoside of biphenyl-type diarylheptanoid.
Nine kinds of constituents, named Sub I, II, III, IV, V, VI, VII, VIII and IX were isolated from MeOH extractives of"Dotuusoo, "Galeolae fructus, Orchidaceae. Three new compounds, Sub IV, V and VI were characterized as 1-[4-(β-D-glucopyranosyloxy) benzyl] (S)-(-)-2-isopropylmalic acid metallic salt, bis [4-[β-D-glucopyranosyloxy) benzyl] (S)-(-)-2-isopropylmalate and 4-methyl ester of Sub IV respectively, together with three known compounds p-hydroxybenzyl methyl ether (Sub I), 4-(β-D-glucopyranosyloxy)-benzyl alcohol (Sub II) and (S)-(-)-2-isopropylmalic acid metallic salt (Sub III).
Daidzein, daidzin and puerarin in MeOH extracts of Pueraria Radix and Species Puerariae were analyzed using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). For standards, daidzein, daidzin and puerarin were isolated from Pueraria Radix. For qualitative analysis, TLC was carried out using silica gel plate and CHCl3-MeOH-H2O (65 : 35 : 10, lower layer). Daidzein and puerarin were detected by exposing to ultraviolet (UV) light, and daidzin was detected by spraying p-anisaldehyde-H2SO4 solution. For determination, HPLC was carried out using Shodex ODS as separation column and CH3CN-H2O (15 : 85) as mobile phase. Daidzein and daidzin were determined using UV detector at 254 nm. Puerarin was determined using fluorescence detector at λEX 254nm. Samples were extracted with MeOH. The extracts were passed through Sep Pak C18 cartridge and applied to TLC and HPLC. Recoveries of these constituents from model preparation were as follows ; daidzein : 86%, daidzin : 96% and puerarin : 99%, respectively. These methods were considered to be useful for one of evaluations of commercial Pueraria Radix and Species Puerariae.
Nux Vomica contains strychnine and brucine which showed a strong pharmacological activity. Therefore, an improved method of analyzing these compounds is of value. For determination of these compounds in Nux Vomica, titrimetry is employed on JPX. Then strychnine and brucine in Nux Vomica and commercial pharmaceutical preparations including Nux Vomica extracts were identified by thin layer chromatography (TLC) and determined using high performance liquid chromatography (HPLC). Samples were extracted with CHCl3-MeOH (2 : 1). The extracts were passed through Sep Pak C18 cartridge and applied to TLC and HPLC. HPLCs of strychnine and brucine were carried out on Shodex ODS column and CH3CN-H2O (25 : 75) to which 1-heptanesulfonic acid was added as a counter ion for the solvent system. Recoveries from model preparations were more than 98%. This method was considered to be useful for the determination of strychnine and brucine in commercial preparations including Nux Vomica extracts.
Multiplicity of DL-glyceraldehyde reductase activity in rabbit lens was investigated. Enzymes with DL-glyceraldehyde reductase activity were separated into two fractions (F-1 : unadsorbed enzyme fraction and F-2 : adsorbed enzyme fraction) by dye-affinity chromatography using Matrex gel orange A. The fraction of F-2 was further separated into four fractions with DL-glyceraldehyde reductase activity (F-2a, F-2b, F-2c and F-2d) by chromatofocusing. The enzyme in F-1 was distinct from enzymes in F-2. The molecular weight of the former was about 60000, and that of the latter was about 33000. The enzyme in F-1 was strongly active with D-erythrose as a substrate, and the enzymes in F-2 were strongly active with DL-glyceraldehyde. The enzyme in F-1 was not appreciably inhibited by aldose reductase inhibitors such as quercitrin, quercetin and 3, 3-tetramethyleneglutaric acid, whereas the enzymes in F-2 were inhibited considerably. Sulfate ion did not activate the enzyme in F-1. Four enzymes in F-2 were subdivided into 2 groups (group A : F-2a and F-2c, group B : F-2b and F-2d) on the basis of their enzymatic properties. The enzymes in both groups A and B were capable of reducing various aldoses, but D-pentose and D-hexose were poor substrates for enzymes of group B. The enzymes in group A were activated by sulfate ion, and the enzymes in group B were not activated. The enzymes in group A were more susceptible to inhibition by aldose reductase inhibitors than those in group B.
In order to clarify the lysosomal change of the rat liver in acute extrahepatic biliary obstruction, the activities of N-acetyl-β-glucosaminidase (NAG) and acid phosphatese (AcP) in the liver and serum were measured over a period until 7 d after the bile duct ligation (BDL). Liver lysosomal change on the biochemical basis was examined by using the liver 3 d after BDL treatment. From the results of the differential centrifugation experiments, the activities of the above enzymes in the nuclear fraction increased significantly, while those in the lysosomal fraction decreased. Sucrose density gradient centrifugation distribution patterns demonstrate that the lysosomal enzyme activities in the nuclear fraction tended to decrease, while those in the lysosomal fraction increased significantly. From kinetic parameters after BDL treatment, Km value in the nuclear fractions was in agreement with that in the lysosomal fractions. On the other hand, a significant increase in Vmax values in the nuclear fraction was found. From these results, it is considered that lysosomal particles are apt to be aggregated and that the size is larger in the liver after the BDL treatment.
Two aldehyde reductases and carbonyl reductases from Japanese monkey liver cytosol have been purified : the two aldehyde reductases obtained were to be homogeneous, but the carbonyl reductases are shown to be heterogeneous poly acrylamide-gel electrophoresis. A major aldehyde reductase, which is a dimer of single subunits with molecular weight of 41000, reduces aromatic and aliphatic aldehydes with nicotinamide adenine dinucleotide (NADH) as a more preferable cofactor than nicotineamide adenine dinucleotide phosphate (NADPH), and is inhibited by pyrazole and o-phenanthroline. The enzyme oxidizes alcohols in the presence of NAD+ as a cofactor. The other monomeric aldehyde reductase with molecular weight of 36000 is a high-Km aldehyde reductase which reduces aldehydes, D-glucuronate and α-diketones and which is sensitive to diphenylhydantoin and phenobarbital. The enzyme is not only immunologically identical with human liver aldehyde reductase, but also its amino acid composition closely resembles that of human liver enzyme. The two carbonyl reductases show similar low substrate specificity for aldehydes, ketones and quinones. One enzyme with molecular weight of 80000 shows dual cofactor specificity for NADH and NADPH and is selectively inhibited by isobutyramide, whereas another enzyme with molecular weight of 32500 exhibits absolute cofactor specificity for NADPH and sensitivity to ethacrynic acid and p-chloromercuribenzoic acid.
Dissolution rates of carbamazepine (CBZ) in four preparations of commercial tablets (200 mg) were measured by the paddle method (pH 1.2, 100rpm) using a cyclindrical vessel of 21, with a spherical bottom. Three tablet preparations (A, B, C) which showed different dissolution rates with each other, and a solution containing 200 mg of CBZ were chosen for the bioavailability test in eight healthy volunteers. The statistically significant differences were observed between a solution and three tablets for the serum levels of CBZ during 1 h after the administration. There was also a significant difference between tablets A and B, or A and C for the serum levels of CBZ at 1 and 2 h. However, there was no significantly difference for the extent of bioavailability. The absorption rate and mean dissolution time (MDT) were calculated by the Loo-Rigelman method and moment method, respectively. The log percent unabsorbed versus time plots of tablet A, B, and a solution yielded straight lines, respectively, but that of tablet C which was the slowest in vitro dissolution rate, was a biphasic curve with an inflexion at 4 h. The time required for 30, 50, 60 and 80% dissolved in vitro were well correlated with absorption rate constant obtained during the initial linear portion. Furthermore, the MDT values of tablet A, B, and C of in vitro were also correlated with those of in vivo, when tablet C is assumed to be absorbed completely with the absorption rate constant obtained during the initial portion. On the basis of these findings, it was suggested that the present in vitro dissolution test might be useful as a method to predict in vivo dissolution rate.
In this study a new size reduction method was applied to slightly soluble medicinal compound having an acidic group in a molecule other than oxolinic acid. Phenytoin (PHT) and phenobarbital (PB) were chosen as medicinal compounds to be reduced, then effects of kinds or concentration of surfactants or polymers on size reduction were investigated. Changes of crystal form by powder X-ray diffraction measurements and dissolution rates of various microparticles were studied. The results obtained were as follows : i) PHT and PB crystallines were reduced so far as 3μm and 4μm (median diameter), respectively. ii) No change in crystal form of PTH microparticles was found, but there were changes in crystal form of PB microparticles in comparison with original crystallines. In the case of PB, original crystallines were anydride, but the microparticles were hydrate. iii) Dissolution rates of both microparticles of PHT and PB were increased remarkably.