Study On High-effective Induction, Extraction And Purification Of Cephalomannine And 7-epi-10-DAT From Taxus Chinensis Var. Mairei

In the present study, the dynamic variations of the contents of cephalomannine and 7-epi-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 cephalomannine and 7-epi-10-DAT in Taxus chinensis var. mairei were also investigated. The optimum picking time and enzyme induction extraction process were obtained. Purification of cephalomannine and 7-epi-10-DAT by macroporous adsorption resins chromatography was first time used, obtaining a good result. Normal-phase and reverse-phase column chromatography was used follwing. Finally, a set of process which employed enzyme induction-vaccum cavitation, macroporous adsorption resins chromatography as well as column chromatography puritification was established for the extraction and purification of Cephalomannine and 7-epi-10-DAT. Results of this study will provide scientific reference for the utilization of renewable Taxus resource.1. RP-HPLC method for simultaneous determination of cephalomannine and 7-epi-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℃,UVat 234 nm.2. The dynamic variations of the contents of cephalomannine and 7-epi-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 cephalomannine and 7-epi-10-DAT contents, all of them showed to be raise then descend and finally raise. Maximum contents in needles and stems was November. Hence, November was confirmed as the best picking time.After comparing the content variations of cephalomannine and 7-epi-10-DAT between new leaves and old ones, it was found that contents of cephalomannine and 7-epi-10-DAT in new leaves was nearly 50% of them in old ones, indicating that cephalomannine and 7-epi-10-DAT can cumulate in Taxus chinensis var. mairei's leaves.3. Effects of UV radiation induction, oxygen vaccum cavitation induction, acid induction, and enzyme induction on the contents of cephalomannine and 7-epi-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 h Induction temperature:45℃After enzyme induction treatment, the contents of cephalomannine and 7-epi-10-DAT were 0.027 mg/g and 0.024 mg/g, which increased 55.21% and 52.13%, respectively, comparing to the untreated ones.4. The vaccum cavitation extraction conditions of cephalomannine and 7-epi-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 45 minNumber of extraction cycles:3Under the above optimum conditions, the extraction yields of cephalomannine and 7-epi-10-DAT from the enzyme-treated plant materials were 0.024 mg/g and 0.021 mg/g, the paste rate was 8.51%, the contents in extracts were 0.967 mg/g and 0.954 mg/g, respectively. After enzyme induction, yield of cephalomannine and 7-epi-10-DAT were 89.61% and 83.24%.5. The separation of by macroporous adsorption resins was first studied in this paper and the optimum adsorption and desorption parameters on the optimal AB-8 resin were obtained.Type of macroporous adsorption resin:AB-8Ration of sample to macroporous adsorption resin:1:15 (wet weight)Eluant:50% ethonal solvent 30BV60% ethonal solvent 10BV65% ethonal solvent 15BVAfter treated by AB-8 resin column under the above conditions, the purity of cephalomannine and 7-epi-10-DAT were 0.875% and 2.581%, respectively. The recovery yields of cephalomannine and 7-epi-10-DAT were 76.54% and 86.78%, respectively.6. Purification of cephalomannine and 7-epi-10-DAT by normal phase and reversed-phase column chromatography.Normal phase column chromatography:Silica gel:300-400 meshRatio of sample to silica gel:1:40Eluant:chloroform:methanol=90:1(v/v)Speed:30 d/minReversed phase column chromatography:Silica gel:ODS C(18)Ratio of sample to silica gel:1:195Eluant:acetonitrile:water=35:65 (v/v)Speed:20 d/min The contents of cephalomannine and 7-epi-10-DAT were 90.38% and 93.47%, the recovery yields were 77.25% and 63.22%, respectively.In conclusion, cephalomannine and 7-epi-10-DAT with the purities of 90.38% and 93.47%, recoveries of 79.82% and 65.67%, respectively, were obtained from the stems and needles of Taxus chinensis var. mairei using the above processes.