Research On The Structure Preliminary Identification And Hypogycemic Effect Of Polysaccharides From Corn Silk

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 .1. The research on three determination methods for polysaccharides yield(1) 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+0.0111,R²=0.998, the equation was linear within the range of 20 140μg, the regression equation of method two was y=6.2161x-0.0292, R²=0.9944, the equation was linear within the range of 20 140μg, the regression equation of method three was y=1.3921x-0.0339, R²=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.(2)Compared three measurement methods for testing the methodology. 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.2. The research on three extraction methods of polysaccharides(1) 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 (℃)> ratio of solid to liquid> time(min) > particle size(mesh) ,and the optimum parameters of technology of extraction are: extraction temperature 90℃, ratio of solid to liquid60:1, time60min, particle size 80 mesh,the yield of polysaccharide was 2.32%。The extraction temperature has significant effects on the polysaccharides extracted (α<0.05)(2) Microwave extraction methodThe Plackett-Burnam design was applied to screening the key factors which would affect the yield of Corn Silk polysaccharides extracted. Key factors were microwave-directed extracting temperature, extracting power, and liquid-solid ratio. The path of steepest climbing was used to approach the optimal region of the extraction condition, and the experiment parameters were further optimized using central composite design and response surface analysis, the optimized condition was obtained. The statistical analysis of the experiment indicated that the extracting temperature, the power had significant effect on the polysaccharides yield. The central composite design showed the polynomial regression models were in good agreement with the experimental results for the polysaccharides yield. The model was Y=7.39+0.4X₁-0.67X₂+0.27X₃-0.37X₁X₂-0.096X₁X₃ +0.13X₂X₃-0.13X₁²-0.49X₂² -0.035 X₃² ,and the optimized condition for polysaccharides yield within the experimental range of the variables studied was extracting temperature 85°C, power 400 W, liquid-solid ratio 80mL/g, time 15 min. At this condition, the predicted polysaccharides yield was 8.93, the real value was 9.36, and the relative deviation between the predicted valued and real value was less than 0.5%.(3) PEF extraction methodWe have made the single factor experiment and response surface methodology (RSM), based on a Corn Silk design (BBD), was utilized to optimize the extraction conditions of polysaccharide from Corn Silk to obtained high polysaccharide yield. Three independent variables including, ratio of liquid to raw material (mL/g) >pulse duration (μs) > electric field intensity (KV/cm) have been investigated. The experimental data obtained were fitted to a second-order polynomial equation and also analyzed by the 3D response surface and contour plots using the Design expert 7.0. The model was Y=7.29+0.31X₁+0.07X₂+ 0.52X₄ +0.44X₁X₂+0.06X₁ X₃ +0.29X₂X₃ -0.92X₁²-0.88X₂²-0.75X₃², the optimum extraction condition was as followed: electric field intensity30 KV/cm , ratio of liquid to raw material 50 mL/g,and pulse duration 6μs. Under the condition, the experimental value of polysaccharide yield was 7.31%, matching well with value predicted. Result suggested that pulsed electric field had potent application for extracting polysaccharide from Corn Silk.3. The research on the anti-diabetic effects of polysaccharidesPolysaccharides could increase food intake for streptozotocin-induced diabetic rats,and which could decrease the daily water consumption in streptozotocin-induced diabetic rats comparable to dimethyl-biguanide, a commercial prevalent anti-diabetic drug. By determination of blood glucose (BG), it revealed that polysaccharides had beneficial effects on the improvement in the control of blood glucose. Daily treatment with 100–500 mg/kg body weight of polysaccharides for four weeks not only brought a significant decrease on blood glucose level in streptozotocin-induced diabetic rats.4. The purification of Corn silk polysaccharides(1) DeproteinizationIt was compared that the deproteinization effect of polysaccharides by Savage with TCA. The deproteinization rate reached 80% of the polysaccharides by Sevage method, and the polysaccharides loss rate was 54.47%.Meantime, TCA method of protein removal rate reached 77.06% when the loss rate up to 72.36% of polysaccharide. To make the polysaccharides were less lost, protein removal rate was high, Sevage method was applied for the deproteinization of the Corn silk polysaccharides.(2) DecolorizedThe polysaccharides were decolorized by reverse micelle solution, the polysaccharides decolorization ratio was 91.43% and the polysaccharides recovery was 77.59%.(3) Molecular weight determinationThe purification of polysaccharides was obtained by SephadexG-100.By the HPLC identifying; there were some other objects except polysaccharides from Corn Silk. The molecular weight were separately 474164Da ,707384Da ,760581Da (41445 Da)by three different extractions, and the molecular weight was 6136811Da(776895 Da)by purified .And these were the foundation of structure determined.4. The structure determined of polysaccharides(1) The Monosaccharide composition determined by GCThe monosaccharide composition of the polysaccharides was mannose, galactose, xylose, fructose, and arabinose. The mole ratio was 0.96:0.75:1.84:1:0.71. There were some different with other papers published.(2) The determination of characteristics of functional groups by FT-IRThe polysaccharide compounds with significant functional groups, which were extracted by boiling, microwave and high voltage pulsed electric field and purified by Sephadex G-100 , the characteristics of functional groups of polysaccharides, namely, O-H stretching vibration, CH₃, CH₂, CH and C-H stretching vibration, etc., angle vibration of C = H, and C = O stretching vibration.In this study, the technology of extraction and purification system of polysaccharides was researched, while the purified technology need further be improved, the best technology in the pilot experiment needs further in-depth study. The relationship between the dose and anti-diabetic effects should be further defined .For other biological activities, such as antioxidant, anticoagulant, anti-tumor, etc. which have been reported the activity should be studied into the depth.