【作者】 刘月涛；

【导师】 彭金咏；

【作者基本信息】 大连医科大学 ， 微生物与生化药学， 2010， 硕士

【摘要】 目的：制备金樱子总黄酮(TFs)并考察其生物活性。方法：本研究主要从以下方面进行：(1)TFs的制备及质量。以总黄酮的含量为测定对象,利用正交试验法优选总黄酮的提取工艺条件；通过D101大孔树脂工艺制备高纯度的总黄酮(TFs)；并通过HPLC指纹图谱和对水解产物黄酮苷元的测定来建立质量标准。(2)总黄酮体内外抗氧化活性实验。在体外考察其对DPPH(1,1-二苯基-2-三硝基苯肼)、·OH(羟基自由基)、·O-2(超氧阴离子)和H2O2(过氧化氢)的清除作用以及还原力的测定,并考察了其对红细胞溶血和脂质过氧化的抑制作用,以维生素C (Vit C)作为阳性对照。此外,通过给药后小鼠血清、肝和肾组织中T-AOC(总抗氧化能力),SOD(超氧化物岐化酶)和MDA(丙二醛)水平的变化来评估它的体内抗氧化能力。(3)总黄酮对小鼠高脂血症的保护作用及其机制研究。正常小鼠分别给予高脂饮食和不同给药剂量总黄酮(25,50 mg/kg, i.g.),并以非诺贝特为阳性对照。实验结束后,通过测定血清的TC(总胆固醇)、TG(甘油三酯)和HDL-C(高密度脂蛋白)的水平评价其降脂活性。通过组织病理学、ALT(谷丙转氨酶)、AST(谷草转氨酶)和ALP(碱性磷酸酶)等指标的测定探讨总黄酮对高脂饮食所致的脂肪肝的保护作用。此外还考察了其对实验动物中体内抗氧化系统的影响。(4)总黄酮对高脂血症大鼠的治疗作用及其血液流变学影响。造模后的高脂大鼠给予总黄酮(100 mg/kg)和阳性对照药非诺贝特4周。实验结束后测定血清的TC、TG和HDL-C水平评价其降脂活性。并通过测定血液流变学的变化来验证其降脂活性。(5)总黄酮对扑热息痛诱导的急性小鼠肝损伤的保护作用及其机制研究。在这项研究中,小鼠分别被灌胃不同剂量的总黄酮(50和200 mg／kg)和联苯双酯(100 mg/kg)5天,在最后一次给药2h后,除空白组所有实验动物腹腔注射扑热息痛(400 mg/kg).实验结束后,测定血清的ALT、AST、MDA、SOD和GSH(谷胱甘肽)等指标来考察TFs对肝损伤的保护作用和体内抗氧化系统的影响。并通过Western blotting、免疫组化和RT-PCR等技术来考察相关蛋白和mRNA的表达水平。(6)通过急性毒性试验来初步考察金樱子总黄酮的毒性。结果：正交试验法优化的金樱子最佳提取条件是用8倍量的60%乙醇回流提取2 h,共2次；D101树脂制备工艺是用4倍树脂柱床体积的水除杂和6倍树脂柱床体积的40%乙醇洗脱得到纯度为78.45%的TFs。产物经水解后经HPLC分析显示槲皮素、山柰素、异鼠李素的总含量为7.37%。体内外抗氧化活性实验显示TFs对DPPH,·OH和·O-2有较强的清除作用,其IC 50值分别为0.01、0.04和0.08 mg/mL,还原力稍弱于阳性对照Vit C。TFs在浓度为0.35 mg/mL和2.00 mg/mL时,还分别具有71.62%和48.45%的红细胞溶血和脂质过氧化的抑制作用。在体内抗氧化方面,TFs(100mg/kg)能明显提高血清和肝和肾组织中T-AOC和SOD的水平,并显著降低MDA；TFs在50 mg/kg的给药剂量时,高脂血症小鼠血清TC、TG和LDL-C水平分别下降了45.02%、33.86%和73.68%,而HDL-C则显著高于模型组,与阳性药相比(分别降低39.22%、36.06%和76.08%)药效相当。组织病理学、ALT、AST和ALP等指标均显示TFs对肝脏具有良好的保护作用,其中,ALT、AST和ALP分别升高了55.85%、29.15%和25.68%。此外,TFs能显著降低MDA水平,提高CAT(过氧化氢酶)、SOD、GSH、GPX(谷胱甘肽过氧化酶)水平；TFs对高脂大鼠的治疗作用及其血液流变学研究显示金樱子总黄酮在给药100 mg/kg时对高脂大鼠的血清TC和TG分别极显著地降低了60.80%和62.39%(p<0.01),略强于非诺贝特(40 mg/kg,分别降低56.82%和56.99%)。TFs能降低全血比粘度(低切、高切)49.59%和63.24%(p<0.01),血浆比粘度、血清比粘度和RBC聚集指数均明显降低(p<0.05),而纤维蛋白含量无明显变化。与模型组相比,在对扑热息痛诱导的小鼠急性肝损伤的保护作用中,高剂量给药组小鼠血清的ALT、AST和MDA分别下降由78.42%、70.85%和30.52%,而SOD和GSH显著高于模型组(p<0.05),强于阳性药的效果。TFs能显著提高Procaspase-3、Procaspase-8、FasL、Prohibitin和Bcl-2的蛋白表达水平,明显降低中Fas、Bax、p53和NF-κB p65的蛋白表达,同时下调TNF-α的表达。在急性毒性实验中,我们发现金樱子总黄酮在给药剂量为20 g／kg时未见任何动物死亡。结论：本实验中从中药金樱子中制备得到的总黄酮,具有制备工艺简单、含量高、质量可控等优点,适合于工业化生产。进一步的药效活性显示制备得到的总黄酮具有显著的体内外抗氧化活性、对高脂血症及心脑血管疾病有着很好的预防和保护作用,同时也对药物性肝损伤有着很好的保护作用。以上结果表明金樱子总黄酮在预防和治疗心脑血管疾病和肝损伤保护等方面具有较大的研究和开发价值,但是有关其长期毒性、物质基础、作用机制、其它模型上的心脑血管活性及肝损伤保护作用还需深入研究。更多还原

【Abstract】 Objective:The aim of the study was to prepare the total flavonoids (TFs) from Rosa Laevigata Michx fruit and further to evaluate its biological activities.Methods:This study was carried out as follows:(1) Preparation and quality control of the TFs. In order to extract the total flavonoids from R. Laevigata Michx fruit, an orthogonal experimental design was applied to optimize the suitable extraction conditions. Then, the TFs was prepared by D101 macroporous resin column chromatography, and its quality was established through HPLC fingerprint analysis and determination of the contents of quercetin, kaempferide and isorhamnetin in its hydrolysis product. (2) In vitro and in vivo antioxidant activities of TFs. The antioxidant activity of TFs in vitro was conducted by scavenging effects on DPPH,·OH (hydroxyl radical),·O2- (superoxide anions) and H2O2 (hydrogen peroxide). And the reducing power, the inhibitory effects on erythrocyte hemolysis and lipid peroxidation were also evaluated. All the results were compared with the positive drug Vit C. As to in vivo investigation, the change of T-AOC (total antioxidative capacity), SOD (superoxide dismutase), and MDA (malondialdehyde) levels in serum, liver and kidney tissues of the TFs-treated mice were investigated and compared with normal mice. (3) The hypolipidemic activity of TFs in hyperlipidemic mice. During the experiment, the mice were administrated high-fat diet, and then given TFs (25,50 mg/kg, i.g.) or the positive drug fenofibrate (50 mg/kg, i.g.) for 4 weeks. After the experiment, the serum TC (total cholesterol), TG (triglycerides) and HDL-C (high density lipoprotein) levels were determined and used to evaluate its hypolipidemic activity. The hepatoprotective effect of TFs was based on the diversity of histopathology, ALT (alanine aminotransferase), AST (aspartate aminotransferase) and ALP (alkaline phosphatase) levels. In addition, the potential effect in the system of antioxidant defense was also discussed. (4) The hypolipidemic activity and the effect in the hemorheology of the TFs in hyperlipidemic rats. Hyperlipoidemic rats were given high-fat diet, and then given TFs (100 mg/kg, i.g.) or the positive drug fenofibrate (40 mg/kg, i.g.) for 4 weeks. The serum levels of TC, TG, HDL-C and hemorheology were used to evaluate its hypolipidemic activity after the experiment. (5) The hepatoprotective activity of TFs in paracetamol-induced liver injury in mice. TFs (50,200 mg/kg) and positive drug (bifendate,100 mg/kg) were administered via gavage daily for 5 days before paracetamol (400 mg/kg, i.p.) treatment. After the experiment, the levels of serum ALT, AST, MDA, SOD and GSH were used to evaluate the hepatoprotective activity and protective effect on the antioxidant system. And then the possible biochemical mechanisms were studied by western blotting, immunohisto-chemistry and RT-PCR (reverse transcriptase polymerase chain reaction) techniques. (6) The acute toxicity of TFs was evaluated.Results:The suitable conditions for the extraction of the TFs from R. Laevigata Michx fruit were optimized as follows:60% ethanol as the solvent, solid-liquid ratio 1:8 for 2 hours refluxing for 2 times. As to D101 macroporous resin column chromatography,4 BV (bed volume of resin) of water was selected to wash some watersoluble chemicals and 6 BV of 40% aqueous ethanol was used to elute the targets and the TFs with the contents of 78.45% were produced. After hydrolysis and HPLC analysis, the total contents of quercetin, kaempferol, and isorhamnetin were 7.37%. In in vitro antioxidant activities, the TFs exhibited a high scavenging effects on DPPH,·OH, and·O2- with the IC50 values of 0.01,0.04 and 0.08 mg/mL, respectively. The samples also showed high potential antioxidant activity in reducing power test. The TFs could exert 71.62% and 48.45% of the inhibitory effects on erythrocyte hemolysis and lipid peroxidation at the concentrations of 0.35 and 2.00 mg/mL, respectively. As to in vivo antioxidant experiment, TFs (100 mg/kg) can significantly improve T-AOC and SOD levels in serum, liver and kidney tissue, and markedly reduce the MDA level. Compared with hyperlipidemic mice, the serum TC, TG and LDL-C levels of TFs (50 mg/kg)-treated group were decreased by 45.02%, 33.86% and 73.68%, respectively. And HDL-C was significantly increased. The results also showed that the TFs exhibited a favorable protective effect on liver induced by high-fat diet, of which the levels of ALT, AST and ALP were decreased by 55.85%,29.15% and 25.68%, respectively. Furthermore, the TFs could significantly decrease the MDA level and improve the levels of CAT, SOD, GSH, and GPX, compared with hyperlipemic mice. During the study of the hypolipidemic activity of TFs in hyperlipidemic rats, TFs at the dose of 100 mg/kg could significantly reduce serum TC and TG by 60.80% and 62.39% (p<0.01), no difference compared with fenofibrate-treated group (56.82% and 56.99%). The TFs could makedly decrease whole blood viscosity (low shear, high shear) by 49.59% and 63.24%(p<0.01). Plasma viscosity, serum viscosity and RBC aggregation index were all significantly decreased (p<0.05). TFs showed good hepatoprotective activity in paracetamol-induced liver injury in mice. After the experiment, the levels of serum ALT, AST and MDA in TFs (200 mg/kg)-traeted group were decreased by 78.42%,70.85% and 30.52%, respectively, while SOD and GSH were significantly increased compared with model group (p<0.05). The ALT and AST levels in positive group were reduced by 62.00% and 50.75%, of which the hepatoprotective activity was lower than the positive drug. Through verification of the possible biochemical mechanisms, the results indicated the TFs could significantly increase the expressions of Procaspase-3, Procaspase-8, FasL, Prohibitin and Bcl-2, and markedly decrease the expressions of Fas, Bax, p53 and NF-κB p65 compared with the mice treated by paracetamol. Meanwhile, the mRNA level of TNF-αwas also down-regulated after TFs treatment. Additionly, we found that no mice dying when the animals were adiministrated the TFs at the high dose of 20 g/kg in the acute toxicity experiment.Conclusion:In the experiment, the protocol for preparation of the total flavonoids with high purity from R. Laevigata Michx fruit was simple and easily for quality control, which was suitable for the industrial production. The futher pharmacodynamic research showed that the product shows significant antioxidant activity, in prevention and protection of cardiovascular and cerebrovascular diseases. It also plays excellent protective effect on the drug-induced liver injury. These results show that the TFs from this medicinal plant has great investigative and developmental significance in the prevention and treatment of cardiovascular and cerebrovascular diseases. Howerer, the long-term toxicity, material basis, mechanism, and the evaluation in cardiovascular activity on other levels will be needed in further study.更多还原