Study On Enzymatic Preparation And Identification Of Anticoagulant Peptides From Whitmania Pigra And Buthus Martensii Karsch Protein Isolation Hydrolysates And Their Antifatigue Effect In Vivo

Nowadays, as work-related stress increases, excessive fatigue condition affects seriously a large number of young people. Meanwhile, due to bad diet and living habits, thrombotic diseases such as cardia-cerebrovascular disease are growing among elderly people. However, no enough safe and reliable edible products have been exploited to help patient improve multiple symptoms. Therefore, this in-depth study on anticoagulant and antifatigue peptides exhibits a practical significance.Both Whitmania pigra(WP) and Buthus martensii Karsch(Bm K) with plenty of protein are excellent materials for preparing bioactive peptides. The present study firstly isolated proteins from WP and Bm K, and then prepared anticoagulant hydrolysates by enzymatic hydrolysis under the optimal process parameters, following by the separation, purification and identification processes. The relationship between the functions and amino acids composition was discussed, and simulated gastrointestinal tract digestion test was brought finally.WP and Bm K proteins isolation were extracted using alkali-solution and acid-isolation process. The extraction rates were 78.52±2.31%(WPI) and 69.56±3.25%(HPI). HPLC displayed that the molecular weight distribution(MWD) of WPI focused on 4230 Da and 92362 Da, while that of HPI was between 27510 Da and 52436 Da.WPI and HPI were hydrolyzed using Alcalase AF 2.4L, Trypsin, Papain, Pepsin, Protamex and Flavourzyme, respectively. However, only when WPI(DH: 19%) and HPI(DH: 18%) were hydrolyzed by Alcalase, the hydrolysates exhibited the highest anticoagulant activities with the IC50 of 12.65±1.53 mg/m L(WA) and 23.65±2.44 mg/m L(HA). The MWD of WA was more than 5733 Da and between 723 Da and 3238 Da, while that of HA was 3024 Da-4872 Da, 989 Da-2053 Da and 377 Da-856 Da.The anticoagulant abilities of WA and HA were evaluated utilizing mouse model. WA-M and HA-M were confirmed to be a positive effect in preventing thrombus induced by the carrageenin injection(p<0.05), and WA-H and HA-H didn’t arouse a remarkable change compared with WA-M and HA-M(p>0.05). WA and HA could prolong easily APTT and PT of mice blood, and WA-H, HA-H could prolong APTT and PT extremely. Hence, the feeding of present WA and HA on mice could signally delay mice blood coagulation.The antioxidatant activities of WA and HA were evaluated using in vitro antioxidatant model. WA and HA had strong scavenging abilities on the radical DPPH· with the IC50 of 0.42±0.03 mg/m L and 0.91±0.08 mg/m L, and WA exhibited stronger scavenging power than HA did(p<0.05). WA and HA also demonstrated conspicuous reducing power of turning Fe3+ to Fe2+. When the concentration of WA and HA reached 1.2 mg/m L, the inhibition ratios on AAPH-induced erythrocyte hemolysis were 82.81% and 60.50%, respectively, which indicated that the free radicals from AAPH were easily scavenged by WA and HA thereby protecting erythrocyte.The antifatigue abilities of WA and HA were evaluated using animal model. The feeding of WA-M, HA-M(8.5 mg/10 g) and WA-H, HA-H(25 mg/10 g) could prolong extremely exhaustive swimming time of the tested mice(p<0.05), WA was better than HA. WA-M, WA-H and HA-H kept hepatic glycogen well. WA-M, WA-H could control lactic acid content in blood serum of tested mice, which decreased 16.55% comparing to control group. The generation of segmental BUN was prevented by WA-L, WA-M å'Œ HA-L. CK activity in blood serum of the tested mice reduced by the feeding of WA-M, HA-M and WA-H, HA-H, respectively, which could protect muscle cell thereby retarding exercise fatigue. Furthermore, only WA-H and HA-H could enhance SOD activity signally(p<0.05) which increased 21.52% and 9.32%, respectively. The antifatigue ability of WA was dramaticlly stronger than HA in present test.The hydrolysates were purified by consecutive chromatographic methods including ion-exchange chromatography, gel filtration chromatography and reverse high-performance liquid chromatography(RP-HPLC), and the higher anticoagulant products of WA3-1(IC50: 0.12 mg/m L) and HA18-3-B-8(IC50: 0.12 mg/m L) whose BCAA, HAA å'Œ NCBB all had a great boost were obtained. The amino acid sequences were identified as NH2-His-Asp-Phe-Leu-Asn-Asn-Lys-Leu-Glu-Tyr-Glu-COOH(WA3-1, Mr: 1422.0 Da) and NH2-Val-Glu-Pro-Val-ThrVal-Asn-Pro-His-Glu-COOH(HA18-3-B-8, Mr: 1119.8 Da) using MALDI- TOF-TOF MS. Furthermore, the anticoagulant activity of WA3-1 kept stable after in vitro digestive simulation. Two novel peptides WP20 and WP21 were synthesized successfully. The results of in vitro absorbing simulation indicated that as the extending of incubation time, the transport amount of WP20 and WP21 had an increasing tendency, whether from UR to DR, or DR to UR.The present paper prepared successfully two anticoagulant peptides with high antifatigue abilities, and the purified anticoagulant peptides were sequenced smoothly. This research not only provided theoretical basis for developing active peptide products, but also enriched the theory of the structure-function of them.