民族药地椒对缺血性心脑血管疾病的效应物质基础和作用机制研究

Folk medicine Thymus quinquecostatus Celak has diaphroretic and wind-eliminating, moving qi to relieve pain, heat-clearing and toxin-resolving, used for anti-inflammatory and analgesic in clinical. The team for the first time from the T. quinquecostatus Celak found several flavonoids and phenolic ingredients, and these ingredients has prominent therapeutic effect of heart head blood-vessel. Early experiments showed that the ethanol part having significantly reduce pathological model mouse infarction range. Accordingly, the applicant for the first time put forward thinking to its ethanol extract has a moderating effect on ischemia cardio-cerebrovascular disease. But the effect of material basis and molecular mechanism of the activity of its extract have not been reported. The project intends to use modern chromatography and chemical analysis techniques and other modern scientific methods for preparing its ethanol extract, which is rich in flavonoids and phenolic components and parts, and analysis its chemical composition; Using two kinds of highly clinical similarity animal models to evaluate the pharmacology and pharmacodynamics of its ethanol extract on the the effect of ischemia cardio-cerebrovascular disease; In molecular biology technology from the level of cells, molecules, enzymes to research the extract and its multiple component effect on ischemic cardiovascular and cerebral vascular diseases related targets and pathways, and to determine the composition of the effect of material; And based on its medicinal substances to establish the quality control method. The project for the interpretation of the folk medicine in the treatment of ischemia cardio-cerebrovascular disease characteristics advantage and development of new drugs lay the foundation.

民族药地椒有祛风解表，行气止痛，清热解毒的功能，临床多用于消炎和镇痛。研究团队首次从地椒中发现多个黄酮和酚类成分，且这些成分拥有突出的心脑血管治疗作用。申请者前期预实验表明地椒乙醇部位能够显著降低病理模型小鼠脑梗死范围。据此，申请者首次提出地椒乙醇提取物对缺血性心脑血管疾病具有调节作用的工作思路。但地椒该活性的效应物质基础及作用机制都未见报道。该项目拟以现代色谱和化学分析技术等现代科学方法制备地椒乙醇提取物富含黄酮和酚类成分部位解析其化学成分；采用与临床相似度高的两种药理动物模型进行地椒提取物缺血性心脑血管疾病效应作用的药理及药效学评价；以分子生物学技术从细胞、分子、酶多个水平研究地椒提取物和其多个组成成分对心脑缺血性疾病作用相关靶点和通路，并确定效应物质成分；从而基于药效物质建立地椒质量控制方法。该项目对于阐释民族药物治疗缺血性心脑血管疾病的特色优势、创制新型药物奠定基础。

地椒为唇形科百里香属植物地椒的全草，广泛分布于中国北方地区，具有祛风解表，行气止痛的功效。从LPE、HPE和PRF中鉴定出43、42和34个酚类化合物，主要有黄酮类化合物和酚酸类化合物。其中，酚酸类化合物以对羟基苯甲酸、丹参素、咖啡酸和迷迭香酸等为主；黄酮类化合物以灯盏乙素、槲皮素、山奈酚、木犀草素等为主；LPE和HPE的总酚含量为168.100 mg/g和224.900 mg/g，PRF总酚含量达到549.500 mg/g。结果表明，地椒中富含酚类成分，为地椒成为一种良好的抗氧化剂提供理论依据。利用AAPH诱导的AB型斑马鱼氧化损伤模型和MTZ诱导的cy17型转基因斑马鱼氧化损伤模型对各提取部位抗氧化活性进行评价，各部位均能显著改善斑马鱼体内的氧化应激损伤，PRF有更好的抗氧化活性。地椒含有丰富酚类成分，是地椒抗氧化活性的主要来源。同时，地椒可以通过直接消除体内的过量ROS和激活Keap1/Nrf2/HO-1通路，减轻大鼠的CIRI，具有良好的抗CIRI作用。同时，结合网络药理学研究，通过异丙肾上腺素（ISO）诱导的小鼠MI模型和结扎大鼠心脏冠状动脉左前降支（LAD）45 min，再灌注24 h致大鼠MIR模型，PRF可以减轻LAD结扎致大鼠MIR损伤，减少心肌梗死面积，具有心肌保护作用，其作用机制可能与抑制内质网应激及细胞凋亡有关。此外，建立后肢缺血小鼠模型，探究地椒乙醇提取物促血管新生作用及其可能的作用机制。地椒可以上调VEGF表达，增加毛细血管密度来促进血管新生，其作用机制与与PI3K/AKT/eNOS信号通路激活有关。