Study On The Isolation, Purification And Hypoglycemic Mechanism Of P-Polypeptide From Momordica Charantia

Momordica charantia, a member of the Cucurbitaceae family and Momordicagenus, is an annually plant distributed in East Africa and Asia and South America andcultivated throughout China. Its powder, juice or organic extracts have been usedtraditionally by people to prevent and cure diabetes, and its hypoglycemiccompositions have been researched for a long time. Now many researchers havefound hypoglycemic compounds in Momordica charantia mainly includingpolypeptides and saponins. In this paper, P-polypeptide, that was so called"Momordica charantia plant insulin" , was chosen as research aim and systematicstudies were carried out on its methods of isolation and purification, hypoglycemicmechanism of type 2 diabetes mellitus, improvement of diabetic complications,physical properties, and the relationship between its molecular structure and thephysiological functions.Momordica charantia P-polypeptide was chosen as research aim. Hypoglycemicpeptides in momordica charantia were primarily screened by ultra-filtration method.Momordica charantia juice was ultra-filtrated into three grades with cut-offmembrances of 10kD, 3kD and 1kD molecular weight and the hypoglycemicfunctions of their filtrated groups and retenated groups were examined by diabeticanimal experiment. Results showed the retenated group between 3kD and 1kDultrafitation membrance could lower blood glucose in normal and alloxan-induceddiabetic rats, the other groups could not do it orally. P-polypeptide could exist inMomordica charantia according to the analysis of composition of Momordicacharantia juice.Momordica charantia P-polypeptide was isolated and purified by gel filtrationand by reversed-phase high performance liquid chromatography (RP-HPLC) with theidentification of animal hypoglycemic experiment and thin-layer chromatography andelectrophoresis and the analysis of amino acid composition. The P-polypeptide wassequenced by Edman method firstly, which showed P-polypeptide was composed of166 amino acid residues. Gel filtration and RP-HPLC was better than formerly usedthin-layer chromatography in a great deal of preparation of P-polypeptide.Hypoglycemic function of P-polypeptide was researched. The rat model of type2 diabetes mellitus was made by intraperitoneal injection with a low dose ofstreptozotozin and feed on a diet enriched in fat and glucose. The result showed thatP-polypeptide injected subcutaneously could lower blood glucose of type 2 fatdiabetic rats and could improve their glucose tolerance, and reduce their serum insulinconcentrations and raise their glycogen in liver and muscle of diabetic rats.P-polypeptide could also lower their cholesterol, triglyceride and lower densitylipoprotein concentrations and increase their high density lipoprotein level in serum,which regulated abnormal metabolism of blood lipids. P-polypeptide could reducetheir H2O2 and MDA concentrations and increase their SOD activity and totalantioxidant activity in serum and liver of type 2 model rats, which was in favor ofimproved complications of type 2 diabetes. The hypoglycemic mechanism ofP-polypeptide was to increase sensitivity of tissue to insulin and improve insulinresistance.The amino acid sequence of P-polypeptide was analyzed and its structure waspredicted by bioinformatic method. The results showed P-polypeptide molecularweight was 17702.7D and molecular formula was C763H1233N217O256S5。P-polypeptidewas soup peptide with its theoretical pI 4.86 and not globularity and could behydrolyzed by enzymes. The result of homologous comparative through BLASTpindicated less than 30% homology between P-polypeptide and others known peptidesand no homology between P-polypeptide and animal insulin. Secondary structure ofP-polypeptide was predicted by PDH and NNPREDICT and GOR servers, whichshowed there were at least three β-strands and one α-helix in P-polypeptide. Domainsfrom 28 to 31(-Cys-Leu-Tyr-Glu-)and from 86 to 88(-Glu-Cys-Ile-)and from 104to 106 ( -Leu-Ile-Val-) were β-strand and fraction from 127 to 133( -Lys-His-Ala-Ser-Glu-Val-Lys-) was α-helix. The ranges from 28 to 31(-Cys-Leu-Tyr-Glu-)and from 86 to 88(-Glu-Cys-Ile-)lied in hydrophobic space.Because of weak similarity, tertiary structure of P-polypeptide colud not be predictedby comparative modeling and by threading method, and this should be researchedfurther by ab initio predition method.Functional peptide fractions of P-polypeptide were researched with enzymatichydrolysis method and the relationship between the molecular structure andphysiological functions was studied with motif search method. Hydrolyzed by pepsinor trypsin, P-polypeptide administrated orally or subcutaneously could not lower theblood glucose of normal and alloxan-induced diabetic rats, which meant peptidefractions of P-polypeptide hydrolyzed had not hypoglycemic function or lowhypoglycemic activity. Motif search results of P-polypeptide in PROSITE databaseindicated that there were phosphorylated sites of cAMP-and cGMP-dependentprotein kinase and Protein kinase C and Casein kinase II and N-myristoylation siteand Amidation site in P-polypeptide. On the base of analysis of function of Proteinkinase C and cAMP-dependent protein kinase in diabetes mellitus and diabeticcomplications, P-polypeptide could hypothetically increase sensitivity of tissue toinsulin and improve insulin resistance in insulin receptor and after insulin receptor tolower blood glucose though protein kinase C and cAMP-dependent protein kinase.