广西民族药当归藤补血活血作用机制与主要化学成分研究

Embelia parviflora Wall., as a medicine commonly used in Zhuang and Yao ethnic groups in Guangxi, is originated from dried aerial parts of Embelia parviflora Wall. exA. DC. from Myrsinaceae family. With efficacies of enriching the blood, regulating the menstrual function and enhancing waist and knee function, it is mainly used for the treatment of anemia, irregular menstruation, lumbar region and leg pain, and other symptoms. Preview Pharmacology research from this project group has shown that Embelia parviflora Wall.exA.DC. has anti-inflammatory, analgesic and anti-coagulation function. This study aims to extract and separate the main chemical components in portions of active efficacy of Embelia parviflora Wall. through modern methods and techniques and identify the monomer structure of isolated compounds. Methods of pharmacology of Chinese materia medica and molecular biology techniques, such as blood deficiency mice bone marrow nucleated cells value-added role, rat bone marrow cells and hematopoietic cytokine genes, red cell membrane adhesion molecules and band 3 protein tyrosine phosphorylation expression effect are used to analyze the effective mechanism of enriching blood. The indicators of plasma viscosity, hematocrit, platelet aggregation rate, LAR, CD18, ET, etc are used to explore function and mechanism of promoting blood circulation of Embelia parviflora Wall. Indicators of the content of NO, TNF alpha, SOD, macrophage of NF-ҝB induced by LPS and the influence of MAPK signal pathway are used to learn its anti-inflammatory action mechanism. This project also aim to find out active ingredients that have the function of anti-inflammatory effects of blood and promoting blood circulation.

当归藤是广西壮、瑶族民间常用药材，为紫金牛科植物小花酸藤子Embelia parvifloraWall.exA.DC.的干燥地上部分，具有补血、调经、强腰膝的功效，主治贫血、月经不调及腰腿酸痛等症。项目组前期研究表明：当归藤具有镇痛、抗炎、抗凝血作用。本项目拟通过现代分离方法和技术，对当归藤药效活性部位化学成分进行提取分离纯化，并对分离的单体成分进行结构鉴定；运用中药药理学和分子生物学技术和方法，以血虚小鼠骨髓有核细胞增值作用、大鼠骨髓细胞与造血相关细胞因子基因以及红细胞膜黏附分子与带3蛋白酪氨酸磷酸化表达作用，分析当归藤补血作用机制；对血浆粘度、血小板聚集率、CD18、ET等指标的检测以探索当归藤活血作用机制；通过测定NO、TNF-α、SOD的含量及对LPS诱导的巨噬细胞NF-ҝB和MAPK信号通路的影响等实验指标，确定当归藤抗炎作用机制；同时筛选出具有补血活血抗炎作用的活性单体。

当归藤为紫金牛科酸藤子属植物小花酸藤子（Embelia parviflora Wall.ex A.DC）的干燥地上部位，具有补血、调经、强腰膝的功效，主治贫血、月经不调、闭经、产后虚弱、腰腿酸痛、跌打损伤等症。课题组研究发现当归藤具有镇痛、抗炎、抗氧化、抗凝血、补血活血等药理作用，其中当归藤石油醚部位具有镇痛作用；乙酸乙酯、正丁醇部位具有明显的活血作用，其作用机制可能与增加大鼠血清中6-keto-PGF1α含量和降低血清中TXB2含量，一方面通过减少TXB2的合成，抑制亢进的血小板功能，另一方面使受损的血管内皮细胞功能恢复，合成6-keto-PGF1α能力提高，维持血液中TXB2/6-keto-PGF1α的平衡，并通过升高t-PA含量、降低PAI-1含量，调节t-PA/ PAI-1的平衡，纠正其平衡失调的作用来达到活血化瘀的目的；当归藤不同极性部位组均可增强血虚证动物的免疫功能及补血作用，其作用机制可能为提高IL-3、EPO、G-CSF、M-CSF、VCMA-1等与造血功能相关的细胞因子的水平有关，其中正丁醇和水部位的补血作用最强；当归藤正丁醇部位在体内体外均具有明显的抗炎作用，其作用机制可能与降低炎症因子NO、TNF-α、IL-6、IL-12、IL-1β的分泌有关，抑制ERK、p38、jnk的磷酸化，抑制Ikkβ 、nf-ka P65蛋白的表达上调ikba蛋白的表达从而抑制MAPK、NF-κB信号通路的活化从而发挥抗炎的，并正丁醇部位及其30%、50%洗脱部位的抗炎作用最强；利用UPLC-Q-Exactive HRMS技术对当归藤化学成分进行筛查发现当归藤中含有没食子酸、原儿茶酸、儿茶素、表儿茶素等化学成分，原花青素B1、木兰花碱、甘露醇、蔗糖、烟酰胺、腺苷、柠檬酸、3,4-二羟基苯甲酸乙酯等是首次在当归藤中被检测出来；从当归藤石油醚部位分离得到4个化合物，经结构鉴定其中的两个分别是豆甾醇、维生素E ；并用气相色谱-质谱法对当归藤化学成分进行鉴定，主要含有1,2,3,5,6,8α-六氢-4,7-二甲基-1-（1-甲基乙基）萘；1,2,3,4,4a,5,6,8α-十八氢-7-甲基-4-亚甲基-1-（1-甲基乙基）萘；Α-毕橙茄醇；十五烷；十七烷；十八烷；1-羟基环己基苯基甲酮；2,4-二叔丁基苯酚相对含量较高，均大于5%，说明挥发油中烷烃类化合物较多。