基于黄嘌呤氧化酶抑制作用的金雀花根抗痛风物质基础及其构效关系研究

Gout is the second metabolic disease caused by hyperuricemia. Xanthine oxidase (XO) is a key enzyme in the production of uric acid. Inhibition of the activity of XO intrinsically could reduce the production of uric acid in the body. Therefore, XO inhibitors have always been the first-line drugs for hyperuricemia. However, the existing inhibitors have prominent adverse reactions and the search for more safe and effective XO inhibitors has been the major task of drug research and discovery. The roots of Caragana sinica (CSR) is a safe and effective anti-gout traditional Chinese medicine, but the material basis for its efficacy in the treatment of gout is still unknown. Previous studies demonstrated that the ethyl acetate and n-butanol extracts of CSR possess strong XO inhibitory activity (IC50 of 8.6 and 18.3 ug/mL, respectively). Ultrafiltration liquid chromatographic analysis showed the presence of a number of potential XO inhibitors. In this study, the potential XO inhibitors will be screened by the combination of ultrafiltration liquid chromatography-mass spectrometry with enzyme channel blocking and metabonomics data analysis methods. XO inhibitors will be obtained through target-orientated separation, structure identification and activity evaluation, and the structure-activity relationship was investigated. Molecular docking analysis will be used to clarify action mode of the inhibitors and XO, and the material basis of CSR for the treatment of gout will be clarified from the perspective of XO inhibition. Based on the discovered XO inhibitors, further molecular design, synthesis, activity test, and preliminary safety evaluation of new XO inhibitors will be carried out, and safe and active XO inhibitors will be expected.

痛风是由高尿酸血症引起的第二大代谢类疾病。黄嘌呤氧化酶(XO)是尿酸生成的关键酶，抑制XO的活性，可从根本上减少体内尿酸的生成，因此XO抑制剂一直是高尿酸血症治疗的一线药物。但现有的抑制剂不良反应突出，寻找更安全、有效的XO抑制剂已成药物研发的重要任务。金雀花根（CSR）是一味安全有效的抗痛风中药，但其治疗痛风的物质基础尚不明确。研究发现，CSR乙酸乙酯和正丁醇萃取物具有很强的XO抑制活性（IC50分别为8.6和18.3微克/毫升）。离心超滤分析发现，其中可能存在大量XO抑制剂。本课题拟采用离心超滤质谱与酶通道阻断、代谢组学分析方法相结合技术，筛选出潜在XO抑制剂；通过定向分离、结构鉴定和活性评价，获得XO抑制剂，结合构效关系和分子对接分析，从XO抑制的角度阐明CSR治疗痛风的物质基础。以所发现的XO抑制剂为基础，开展分子设计、合成、活性和安全性初步评价，期望发现安全有效的XO抑制剂。

痛风是由高尿酸血症引起的第二大代谢类疾病。黄嘌呤氧化酶(Xanthine Oxidase, XO)是尿酸生成的关键酶，抑制XO的活性,可从根本上减少体内尿酸的生成，是抗痛风及高尿酸血症的有效手段。由于现有的抑制剂不良反应突出，寻找更安全、有效的XO抑制剂已成药物研发的重要任务。金雀花根（Caragana sinica roots, CSR）是一味安全有效的抗痛风中药，但其治疗痛风的物质基础尚不明确。本课题从其乙酸乙酯萃取物（IC50为8.6 ug/mL）中分离、鉴定出30个化合物，获得了14个不同作用强度的 XO 抑制剂，从抑制尿酸生成的角度阐明金雀花根治疗痛风的物质基础；获得了9个抑制活性突出的天然 XO 抑制剂。其中，除了(+)-a-Viniferin 和(+)-ε-viniferin的活性与阳性药别嘌呤醇（IC50=71.7 umol/L）相当，7个化合物的抑制活性均优于阳性药别嘌呤醇（IC50<71.7 umol/L）。白皮杉醇和Viniferifuran抑制活性最为突出（IC50分别为12.4和13.9 umol/L）。体内实验发现，(+)-a-Viniferin与Viniferifuran能将高尿酸血症小鼠体内尿酸水平恢复至接近正常水平，并能较大程度的改善模型组小鼠肾小管间质病变，肾小管扩张和轻度肾水肿的病理状态。机制研究表明，(+)-a-Viniferin与Viniferifuran降尿酸作用主要与抑制肝组织的蛋白表达以抑制尿酸的生成，下调肾组织URAT1和GluT9、上调OAT1和ABCG2的表达从而促进尿酸排泄有关。初步的安全性实验表明，二者的安全性均较阳性药好。以上结果表明，a-Viniferin与Viniferifuran等多个单体均可能是极有开发价值的XO抑制剂。