The Purfication And Properties Of The Hypoglycemic Peptide From Flax Seeds

Diabetes mellitus?DM? is a serious chronic disease of metabolic dysregulation which has many complications,for example,retinopathy,nephropathy,neuropathy,and so on.The best treatment for DM is insulin injection and oral hypoglycemic drugs.Recently years,researchers have isolated many different kinds of hypoglycemic peptides from pumpkin,bitter gourd and leguminous plants.The hypoglycemic peptides isolated from natural products were more safer,economic and better environmental compatibility than chemical synthesis.Flax,as a kind of flax linen genera annual herb,was planted widely in our country.Flax seeds contained a variety of biological active substances:flax protein,?-linolenic acid,lignans,flax gum and so on.At present,two components?-linolenic acid and secoisolariciresinol diglucoside?SDG?in flax seeds have been found with hypoglycemic effect.The protein of flax seeds can be up to 20-25%,main components was globulin and albumin which had edible and medical values.The main product of flax was linseed oil,however,the flax meal,after oil extraction,also contained a high quantity of flax protein,but the peptide which has been used as animal feed ingredients and sometimes as a fertilizer,which made a big waste of resources.In order to improve the utility of flax meal,we regarded the?-glucosdiase inhibitory activity as an index to isolate the flax hypoglycemic peptide.The crude protein was loaded on anion exchange column Q-Sepharsoe4B,equilibrated with the same buffer,collected the different fractions,then analyzed the?-glucosdiase inhibitory activity.The hypoglycemic peptide which had the activity to inhibit?-glucosdiase activity has been isolated from flax meal by acetone fractionation,Tris-HCl buffer extraction and Q-Sepharose4B.With the purification steps mentioned above,the overallrecovery of?-glucosdiase enzymatic inhibitory activity of 10.95%,the specific activity of 140.76U/mg and the purification fold of 5.10 for the hypoglycemic peptide from crude extraction were achieved.The LC-ESI-MS showed the peptide was a kind of trypsin inhibitor,belonged to Potato trypsin inhibitor family and the relative molecular weight was8k Da by Tricine SDS-PAGE.In addition,it was a double-function peptide and the two inhibitory activities were stable below 70?,as well as p H 2.0-7.0.Compared to the hypoglycemic drug acarbose,for?-glucosdiase inhibitory activity,the IC₅₀ of acarbose was 592.35?mol/L,whereas hypoglycemic peptide was 113.92?mol/L,as for the trypsin inhibitory activity,the IC₅₀ of the soybean trypsin inhibitor?SBTI?was1.84?mol/L,the IC₅₀ of the buckwheat trypsin inhibitor?BTI?was0.228?mol/L,but the hypoglycemic peptide was 6.17?mol/L.As well as the inhibitory models,it was a competitive inhibitor for?-glucosidase and a non-competitive type for trypsin.What's more,the peptide contained a pair of disulfide bond which linked the N-termius and C-termius.The?-glucosdiase inhibitory activity descended 21.2%,as well as the trypsin inhibitory declined 40.32%when incubated it with the reducing agent DTT for 60min.In addition,The CD spectra showed the 208nm and222nm negative peaks disappeared with the increase of time.This indicated that the change of the secondary structure may lead to the two inhibitory activities declare.The hypoglycemic peptide,whose concentration was lower than 50?mo/L,had almost no effect on the growth of Caco-2 and Hep G2 cell lines by MTT assay.Cytology experiments demonstrated that the consumption of glucose and production of lactic acid increased rapidly when incubated hypoglycemic peptide in Caco-2 and Hep G2 cells.In addition,the consumption of glucose and the production of lactic acid had a dosage dependent with the hypoglycemic peptide.To our knowledge,the peptide,which was found with hypoglycemic effect of flax meal for the first time,was a double-function peptide and it had the ability to endure the digestive enzyme and inhibit the?-glucosdiase activity.Therefore,the flax hypoglycemic peptide may have applications as do other?-glucosidase inhibitors and may be a new choice for disease control.