薬用人参成分のイースタンブロッティング

イースタンブロッティングは TLC で分離した薬用成分を PVDF 膜へ転写し、抗原であるターゲット低分子のモノクローナル抗体で染色する手法で、目で見ることが出来る説得力の大きな手法である。新たに開発したイースタンブロッティングでは親水性ポリエーテルスルホン膜を用いることで、ジンセノシド類を膜上で展開・分離することが可能となり、転写プロセスを省略し、抗体染色が行える。さらに染色結果をスキャナーで取り込み、NIH Image 画像解析ソフトウェアを用いてスポットを数値化し、定量分析を完結させた。HPLC による分析値との相関も良好であった。In this review, we summarize studies from Asian and Caucasian countries and highlight the importance of the APC anticoagulant system in coagulation regulation in vivo. Thrombophilia is defined as an increased tendency to thrombosis, and can be either inherited or acquired. It has been recognized that in Caucasians, a polymorphism of coagulation factor V, factor V Leiden (R506Q), is a major risk factor for venous thrombosis. However, this does not appear to be the case for Asian thrombophilia. Accumulated studies from Asian and Caucasian countries indicate that the frequencies of protein S deficiency and of protein C deficiency were higher in Asian patients suffering from deep vein thrombosis than in Caucasian patients, indicating that deficiency of the activated protein C (APC) anticoagulant system is more common in Asain than in Caucasian patients. This would suggest that these gene variants are responsible for the deficiency of the activated protein C anticoagulant system and that the deficiency of the activated protein C anticoagulant system is a major risk factor for venous thrombosis

センノサイドＡ、Ｂの免疫化学的測定法の開発について

センノサイドＡおよびセンノサイドＢは、大黄やセンナの主有効成分で瀉下作用をもつ。腸内細菌はセンノサイドをレインアンスロンに代謝し、レインアンスロンが下剤効果を発揮する。大黄やセンナの品質を管理するために、センノサイドＡ、Ｂの簡便な分析方法が必要である。モノクローナル抗体を用いたイムノアッセイは迅速、簡便かつ高感度であり、様々な医薬品成分の分析方法として重要である。著者らはこれまでに、天然由来の生理活性成分に対するモノクローナル抗体作製の一環として、センノサイドＡ、Ｂに対するモノクローナル抗体の作製を行ってきた。その応用として、以下に示すセンノサイドＡ、Ｂの免疫化学的測定法の開発を行ったので報告する。　１.　ELISA 法やイムノクロマトグラフ法を用いたセンノサイドＡ、Ｂのアッセイ系の確立　２.　センノサイドＡ、Ｂのイースタンブロッティング法の開発　３.　免疫組織染色による大黄切片中のセンノサイドＡの分布Sennoside A（SA）and sennoside B（SB）are the major purgative constituents of rhubarb and senna. Intestinal bacteria metabolize sennosides into rheinanthrone, which acts directly on the intestines as a purgative, functioning in the same way as natural prodrugs. Therefore, phytochemical studies of such plants require simple assays for the analysis of SA and SB, such as immunoassay systems that use monoclonal antibodies (MAbs) against bioactive compounds of low molecular weight. In fact, these methods are important tools for the qualitative and quantitative analysis of active constituents from natural medicines and plants. The production and characterization of anti-SA and anti-SB MAbs have been performed in our ongoing study of the production of MAbs against naturally occurring bioactive compounds. As an extension of antibodies generation, we report here the immunoassay systems as indicated below. 1. Establishment of an ELISA and immunochromatographic assays for SA and SB. 2. Development of eastern blotting technique for identification of SA and SB. 3. Immunohistochemical localization of SA in fresh rhubarb root

Molecular Mechanisms Underlying Anti-Inflammatory Actions of 6-(Methylsulfinyl)hexyl Isothiocyanate Derived from Wasabi (<i>Wasabia japonica</i>)

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC) is a major bioactive compound in wasabi (Wasabia japonica), which is a typical Japanese pungent spice. Recently,in vivoandin vitrostudies demonstrated that 6-MSITC has several biological properties, including anti-inflammatory, antimicrobial, antiplatelet, and anticancer effects. We previously reported that 6-MSITC strongly suppresses cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and cytokines, which are important factors that mediate inflammatory processes. Moreover, molecular analysis demonstrated that 6-MSITC blocks the expressions of these factors by suppressing multiple signal transduction pathways to attenuate the activation of transcriptional factors. Structure-activity relationships of 6-MSITC and its analogues containing an isothiocyanate group revealed that methylsulfinyl group and the length of alkyl chain of 6-MSITC might be related to high inhibitory potency. In this paper, we review the anti-inflammatory properties of 6-MSITC and discuss potential molecular mechanisms focusing on inflammatory responses by macrophages.</jats:p

Application of Monoclonal Antibodies against Bioactive Natural Products: Eastern Blotting and Preparation of Knockout Extract

Matrix-assisted laser desorption/ionization (MALDI) tof mass spectrometry was used for the confirmation of hapten number in synthesized antigen. As application of MAb, the MAbs against ginsenosides and glycyrrhizin have been prepared resulting in the development of two new techniques that we named the eastern blotting method and the knockout extract preparation. In eastern blotting technique, glycosides like ginsenosides and glycyrrhizin separated by silica gel TLC were blotted to PVDF membrane that was treated with a NaIO4 solution followed by BSA resulted in glycoside-BSA conjugate on a PVDF membrane. The blotted spots were stained by MAb. Double staining of eastern blotting for ginsenosides using antiginsenoside Rb1 and Rg1 MAbs promoted complete identification of ginsenosides in Panax species. The immunoaffinity concentration of glycyrrhizin was determined by immunoaffinity column conjugated with antiglycyrrhizin MAb resulting in the glycyrrhizin-knockout extract, which was determined by the synergic effect with glycyrrhizin on NO production using the cell line

Pharmacological Effects of Ginseng on Liver Functions and Diseases: A Minireview

Ginseng, an ancient and famous medicinal herb in the Orient, has been used as a valuable tonic and for the treatment of various diseases including hepatic disorders. Ginseng saponins, commonly known as ginsenosides, are principal constituents and have believed to be responsible for multiple ginseng health benefits. There are more 40 ginsenosides isolated from ginseng. To date, treatment options for common liver diseases such as cirrhosis, fatty liver, and chronic hepatitis remain problematic. In this regard, ginseng extracts and individual ginsenosides have shown a wide array of beneficial role in the regulation of regular liver functions and the treatment of liver disorders of acute/chronic hepatotoxicity, hepatitis, hepatic fibrosis/cirrhosis, hepatocellular carcinoma, and so on in various pathways and mechanisms. In this paper, we first outline the pharmacological effects of ginseng and ginsenosides on the liver functions.</jats:p

Utilization of a Biodegradable Mulch Sheet Produced from Poly(Lactic Acid)/Ecoflex®/Modified Starch in Mandarin Orange Groves

We have developed a mulch sheet made by inflation molding of PLA, Ecoflex® and modified starch, which all have different biodegradabilities. A field test of use as an agricultural mulch sheet for mandarin oranges was carried out over two years. The mechanical properties of the mulch sheet were weakened with time during the field test, but the quality of the mandarin oranges increased, a result of the controlled degradation of the sheet. The most degradable modified starch degraded first, allowing control of the moisture on the soil. Accelerator mass spectroscopy was used for evaluation of the biomass carbon ratio. The biomass carbon ratio decreased by degradation of the biobased materials, PLA and modified starch in the mulch sheet

Reconstruction of primary vertices at the ATLAS experiment in Run 1 proton–proton collisions at the LHC

This paper presents the method and performance of primary vertex reconstruction in proton–proton collision data recorded by the ATLAS experiment during Run 1 of the LHC. The studies presented focus on data taken during 2012 at a centre-of-mass energy of √s=8 TeV. The performance has been measured as a function of the number of interactions per bunch crossing over a wide range, from one to seventy. The measurement of the position and size of the luminous region and its use as a constraint to improve the primary vertex resolution are discussed. A longitudinal vertex position resolution of about 30μm is achieved for events with high multiplicity of reconstructed tracks. The transverse position resolution is better than 20μm and is dominated by the precision on the size of the luminous region. An analytical model is proposed to describe the primary vertex reconstruction efficiency as a function of the number of interactions per bunch crossing and of the longitudinal size of the luminous region. Agreement between the data and the predictions of this model is better than 3% up to seventy interactions per bunch crossing