基于褪黑素受体的传统中药钩藤中镇静催眠活性物质的发现及构效关系研究

Insomnia is a symptom characteristic by difficulty initiating and maintaining sleep and associated with daytime consequences. Many factors can cause insomnia which is typically secondary to medical, psychiatric, circadian, or sleep disorders, and can also be a primary disorder. Sedative and hypnotic medicines are the main therapeutic means for insomnia in clinical, however their efficacy is unsatisfied due to the obvious side effects including dependence and withdrawal syndrome. Increasing clinical evidences suggest that melatonin receptor (MT1 and MT2) agonists represent a novel therapeutic approach for the treatment of sleep disturbances. However, the currently reported melatonin receptor agonists are exclusively synthetic compounds which show highly structural similarity with melatonin. Therefore, naturally derived melatonin receptor agonists with diverse skeletons are urgently needed. Gou-Teng (Uncaria rhynchophylla) is a famous traditional Chinese herb used for the treatment of convulsion, hypertension, epilepsy, eclampsia, and cerebral diseases. The main chemical constituents of Gou-Teng are indole alkaloids, flavonoids, triterpenoids and organic acids, of which the indole alkaloids as the characteristic constituents are correspondent to the hypotensive effect. In contrast, the sedative and hypnotic constituents of Gou-Teng are still disputed. Our previous investigation manifested that the total extract of U. rhynchophylla showed agonistic effect on MT1 receptor with an agonistic rate of 100.48 % (400 μg/mL). In order to clarify the active constituents, the subsequent investigation resulted in five flavanols, three triterpenoids, and two steroids from A-1-3, and eight indole alkaloids from A-2. The following bioassay on HEK-293 cell line in vitro indicated that the flavanols showed high agonistic effect on MT1 receptor, of which catechin (1) and 3-O-methylcatechin (3) exhibited the highest agonistic rates with the EC50 values of 12.90 and 29.40 μM. Preliminary structure-activity relationship (SAR) investigation suggested that the 2,3-cis form was prior to the trans-form. This is the first time to characterize the agonistic effects of Gou-Teng and the correspondent active constituents. Therefore, further investigation on Gou-Teng will be very significant for searching natural melatonin receptor agonists. In this project, systematically bioassay and LCMS guided isolation, chemical modification and SAR investigation will be conducted on the active constituents of Gou-Teng, by which the high active and selective leading compounds (MT1/MT2 agonists) will be further evaluated for the sedative and hypnotic efficacy in vivo. The successful implementation of this project (if granted) will provide scientific basis for developing natural melatonin receptor agonists as new sedative and hypnotic leading compounds.

失眠是现代社会常见病，现有药物有一定疗效但副作用明显，不能满足临床需要。褪黑素(MT)受体激动剂是新型镇静催眠药物研究热点，可避免苯二氮卓类药物的依赖性和戒断症状。已有MT受体激动剂均为合成化合物，结构类似，亟需开发结构类型多样的天然来源激动剂。中药钩藤镇静催眠疗效确切，当前研究主要针对降压活性和生物碱成分，未见其关于MT受体活性报道。我们首次发现钩藤提取物具MT1受体激动活性，并从一活性部位分离得到系列活性黄烷醇，特别是儿茶素和3-O-甲基儿茶素对MT1受体的激动率为287.97%和157.35%，且剂量依赖性好，EC50分别为12.9和29.4μM，值得深入研究。本研究在此基础上，通过活性跟踪和LCMS分析继续开展钩藤活性成分的导向分离和结构优化，以期发现MT1/MT2受体高活性/高选择性激动剂，进行动物体内镇静催眠药效评价，为天然来源新型镇静催眠先导化合物的发现提供化学和药理学基础。

传统中药钩藤为茜草科Rubiaceae 钩藤属Uncaria 植物钩藤U. rhynchophylla，大叶钩藤U. macrophylla，毛钩藤U. hirsuta，华钩藤U. sinensis 或无柄果钩藤U. sessilifructus 的干燥带钩茎枝。为了揭示中药钩藤的改善睡眠及抗抑郁等活性成分，在LCMS的导向下，利用稳转MT1、MT2、5-HT1A和5-HT2C受体的体外筛选模型，从钩藤活性部位分离鉴定75个化合物，结构类型包括吲哚生物碱、三萜、黄酮、有机酸等，发现10个新化合物，20个化合物具有MT1和MT2受体激动活性。合成儿茶素、吲哚类等各类衍生物120个，发现高活性化合物30个。采用协同戊巴比妥钠诱导小鼠睡眠试验（睡眠潜伏期和睡眠时间）、协同阈下剂量戊巴比妥钠诱导小鼠睡眠试验、小鼠悬尾（TST）、小鼠强迫游泳（FST）、小鼠自主活动（OFT）等动物模型，开展了4个单体化合物的动物体内药效评价，发现2个高活性化合物具有显著的改善睡眠和抗抑郁活性，并开展了其体内代谢产物研究。对具有成药前景的衍生物A20开展了初步成药性研究，包括合成工艺、质量标准、稳定性、初步安全性等。进一步开展了7种钩藤属植物和钩藤4个部位的化学成分和神经递质受体激动活性的比较研究。本研究首次揭示了中药钩藤的褪黑素受体激动活性成分，及活性成分的改善睡眠和抗抑郁药理活性，为基于褪黑素受体的创新天然药物研发奠定了基础。本项目发表研究论文9篇（SCI论文7篇），其中第一标注论文7篇，第二标注论文1篇，第三标注论文1篇。申请中国发明专利8项，2项已获授权（ZL201611199960.0、ZL201710091808.9）。协助培养博士研究生1名和硕士研究生2名。期间参加2次国内学术会议并作会议交流报告。项目负责人入选中科院“青年创新促进会”优秀会员（2017），云南省中青年学术和技术带头人后备人才（2017）、云南省“万人计划”青年拔尖人才（2018），获得云南省优秀青年基金资助（2019）。

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