Study On Cell Suspension Culture And Extraction And Separation Of Taxol From Taxus Cuspidata

Taxol is one of the most widely used natural anticancer drugs,but it is difficult to extract taxol directly because of its low content in Taxus.Therefore,it is necessary to use plant cell culture method to produce taxol efficiently,and then use effective extraction and separation methods to extract taxol and its analogues,in order to fundamentally solve the problem of raw material shortage of Taxus.Therefore,in this paper,the callus of Taxus cuspidata was induced,subcultured and high-yield cell lines were screened,and the cell suspension culture conditions were optimized.The effect of cell secretion enhancers on the grow than The effect of cell secretion enhancers on the growth and taxol synthesis and accumulation of Taxus cuspidata cells was investigated;the synergistic effect of them and regulatory factors on taxol production was explored:and the best culture scheme for efficient taxol production was obtained.At the same time,magnetic ionic liquid was used as an additive to assist ultrasonic extraction of taxol.Finally,the molecularly imprinted polymer of harringtonine(paclitaxel analogues)was synthesized by bulk polymerization to separate harringtonine and simplify the difficulty of paclitaxel separation.It lays the foundation for the production of taxol by cell culture of Taxus chinensis and provides the theoretical basis for the efficient development of medicinal plant resources.The conclusions obtained through experimental research in this paper are as follows:(1)To explore the effects of four different types of cell secretion promoters on taxol synthesis and accumulation,respectively,to investigate their effects on cell growth,cell membrane permeability,malondialdehyde(MDA)and taxol synthesis and release.Finally,it is concluded that four kinds of cell secretion promoters can significantly improve taxol production and permeability.The optimum concentrations of DMSO,Tween-80,Mg Cl2 and mannitol were 4%,300μg·g-1,12mmol·L-1 and 300mmol·L-1,respectively.(2)Using box Behnken design,the synergistic effect of DMSO and regulatory factors(methyl jasmonate and Ethrel)was optimized.The optimal concentration combination of DMSO and methyl jasmonate and Ethrel was as follows:DMSO concentration was 4.721%,methyl jasmonate concentration was 103.389μmol/L,Ethrel concentration was73.771μmol/L,At this time,the total taxol content of suspension cultured Taxus cells reached the maximum of 15.705mg/L.(3)In the experiment of ultrasonic extraction of paclitaxel assisted by magnetic ionic liquid,Box Behnken design was used to explore the interaction of three factors(solid-liquid ratio,ultrasonic time and {C4(MIM)2}Fe Cl3/Br2 addition)on the extraction of paclitaxel.The optimal extraction process was obtained as follows:solid-liquid ratio(G/ml)1:43.5,ultrasonic time 50 minutes,The addition amount of {C4(MIM)2} Fe Cl3/Br2 was 1.5wt% and the extraction amount of paclitaxel was 2.828mg/g.(4)In the optimization experiment of synthesizing the molecularly imprinted polymer of harringtonine by bulk polymerization,it was found that the molecularly imprinted polymer with 2-vp as the functional monomer(harringtonine:functional monomer = 1:8)had the best adsorption performance when chloroform was used as the porogen,and its adsorption capacity and imprinting factor were 1.18 mg/g and 2.23,respectively.