Screening Of Strains For Main Saponins Transformation From Flower And Root Of Panax Notoginseng And Identification Of Its Transformation Products

Panax notoginseng（Burk.）F.H.Chen（Araliaceae）is a precious traditional Chinese herbal medicine that mainly grows in the Yunnan and Guangxi province s.The roots and flowers of P.notoginseng contain a high concentration of saponins,which are the main bioactive ingredients in different parts of the plant.The P.notoginseng flower（PNF）contains more than 20%,with ginsenosides Rb₁,Rb₂,Rb₃,and Rc,and notoginsenosides Fa and Fc being the major saponins in the PNF.These saponins belong to the protopanaxadiol（PPD）ginsenosides.The primary saponins found in the root of P.notoginseng are ginsenosides Rb₁,Rg₁,Re,Rd,and notoginsenoside R₁.These saponins constitute more than 80%of the total saponin content in the root of P.notoginseng.P.notoginseng root contains both PPD and protopanaxatriol（PPT）.P.notoginseng saponins can be classified as damaran-type ginsenosides according to their aglycone skeleton,The damalkane ginsenosides can be divided into PPD ginsenosides without hydroxyl substitution at C-6 and PPT ginsenosides with hydroxyl substitution at C-6.Rare ginsenosides（such as Rg₃,Rg₅,Rk₁,Rh₁,Rh₂,Rd₂,Fe）exhibit advantages of better cell permeability and higher bioavailability due to less sugar content,so rare ginsenosides have higher pharmacological activity.At present,the main methods to obtain trace ginsenosides are physical transformation,chemical transformation,biological transformation and cloning ginsenoside enzyme transformation.Biological transformation is one of the main means to obtain rare ginsenosides.Compared with the other two methods,it has more advantages.In this study,we isolated and purified a large number of fungi from panax notoginseng soil and flowers,and screened for saponin conversion activity,aiming to find strains with high substrate conversion rate,few by-products and high pharmacological activity of conversion products,which will provide a new source of strains for microbial transformation of rare ginsenosides and provide a theoretical basis for its large-scale production.The specific research contents and results include the following aspects:1.Multiple strains were isolated from rhizosphere soil and flowers of P.notoginseng,and three strains（including strains F17,S7,S3）with obvious saponin conversion activity to ginsenoside Rb₁ and Rg₁ with high content in P.notoginseng were obtained through primary screening and rescreening.Morphological and molecular biological identification of the selected target strains were carried out.The results showed that strain F17 was Pestalotiopsis biciliata,strain S7 was Cladosporium xylophilum,and strain S3 was Aspergilus tubingensis.2.The total saponins of root and flower from P.notoginseng were extracted by ethanol reflux,and the extraction rate was 30%.C.xylophilum was used to transform total saponins of P.notoginseng flower and single saponins with high content（ginsenosides Rb₁,Rb₂,Rb₃,and Rc and notoginsenosides Fa and Fc）,and qualitative and quantitative analysis of the transformed products was conducted by HPLC,TLC and MS.In addition,during the transformation process,the sequential order and content changes of the transformation products were regularly monitored,and the transformation path of each saponin was analyzed combined with the structural differences with the substrate.The results showed that:the main ginsenosides Rb₁,Rb₂,Rb₃,and Rc and notoginsenoside Fa were converted by C.xylophilum into ginsenosides F₂,Rd₂,notoginsenosides Fd,Fe and ginsenoside R₇,with the conversion rates of 100,100,100,88.5 and 55.3%,respectively.The transformation paths of saponins were also determined.Three monomeric ginsenosides（ginsenoside Rd₂,notoginsenosides Fe and Fd）were isolated and identified from the transformed products after the cultivation of PNF saponins transformed by C.xylophilum.The crude enzyme produced by C.xylophilum was studied.The results showed that the optimal p H of crude enzyme for biotransformation of monomer saponin was 5～6,the optimal temperature was 50℃,the molecular weight of purifiedβ-glucosidase was 66.4 k Da,and the activity ofβ-glucosidase was 129 U/m L.3.In this study,A.tubingensis was used to transform the main single saponins（ginsenosides Rb₁,Rg₁,Re,Rd and notoginsenoside R₁）in the main root of P.notoginseng.HPLC,TLC and LC-MS were used to qualitative and quantitative analysis of the transformed products,and the transformation paths of each saponins were analyzed.The results showed that A.tubingensis can convert the 5 main saponins in the root of P.notoginseng into 16 rare saponins,and the transformation products are relatively abundant.The transformation reactions include deglycosylation,differential isomerization and dehydration.The transformation paths of ginsenosides Rb₁,Rg₁,Re,Rd and notoginsenoside R₁ are as follows:Rb₁→Rd→（R/S）Rg₃→Rk₁,Rg₅;Rd→（R/S）Rg₃→Rk₁,Rg₅和Rd→F₂;Rg₁→（R/S）Rh₁→Rk₃、Rh₄;Re→（R/S）Rg₂→F₄,Rg₆;R₁→（R/S）R₂.Meanwhile,theβ-glucosidase produced by A.tubingensis was also analyzed.The results show that:the optimal p H value of the enzyme was 5,and the activity ofβ-glucosidase was more than 70%at p H 5～8.The optimum temperature of enzyme was 50℃,which was particularly stable at 30℃～70℃,and the enzyme activity remained above 70%.After purification,the molecular weight of the purifiedβ-glucosidase was 66 k Da.In the process of enzyme purification,the yield of pure enzyme was 3.4%,and the enzyme specific activity increased by 4.8 times.4.In this study,P.biciliata was used for the transformation of the main saponins(ginsenoside Rb₁)in the main root of P.notoginseng.HPLC,TLC and MS were used for qualitative and quantitative analysis of the transformation products and analysis of the transformation path of ginsenoside Rb₁.The experimental results showed that P.biciliata could efficiently convert ginsenoside Rb₁ into rare ginsenoside F₂,C-K and ginsenoside Rd with less content but higher activity of P.notoginseng,with the conversion rates of53%,11%and 10%,respectively.