【作者】 魏媛媛；

【导师】 帕尔哈提·克热木； 阿吉艾克拜尔·艾萨；

【作者基本信息】 新疆医科大学 ， 药理学， 2011， 博士

【摘要】 目的:石榴花是传统维吾尔药材,用于治疗具有“三多一少”特征的疾病,现代医学研究发现石榴花提取物具有良好的降糖活性。本研究通过提取石榴花多酚,建立糖尿病大鼠模型,观察其对糖尿病大鼠血糖、血脂以及胰岛素抵抗的影响,并进一步探讨石榴花多酚降糖及缓解胰岛素抵抗的分子机制,为开发和有效利用维吾尔地方药材提供理论依据。方法与结果:第一部分:石榴花多酚制备选用D101大孔树脂纯化方法提取石榴花,以总酚含量为评价指标,获得多酚得率为68%的石榴花多酚。并通过正交试验,研究不同因素对石榴花多酚物质浸出率的影响,确定最佳的提取方法为:以60%乙醇作溶液,料液比为（g∶ml）1∶20,在50℃下提取2次,每次提取时间为1h,此条件下稳定性较好。第二部分:石榴花多酚对糖尿病大鼠药效学研究1.制备糖尿病大鼠模型高脂饲料加腹腔注射链脲佐菌素（STZ）45mg·kg^-1复制糖尿病大鼠模型,空腹血糖（FPG）≥11.1mmol·L^-1,确定为糖尿病成模大鼠。所得模型鼠明显多饮、多食、多尿、喜卧、毛发灰暗、生长缓慢;并且具备高空腹血糖,高胰岛素水平及胰岛素抵抗等特点,接近人类2型糖尿病发病特征。2.分组给药并检测指标以二甲双胍(100mg·kg^-1)为阳性对照药物,石榴花多酚(100mg·kg^-1,300mg·kg^-1)给药四周后,分别测定各项指标,结果如下:石榴花多酚两剂量组和二甲双胍组均能有效降低糖尿病大鼠血清中的FPG、TC、TG、LDL-C;显著升高HDL-C/TC比值（P<0.05,P<0.01）;对体重无明显影响;石榴花多酚两剂量组和二甲双胍组均能有效降低糖尿病大鼠FIns水平,降低IRI,并增加ISI（P<0.05,P<0.01）。光镜下分别观察肝脏和胰岛细胞的病理学改变显示石榴花多酚两剂量组对糖尿病大鼠肝脏组织和胰岛细胞有一定的保护作用。第三部分:石榴花多酚降糖机制的研究1.石榴花多酚对糖尿病大鼠糖原合成的影响及机制测定大鼠肌、肝糖原含量（MG、HG）含量;骨骼肌己糖激酶（HK）活性、肝脏葡萄糖激酶（GK）活性;Western Blot法检测大鼠骨骼肌中糖原合酶激酶-3β（GSK-3β）和AMP活化蛋白激酶α（AMPKα）和磷酸化AMP活化蛋白激酶α（P-AMPKα）的蛋白含量。结果显示:石榴花多酚可有效增加MG含量（P<0.05,P<0.01）,PFPⅡ组HK活性明显增强（P<0.01）;但肝脏HG含量和GK活性无明显改变（P>0.05）;PFP I组中GSK-3β的蛋白表达降低（P<0.01）,P-AMPKα的表达量（P<0.01）及P-AMPKα/AMPKα（P<0.05）则显著增加。2.石榴花多酚对糖尿病大鼠胰岛素抵抗水平的影响分光光度法测定血清游离脂肪酸（FFA）浓度;ELISA法测定血清白介素-6（IL-6）和白介素-18（IL-18）含量。分光光度法观察石榴花多酚溶液在Mn²⁺-H₂O₂-PAR体系中对·OH的抑制率和对DPPH·的清除率;分别检测大鼠肝脏和骨骼肌中超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、还原型谷胱甘肽（GSH）和丙二醛（MDA）。RT-PCR法测定大鼠肝脏中PPARα/γmRNA水平;Western Blot法检测PPARα/γ蛋白表达水平。结果显示:1)石榴花多酚能有效降低糖尿病大鼠血清FFA和IL-6,两个剂量组均（P<0.05）。但对IL-18无显著影响（P>0.05）;2)体外抗氧化实验中石榴花多酚(0.08mg·ml^-1)在Mn²⁺-H₂O₂-PAR体系中对·OH的清除率46%,相当于Vc(0.2mg·ml^-1)的3倍;石榴花多酚(0.02mg·ml^-1)对DPPH·的清除率87%,约为Vc(0.2mg·ml^-1)的5倍。石榴花多酚能有效增加糖尿病大鼠肝脏CAT活力（P<0.05）;PFPⅡ组SOD显著升高（P<0.01）;PFPⅠ组MDA则显著降低（P<0.05）;骨骼肌中,PFPⅡ组大鼠SOD显著升高（P<0.01）;两剂量组中MDA明显降低,GSH则显著增加（P<0.05,P<0.01）;3)石榴花多酚两个剂量组中PPARγmRNA的表达增强（P<0.01）,PFPⅠ组中PPARαmRNA的表达量增加;PFPⅠ组中PPARγ蛋白表达量增加（P<0.05）,两剂量组中PPARα蛋白表达量均上调（P<0.05）。3)初探石榴花多酚对糖尿病大鼠血管内皮功能的影响ELISA法测定大鼠血浆中以下各指标,结果显示:石榴花多酚两个剂量组中ET-1、ATⅡ和TXB2浓度均显著降低,6-Keto-PGF1a显著增加,TXB2/6-Keto-PGF1a均明显降低（P<0.01,P<0.05）。结论:1.石榴花多酚具有良好的降糖作用:通过增加糖尿病大鼠骨骼肌糖原含量降低血糖;机制可能与增强骨骼肌组织中己糖激酶活性,下调糖原合酶激酶-3的蛋白表达及增强骨骼肌AMPK活性有关。2.石榴花多酚可以缓解胰岛素抵抗水平:可能与降低血清中TG、FFA和血清炎症介质IL-6含量;清除糖尿病大鼠体内自由基有关;还可通过上调肝脏PPARα/γ的蛋白表达,提高肝脏组织对胰岛素敏感性;通过降低骨骼肌中GSK-3β的表达,增加AMPK活性,提高骨骼肌组织对胰岛素的敏感性。3.石榴花多酚对糖尿病大鼠的血管内皮有一定的保护作用:血浆中ET-1和ATⅡ浓度及TXB2和6-Keto-PGF1a比值下降提示石榴花多酚对糖尿病大鼠的血管内皮起到一定的保护作用。更多还原

【Abstract】 Objective:Pomegranate flower in traditional Uygur medicine is wildly used for the treatment of the disease with a little "as" san-duo symptom, modern medical research have found that pomegranate flower extract has effective hypoglycemic activity, In this study, effect of PFP on blood glucose, blood lipids and insulin resistance of diabetic rats were observed through extraction of pomegranate flower polyphenols （PFP） and establishment of diabetic rats, then hypoglycemic mechanism of PFP was further explored, which provide a theoretical data and basic information for the development and effective use of Uighur local herbs.Methods:PARTⅠPomegranate flower polyphenol extractionD101 macroporous resin purification method was used to extract pomegranate polyphenols; with total phenol content as evaluation index, finally 68% of pomegranate flower polyphenols （PFP） were got from the effective part of pomegranate flower. And orthogonal experiment was used to study different factors on the leaching rate of pomegranate flower polyphenols in order to get optimum extraction process as follows: pomegranate flower was extracted for 2 times at 50℃, each extraction time covered 1h; the solution with 60% ethanol and solid-liquid was mixed as 1∶20 （g∶ml） .PARTⅡPharmacodynamic studies of PFP on diabetic rats1.Establishment of diabetic rats modelsThe models is established by feeding with high fat diet plus intraperitoneal injection of streptozotocin （STZ） 45mg·kg^-1.The rats whose fasting plasma glucose （FPG） ≥11.1mmol·L^-1 are identified as diabetic model rats. Diabetic model rats tend to drink, eat and urinate more than normal and like lying down, whose hair become gloomy and FPG, insulin levels insulin resistance become higher. Characteristics of model rats close to the human type 2 diabetes.2.Administration and Measurement of indexesWith metformin (100mg·kg^-1) as a positive control, diabetes rats fed with pomegranate flower polyphenols (100mg·kg^-1, 300mg·kg^-1) dose respectively for four weeks, then Weight, FPG, GLR, Fins, IRI, lipid profiles were detected.Results: Compared with diabetic models rats, fasting plasma glucose of rats in two-dose group and metformin group effectively reduced, as well as plasma triglycerides, low-density lipoprotein cholesterol, total cholesterol. while ratio of high-density lipoprotein to total cholesterol （HDL-C/TC） was higher. （P<0.05, P<0.01）; Serum insulin levels as well as IRI were reduced significantly, while ISI was effectively elevated in two dose group. （P<0.05, P<0.01）. In addition, PFP showed a protective effect on liver tissue and islet B cells of diabetic rats by pathology examination under light microscopy.PARTⅢHypoglycemic mechanism of PFP1.Effects of PFP on glycogen synthesis and mechanism research Effects of PFP on glycogen synthesis were evaluated by measuring the following items: MG and HG content, skeletal muscle HK activity ,GK activity; then Western Blot assay was applied for checking GSK-3βand AMPKαandP-AMPKαprotein expression in skeletal muscle .Results: Compared with diabetic models rats, MG level in both group （P<0.05, P<0.01） and HK activity in PFPⅡgroup （P<0.05） were effectively elevated; however HG and GK activity in liver showed no significant change （P>0.05）; GSK-3βprotein expression effectively reduced in PFPⅠgroup （P<0.01）; P-AMPK/AMPK significantly increased in PFPⅠgroup （P<0.05）.2.Effect of PFP on IR of diabetic ratsSerum FFA was measured by UV-visible spectrophotometry; serum IL-6 and IL-18 were checked by ELISA method; antioxidant activity of PFP was evaluated by clearance of DPPH·and·OH in Mn²⁺-H₂O₂-PAR system in Vitro; then SOD、CAT、GSH and MDA in liver and skeletal muscle were detected respectively; PPARα/γmRNA level by RT-PCR and PPARα/γprotein expression by Western blot were measured.Results: Compared with model rats, 1)Serum FFA （P<0.05） and IL-6 （P<0.01） were significantly higher in two-dose group; however IL-18 had no significant change （P>0.05）. 2) In vitro, PFP (0.08mg·ml^-1) in the clearance rate of·OH 46%is about three times of Vc (0.2mg·ml^-1) in Mn²⁺-H₂O₂-PAR system. PFP (0.02mg·ml^-1) with scavenging rate of DPPH·87%is about 5 times of Vc (0.2mg·ml^-1). Antioxidant activity of PFP in liver: Compared with model rats,CAT significantly increased in both PFP group （P<0.05）; SOD was significant higher （P<0.01） in PFPⅡgroup; MDA was significant lower in PFPⅠgroup （P<0.05）. In skeletal muscle : SOD were significantly elevated in PFPⅡgroup （P<0.01）; MDA was significantly reduced, GSH was significant increased in both PFP group. （P<0.05, P<0.01）. 3) Compared with model rats,PPARγmRNA （P<0.01） in both dose group and PPARαmRNA in PFPⅠgroup （P<0.01） significantly increased; PPARγprotein expression in PFPⅠgroup （P<0.05） and PPARαprotein expression in both PFP group （P<0.05）, were effectively upgrulated.3.Effect of PFP on vascular endothelium of diabtic ratsPlasma ET-1, ATⅡ, TXB2 and 6-Keto-PGF1a were detected by ELISA. Results: compared with model rats, ATⅡand ET-1 concentrations were significantly reduced in two-dose group （P<0.05）, and significant reduction of TXB2 （P<0.01） and increment of 6-Keto-PGF1a （P<0.01; P<0.05） caused a significant reduction in TXB2 and 6-Keto-PGF1a ratio （P<0.01）.Conclusion:1) PFP perform hypoglycemic function in diabetic rats, possibly related to the following aspects: reduction of blood glucose in diabetic rats was probably induced by incremental glycogen content of skeletal muscle.and incremental skeletal muscle glycogen content was possibly caused by enhancement of hexokinase activity, which is the rate-limiting enzyme in glycogen synthesis process. In addition, another possible hypoglycemic mechanism was related to reduction of the protein expression of GSK-3βand significant enhancement of AMPK activity. 2) PFP are able to alleviate insulin resistance level in diabetic rats: the possible mechanism related to the following aspects: reducution of serum TG, FFA and serum inflammatory mediators IL-6 in diabetic rats and antioxidant activity; moreover, PFP are able to improve insulin sensitivity in liver tissue of diabetic rats by enhancing both protein expression of PPARγand PPARα, function as dual PPARα/γagonists and improve insulin sensitivity in skeletal muscle by reducing GSK-3βexpression and enhancing AMPK activity in skeletal muscle. 3) Protective effect of PFP on vascular endothelial of diabetic rats: the results of plasma ET-1, ATⅡ, TXB2 and 6-Keto-PGF1a suggested that: PFP may have preventative and protective effect on vascular complications of type 2 diabetes.更多还原