两种植物药中抗慢性支气管炎活性成分及其作用机制研究

The morbidity and mortality of chronic bronchitis is globally very high because of thronic irritation of the conducting airways by inhaled substances, most importantly cigarette smoke, air pollution, and occupational exposures. Especially, incidence rate of chronic bronchitis has increased in the north parts of our country owing to serious air polution. There has presently been lack of medicine with significant effect and non-toxic side effect in treatment of chronic bronchitis in the market. The results of our previous research showed that the ethanol extracts of Aralia melanocarpa (Lévl.)Lauener and Illigera cordata Dunn had good activities against chronic inflammation of the bronchial tubes in experiment in vivo and in vitro. Moreover, further guided by bioactivities, the ethyl acetate extracts which were extracted from the ethanol extracts of these two plants showed significant anti-inflammatory activity in inflammatory model by LPS- stimulated murine RAW 264.7 macrophages in vitro, compared with those of petroleum ether extracts and n-butanol extracts. This project aims to: (1) guided by in vitro anti-inflammatory activity which was tested using in vitro inflammatory model by LPS-stimulated murine RAW 264.7 macrophages, isolate bioactive chemical constituents from the ethyl acetate extracts of Aralia melanocarpa (Lévl.) Lauener and Illigera cordata Dunn using modern separation technology, including HPLC, sephadex LH-20 column chromatography and so on; (2) elucidate the structure of the natural products isolated on the basis of the spectroscopic analysis and chemical evidence; (3) through bioassay-guided fractionation way, purify anti-inflammatory substance groups from these two plants by chromatography on silica gel and resin, further isolate chemical composition of active substance groups by chromatography on Rp-18, Sephadex LH-20, HPLC, et al, and elucidate the sutrctures of the isolates based on MS, 1D and 2D NMR spectroscopic data; (4) synthesize natural products with significant anti-inflammatory activities and their derivatives, evaluate their anti-inflammatory activities, study their structure-activity relationship, then design and synthesize the targeted derivatives with stronger anti-inflammatory activity. (5) further confirm the therapeutic effect of active substance groups and main natural products from these two plants on chronic bronchitis induced by lipopolysaccharide in rats, thus discover their active constituents and explore possible mechanism of the bioactive compounds, which will lay the foundation for developing novel medicine for treating chronic bronchitis. The results of this research can be expected to has directive functions of these two plants on clinic and promote development and utilization of these two plant resources in Yunnan province.

慢性支气管炎发病率和死亡率非常高，但是目前市面上缺乏疗效好、副作用小的慢性支气管炎治疗药。本课题组前期研究发现，丛枝葱木和心叶青藤两种植物药具有明显的抗慢性支气管炎活性。在脂多糖(LPS)至小鼠腹腔巨噬细胞RAW 264.7体外炎症模型指导下，发现两种植物药中抗慢性支气管炎的活性组分主要集中在乙醇提取物的醋酸乙酯萃取部位。本项目将通过体外活性跟踪，借助各种色谱分离技术和结构鉴定方法，从已锁定的活性部位和组分中，明确抗慢性支气管炎的活性成分；对活性成分进行合成或结构修饰，比较、分析其结构与活性之间的关联，探讨其构效关系；采用脂多糖致慢性支气管炎大鼠模型，验证前期体外模型筛选出的活性优良的物质群和单体成分的体内抗炎作用，并初步探讨其作用机理，为将其研发为新型慢性支气管炎药物提供前期研究基础。该研究不仅对两种植物药在治疗慢性支气管炎的临床应用有指导作用，还有利于推动该两种药物资源的深入开发利用。

慢性支气管炎发病率和死亡率非常高，尤其是我国北方空气污染严重，雾霾天气增多，发病率比以往显著增加。目前市面上缺乏疗效好、副作用小的慢性支气管炎治疗药。本项目以脂多糖诱导小鼠单核巨噬细胞RAW264.7炎症模型为指导，采用现代分离技术，分离、鉴定心叶青藤和丛枝楤木2种彝族植物药提取物中的活性成分，阐明其抗慢性支气管炎活性物质基础；对活性化合物进行合成或结构修饰，分析其结构与活性之间的关联，探讨构效关系；对优选的活性化合物开展体内动物实验，并初步探讨其作用机理。通过该项目的实施，从心叶青藤和丛枝楤木2种植物药提取物的活性部位中分离、鉴定了129个化合物，含新化合物37个，新颖结构化合物7个；确定了心叶青藤中新颖结构二萜类化合物的立体构型，为该类化合物的化学性质及相关生物活性研究奠定了基础；完成了所分离化合物的活性测试，阐释了2种植物药中的主要抗慢性支气管炎活性物质基础。研究结果表明心叶青藤中的主要抗慢性支气管炎成分为单萜、二萜及生物碱类化合物，丛枝楤木中的主要抗慢性支气管炎成分为贝壳杉烷型二萜和黄酮类化合物；研究了心叶青藤中的单萜类化合物及丛枝楤木中的二萜类化合物的构效关系，明确了两类化合物中的效用基团；对丛枝楤木中的活性黄酮类化合物的合成工艺进行了探索与优化，解决了该类化合物的来源问题；通过构效关系研究发现5,7,4'-三甲氧基二氢黄酮和5,7,3'-三甲氧基二氢黄酮的活性较为突出，其对LPS诱导RAW264.7细胞产生NO的半数抑制浓度分别为5.3 ± 0.4和3.4 ± 0.8 μg/mL，具有良好的应用前景和深入研究的价值；对心叶青藤中的单萜、二萜及生物碱类化合物和丛枝楤木中的贝壳杉烷型二萜开展了初步的作用机理探索。本项目的研究结果不仅有利于2种彝药在临床上的广泛应用，而且为其代谢产物及相关衍生物在抗慢性支气管炎方面的深入开发应用提供了数据支持。