Pharmacognosy, Department of Medicinal Chemistry, Uppsala University, Biomedical Centre, SE-751 23, Uppsala, Sweden; Department of Chemistry, Faculty of Science, University of Colombo, Thurston Rd, Colombo 03, Sri Lanka.
Pharmacognosy, Department of Medicinal Chemistry, Uppsala University, Biomedical Centre, SE-751 23, Uppsala, Sweden.
J Ethnopharmacol. 2020 Jan 10;246:112158. doi: 10.1016/j.jep.2019.112158. Epub 2019 Aug 14.
Sri Lanka is known to have very diverse flora. Many of these species are used for plant-based remedies, which form the integral part of two Sri Lankan systems of traditional medicine, Ayurveda and Deshiya Chikitsa. Despite their widespread use, only a limited number of studies have probed into the scientific evidence for bioactivity of these medicinal plants. Such studies rarely progress to the identification of bioactive natural products.
The primary aim was to develop a bioactivity screening method and apply it to 50 Sri Lankan medicinal plants where antimicrobial properties could be relevant for its traditional use. The subsequent aim was the progression into defining and characterising potent isolates within targeted compound classes from such plants, i.e. Derris scandens and its antimicrobial flavonoids.
The plant collection comprised 24 species of Fabaceae, 15 Rubiaceae, 7 Solanaceae and 4 Cucurbitaceae plants. These 50 species were collected based on their ethnopharmacological importance and use in Sri Lankan traditional medicine. Crude extracts from each species were initially subjected to radial disc diffusion and microdilution assays. Subsequently, aqueous extracts of all plants were microfractionated in deep well plates using reversed-phase HPLC. Fractions were tested for antibacterial and cytotoxic activities and masses of target bioactive compounds were identified using mass spectrometry. Bioactive compounds with the masses identified through microfractions were isolated from Derris scandens using reversed-phase HPLC. The isolated pure compounds were characterised using LC-MS and NMR.
Crude aqueous extracts from 19 species showed activity against Gram-positive bacteria (Staphylococcus aureus and Bacillus cereus) in the radial disc diffusion assay. Crude aqueous extracts from 34 plant species and organic extracts from 46 plant species were active against S. aureus (≤4 mg mL) in the microdilution assay. Microfractionation demonstrated antibacterial activity for 19 plants and cytotoxicity for 6 plants. Furthermore, target bioactive compounds and their molecular ions were identified during microfractionation. Dalpanitin and vicenin-3, two of the flavonoids isolated from Derris scandens gave MICs of 23 μg mL against S. aureus. Dalpanitin also exhibited relevant MICs on Gram-negative bacteria (94 μg mL against Escherichia coli and Pseudomonas aeruginosa).
The microfractionation protocol developed in this study enabled time-efficient screening of many plants species, using a small quantity of sample material. In addition, microfractionation served as a guiding tool for identifying individual antimicrobial compounds. Through this process, flavonoids were isolated from Derris scandens, out of which dalpanitin and vicenin-3 showed activity in the low micromolar range. The high hit rate for in vitro antibacterial properties from this ethnopharmacologically guided sample collection gives credence to Sri Lankan traditional herbal medicine as a source for drug discovery.
斯里兰卡拥有非常多样化的植物群。其中许多物种被用于植物性药物，这些药物构成了斯里兰卡两种传统医学体系——阿育吠陀和 Deshiya Chikitsa 的重要组成部分。尽管这些药物被广泛使用，但只有少数研究探讨了这些药用植物的生物活性的科学证据。这些研究很少能进展到鉴定生物活性天然产物。
主要目的是开发一种生物活性筛选方法，并将其应用于 50 种斯里兰卡药用植物中，这些植物的抗菌特性可能与其传统用途有关。随后的目标是在靶向化合物类别中从这些植物（即鸡血藤和其抗菌类黄酮）中确定和表征有效分离物。
植物采集包括 24 种豆科、15 种茜草科、7 种茄科和 4 种葫芦科植物。这些 50 种植物是根据其在民族药理学中的重要性和在斯里兰卡传统医学中的用途而采集的。从每种植物中提取粗提取物，首先进行放射圆盘扩散和微量稀释测定。随后，使用反相高效液相色谱法（HPLC）在深孔板中对所有植物的水提取物进行微分级。对各馏分进行抗细菌和细胞毒性活性测试，并通过质谱鉴定目标生物活性化合物的质量。使用反相高效液相色谱法从鸡血藤中分离出具有通过微分级鉴定出的质量的生物活性化合物。使用 LC-MS 和 NMR 对分离出的纯化合物进行表征。
19 种植物的粗水提物在放射圆盘扩散试验中对革兰氏阳性菌（金黄色葡萄球菌和蜡样芽孢杆菌）显示出活性。在微量稀释试验中，34 种植物的粗水提取物和 46 种植物的有机提取物对金黄色葡萄球菌均有活性（≤4mg/mL）。微分级显示 19 种植物具有抗菌活性，6 种植物具有细胞毒性。此外，在微分级过程中鉴定出了目标生物活性化合物及其分子离子。从鸡血藤中分离出的两种类黄酮，dalpanitin 和 vicenin-3，对金黄色葡萄球菌的 MIC 分别为 23μg/mL。Dalpanitin 对革兰氏阴性菌（大肠杆菌和铜绿假单胞菌的 MIC 分别为 94μg/mL）也表现出相关的 MIC。
本研究中开发的微分级方案使我们能够使用少量样品材料，对许多植物物种进行高效快速的筛选。此外，微分级还可以作为一种指导工具，用于识别单个抗菌化合物。通过这一过程，从鸡血藤中分离出了类黄酮，其中 dalpanitin 和 vicenin-3 在低微摩尔范围内表现出活性。从这种基于民族药理学的样本采集中获得的体外抗菌特性高命中率为斯里兰卡传统草药作为药物发现的来源提供了依据。
