Antitumor Agents: Taxol and Taxanes – Production by Yew Cell Culture
423
Cell culture
Preweighed
tube with cells
after medium
removal
Homogenize
in hexane,
1 h sonication
Centrifuge,
discard
pellet
Extracted
dry weight
Dry pellet
Centrifuge
Homogenize in
MeOH : CH
2
Cl
2
,
2 h sonication
Evaporate supernatant
Resuspend in
exact volume HPLC
grade MeOH, filter
and proceed to HPLC
analysis
Discard aqueous
phase, evaporate
organic phase
Partition
with H
2
O,
mix and
centrifuge
Resuspend
in CH
2
Cl
2
1
2
3
4
56
8
91
0 1
1
1
2
7
Fig. 2
Extraction of Taxol
and taxanes from plant material.
PVP in the media. It may be necessary
to use larger flters (keeping the pore size
at 0.45
µ
m) and larger volumes oF HPLC
grade methanol to flter and resuspend ex-
tracts oF large volumes oF medium. Once
fltered, these extracts can be Further con-
centrated prior to HPLC analysis. Other
alternatives to Facilitate medium sample
preparation are possible: For example, the
removal oF PVP by pore size exclusion prior
to medium extraction or the substitution
oF the last fltration step by centriFugation
prior to HPLC analysis.
3.2
Separation, Quantifcation and
Identifcation
Thetaxaneanalysesarecommonlydoneby
HPLC. There are various chromatographic
methods to carry out such analyses. One
oF these methods is described below.
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