摘要
糖尿病发病率在世界范围内逐年上升,引起了世界各国的高度重视。目前治疗方式主要是利用药物控制血糖升高及防治并发症。由于中药具有低毒、降糖机制多元化的特点,成为降糖药物研究的热点。番石榴Psidiumguajava L.叶在我国有广泛分布,民间常用于防治糖尿病。研究证明番石榴叶富含黄酮类物质,而其降血糖作用与所含黄酮类物质有关。为了完善对番石榴叶降血糖有效部位及降血糖机制的研究,本论文摸索了番石榴叶有效部位总黄酮的提取纯化工艺,并考察了所提取的番石榴叶总黄酮对STZ糖尿病小鼠和STZ糖尿病大鼠血糖水平的影响;在此基础上,以STZ糖尿病大鼠为对象,从番石榴叶总黄酮对大鼠小肠黏膜α-葡萄糖苷酶活力、抗氧化能力、葡萄糖代谢能力、胰岛素抵抗的影响等方面对番石榴叶总黄酮降血糖机制进行了探讨。旨在为扩大番石榴叶在临床的应用提供理论基础和技术支持。
1.番石榴叶总黄酮提取纯化工艺的研究
1.1经正交设计,以总黄酮含量为指标,确定了番石榴叶总黄酮最佳提取工艺条件,发现影响番石榴叶总黄酮提取效果的主次因素为:乙醇浓度＞回流时间＞提取次数＞溶剂用量,而番石榴叶总黄酮最佳提取工艺为:10倍量70%乙醇加热回流提取2次,每次2小时。
1.2番石榴叶总黄酮提取液浓缩后,经石油醚、乙酸乙酯初步纯化,用大孔吸附树脂进一步纯化。以动态饱和吸附量、动态洗脱率为考察指标,比较了D_(101)、AB-8两种大孔树脂分离纯化番石榴叶总黄酮能力的优劣。又以总黄酮回收率为指标,从溶剂系统、洗脱速度、洗脱剂用量方面对筛选出的最佳树脂的吸附工艺参数进行了研究。发现在考察的2种树脂中,AB-8型树脂最适于番石榴叶总黄酮的分离纯化,其优化工艺条件为:4倍树脂体积50%乙醇洗脱,速度2 ml/min。经梯度洗脱及薄层层析分析发现,收集30%～50%乙醇区间洗脱液可进一步纯化番石榴叶总黄酮(TFPL)。
1.3按所确定的最佳提取纯化工艺提取纯化的TFPL经定性分析,证实为黄酮类物质;薄层层析可见4个黄酮类物质斑点,其Rf值分别为0.84;0.67;0.55;0.41。其中R_f值0.84的斑点可能为槲皮素。
按以上所确定的提取及纯化工艺进行提取,TFPL的提取率为0.31%±0.07(n=23),所制备TFPL的平均纯度为89.46%±1.28;RSD=1.40%(n=23)。
2.番石榴叶总黄酮对STZ糖尿病小鼠、大鼠血糖水平的影响
2.1在STZ糖尿病小鼠模型上,观察TFPL对空腹血糖水平的影响,比较体重及心脏、肝脏、肾脏、和胰腺的脏器指数变化。共设糖尿病模型组、糖尿病+阳性降糖药物组、糖尿病+TFPL低(140mg/kg)、高剂量组(210mg/kg)、正常对照组等共5组,治疗14d后,模型组小鼠空腹血糖值达29.49±6.67mmol/L,TFPL低、高剂量组小鼠空腹血糖值分别降低至18.12±2.91、16.95±3.57 mmol/L;差异有显著性意义。说明TFPL可降低STZ糖尿病小鼠空腹血糖。但在各组STZ糖尿病小鼠的体重及脏器指数方面没有表现出显著性差异。
2.2在STZ糖尿病大鼠模型上,考察TFPL对空腹血糖、淀粉负荷后血糖水平的影响,并检测了糖化血清蛋白、糖化血红蛋白的含量。共设糖尿病模型组、糖尿病+阳性降糖药物组、糖尿病+TFPL低(900mg/kg)、高剂量组(120mg/kg)、正常对照组等共5组,治疗8周后,模型组大鼠空腹血糖值达27.89±4.89mmol/L,TFPL低、高剂量组大鼠空腹血糖值分别显著性降低至21.76±2.27、17.27±2.43mmol/L。淀粉负荷1h、2h血糖分别从模型组大鼠的28.10±1.87、26.68±1.98 mmol/L显著性降低至TFPL低剂量组大鼠的24.20±1.60、19.55±1.18mmol/L,TFPL高剂量组大鼠的22.88±1.60、17.29±1.58mmol/L。TFPL低、高剂量组大鼠糖化血清蛋白、糖化血红蛋白的含量比模型组大鼠也均有显著性降低。说明TFPL可降低STZ糖尿病大鼠空腹、淀粉负荷后血糖,降低STZ糖尿病大鼠糖化血清蛋白、糖化血红蛋白的含量,对长期血糖控制有一定效果。
3.番石榴叶总黄酮降血糖机制研究
3.1提取正常SD大鼠小肠黏膜α-葡萄糖苷酶,以蔗糖、麦芽糖为底物,考察了TFPL对所提取小肠黏膜α-葡萄糖苷酶的体外抑制作用,发现TFPL可抑制正常SD大鼠小肠黏膜α-葡萄糖苷酶活力,对蔗糖酶及麦芽糖酶的抑制率分别达70%和60%以上,其IC_(50)分别为2.01、1.96g/L。
测定了药物治疗8周后的各组大鼠小肠黏膜α-葡萄糖苷酶活性,发现模型组大鼠蔗糖酶及麦芽糖酶活性均较正常组大鼠有显著升高,而TFPL低、高剂量组大鼠中两种酶活性较模型组均有下降。
以上实验说明TFPL可抑制正常及STZ糖尿病大鼠小肠黏膜α-葡萄糖苷酶活性,从而延缓碳水化合物的消化吸收,达到降低血糖的目的。
3.2增强机体的抗氧化能力是防治糖尿病及其并发症的一个重要方向。本实验以过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-PX)、超氧化物歧化酶(SOD)、一氧化氮和酶(NOS)活性及丙二醛(MDA)含量为指标,考察了TFPL对STZ糖尿病大鼠血清、肝组织、心肌组织抗氧化能力的影响。发现治疗8周后,模型组大鼠较正常组大鼠的CAT、GSH-PX、SOD活力均降低,而NOS活力及MDA含量增加,说明其抗氧化能力被损伤;与模型组比较,TFPL治疗组大鼠的CAT、GSH-PX、SOD的活力增高,NOS活力及MDA含量降低。说明TFPL可减少STZ糖尿病大鼠抗氧化能力的损伤。
3.3糖尿病以葡萄糖代谢障碍为特点,增强糖代谢能力可改善高血糖及其引起的损害。本实验以肝、肌糖元含量及血清、肝丙酮酸激酶活性为指标,考察了TFPL对STZ糖尿病大鼠葡萄糖代谢能力的影响:模型组大鼠与正常组大鼠比较,肝、肌糖元含量下降,血清及肝组织PK活性下降,表现出葡萄糖代谢能力低下;而TFPL治疗组大鼠与模型组大鼠比较,其肝、肌糖元含量增加,血清及肝组织PK活性增加,说明TFPL可增强STZ糖尿病大鼠的葡萄糖代谢能力。
3.4为了研究TFPL对STZ糖尿病大鼠胰岛素抵抗的影响,提取了各组大鼠肝、骨骼肌总RNA,合成相应cDNA,利用RT-PCR方法观察TFPL对大鼠肝胰岛素受体(ISR)、骨骼肌ISR、骨骼肌葡萄糖转运蛋白4(GLUT4)mRNA表达的影响,发现模型组大鼠与正常组大鼠比较,肝ISR、骨骼肌ISR、骨骼肌GLUT4 mRNA表达均下降,而TFPL治疗组大鼠较模型组大鼠肝ISR、骨骼肌ISR、骨骼肌GLUT4 mRNA表达都有上调,说明TFPL可改善STZ糖尿病大鼠的胰岛素抵抗。
综上所述,本实验所确定的番石榴叶总黄酮提取纯化工艺稳定,所提纯的TFPL纯度高;经动物实验发现,所提纯TFPL可降低STZ糖尿病小鼠空腹血糖,降低STZ糖尿病大鼠空腹、淀粉负荷后血糖,长期血糖控制能力好。此外,实验发现TFPL可抑制正常大鼠和STZ糖尿病大鼠小肠黏膜α-葡萄糖苷酶活性、抑制STZ糖尿病大鼠血清、肝、心肌组织CAT、GSH-PX、SOD、PK活力的下调、抑制STZ糖尿病大鼠肝糖元、肌糖元含量的下降、抑制STZ糖尿病大鼠NOS活力及MDA含量的增加,抑制STZ糖尿病大鼠肝ISR、骨骼肌ISR、骨骼肌GLUT4 mRNA表达的下调。说明TFPL可能通过以上机制发挥降血糖作用。
The rate of Diabetes mellitus rises year by year worldwide,which aroused highly attention from every country in the world.The present treatment is to use medication to prevent the blood glucose level from increasing and avoid complication.The traditional Chinese medicine has become very popular, because there is very little poison contained in it and it has a diversity of blood glucose decreasing mechanism.Psidium guajava L.leaves are widely distributed in our country.They are generally used for the treatment of diabetes. It was proved by research that constituents containing flavones were found in Psidium guajava L.leaves,which was related to the function of decreasing blood glucose level.For the purpose of optimizing the research of blood glucose decreasing functional parts of Psidium guajava L.leaves and its functional mechanism,the present study dealed with the technology of extraction and purification of the total flavones,which are the effective part of Psidium guajava L.leaves,and investigated the influence of the total flavones of Psidium guajava L.leaves on the blood glucose level of streptozotocin(STZ)-induced diabetic mice and rats.It served as a base for the investigation of blood glucose decreasing functional mechanism of the total flavones of Psidium guajava L. leaves with STZ diabetic rats as the object,which carried out on the influence of the total flavones of Psidium guajava L.leaves to the activity ofα-glucosidase in the small intestinal mucosa of rats,the influence to the anti-oxidation capacity,the influence to the glucose metabolism capacity,the influence to the insulin resistence of rats aspects.It would provide a theoretical and technical base for spreading the clinical application of Psidium guajava L.leaves.
1.Research in extraction and purification technology
1.1 Through an orthogonal design,with the content of flavones as an index,the optimun technical prcess of extracting the total flavones from Psidium guajava L.leaves was determined.The factors influencing the extraction of the total flavones from Psidium guajava L.leaves are:the concentration of ethanol＞heating times＞extraction times＞the dosage of ethanol.The optimum process of the extraction technology was:70%ethanol,ten-fold solvent,two times extraction,two hours each time.
1.2 After the primitive purification of the total flavones of Psidium guajava L. leaves by petroleun ether and ethyl acetate,with the dynamic saturation adsorption and dynamic elution ratio served as indexes,the separating and purifying quality of the D_(101)and AB-8 macroreticular resins was compared. Then with the reclaim ratio of the total flavones as an index,the best technical absorption parameter was investigated on the eluent system,eluenting speed, eluent dosage aspects.It was found that to the two kinds of resins,AB-8 resin was more favorable in the separation and purification of the total flavones of Psidium guajava L.leaves,and the technical conditions were:eluted with 4BV of 50%ethanol,at the speed of 2ml/min.
Through an analysis of gradient elution and thin layer chromatography,it was found the collecting 30%-50%ethanol elution section could further purify the total flavones of Psidium guajava L.leaves(TFPL).
1.3 With some determining analysis,the extracted and purified TFPL according the optimum technical process was proved to be constituents of flavones.Four spotted constituents of flavones could be seen after thin layer chromatography, the R_f value of which was 0.84,0.67,0.55,0.41,respectively.The one with Rf 0.84 maybe quercetin.
Extracting with determined extraction and purification technology,the TFPL extraction ration was 0.31%±0.07(n=23),the average TFPL purity quotient was 89.46%±1.28;RSD=1.40%(n=23)
2.The influence of the total flavones of Psidium guajava L.leaves on blood glucose level of STZ diabetic mice and rats.
2.1 the TFPL's influence on blood glucose in STZ diabetic mice model was observed,and it was compared that the weight and the change of organ index in heart,liver,kidney,and pancreas.Five group was seted:diabetes model group, diabetes+positive blood glucose decreasing drug group,diabetes+low (140mg/kg)and high(210mg/kg)TFPL dosage groups,and normal control group.After a 14-day treatment,the fasting blood glucose in model group reached 29.49±6.67mmol/L,and 18.12±2.91、16.95±3.57 mmol/L in low and high TFPL dosage group,which showed that TEPL could decrease the fasting blood glucose in STZ diabetes mice.But to the body weight and the organ index, there were not remarkable different be found.
2.2 The influence of TFPL on the fasting blood glucose and the blood glucose level after loading starch was investigated,and the concentration of glycosylated serum protein and glycosylated hemoglobin was checked.Five diabetic model group was seted:diabetes+positive blood glucose decreasing drug group, diabetes+low(900mg/kg)and high(120mg/kg)TFPL dosage groups,and normal control group.After an 8-week treatment,the fasting blood glucose in model group reached 27.89±4.89mmol/L,and 21.76±2.27,17.27±2.43mmol/L in low and high TFPL dosage group.The level of blood glucose at 1h,2h after starch loading reduced from 28.10±1.87,26.68±1.98 mmol/L in model group to 24.20±1.60,19.55±1.18mmol/L in low TFPL dosage group and 22.88±1.60, 17.29±1.58mmol/L in high TFPL dosage group.The concentration of glycosylated serum protein、glycosylated hemoglobin in low and high TFPL dosage groups reduced remarkably,compared to model group.It showed that TFPL could reduce the fasting blood glucose and the blood glucose level after starch loading in STZ diabetic rats,and had certain effect on long term blood glucose controlling.
3.Research in the blood glucose decreasing mechanism of the total flavones Of Psidium guajava L.leaves.
3.1 Theα-glucosidase was extracted from the small intestinal mucosa of normal SD rats,after that,with the sucrose and maltose as substrate,investigated the inhibiting function of TFPL on which ex vivo.It was found that TFPL could inhibit the activity ofα-glucosidase in the small intestinal mucosa of normal SD rats.The sucrase or maltase inhibiting rates weree over 70%and 60%,with 2.01 and 1.96g/L of IC_(50)respectively.The activity ofα-glucosidase in the small intestinal mucosa of each group of STZ diabetic rats after 8 weeks medication was examined.The sucrase or maltase of diabetic rats from model group increased,compared to normal group,while decreased in low and high dosage group,which showed that TFPL could inhibit the activity ofα-glucosidase in the small intestinal mucosa of normal and STZ diabetic rats,which would restrain the digestion and absorption of carbohydrates,then decresed the blood glucose lever of rats.
3.2.Improving the anti-oxidation ability was one treatment to diabetes mellitus and its complication.With the activity of catalase(CAT),Glutathione peroxidase(GSH-PX),Superoxide dismutase(SOD),NO synthase(NOS)and the concentration of Malondialdehyde(MDA)as the index,it was investigated that the influence of TFPL on anti-oxidation ability of the serum,liver,and cardiac muscle in STZ diabetic rats,it was found that the activity of CAT,GSH-PX, SOD decreased in model group,compared to normal group,while the activity of NOS and the concentration of MDA increased,which showed that the anti-oxidation capacity was destroyed.
Compared to model group,TFPL could raise the activity of CAT、GSH-PX、SOD,and reduced the activity of NOS and the concentration of MDA. It proved that TFPL could prevent the anti-oxidation capacity of STZ diabetic rats from being destroyed.
3.3 The metabolism of glucose was abnormal in diabetes mellitus,increasing the capacity of glucose metabolism could improve the high blood glucose level and the damage induced.In the examination of the influence of TFPL on glucose metabolic capacity of STZ diabetic rats,it was found that the concentration of glycogen of liver and muscle were decreased in model group,compared to normal group,and the PK activity of serum and liver organ were also decreased, while the concentration of glycogen of liver and muscle were increased and the PK activity of serum and liver increased in TFPL treatment group,compared to model group.It proved that TFPL could strengthen the glucose metabolic capacity of STZ diabetic rats.
3.4 To investigate the influence of TFPL to the insulin resistance of STZ diabetic rats,the study extracted the general RNA in liver and skeletal muscle of rats in each group;synthesized the corresponding cDNA,observed the expression of liver and skeletal muscle ISR mRNA and skeletal muscle GLUT4 mRNA of diabetic rats with the method of RT-PCR.It was found that their expression were all decreased in model group,compared to normal group,while increased in TFPL group,compared to model group.It proved that TFPL can improve the insulin resistance of STZ diabetic rats.
As stated above,the total flavones of Psidium guajava L.leaves extraction and purification technology determined in this experiment was stable,and the extracted TFPL had a high purity quotient,which could decrease the fasting blood glucose lever in STZ diabetic mice,the fasting blood glucose and blood glucose level after starch loading in STZ diabetic rats.Its blood glucose decreasing mechanism might relate to its function on inhibiting the activity ofα-glucosidase in the small intestinal mucosa of rats,inhibiting the CAT,GSH-PX, SOD,PK activity,decreasing the concentration of glycogen of liver and skeletal muscle,increasing the NOS activity and the concentration of MDA,and restraining the expression of liver and skeletal muscle ISR mRNA,skeletal muscle GLUT_4 mRNA of STZ diabetic rats.
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