Study On High-effective Induction And Extraction Of Paclitaxel And 7-xyl-10-DAT From Taxus Chinensis Var. Mairei

In the present study,the dynamic variations of the contents of Paclitaxel and 7-xyl-10-DAT from Taxus chinensis var.mairei were studied.UV radiation induction,oxygen vaccum cavitation induction,acid induction,and enzyme induction on the contents of Paclitaxel and 7-xyl-10-DAT in Taxus chinensis var.mairei were also investigated.The optimum picking time and enzyme induction extraction process were obtained,and the purification of Paclitaxel and 7-xyl-10-DAT by macroporous adsorption resins and column chromatography from enzymetreated plant materials were optimized.Finally,a set of process which employed enzyme induction-vaccum cavitation,macroporous adsorption resins enrichment as well as column chromatography puritification was established for the extraction and purification of Paclitaxel and 7-xyl-10-DAT.Results of this study will provide scientific reference for the utilization of renewable Taxus resource.1.An RP-HPLC method for simultaneous determination of Paclitaxel and 7-xyl-10-DAT was developed as follows:Chromatographic column Curosil-PFP 5μ(250 mm×4.6 mm I.D.),mobile phase acetonitrile-water(42:58,v/v),flow rate 1 mL/min,injection volume 10μL,column temperature 35℃,UV at 234 nm.A good linearity was achieved for Paclitaxel and 7-xyl-10-DAT in concentration ranges of 0.30-300μg/mL and 0.25-500μg/mL,respectively.The developed method was validated with satisfactory repeatability,precision and stability(RSD＜3.0%).2.The dynamic variations of the contents of Paclitaxel and 7-xyl-10-DAT from stems and needles of Taxus chinensis var.mairei at different periods were studied,and the best picking time were obtained.Stems and needles showed a similar trend of Paclitaxel and 7-xyl-10-DAT contents,the maximum contents in needles and stems were in July and November.Because of stronger photosynthesis in July,plants were in vigorous growth period,and lesser biomass was accumulated.Plants began to enter dormant period in November,and more biomass was accumulated.Hence,November was confirmed as the best picking time.3.Effects of UV radiation induction,oxygen vaccum cavitation induction,acid induction, and enzyme induction on the contents of Paclitaxel and 7-xyl-10-DAT in Taxus chinensis var. mairei were investigated.The optimum process of enzyme induction was confirmed for the first time,and the main parameters affecting the contents were optimized as follows:Enzyme induction:Enzyme type:cellulaseConcentration of pectinase:1 mg/mLInduction time:24 hInduction temperature:45℃ After enzyme induction treatment,the contents of Paclitaxel and 7-xyl-10-DAT were 0.044 mg/g and 0.565 mg/g,which increased 35.19%and 31.31%,respectively,comparing to the untreated ones.4.The vaccum cavitation extraction conditions of Paclitaxel and 7-xyl-10-DAT from the enzyme-treated plant materials were optimized,and the optimum extraction parameters were as follows:Extraction solvent:ethanol-water(80:20,v/v) solutionRatio of liquid to solid:30:1Extraction time:each 30 min,total 1.5 hNumber of extraction cycles:3Under the above optimum conditions,the extraction yields of Paclitaxel and 7-xyl-10-DAT from the enzyme-treated plant materials were 0.043 mg/g and 0.564 mg/g,the paste rate was 8.51%,the contents in extracts were 0.505 mg/g and 6.627 mg/g,respectively.After enzyme induction,yield of Paclitaxel and 7-xyl-10-DAT were 132.12%and 131.08%.5.The separation of 7-xyl-10-DAT by macroporous adsorption resins was studied for the first time and the optimum adsorption and desorption parameters on the optimal AB-8 resin were obtained.Sample concentration:0.0657 mg/mLProcessing volume:15 BVAdsorption flow rate:1 mL/minDesorption solution and volume:ethanol-water(30:70,v/v) solution 3 BV and followedby ethanol-water(80:20,v/v) solution 6 BVDesorption flow rate:1 mL/minOperating temperature:35℃After treated by AB-8 resin under the above conditions,the contents of 7-xyl-10-DAT in paste(obtained by the combination of the solution after enzyme induction and the filtrate after water precipitation) were increased 62.43-fold,from 0.053%to 3.34%,the recovery yield was 85.85%.6.Purification of Paclitaxel and 7-xyl-10-DAT by normal phase and reversed-phase column chromatography.The contents of paclitaxel and 7-xyl-10-DAT were 91%and 93%, the recovery yields were 72.72%and 43.02%,respectively.In conclusion,Paclitaxel and 7-xyl-10-DAT with the purities of 91%and 93%and the yields of 0.041 mg/g and 0.205 mg/g were obtained from the stems and needles of Taxus chinensis var.mairei using the above processes.Results of this study will provide scientific reference for the protection of natural Taxus resource and the deep processing and using of artificial planting taxus,which possesses important theoretical signification and application value.