Extraction Of Kaempferia Elegans Polysaccharides By PEF Pretreatment And Their Effect On Diabetic Mice

Kaempferia elegans,a characteristic plant in Guangdong Province,belongs to Kaempferia,Zingiberaceae.At present,domestic researchers and foreign researchers mainly focus on the isolation of chemical substances and on the in vitro activity of Kaempferia elegans.However,the yield of Kaempferia elegans is limited and the structural characteristics & biological activity are still unclear.This paper aims to make a systematic study on polysaccharide from the root of Kaempferia elegans.Firstly,in terms of the polysaccharide extraction,polysaccharides were obtained by hot water extraction and pulsed electric field assisted hot water extraction.Single factor analysis and response surface optimization were adopted for their own optimum technologic parameters respectively,then comparing these two extractions yields to find a better way of getting a higher extraction yield for Kaempferia elegans polysaccharide.And the influence caused by pulsed electric field pretreatment on raw materials was also analyzed.Secondly,the optimal extraction technology was adopted to extract the polysaccharides from Kaempferia elegans,and the polysaccharides were preliminarily purified.Then analyzing and identifying the chemical composition,molecular weight and monosaccharide components of the crude polysaccharides of Kaempferia elegans were also processed.Moreover,the structure of polysaccharides was analyzed and verified by ultraviolet spectrum,infrared spectrum and scanning electron microscopy.In addition,the antioxidant activity and hypoglycemic activity of Kaempferia elegans crude polysaccharides were investigated in vitro.Finally,to study its intervention effect on diabetic mice and potential hypoglycemic mechanism,the crude polysaccharide was introduced into animal experiment.The main research results are as follows:(1)Extraction of polysaccharide from Kaempferia elegans.Hot water extraction method is a traditional method of extracting polysaccharides.According to the response surface optimization,the extraction yield was 264 mg/g under the condition of 100 ℃(extraction temperature),2.7 h(extraction time)and 1:18(solid-liquid ratio).While under the condition of31 k V/cm electric field intensity,64 pulse number,98 ℃ extraction temperature,2.5 h extraction time and 1:18 solid-liquid ratio,the extraction yield of polysaccharide increased to405.04 mg/g,which increased by 1.53 times compared with the hot water extraction yield.The result indicates that pulsed electric field pretreatment could significantly improve the extraction yield of polysaccharides.Moreover,the reason why PEF could improve the polysaccharide extraction yield might be the loose structure caused by electric fields,which was originally tight structure,but into many cell wall fragments caused by electroporation effects.This process is helpful to spread the solvent and beneficial for a fully combination of the solvent,so as to enhance the mass transfer process and improve the polysaccharide extraction yield.(2)Identification and analysis of crude polysaccharides from Kaempferia elegans.The polysaccharide was extracted by PEF assisted hot water extraction,and purified by alcohol precipitation and deproteinization.After being purified,the main components are of 887.73 ±8.73 mg/g polysaccharide,4.95 ± 1.10 mg/g protein,27.86 ± 0.91 mg/g sulfate,75.54 ± 11.08mg/g uronic acid,3.71 ± 1.20 mg/g total polyphenols.The two main different molecular weight of consisting the Kaempferia elegans crude polysaccharides are 1268 k Da and 6 k Da.The monosaccharides are mainly composed of glucose,rhamnose,arabinose,galactose,xylose and fucose,and their molar percentages were 84.44%,6.52%,3.39%,2.27%,1.97% and 1.41%respectively.FTIR spectra shows that the polysaccharide contains uronic acid and α-pyranoside,which are in a shape of spherical particles packed into sheets under electron microscope.(3)In vitro activity research of Kaempferia elegans crude polysaccharide.In vitro antioxidant experiments shows that the Kaempferia elegans crude polysaccharide has a certain degree DPPH free radical scavenging ability and ABTS free radical scavenging ability,but it only works with effective scavenging effect when a certain dosage was reached.And in vitro hypoglycemic experiments shows that good α-glucosidase inhibition ability which is dosedependent is also found in.This demonstrates Kaempferia elegans crude polysaccharides is with the potential to improve the blood glucose of diabetic.(4)Evaluation for the effect of crude polysaccharide on diabetic mice.Established diabetic animal model induced by STZ and the six-week lavage intervention were done on the diabetic mice.By comparing predesigned set of models,it turns out that the crude polysaccharide could improve the situation about the rising trend of fasting plasma glucose,and it could also reduce the drinking intake and food intake of diabetic mice.In addition,it could improve the typical symptoms of diabetes,and no side effects on body weight.Furthermore,the organ index shows that the liver,kidney and pancreas of STZ-induced diabetic mice were damaged to a certain extent,but the Kaempferia elegans crude polysaccharide could protect the organs.(5)The potential hpyerglycemic mechanism of Kaempferia elegans crude polysaccharides.After the serum and liver index determination of diabetes mice under Kaempferia elegans crude polysaccharides intervention,the mechanism of the mice blood glucose was explored in these three aspects: Ⅰ.Increasing the content of GCK and CP and decreasing the content of G6 Pase.By adjusting the content of key enzymes in the glucose metabolism pathway,the consumption of glucose in the process of glycolysis could be promoted,or the production of endogenous glucose in the gluconeogenic pathway or promote the production of insulin could be inhibited,and the body’s glucose level could finally be regulated;Ⅱ.The content of TG,TCHO & LDL was reduced,while the content of HDL was increased,to adjust the blood lipid indexes to a normal level,and to break the vicious circle,followed by a prevention of any further aggravation of diabetes;Ⅲ.The Kaempferia elegans crude polysaccharides have good free radicals scavenging ability which could increase the activities of CAT,GAH-Px and T-SOD.This means the ability of Kaempferia elegans crude polysaccharides is useful for enhancing the antioxidant defense system capacity of the living organisms,in the end to achieve the effect of reducing blood glucose.