| Objective:Previous studies have obtained the extraction and purification process of the active fraction anti-liver fibrosis of Periplaneta americana,but the extraction rate is low.In this paper,the crude extracts of Periplaneta americana were used as raw material,and the enzymolysis technology was used to prepare the active fraction of anti-liver fibrosis with high yield and high activity,which laid a foundation for the follow-up research on anti-liver fibrosis of Periplaneta americana.Methods:1.Screening of proteaseThe protein content in crude extract of Periplaneta americana was determined by Folin-Ciocalteu method and BCA（2,2’-biquinoline-4,4-dicarboxylic acid disodium salt）.The hydrolysis degree,elution fraction yield and inhibition rate of rat hepatic stellate cells（HSC-T6）were used as evaluation indexes,and compared with the original process,to evaluate the enzymolysis effects of Trypsin（TR）,Pepsin（PE）,Alkaline（AL）,Papain（PA）and Neutral protease（NE）on the crude extract from Periplaneta americana.N-Tris[hydroxymethyl] methylglycine-sodium dodecyl sulfat-polyacryla-mide gel electrophoresis（Tricine-SDS-PAGE）was used to detect the molecular weight change of crude extracts by enzymolysis of different proteases.2.Process optimization of preparation of anti-liver fibrosis active components from Periplaneta americana by neutral and papain proteasesTaking hydrolysis degree,yield of active components and activity in vitro as evaluation indexes,and on the basis of single factor investigation（the dosage of enzyme,enzymatic hydrolysis time,p H value,enzymatic hydrolysis temperature,substrate concentration）,L9（34）orthogonal experiment was used to optimize the preparation process of active components of Periplaneta americana anti-liver fibrosis by neutral and papain proteases.Results:1.Screening of proteaseThe protein content in crude extract of Periplaneta americana is more than 45%.The anti-liver fibrosis active components of Periplaneta americana were prepared by enzymolysis method.The hydrolysis degree of different proteases to crude extract was NE > PA > AL >TR> PE.Using the yield of component D as the evaluation index,the results showed that PA > NE > AL > original process > PE > TR.Compared with the original process,the yield of D component obtained by enzymolysis of PA,NE and AL increased by 30.36%,16.07% and14.29% respectively.Compared with the original process,the inhibitory effect of components A,B and C on HSC-T6 was not significantly improved after enzymolysis of different proteases,but the IC50 of component D（active component） obtained by enzymolysis of PA,NE,AL and TR on HSC-T6 cells was relatively small at 24,48 and 72 hours.The results showed that the molecular weight of the crude extract of Periplaneta americana was mainly distributed between 16 and 45 KDa,and the molecular weight decreased after hydrolysis by different proteases.2.Process optimization of preparation of anti-liver fibrosis active components from Periplaneta americana by neutral proteaseThe results showed that the correlation between hydrolysis degree and the yield of active components was poor,so the optimal conditions of enzymolysis were determined mainly according to the yield of active components and the activity in vitro.The results of single factor experiment and orthogonal experiment showed that the effects of various factors on the yield of active components were the dosage of enzyme > p H of enzymolysis> enzymolysis temperature >enzymolysis time,and the effects on the activity in vitro were the dosage of enzyme > p H of enzymolysis > time of enzymolysis > temperature of enzymolysis.The optimum enzymolysis technology of neutral protease was determined as follows: 0.3%enzymatic dosage,2h enzymolysis time,8.5 p H value,45℃ enzymolysis temperature and1.07 substrate density.Verified this process,it showed that the hydrolysis degree of the crude extract Periplaneta americana was 15.8%,the average yield of active components was 0.72%,RSD was 2.27%,and the increase rate of active components was 33.33% compared with the original process（0.54%）.The IC50 values of the active components were 122.10 μg·mL-1,122.1 μg·mL-1,and 109.81 μg·mL-1 at 24,48,and 72 h by the original process.The IC50 values of the active ingredients obtained from the verification process were 116.02-122.77μg·mL-1,111.75-117.57μg·mL-1 and 99.62-113.91 μg·mL-1 at 24,48 and 72 h.Compared with the original process,the IC50 values were reduced and had significant difference（P <0.05）.3.Optimizing the preparation of active components of Periplaneta americana anti-liver fibrosis by papain proteaseThe enzymolysis process of papain was optimized by single factor experiment and orthogonal experiment: the amount of papain was 0.6%,the p H value was 7.0,the time of enzymolysis was 1 h,and the temperature of enzymolysis was 55℃.Verified this process,it showed that the hydrolysis degree of the crude extract of Periplaneta americana was 11.49%,the average yield of active components was 0.70%,RSD was 2.2%,and the increase rate of active components was 32.16% compared with the original process（0.53%）.The IC50 values of the active components obtained by the original process were 123.66,113.46 and 108.11μg·mL-1 at 24,48 and 72 hours,and the IC50 values of the active components from the verification process were 114.41-118.54 μg·mL-1,104.46-114.29 μg·mL-1,105.85-107.05μg·mL-1 at 24,48 and 72 hours.There was no significant difference in the activity in vitro compared with the original process（P>0.05）.Conclusion:With the degree of hydrolysis,the yield of active components and the activity in vitro as evaluation indexes,the optimum protease was selected to prepare the anti-liver fibrosis active components of Periplaneta americana,and the optimum technology of preparing the anti-liver fibrosis active components of Periplaneta americana by neutral protease and papain protease were obtained.Considering the cost,neutral protease is better than papain to prepare active components of anti-liver fibrosis from Periplaneta americana. |
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