赤芍和牡丹皮中褪黑素受体天然/类天然激动剂的发现及抗抑郁药效评价

Melatonin (MT) receptors mainly including MT1 and MT2 subtypes are the action sites of the endogenous hormone, melatonin. MT1 and MT2 receptors widely distributed in the central nervous system and peripheral tissues are promising targets for treating central nervous system diseases (e.g. depression, anxiety, sleep disorders), type 2 diabetes mellitus, tumors and pains. With the MT1 and MT2 receptors being cloned in the mid-1990s, hundreds of ligands were synthesized and evaluated for agonistic activities. Currently, several MT receptor-mediated drugs, e.g., ramelteon, tasimelteon, and agomelatine have been on the market for the treatment of depression and sleep disorders. However, the currently reported melatonin receptor agonists are almost synthetic compounds which show highly structural similarity with melatonin. Therefore, naturally derived melatonin receptor agonists with diverse skeletons are urgently needed. Traditional Chinese herbs have been used for medicinal purposes in China for thousands of years, which are fascinating sources for drug discovery. In our previous investigation, the total extracts of Chi-Shao (Paeonia veitchii) and Mu-Dan-Pi (P. suffruticosa) were revealed with agonistic activities on MT1 and MT2 receptors. Subsequent bioassay- and LCMS-guided investigation resulted in two novel norditerpenes, (±)-paeoveitol and a series of pinane-type monoterpenes from the active parts of P. veitchii. In particular, albiflorin showed potent activities on both MT1 and MT2 receptors, with agonistic rates of 120.5 and 89.2% at the concentration of 1mM, according to the assay on HEK293 cell line in vitro. The following dose-effect studies provided the EC50 values of 75.8 and 116.9 μM for MT1 and MT2 receptors, respectively. The preliminary structure–activity relationships study indicated the importance of the lactone unit. In this project, systematically bioassay and LCMS guided isolation will be applied to reveal the active constituents of Chi-Shao and Mu-Dan-Pi. Furthermore, diverse derivatives will be synthesized by chemical modification, liver microsomes and intestinal microbiota to establish the drug-like libraries. The most potent compounds will be further evaluated for anti-depressant efficacy in vivo. The successful implementation of this project (if granted) will provide scientific basis for the discovery of new anti-depressant leading compounds from natural resources.

褪黑素（MT）受体主要包括MT1和MT2两种亚型，是多种疾病的潜在治疗靶点。MT受体激动剂成药前景广阔，然而当前报道的激动剂主要为人工合成化合物，结构类似，受体选择性（特别是MT1）不理想。立足我国丰富的中药资源，发现类型多样的MT受体天然激动剂具有较高的可行性。我们首次揭示赤芍和牡丹皮具有MT受体激动活性，并从活性部位分离得到一对新颖降二萜和系列活性单萜，首次发现蒎烷型单萜芍药内酯苷在1mM浓度下，对MT1和MT2受体的激动率分别为120%和89%，EC50值分别为76和117μM，提示五元内酯环是重要活性位点。本研究将在前期基础上，借助活性跟踪和LCMS分析继续开展赤芍和牡丹皮中活性成分的导向分离，综合运用有机合成、肝微粒体转化、肠道菌群代谢等手段，构建MT受体天然/类天然激动剂化合物库，对高活性化合物开展抗抑郁药效评价，为基于MT受体的新型抗抑郁先导化合物的发现奠定化学和药理学基础。

为了发现抗抑郁和降血糖活性成分，对芍药、牡丹和黄牡丹不同部位进行液质分析和活性比较，从芍药不同部位指认58个化学成分，从牡丹不同部位指认55个化学成分，从滇牡丹不同部位指认57个化学成分，结构类型主要包括单萜苷、没食子酸衍生物、黄酮、苯乙酮等，揭示了不同类型化合物的分布规律，开展了主要化合物的含量测定。活性研究表明牡丹茎对4种神经递质受体具有激动活性，在250 μg/mL时对MT2的激动率为128.7%和140.6%；根皮和根芯对MT1、MT2和5-HT1A具有较强的激动作用；叶对5-HT1A具有激动活性。所有部位对α-葡萄糖苷酶和PTP1B均具有显著抑制活性，提示具有潜在的降血糖活性。进一步从芍药、牡丹和黄牡丹中分离105个化合物，活性研究发现二苯乙烯二聚体具有显著的5-HT和MT受体激动活性，三聚体具有MT中等激动活性。特别是化合物7(0.50 mM)对MT2的激动率为91.0%，对MT1的激动率为13.2%，对5-HT1A的激动率为89.2%，EC50为0.18mM。大部分二苯乙烯对PTP1B和α-糖苷酶显示较好的抑制活性，化合物1、11和16对PTP1B的IC50为27.2、27.8和53.9 μM，对α-糖苷酶的IC50为13.6、14.4和10.8 μM，化合物5和7为酶非竞争性抑制剂，11和16为混合抑制剂。三萜类化合物(2、5、6、8-12)抑制PTP1B的IC50为47.9~192.6μM，对TCPTP无抑制作用，为PTP1B的选择性抑制剂；5个三萜(2、5、8、9、11)对GPa具有显著的抑制作用，IC50为39.8~92.4μM。对活性化合物进行结构修饰，合成衍生物110个，发现高活性分子10余个，完成3个活性分子的动物体内药效评价。发现MA葡萄糖苷对MT1和MT2受体的激动作用增强2.5-7.5倍，EC50分别为0.40和0.38 mM，动物实验表明，该化合物在高、中剂量下，可显著缩短小鼠悬尾和强迫游泳不动时间，与氟西汀(20mg/kg)药效相当，作用机制研究表明，可以通过影响小鼠脑内NE和DA水平发挥抗抑郁的作用，完成该分子的初步成药性研究。项目的实施为芍药科药用植物的综合利用和抗抑郁和降血糖活性先导分子的发现提供了科学依据。项目已发表第一标注论文5篇，另投稿1篇，申请发明专利6项，授权1项，（协助）培养硕士研究生3名。