Our studies on the elucidation of structure and stereochemistry of the palytoxin including minor toxins, which were named homopalytoxin, bishomopalytoxin, neopalytoxin and deoxypalytoxin have been reported. The conformation of palytoxin has been further discussed.
A regiospecific synthesis of emodin trimethyl ether (12a), 7-hydroxyemodin tetramethyl ether (12b), helminthosporin trimethyl ether (12c), and chrysophanol dimethyl ether (12d) by use of directed lithiation strategy as a key step is described. The pathalides (7 and 9) were prepared by the condensation of the corresponding o-lithio benzamides (2) with benzaldehydes (3a and 3c) which were synthesized from m-cresol methoxymethyl ether by directed lithiation reaction. The phthalides (9) were then reduced to o-benzylbenzoic acids (10), which were easily converted to anthraquinones (12) by cyclisation with trifluoroacetic anhydride and subsequent oxidation with chromium trioxide.
Paterno-Buchi reaction of furan and benzaldehyde was studied. Four 2 : 1 adducts were isolated and their structures were solved by chemical conversion, spectral resolution, and X-ray analysis. Two of them (B and C) has 4, 6, 8-trioxatricyclo [5.2.0.02, 5] nonane skelton and the other (D and E) had 3, 6, 8-trioxatricyclo [5.2.0.02, 5] nonane skelton. The former gave dimethoxy derivatives (F and G) and the latter gave mono methoxy one (H and J) by the treatment with acetic acid in methanol. Since C was presumed to be a novel sym-4, 6, 8-trioxatricyclo [5.2.0.02, 5] nonane skelton, it was solved by X-ray analysis.
Effects of 70% methanol extract obtained from Moutan Cortex (the root cortex of Paeonia moutan SIM., Paeoniaceae), which is one of the most important crude drugs in traditional Chinese medicine, on the phagocytic activity of the mouse resiculoendotherial system were studied by the carbon clearance method in vivo. Cell numbers of peritoneal exudate cells solution and phagocytosis of latex by peritoneal macrophage (Mφ) were also examined in vitro. The clearance-rate of carbon was significantly shortened 1 h after the administration of 70% methanol extract (400mg/kg, p.o.). Microscopically, carbon particles were internalized by Mφ of marginal zones between white and red pulp in the spleen, and in the liver, mainly by Kupffer cells. The 70% methanol extract activated the phagocytosis of carbon by Kupffer cells in the liver. Cell numbers of peritoneal exudate cells solution increased 24 h after the administration of 70% methanol extract (400mg/kg, p.o.). The incubation of 70% methanol extract (100μg/ml) with peritoneal Mφ promoted the phagocytosis of latex by peritoneal Mφ. These results suggest that 70% methanol extract obtained from Moutan Cortex promotes the phagocytic activity on the reticuloendotherial system in mice and that it has a stimulatory effect on Mφ because of an increase in cell numbers of peritoneal exudate cells solution and of phagocytic activity of peritoneal Mφ.
A high performance liquid chromatographic (HPLC) method was established for the quantitative determination of pheophorbide a (PB-a) in chlorella tablets. PB-a was extracted from chlorella tablets with 85% acetone. The acetone aqueous extract was shaken with diethyl ether, the ether extract was washed with 5% Na2SO4 and shaken with 17% HCl. The acid extract was diluted with water and re-extracted with diethyl ether. The ether extract was evaporated to dryness, the residue of which was dissolved in methanol and applied to HPLC. PB-a was separated on Radial PAK μBondapak C18 column with methanol-0.025M ammonium acetate (88 : 12, v/v) as a mobile phase. The average recovery of PB-a added to chlorella tablets was 82.9% with coefficients of variation of 6.72%. The official method (spectrophotometric determination) was used to determine PB-a content of 19 commercial chlorella tablets in order to compare it with the proposed HPLC method. The PB-a contents obtained by the official method (8.0-111.8mg%, an average 36.4mg%) were higher than those by the proposed one (2.9-80.6mg%, an average 22.4mg%). As PB-a was separated from other chlorophyll degradative products, the proposed method was more specific and accurate than the official one.
The optimum conditions for the determination of vitamin K3 (VK3) in the plasma by differential pulse polarography were examined. This technique, after ether extraction, was found to be useful for the determination down to a level of 3×10-7M of VK8. The recovery of VK8 added to the plasma (4.0×10-6M) was more than 90%. At this concentration the coefficient of variation (n=4) was 5.5%.
Conditioned medium prepared from adherent cells in long-term culture of mouse bone marrow stimulated in vitro colony formation by mouse bone marrow cells. Colonystimulating activity (CSA) was detected in the medium from 12-week-old cultures, and increased weekly to reach a plateu level 25 weeks after initiation of cultures. The CSA level was maintained over a 40-week period. The cultures were not recharged with fresh bone marrow cells throughout the experiment, and then no non-adherent cell production was observed in them. Two types of colony-stimulating factor were separated by gel filtration on Ultro-gel AcA 34 column. The larger molecular weight component that was heat-stable and trypsinresistant stimulated macrophage colony formation. The smaller molecular weight component that was heat-labile and trypsin-sensitive stimulated granulocyte colony formation.
Two forms of nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P-450 reductase, reductase I (MW 78000) and reductase II (MW 72000), were purified as a single component on SDS-polyacrylamide gel electrophoresis from the porcine adrenal microsomes. When cytochrome c was used as an electron acceptor, specific activities of these reductases were 51.8μmol/min/mg for reductase I and 53.1μmol/min/mg for reductase II, respectively. These reductases were able to reduce artificial electron acceptors, such as ferricyanide, dichlorophenol indophenol, nitroblue tetrazolium and neotetrazolium. Two forms of reductase were optically flavoprotein. On the kinetic analysis, the apparent Km values of cytochrome c for reductase I and reductase II were 40.8 and 21.3μM ; those of NADPH for reductase I and reductase II were 35.7 and 22.7μM, respectively. Reductase I was able to support cytochrome P-450 monooxygenase reactions on the reconstituted system, such as 21-hydroxylation, 17α-hydroxylation and cleavage reaction of C17-C20 bond by highly purified cytochrome P-450 (21-hydroxylase), P-450 (17α-hydroxylase-C17, 20 lyase). The apparent Km values were 0.33μM for P-450 (21-hydroxylase) and 0.58μM for P-450 (17α-hydroxylase-C17, 20 lyase), when progesterone was used as substrate. However, reductase II was unable to support the monooxygenase reaction. From these observations, it is discussed that reductase II which retains the flavin prosthetic group is a cytochrome P-450 reductase lacked a small hydrophobic peptide (MW 6000) which is specific binding site to cytochrome P-450 for the electron transfer.
Hyaluronidase (HAase) [EC 18.104.22.168] was partially purified about 500-fold from pig testis by gel filtration on Sephadex G-100 and ion-exchange chromatographies on CM-52 and DE-52 cellulose. The partially purified HAase hydrolyzed both hyaluronic acid and chondroitin 4-sulfate in the optimal pH range from 5.0 to 5.2, but the enzyme did not have chondro-4- and -6-sulfatase activities. For the maximal hydrolysis of hyaluronic acid, the enzyme required the presence of NaCl in the reaction system, whereas high concentration of the salt inhibited significantly the enzyme. Although partially purified HAase was eluted in a single peak corresponding to mol. wt of 61 K from the column of Sephadex G-100, the enzyme was separated into several moieties with different isoelectric points (about 8, 6.9, 6.7, 6.5 and less than 6.5) by the chromatofocusing, indicating the presence of isoenzymes of HAase.
The presence of seven crystal forms of cianidanol (tetrahydrate·I, monohydrate·I, II, anhydrate·I, II, III, IV) was confirmed by X-ray diffractometry, thermal analysis and by the measurement of water content. The effect of temperature and humidity on the transformation among the seven crystals were investigated. Anhydrate·I transformed to monohydrate·I in low humidity of 11-44% of relative humidity (R.H.) and tetrahydrate·I in high humidity above 68% of R.H. at 20°C. Monohydrate·I transformed to anhydrate·II in low humidity under 30% of R.H. and monohydrate·II at 75% of R.H., 40°C. Anhydrate·II, III, IV transformed to tetrahydrate·I at 94% of R.H., 20°C. Monohydrate·II did not transform to any other form at 0-94% R.H., 20°C, it was the most stable form at room temperature. Dissolution profiles of the seven crystals measured in water were almost the same.
The biological fate of N, N'-diphenyl-p-phenylenediamine (DPPD) was examined in rabbits and rats after intravenous, intraduodenal, and multiple (6 d) oral administration. It was found that this compound was metabolized to DPPD glucuronide and hydroxylated DPPD. Unchanged DPPD and its glucuronide in the biological fluids and tissues were determined by high performance liquid chromatography and gas chromatography which were newly developed. Unchanged DPPD and its glucuronide were excreted in the urine and bile after intravenous and intraduodenal administration of DPPD. In this experiments the excretion of DPPD glucuronide was superior to that of unchanged form in the both fluids, though their amounts were not so large. After multiple oral administration, total fecal excretion of DPPD in rabbits and rats were 72.2 and 55.4% of the dose, respectively. In rabbits, urinary excretion of DPPD and its glucuronide were 2.4 and 3.4% of the dose, respectively. On the other hand, in rats, they were only 0.04% of the dose. Tissue distribution of DPPD in rats was also examined. Unchanged DPPD was detected considerably only in fat tissue.
The red mold toxicosis, the major symptoms of which are diarrhea, vomiting and nausea, has been suggested to be induced by trichothecenes produced by Fusarium fungi. Fusarenon-X (F-X) is one of the trichothecene mycotoxins. In this study, we measured the blood glucose level, intestinal glucose contents and intestinal starch contents after the mouse small intestine was loaded with starch, in order to observe the effect of F-X and various cathartics on the digestive and absorptive abilities of the mouse small intestine. Mannitol, castor oil and magnesium sulfate tended to inhibit the dietary increase of blood glucose, the increase of intestinal glucose contents and the decrease of intestinal starch contents after the administration of starch. These results suggest that these cathartics may slightly inhibit the digestion or the absorption in the intestine, but their inhibitory effects seem to be too weak to induce a significant increase of osmotic pressure in the intestinal lumen. Dioctyl sodium sulfosuccinate (DSS) significantly inhibited the increase of blood glucose level, the increase of intestinal glucose contents and the decrease of intestinal starch contents after the starch administration, suggesting that DSS may induce malabsorption and maldigestion in the intestine which may be one of the factors of induction of DSS-induced diarrhea. In contrast, F-X induced no change on blood glucose level, intestinal glucose and starch contents. Therefore, F-X-induced diarrhea can not be related to the increase of osmotic pressure of the intestinal lumen which is induced by malabsorption and maldigestion. Present results support the mechanism of F-X-induced diarrhea proposed previously by us that F-X causes diarrhea through increasing the permeability of intestinal epithelium cells and resulting in the exudation of plasma contents and an eventual increase in the intestinal fluids.
Highly purified 17α-hydroxylase-C17, 20 lyase (cytochrome P-450) from porcine adrenocortical microsomes was incubated with [4-14C]-C21-steroid substrate in the presence of cytochrome P-450 reductase and nicotinamide adenine dinucleotide phosphate as a cofactor. On the kinetic analysis, C17, 20 lyase activity was strongly inhibited by progesterone. The inhibition was competitive, and K1 value for progesterone was 0.9μM. In addition, 17α-hydroxylase activity was inhibited by 17α-hydroxyprogesterone. The inhibition was competitive, and K1 value for 17α-hydroxyprogesterone was 7.5μM. These results suggest that both hydroxylase and lyase reactions are catalyzed on a single active site.
Substrate specificity of bilirubin oxidase (EC 22.214.171.124) and phenol oxidase (EC 126.96.36.199) against benzoic acid derivatives was investigated by the enzyme catalyzed coloration reaction. As a result, it was found that the both enzymes individually react with benzoic acid derivatives to give characteristic colored compounds. Especially, in the presence of 4-aminoantipyrine or 2, 2'-azino-di-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt as coupler, sensitive, conspicuous and versatile chromogenic reactions were catalyzed by the enzyme.