Bioassay-Guided Fractionation of Extracts from Codiaeum variegatum against Entamoeba histolytica Discovers Compounds That Modify Expression of Ceramide Biosynthesis Related Genes

Leaves of Codiaeum variegatum (“garden croton”) are used against bloody diarrhoea by local populations in Cameroon. This study aims to search for the active components from C. variegatum against Entamoeba histolytica, and thereby initiate the study of their mechanism of action. A bioassay-guided screening of the aqueous extracts from C. variegatum leaves and various fractions was carried out against trophozoites of E. histolytica axenic culture. We found that the anti-amoebic activity of extracts changed with respect to the collection criteria of leaves. Thereby, optimal conditions were defined for leaves' collection to maximise the anti-amoebic activity of the extracts. A fractionation process was performed, and we identified several sub-fractions (or isolated compounds) with significantly higher anti-amoebic activity compared to the unfractionated aqueous extract. Anti-amoebic activity of the most potent fraction was confirmed with the morphological characteristics of induced death in trophozoites, including cell rounding and lysis. Differential gene expression analysis using high-throughput RNA sequencing implies the potential mechanism of its anti-amoebic activity by targeting ceramide, a bioactive lipid involved in disturbance of biochemical processes within the cell membrane including differentiation, proliferation, cell growth arrest and apoptosis. Regulation of ceramide biosynthesis pathway as a target for anti-amoebic compounds is a novel finding which could be an alternative for drug development against E. histolytica.喀麦隆当地居民使用变叶木（Codiaeum variegatum，俗称花园变叶木（garden croton））的叶片治疗血性腹泻。本研究旨在从变叶木中筛选抗溶组织内阿米巴（Entamoeba histolytica）的活性成分，并由此探究其作用机制。我们采用生物活性导向的筛选方法，对变叶木叶片的水提取物及其不同组分开展了针对无菌培养的溶组织内阿米巴滋养体的活性检测。研究发现，提取物的抗阿米巴活性会随叶片采集标准的不同发生变化，据此确定了可最大化提取物抗阿米巴活性的最优叶片采集条件。通过分级分离流程，我们获得了多个次级组分（或单体化合物），其抗阿米巴活性显著高于未分级的粗水提取物。通过观察滋养体的诱导死亡形态特征（包括细胞圆缩、裂解），验证了活性最强组分的抗阿米巴活性。利用高通量RNA测序开展差异基因表达分析后发现，该活性成分的抗阿米巴机制可能靶向神经酰胺——一种参与干扰细胞膜内生化过程（包括细胞分化、增殖、生长阻滞及凋亡）的生物活性脂质。将神经酰胺生物合成通路作为抗阿米巴化合物的作用靶点，这一全新发现可为抗溶组织内阿米巴药物开发提供新的替代方向。