In this study we carried out the pharmacological elucidation of the principle of drug action in the Japanese traditional Sino-medicine system, which could have contributed to the modern pharmacology and therapeutics through the discovery of a novel drug design and a new mechanism of drug action. The aim of this study was to focus on the elucidation of the supporting moiety in the drug design for the drug having affinity for a patho-receptor using a quite unique strategy and a new approach based on the natural products, Japanese traditional Sino-medicines relating to disease state, because of enormous clinical experiences with a long history. The characteristics of the pharmacological effects of Japanese traditional Sino-medicines were experimentally demonstrated by the following three key compartments, 1) the combined effect of drugs, 2) the selective activity of drugs to the disease state, 3) the possibility of individual symptomatological patterns (sho) evaluated by autonomic and immunopharmacological components. The success of the investigation on the principle of drug action of Japanese traditional Sino-medicines induced not only to obtain many novel compounds and unknown new mechanisms of drug action, but also to find a new salivary peptide for anti-hyperglycemics and a fresh mechanism of ACh receptor desensitization in neuromuscular synapse in the modern pharmacology. The results have brought the interchangeability of Japanese traditional Sino-medicine system to modern medicinal sciences, as described in the following contents, I. On the elucidation of a principle of drug action in the Japanese traditional Sino-medicine system. II. On the developmental frontier of drug design based on Japanese traditional Sino-medicines. III. On the interchangeability between the Japanese traditional Sino-medicine system and the modern medicinal sciences.
The study of the participation of T cells and the kinetic studies of cytokines by the RT-PCR method in the challenge phase of contact hypersensitivity reaction (CHR) were performed using skin tissues collected at regular intervals from mice sensitized with 2, 4-dinitro-1-fluorobenzene (DNFB). The results obtained from these studies and the further analyses using anti-CD4 and CD8 antibodies showed that the revelation of phenotype of lymph node cells at the reaction site and T lymphocytes at the CHR site were found to play important roles. Furthermore, we examined cytokines formed at the CHR site on the mRNA level. Consequently, it was found that 24 h after the challenge IFN-γ, IL-6 and IL-1β were expressed and 48 h after the challenge IL-2, IL-4 and TNF-α were expressed in addition to these cytokines. The results of the expression of IL-12 and those of the examination using IFN-γ knock out mice suggested that the expression of IFN-γ among several Th1 types of cytokine is especially important for the formation of CHR.
We examined the mechanism of the onset of contact hypersensitivity reaction (CHR) induced by several kinds of chemical substances in mice, focusing on cytokines. Before doing this experiment, we investigated the involvement of T cells in the formation of CHR using each primer of CD4 and CD8 (surface markers of lymphocytes) positive cells. It was suggested that both cells participate in the formation of CHR. On the basis of these results we analyzed cytokines produced in the reaction site on the mRNA level in with or without the presensitization of two kinds of strong contactants. Consequently, there was a tendency that the expression of mRNA of Th1 types of cytokines was commonly observed around 24 h after the reaction irrespective of the presence or the absence of presensitization. These date suggested that CHR can be presumed on the genetic level by the only one priming of strong contactants.
Dihydrodiol dehydrogenase (DD) oxidizes naphthalene dihydrodiol to 1, 2-dihydroxynaphthalene, which is immediately autoxidized to 1, 2-naphthoquinone. Here we established a fluorometric assay for the enzyme, which is based on the conversion of 1, 2-naphthoquinone to fluorescent compounds by reacting with ethylenediamine. The formed fluorescent compounds were synthetically identified as 6-(2-aminoethylamino)benzo[ƒ]quinoxaline and 2, 6- or 3, 6-bis(2-aminoethylamino)benzo[ƒ]quinoxaline, which showed the same fluorescence at 550 nm at an excitation wavelength of 420 nm. This method provides a 9000-fold increase in sensitivity over a currently available assay which measures the change in the absorbance of a cofactor, NADPH. Since this simple and sensitive method allowed many samples to be assayed simultaneously, we applied it to the analysis of multiple forms of DD, separated by an anion-exchange chromatography, from six human liver specimens.
A new syringe-type minicolumn, called Extrashot-Silica (EXS-Silica), containing diatomaceous earth granules was described. The EXS-Silica differs from the conventional pretreatment column. Using the EXS-Silica we can execute the simultaneous extraction-injection to HPLC, column. Therefore, an analysis using the EXS-Silica is an easier and faster method than the general HPLC analysis method. In this study, we carried out the simultaneous determination of four xanthine derivatives, such as caffeine, theobromine, theophylline and paraxanthine, in serum specimens. We used dichloromethane containing 4% ethanol (v/v) for the extraction-injection and water-acetic acid-ethanol-dichloromethane (0.2 : 0.2 : 4 : 95.6, v/v) for the mobile phase of HPLC. The eluent was monitored with a UV detector at 275 nm. A linear relationship between the amount of drug and the peak height was confirmed in the range of 1-40μg/ml for the abovementioned four xanthine derivatives in the serum. When a 5μl aliquot of the serum was subjected to this method, the observed detection limits of the drug were far less than therapeutic concentrations. The analytical accuracy of our method was finally confirmed by comparing the obtained analytical data by the new method with those obtained using the fluorescense polarization immunoassay method. Serum concentrations of theophylline obtained by these two methods correlate satisfactorily. Except for minor modifications in the injector, the existing liquid-chromatographic equipment can be used.
We investigate the method for the determination of an appropriate mixing region of three kinds of antacid, magnesium hydroxide, dried aluminum hydroxide gel and calcium carbonate using trianglular coordinates. Using the trianglular coordinates, the appropriate mixing region of satisfying the following categories was determined ; 1) acid neutralizing capacity (200 ml), 2) immediate action (pH 6-8 range obtained 10 min after pouring an antacid into 50 ml of 0.1mol/1 HCl (aq) sol.), 3) bowel property (mol ratio of Mg/Al=1-3), and 4) quantities of combination of three antacids (under 700 mg of total quantities). Modified Fuchs Test was performed at some points which met the above conditions. The durability of the combination of these three antacids was evaluated. The following regions have long durability of antacids (about 60 min for persisting pH 3.5-5); magnesium hydroxide 40-60%, dried aluminum hydroxide gel 30-50% and calcium carbonate 10%. In the cases of changing the above mentioned conditions, and of using different kinds of antacid, the determination of the appropriate mixing region using triangular coordinates is effective for the pharmaceutical technology. In this study we emphasize the following striking features ; utilization of triangular coordinates, fixing the combination of antacid, and incorporation of three kinds of antacid with aluminum, magnesium and calcium.