Mechanisms and active compounds in the cognitive effects of Centella asiatica

描述(申请人提供)：积雪草Centella asiatica(L.)城市草本植物(CA)，是一种来自阿育吠陀传统的中草药，用作记忆增强剂和神经补充剂。在人类和许多啮齿动物模型上的研究支持CA改善记忆和学习的能力。在我们的初步研究中，CA水提取物可以逆转老年Tg2576小鼠的学习障碍，Tg2576是一种阿尔茨海默病(AD)高β淀粉样蛋白(Aβ)斑块负荷模型。这并没有伴随着大脑Aβ水平的变化，这表明CAW减弱了Aβ的毒性效应，而不是改变其形成或沉积。这一结论得到了来自MC65人神经母细胞瘤细胞系的数据的支持，在该细胞系中，CAW防止了细胞内Aβ积聚导致的细胞死亡，同样不改变Aβ水平。导致这种作用的活性化合物似乎不是CA众所周知的三萜(积雪草酸、马迪卡酸及其糖苷)，因为我们在Tg2576小鼠和MC65细胞模型中观察到了强劲的活性，尽管CAW中没有这些化合物。当在MC65细胞中测试时，三萜类化合物也是无效的。相反，CAW含有丰富的酚类化合物，包括咖啡酰奎宁酸，这表明对这些CA的成分有重要的治疗作用，但研究不足。尽管有明确的证据表明CA具有促进认知的作用，但对于CA的活性成分、其作用的生化机制(S)以及认知改善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A-β毒性的机制，确定关键的活性化合物，并确定这些物质的治疗血浆水平。具体目的1是通过靶向和无偏倚两种方法确定CAW减轻A-β对MC65细胞毒性的机制(S)。有待研究的靶向机制是对抗氧化状态、钙稳态和线粒体功能的影响。这种无偏见的方法将利用代谢组学技术来识别Aβ引起的变化，并探索CAW对这些变化的减弱作用。具体目的2是分离和鉴定CAW中对体外培养的MC65细胞和原代皮层神经元的Aβ神经毒性具有减弱作用的活性化合物，并探讨这些化合物之间的协同作用和机制。在具体目标3中，将利用Tg2576小鼠模型和野生型对照，在体内验证目标1和目标2中体外鉴定的CAW的作用机制和活性化合物。这些化合物的治疗血药浓度将通过CAW的剂量效应研究来确定。这项研究中要探索的机制与神经元健康和认知能力的衰老有关，并不局限于与Aβ毒性有关的机制。这项研究的成功结果将支持开发一种标准化的CA产品，该产品包含适当水平的活性化合物，具有明确的作用机制，并具有目标治疗血浆水平，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.