黑果枸杞矮牵牛素类化合物抗痛风作用及其机制研究

Lycium ruthenicum Murr, Tibetan medicine called ‘pangma’， has a wide range of medicinal value and is one characteristic berry plant in Qinghai-Tibet Plateau. It enjoys the reputation of “the king of anthocyanins”, up to 95% anthocyanins are petunidin compounds. Among them, its main component petunidin-3-O-rutinoside (trans-p-coumaroyl) -5-O-glucoside accounting for 80% of the total anthocyanin, this phenomenon of accumulating one anthocyanin compounds is rarely seen in berries. We carried out previous study to explore the effect of Lycium ruthenicum Murr and found that Lycium ruthenicum Murr anthocyanins have a significant effect in reducing uric acid levels of hyperuricemia mice and foot swelling of gout rats. It is even better than the positive drug colchicine in the aspect of anti-inflammatory effect. This project takes the petunidin compounds as research objective, which was isolated from Lycium ruthenicum Murr anthocyanins by modern chromatographic separation technology. On the basis of mouse hyperuricemia model, antioxidant and related biochemical indexes in serum and liver will be measured and anti-uric acid effect as well as its mechanism could be clear. In addition, rat acute gout models will be established. Through of the determination of related biochemical indicators, oxidase activity, cytokines, inflammatory factors, pain mediators, observation and analysis of histopathological sections, we will systematically study the anti-gout effects of petunidin compounds and material basis. Based on a series of inflammatory responses by activating NF-κB pathway, the molecular biology means such as qPCR and western blot will also be used to find the downstream targets and elucidate the mechanism. The results of this study will provide a new way for the prevention and treatment of gout and open up new uses for the rich resources of Tibetan medicinal materials.

黑果枸杞，藏药名为“旁玛”，具有广泛的药用价值，是青藏高原一种富含花青素的特色浆果植物。其素有“花青素之王”的美称，95%的花青素为矮牵牛素的衍生物，其中主要的单一成分占黑果枸杞总花青素的80%。前期研究发现黑果枸杞花青素具有明显降低高尿酸血症小鼠体内尿酸水平和痛风大鼠足肿胀的作用，其抗炎效果甚至还优于阳性药物秋水仙碱。本项目采用现代色谱分离技术从黑果枸杞花青素中分离得到矮牵牛素类衍生物。建立小鼠高尿酸血症模型，对血清和肝脏中抗氧化和相关指标测定，明确其降尿酸作用及机制。在建立大鼠急性痛风模型基础上，通过相关氧化酶活性、细胞因子、炎症因子、疼痛介质等指标的测定，相关病理切片的观察与分析，明确其抗痛风作用的物质基础。从NF-κB通路表达产生一系列炎症反应的角度出发，辅助以分子生物学方法qPCR和western blot寻找下游相关作用靶点，探讨其作用机制，从而为痛风病的防治提供一条新的途径。

痛风是长期嘌呤代谢障碍、血尿酸增高引起组织损伤的一组异质性疾病。研究表明从天然植物中提取分离的一些抗氧化活性物质均能显著性的抑制黄嘌呤氧化酶的活性，降低小鼠体内的尿酸水平。许多天然药用植物活性成分在体内外均表现出较好的抗炎活性，其机制可能是抑制环氧化酶COX-2、脂氧合酶5-LOX、iNOS等的活性有关。黑果枸杞富含多种活性物质，具有有别于枸杞独特的功效成分-花青素。黑果枸杞花青素具有多种药理学作用，然而最为显著的是其抗氧化作用。鉴于黑果枸杞花青素较为突出的抗氧化作用，结合氧化应激与高尿酸和痛风之间的理论关系。前期对黑果枸杞功能性开发和研究过程中发现，黑果枸杞花青素提取物能够显著地降低高尿酸血症小鼠体内尿酸水平，同时对急性痛风大鼠的足肿胀有显著的抗炎镇痛作用。.在此基础上，本项目进一步开展了系统和深入的研究。在对黑果枸杞花青素进行提取纯化富集获得黑果枸杞花青素富集物的基础上，采用现代色谱分离方法获得了黑果枸杞花青素的主成分矮牵牛素类化合物。首先利用腹腔注射氧嗪酸钾建立高尿酸血症模型，同时对模型进行了优化，验证了黑果枸杞花青素的降尿酸效果。采用大鼠右后足注射尿酸钠结晶来诱导急性痛风模型，并对所建立的模型进行优化，确定了诱导药物的最适浓度和剂量。考察了黑果枸杞花青素富集物和矮牵牛素类化合物的抗痛风作用，检测NF-κB通路表达产生一系列炎症反应相关因子，采用PCR和western blotting 等手段对其下游的相关酶5-LOX、COX-2以及iNOS等的RNA和蛋白质表达水平进行分析，从而对其抗痛风作用机制进行探究。.综上，本项目分别利用高尿酸血症和急性痛风模型实验对黑果枸杞花青素富集物和矮牵牛素类化合物的降尿酸和抗痛风作用进行了比较系统的研究，并进一步明确了其物质基础和作用机制，为黑果枸杞防治高尿酸和痛风的研究和产品开发提供了理论依据和研究思路。