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Isolation and identification of secondary metabolites from taxus media suspension cell culture Pawlak, Sonia Kristin


The diterpenoid taxol, a compound isolated from the bark of the Pacific Yew tree, Taxus brevefolia, has shown remarkable success in the treatment of several types of cancer. Limited supply of taxol, due to low concentrations produced naturally and difficulty in synthesis of the drug, have spurred research attempts to develop alternative methods of producing taxane compounds in vitro using suspension cell cultures. Suspension cell culture propagated from the stems of Taxus media were grown in airlift type bioreactors for 28 days using a modified B5 medium. The cells and broth from multiple bioreactor experiments were combined and extracted with ethyl acetate and methanol. The ethyl acetate extracts were roughly separated by partitioning with solvents before high pressure liquid chromatography (HPLC) analysis was performed to determine if taxol, baccatin III, 10 deacetylbaccatin, 14-hydroxy-10-deacetylbaccatin, 14- hydroxybaccatin, cephlomannine, 7-epitaxol or 7-methyltaxol were present. Additionally, the partitioned extract was checked agains the taxane standards by thin layer chromatography (TLC). There was no indication that the taxane compounds used as standards were present in the cell extract above the detectable limits for these methods. To determine what types of compounds the cell culture was producing, the cell extract was separated using column chromatography, preparative TLC and recrystallization. Five compounds were purified and identified as 3-β-0[P- β -glucoside] β -sitosterol, 3B-6B, 23-trihydroxyolean-28-oic acid, cyclo (L-proline-L- valine), by comparing mass spectral (MS) and nuclear magnetic resonance (NMR) spectroscopy with that published in the literature. Two novel compounds 9,10 epoxy, 11-hydroxy 12,13 linoleic acid and 4,6- dihydroxy,-2-methoxy, 8-methyl xanthone were found in cell culture. They were identified using NMR and MS data and by comparison with compounds of like structure in the literature.

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