Studies On Hypoglycemic And ACE Inhibitory Activity Of The Bioactive Peptides From Oyster

Part one Preparation of the Bioactive Peptides from Oyster and Analysis of their Nutrient CompositionObjective To prepare the Bioactive Peptides from Oyster and analysis of their nutrient composition.Methods The collection of bioactive peptides, which were from separation of enzymatic hydrolysate from oyster by using Sephadex G-50,and then freeze-dried.Using Gel chromatography to determine the distribution of bioactive peptides'relative molecular weight,using high performance liquid chromatography to determine the content of taurine, measuring amino acid by amino acid analyzer, using phenol-sulphuric acid method to determine the content of glycogen, and using biuret method to determine protein content.Results Using Sephadex G-50 to achieve better separation results, their relative molecular weight mainly concentrated in the distribution which was less than 5kD, taurine content was 3.33%, the total amino acids were 44.30%, glycogen content was 26.12%, protein content was 27.16%.Conclusion The method is simple, its effect for separation is good. BPO include various amino acids which are required for the human body, and contain higher glycogen and protein, which can be used as functional foods but need more further study.Part two The Hyperglycemia Lowing Effects of the Bioactive Peptides from Oyster on Diabetic miceObjective To investigate the roles of the Bioactive Peptides from Oyster in lowing hyperglycemia and possible mechanisms.Methods Healthy Kunming mice 220, which contain male and female, divided into normal experiment, prevention experiment and therapy experiment parts. In the normal experiment, including NC group and BPO low, middle and high-dose groups. In the prevention experiment, including NC, model control group (MC), BPO low, middle and high-dose groups. In the treatment experiment, including NC, MC, the positive drug control group and BPO low, middle and high-dose groups. The mice were administrated with alloxan 200mg/kg(according to mouse body weight) by intraperitoneal injection to build diabetic mice model. NC and MC were administrated with saline, BPO low, middle and high-dose groups 0.5g/kg, 1g/kg, 2g/kg (according to mouse body weight), metformin tables 0.5g/kg.The end of the experiment, mice in each group were weighed, drawed by the eyeball to determine fasting blood glucose by GOD-PAD method, after death which were removed thymus,spleen, liver and kidney organ to weight. Then calculated the organ index, and observed the morphological changes by pathological sections of pancreas. Using enzymatic method, UV-spectrophotometry method to determine triglyceride (TG), cholesterol (CHO),high density lipoprotein (HDLC), malondialdehyde(MDA), hydroxyl radical (Fenton reaction),blood urea nitrogen (BUN), serum calcium (MTB) and other biochemical markers.Results In the normal experiment, comparing with NC group, the FBG, body weight, liver index and kidney index of the mice in the BPO groups were no significant difference, the thymus index and spleen index of BPO high-dose group were significantly increased. In the prevention experiments, mice thymus index and spleen index of the MC group and all BPO groups decreased. Comparing with the MC group, thymus index and spleen index of BPO the high-dose group mice were significantly different (P<0.05). The level of FBG in the middle and high dose groups was markedly reduced (P<0.05).In the treatment experiment, the level of FBG in the middle and high dose groups was significantly lower than MC(P<0.01), which were closed to the level of the positive drug control group. Comparing with MC, each group of BPO improved the contents of HDL-C and serum calcium, and reduced the level of TG and CHO, middle and high dose groups significantly enhanced mouse thymus index and spleen index (P<0.05), significantly improved the body's ability to remove serum hydroxyl radical (P<0.01), significantly decreased the content of serum MDA and BUN (P<0.01). Pathology observation showed that the damage from islet in mice of BPO group was lightter than MC,islet of high-dose group had a higher degree of improvement, which improved markedly in positive drug control group.Conclusion 1.BPO have no significant effect on FBG, body weight, organ weight of normal mice.2. BPO can prevent alloxan-induced diabetic mice,which prompts that BPO have some effects on protecting alloxan-induced diabetic mice from formation and islet injury.3. BPO can reduce the levels of FBG for diabetic mice, which shows that BPO promopt tissue repairation and restore function of islet.4. BPO can reduce the serum MAD, TG, CHO, BUN, and improve thymus index and spleen index of hyperglycemia mice, which indicate that BPO can regulate blood lipids, remove free radicals, improve antioxidation ability and enhance immune function.Part three Study on extraction and activity determination of ACE inhibitory peptides from OysterObjective To extract ACE inhibitory peptides from oyster and determine activity.Methods Appling salting-out, sephadex G-100 gel chromatography and ion-exchange DEAE Sepharose CL-6B to purify ACE from lung. Fresh oyster as raw material, using enzymatic method to prepare ACE inhibitory peptides from oyster, to chose the best enzymatic conditions from bovine trypsin, alkaline protease and animal protein hydrolase, separate enzyme solution by Sephadex G-25. Using RP-HPLC to determine the inhibitory activity of ACE inhibitory peptides from oyster.Results The purified ACE reached a pure electrophoresis, could be used for HPLC determination. Selecting the best enzymatic conditions: using alkaline protease, pH of 9.0, hydrolysis time at 4 hours, hydrolysis temperature at 55℃. Isolated from the four components(0.48mg/ml),ACE inhibition rate was 25.02%,41.78%,60.44%,17.45%. Conclusion By alkaline protease enzyme, activity of ACE inhibitory peptides is strongest.HPLC is applied to detect the inhibitory activity of ACE inhibitory peptides, which is simple, high precision and accuracy.