在多诺瓦尼利什曼尼亚,内脏利什曼病的致病原虫,核苷水解酶(NH)是其DNA和RNA生物合成的基础。因此,LdNH被认为是发展新的利什曼病化疗的潜在目标。辣木是一种原产于印度东北部的药用植物,具有许多药理特性,包括抗利什曼的活动。因此,本研究旨在探讨油茶叶和花不同提取物对LdNH的抑制活性。使用共价固定在磁性颗粒上的LdNH(LdNH-MPs),基于通过HPLC-DAD对形成的产物的直接定量,开发了一种新的活性测定。本研究采用不同的提取方法从油茶叶和花中筛选出12种提取物。从花中提取的乙醇(70%乙醇),通过输注(FIEH)或超声辅助提取(FUEH)获得,IC50值分别为26.2±4.63µg/mL和4.96±0.52µg/mL。通过高分辨率LdNH抑制谱研究了最有前途的提取物(FUEH),在生物色谱图中显示了不同的抑制区域。尝试进行配体捕捞测定以查明生物活性化合物。配体打捞测定的洗脱步骤中采用的实验条件没有导致配体分离。然而,与活性和非活性LdNH-MP孵育后的粗提物溶液和上清液的分析显示缺失峰,涉及选择性保留在活性LdNH-MP孵育中的化合物。缺失的峰在高分辨率LdNH抑制谱中表现出抑制的相同区域中洗脱。通过UHPLC-MS/MS鉴定配体为帕拉金糖,腺苷,3-对-香豆酰基奎尼酸,4-对-香豆酰基奎尼酸,金丝桃苷,槲皮素-3-O-丙二酰糖苷,和山奈酚-3-O-半乳糖苷.
In Leishmania donovani, the causative protozoan of visceral leishmaniasis, nucleoside hydrolase enzyme (NH) is fundamental for the biosynthesis of its DNA and RNA. Therefore, LdNH is considered a potential target for the development of new leishmaniasis chemotherapy. Moringa oleifera Lamarck is a medicinal plant native to northeastern India with numerous pharmacological properties, including antileishmanial activity. Thus, this study aimed to explore the inhibitory activity of different extracts from M. oleifera leaves and flowers on LdNH. Using LdNH covalently immobilized on magnetic particles (LdNH-MPs), a novel activity assay was developed based on the direct quantification of the formed product by HPLC-DAD. This study screened 12 extracts from leaves and flowers of M. oleifera using different extraction methods. The hydroethanolic (70% ethanol) extract from flowers, obtained by infusion (FIEH) or ultrasound-assisted extraction (FUEH), exhibited respectively IC50 values of 26.2 ± 4.63 µg/mL and 4.96 ± 0.52 µg/mL. The most promising extract (FUEH) was investigated by high-resolution LdNH inhibition profiling, which showed different regions of inhibition in the biochromatogram. A ligand fishing assay was attempted to pinpoint the bioactive compounds. Experimental conditions employed in the elution step of the ligand fishing assay did not result in ligands isolation. However, the analyses of the crude extract solution and the supernatants after the incubation with the active and inactive LdNH-MPs indicated missing peaks referring to compounds selectively retained in the active LdNH-MPs incubation. The missing peaks eluted in the same region that exhibits inhibition in the high-resolution LdNH inhibition profiling. The ligands were identified by UHPLC-MS/MS as palatinose, adenosine, 3-p-coumaroylquinic acid, 4-p-coumaroylquinic acid, hyperoside, quercetin-3-O-malonyl glycoside, and kaempferol-3-O-galactoside.