| 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 technology1.1 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 D101and 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 Rf 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 IC50respectively.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 GLUT4 mRNA of STZ diabetic rats.
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