Study On The Discovery Of Specific Oxidases Toward The Skeleton Of Damarane-type Ginsenosides,the Preparation Of Their Derivatives And The Elucidation Of The Anti-myocardial Injury Effects

Objective:The structure modification of series of dama-alkane ginsenosides is carried out by biotransformation to generate rare saponin oxidized derivatives.The anti-myocardial injury activity and molecular mechanism of damaane-type ginsenoside oxidized derivatives are studied based on genomic technology.Fungal samples are extracted at different transformation stages for transcriptome sequencing,and then the differentially expressed genes/transcripts are systematically analyzed,combined with the time course experiment of ginsenoside conversion,the potential key enzymes of the oxidation reaction of ginsenoside were screened,which provided a theoretical basis for the biotransformation of structurally modified ginsenosides,and laid a foundation for the creation of new drugs and the development of new dosage forms of traditional Chinese medicine.Methods:1.Target microorganisms are screened from potential oxidative functions by targeted oxidation of the dama-type ginsenoside backbone oxidation reaction.The optimal fungal biocatalytic system was used to convert 10 Dama alkane ginsenosides.The selection range of substrates for fungal MS-to-dama diffrane ginsenosides is explored,and the transformation derivatives are analyzed using LCMS-IT-TOF and HPLC-QQQ-MS combined with saponins.The medium composition and substrate concentration of fungal MS biotransformation of dama seponin are screened by univariate investigatio,the growth curve of fungal MS is drawn,the time of substrate addition is determined,and the time course experiment is carried out to determine the transformation law.2.Based on solid phase extraction（SPE）,the separation and purification of dammarane-type ginsenosides is optimized.The ginsenosides Rh₁,Rh₂,Rg₂,Rg₃and F₂are amplified and transformed,the target oxidation products are separated and purified,and the structures of the compounds are identified by combining HPLC-MS and ¹³C-NMR.3.Preliminary evaluation of the antioxidant activity of ginsenoside oxidative derivatives are explored by DPPH and SOD;DOX-induced AC16 human cardiomyocytes are used as a model of myocardial injury.The effects of Rh₁,Rh₂,Rg₂,Rg₃and F₂and their24,25-dihydroxy derivatives on cell viability in the concentration range of 25～100μg/m L are investigated by CCK-8 method.Levels of SOD,LDH,MDA and ROS before and after administration are determined,the stained morphology of cells is observed,their anti-myocardial injury activity is evaluated,and the molecular mechanism of action is preliminarily constructed by genomics technology,and then the results are constructed by western-blot technology verification mechanism.4.Full-length transcriptome sequencing is used to analyze the expressed genes/transcripts of fungal MS at different induction stages,screening the differentially expressed genes associated with oxidases and discovering key oxidase and biocatalytic enzyme clusters through chemical-transcript trend fitting.Results:The best culture microorganisms for the biotransformation of dammarane ginsenosides is fungal MS,and the most suitable medium composition is:potato powder5g/L,20g/L glucose,10g/L peptone,1g/L Nacl.The optimal substrate concentration of fungal MS-converting dammarane ginsenoside conversion medium is 150 mg/L.Under these conditions,the conversion of different dama diffrane ginsenosides shows that the pathway of ginsenoside Rg₁conversion is:Rg₂→24,25-dihydroxy Rg₂,the rate is:97%,and the purity of derivative separation is 96%.The pathway of ginsenoside Rh₁conversion is:Rh₁→24,25-dihydroxy Rh₁,the rate is:89%,and the separation purity is 91%.The pathway of ginsenoside Rh₂conversion is:Rh₂→24,25-dihydroxy Rh₂,the rate is:99%,and the separation purity is 99%.The pathway of ginsenoside Rg₃is conversion:Rg₃→24（S/R）,25-dihydroxy Rg₃,the rate was:98%,and the separation purity was 96%and 95%,respectively.The pathway of Ginsenoside F₂conversion is:F₂→Rh₂→24,25-dihydroxy Rh₂,the conversion rate is 96%,the separation purity is 98%,and the p H value of different stages of the medium is determined,which verified that fungal MS conversion dama marane ginsenoside with enzymes is produced by its own growth and metabolism to modify the structure of ginsenosides,rather than acidic or basic substances,resulting in changes in the structure of ginsenosides.The scavenging rate of DPPH radicals,the activity of superoxide dismutase（SOD）of ginsenosides and their oxidized derivatives increases with the increase of sample concentration（range 25～100μg/m L）,among which Rg₃derivatives are the most prominent.The concentration of ten saponins is not cytotoxic to AC16 human cardiomyocytes in the range of 25～100μg/m L,and in the DOX-induced AC16 human cardiomyocyte injury model,all ten saponins could significantly improve the survival rate of injured AC16 human cardiomyocytes.The effect of Rg₃derivatives and Rh₁derivatives is even higher than that of active control drugs,especially therapeutic administration in which the therapeutic administration of ginsenoside 24（R）,25-dihydroxy Rg₃is about 29%higher than that of prototype saponins.Rg₃and its derivatives can significantly increase the SOD content of AC16 human cardiomyocytes,induced by DOX,and reduce the levels of LDH,MDA and ROS.the apoptosis experiment of Hoechst 33258 staining shows that Rg₃and 24（R）,25-dihydroxy Rg₃can improve the apoptosis of AC16 human cardiomyocytes caused by DOX,and the role of ginsenosides modified by oxidative structure is significantly enhanced in this regard.Analysis of GO enrichment and KEGG signaling pathway enrichment are carried out on the differential transcriptome genes of the Rg₃oxidative derivatives group and the DOX model,and verified by Western-blot experiment,finding that24（R）,25-dihydroxy Rg₃can regulate downstream NF-κB expression through the PI3K/Akt pathway and the MAPK pathway,thereby doxorubicin（DOX）-induced apoptosis of cardiomyocytes is inhibited.Comparing with the blank culture,there are mainly 7 oxidases with expression changes in fungal MS transcripts induced by saponin substrates,among which cytochrome P450（ONT.10421.5）,NADH:flavin oxidoreductase/NADH oxidase family are consistent with the content trend of saponin derivatives protein（ONT.5329.1）and Cupredoxin（ONT.2555.5）;thereinto,the 3-hydroxyphenylacetate 6-hydroxylase[EC:1.14.13.63]enzyme corresponding to the cytochrome P450（ONT.10421.5）gene may be the key enzyme for the directed oxidation of dammarane-type saponins,while the other two enzymes are coenzymes in which the reaction occurs.Conclusion:Fungal MS is a promising ginsenoside structure-modifying strain that can perform biocatalytic reactions directly on substrates under suitable conditions.Experiments show that fungal MS can produce 24,25-dihydroxy oxidative derivatives by directional oxidation of ginsenoside type ginsenosides,and through the optimization of fermentation conditions such as medium composition and substrate concentration,the conversion rate and selectivity of the reaction can be significantly improved,the formation of by-products can be reduced,and the fermentation products with simple chemical background can be obtained,conducive to the separation and purification of target products in subsequent experiments.The reaction can be used as a powerful means for the preparation of ginsenoside series oxidation derivatives and the mining of new compounds.The hydroxylation modification of dammadine-type saponins increases the biological activity of saponin myocardial injury repair and can be used as a potential drug for the treatment of myocardial injury-related diseases.Furthermore,transcriptomics is used to track the expression changes of enzyme genes in the catalytic process,and the target oxidase is successfully excavated,providing a large theoretical basis for the subsequent elucidating of substrate selectivity and catalytic mechanism of the reaction.