Suresh Awale

University of Toyama Repository

― 153 ―新興ウイルス感染症に対する創薬 安田 二朗 （長崎大学感染症共同研究拠点）  直近半世紀の間に新たに人類の脅威として出現した新興ウイルス感染症の数は 30を超える。それらの中には、エボラウイルス病やラッサ熱など極めて高い致死率を示すものも少なくないが、これらの新興ウイルス感染症に対する治療薬（抗ウイルス剤）の開発は遅れている。 重症熱性血小板減少症候群（SFTS）は、2013 年以降わが国でも毎年患者が報告されている新興ウイルス感染症で致死率も極めて高い。SFTS に対する確立された治療法はなく、ワクチンや抗ウイルス剤も現時点では存在しない。我々は、現在複数の手法を使って、SFTSウイルスの増殖を阻害する化合物のスクリーニングを行っており、抗 SFTS 薬の開発を目指している。今回のセミナーではその成果の一部を紹介する。 Application of NMR metabolomics in understanding the physiological target of Kampo medicineSuresh AwaleInstitute of Natural Medicine, University of Toyama Nuclear Magnetic Resonance (NMR) Spectroscopy is an indispensable tool for the structure elucidation of the compounds in natural products and organic chemistry. Recently, NMR is also becoming a major technique for the analysis of metabolites in bio-samples, and the biomarker discovery.  A key advantage of NMR metabolomics is its high reproducibility and direct identification of the metabolites. Furthermore, it is a non-destructive technique, which can rigorously quantify major compounds present in both in vitro and in vivo bio samples. Given its simplicity of methodology, direct identification and quantitative information of the metabolites, we applied NMR metabolomics as a novel and powerful tool to determine the physiological target of the Kampo medicine in vivo. For this purpose, a Kampo medicine ‘Gorei san’  (五苓散) was used as the exploratory example in this study.  Gorei san  (GRS)is one of the traditional Kampo medicine composed of five crude drugs (Alisma Rhizome, Chuling (猪苓), Poria Sclerotium (茯苓), Cassia (桂皮) and AtractylodesRhizome (白朮). It is regarded as a typical water-retaining agent, and it has the function of regulating the metabolic abnormality of moisture in the body and returning it to normal. In the present study, male Wistar rats of 8 weeks of age were fed for four weeks with the regular diet (Cont group, n = 6) or a feed containing 1% GRS extract (1% GRS group, n = 6). Measurement of food intake, drinking water, body weight and urine volume was monitored each week. After the administration period, various organs and bio-fluids such as blood and urine were collected for the NMR analysis. In the present investigation, 26 primary fluctuating metabolites were identified and quantified.  Administration of GRS was found to significantly alter the metabolites involved in pyruvate metabolism pathways, such as lactate, acetate, succinate, and citrate.  Acknowledgement: This study was supported by the President's discretionary expenses (Year 2016-2017), University of Toyama for the project「次世代 KAMPO 科学の創成を志向した漢方オミックス(Kampo Omics)の基盤構築」. ― 154 ― Japanese Herbal Medicine (Kampo) Based Antimalarial Drug Development  Awet Teklemichael1,2, Shusaku Mizukami1,3, Kazufumi Toume4, Farhana Mosaddeque1,2,Michiko Fukuda1, Mahamoud Sama Cherif1, Nguyen Huy Tien1,3, Katsuko Komatsu4, Kenji Hirayama1  1Department of Immunogenetics, Institute of Tropical Medicine (NEKKEN), 2Leading Graduate School Program, and Graduate School of Biomedical Sciences, 3Department of Clinical Product Development, NEKKEN, Nagasaki University, 4Division of Pharmacognosy, Department of Medicinal Resources, Institute of Natural Medicine, University of Toyama.Introduction: Malaria is critical global health issue especially tropical and subtropical countries. The emergence of resistance to the available antimalarial drug requires the urgent development of new medicine with new mechanisms of action. Herbal medicine, includingJapanese (kampo) one, is still an attractive source of the antimalarial drug since the isolation of quinine and artemisinin. The aim of the study is to examine kampo as the source of new antimalarial drug utilizing a compound and crude extract library. In previous joint seminar, we showed our finding of antimalarial kampo compounds and crude drug extract. According to the finding, in parallel with the compound based drug development strategy, we sought a way of drug discovery based on already approved kampo formula which contains the effective kampo crude extract because it is proven to be enough safe.  Methods and Material: As reported in previous seminar, the antimalarial activity of 96 compounds and 120 extracts in the library provided by Institute of Natural Medicine, University of Toyama were evaluated using in vitro antimalarial assay against chloroquine/mefloquine sensitive (3D7A) and -resistant (Dd2) strain of Plasmodium falciparum in erythrocytic cycle.After the drug treatment, the red blood cells were stained with SYBR Green to detect the parasite. In addition, the cytotoxicity was also examined. We select the kampo formula based on the content of active crude drug extract, and evaluated using the in vitro assay. Moreover, active kampo crude drug extract and formula were further examined against mouse malariaP. yoelii. In vivo parasite growth suppression by them were evaluated in mice inoculated with parasite (1×104/mouse). This in vivo antimalarial effect was examined in a 5-day suppressive test (drug treatment started 48 hr before and continued until 48 hr after challenge infection). Result: As the result of in vitro assay with the library, coptisine chloride (compound, IC50=1.1 µM, SI (selective index) =37.8) and coptise rhizome (crude drug extract, IC50=5 µg/mL, SI>200) showed high antimalarial activity and its safety. Then, we examined two approved kampo formula, Sanohshashinto and Ohrengedokuto, containing coptis rhizome by the in vitro assay, and those exhibited good antimalarial activities (IC50 36 and 40 µg/mL, respectively). Moreover, even in a preliminary experiment, both Ohrengedokuto and coptis rhizome showed a suppression of parasitemia in malaria mouse model. Conclusion: High antimalarial activity with low cytotoxicity was observed from kampo formula which contain antimalarial kampo compounds and crude drug extract in vitro experiments. Furthermore, even as preliminary experiment, antimalarial effect of kampo crude drug extract and formula were observed in vivo experiment, suggesting kampo formula as a potential antimalarial drug. This study is an important input for the development of an antimalarial drug from kampo.

Application of NMR metabolomics in understanding the pjysiological target of Kampo medicine

Suresh, Awale

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Application of NMR metabolomics in understanding the pjysiological target of Kampo medicine

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