Mechanisms and active compounds in the cognitive effects of Centella asiatica

描述（申请人提供）：积雪草（L.）城市草药（CA），是一种中草药-rasayana（神经恢复）草药从阿育吠陀传统，用作记忆增强剂和神经滋补。对人类和许多啮齿动物模型的研究支持CA改善记忆和学习的能力。在我们的初步研究中，CA水提取物（CAW）可以逆转老年Tg2576小鼠的学习缺陷，Tg2576是一种具有高β淀粉样蛋白（a β）斑块负担的阿尔茨海默病（AD）模型。这并不伴随着大脑Aβ水平的变化，这表明CAW减轻了Aβ的毒性作用，而不是改变其形成或沉积。这一结论得到了MC65人神经母细胞瘤细胞系数据的支持，其中CAW阻止了细胞内Aβ积累引起的细胞死亡，同样没有改变Aβ水平。产生这种效果的活性化合物似乎不是CA中众所周知的三萜（亚洲酸、马来酸及其苷），因为我们在Tg2576小鼠和MC65细胞模型中观察到强大的活性，尽管CAW中缺乏这些化合物。在MC65细胞中测试时，三萜化合物也没有活性。相反，CAW富含包括咖啡酰奎宁酸在内的酚类化合物，这表明这些酚类化合物具有重要的治疗作用，但对CA成分的研究并不充分。尽管有明确的证据表明CA具有增强认知的作用，但对CA的活性成分、它们的生化作用机制以及改善认知所需的有效剂量还没有系统的研究。本研究的目的是详细探讨CAW保护神经元免受Aβ毒性的机制，确定关键活性化合物，并确定这些物质的治疗血浆水平。特异性目的1是确定CAW在MC65细胞中减弱Aβ毒性的机制，使用靶向和无偏倚方法。目标机制有待研究的影响是抗氧化状态，钙稳态和线粒体功能。无偏倚方法将利用代谢组学技术鉴定Aβ诱导的变化，并探索CAW对这些变化的衰减。特异性目的2是在体外培养MC65细胞和原代皮质神经元中分离鉴定CAW中抑制Aβ神经毒性的活性化合物，并探讨这些化合物的协同作用及其机制。在特异性目标3中，Tg2576小鼠模型和野生型对照将用于验证Aims 1和2中体外鉴定的CAW的体内机制和活性化合物。这些化合物的治疗血浆水平将通过CAW的剂量反应研究来确定。在这项研究中要探索的机制与年龄相关的神经元健康和认知能力下降有关，而不限于与Aβ毒性相关的机制。这项研究的成功结果将支持标准化CA产品的开发，该产品含有适当水平的活性化合物，具有明确的作用机制和目标治疗血浆水平，所有这些都将直接导致CA在与年龄相关的认知能力下降的人类中的临床试验。

DESCRIPTION (provided by applicant): Centella asiatica (L.) Urban herb (CA), is a medhya-rasayana (nerve rejuvenating) herb from the Ayurvedic tradition, used as a memory enhancer and nerve tonic. Studies in humans, and numerous rodent models, support CA's ability to improve memory and learning. In our preliminary studies, CA water extract (CAW), reversed learning deficits in aged Tg2576 mice, a model of Alzheimer's disease (AD) with high beta amyloid (Aβ) plaque burden. This was not accompanied by changes in brain Aβ levels, suggesting that CAW attenuates the toxic effects of Aβ rather than altering its formation or deposition. This conclusion is supported by data from the MC65 human neuroblastoma cell line, where CAW prevented cell death caused by intracellular Aβ accumulation, again without altering Aβ levels. The active compounds responsible for this effect appear to be other than CA's well known triterpenes (asiatic acid, madecassic acid and their glycosides), since we observed robust activity in both the Tg2576 mouse and MC65 cell models despite the absence of these compounds in CAW. The triterpene compounds were also inactive when tested in MC65 cells. CAW is, instead, rich in phenolic compounds including caffeoylquinic acids, suggesting an important therapeutic role for these, and inadequately studied components of CA. Despite clear evidence of a cognitive enhancing effect of CA, there has been no systematic investigation of CA's active components, their biochemical mechanism(s) of action, and effective doses required for cognitive improvement. The goal of the present study is to explore in detail, the mechanisms by which CAW protects neurons from Aβ toxicity, identify the critical active compounds, and determine therapeutic plasma levels of these substances. Specific Aim 1 is to determine the mechanism(s) by which CAW attenuates Aβ toxicity in MC65 cells, using both targeted and unbiased approaches. Target mechanisms to be investigated are effects on anti-oxidant status, calcium homeostasis and mitochondrial function. The unbiased approach will utilize metabolomics technology to identify changes induced by Aβ, and explore attenuation of these changes by CAW. Specific Aim 2 is to isolate and identify active compounds in CAW responsible for attenuation of Aβ neurotoxicity in MC65 cells and primary cortical neurons in vitro, and to explore synergistic interactions and mechanisms of these compounds. In Specific Aim 3 the Tg2576 mouse model and wild type controls will be used to validate in vivo, the mechanisms and active compounds of CAW, identified, in vitro, in Aims 1 and 2. The therapeutic plasma levels of these compounds will be determined through a dose response study of CAW. The mechanisms to be explored in this study are relevant to age- related decline in neuronal health and cognition in general, and are not limited to those associated with Aβ toxicity. A successful outcome to this study will support the development of a standardized CA product containing appropriate levels of active compounds, with defined mechanisms of action, and target therapeutic plasma levels, all of which will lead directly to clinical trials of CA in humans with age-related cognitive decline.