| Momordica charantia L.Var.abbreviata Ser.(MCV) is a member of Cucurbitaceae family. Momordica charantia variety was originally growing wild in eastern India.It was domesticated and improved by Jiangsu High-quality Farm Product Development Center,China and has been successfully planted over large areas by Wuxi Furen Biological Science and Technology Co., Ltd.It is reported that Momordica Charantia L.has many pharmacological benefits such as antidiabetic,hypocholesterolemic,hypo-triglyceridemic,antitumor,antibacterial,anti-HIV, anti-inflammatory,antioxidant,etc.In this dissertation,the hypoglycemic effect of an aqueous extract from MCV and a purified peptide called MC2-1-5 with hypoglycemic effect from aqueous extract was stuied.At the same time,MCV protein hydrolysates(MCPHs) with hypoglycemic effect were prepared by Alcalase.The hypoglycemic mechanism of MC2-1-5 and MCPHs was studied in alloxan-induced diabetic mice.The chemical composition of the fresh MCV fruit were analyzed and found to be as follows:92.28%moisture,0.48%ash,1.42%protein,0.93%fat,1.48%reducing sugar,1.55% of polysaccharide and 0.78%crude fiber.The effect of MCV fruit to water ratio,salt concentration,extraction temperature and extraction time on extraction rate of MCV protein were studied.The parameters affecting protein extraction were optimized by an orthogonal experimental design,that is to say,MCV fruit to water ratio of 1:2,salt concentration of 0.05 mol/L,extraction temperature of 40℃and extraction time of 4 h.Amino acid(AA) composition profile of MCV aqueous extract showed that the content of Arginine was highest, followed by Glutamic acid and Aspartic acid of 22.06%,9.75%and 9.54%respectively.The total percentage composition of the three AA was 41.35%.The AA composition profile of fresh MCV showed that the content of Arginine,Glutamic acid and Aspartic acid was 20.13%, 12.51%and 9.63%respectively.The total percentage composition of the three AA was 42.27%.The acute(a single dose) and chronic(15 d) hypoglycemic effects of aqueous extract were studied in alloxan-induced diabetic mice.The results showed that an aqueous extract from MCV fruit at different doses(250,500 and 750 mg/kg) produced a significant hypoglycemic effect in alloxan-induced diabetic mice within 2 h after oral administration,which was maintained for 4 h.As far as the most effective dose under the experimental conditions stuied is concerned,it was found to be 500 mg/kg in all groups.The hypoglycemic effect of the aqueous extract administered at a dose of 500 mg/kg was more effective than that of glibenclamide administered at a dose of 100 mg/kg.However,the aqueous extract didn't have significant effect on blood glucose level(BGL) in normal mice.In addition,the hypoglycemic effect of aqueous extract was further confirmed by the oral glucose tolerance test(OGTT).A 15-day treatment of the aqueous extract at a dose of 500 mg/kg in diabetic mice continuously lowered the fasting blood glucose(FBG) level.Treatment of alloxan-induced mice for 15 days with the aqueous extract at a dose of 500 mg/kg produced a 59.88%fall in FBG level.During the treatment period,the aqueous extract significantly increased the body weight(BW),lowered the food and water intake in diabetic mice. Using BGL in diabetic mice as an index,the peptide with hypoglycemic effect was purified by ultrafiltration(UF),Sephadex G-25 and semi-RP-HPLC.The influence of input flow rate,operating pressure,operating temperature and sample concentration on flux was studied.The UF conditions used were:input flow rate 200 L/h,temperature 20℃,pressure 0.1 MPa and sample concentration 20 g/L.On separating MCV aqueous extract into two fractions (MC1 and MC2) by UF,MC2 showed significant hypoglycemic effect in alloxan-induced mice. It produced a reduction of 46.15%and 52.59%in BGL after 2 and 4 h respectively of oral administration.MC1 didn't show hypoglycemic effect in diabetic mice.Six different fractions of MC2 were obtained by Sephadex G-25 gel filtration chromatography.The in vivo test showed MC 2-1 at a dose of 20 mg/kg could lower the BGL in diabetic mice.It produced a reduction of 47.43%and 51.87%in BGL after 2 and 4 h respectively of oral administration.Other fractions had no significant effect on BGL in diabetic mice.A further ten different fractions of MC 2-1 were obtained through Semi-HPLC.The in vivo test showed MC 2-1-1 and MC 2-1-5 at a dose of 2 mg/kg could lower the BGL in diabetic mice,while other fractions had no significant effect on BGL in diabetic mice.MC2-1-1 produced a reduction of 43.84%and 54.57%in BGL after 2 and 4 h respectively of oral administration.MC2-1-5 produced a reduction of 61.70%and 69.18%in BGL after 2 and 4 h respectively of oral administration.In addition,MC2-1-5 improved the oral glucose tolerance in diabetic mice.An Infrared spectrograph showed MC2-1-5 to have characteristic IR absorption peak of peptide in the spectral region 500～4000 cm-1.MALDI TOF-MS showed the molecular weight of MC2-1-5 was 3405.5174 Da.Protein Sequencer showed the N terminal frist ten AAs of MC2-1-5 were GHPYYSIKKS.MCV souble protein did not have hypoglycemic effect.In order to make use of the MCV soluble protein,the protein was hydrolyzed to produce MCPHs with hypoglycemic effect.The MCV protein was hydrolyzed using six different proteases.The results showed Alcalase 2.4L to have the best hydrolysing capacity.The protein recovery was also highest with Alcalase.The hydrolysis parameters using Alcalase were optimized as follows:enzyme to substrate ratio 2.37%,pH 9.2 and hydrolysis temperature 57℃.Three hydrolysates with different DH(8.5%, 11%and 13.5%) were obtained using Alcalase under the optimized conditions.The hypoglycemic effect of the three hydrolysates was stuied in alloxan-induced diabetic mice.The results showed that the MCPHs at DH 11%had the best hypoglycemic effect,followed by MCPHs at DH 13.5%.MCPHs at DH 8.5%didn't have any hypoglycemic effect.DA201-C Macroporous resin was chosen to desalt MCPHs by comparing the adsorbing and desorbing capacity.The optimal conditions for desalting using DA201-C macroporous resin were determined.Under the optimal conditions,the desalting rate and recovery of MCPHs was 92.49%and 82.38%respectively.The content of MCPHs increased from 76.67%to 85.69%.The hypoglycemic effect of desalted MCPHs was better than that of MCPHs before desalting.The hypoglycemic mechanism of MC2-1 and MCPHs was studied.Compared with the diabetic control,a 28-d treatment with MC2-1 and MCPHs increased the BW and hepatic glycogen content,lowered food intake and water intake,made the organ/body weight near to normal and improved the oral glucose test in diabetic mice.In addition,MC2-1 in particular, increased the serum insulin level in diabetic mice.MC2-1 and MCPHs could improve the lipid profile in diabetic mice.After a 28-d treatment with MC2-1 and MCPHs,free amino acid(FFA),total cholesterol(TC),triglyceride (TG) and low density lipoprotein cholesterol(LDL-C) lowered significantly,while high density lipoprotein cholesterol(HDL-C) increased significantly.MC2-1 and MCPHs could improve the mechanisms of oxidative stress and antioxidant capacity in diabetic mice.After a 28-d treatment with MC2-1 and MCPHs,malondialdehyde(MDA) content lowered while SOD activity and GSH-Px activity increased.Tissue morphology showed that MC2-1 and MCPHs could improve the cell injury in islet,liver and kidney induced by alloxan in diabetic mice.In summary,the hypoglycemic mechanism of MC2-1 is to recover the isletβcell,so as to stimulate insulin secretion,to increase the hepatic glycogen content and improve antioxidant capacity in diabetic mice.The hypoglycemic mechanism of MCPHs is similar to that of MC2-1, except that it couldn't stimulate insulin secretion.Since MC2-1-5 is the main hypoglycemic constituent in MC2-1,it is speculated that the hypoglycemic effect of MC2-1-5 was similar to that of MC2-1.
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