| In recent years,the health problems caused by excessive drinking are increasing prominently with the increasing consumption of alcohol products in China.Alcoholic liver disease?ALD?is one of the typical cases.Alcohol-induced liver dysfunction involves the generation of free radicals,oxidative stress,and lipid peroxidation.At present,drug and nutritional therapy are the primary methods to prevent or cure ALD.However,the existing drugs have potential side effects.Bioactive peptides are widely favored for their advantages of small molecular weight,easy absorption,ample source of food protein and good safety.Therefore,it is a hotspot of research and attention to seek a safe and effective hepatoprotective peptide to prevent or treat ALD.Hyriopsis cumingii,is an unique and high-quality freshwater mussel in China.Its meat is rich in protein,amino acid and other bioactive substances,which could be a source of high-quality protein and an ideal source for extracting bioactive peptides.According to the records of“Diet therapy materia medica”and“Compendium of materia medica”,Hyriopsis cumingii meat was mainly used to“alcohol detoxification”in ancient folk.Both its peptide and peptide-zinc chelate have hepatoprotective effect.Therefore,Hyriopsis cumingii peptides?Hcp?was prepared from its meat by enzymatic hydrolysis based on the index of alcohol dehydrogenase?ADH?activation rate in vitro,and then evaluated the anti-hangover activity of a mice drunk model.Secondly,on the basis of analyzing the basic characteristics of Hcp and its ultrafiltration components?Hcp-?,Hcp-?and Hcp-??,the anti-hepatic injury activity in vitro of them were evaluated.Then,the hepatoprotective effect and mechanism of its ultrafiltration components were determined by an mice acute ALD model.Furthermore,the hepatoprotective peptides were isolated from the highest anti-ALD activity ultrafiltration component by Gel-filtration and RP-HPLC.Finally,its primary structures were characterized by UPLC-ESI-Q-TOF-MS/MS.It will provide a theoretical basis for high-value utilization of Hyriopsis cumingii meat and its theoretical basis on the prevention or treatment of ALD.The main research contents and results are as follows:?1?The neutral protease was chose from 6 kinds of proteases as the protease for preparing Hcp with the indicator of ADH activation rate in vitro.The enzyme hydrolysis temperature,enzyme hydrolysis time,ratio of material-liquid and enzyme dosage were used to investigate their effects on the ADH activity in vitro of Hcp.On this basis,the response surface method was used to optimize the enzymatic hydrolysis process of Hcp.The optimal conditions are as follows:enzyme hydrolysis temperature is 50.0?,enzyme hydrolysis time is 2.0 h,ratio of material-liquid is 1:3,and enzyme dosage is 1000 U/g.Under these conditions,the ADH activation rate in vitro of Hcp is 18.30%.?2?The anti-hangover activity of Hcp was studied preliminarily by establishing a mice drunk model.Compared with the model group,each dose of Hcp groups could significantly prolong the drunken time,shorten the sleep time,reduce the alcohol concentration of mice blood and enhance the ADH activity of mice liver?P<0.05?.The alcohol concentration of mice blood was decreased from 20.31 mmol/L to 8.80mmol/L after drinking 90 min.The ADH activity in mice liver was increased from1.92 U/mg to 7.64 U/mg.It indicated that Hcp has a certain anti-alcoholic and anti-drunk effect on the drunk mice,especially in the high dose group of Hcp.?3?Firstly,the basic nutritional components,amino acid composition,molecular weight distribution of Hcp and its ultrafiltration components were analyzed,and then the in vitro anti-hepatic injury activity were evaluated by determined the antioxidant activity and ADH activation rate in vitro.The basic characteristics analysis showed that Hcp has a high content of protein?57.60%?,small content of fat?2.62%?,and complete amino acid composition.The molecular weight is concentrated in the range of 450-6500 KDa?83.54%?mainly.After ultrafiltration,there is a significant difference between Hcp and its ultrafiltration components on crude protein,amino acid content and yield of small molecule peptide?P<0.05?.It showed that ultrafiltration could effectively separate peptides with different molecular weight.Secondly,the content of amino acids related to anti-hangover activity in Hcp-?could increase significantly compared with Hcp-?and Hcp-?.The anti-hepatic injury activity in vitro showed that Hcp and its ultrafiltration components could scavenge ABTS and DPPH free radicals to a certain extent,and has a strong reducing power and active ADH in vitro?P<0.05?.It indicated that Hcp and its ultrafiltration components has a certain anti-hepatic injury activity in vitro,especially the strongest activity of Hcp-?.?4?The hepatoprotective effects and mechanisms of Hcp ultrafiltration components on anti acute alcohol-induced liver injury were investigated by establishing an acute ALD model of mice.Compared with the alcohol model group,the intervention of each ultrafiltration component of Hcp could significantly reduce the Liver index,ALT and AST activity of serum,MDA,TG and cytochrome P4502E1content of liver?P<0.05?;significantly increase the GSH content,SOD,ADH and ALDH activity of liver?P<0.05?;and up-regulate the m RNA expression of ADH2,ALDH2,and down-regulate the m RNA expression of CYP2E1,Fabp5,G6pc,Idi1,?-catenin,TGF-?1 and TGF-?,which related to alcohol metabolism,lipid metabolism and liver fibrosis.It could activate wnt/?-catenin pathway and promote?-catenin entry into the nucleus.The above results indicated that Hcp could reduce the alcohol-induced liver acute injury in mice by regulating fatty acid metabolism and inhibit the formation of liver fibrosis.Its metabolism involved in antioxidant stress and activating alcohol metabolism enzyme,especially the anti ALD effect of Hcp-?.It indicated that the high anti ALD activity peptide is mainly in the range of MW=3?10 KDa.?5?On the basis of ADH activation rate in vitro to tracking,Hcp-??MW=3?10KDa?was separated by Sephadex G-25 and RP-HPLC,and the polypeptide sequence of high ADH activation activity in vitro was identify by UPLC-ESI-Q-TOF-MS/MS.Among the F1,F2,F3,F4 and F5 with Sephadex G-25 separation of Hcp-II,F3 shows the highest ADH activation rate in vitro?68.72%?.Moreover,among F3-1,F3-2,F3-3and F3-4 with semi-preparative RP-HPLC(XBridge TMBEH130 C18)of F3,F3-2shows the highest ADH activation rate in vitro?55.97%?.Finally,there are two functional active peptide from F3-2 were identified by UPLC-ESI-Q-TOF-MS/MS.The amino acid sequence is tetrapeptide of Val-Leu?Ile?-Ala-Pro and pentapeptide of Val-Asn-Leu?Ile?-Leu?Ile?-Glu,respectively. |
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