| BackgroundDiabetes mellitus (DM),which is caused by genetic and environmental factors,is a group of clinical syndrome characterized by common disorders of glucose metabolism. Insulin deficiency and insulin function disorders alone or both give rise to sugars, fats, proteins, water and electrolyte metabolism disorders, clinical chronic high blood glucose as the main character.Acute complications,such as Ketoacidosis, hyperosmolar hyperglycemia and lactic acidosis.Diabetes mellitus can be complicated with a variety of chronic complications,leading to organ dysfunction and failure, being maimed and even killed, placing heavy burdens on society, and the rising incidence of diabetes, patients with diabetes by2025is expected to increase to300million in the world.Especially,the incidence increase rapidly in developing countries, the number of diabetic patients in China has reached more than90million visitors,only less than in india. The chronic complications of diabetes are serious, with the extension of course, mainly for cerebrovascular and coronary heart disease, peripheral vascular disease, kidney disease and retinopathy, et al. Type2diabetes mellitus accounts for90%of all the DM.The basic characteristics of type2diabetes is the lack of insulin secretion and insulin resistance, which is associated with obesity and metabolic syndrome. Pre-diabetes, that is,the state of impaired fasting glucose and impaired glucose tolerance, accounted for more than10%of all population groups, mainly for the rise in postprandial blood glucose, early detection、 early intervention to reduce postprandial blood glucose, we can reverse this state to prevent the development of diabetes. According to two cross-sectional surveys in China from1998and2006, indicates that,90%and73%of glycosylated hemoglobin(HbAlc) in type2diabetic patients did not achieve the desired control objectives [1]. Although traditional Western medicines has gained effective results in controlling blood glucose and blood lipid, but they often produce many side-effects, and cannot cure the diseases, so long-term, effective prevention and control of chronic disease are important measures to delay occurrence of complications and improve patients’quality of health and life.To the patients with impaired glucose tolerance, pre-diabetes and elevated blood lipid, glucose and lipid-lowering health food will play a positive role in preventive health care. In view of this, the project team has proposed topics of making use of natural peach gum auxiliary to develop blood glucose and lipid-lowering health food.Polysaccharide is a class of complex macromolecular compounds which is made up of ten and above monosaccharide, its sources in the natural world is very diverse, for instance, polysaccharide in bacteriums, plants,fungi,animals and so on. Polysaccharides exhibit immune regulation, anti-tumor, anti-inflammatory, antiviral and hypoglycemic and lipid-lowering role, and many other biological functions.Over the past several decades, more and more studies have concentrated in the hypoglycemic role of polysaccharides,polysaccharide hypoglycemic mechanism is mainly focused on the following aspects [2-8]:①improve islet cell morphology and function, and promote insulin secretion;②raise insulin sensitivity;③make the glucose metabolism better, promote the use of sugar by liver, muscle and external peripheral tissues and the target organ;④regulate autoimmune, scavenge free radicals, prevent lipid peroxidation;(5) improve the activity of glucose metabolism enzymes;⑥inhibit the activity of a glucose glycosides, delay the absorption of glucose.Peach resin is a kind of semi-transparent gum which is from the trunk of rosaceae by the mechanical damage (such as insect bites, cuts, etc.), or some disease. Its main compositions are polysaccharides and protein. Also contain few of other materials. The polysaccharides constitute of galactose, arabinose, rhamnose, glucuronic acid and so on. DingTing[9] with Peach resin powder such as to four oxygen pyrimidine diabetic rats irrigation Peach resin can improve the stomach study found that diabetic rats insulin levels, reduce blood sugar, also can reduce low-density lipoprotein cholesterol, triglyceride and total cholesterol, and a certain amount of activity relationship. HongYuZhi[10] used30g of the original peach resin and two steamed bread simultaneously in30cases of patients with diabetes,2hours after the meal, compared to the fasting blood sugar,patients’blood sugar, c-peptide, insulin/blood-sugar ratio showed no obvious difference, considered that the mechanism of the Peach resin in lowering blood sugar is similar to other plant dietary fiber. That is,the original Peach resin can delay the absorption of sugar in the upper intestinal tract, causing secretion of gastric inhibitory polypeptide (GIP) decreased, blood insulin levels droped. When Peach resin improves glucose tolerance after a meal, but do not increase the blood insulin levels at the same time, rather than increased insulin resistance in type2diabetes patients with high insulin hyperinsulinemia, and the dose of insulin or oral medications can be reduced.Caco-2cell,which is differentiated from human intestinal epithelial tumor cell, is currently one of the main model to study drug intestinal absorption,highly expresses the main carrier and related enzymes for the absorption of nutrients, including the main glucose absorption carrier in the intestinal brush border side(SGLT-1), has become a standard screening tools in vitro that predict drug absorption studies in human intestinal absorption and drug carriers absorption[12-13].Our country richs in Peach resin resources,widely distributed in guangdong, hebei, shanxi, gansu, jiangxi, jiangsu, etc.Peach resin yield great, and the price is cheap, about2500yuan/ton.The hypoglycemic research of Peach resin polysaccharide can provide the theory basis for the development of new glucose-lowering drugs and health care products. By using the method of heavy water and alcohol extraction to extract Peach resin polysaccharide, by means of Caco-2cell monolayer model to simulated intestinal epithelial glucose absorption process, adding peach gum polysaccharide intervention,aimed to study the relationship between peach gum polysaccharide and glucose absorption, to provide a theoretical basis for the intestinal hypoglycemic mechanisms of peach gum polysaccharide。2.Methods2.1. The extraction and separation of peach gum polysaccharideCollected the fresh Peach resin to miscellaneous,rinsed after freeze-drying, dry Peach resin through ultramicro-pulverization, got the original Peach resin powder. Joined the original Peach resin powder to distilled water,90℃water bath for4h,mixing extracts, repeated again. Merged two soaks raises liquid, collected supernatant in4000r/separation heart on20minutes clear liquid. Put the supernatant in60℃vacuum,evaporated to the original volume of one-fifth. Deproteinization: the proportion of chloroform and butanol solution(v:v=5:1), added to the concentrated liquid,keen to shake mixture for5min after stalling extraction,divided into water and solution layer of the intersection,got the protein liquid sugar from deformed protein, repeated several times until the junction of the water and solution layer has no obvious protein precipitation. Used the gradient of alcohol precipitated method in the deproteinization solution by adding ethanol to80%,repeated several times until there was no precipitation generated, freeze drying, finally we could gain Peach resin polysaccharide by precipitated method of ethanol.2.2. General cell culture2.3. Determination of the cytotoxicity2.4. The establishment of caco-2cell modelwhen the cells were transmitted to DI20-30,taking the well-growing cells in logarithmic growth phase,discard the medium,three times rinsed by HBSS,trypsin digested, used moderate full-medium to end the digestion, transferred to the15ml centrifuge tube and placed it in a centrifuge machine,800rpm centrifugaled3-5min. Discarded the supernatant, and added a complete medium, counted the cell counts under the inverted microscope, adjusted the concentration of cell suspension to1x105-5x105/mL. The cells were seeded in the Transwell polycarbonate membrane on the culture plate according to a certain amount of inoculation of cell concentration each bore (Corning,3413). Medium was changed every other day during the first week, afterward,once a day, while we changed the medium,we should be careful that do not touch the membrane of cells,ensuring cells’ fully adherent to form the complete structure. Trained for21-23days.2.5. The identification of the caco-2cell modelAfter inoculation on transwell board,we used cell resistance meter to detect the value of the transmembrane resistance(TEER),to understand the process of the integrity and connection tightness in the development of the cell monoplayer.2.6. The methods to study the effect of peach gum polysaccharide to the glucose absorption on caco-2cellTaking the qualified caco-2cell monoplayer model,firstly,we diacarded the medium in the AP side and the BL side of the tranwell system,washed two times on each side by HBSS,then placed the cells in a CO2incubator,balanced for20min.According to the experimental group,took moderate glucosamine and peach gum polysaccharide,soluted in HBSS,filtrated and sterilized,heated to37°c before use.Prior to dosing,used MTT to test the toxicity of peach gum polysaccharide to caco-2cells.Mixed the glucose and (or) peach gum polysaccharide with HBSS joined0.1ml to AP side, added0.7ml HBSS to BL side as incoming fluid,and placed them in the incubator incubation. During this period,draw the liquid of AP side to detect the excess glucose. By the amount of lessons, put a equivalent amount of liquid into the AP side again in the small room.2.6.1The effect of a certain concentration of peach gum polysaccharide on the absorption of different concentrations of glucose.2.6.2The effect of different concentrations of peach gum polysaccharide on the absorption of a certain concentration of glucose.2.7Statistical AnalysisThe set of data represented by mean±standard deviation,and a SPSS13.0tatistical software was used to analyze.The comparison between MTT experimental groups analysed with single-factor analysis of variance(ONE-WAY ANOVA),and the rest with a analysis of variance of a single repeated measures design information.3.Results3.1. The extraction and separation of peach gum polysaccharideThrough the use of water baths, Sevage deproteinized (removal of protein or papain)and ethanol precipitation methods,we got relatively pure polysaccharides from peach gum, then purificated by sephadex gel chromatography, and grinded after crushing, grayish-brown powder, yield is about50%.3.2. The morphology of caco-2cells Caco-2cells cultured in vitro for adherent growth cells. In the flask, a single cell growed flat, angular, Central round the nucleus was visible in the cell body, and a gradual growth as film-like layer of cells, closer connections between cells, the cells form changed into neat square or circle, the overall status liked a "paving stone-like" form.DI20-30cultivation of cells inoculated in the Transwell polycarbonate membrane,after trained on the hole, about21days of culture, celsl differentiated to form microvilli junctional complex, built stronger connections and a good brush border, became a reliable monolayer absorption model.3.3.The result of the cytotoxicityPeach gum polysaccharide solution was infugrated by HBSS, the concentration was (4,2,1,0.5,0.25,0.125ug/ml) respectivelyjoined into the96orifice plate to take MTT test, to study its toxic effect on caco-2cell. The results showed that when the concentration of peach gum polysaccharide solution was between0.125-1ug/ml, compared with the control group, no significant difference existed (P>0.05,n=6), that is,0.125-lug/ml of peach gum polysaccharide had no apparent toxic effects on caco-2cells.3.4. The identification of the caco-2cell modelAfter inoculation on Transwell boards,we used cell resistance meter to detect the value of cellular transmembrane resistance (TEER), TEER value rised with the increase of the training days, which, TEER values were greater than60021days later, met the test requirement.3.5. The effect of a certain concentration of peach gum polysaccharide on the absorption of different concentrations of glucose.After joined lug/ml peach gum polysaccharide to the experimental group, the control group did not join the polysaccharides from peach gum, the concentration of glucose was10,15,20,25mmol/L, detected the glucose values on30,60,90,120min, respectively. There was significant differences in the absorption of glucose when compared10mmol/L glucose plus1ug/ml peach gum polysaccharide group with the control group,(P=0.000); There was significant differences in the absorption of glucose when compared15mmol/L glucose plus lug/ml peach gum polysaccharide group with the control group,(P=0.000); There was significant differences in the absorption of glucose when compared20mmol/L glucose plus lug/ml peach gum polysaccharide group with the control group,(P=0.001); There was significant differences in the absorption of glucose when compared25mmol/L glucose plus lug/ml peach gum polysaccharide group with the control group,(P=0.009). It can be seen that peach gum polysaccharide could inhibit uptake of glucose at different concentrations.Compared experimental group with each other of the absorption of glucose over time, he results showed that glucose uptake were significantly different between different experimental groups (P=0.000), that is, the smaller the glucose concentration of experimental group was,the better the effect of peach gum polysaccharides inhibited the glucose absorption. Mauchly sphericity test statistic, W=0.507,P=0.076,did not deny the ball suppose, when applied single-variable test methods we do not need to take ε correction.3.6. The effect of different concentrations of peach gum polysaccharide on the absorption of a certain concentration of glucose.Compared with the control group, different concentrations of peach gum polysaccharides solution (0.5,0.25,0.125ug/ml) inhibited the absorption of certain concentration of glucose (20mmol/L) signficantly(P<0.05), the result is shown in table2. Effects of polysaccharides from peach gum concentration group of glucose uptake over time relationships are shown in Figure2, shows that the higher the concentrations of peach gum polysaccharide was,the less the glucose uptake was, so the better effect to inhibit the glucose uptake (P<0.05). Mauchly sphericity test statistic,W=0.256,P=0.001, denial of spherical assumptions, Greenhouse-Geisser correction,4points of glucose absorption in different experimental groups had significant difference (F=934.31,P=0.000).Conclusion1. peach gum polysaccharide cannot be digested and absorbed by upper digestive tract, since it was a macromolecular compound combined with more than ten monosaccharide through Glycoside keys closely, so it can not come into the blood to play a role.2.Experimental research group of this study confirmed that peach gum polysaccharide has a certain lower postprandial blood glucose effect in alloxan-induced diabetic model rats; they can also inhibit amylase and maltase activity in the digestive tract, delayed the digestion and absorption of carbohydrates. In a conclusion,the part of peach gum polysaccharides played a role in the hypoglycemic effect is within the intestines.3.Peach gum polysaccharide expressed a concentration-dependent effects on glucose absorption,indicated that the there is a role of competitive inhibition between the glucose absorption and peach gum polysaccharide.4.Established a caco-2model in vitro, excluded the interference of intestinal digestive enzyme and intestinal basement side GLUT-2and intestinal bacteria flora factors on polysaccharide decomposition and monosaccharide absorption, mainly effected the upper digestive tract brush margin side of glucose absorption of important factors is trans-protein of glucose, SGLT-1, and polysaccharide essentially has similar unit structure to monosaccharide, prompted that peach gum polysaccharide may combined through bit points of glucose absorption, thereby delayed the absorption of glucose, reduced the postprandial blood glucose.5.Peach gum polysaccharide itself does not have clear toxicity to caco-2cells. Its low price, wide distribution, nonpoisonous, without being absorbed, reducing postprandial blood glucose and other excellent characteristics offer good prospects for further development of health food and drug based on it. |
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