2020 Fiscal Year Annual Research Report
植物内生真菌を用いたケミカルバイオロジーによる抗トキソプラズマ薬の探索
| Project/Area Number |
20F20402
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| Research Institution | Obihiro University of Agriculture and Veterinary Medicine |
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Principal Investigator |
西川 義文 帯広畜産大学, 原虫病研究センター, 教授 (90431395)
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| Co-Investigator(Kenkyū-buntansha) |
ARIEFTA NANANG 帯広畜産大学, 原虫病研究センター, 外国人特別研究員
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| Project Period (FY) |
2020-11-13 – 2023-03-31
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| Keywords | 内生菌 / カビ / トキソプラズマ / マラリア / 細胞毒性 |
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| Outline of Annual Research Achievements |
The main goal for this first year is to collect and screen the endophytic fungi. Intensive collection, identification, and extraction of endophytic fungi are essential to depository promising fungal extracts for anti-Toxoplasma and antiplasmodial screenings. The Ainu people who once dominated Hokkaido used the local flora in their folk remedies. The plants used by the Ainu were quite different from those in the traditional Japanese pharmacopeia due to the presence of temperate eastern Asiatic elements. Several endophytes can synthesize bioactive compounds, which the plant can use to defend against pathogenic fungi and bacteria. These compounds also proved useful and represented a promising source of new bioactive compounds. Therefore, we explore Hokkaido medicinal plant-associated fungi to be screened and used against protozoan diseases, including toxoplasmosis and malaria. Sixteen plants were used as sources of the fungi isolation, resulting in the isolation of 130 endophytic fungi. The 130 methanolic extracts of these fungi were in vitro screened against Toxoplasma gondii RH-GFP and Plasmodium falciparum 3D7. From the screening processes of these 130 fungi, we find that 6 fungi exhibit inhibition against both parasites, 2 fungi inhibit specific to T. gondii RH-GFP, and 9 fungi inhibit specific to P. falciparum 3D7.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
The screening processes showed that extracts derived from fungal endophytes have in vitro anti-Toxoplasma and antiplasmodial activities. Of 130 isolated fungi, 6 (4.6%) were active against both parasites, 2 (1.5%) were active specific against T. gondii RH-GFP, and 9 (6.9%) were active specific against P. falciparum 3D7. These total of 17 fungi are potential starting points in the exploration of active compounds through the isolation process. Bioactivity-guided isolation (against T. gondii RH-GFP and P. falciparum 3D7) is an essential primary screening method for active compounds and needs more extensive study.
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| Strategy for Future Research Activity |
The potent fungi from the screening processes will be applied to the isolation guided by anti-Toxoplasma and antimalaria activities (in vitro). These research steps are intended to confirm and trace the active agent(s) responsible for the anti-Toxoplasma and antimalaria activities. Firstly, each moldy media from scale-up fermentation (600 grams rice) will be extracted using methanol. The dried methanol extract will be suspended in distilled water and partitioned with hexane, chloroform, and ethyl acetate. Each fraction will be evaluated for anti-Toxoplasma activity and antimalaria. The active fraction(s) will be applied to various chromatography methods for further separations. Every step of column chromatography will be guided by anti-Toxoplasma and antimalarial activities, and only the active fractions will be further separated. LC-MS/MS profiling and molecular networking analyses for active fractions will be conducted to generate the compound's map, trace, and visualize the active components' cluster. The structures of isolated active compounds will be determined by spectroscopic methods, including nuclear magnetic resonance (NMR), high-resolution mass spectroscopy (HR-MS), and chemical derivatization. Anti-Toxoplasma and antimalaria effects on growth and cytotoxicity of isolated active compounds will also be evaluated in vitro. The efficacy of the most promising bioactive compounds will be evaluated using the mouse model. Furthermore, the potent fungi will be identified based on the ITS rDNA region.
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