In search of new biologically active compounds in nature, we have been investigating naturally occurring substances on the following subjects : I. chemical studies on naturally occurring drug materials [a) elucidation of bioactive constituents in natural drugs, b) elucidation of scientific basis for crude drug processing, and c) investigation of bioactive constituents in food materials], II. exploitation of new pharmaceuticals in nature [a) investigation of marine natural products and b) exploitation of Indonesian medicinal plants], and III. synthetic studies on bioactive natural products [a) chemical modification of naturally abundant carbohydrates and terpenoids and b) synthetic studies of complex lipids]. This article reviews the results obtained in our laboratory since 1978 on the subjects of I-a, b, c, II-a, and III-a.
By the reaction of 2-(1-cyano-1-methylthio) methyleneindan-1, 3-dione (1) with 7-dimethylaminoindolizine derivatives (8c, 9a, b), the corresponding substituted compounds (11, 12a, b) were obtained. Novel methine dyes (13-16) exhibiting their absorption maxima in the near-IR region (692-828 nm) were synthesized by the condensation of the compounds (11, 12a, b) with malononitrile in the presence of TiCl4 and pyridine.
A sensitive flow injection analysis using luminol/peroxidase chemiluminescence was developed for the determination of choline-containing phospholipids in serum. Flow injection manifold was composed of two channel system with an enzyme column, in which phospholipase D was immobilized together with choline oxidase. The serum sample (5 μl) was pretreated by Extrelut column (diatomite column) extraction with chloroform-methanol (95 : 5). The extract (20μl) was injected into a sample carrier at 38°C and passed through the enzyme column, which converted phospholipid to choline and subsequently to hydrogen peroxide. Produced hydrogen peroxide was monitored by measuring the chemiluminescence intensity of luminol/peroxidase system at 5°C. The response was linear against the amount of phospholipids ranging from 2 to 2000 pmol/test, and the relative standard deviation was less than 2%. In the determination of phospholipids in the serum, a correlation coefficient (r) between 4-aminoantipyrine/phenol and the proposed methods was found to be 0.983 (Y=1.035X-6.2). The throughput rate was 15 samples/h.
Fresh lamprey (F-La) or sardine (F-Sa) oil is known to contain a large amount of n-3 polyunsaturated fatty acids such as eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. When F-La or F-Sa was deodorized with steam at 280°C under 1 mmHg for 1 h (H-La and H-Sa, respectively), the contents of EPA and DHA were reduced and unidentified peaks were newly detected by gas-liquid chromatography. To know the biological influence of these high-temperature deodorized oils, the sterilizing function of the macrophage against Listeria monocytogenes was investigated in male ddY mice fed H-La or H-Sa. One week feeding of H-La or H-Sa lowered the LD50 values of the bacteria injected intravenously. Numbers of the viable bacteria on the day 3 after intravenous injection were about 10 times higher in the liver and 5 times higher in the spleen of mice fed H-La or H-Sa as compared with those of the control group. These results suggest that the sterilizing function of fixed macrophages both in the liver and the spleen was suppressed in mice fed H-La or H-Sa.
The oxidative metabolism of disopyramide (DIS) enantiomers to mono-N-dealkyldisopyramide in mouse hepatic microsomes was studied in vitro. When (R-)-DIS was used as a substrate Lineweaver-Bulk plot was a single line with the apparent Km value of 0.23 mM and Vmax value of 1.05 nmol/mg protein/min. When S(+)-DIS was used as a substrate the plot was biphasic, and two apparent Km values (Km1=0.04 and Km2=0.77, mM) and two Vmax values (Vmax1=0.58 and Vmax2=1.53, nmol/mg protein/min) were obtained. When racemic DIS was used as a substrate the plot was also biphasic, while the plot observed at the lower concentration region was shifted to the upper region in comparison with that estimated from the sum of the individual N-dealkylase activities of each enantiomer. These results suggest that the N-dealkylation of R(-)-DIS is carried out by very limited cytochrome P-450 isozyme (s), but that of S(+)-DIS is carried out by multiple cytochrome P-450 isozymes ; that is, N-dealkylations of R(-)-and S(+)-DIS are involved in the different cytochrome P-450 isozymes each other, particularly at relatively higher concentration of the substrates.
To improve the rectal delivery of an anti-inflammatory drug, biphenylylacetic acid (BPAA), the use of 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD) and heptakis (2, 6-di-O-methyl)-β-cyclodextrin (DM-β-CyD) was investigated. Inclusion complex formations of BPAA with both β-CyDs in a molar ratio of 1 : 1 in water were ascertained, and their stability constants were determined. The dissolution of BPAA in water and the release of BPAA from an oleaginous suppository (Witepsol H-5) were significantly increased by β-CyDs, depending on the magnitude of the stability constants of the water-soluble complexes. However, the serum levels of BPAA after rectal administration of the suppositories containing BPAA or its β-CyDs complexes in rats increased in the order of BPAA alone «DM-β-CyD⪈HP-β-CyD complex. The in situ recirculation study revealed that the greater the stability constant of the complex, the lesser was the absorption of BPAA from the rectal lumen of rats under the solution state. Both in vivo and in situ studies demonstrated that rather high amount of HP-β-CyD (about 20% of dose) was absorbable from the rat's rectum, compared with DM-β-CyD (less than 5% of dose), suggesting the possibility of the permeation of BPAA through the rectal membrane in the form of HP-β-CyD complex. Furthermore, DM-β-CyD and HP-β-CyD significantly reduced the irritation of the rectal mucosa caused by BPAA after the administration of the suppositories to rats. The above data suggest that water-soluble β-CyD derivatives are useful in the rectal formulation of BPAA, improving the bioavailability and local irritancy.
The stable water-in-oil-in-water (W/O/W) multiple emulsion was prepared by a two-step procedure for emulsification using glyceryl tricaprylate (Panasate-800) as the oil phase. The water-soluble drugs such as cefadroxil, cephradine, 4-aminoantipyrine, and antipyrine were selected and entrapped separately in the inner aqueous phase of W/O/W multiple emulsion. In consideration of parenteral administration, pH 7.4 phosphate buffered saline was used in both inner and outer aqueous phases. Moreover, these multiple emulsions could be significantly stable for a month at room temperature by the addition of hydrophilic polymer like gelatin and of amino acid like lysine to the inner aqueous phase.