Preparation Of Antioxidant Peptides From Mulberry Leaves And Their Protective Effects On Oxidative Damage In BRL-3A Hepatocytes

Mulberry leaves,also known as "iron fan",were first published in " Sheng Nong’s herbal classic".It has the effect of dispersing wind-heat,clearing the liver and brightening the eyes.Mulberry leaves contains a lot of active ingredients,such as protein,carbohydrates,vitamins,minerals,alkaloids,etc.The protein content is more than 20%,and there is a rich variety of amino acids,which has the value of development and utilization.According to the research,the peptides prepared by enzymatic digestion of proteins can produce more functional activities,such as hypotension,hypoglycemia,anti-inflammatory,antioxidant,anti-aging,et al.Protein content is the basis of preparation of high functional active polypeptides,and increasing protein dissolution is the premise of preparation of high protein content samples.The results show that the extraction conditions have a great influence on the amount of plant protein leaching,and the commonly used alkali-soluble extraction method mainly has the problems of inadequate protein dissolution and low yield.In order to further improve the dissolution amount of protein,physical assisted extraction and biological enzymatic extraction are mainly used at present,which can promote the release of protein to a certain level.In recent years,in order to improve the protein content to a greater extent,some scholars have combined ultrasonic assistance with cellulolytic hydrolysis to synergistically degrade cellulase components in the substrate and increase the contact area between the substrate and the solvent,which can further promote the dissolution of plant proteins.The main problems of traditional proteolytic methods are low product activity and long enzymatic digestion time.In order to make up for these deficiencies,the commonly applied methods include chemically assisted enzymatic digestion and multi-enzyme synergistic enzymatic digestion,which improve the functional activity of peptides and shorten the enzymatic digestion time to different degrees.In recent years,the physical techniques include microwave,ultrasound and critical extraction have been widely used in the auxiliary preparation of peptides,which the microwave can make the peptide bonds of proteins and amino acid side chains produce orderly vibrations,changes the advanced structure of protein,exposes more enzymatic cleavage sites and increase the binding of proteases and substrate,the microwave has the characteristics of strong penetration and high heating efficiency.According to the recent studies,the microwave pretreatment of proteins can shorten the digestion time by proteases and obtain higher antioxidant activity of peptides than other auxiliary enzymatic means.In addition,proteases in the gastrointestinal tract are also common enzymes for the preparation of peptides.Based on the in vitro enzymatic digestion,transferring part of the enzymatic process to the gastrointestinal tract within digestion to shorten the in vitro enzymatic digestion time.In order to further verify the antioxidant effect of mulberry leaves peptides,a model of oxidative damage to liver cells was established in vitro,and mulberry leaves antioxidant peptides（MLAP）was used to intervene the damage model.To investigate the protective effect of MLAP on oxidative stress induced hepatocyte injury by measured antioxidant and hepatocyte function indexes.Therefore,the dissolution amount of mulberry leaves was used as an index and extracted the mulberry leaves protein by ultrasonic-assisted cellulase degradation to explore the optimum extraction conditions of mulberry leaves protein in this experiment.The antioxidant activity of mulberry leaves peptides after gastrointestinal simulation digestion was used as an index,seeking the optimum preparation conditions of mulberry leaves antioxidant peptides（MLAP）by microwave pretreatment with proteinbinding enzyme hydrolysis method.To investigate the protective effect of MLAP on oxidative damage of hepatocytes by measuring the cytokines related to oxidative stress.Objective: To optimize the preparation process of MLAP and investigate its protective effect against oxidative damage in BRL-3A hepatocytes.Methods: 1.Screening the extraction process of mulberry leaves protein based on the the protein dissolution amount（1）The solvent parameters for the protein dissolution amount of mulberry leaves.were screened,which used a single-factor test with the following factors and level designs: NaOH concentration（0.1%,0.3%,0.5%,0.7%,0.9%）,extraction time（3,3.5,4,4.5,5 h）,extraction temperature（50°C,60°C,70°C,80°C,90°C）and feed-to-liquid ratio（5%,6%,7%,8%,9%）.（2）The extraction conditions of mulberry leaves protein by ultrasoundassisted cellulase degradation were screened,which used a single-factor stepwise optimization method with the following factors and level designs: pH（5,6,7,8,9）,ultrasound temperature（30°C,35°C,40°C,45°C,50°C）,enzyme addition（3%,4%,5%,6%,7%）and ultrasound time（5,10,15,20,25 min）as indicators,15,20,25 min).And on this basis,the Box-Behnken response surface method was used to further optimize the extraction conditions of mulberry leaves protein by ultrasound-assisted cellulase degradation.2.Screening the preparation process of MLAP based on antioxidant activity（1）Based on the combined rank-sum ratio（RSR）of five antioxidant indexes（DPPH radical scavenging rate,-OH radical scavenging rate,total reducing capacity,superoxide anion scavenging rate and total antioxidant capacity）of MLAP after gastrointestinal simulated digestion.Neutral protease,papain,flavor protease,complex protease and alkaline protease were used to prepare the MLAP and screen the best proteases.（2）Based on the RSR of MLAP antioxidant index after gastrointestinal simulated digestion,a single factor step by step optimization test was used to screen the enzymolysis conditions of the proteases identified in the previous step.Including pH（4,5,6,7,8）,temperature（40℃,45℃,50℃,55℃,60℃）,time（80,100,120,140,180 min）,enzyme addition amount（8%,9%,10%,11%,12%）,solid-liquid ratio（1%,2%,3%,4%,5%）.（3）Based on the RSR of antioxidant index of MLAP after gastrointestinal simulation digestion,the conditions of microwave pretreatment mulberry leaves protein were screened,including microwave time（10,20,30,40,60 s）and microwave power（300,400,500,800,1000 W）,and the preparation process of MLAP was finally determined.3.Establishing the hepatocytes injury model induced by H₂O₂Well-grown BRL-3A hepatocytes were randomly divided into blank group and different concentrations of H₂O₂ groups（100,120,130,140,150 μmol/L）,and the cell morphology was observed under the inverted microscope,the cell survival rate was determined by CCK8 method and the dehydrogenase（LDH）release in the cell culture was measured after 4 h of H₂O₂ effect.4.Determining the concentration of MLAP damage to BRL-3A hepatocytesWell-grown BRL-3A hepatocytes were randomly divided into blank group and the different concentrations of MLAP groups（400,500,600,700 and 800 mg/L）,and the cell morphology was observed under the inverted microscope,the survival rate of the cells was determined by CCK8 method,and the amount of LDH released from the cell culture medium was also measured after 20 h of MLAP effect.5.Determining the protective concentration of MLAP against H₂O₂ induced damage in BRL-3A hepatocytesWell-grown BRL-3A hepatocytes were randomly divided into blank group,model group,and the different concentrations of MLAP groups（50,80,100,120,130 mg/L）.The different concentrations of MLAP group acted on the cells for 20 h,the model group and the different concentrations of MLAP group were acted on the cells for 4 h with H₂O₂ at a final concentration of 130 μmol/L.The cell morphology was observed under the inverted microscope,the survival rate of the cells was determined by CCK8 method,and the amount of LDH released from the cell culture medium was also measured.6.Protective effect of MLAP on H₂O₂ induced damage in BRL-3A hepatocytesWell-grown BRL-3A hepatocytes were randomly divided into blank group,model group,and the different concentrations of MLAP groups（100,200,400 mg/L）,the different concentrations of MLAP groups acted on the cells for 20 h,the model group and the different concentrations of MLAP group were acted on the cells for 4 h.Cell morphology,cell survival rate,superoxide dismutase（SOD）activity and malondialdehyde（MDA）content in cells were measured;LDH,aspartate aminotransferase（AST）and alanine aminotransferase（ALT）were released in cell culture medium.Results: 1.Optimization of the extraction process of mulberry leaves protein（1）Single-factor screening of the solvent parameters for the extraction of mulberry leaves protein are: NaOH concentration 0.5%,extraction time 3.5 h,extraction temperature 60°C,and material-to-liquid ratio 7%.（2）Single-factor step-by-step optimization of the extraction conditions of mulberry leaves protein by ultrasound-assisted cellulase degradation: pH 7,ultrasound temperature 35℃,enzyme addition 4%,and ultrasound time 10 min.（3）The response surface method is used to optimize the conditions for the extraction of mulberry leaves protein by ultrasound-assisted cellulase degradation: pH 7.18,sonication temperature 35.26 ℃,enzyme addition amount 4.15%,sonication time 10.08 min,the theoretical protein dissolution amount of mulberry leaves protein is 13.54 mg/m L.For the feasibility of the experimental operation,the operating conditions of the response surface method were adjusted as follows: pH 7.2,sonication temperature 35 ℃,enzyme addition amount 4.2%,sonication time 10 min.Under these conditions,the dissolution of mulberry leaves protein was 13.78 mg/m L,the content of mulberry leaves protein was 62.69%.2.Optimization of the preparation process of MLAP（1）After the enzymatic digestion of mulberry leaves protein by neutral protease,papain,flavor protease,complex protease and alkaline protease,the RSR of antioxidant activity of MLAP were obtained as 0.56,0.8,0.72,0.68 and 0.2,which were ranked as: papain>flavor protease>complex protease >neutral protease >alkaline protease,according to the sequencing results,the enzymatic hydrolysis of papain is the best.（2）The enzymatic conditions for the preparation of MLAP by singlefactor stepwise optimization are: pH 5,temperature 50 ℃,time 100 min,10% enzyme addition,and 2% material-to-liquid ratio.（3）The microwave conditions for preparing MLAP are: microwave time 30 s,microwave power 600 W.3.Establishing the hepatocytes injury model induced by H₂O₂After the cells were treated with different concentrations of H₂O₂（100,120,130,140 and 150 μmol/L）for 4 h,compared with the blank group,with the increase of H₂O₂ concentration,the degree of cell damage increased,some cells showed agglomeration,refractive index decreased,cell survival rate decreased,and the release of LDH in the cell culture medium increased,when the concentration of H₂O₂ reached 130 μmol/L,the cell refractive index decreased,some cells appeared rupture,cell survival rate decreased,and the release of LDH in the culture medium increased.Therefore,the damage concentration of H₂O₂ in BRL-3A hepatocytes was 130 μmol/L.4.Determining the concentration of MLAP damage to BRL-3A hepatocytesAfter different concentrations of MLAP groups（400,500,600,700 and 800 mg/L）acted on the cells for 20 h,compared with the blank group,when the concentration of MLAP reached 700 mg/L,the cells showed partial clustering,unclear boundary,cell membrane damage,cell survival rate decreased by 27.97%,and the release of LDH in the culture medium increased significantly by 14.07%.Therefore,the damage concentration of MLAP in BRL-3A hepatocytes was 700 mg/L.5.Determining the protective concentration of MLAP against H₂O₂ induced damage in BRL-3A hepatocytesAfter acted on the cells for 4 h of H₂O₂,compared with the blank group,the cell boundary of the model group was unclear,some of the protrusions disappeared,the cell survival rate decreased,and the release of LDH in the culture medium increased.After acted on the cells for 20 h of different concentrations of MLAP groups（50,80,100,120,130 mg/L）in H₂O₂ induced hepatocytes,compared with the model group,when the concentration of MLAP was in the range of 50⁸0 mg/L,the cell boundary was not clear,part of the protrusion disappeared,the cell survival rate did not change significantly,the LDH release amount in the medium did not decrease significantly,when the concentration of MLAP reached 100 mg/L,the cell protrusion increased,the refraction became strong,the cell survival rate increased by 24.32%,the LDH release amount in the medium decreased by 45.52%.Therefore,the protective concentration of MLAP against BRL-3A hepatocytes damage caused by H₂O₂ is 100 mg/L,the final determination of MLAP concentration range is 100⁷00 mg/L.6.Protective effect of MLAP on H₂O₂ induced damage in BRL-3A hepatocytesAfter acted on the cells for 4 h of H₂O₂,compared with the blank group,the model group showed reduced cell protrusion,reduced refractive index,incomplete cell damage,the cell survival rate decrease by 38.21%,the LDH release amount in the medium increased by 214.97%,the intracellular MDA content was increased by 354.22%,and the intracellular SOD activity was decreased by 27.49%.The release of AST and ALT in culture medium respectively increased by 58.51% and 58.10%,Compared with the model group,low,medium and high concentration MLAP（100,200,400 mg/L）reduced the damage to cell structure caused by H₂O₂ to different degrees,increased cell processes,increased refraction,and respectively increased cell survival rate by 19.42%,27.67%,36.72%,the release of LDH in culture medium decreased by 18.61%,29.93%,51.46%,AST decreased by 26.18%,41.95%,36.75%,and ALT decreased by 28.38%,35.88%,37.63%,respectively.Intracellular MDA content decreased by 48.43%,62.52% and 60.18%,respectively,while intracellular SOD activity increased by 6.50%,38.09% and 32.49%,respectively.Conclusion: 1.The extraction process of mulberry leaves protein is: the material to liquid 7%,pH 7.2,cellulase 4.2%,sonication at 35°C for 10 min,0.5% NaOH extraction solvent at a constant temperature of 60°C for 3.5 h.The protein dissolution amount of mulberry leaves protein obtained under this condition was 13.78 mg/m L,The protein content of mulberry leaves after acid precipitation is 62.69%.2.MLAP is prepared by the following process: the material to liquid 2%,600 W microwave for 30 s,pH 5,10% papain,and 100 min of enzymatic digestion at a constant temperature of 50°C.3.MLAP can protect the H₂O₂ induced BRL-3A hepatocytes injury: MLAP can alleviate the abnormal morphology of BRL-3A hepatocytes damage caused by H₂O₂,improve cell survival rate,reduce the release of LDH,AST and ALT in culture medium,reduce the content of MDA in cells,and increase the activity of SOD.