Study On Extraction And Purification Of Lipu Taro Globulin And Its Blood Glucose Regulation Activity In Vitro

Taro is a herbaceous plant of the Araceae family,which is rich of active substances,such as polysaccharides,protein,flavonoids and saponins.In this paper,lipu taro globulin was extracted,isolated and purified.The relative molecular weight,purity,amino acid composition,isoelectric point and secondary structure of lipu globulin were determined.The lipu globulin inhibitory activities onα-amylase andα-glucosidase,and the effects of lipu globulin on glucose consumption,glycogen synthesis,HK and PK activities,IRS2,GLUT4,PCK2 and G6Pase protein expression of IR-Hep G2 cells,these were determined to study of its effect on glucose metabolism in vitro,and providing reference for special dietary development of specific population.The main findings were as follows:1.Lipu taro globulin was extracted with phosphate buffer solution（0.1mol/L,p H=6.8）containing 0.3 mol/L Na Cl.The optimized extraction process was determined:the material-liquid ratio was 15 m L/g,the temperature was40℃,and the time was 120 min.The extraction rate of globulin was36.75±0.31%,and the purity was 76.72±0.47%.2.The globulin was separated by 80%ammonium sulfate and purified by DEAE-52 column chromatography.Five elution peaks were obtained,0.1mol/L Na Cl elution globulin with the highest content and activity,and its purity was 93.58±0.29%,the relative molecular mass was 22 k Da,the highest amino acid content were aspartic acid,glutamic acid and leucine,and the isoelectric point was 5.6±0.2.The secondary structure consisted of 39.42%～42.61%ofβ-folding,26.34%～31.38%of random criping,18.25%～24.16%ofβ-turning,and 5.04%～13.27%ofα-helix.3.According to double reciprocal curve method,globulin showed non-competitive inhibition ofα-amylase andα-glucosidase,with IC₅₀of 0.75±0.10mg/m L forα-amylase and 2.09±0.19 mg/m L forα-glucosidase.K_iforα-amylase was 11.47±0.57 mg/m L and K_iforα-glucosidase was 39.67±1.20mg/m L,which indicated that the globulin had the potential of regulating blood glucose.4.To further study the effects of globulin on glucose consumption,glycogen synthesis,HK and PK activities,IRS2,GLUT4,PCK2 and G6Pase protein expression in IR-Hep G2 cells were studied.Results:Lipu taro globulin could significantly increased the glucose consumption of IR-Hep G2by 40.62±0.98%,and effectively promoted the absorption of glucose.The content of liver glycogen was significantly increased by 28.80±1.26%.The activity of HK and PK were increased by 54.63±2.48%and 44.29±2.64%respectively.The expression regularity of related proteins determined by the WB experiment was not obvious.The protein expression of the signal elements in the PI3K/Akt signal pathway initially showed a certain regularity,but it was not enough to explain the mechanism.More specific key enzymes in the relevant signal pathway should be selected to providing supporting evidence to determine the signal pathway.In conclusion,the globulin can improve insulin resistance of Hep G2 cells,promote glucose absorption and liver glycogen production,and enhance glycoly-related enzyme activities.5.The processing characteristics of Lipu taro globulin were determined,laid the foundation for its application in blood sugar regulating functional food.Results:The largest solubility of the globulin was at 65℃,the minimum was near the isoelectric point,and solubility was higher in alkaline conditions,the water binding capacity was maximum at 50℃,was minimum during p H4.5～6.0,the oil binding capacity was maximum at 50℃,foamability was similar to the law of solubility,stability and sparkling at the isoelectric point was highest,emulsification and emulsion stability were minimumin at isoelectric point and higher in alkaline conditions,DSC analysis showed that the globulin of denaturation temperature was79.30±1.36℃and enthalpy was 7.46±0.53 J/g.