全 文 ：Received 22 Jan. 2004 Accepted 18 May 2004
Supported by the Fonds der Chemischen Industrie (Frankfurt/Main) and Alexander von Humboldt Fellowship of Germany.
* Author for correspondence. Tel (Fax): +86 (0)20 85228205; E-mail: .
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Acta Botanica Sinica
植 物 学 报 2004, 46 (11): 1383-1386
Biotransformation of Paclitaxel (Taxolâ) by the Cell Suspension
Cultures of Rauwolfia serpentina
ZHAO Yu1, 2, YU Rong-Min3*, Cornelia SCHROEDER2, Ian SADLER4, Matthias UNGER2, SUN Xian-Feng1, 2,
David W. H. RANKIN4, Joachim STöCKIGT2
(1. Department of Traditional Chinese Medicine and Natural Drug Research, College of Pharmaceutical Sciences,
Zhejiang University, Hangzhou 310031, China;
2. Lehrstuhl für Pharmazeutische Biologie, Institut für Pharmazie, Johannes Gutenberg-Universität Mainz, D-55099 Mainz, Germany;
3. College of Pharmacy, Jinan University, Guangzhou 510632, China;
4. Department of Chemistry, The University of Edinburgh, Edinburgh EH9 3JJ, U.K.)
Abstract: The biotransformation of paclitaxel (Taxolâ) by the cell suspension cultures of Rauwolfia
serpentina (L.) Benth. et Kurz. were investigated. Three Paclitaxel-based intracellular metabolites were
detected from the cell filter cake and were, by high field 1H-NMR and MS data, identified as 10-deacetyltaxol,
baccatin Ⅲ, and 10-deacetylbaccatin Ⅲ. No glucosidated or hydroxylated derivatives were checked out in
this incubation experiment.
Key words: biotransformation; cell culture; Paclitaxel; taxanes; Rauwolfia serpentina
Among many antimitotic natural anticancer agents,
which induce G2/M phase cell cycle arrest of tumor cells,
Paclitaxel (Taxolâ) is the first compound to cause cell cycle
arrest by promoting the assembly of microtubules in vitro
(Wani et al., 1971; Schiff and Horwitz, 1979; Suffness, 1993).
Both chemists and biologists have been drawn to this
diterpene derivative due to its promising spectrum of anti-
neoplastic activity, its unique mechanism of action and the
synthetic challenge arose from complex and densely
functionalized ring system features.
However, the poor water solubility of Paclitaxel (0.03
g/L) (Swindell et al., 1991) and acute hypersensitivity reac-
tions due to the utilization of Cremaphor (Weiss et al., 1990)
restricted to clinical application. A number of attempts have
been done to seek the possibilities of improving the solu-
bility and activity. Taxotere (Docetaxelâ) and continuously
increasing Protaxol were developed to overcome these
imperfections. They are more water-soluble and contain no
hypersensitivity-inducing Cremophor (Pazdur et al., 1992;
Bissett et al., 1993; Lavelle et al., 1993; Greenwald et al.,
1994; Nicolaou et al., 1994; Bissery et al., 1995; Greenwald
et al., 1995; van Oosterom and Schriivers, 1995; Golik et al.,
1996).
Recently, several groups have studied the biotransfor-
mation of Paclitaxel by rat (Monsarrat et al., 1990), by hu-
man cancer cell (jurkat) (Hamada et al., 1996a), and by plant
cell suspension cultures of Eucalyptus species (Hamada et
al., 1996b), by which Paclitaxel is bioconverted into 10-
deacetylbaccatin Ⅲ, baccatin Ⅲ and 2-debenzoyltaxol.
The cell suspensions of Rauwolfia serpentina (L.)
Benth. et Kurz. has been established since 1980, and the
culture is presently optimized as one of the most efficient
and best characterized systems when the phytochemistry
and enzymology of indole alkaloids, the biotransformation
capabilities, and the application for hybrid generation is
concerned (Stöckigt, 1995a). A series of enzymes were pu-
rified and characterized, which exhibit different functions
in alkaloid biosynthesis and biotransformation
(Falkenhagen et al., 1995; Lutterbach and Stöckigt, 1995;
Stöckigt, 1995; Stöckigt et al.,1995; Schröder et al., 1996).
All these information prompted us to utilize cell suspen-
sion to biotransform Paclitaxel, which might lead to much
polar hydroxylated or glycosidated Paclitaxel derivatives.
1 Materials and Methods
The cultured suspension cells of Rauwolfia serpentina
(L.) Beath. et Kurz. were induced and incubated as previ-
ous described (Lutterbach and Stöckigt, 1992). One hun-
dred mg of Paclitaxel (1) was dissolved in 100 mL of LS-
medium with ca. 20 mL of EtOH, followed by continuous
feeding (at a rate of 0.02 mL/min) to suspension cells (16 g
DW) in a 1-liter Erlenmeyer in LS-medium, with a shaking
rate of 100 r/min at 25 ℃ under continuous illumination
(600 lx). Samples were fetched aseptically every 24 h and
Acta Botanica Sinica 植物学报 Vol.46 No.11 20041384
were checked, after suck filtration and supersonic treatment,
by HPLC before comparing with that of blank LS-medium
and with standard samples. The newly-emerged peaks re-
sponding to the intracellular metabolites were isolated and,
after evaporation, were isolated by HPLC separation using
MeCN-H2O solvent system. Their FABMS spectra and 600
MHz 1H-NMR spectra were measured, and were compared
with literature data (Chauviere et al., 1981; McLaughlin
et al., 1981; Kingston et al., 1982; Kingston et al., 1982;
Senilh et al., 1984; Kerns et al., 1994), and then direct with
authentic samples. No glucosidated or hydroxylated taxanes
were obtained. Nevertheless, hydrolyzed Paclitaxel metabo-
lites (2-4) were found in this feeding experiment. A time-
course investigation was performed and the results are
shown in Table 1.
2 Results and Discussion
The results of the biotransformation shown in Table 1
suggested that at a lower concentration, Paclitaxel cleav-
ages its C-13 ester group to form baccatin III (3) at the
beginning of the feeding. Approximately 60% of the pre-
cursor Paclitaxel was hydrolyzed to compounds 3 and 2
when the relative concentration of precursor added up to
50 g/L (Fig.1).
Further elevation of the relative concentration of
Paclitaxel would conversely lower down the conversion
rate from compound 1 to 3, as well as compound 2 which
was undetectable at this moment. The result may be due to
the transformation capacity attended saturation, and thus
negatively influenced the further transformation of com-
pound 1. Meanwhile, the already transformed compound 2
was enzymatically hydrolyzed its C-13 ester side chain and
formed the final product compound 4. However, there ex-
ists another possibility that the intracellular deacetylase
release acetoxy group of C-10 of compound 3 which would
also form compound 4.
It could be noticed that in the transforming period of 72
h to 96 h, the amount of the metabolite 3 decreased while
residual Paclitaxel increased. This could be caused by two
reasons: firstly, the Paclitaxel of over-saturated may inhib-
its the growth of suspension cells and decreases the activ-
ity of enzymes; secondly, alcohol used as adjuvant agent
of Paclitaxel in LS-medium may poison suspension cells,
and also cause the decrease of metabolite products and
increase of residual Paclitaxel. However, the transforma-
tion of 4 from 3 will be increased interestingly as the amount
of Paclitaxel reaches 100 mg/L (Table 1).
At last, in a high concentration of Paclitaxel was added
Fig.1. The biotransformation pathway of Paclitaxel.
Table 1 Relative conversion rate of Paclitaxel
Time (h) Compound 1* (mg/L) Compound 2 (%) Compound 3 (%) Compound 4 (%) Residual 1 (mg/L)
24 25 - 22 - 12
48 50 12 48 - 9
72 75 - 22 2 36
96 100 - 9 10 55
*, relative concentration of Paclitaxel in the whole cell suspension. -, not detected.
ZHAO Yu et al.: Biotransformation of Paclitaxel (Taxolâ) by the Cell Suspension Cultures of Rauwolfia serpentina 1385
to the cultures, the contents of compound 3 further de-
scend while the relative content of the final product 10-
deacetylbaccatin III (4) increased to 10% (Table 1). No other
taxoids was detected by repeated HPLC and TLC analyses.
Feeding Paclitaxel in the cell-free LS-medium showed that
there was no conversion occurred in that system.
Acknowledgements: One of authors (ZHAO Yu) also
wants to express his gratitude to Alexander von Humboldt
Fellowship for their affording an opportunity to carry out
this work in Germany. Dr. Francoise Gueritte-Voegelein (Gif-
sur-Yvette, Centre National de La Recherche Scientifique,
France) and Prof. Bruno Danieli (Milan, Italy) are gratefully
acknowledged for their kindly providing standard samples.
References:
Bissett D, Setanoians A, Cassidy J, Graham M A, Chadwick G
A, Wilson P, Uzannet V, Le Bail N, Kaye S B. 1993. Phase I
and pharmacokinetic study of Taxotere (RP56976) adminis-
trated as a 24-hour infusion. Cancer Res, 53: 523-527.
Bissery M C, Nohynek G, Sanderink G J, Lavelle F. 1995.
Docetaxel (Taxotere): a review of preclinical and clinical
experience. Part I: preclinical experience. Anticancer Drugs,
9: 339-355.
Chauviere G, Guenard D, Picot F, Senilh V, Potier P. 1981. Struc-
tural analysis and biochemical study of isolated products of
the yew: Taxus baccata L. (Taxaceae). CR Acad Sci Paris,
Serie II, 293: 501-503.
Falkenhagen H, Polz L, Takayama H, Kitajima M, Sakai S I, Aimi
N, Stöckigt J. 1995. Substrate specificity of vinovine
hydroxylase. A novel membrane-bound key enzyme or Rau-
wolfia indole alkaloids biosynthesis. Heterocycles, 41: 2683-
2690.
Golik J, Wong H S L, Chen S H, Doyle T W, Wright J J, Knipe J,
Rose W C, Casazza A M, Vyas D M. 1996. Synthesis and
antitumor evaluation of paclitaxel phosphorooxymethyl
ethers: a novel class of water soluble paclitaxel prodrugs. Bioorg
Med Chem Lett, 6: 1837-1842.
Greenwald R B, Pendri A, Bolikal D, Gilbert C W. 1994. Highly
water soluble taxol derivatives: 2-polyethylene glycol esters
as potential prodrugs. Bioorg Med Chem Lett, 4: 2465-2470.
Greenwald R B, Pendri A, Bolikal D. 1995. Highly water soluble
taxol derivatives: 7-polyethylene glycol carbamates and
carbonates. J Org Chem, 60: 331-336.
Hamada H, Sanada K, Ikeda S, Oda T, Williams H J, Moyna G,
Scott A I. 1996. Epimerization of taxol in human cancer cells.
Nat Prod Lett, 8: 113-117.
Hamada H, Sanada K, Furuya T, Kawabe S, Jaziri M. 1996.
Biotransformation of paclitaxel by cell suspension cultures of
Eucalyptus perriniana. Nat Prod Lett, 9: 47-52.
Kerns E H, Volk K J, Hill S E, Lee M S. 1994. Profiling taxanes in
taxane extracts using LC/MS and LC/MS/MS techniques. J
Nat Prod, 57: 1391-1403.
Klein L L, Maring C J. International Patent WO 02400.
Kingston D G I, Hawkins D R, Ovington L. 1982. New taxanes
from Taxus brevifolia. J Nat Prod, 45: 466-470.
Kingston D G I, Molinero A A, Rimoldi J M. 1994. The taxane
diterpenoids. Prog Chem Org Nat Prod, 61: 1-206.
Lavelle F, Gueritte-Voegelein F, Guenard D. 1993. Taxotere: from
yew’s needles to clinical practice. Bull Cancer, 80: 326-338.
Lutterbach R, Stoockigt J. 1992. High-yield formation of arbutin
from hydroquinone by cell-suspension cultures of Rauwolfia
serpentina. Helv Chim Acta, 75: 2009-2011.
Lutterbach R, Stockigt J. 1995. Dynamics of the biosynthesis of
methyl ursubin in plant cells emplying in vivo 13C NMR
without labelling. Phytochemistry, 40: 801-806.
McLaughlin J L, Miller R W, Powell R G, Smith C R Jr. 1981. 10-
Hydroxybaccatin III, 10-deacetylcephalomannine, and 10-
deacetyltaxol: new antitumor taxanes from Taxus wallichiana.
J Nat Prod, 44: 312-319.
Monsarrat B, Mariel E, Cros S, Gares M, Guenard D, Voegelein F
G, Wright M. 1990. Taxol metabolism. Isolation and identifi-
cation of three major metabolites of taxol in rat bile. Drug
Metab Dispos, 18: 895-901.
Nicolaou K C, Guy R K, Pistons E N, Wrasidlo W. 1994. A water
soluble prodrug of taxol with self-assembling properties. Angew
Chem, 106: 1672-1675.
Nicolaou K C, Wrasidlo W, Guy R K, Pitsinos E. 1994. Interna-
tional Patent WO 18798.
Nicolaou K C, Wrasidlo W, Guy R K, Pitsinos E. 1995. Interna-
tional Patent WO 18804.
Pazdur R, Newman R A, Newman B M, Fuentes A, Benvenuto J,
Bready B, Moore D Jr, Jaiyesimi I, Vreeland F, Bayssas M M
G, Raber M N. 1992. Phase I trial of Taxotere: five-day
schedule. J Nat Cancer Inst, 84: 1781-1788.
Schiff P B, Horwitz S B. 1979. Promotion of microtubules as-
sembly in vitro by taxol. Nature, 277: 665-667.
Schröder C, Lutterbach R, Stöckigt J. 1996. Preparative biosyn-
thesis of natural glucosides and fluorogenic substances for b-
glucosidases followed by in vivo 13C NMR with high density
plant cell cultures. Tetrahedron, 52: 925-934.
Senilh V, Blechert S, Colin M, Guenard D, Picot F, Potier P,
Varenne P. 1984. New analogues of taxol extracts from Taxus
baccata. J Nat Prod, 47: 131-137.
Stöckigt J. 1995. Biosynthesis in Rauwolfia serpentina, modern
aspects of an old medicinal plant. Alkaloids, 47: 115-172.
Stöckigt, J, Obitz P, Falkenhagen H, Lutterbach R, Endreß S.
1995. Natural products and enzymes from plant cell cultures.
Plant Cell Tiss Org Cult, 43: 97-109.
Acta Botanica Sinica 植物学报 Vol.46 No.11 20041386
Suffness M. 1993. Taxol: from discovery to therapeutic use. Ann
Rep Med Chem, 28: 305-314.
Swindell C S, Krauss N E, Horwitz S B, Ringel I. 1991. Biological
active taxol analogues with deleted A-ring side chain substitu-
ents and variable C-2 configurations. J Med Chem, 34: 1176-
1184.
Ueda Y, Matiskella J D, Mikkilineni A B, Farina V, Knipe J O,
Rose W C, Casazza A M, Vyas D M. 1995. Novel, water-
soluble phosphate derivatives of 2-ethoxycarbonylpaclitaxel
as potential prodrugs of paclitaxel. Synthesis and antitumor
evaluation. Bioorg Med Chem Lett, 5: 247-252.
van Oosterom A T, Schriivers D. 1995. Docetaxel (Taxotere
registrated): a review of preclinical and clinical experience.
Part II: clinical experience. Anticancer Drugs, 6: 356-368.
Wani M C, Taylor H L, Wall M E, Coggon P, Mcphail A T. 1971.
Plant antitumor agents. VI. The isolation and structure of
Taxol, a novel antileukemic and antitumour agents from Taxus
brevifolia. J Am Chem Soc, 93: 2325-2327.
Weiss R B, Donehowr R C, Wiernik P H, Ohnuma T, Gralla R J,
Trump D L, Baker J R, van Echo D A, Von Hoff D D, Leyland-
Jones B. 1990. Hypersensitivity reactions from taxol. J Clin
Oncol, 8: 1263-1268.
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