Bioassay Guided Isolation of Hypoglycemic Compounds from Philippine Medicinal Pla

描述（申请人提供）：菲律宾是地球上生物多样性的热点地区之一。在6000种地方性植物中，据信传统医生和草药学家使用约1500种作为药用植物，其中只有1%的植物具有活性成分。尽管信息匮乏，但在菲律宾等发展中国家，从发展中国家进口的药物超出了大多数人的购买力，草药仍然是治疗许多疾病的主要来源。因此，本研究的长期目标是基于代谢物水平的生物测定来明确鉴定抗糖尿病植物，并提供一种基于生物测定指导实验动物反应的草药疗效量化的新方法。前者将对菲律宾提供有效和负担得起的糖尿病治疗药物产生直接影响。后者将提供一种基于事实的草药质量控制手段，目前即使在美国也并不总是可用。为了达到这些目标，我们建议筛选10+已报道具有抗糖尿病特性的本土植物。筛选将在菲律宾迪利曼大学进行。粗提取物、馏分和分离物的筛选将使用一套葡萄糖、谷氨酰胺和脂肪酸的生物传感器进行生物测定。这些生物传感器目前正在Tolosa实验室开发，项目资助为R01DK072465，用于连续监测代谢物。虽然最终的设备还没有准备好，但基于单个蛋白质的传感器可以用于检测血液中的代谢物。因此，我们建议开发一种方案来测量雄性瑞士韦伯斯特小鼠在高葡萄糖负荷下的代谢物水平。使用这种生物测定方案，我们将从已鉴定的菲律宾药用植物中筛选和鉴定两种具有最有效的降糖活性的植物，这些植物已被民间报道用于治疗糖尿病。粗提取物将被连续分离，直到从两种最有效的菲律宾药用植物中分离出降糖成分。这些纯化合物的化学结构将通过标准光谱技术来确定。这个项目的新颖之处在于，我们将多重测量葡萄糖、谷氨酰胺和脂肪酸，而不是测量单一的代谢物（葡萄糖），以观察这些代谢物在高葡萄糖负荷下的比例和趋势，然后是抗糖尿病原理。看看是否可以从这些趋势中收集到模式，进一步根据它们的活性将抗糖尿病化合物分类，这应该是很有趣的。小灵通398/2590 （Rev. 11/07）页延续格式页

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