【作者】 赵启鹏；

【导师】 张艺；

【作者基本信息】 成都中医药大学 ， 中药学， 2010， 博士

【摘要】 糖尿病已是世界性公共卫生问题,给患者家庭带来沉重的经济负担,严重影响患者的生活质量。研究治疗2型糖尿病(T2DM)的新药是世界各国药物研发公司追逐的热点。糖脉康颗粒具有养阴清热,活血化瘀,益气固肾之功效。用于气阴两虚兼血瘀的T2DM等。目前对糖脉康颗粒的报道主要集中在临床效果观察上,对其治疗T2DM的机制研究较少,不利于中药在治疗糖尿病上的开展,有必要进行其确切机制研究。目的在中医药理论指导下,以氧化应激为切入点,采用多学科相结合的方法,探索糖脉康颗粒治疗糖尿病的机制及物质基础。方法1、采用体外清除1,1-二苯基苦基苯肼(DPPH)法、芬顿反应产生羟基自由基(·OH),邻苯三酚自氧化产生超氧阴离子(O2-·)法,对糖脉康颗粒进行抗氧化研究；2、分别采用HPLC-DAD、HPLC-ELSD方法,初步建立糖脉康颗粒的指纹图谱,使用化学对照品和药材对色谱峰进行定性、归属,采用中药色谱指纹图谱相似度评价系统进行相似度分析,采用SPSS软件对样品进行聚类分析；3、以SPSS、SAS、DAS软件对指纹图谱-体外抗氧化指标进行相关性分析。4、采用高脂加35mg/kg链脲佐菌素诱导大鼠糖尿病模型,评价糖脉康颗粒的有效性；5、基于3T3-L1前脂肪细胞模型,采用蛋白免疫印迹(Western blotting, WB)法研究糖脉康颗粒对PPARs的影响；结果1、14批糖脉康颗粒对DPPH的平均IC50=0.71mg(生药量),40 mg(生药量)对·OH自由基的平均清除率为77.90%,13.33 mg生药量对O2-·的平均清除率为53.77%;2、分别建立了糖脉康颗粒的HPLC-DAD、HPLC-ELSD指纹图谱,HPLC-DAD标定共有色谱峰21个,标定5个对照品色谱峰,14批样品相似度大于0.80,14批样品可分为3类；HPLC-ELSD标定共有色谱峰24个,标定3个对照品色谱峰,12批样品相似度均大于0.80,两批相似度小于0.80；3、SAS软件对HPLC-DAD、HPLC-ELSD指纹图谱-抗氧化分析结果,前者清除DPPH与葛根素等12个色谱峰相关；清除·OH与原儿茶醛等10个色谱峰相关；清除O2-·与淫羊藿苷7个色谱峰相关；后者清除DPPH与黄芪甲苷等8个色谱峰相关；清除·OH与淫羊藿苷等9个色谱峰相关；清除O2-·与淫羊藿苷10个色谱峰相关。4、糖脉康颗粒可以降低血糖、大鼠饲料消耗量、饮水量、尿量,增加体质量,改善血液流变学指标及血液生化学指标,对胰腺有保护作用；可以明显提高总抗氧化能力(T-AOC)和超氧化物歧化酶(SOD)的活力,降低丙二醛(MDA)、癌基因(c-Myc)、肿瘤坏死因子α(TNF-α)、核因子-kappa B (NF-κB)含量；5、糖脉康颗粒可以增加3T3-L1中过氧化物酶体增殖物激活受体α、δ、γ(PPARα、PPARδ、PPARγ)的蛋白表达；与模型组比较,糖脉康颗粒可以增加糖尿病大鼠肝脏组织中PPARβ的蛋白表达,降低PPARδ表达,高剂量增加PPARα表达,中剂量降低PPARα表达；结论1、糖脉康颗粒体内外均具有较好的抗氧化活性,其改善大鼠糖尿病症状机制可能与其抗氧化作用相关；2、糖脉康颗粒体外抗氧化作用主要与组方中淫羊藿(淫羊藿苷)、葛根(葛根素)、黄芪(黄芪甲苷)、丹参(丹酚酸B、原儿茶醛)与赤芍(芍药苷)等药材的化学成分相关；3、糖脉康颗粒对3T3-L1前脂肪细胞和糖尿病大鼠肝组织中的PPARs蛋白表达有影响。更多还原

【Abstract】 Diabetes is a worldwide public health problem, bringing a heavy financial burden to the patients and the whole society, extremely diminishing patients’ living standard. Research of new drug on the treatment of type 2 diabetes is one of the hotspots of worldwide pharmaceutical research and development.Tangmaikang has the effect on clearing heat, promoting blood circulation, enrich Qi and benefit the kidney, applied on both Qi-Yin deficiencies and blood stasis of type 2 diabetes. Current report of Tangmaikang mainly concentrated on the observation of clinical results, rarely pay attention to the mechanism research of treatment of type 2 diabetes, which gave less advantage to the development of traditional medicines on treatment of diabetes, so as to that the research of its exact mechanism is necessary.ObjectiveUnder the guidance of the theory of traditional chinese medicine, regarding oxidative stress as an entry point, using combining methods to explore Tangmaikang’s remedying mechanism and material basis of diabetes.Methods1. Using methods of in-vitro DPPH,·OH Fenton reaction, pyrogallol autoxidation generate O2-·, to approach the anti-oxidation study of Tangmaikang.Process the establishment of Tangmaikang HPLC-DAD fingerprint, and assign the major peaks.2. Respectively using HPLC-DAD, HPLC-ELSD methods initially established Tangmaikang’s Fingerprint, using standard and herbs on the qualitative and attribution of the peaks, using chromatographic Fingerprint evaluation system for similarity analysis, using SPSS software to cluster analysis of samples;3. Using software of spss, sas, das, to analysis the correlation of the fingerprint and antioxidant.4. Taking high-fat plus 35mg/kg streptozotocin (STZ) induced diabetic rats as models, access the effectiveness of Tangmaikang.5. Based on 3T3-L1 cell model, study the effect of Tangmaikang on PPARs by the method of western blotting (WB).Results1.14 batches of Tangmaikang on the average of DPPH is IC50= 0.71mg (crude drug content),40 mg (crude drug content) of the averagely·OH radical clearance rate was 77.90%,13.33 mg (crude drug content) of the averagely O2-·clearance rate was 53.77%.2. Respectively established Tangmaikang’s HPLC-DAD, HPLC-ELSD Fingerprint. The HPLC-DAD calibrate a total of 21 common peaks and 5 reference standard peak; the similarity of 14 batches of samples were higher than 0.80,14 batches of samples can be divided into three categories. The HPLC-ELSD calibrate a total of 24 peaks and 3 reference standard peak; the similarity of 12 batches of samples were higher than 0.80, two groups’similarity is lower than 0.80.3. The HPLC-DAD, HPLC-ELSD Fingerprint-Antioxidant SAS analysis presented the correlation between the former clearance of DPPH and 12 peaks, the clearance of·OH was associated with 10 peaks, the clearance of O2-·was associated with 7 peaks, meanwhile the latter clearance of DPPH was associated with 8 peaks, the clearance of·OH was associated with 9 peaks, and the clearance of O2-·was associated with 10 peaks.4. Tangmaikang can reduce the consumption of feed, water, and the urine output of rats, and increase the rats’weight, lower their blood glucose, improve the indicators of hemorheology and blood biochemical, give a protective effect on pancreatic of them. It is also significantly improve the activities of T-AOC, SOD, descending contents of MDA, c-Myc, TNF-α, NF-κB.5. Tangmaikang can increase the the peroxisome proliferator-activated receptorα,δ,γ(PPARα, PPARδ, PPARγ) protein expression in 3T3-L1; cmpared with model group, Tangmaikang can increase the liver tissue of diabetic rats the protein expression of PPARy, decreased the expression of PPARδ; increase expression of PPARa in high-dose, while decreased it in mid-dose;Conclusions1. Tangmaikang has well antioxidant activity both in-vivo and in-vitro, the remedy mechanism of diabetes on rats is closely related to its antioxidant;2. Tangmaikang’s in vitro antioxidant activity mainly related to the prescription where Herba Epimedh (icariin), radix puerariae lobatae (puerarin), radix astragali (astragaloside), Radix Et Rhizoma Salviae Miltiorrhizae (Dan acid B, protocatechuic aldehyde) and Radiz Paeoniae Rubra (paeoniflorin), etc. the chemical constituents of medicinal herbs presented.3. Tangmaikang has an effection on 3T3-L1 pre-adipocytes and the PPARs protein expression of diabetic rats’liver tissue.更多还原