Bioassay Guided Isolation of Hypoglycemic Compounds from Philippine Medicinal Pla

描述（由申请人提供）：菲律宾是地球上生物多样性的热点之一。在6，000种地方植物中，据信传统医生和草药医生使用约1，500种作为药用植物，其中只有1%的活性成分得到了鉴定。尽管缺乏这方面的信息，但在菲律宾等发展中国家，从发展中世界进口的药物超出了大多数人的购买力，草药仍然是许多疾病的主要治疗来源。因此，本研究的长期目标是基于代谢物水平的生物测定来确定抗糖尿病的植物，以及提供基于生物测定指导的实验动物反应来量化草药功效的新方法。前者将对菲律宾提供有效和负担得起的糖尿病补救措施产生直接影响。后者将提供一种基于事实的草药质量控制手段，目前即使在美国也不总是可用。为了达到这些目标，我们建议筛选10+土著植物，据报道，具有抗糖尿病的特性。筛选将在菲律宾大学Diliman进行。粗提物、馏分和分离物的筛选将使用一组葡萄糖、谷氨酰胺和脂肪酸生物传感器进行生物测定。这些生物传感器目前正在Tolosa实验室根据项目资助R 01 DK 072465开发，用于连续代谢物监测设备。虽然最终的设备还没有准备好，但基于蛋白质的单个传感器可以用于检测血液中的代谢物。因此，我们建议开发用于测量响应于高葡萄糖负荷的雄性瑞士韦伯斯特小鼠中的代谢物水平的方案。使用该生物测定方案，我们将筛选和鉴定两种植物具有最有效的降血糖活性，从已被民间报道用于治疗糖尿病的菲律宾药用植物中。粗提物将连续分馏，直到降血糖成分从两种最有效的菲律宾药用植物中分离出来。这些纯化合物的化学结构将通过标准光谱技术确定。该项目的新奇在于，我们将多路复用葡萄糖、谷氨酰胺和脂肪酸，而不是测量单一代谢物（葡萄糖），以观察这些代谢物对高葡萄糖负荷的响应比例和趋势，然后是抗糖尿病原则。这应该是有趣的，看看是否可以从这些趋势中收集模式，以进一步将抗糖尿病化合物根据其活性分类。PHS 398/2590（Rev. 11/07） 公共卫生相关性：菲律宾群岛的地理位置使其成为地球上少数几个拥有丰富多样的陆地和海洋物种的地方之一，其中许多物种尚未被发现或描述。正因为如此，菲律宾人有很长的历史，使用许多天然产品用于各种用途，包括药用目的。由于从发达国家进口的药品越来越昂贵，超出了大多数菲律宾人的购买能力，因此必须根据事实确定现有草药的功效。在这里，我们建议筛选几种菲律宾本土植物用于菲律宾传统医学治疗糖尿病及其症状。这些植物将根据其降低血糖水平和影响其他两种代谢物谷氨酰胺和脂肪酸的能力进行筛选。在项目结束时，我们希望分离，纯化和鉴定两种最活跃的植物物种中的活性成分。这项研究的直接影响是确定了菲律宾人在预防或维持糖尿病护理方面可以随时利用的植物。从长远来看，这些植物活性成分的化学结构鉴定将为进一步的药物开发提供先导化合物。PHS 398/2590（Rev. 11/07）

DESCRIPTION (provided by applicant): The Philippines is one of the planet's biodiversity hot spots. Of 6,000 endemic plants, it is believed that traditional doctors and herbalists use about 1,500 as medicinal plants of which only 1% has been characterized for their active components. Despite this paucity of information, in developing countries such as the Philippines where drugs imported from the developing world are beyond the purchasing power of the majority, herbal medicines remain as the main source of remedies for many ailments. Thus, the long-term objectives of this study is to definitively identify plants that are anti-diabetic based on bioassays for metabolite levels, as well as provide a novel method of quantifying efficacy of herbal medicines based on bioassay guided responses of experimental animals. The former will have an immediate impact in the availability of effective and affordable remedies for diabetes in the Philippines. The latter will provide a means of fact-based quality control of herbal medicines that at present is not always available even in the US. In order to arrive at these objectives, we propose to screen 10+ indigenous plants that have been reported to possess anti-diabetic properties. Screening will be executed at the University of the Philippines Diliman. The screening of crude extracts, fractions and isolates will be bioassay directed using a set of biosensors for glucose, glutamine and fatty acids. These biosensors are currently being developed in the Tolosa lab under project grant R01DK072465 for a device for continuous metabolite monitoring. Although the final device is not yet ready, the individual protein- based sensors can be used in an assay format to test for metabolites in blood. Thus, we propose to develop the protocols for measuring metabolite levels in Male Swiss Webster mice in response to a high glucose load. Using this bioassay protocol, we will screen and identify two plants with the most potent hypoglycemic activity from identified Philippine medicinal plants that have been folkloric ally reported for the treatment of diabetes. Crude extracts will be successively fractionated until the hypoglycemic constituents(s) are isolated from the two most potent Philippine medicinal plants. The chemical structures of these pure compounds will be determined by standard spectroscopic techniques. The novelty of this project is that rather than measuring a single metabolite (glucose), we will multiplex glucose, glutamine and fatty acids to see ratios and trends of these metabolites in response to a high glucose load, and then to the anti-diabetic principle(s). It should be interesting to see if patterns can be gleaned from these trends to further classify anti-diabetic compounds into groups based on their activity. PHS 398/2590 (Rev. 11/07) Page Continuation Format Page PUBLIC HEALTH RELEVANCE: The geography of the Philippine archipelago makes it one of few places on earth with a rich diversity of terrestrial and marine species, many of which have yet to be discovered or characterized. Because of this, Filipinos have a very long history of employing many natural products for various uses including for medicinal purposes. As pharmaceuticals imported from developed countries become more expensive and beyond the purchasing capacity of most Filipinos, fact-based determination of efficacy of readily available herbal medicines is an imperative. Here we propose to screen several Philippine indigenous plants used in traditional Filipino medicine for the treatment of diabetes and its symptoms. These plants will be screened based on their capacity to lower blood glucose levels and affect two other metabolites, glutamine and fatty acids. At the end of the project, we hope to isolate, purify and identify the active principle(s) in the two most active plant species. An immediate impact of this study is the definitive identification of plants that can be readily availed by Filipinos in preventative or maintenance care of diabetes. On the long term, identification of the chemical structures of the active components of these plants will provide lead compounds for further drug development. PHS 398/2590 (Rev. 11/07) Page Continuation Format Page