Enzymatic reaction mechanisms are studied from a quantum chemical point of view. Calculations were performed by using ab initio molecular orbital method. The role of Asp 102 in the catalytic triad of trypsin, roles of three arginine residues, Glu 270 and zinc in the active site of carboxypeptidase A, the charged state of cysteine residue in the catalytic site of cysteine protease, a histidine-aspartic acid hydrogen bonding system in the active site of ribonuclease A, and conversion from L-lactate to pyruvate in the enzymatic reaction for lactate dehydrogenase are reported.
Both association constant and bound chemical shift are estimated by a simple graphic method under equimolar conditions for the association between Lewis base and tris (dipivaloylmethanato) europium in CCl4 or cyclohexane by using 1n Δ=-K1-1/2·CBO-1/2+1n Δ1. This method can be applied in the conditions of K1CBO≳0.45 and the result is correlated successfully with the value estimated from the computer simulation.
We have investigated the alkaloidal constituents of the root of an Aconitum species collected at Mt. Mineoka (Chiba Pref.). In addition to the known nine compounds, i.e. mesaconitine, hypaconitine, aconitine, deoxyaconitine, isotalatizidine, 14-O-acetyltalatizamine, neoline, takaosamine, and ignavine, a new diterpene alkaloid was isolated. The structure of this new base was established to be 15-α-hydroxyneoline by the spectroscopic evidence and by the partial synthesis from neoline.
A new acylated sugar, named ligustrin A, was isolated from flowers of Ligustrum obtusifolium SIEB. et ZUCC., L. ovalifolium HASSK., and L. japonicum THUNB. Ligustrin A consists of oleanolic acid (1 mol), palmitic acid (3 mol), and L-arabinose (1 mol). Its structure was elucidated to be 3, 4-di-O-palmitoyl-1-O-(3-O-palmitoyloleanoyl)-α-L-arabinopyranose on the basis of the spectroscopic and chemical evidence.
A new genuine natural product of Cimicifuga triterpenol glycoside, named shengmanol xyloside (7), C35H56O9, mp 244-245.5°C, [α]22D-10.2°, was isolated from the fresh underground parts of Cimicifuga japonica. The xyloside (7) turned, on a mild treatment with p-toluenesulfonic acid in methanol, into 25-O-methylcimigenol xyloside (6). On enzymatic hydrolysis, 7 yielded its genuine genin, shengmanol (14), C30H48O5·1/2H2O, mp 153.5-154.5°C, [α]26D+0.5°, which afforded cimigol (17) on alkali treatment. The xyloside (7) afforded a pentaacetate (16), which was identical with the peracetate of acetyl shengmanol xyloside (2). On the basis of chemical and spectral data, the structure of shengmanol xyloside (7) was proposed to be (23R, 24S)-3β, 15α, 16α-trihydroxy-16, 23 ; 24, 25-diepoxy-9, 19-cyclolanostane 3-O-β-D-xylopyranoside. The contents of cimigenol xyloside (1), acetyl shengmanol xyloside (2), 24-O-acetylhydroshengmanol xyloside (3) and shengmanol xyloside (7) in the fresh underground parts of C. japonica were estimated on the basis of the high-performance liquid chromatography equipped with a RI detector. The total content of 2, 3, and 7 was found to be about two and a half times as much as that of 1, suggesting that the chemically unstable xylosides 2, 3, and 7 are genuine glycosides of some Cimicifuga triterpenol xylosides such as 1 and 6.
From the whole plant of Erythraea centaurium four xanthones (I-IV) were isolated. On the basis of chemical and spectral evidences, they were shown to be methylbellidifolin, methylswertianin, 1, 8-dihydroxy-3, 4, 6-trimethoxyxanthone and 1, 8-dihydroxy-2, 3, 4, 6-tetramethoxyxanthone, respectively.
A method for the quantitative determination of 10-hydroxy-2-decenoic acid (10-HDA) in royal jelly was establishd by high-performance liquid chromatography (HPLC) by use of a reversed-phase stationary phase. The 10-HDA extracted with CH2Cl2 from royal jelly was determined by HPLC under the following operating conditions : column, LiChrosorb RP-18 ; mobile phase, 55% methanol in water (pH 2.5) ; monitoring, ultraviolet absorption at 205 nm : internal standard, suberic acid ; temperature, 50°C. This method was simpler and more rapid than gas chromatographic method. It was successfully applied to the determination of 10-HDA contents in raw royal jelly, granules and tablets containing royal jelly.
Effects of total saponin preparations extracted from panacis japonici rhizoma, saponariae radix alba, sapindus pericarpium, senegae radix, thea semen, bupleuri radix, hippocastani semen, and styrax pericarpium, which contain oleanane-type aglycone on plasma levels of ACTH, corticosterone, and glucose in rat were determined by the radioimmunoassay method, the competitive protein binding method, and by the glucose oxidase method, respectively. All of saponins tested were found to increase plasma ACTH, corticosterone, and glucose at 30 minutes after the intraperitoneal administration. ED50 Value for the corticosterone secretion-inducing activity of saponins was estimated. Among 8 saponins, styrax saponin was the most effective (0.7 mg/kg) and panacis japonici saponin was the least (54 mg/kg). The increase in plasma glucose by these saponins was similar to that of isolated saikosaponin a or d but not to that of ginseng saponin. Hemolytic activities of these saponins were also determined, and the order of the activity was almost parallel to the order of ED50.
One of the TAME hydrolases of Trimeresurus mucrosquamatus venom was isolated by a combination of gel filtration on Sephadex G-100 and ion exchange chromatographies on CM-Sephadex C-50, Aminohexyl-Sepharose 4B and DEAE-Sephacel. From 1 g of the lyophilized venom 1.5 mg of purified arginine ester hydrolase was obtained. The substrate specificity of this enzyme was strictly directed to the hydrolysis of ester linkages to which an arginine residue contributed the carbonyl group. The esterolytic activity was inhibited by benzamidine, N-(p-tosyl)-L-phenylalanine chloromethyl ketone (TPCK) or diisopropyl fluorophosphate (DFP). The purified enzyme was proved to be homogeneous by electrophoresis on polyacrylamide gel and isoelectric focusing. The molecular weight was found to be about 33000. Its isoelectric point was 6.93. The esterolytic activity of the final preparation was 125.7 unit/mg. This enzyme had capillary permeability-increasing activity, but not kininreleasing and clotting activities. This protein was stable to heat treatment, and between pH 4-9. Its Michaelis constant (Km) for TAME and inhibition constants (K1) for benzamidine, TPCK, and DFP were found to be 7.81×10-3 M, 0.67×10-3 M, 0.43×10-3 M, and 3.66×10-3 M, respectively. This enzyme was a glycoprotein.
The microvillus fraction isolated from rabbit renal cortex contained about 5% of the total carbonic anhydrase activity in the homogenate, which could not be explained by contamination with the cytoplasm or erythrocyte soluble fractions. The carbonic anhydrase of the microvillus fraction showed two K1 values for each of the three sulfonamide inhibitors tested (sulfanilamide, acetazolamide, and ethoxzolamide). The values were essentially the same as those shown by the cytoplasm and erythrocyte soluble fractions, though the activity ratio of low to high K1 isozymes of the microvillus fraction seemed to be different from those of the other two fractions.
The hemolytic action of sodium alginate solution on human red blood cells (RBC), and the aggregations of human and rat RBC in the presence of sodium alginate were studied. The human RBC in sodium alginate solution did not indicate any morphological change of RBC. The hemoglobin was not detected in the supernatant of incubation medium. These results show that sodium alginate has no hemolytic action on human RBC. Human and rat RBC showed a marked aggregation by sodium alginate in the neutral medium. The rouleaux formations of RBC in various concentrations of sodium alginate having different molecular weights were examined. In addition, the concentration of sodium alginate required to induce a discernible aggregation of RBC was determined. Sodium alginates having larger molecular weights showed more pronounced activities for aggregation of RBC as compared with those having smaller molecular weights, and the aggregation of RBC increased with an increase in the concentration of sodium alginate. The aggregation of RBC in sodium alginate solution was inhibited by urea, suggesting that the aggregation of RBC is mainly caused by hydrogen bonding. The rouleaux of RBC covered with fibrin net was observed in the coagulation cruor by means of scanning electron microscopy, when sodium alginate was added to human or rat blood.
Effects of gomisin A, schizandrin, and some analogues, which are lignan components of schizandra fruits, on the liver damages induced by various drugs were examined biochemically and histologically. Pretreatment of gomisin A markedly reduced the activities of GOT, GPT, LDH, T-BIL, and T-CHO in the serum elevated by CCl4 and slightly decreased ALP activity. Histologically, hepatocellular necrosis by CCl4 was inhibited by pretreatment of gomisin A. Schizandrin, (+)-deoxyschizandrin, deoxygomisin A, and dimethylgomisin J also biochemically protected the liver from the injury by CCl4, but the potency of each compound was weaker than that of gomisin A. Gomisin A and schizandrin lowered the elevation of GOT and GPT in the serum induced by galactosamine. The action of gomisin A was stronger than that of schizandrin. Under microscopic observation, gomisin A depressed the appearance of vacuoles. Gomisin A prevented balooming of liver cells and fat droplets from 1% orotic acid, but not from ANIT induced injury biochemically and histologically. By considering chemical structures, it is necessary for lignan compounds to possess a methylenedioxyl moiety at C-12 and 13 positions in exerting the strong inhibitory effect on the liver damage.
In order to compare the structure-activity relationship of C-homobenzomorphan derivatives with that of 6, 7-benzomorphan derivatives, N-allyl ((+)-7, (-)-7, (±)-7, 8), N-(3, 3-dimethylallyl) ((+)-9, (-)-9, (±)-9, 10) and N-cyclopropylmethyl derivatives ((+)-11, (-)-11, (±)-11, 12) of (-)-1-methyl-, (+)-1-methyl-, (±)-1-methyl-and 1, 12α-dimethyl-10-hydroxy-2, 3, 4, 5, 6, 7-hexahydro-1, 6-methanol-1H-4-benzazonine have been synthesizad as shown in Chart 1. These compounds showed no significant analgesic activity.
Extracts of 104 crude drugs have been tested for mutagenicity by a modified Ames' method, by using Salmonella typhimurium TA 98 and TA 100 in the presence or absence of rat liver S-9 Mix. The crude drugs were extracted with water or methanol. The extracts were chromatographed on an XAD-2 column to remove histidine, if any, and subjected to the test. Of the 104 crude drugs 87 showed no mutagenicity and 10 were cytotoxic to S. typhimurium. Mutagenic activity was observed in 7 crude drugs, among which Dictamini Radicis Cortex, Zingiberis Siccatum Rhizoma and Chamomillae Flos showed relatively high activity.