Isolation And Biotransformation Of Triterpenoid Saponins From The Aerial Parts Of Agriophyllum Squarrosum Mongolica

Agriophyllum squarrosum is widely distributed in the desert areas of northwest and northeast China.It contains abundant alkaloids,flavonoids and saponins.It is also the main pharmacodynamic ingredient.There are also records of Agriophyllum squarrosum and its seeds（Dongyizi）in Chinese Pharmacopoeia.Saponins,as the main active ingredients of many traditional Chinese medicines,often contain large glycosyl substituents in their structures,while saponins with large molecular structure are not conducive to human absorption and utilization.Some studies have shown that triterpenoid saponins are the main active ingredients of Agriophyllum squarrosum.Their structures are linked by larger glycosyl groups.However,the study on the biotransformation of the active ingredients of Agriophyllum squarrosum had not been reported.In this paper,the extraction of triterpenoid saponins,the main active ingredient of Agriophyllum squarrosum,was studied,and the biotransformation of them was studied,which was beneficial to the development and utilization of Agriophyllum squarrosum as an economic medicinal plant.In this paper,Kjeldahl method,Coomassie blue colorimetric method,phenol-concentrated sulfuric acid colorimetric colorimetric method and alkaline hydrolysis method were used to study the total egg content,soluble protein content,soluble sugar content and fat content of Agriophyllum squarrosum.Determination,the determination of these substances is conducive to the design of the separation and purification process of the medicinal components of the cannula;this experiment also uses the Na NO₂-Al（NO₃）₃-Na OH colorimetric method,vanillin colorimetric method and Acid-base titration,to detect the content of flavonoids saponins and alkaloids,which are the main medicinal components of Agriophyllum squarrosum.It can provide reference for the expansion of Agriophyllum squarrosum.In this paper,the traditional ethanol extraction method was used to extract the saponin,and the extraction conditions were optimized.After a large amount of extraction,the saponin extract was concentrated and removed,and the AB-8 resin column and D280 resin column were used respectively.It is subjected to de-sugarization and decolorization treatment.The soil samples from the roots of Agriophyllum squarrosum grown in the Alashan Desert region of Inner Mongolia were screened,and the preliminary biotransformation experiments were carried out on the extracted total saponins of Agriophyllum squarrosum by the strains after primary screening and rescreening.Thin layer chromatography was carried out,and the selected strains for biotransformation of saponin were preserved.The experiment also optimized the fermentation conditions of the strains that have a transformation effect on saponin,and determined the most Excellent culture temperature,culture time,inoculum size,medium water content and medium ratio;the experiment finally determined the optimum conversion time and p H of the enzyme solution obtained by transforming the saponin saponin under the optimal fermentation conditions and time.The total protein content of the above part of Agriophyllum squarrosum was 8.62%,the soluble protein content was 0.063‰,the soluble sugar content was 0.032‰,and the fat content was 6.73‰.The content of flavonoids in Agriophyllum squarrosum was 1.07%and the total saponin content was 3.10%,the alkaloid content is about 5.50%.The optimal extraction conditions of saponin are 70%ethanol,heated in a water bath at 65°C for 2 h,and soaked for 3 times;the saponin obtained by deodorization,de-sugarization and decolorization of the saponin extract The yellowish amorphous powder was 3.36 g,and the yield was 36.1%based on the content of the saponin of the Agriophyllum squarrosum extract,the extracted crude saponins were detected by TLC with at least four fluorescent spots in each band under ultraviolet light.After screening and rescreening the experimental sieve soil samples,a total of 2 strains of fungi with clear morphology,single colony and transformation of Agriophyllum squarrosum saponin were obtained,and the crude enzyme extract and the total saponin of Agriophyllum squarrosum were subjected to biotransformation reaction and then detected by thin layer chromatography.The fluorescent spots were observed to increase or decrease or change the brightness and darkness under the ultraviolet light of the substrate..Condition optimization,the optimum fermentation conditions of strain 1 were determined as follows:15%inoculation（spore content 10⁷/ml）at 14:6 and 100%water content in the medium of bran-dried Sophora japonica culture at 35℃for 3 days,The optimum fermentation conditions of Bacillus 2 were as follows:10%inoculation（spore content 10⁷/ml）at 16:4 and 50%water content in the medium of bran-dried Sophora japonica culture at 35℃for 3 days.The preserved strains were cultured under the optimum fermentation conditions,then treated with acetic acid-sodium acetate buffer at p H 5.2 and centrifuged at 9000 r/min for 10 minutes to retain the supernatant as crude enzyme solution,the optimal fermentation conditions for the crude enzyme extract of the strain 1 were constant temperature reaction in the environment of p H 5.2 for 18 h,and the crude enzyme extract of the bacteria 2 was The optimal fermentation conditions were a constant temperature reaction for 24 h in an environment of p H 5.6.