淫羊藿黄酮类化合物活性物质的体外模型筛选及抗氧化研究

【作者】 包宇；

【导师】 孙润广；

【作者基本信息】 陕西师范大学 ， 声学， 2012， 博士

【摘要】 本论文从化学成分和细胞生物学层面分析了中药淫羊藿黄酮类化合物中的活性成分,建立了体外氧化应激模型和血脑屏障模型,利用模型对淫羊藿黄酮类化合物进行筛选,并对筛选出的活性成分进行分析、鉴定和体外抗氧化研究,为与心血管疾病和中枢神经系统疾病相关的药物筛选提供了新的研究思路。本论文的主要研究内容有：1.体外氧化应激筛选模型的建立并应用该模型从淫羊藿黄酮类化合物中筛选活性成分以筛选活性化合物为目的,建立了从中药中筛选与心血管疾病相关的活性化合物的体外氧化应激模型。该方法是利用中药中活性成分与氧化损伤人脐静脉内皮细胞上的效应靶点特异性结合的特性,使中药提取物与内皮细胞相互作用,活性成分与之结合,未结合的成分被洗掉。再通过细胞膜受体失活的方式,使与细胞受体特异性结合的成分解离下来,应用液相-质谱联用技术分析鉴定筛选出的活性成分。将该模型应用于淫羊藿黄酮类化合物中,筛选与心血管疾病相关的活性成分。研究结果表明,淫羊藿黄酮类化合物中至少有6种活性成分能与人脐静脉内皮细胞受体特异性结合,其中3种活性成分被鉴定分别为朝藿定B、淫羊藿苷和淫羊藿次苷Ⅱ。2.体外血脑屏障筛选模型的建立并应用该模型从淫羊藿黄酮类化合物中筛选活性成分中枢神经系统对外界相对封闭,具有免受多种物质影响的独特结构,致使许多药物不能进入中枢神经系统发挥作用,利用体外血脑屏障模型可筛选出能够作用于中枢神经系统的活性物质。本论文利用ECV304/C6细胞共培养模式来模拟在体血脑屏障建立药物筛选模型,通过跨膜电阻和小分子物质渗透率分析证明,ECV304细胞在C6细胞诱导下所建模型具有体内血脑屏障的特点。应用所建血脑屏障模型,从淫羊藿黄酮类化合物中筛选出能够通过血脑屏障的活性成分,并进-步利用液相-质谱联用技术对筛选出来的药物进行分析和鉴定。研究结果表明,淫羊藿黄酮类化合物中至少有2种化合物能够通过血脑屏障,它们分别是淫羊藿苷和淫羊藿次苷Ⅱ。3.淫羊藿苷和淫羊藿次苷Ⅱ的体外抗氧化研究淫羊藿苷是淫羊藿中主要的活性成分,属于黄酮类化合物,具有保护心血管系统、调节免疫、抗癌、抗骨质疏松和改善性功能紊乱等多种重要的生物活性。淫羊藿次苷Ⅱ是淫羊藿苷的代谢产物,对其功能知之甚少。本实验建立了体外自由基发生体系：DPPH?、NBT-NADH-PMS, OH?、卵黄脂质过氧化模型反应体系、还原铁氰化钾以及β-胡萝卜素-亚油酸自氧化体系分别测定了淫羊藿苷和淫羊藿次苷Ⅱ对DPPH?、 O2-?、 OH三种自由基的清除能力,它们对脂质过氧化的抑制能力,还原力以及总抗氧化能力,以此来研究待测样品淫羊藿苷和淫羊藿次苷Ⅱ的体外抗氧化活性,并以BHT和Vc作为阳性对照。实验结果表明,淫羊藿苷具有很强的清除DPPH?、 O2-?、还原能力和总抗氧化的能力,有较强的清除OH·的能力,且均呈剂量依赖性。淫羊藿次苷Ⅱ对DPPH的清除能力很强,在清除OH·,02-·和抑制脂质过氧化方面也有较强的作用。淫羊藿苷和淫羊藿次苷Ⅱ抗氧化效果在一定范围内与其浓度呈正相关,即随着浓度的升高,抗氧化能力逐渐增强。但当抑制率或清除率达到一定水平时,剂量的加大对其抗氧化效果影响不大。淫羊藿苷和淫羊藿次苷Ⅱ对同一种自由基的清除能力也不相同。究其原因可能是因为两者酚羟基数目、所处位置和糖基的数目、所处位置的不同。本实验的研究结果对于阐明淫羊藿苷和淫羊藿次苷Ⅱ药理活性的作用机制具有一定的参考价值,为潜在抗氧化剂的开发和利用奠定实验基础。更多还原

【Abstract】 This paper analyses bioactive components in flavonoids extracts from Herba Epimedii at the level of chemical composition and cell biology, and builds the models of oxidative stress and blood-brain barrier in vitro. At the same time, this paper processes screening, analysis, identification activity and antioxidant activities in vitro of bioactive compounds in flavonoids extracts from Herba Epimedii, in order to offer new ideas for modernization and screening cardiovascular and central nervous system (CNS) related diseases drugs.The research works are as follows:1. The foundation of oxidative stress screening model, and screening of active compounds from flavonoids extracts from Herba Epimedii.In order to screen active compounds, oxidative stress model is used to screen active compounds related cardiovascular diseases from Chinese herbal medicine. The principle of the oxidative stress model is the oxidative-damaged ECV304cells induced by H2O2combine active compounds which are screened from Chinese herbal medicines, and the unbound components and superfluous components are washed and discarded from cell surface of ECV304cells. Then the special binding components release from cell membrane receptors by changing pH. The oxidative stress model combines with LC-MS technique is applied in screening bioactive compounds from flavonoids from Herba Epimedii extracts. The results show that there are6active components which are bound and captured by ECV304membrane receptors, and the3active compounds are identified as epimedin B, icariin and icariside II.2. The foundation of blood-brain barrier screening model, and screening of active compounds from flavonoids extracts from Herba Epimedii.The CNS has formed the unique structure in the human evolution, and the CNS avoids the influence of many kinds of molecular drugs, simultaneously this kind of isolation influences the function of medicine to cure CNS diseases. The active compounds which can permeate into blood-brain barrier model in vitro are screened from Chinese herbal medicine. The ECV304/C6co-culture model is applied to simulate the blood-brain barrier in vivo, and the trans-epithelial electrical resistance (TEER) and permeability coefficient of ECV304/C6co-culture are analyzed. The results show that ECV304/C6co-culture has the characters of blood-brain barrier in vivo. The blood-brain barrier model combines with LC-MS technique is applied to screen activity compounds which can go through the blood-brain barrier from flavonoids extracts from Herba Epimedii. The screened candidate drugs are analyzed and identified with LC-MS technique. The result shows that2candidate drugs can go through blood-brain barrier and confirmed by LC-MS, the2screened bioactive compounds are icariin and icariside II. Further more, the results prove that ECV304/C6co-culture model can screen the active compounds which can go through blood-brain barrier from Chinese herbal medicine extracts, and the method is helpful in guiding screening and developing new drugs of CNS targets.3. Antioxidant properties of icariin and icariside II in vitro.Icariin, a major active flavonoid isolated from Herba Epimedii, possesses wide pharmacological actions, especially in protecting cardiovascular system, immunological function modulation, anti-tumor, anti-osteoporosis and improvement of sexual dysfunction. Icariside II is metabolites of icariin, and its function is hardly understood. We analysed the antioxidant activity of the icariin and icariside II using different assays including:total antioxidant activity and reducing power, hydroxyl radical scavenging assay, superoxide radical scavenging assay,1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging assay and lipid peroxidation assay which uses egg yolk as the lipid-rich source. Total antioxidant capacity was determined by the assay based on the decrease in absorbance of β-carotene by the sample. These various antioxidant activities were compared to standard antioxidants such as BHT and Vc.The experiment results show icariin exhibites strong DPPH radical, superoxide radical scavenging activity, reducing power and total antioxidant activity, icariside II has stronger DPPH radical scavenging capacity than icariin, and effective inhibition of lipid peroxidation. The radical-scavenging and inhibition effects of icariin and icariside II are in a dose-dependent manner. A positive correlation is found between dose of the drugs and antioxidant effects, and the effects change unobviousely once the scavenging or inhibition ratio reaches some level. The effects of radical scavenging of icariin and icariside II are different, and the reason probably is the difference of number and position of two compounds’phenolic hydroxyl and glycosyl. The experiment provided the reference for the study of the pharmacological activity mechanism of icariin and icariside II, and experiment foundation for potential antioxidants’exploitation and use.更多还原