电化学生物传感靶向识别车前子中黄嘌呤氧化酶抑制剂研究

The xanthine oxidase inhibitors have been found in a wide variety of plants used in traditional herbal medicines for treatment of gout and rheumatism. This has attracted enormous interest over the past years due to those herbal medicines marked with a long history of proven safety and efficacy. .However, the further research of xanthine oxidase inhibitors from traditional herbal medicines has been limited by the present research methods since they are complex and time-consuming, require a relatively large amount of monomer, and lack of real-time kinetics information..The plantain seed of P. asiatica L. is a genuine medicinal materials of Jiangxi provinces. It can inhibit the activity of xanthine oxidase in hyperuricemia mice obviously in our previous study, but the effective substance is still unidentified..In this study, taking the interaction target of xanthine oxidase as bio-recognition component, we firstly use the electrochemical biosensing method based on carbon nanotubes composite possessing synergistic electrocatalytic effects in vitro combined with modern separation technology and the animal experiment in vivo to recognize xanthine oxidase inhibitors in the plantain seed of P. asiatica L.。.The new method can not only recognize the inhibitors fastly but also monitor the kinetics process in real-time, which obtains the properties of high performance, sensitive, fast, real-time and economic analysis. .The new method is very attractive as firstly, it can accumulate experimental data and effective substance for development of safe and effective drugs for treatment of gout from plantain seed and facilitate the development of the economic and society of Jiangxi provinces; Secondly, it can provide a new method for the research of xanthine oxidase inhibitors which is separated and prepared from traditional herbal plants and may finally promote the modernization and internationalization of traditional herbal medicines for treatment of gout and rheumatism.

从传统抗痛风及风湿病等药用植物中寻找新的安全有效的黄嘌呤氧化酶抑制剂（XODI）对痛风的防治具有重大意义。现有XODI研究方法繁琐耗时单体成分需求量相对较大且缺少实时动力学信息，难以满足经植物化学分离得到的XODI高效快速研究需要。车前子为江西道地药材，本课题组前期研究表明其提取物能明显抑制急性高尿酸血症小鼠体内黄嘌呤氧化酶活性。本研究首次以黄嘌呤氧化酶为抑制剂靶向识别元件，利用碳纳米管纳米复合物协同电催化特性，结合现代分离制备技术及体内药效学研究，建立体内法与体外酶抑制型电化学生物传感相结合的新方法靶向识别车前子中单体成分抑制剂。该方法不仅能对量少单体抑制剂进行快速灵敏的靶向识别，还能对整个靶向识别动力学过程进行实时监测，适合于经中药化学分离制备的XODI高效快速研究。本研究能为基于车前子安全有效的抗痛风中药研制奠定物质与理论基础，并为抗痛风及风湿病等中草药的现代化、国际化开辟新的途径。

从车前子中筛选黄嘌呤氧化酶抑制剂具有重要研究价值。项目采用LH-20大孔树脂、利用硅胶、ODS柱层析、Sephadex LH-20凝胶、高压制备技术与功能材料对车前子化学成分进行分离纯化，共获得毛蕊花糖苷，异毛蕊花糖苷，去咖啡酰基毛蕊花糖苷，肉豆蔻酸，bis(2-ethythexyl)benzene-1, 2-dicarboxylate，β-胡萝卜苷，α-亚油酸，α-亚麻酸，(+)-(7R，7'R，8S，8'S)-neo-olivil，去氢催吐萝芙木醇，Cyclo-(Phe-Pro)，Blumenol A，(+)- erythro-guaiacylglycerol-β-ferulic acid ether，γ-亚麻酸，油酸，吲哚-3-甲醛，圣草酚，木犀草素，羟基酪醇，( E) -3，4-二羟基苯亚甲基丙酮，阿魏酸，5，7-二羟基色原酮，金圣草素等23个化合物。. 以XOD为靶向识别元件，应用碳纳米管建立了电化学生物传感靶向识别XOD抑制剂方法，并识别得到木犀草素，毛蕊花糖苷，金圣草黄素，吲哚-3-甲醛，圣草酚，(E)-3,4-二羟基苯亚甲基丙酮及二(2-乙基己基)-苯-1,2-羧酸酯7个XOD抑制剂成分，且均为竞争性抑制类型，其抑制动力学常数分别为4.3、10.1、90.2、68.2、73.0、136.4、141.7 μg/mL。其中吲哚-3-甲醛， (E)-3,4-二羟基苯亚甲基丙酮及二(2-乙基己基)-苯-1,2-羧酸酯为新发现的XOD抑制剂，而木犀草素、毛蕊花糖苷抑制活性最接近别嘌呤醇，在可操作条件下选择毛蕊花糖苷进行体内活性研究。. 应用氧嗪酸钾盐诱导的急性高尿酸血症模型，对毛蕊花糖苷进行了与提取物降尿酸功效的等效性评估，发现毛蕊花糖苷降尿酸率达到车前子提取物的67.8%-85.2%；对XOD的抑制等效率达到车前子提取物的66.1%-82.2%；毛蕊花糖苷对肾mURAT1及mGLUT9mRNA表达下调等效率分别达车前子提取物的55.8%-83.2%与56.0%-76.2%。从而阐明了车前子主要XOD抑制剂成分及降尿酸成分为毛蕊花糖苷。. 项目建立了一种全新的电化学生物传感靶向识别XOD抑制剂方法，阐明了车前子主要降尿酸成分与XOD抑制剂成分为毛蕊花糖苷，为车前子抗痛风药用开发奠定了理论与物质基础。