| BackgroundAs an important phase of the natural course of diabetes, impaired glucose tolerance refers to the abnormal metabolism condition between normal glucose steady state and diabetes. In1985, WHO defined clinic IGT as the metabolism condition of limosis blood sugar being less than7.0mmoI/L and being between7.8mmol/L and11.lmmol/L2hours after being loaded. It has been proved by research that obvious insulin resistance and secretion defects of pancreatic β cells have appeared in this stage, leading to the obvious increase of incidence rate of diabetes. Clinic research shows that unhealthy life style and environmental factors lead to overweight or obesity, and then insulin resistance, increased load of β cells, defects of pancreas islet function, amd finally IGT. According to existing research, the abnormality of pancreas islet β cells and IR paly a decisive role in the appearance of T2MD. The function of β cells of IGT patients is in the compensatory stage with relatively mild damage. It can be concluded that with positive intervention of IGT, patients can be cured. According to the research report of International Diabetes Federation(IDF) and American Diabetes Association(ADA), nearly all diabetes patients will go through the stage of IGT. The report from Evidence-based Practice Center of McMaster University points out that to those people with impaired glucose tolerance, the relative danger level of impaired glucose tolerance turning into diabetes is6times higher than that of those with normal sugar tolerance. In terms of the relative danger of dying from various reasons, the probability of the former is1.5times higher than that of the latter and in terms of death caused by cardiovascular diseases, the former is1.7times higher than that of the latter.According to the epidemiological research, there are about92.4million diabetes patients and about148million IGT patients. In recent years, the prevalence rate of IGT keep increasing and10%-15%of IGT patients entering the stage of Type2Diabetes Mellitus. According to investigation results, among Chinese IGT patients, the rate of turning into diabetes is about8%-10%, ranking among the front row around the world. Therefore, IGT patients are highly inclined to become diabetes patients. It is imperative for us to improve and guard against IGT and then prevent it from developing into diabetes.Prebiotics refers to the matters that are undigested or difficult to be digested. Also called as bifidus factors, they can selectively stimulate the growth and activity of intestinal probiotic bacteria and promote the growth of e-host. Through promoting the proliferation of intestinal probiotic bacteria like bifid bacteria and lactobacillus prebiotics can improve the environment of intestinal flora. Prebiotics can be found in mostly undigested matters like oligosaccharide, polysaccharide, resistant starch, peptides, and protein.Abundant documents have revealed that oligosaccharide, polysaccharide, and resistant starch can not only stabilize blood glucose, but also promote the growth of intestinal probiotic bacteria. However, the comparative study of the difference of treatment effects of various breeds can rarely be found. This experiment aims to, through inducing rats to develop IGT after feeding them high-sugar and high-fat diet and injecting small dose alloxan, the clinical characters of IGT, and establishing the model of IGT rats, observe the effects of peach gum polysaccharide, resistant starch, and Fructooligosaccharide on the IGT rats and the growth of intestinal probiotic bacteria of normal rats and provide experimental basis for developing new healthcare food.Objective1. After establishing the model of IGT rats through feeding high-sugar and high-fat food and the injection of small dose alloxan, this thesis will observe the effects of peach gum polysaccharide, resistant starch, and fructooligosacch-ride on the fasting blood-glucose,blood tolerance and other experimental indicators of IGT rats.2. Compare the hypoglycemic effects of those three healthcare food and provide experimental basis for developing new healthcare drugs.3. Compare the effects of peach gum polysaccharide, resistant starch, and fructo-oligosaccharide on the bifid bacteria and lactobacillus prebiotics of normal rats.Methods1The distraction and separation of peach gum polysaccharideAfter being rouged and rinsed, fresh peach gum will be frozen dried, grinded up, extracted in the water with the temperature of90℃, and deproteinized through Sevage method. With the method of gradient alcohol sinking, the deproteinized liquid glucose will be deposited with the mixing of absolute ethyl alcohol, reaching the concentration of90%. After extracting the concentration through centrifuge, we can get peach gum polysaccharide, which will be purified by glucan gel column chromatography.2Establishing the model of IGT rats44male SD rats, with weight ranging from160g to200g, will be randomly divided into two groups, the normal group and the model group. During the experiment, the normal group will be given with basic feed and the model group will be given with high-sugar and high-fat feed made up of a mixture of75%of basic feed,10%of palm oil and15%of cane sugar. After three weeks’feeding, the rats of the model group will be injected with alloxan and restricted from eating(water not included) for12hours. The alloxan will be made into2%alloxan and the amount will be80mg/kg in the first day,80mg/kg in the third day and40mg/kg in the fifth day. One week after the injection, the rats will receive oral glucose tolerance test(OGTT) and be gavaged with20%of sugar with the standard of2g/kg. Then the blood of the vein will be collected after0,30,60,90,120minutes. The blood glucose levels of different time point were calculated through glucose method to calculate the Area Under the Curve(AUC). The calculating method can be listed as follows:mmol/L=1/2(G0+G30)×30+1/2(G3o+G60)×30+1/2(G60+G90)×30+1/2(G9o+G120) X30(Go,G3o,G6o,G120represent the blood level after0,30,60,90,and120minutes respectively).The standard of assessing the bood glucose level is the limosis sugar level of the model group is similar to that of the normal group but the area below the curve is larger than that of the normal group, which is meaningful in statistics.During the process of establishing model, one rat from the normal group,3rats from the model group died. Apart from the dead rats mentioned above, about32rats fitting the standard of IGT rat model will be selected and divided into three groups according to the area under the curve of blood glucose level:IGT rat model group, Peach gum polysaccharide group, resistant starch group, oligosacchridee group, each consisting of8rats.3Oral glucose tolerance test (OGTT)After successfully establishing the model of IGT rat model,32rats fitting the standard of IGT rat model will be selected and divided into three groups according to the area under the curve of blood glucose level:IGT rat model group, Peach gum polysaccharide group, resistant starch group, oligosaccharide group, each consisting of8rats. The normal group and the model group will be treated with normal saline respectively and the third group will be treated with peach gum polysaccharide (5g/kg),the forth group with fructooligosaccharide (5g/kg),the fifth group with banana resistant starch(5g/kg). The latter three groups will be fed with high-fat forage and20days later, receive glucose tolerance test. The area under the curve of blood glucose level and the fluctuation of blood glucose level at different time point will be compared.4Intestinal flora analysis The asepsis sample of a small amount of fresh dejection will be made and weighed before feeding and24hours after the last time feeding. The sample will be put into the normal saline whose weight is ten times of the dejection. Then it will be put into the dry sterilized small tube and diluted with sterilized diluent to turn the proportion into10-8, which will be transferred to the designated medium by a pipette. The bifidobacterium(anaerobic cultured with37℃in72hours), lactobacillus(slight anaerobic cultured with37℃in48hours) of the dejection will be monitored to compare the change before and after the feeding of foods. The number of bacterium will be calculated in terms of colony forming unit(CFU).5Statistics analysisThe data will be analyzed with SPSS13.0and the result will be listed as follows: Mean±Standard deviation(X±S). Separate samples will be compared through one-way ANOVA. With variance being the same, LSD is available. With different variances, Welch method is available and two numbers will be compared through the LSD method and Dunnett T3method, which is also adopted to compare the impact of different control groups on glucose tolerance.Result1The extraction and separation of Peach gum polysaccharideThree kinds of PPR with different molecular weight were extracted by hot water extraction, Sevage method to take off the protein, Gradient alcohol sink, purification by Glucan gel chromatography column. PPR status to beige solid powder, yield is29.8%.2The condition of the model ratsNormal rats are in good condition with normal eating, drinking, urination and defecation, sensitive reaction, glossy hairy back and obvious increase of weight. The model group suffer from polyuria and polydipsia with normal psychological condition and activity, less glossy hair.4rats died from probable alloxan damage or hypoglycemia. After being injected with a small amount of alloxan and fed with high-fat and high-sugar forage, the rats will receive OGTT (F=5.206,P=0.028). The areas, examined by Levene, below the IFG group are variant in heterogeneity. Obvious difference can be found through Satterthwaite method(t=-8.232, P=0.000). It can be concluded from the mentioned above that the IGT model is successful.3The therapeutical effects of peach gum polysaccharide, resistant starch,and fructooligosaccharide on IGT model rats3.1The condition of rats receiving the treatment of peach gum polysaccharide, resistant starch,and fructooligosaccharideThe rats, after receiving the treatment of peach gum polysaccharide, resistant starch,and fructooligosaccharide, the symptoms of polyuria and polydipsia, the hair and condition get improved and they also become more sensitive and active.3.2The therapeutical effects of Peach gum polysaccharide,resistant starch, and fructooligosaccharide on IGT model ratsAt the beginning of the experiment, the difference in weight of the rats are unmeaning in statistics. After8weeks’ feeding, the weight of rats increases obviously. Compared with rats of the normal group, the weight of the rats of peach gum polysaccharide, resistant starch,and fructooligosaccharide of IGT model drop slightly but the difference is unmeaning in statistics(.P>0.05). After examined through Levene and analysed through ONE-WAY ANOVA, the difference is unmeaning in statistics (F=0.383, P=0.81).3.3Effects of peach gum polysaccharide, resistant starch, and fructooligosaccharide on the glucose tolerance of IGT model ratsWith OGTT experiment, the blood glucose level of the rats will be measured at5time points, namely0minutes,30minutes,60minutes,90minutes and120minutes. Compared with normal rats, the blood glucose level of the model group30minutes,60minutes, and90minutes after being fed with glucose(P<0.05). The area under the curve of blood glucose is higher than that of the normal group(P<0.05) and glucose tolerance level begins to decline. The difference of the limosis blood sugar level of the normal group, the model group, and three control groups is unmeaning in statistics(P>0.05). Compared with the model group, the peach gum polysaccharide, resistant starch,and fructooligosaccharide can obviously lower blood sugar level30 minutes after being fed (p<0.01) but the difference among three control groups are unmeaning in statistics. This also correspondences to the sugar level60minutes,90minutes and120minutes after being fed. In terms of the area under curve of the blood glucose tolerance, the differences among those groups are quite obvious (F=5.939, F=0.001). Compared with the model group, the area under curve of the blood glucose tolerance of the other three groups is less. But the comparison between the peach gum polysaccharide group, resistant starch group,and fructooligosaccharide group and three control groups is unmeaning in statistics.4. the effects of the peach gum polysaccharide group, resistant starch group,and fructooligosaccharide group on the growth of intestinal probiotics of normal ratsBefore the feeding of food, the number of bifidobacterium and lactobacillus are equivalent, the difference of which is unmeaning in statistics (F=0.781,1.033; P=0.518, P=0.399). after20d of successive gavage, the number of the rats of the peach gum polysaccharide group, resistant starch group,and fructooligosaccharide group increase obviously compare with that of the normal group(P<0.01). The difference between the number of bifidobacterium and lactobacillus between peach gum polysaccharide group and resistant starch group is unmeaning in statistic (P>0.05). The number of bifidobacterium and lactobacillus of the oligosaccharide group is higher than that of the peach gum polysaccharide group and the resistant starch group (P<0.01).Conclusion1. With limosis injection of a small amount of alloxan every two days and3weeks’ feeding of high-fat and high-sugar food, the IGT rat model can be established.2. Peach gum polysaccharide, resistant starch,and fructooligosaccharide have no bad effects on the weight of rats but they can improve the condition of the IGT rats.3. Peach gum polysaccharide, resistant starch,and fructooligosaccharide can reduce the blood sugar level30minutes after meals and delay the absorption of the blood sugar after meals.4. Peach gum polysaccharide, resistant starch,and fructooligosaccharide can improve the area below the sugar tolerance curve and thus improve glucose tolerance but the effect of reducing blood sugar level is not obvious.5. Peach gum polysaccharide, resistant starch,and fructooligosaccharide can obviously promote the growth of bifidobacterium and lactobacillus, among which the effect of the fructooligosaccharide is better.6. Peach gum polysaccharide, resistant starch,and fructooligosaccharide all can reduce the blood sugar level and promote the proliferation of the intestinal probiotics. China is processed with rich peach gum which is tremendous in output, low in price and cost. The conclusions above can provide theoretical basis for developing peach gum polysaccharide into the healthcare food which can lower blood sugar level and improve the intestinal probiotics. |
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