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

筛选过程中产生的强效真菌将用于体外抗弓形虫和抗疟疾活性指导下的分离。这些研究步骤旨在确认和追踪负责抗弓形虫和抗疟疾活性的活性剂。首先，每个发霉培养基的规模发酵（600克大米）将使用甲醇提取。将干燥的甲醇提取物悬浮在蒸馏水中，用己烷、氯仿和乙酸乙酯进行分区。将对每个部分的抗弓形虫活性和抗疟疾活性进行评估。活性组分将应用于各种色谱法进行进一步分离。柱层析的每一步都以抗弓形虫和抗疟活性为指导，只进一步分离活性组分。将对活性组分进行LC-MS/MS分析和分子网络分析，以生成化合物的图谱，跟踪和可视化活性组分簇。分离活性化合物的结构将通过光谱方法确定，包括核磁共振（NMR）、高分辨率质谱（HR-MS）和化学衍生化。抗弓形虫和抗疟疾对分离活性化合物生长和细胞毒性的影响也将在体外进行评估。最有希望的生物活性化合物的功效将通过小鼠模型进行评估。此外，还将根据ITS rDNA区域鉴定强效真菌。

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.