【作者】 王春怡；

【导师】 李卫民；

【作者基本信息】 广州中医药大学 ， 中药学， 2009， 博士

【摘要】 胰岛素抵抗是2型糖尿病(T2DM)发生的重要因素和显著特征。因此针对胰岛素抵抗(IR)的治疗是防治糖尿病(DM)及代谢综合征的重中之重。中药防治疾病时主要采用复方的形式,体现了整体调节,综合治疗的特点。中药复方发挥其疗效的物质基础来自于其化学成分或活性部位,然而仅仅的几个单体化合物无法代表全方,应更重视化学成分群体或有效部位的作用。将复方有效部位进行配伍应用,则有可能使全方药效呈现增效协同的作用,同时降低不良反应的程度。因此,以中药有效部位进行组方,研制创新药物,创建在中医药理论指导下具有示范效应的创新中药的研究模式,提高中药产业的整体素质和竞争力。黄芪散古方源自《圣济总录》,组方为:葛根、黄芪、桑白皮。三药相伍,益气健脾,益气清热,生津止渴,三消并治,共奏标本兼顾之效。大量研究表明三药对T2DM或IR具有良好的疗效,其药效物质基础及作用机制各有所不同,因此认为该方可能通过多途径、多靶点的综合效应来改善T2DM-IR。目前基于黄芪散整方的研究未见有报道,这使得该方的临床应用及开发具有一定的局限性。本研究首先立足于整方,试图阐明黄芪散干预T2DM-IR的作用及作用机制,并采用一系列现代中药制剂技术,富集各药有效部位,为黄芪散的进一步制剂开发奠定基础。第一部分文献研究文献研究方面全面查阅了近年来国内外T2DM-IR的现代医学研究进展,包括IR的流行病学研究、病因研究以及发病机制等相关文献资料,同时对DM的中医认识、黄芪散各药的化学成分和药理学研究进展进行文献研究。对目前现代医学和中医学对于T2DM-IR的发病机理、治疗用药,T2DM-IR分子机制的近期研究成果,以及动物模型的建立及研究方法等概况进行了较为系统的总结和综述,掌握了相关项目科研的前沿动态。第二部分实验研究1.黄芪散干预实验性2型糖尿病胰岛素抵抗的作用及机制研究通过建立STZ所致DM小鼠模型、氢化可的松琥珀酸钠(HCSS)诱导的IR小鼠模型初步评价黄芪散对DM及IR的干预作用。结果表明黄芪散降低DM小鼠的空腹血糖(FBG),增加糖耐量,同时提高糖耐量异常而尚未形成DM的小鼠对胰岛素的敏感性,改善胰岛素抵抗。采用STZ及高脂饮食建立T2DM-IR大鼠模型。通过对模型大鼠FBG、空腹胰岛素水平(FINS)、糖耐量、胰岛素耐量、胰岛素敏感指数(ISI)和抵抗指数(HOMA-IR)、血清和肝脏的脂质代谢及游离脂肪酸(FFA)水平测定、胰腺HE染色病理形态学检查,探讨黄芪散对T2DM-IR的糖脂代谢和IR的影响。结果表明黄芪散显著降低T2DM-IR大鼠的FBG,改善糖耐量,降低IR所形成的高FINS水平,提高胰岛素耐量,增加ISI指数和降低HOMA-IR指数,对胰腺组织结构的病变具有保护作用;同时黄芪散可调节血脂、肝脂、下调血清及肝脏中FFA的水平。提示黄芪散可改善糖脂代谢、提高胰岛素的敏感性发挥其治疗IR的作用。在建立T2DM-IR模型大鼠的基础上,通过采用放免法测定血清中肿瘤坏死因子(TNF-α)、瘦素(leptin)水平,RT-PCR和免疫组化法测定肝脏、骨骼肌和脂肪组织中GLUT-4、IRS-1的mRNA和蛋白的表达,RT-PCR法测定脂肪组织中PPAR-γmRNA,探讨黄芪散改善IR的机制。结果表明黄芪散改善IR的机制表现在:①上调T2DM-IR大鼠骨骼肌和脂肪组织中GLUT-4的mRNA和蛋白的表达,提高肝脏、骨骼肌和脂肪组织中IRS-1的mRNA和蛋白的表达,从而调节胰岛素信号转导通路,促进葡萄糖的转运,增加外周组织对胰岛素的敏感性;②显著下调TNF-α,leptin的水平,改善胰岛素信号通路转导,减少脂肪分解,纠正脂肪-胰岛内分泌轴的反馈机制,改善机体对瘦素的敏感性,从而改善机体对胰岛素的敏感性;③促进脂肪组织中调控胰岛素敏感性的重要受体PPAR-γmRNA的表达,间接发挥其改善IR的作用。2.黄芪散的制剂基础研究对不同产地葛根、黄芪药材进行质量考察,筛选质量中等的药材进行工艺考察。对葛根总黄酮的提取纯化工艺进行了研究,考察不同乙醇浓度,溶媒倍量,提取时间、提取次数和提取温度对总黄酮转移率和纯度的影响,建立了四因素三水平的正交试验,最终确立了最佳的提取工艺为:10倍量70%乙醇加热回流提取2次,每次2小时。采用大孔吸附树脂对总黄酮粗提物进行了纯化处理,筛选了不同型号的树脂,考察树脂的静态、动态吸附性能,并对上样液浓度和流速,洗脱溶剂浓度和体积等相关因素进行优化,确立了最佳的纯化工艺为:AB-8型大孔吸附树脂,药材与干树脂重量比2:1,上样液浓度为0.10g生药/ml,依次用10BV蒸馏水、10BV50%乙醇以1～3ml/min的洗脱速度洗脱,收集50%乙醇部分,减压回收乙醇,冷冻干燥,得到的总黄酮纯度在62.0%以上。对10个批次的葛根药材进行总黄酮提取物的制备,建立薄层鉴别方法;确定提取物干燥失重限度;建立紫外—可见分光光度法以及高效液相色谱法对提取物中总黄酮和葛根素的含量测定方法,初步建立葛根总黄酮提取物的质量标准。对黄芪总皂苷的提取纯化工艺进行了研究,考察不同乙醇浓度,溶媒倍量,提取时间、提取次数和提取温度对总皂苷转移率和纯度的影响,建立了四因素三水平的正交试验,最终确立最佳提取工艺为:8倍量70%乙醇加热回流提取3次,每次1.5小时。采用大孔吸附树脂对总皂苷粗提物进行了纯化处理,筛选了不同型号的树脂,考察了树脂的静态、动态吸附性能,并对上样液浓度和流速,洗脱溶剂浓度和体积等相关因素进行了优化,确立了最佳的纯化工艺为:药材与干树脂重量比3:1,上样液浓度为0.075g生药/ml,依次用10BV蒸馏水、10BV0.5%NaOH、10BV10%乙醇、10BV70%乙醇以1～3ml/min的洗脱速度洗脱,收集70%乙醇部分,减压回收乙醇,冷冻干燥,得到的总皂苷纯度在68.0%以上。对10个批次的黄芪药材进行总皂苷提取物的制备,建立薄层鉴别方法;确定提取物干燥失重限度;建立紫外—可见分光光度法以及高效液相色谱法对提取物中总皂苷和黄芪甲苷的含量测定方法,初步建立黄芪总皂苷提取物的质量标准。更多还原

【Abstract】 Insulin resistance（IR） is important factor and dominant feature in type 2 diabetes mellitus pathogenesis.Therefore IR treatment is the most important in prevention and treating diabetes mellitus and metabolic syndrome.Chinese medicine mainly apply compound recipe to treat disease,displaying features of integral regulation and combined therapy.Chinese herbal compound effective material basis are its chemical compositions or active sites,however just a few monomer compounds cannot be representing the whole recipe,should attach more important to the role of of chemical constituents group or active sites.Active sites effective application compatibility,there maybe present synergy,while reducing theextent of adverse reactions.Therefore,under the guidance of TCM theory,compatibility apply Chinese medicine active sites to develop innovative drugs,set up innovative Chinese medicine studying mode with demonstration effects,to improve the overall quality and competitiveness of Chinese medicine industry.Huang QI San ancient recipe origin from《Shengji Zonglu》,composed by radix Pueraria,Radix Astragali,Cortex Mori.rhe three medicines compatible application,could be nourishing qi to invigorate spleen,benefiting vital energy and clean fever,nourishing qi to invigorate spleen,treating triple diabetes together,to play the role of treating appearance and substance together.Much research indicate that the three medicines have the good curative effect to T2DM or IR,which active material and mechanism of effect are all different,therefore the recipe is presumed be through multi-ways, and multi-target targets comprehensive effect improve T2DM-IR.At present, the study based on Huang Qi San has not been reported,so its clinical application and development has certain limitation.The research firstly based on the whole recipe,attempted to interpret the effect and mechanism of Huang Qi San intervention in T2DM-IR,then apply a series of modern Chinese medicine purification and pharmaceutical production technology,to gather each medicine active sites,to make foundation for further preparations development.PartⅠ:Literature reviewAll recent relevant literature concerning T2DM-IR modern medicine research progression,concerning epidemiology,etiopathogenisis,and pathogenesis studies are documented.We also review the literature concerning TCM recognition in DM,chemical composition and Huang Qi San each medicine pharmacology research progression.So this study made a systematic review of mechanism,pathogenesis and allopathic medicine studies of IR in T2DM into consideration both TCM and modern medicine,latest molecule pathogenesis study achievement,and animal model establish and experiments study,to prehensile related advancing front research developments.PartⅡ:Laboratory studies1.The studies on effects and mechanisms of Huang Qi San interfere in laboratory IR in type 2 diabetes.To assess the effects of Huang Qi San on streptozocin（STZ） inducing DM mouse model,Hydrocortisone Sodium Succinate（HSCC） inducing IR mouse model. The results showed that Hang Qi San can lower fasting blood sugar（FBG） and increase sugar tolerance,and increase insulin sensibility of mice with abnormal sugar tolerance but not DM,to improve insulin resistance.Use STZ with high fat diet to set up T2DM-IR rat model.We determine model rat FBG,fasting insulin（FINS）,sugar tolerance,insulin tolerance,insulin sensitivity index（ISI）,and insulin resistance index（HOMA-IR）,lipid metabolism and free fatty acids content of serum and hepatic,HE staining of pancreas,to observe the effects of Huang Qi San on glycolipid metabolism and insulin resistance in T2DM-IR.The results showed Huang Qi San can significant lower FBG,improve sugar tolerance,lower serum hyperinsulinism induced by IR,increase insulin tolerance,ISI,lower HOMA-IR index,and protect pancreatic tissue structure.Meanwhile,Huang Qi San can regulate blood fat, hepatic lipid,down regulate serum and hepatic FFA level.That indicate Huang Qi San can improve glycolipid metabolism,increase insulin sensitivity to cure IR.On the basis of T2DM-IR model rats,apply radioimmunity（RI） method to determine TNF-α,leptin content in serum,apply RT-PCR method and immunohistochemical method to observe mRNA and protein expression of GLUT-4, IRS-lin liver,skeletal muscle and adipose tissue,apply RT-PCR method determine adipose tissue PPAR-γmRNA expression,to illuminate the mechanisms of Huang Qi San improve IR.The results showed the mechanisms of Huang Qi San improving IR followed as:①Up-regulate T2DM-IR rats skeletal muscle and adipose tissue GLUT-4 mRNA and protein expression,increase liver, skeletal muscle and adipose tissue IRS-1mRNA and protein expression,to regulate insulin signal transduction,promot glucose transportation,increase periphery tissue insulin sensitivity.②Down-regulate significantly TNF-α, leptin content in serum,improve organism leptin sensitivity,to improve organism insulin sensitivity.③Promote adipose tissue PPAR-γmRNA expressions,which are important receptor for controlling insulin sensitivity, improve indirectly IR.2.The Preparation of Basic Research on Huang Qi SanExamine quality of radix puerariae and radix astragali from different habitats,to sieve mediocre quality medical material for technology study. Studies were carried on the extraction and purification process of total flavonoids by comparing total flavonoids extract yield and purity of the impact of ethanol concentration,ratio of herb to solvent,extraction time,extraction times and extraction temperature.Set up the L₉（3⁴） orthogonal test and confirmed the best extraction process as 10BV 70%ethanol distilling two times, 2h per time.Extract of total flavonoids was purified with macroeticular resins. Different kinds of resins were applied to inspect the static and dynamic adsorption ability,to optimize the concentration,volume and flow rate of eluant.It was confirmed that the best purification process was AB-8 macroeticular resins,medical material dried resins being 2:1,sample fluid concentration being 0.10g dried medicinal herb per milliliter,eluted in order by 10BV distilled water,10BV 50%ethanol with flow rate of 1～3ml/min.50% ethanol part was collected,dried under vacuum and freeze drying resulting in the total flavonoids purity above 62.0%.Total flavonoids extract were prepared by 10 lots radix puerariae.The studies established TLC method. confirmed the limits of loss on drying,established quantitative analysis method of total flavonoids by ultra-voilet spectrophotometry and puerarin by RP-HPLC,for establishing the criteria of quality control of radix puerariae total flavonoids extract.Studies were carried on the extraction and purification process of radix astragali total saponins by comparing total saponins extract yield and purity of the impact of ethanol concentration,ratio of herb to solvent,extraction time,extraction times and extraction temperature.Set up the L₉（3⁴） orthogonal test and confirmed the best extraction process as 8BV 70%ethanol distilling three times,1.5h per time.Extract of total saponins was purified with macroeticular resins.Different kinds of resins were applied to inspect the static and dynamic adsorption ability,to optimize the concentration,volume and flow rate of ehant.It was confirmed that the best purification process was AB-8 macroeticular resins,medical material dried resins being 3:1,sample fluid concentration being 0.075g dried medicinal herb per milliliter,eluted in order by 10BV distilled water,10BV 0.5%NaOH,10BV 10%ethanol,10BV 70% ethanol with flow rate of 1～3ml/min.70%ethanol part was collected,dried under vacuum and freeze drying resulting in the total saponins purity above 68.0%.Total saponins extract were prepared by 10 lots radix astragali.The studies established TLC method,confirmed the limits of loss on drying, established quantitative analysis method of total saponins by ultra-voilet spectrophotometry and astragalosideⅣby RP-HPLC-ELSD for establishing the criteria of quality control of radix astragali total saponins extract.更多还原