Note: Descriptions are shown in the official language in which they were submitted.
~~.~8?~
WO 92/18492 ~CT/US92/030$8 ,
TECHNICAL FIELD OF THE INVENTION
This invention relates to methods and
compositions for obtaining crude taxane mixtures from
renewable sources of plant matter and a method of
purifying such taxane mixtures and compositions to
obtain specific taxanes. More particularly, this
invention relates to a method of treating plant matter
in a manner which preserves taxane content. This
invention also relates to a method of extracting
taxanes from plant matter with solvents to produce a
crude mixture of taxanes wherein the crude taxane
mixture is present in an aqueous solvent.
Additionally, this invention relates to a method of
purifying specific taxanes from a crude taxane mixture
i
using normal phase chromatography.
BACICGROUND OF THE INVENTION
Taxanes are alkaloids possessing a taxane
nucleus. The taxane nucleus comprises the three ring
structure shown below which is also identified as
~,8,12,15,15-pentamethyl-tricyclo (9.3.1.03~$]
pentadecane.
35
CA 02108265 2003-09-22
'~'O 92/1&t92 PCT/LS92/03088
- 3 -
V
At least 56 different taxanes have been
identified in the literature. For example, in 1980,
R.W. Miller authored an article which surveyed known
taxus alkaloids and other taxane derivatives and
reported for these compounds formulas, HNMR, MS, and x-
ray data. The article by R.W. Miller, appearing at J.
Nat. Prod. 43(4): 425-437 (1980)a
M.G. Begley, E.A. Freeknall, and G.
Pattenden, Acta Crystalloqr, 40; 1745 (1984); D.P.
Della Casa de Marcano and T.G. Halsall, Chem. Comm.
1382 (1970); D.G.I. Kingston, D.A. Hawkins, and L.
Ovington, J. Nat. Prod. 45; 466 (1982); F. Gueritte-
Voeglein, D. Guenard, and P. Potter. J. Nat. Prod.
50:(1):9-11 (1987): B. Lythgoe in "The Alkaloids" Ed.
by R.H.F. Manske, Vol. 10, Academic Press, New~York
1968, pp. 597-626; D.P. Della Casa de Marcano and T.G.
~_t08~65
W~ 92/18492 ' PC.'TI~J~92103J8~ ~r~.~,
- 4 -
Halsall, Chem. Commun. 1381 (1970) V. Senilh, S.
Hlechert, M. Colin, D. Guenard, F. Picot, P. Potier,
arid P. Varenne, J. Nat. Prod. 47(1) pp. 131-137 (1984);
J.L. McLaughlin, R.W. Miller, R.G. Powell and C.R.
Smith, Jr., ~. Nat. Prod. 44:312-19 (1981); D.P. Della
Case de Marcano and T.G. Halsall, Chem. Comm. 365
(1975); C.H. Oliver ~Iuang, David G.I. Kingston, Neal F.
Magri, G. Samaranayake and F.E. Boetner, J. Nat. Prod.
49(4): 665-669 (1986).
The taxane series of molecules possess potent
antitumor activity. Generally, the taxanes which have
been studied for their antitumor activity have found
use in the treatment of ovarian cancer and leukemia
(W. P. MacGuire et al., Annals of Internal Medicine,
vol. 111, pg. 273 (1989)). Taxanes are believed to
exert their antitumor activity by inducing tubulin
polymerization and forming extremely stable and
nonfunctional microtubules which has an
antiproliferative effect on taxane sensitive cells.
(Eric K. Rowinsky et al., Journal of the National
Cancer Institute, Vol. 82, No. 15, pp. 1247-1259
(1990)). Among the taxane molecules which have been
studied most with respect to their antitumor activity
are taxol, cephalomannine, desacetylcephalomannine,
baccatin III, 10-desacetyl baccatin III and 10-
desacetyltaxol: The structures of these taxanes are
Shown belowt
CA 02108265 2003-09-22
- 5 -
J
"""
R~ - AC 8aoatln III
~ 4~0~1 b~oo~h pl
01~
O
Rj-~-NH O
2 0 ~~~ _ O
OH
2 5 ~ -,~, p~: CaHb Taoool*
I~ - ofl, I~ - ca H5 ~ a.o...o~n T.~I
R, - ~ ~ ~ ~ s ~ - aa~s W.yvMro~r.,r,.
Rt - aH, A2 ~ ai aai - ass ~ 'o°,
35
trade-mark
A
CA 02108265 2003-09-22
- 6 -
The taxane compound known as taxol, was first
reported to be isolated from the stem bark of the
western yew Taxus*brevifolia, a slow growing conifer.
Its structure was elucidated by M.C. Wani et al.,
Journal of the Americas Chemical Society, Vol. 93, pp.
2325-2327, (1971).
Taxanes are commonly isolated from the bark
of T. brevifolia collected in the wild. Because the
concentration of specific taxanes in T. brevifolia is
extremely low (for example, taxol is present in a
concentration of between about 0.004% to about 0.02%
based upon the dry weight of bark), large quantities of
trees must be harvested and processed to produce even
modest amounts of taxanes needed for research purposes.
Furthermore, wild trees grow under very different
conditions resulting in highly variable levels of
taxanes produced in the bark. In addition, wild
populations of trees are an unreliable source for
taxanes because they are plagued with many
uncertainties and risks such as forest fires, annual
climatic variations, natural variations in taxo~
content in the different chemotypes of wild
populations. Increased criticism from
environmentalists concerning harvesting of wild plants
also threatens the availability of T. brevifolia as an
adequate source of taxanes. T. brevifolia, therefore,
* trade-mark
CA 02108265 2003-09-22
represents a nonrenewable and inconsistent source of
taxanes.
Even more critical is the fact that extensive
harvesting of wild trees risks the destruction of the
genaplasm essential for the future cultivation of T.
brevifolia. Such harvesting could result in the loss
of wild genes coding for proteins providing for such
characteristics as disease and pest resistance, cold
hardiness, high growth rates and tolerance to full
sunlight and the extremes of drought and flooding and
high taxol*/taxane content. The preservation of these
wild genes will be critical to long-term development of
cost-effective taxol* and other taxane production
whether produced from cultivated plants, tissue culture
or genetically modified microorganisms. Because of the
critical role wild germplasm will serve in future
production strategies, the preservation of wild
populations should be considered an essential component
of the development strategy for taxol* and other taxane
production.
Since the harvesting of wild populations of
- ~ brevifolia yields such a limited supply of taxol*,
clinical experiments of taxol have been restricted to
only a few specific chemotherapeutic applications.
Lack of a stable and reliable source of taxol* at a
predictable cost will also significantly impede
clinical utilization of the agent. Development of a
* trade-mark
CA 02108265 2003-09-22
sustainable, economic and reliable source for taxanes
is imperative.
The potential of taxol* as a cancer
chemotherapeutic agent and the structural complexity of
the taxol molecule has prompted a large effort directed
toward its de novo synthesis. However, the molecular
complexity of taxol* suggests that a total synthesis of
taxol from readily available raw material is not likely
to be economically feasible.
A synthesis of the taxane ring skeleton is
reported by R.A. Holton et al., at Journal of the
~°erican Chemical Society, ~ø 5731 (1984), and 'b d,
Vol. 110, pp. b558-6560 (1988). However, these
syntheses are deficient in that the final product lacks
sufficient pharmacological activity to serve as an
effective antitumor agent.
Semi-synthesis of taxol using 10-desacetyl
baccatin III, a more abundant precursor isolated from
the leaves of T. baccata (lg/Kg fresh leaves), has been
reported by French workers. Additionally, V. Senilh et
al., C.R. Seances Acad. Sci. Ser. 2, Vol. 299, pp.
1039-1043 (1984); F. Gueritte-Voegelin, Tetrahedron,
Vol. 42, pp. 4451-4460 (1986): Colin et al. European
patent application 0 253 278 and Colin et al. European
patent application 0 253 739 refer to the semisynthesis
of taxol*from 10-desacetyl baccatin III. These methods
use taxane derivatives as the starting materials, and,
* trade-mark
CA 02108265 2003-09-22
_ g _
therefore, suffer from the disadvantage that the
approach requires isolation and purification of taxanes
from a plant source followed by conversion of the
purified taxane to taxo~': this multistage preparation
of taxol is more expensive than isolation of taxol*
directly from the plant material.
There have been efforts to develop new
techniques for isolating taxanes such as taxol~ from
plant matter. However, none of the methods reported to
date provide for the adequate extraction of taxanes
from a renewable source. For example, M.C. Wani et
al., Journal of the American Chemical Society, Vol. 93,
pp. 2325-2327 (1971), refers to the purification of
taxanes l-from stem bark of a u* brevifolia using normal
phase column chromatography protocols. Normal phase
column chromatography entails the use of a polar column
packing to effect molecular separation.
National Cancer Institute Natural Products
Branch paper NSC #125973 dated July 15, 1983 also
refers to the purification of taxol from bark, wood
without bark, branches, twigs, needles, seeds/fruits
and roots also from Maxus brevifolia.
A disadvantage of the method for isolating
taxol described in the NSC #125973 paper and other
publications is the reliance on the use.of methylene
chloride in the extraction process. Methylene chloride
°nd other chlorinated hydrocarbons, such as chloroform,
* trade-mark
CA 02108265 2003-09-22
- 1~ -
are recognized to be toxic and potentially
carcinogenic. It is, therefore, desirable to avoid
utilizing these solvents in the extraction and
purification of taxar_es from plant matter, both from
the standpoint of exposure of these chemicals to
personnel who carry out these procedures and from the
standpoint of the potential exposure of patients to
these chemicals via trace amounts not removed from the
purified taxanes in the final dosage form of the taxane
medications.
V. Senilh et al, Journal of Natural Products,
Vol. 46, No. 1, pp. 131-137 (1984) refers to the
purification of taxol*and cephalomannine from the trunk
of a s'~baccata L. The purification method which is
reported by Senilh et al. is deficient in that further
purification of specific taxanes using IiPLC or
crystallization is required even after multiple
elutions of taxanes on a variety of normal phase
chromatography packings has been performed.
There have been attempts to isolate taxanes
from plant matter using reverse phase column
chromatography protocols. See, e.g., Keith M. Witherup
et al., Journal of Natural Products, Vol. 53, No. 5,
pp. 1249-1253 (1990): Keith M. Witherup et al., Journal
o_f Liquid Chromatography, Vol. 12, No. 11, pp. 2117-
2132 (1989). These attempts are undesirable in that
the taxanes are isolated in relatively low yield,
* trade-mark
~~! (~~;~~;i
,.;,.."W~ 92/18492 PCT/US92/03088
- 11 -
approximately 50~ based. upon the theoretical yield of
taxanes.
Reverse phase column chromatography entails
the use of a nonpalar column packing to effect
molecular separation. Another disadvantage of reverse
phase chromatography is the difficulty in separating
taxanes from the aqueous elution medium. The
evaporation of an aqueous medium is both expensive and
time consuming. Furthermore, the use of an aqueous
medium hydrolyzes labile bonds, thereby lowering the
yield of taxanes. Also, the aqueous medium used in the
reverse phase chromatography step partially epimerizes
the c-7 stereocenter. The epimers so produced are
undesirable in that they have diminished
pharmacological properties. Furthermore, these
undesirable epimers are separated from
pharmacologically useful taxanes only with additional
expense arid difficulty.
Separation is further complicated because
desired taxane products coelute during the reverse
phase chromatography step. Additional chromatography
steps must therefore be performed to completely
separate the taxanes, resulting in increased expense
and effort.
Another shortcoming of known methods for
isolating taxanes from plant matter is that the methods
require that the plant matter be in substantially
CA 02108265 2003-09-22
- 12 -
desiccated form. Present methods of drying plant
matter promote the degradation of taxanes contained
within the plant matter. Drying plant matter,
therefore, contributes to decreased taxane yields.
SUMMARY OF THE INVENTION
In accordance with the present invention,
there are provided novel renewable sources of taxanes
from cultivated varieties of Taxus plants in contrast
to wild plants which are a limited resource.
Preferably, the plants to be processed for taxane
extraction are provided as intact clippings wherein a
substantial amount of foliage is attached to the stems.
The plant matter is subsequently treated with one or
more solvents to obtain a taxane-rich composition.
This invention also provides a taxane-rich
composition which is prepared from one or more
cultivars of ornamental Taxus*plants. The taxane-rich
composition is prepared by treating the plant matter
from one or more cultivars, provided as intact
clippings, with one or more solvents to obtain the
taxane-rich product. Prior to solvent treatment the
plant matter optionally may be subjected to a drying
step or may be ground.
In addition, this invention provides a
process for preparing a plant material which can be
used as a source for subsequent extraction of taxanes
* trade-mark
...,,Vb'~ 92/18492 ? ~. ~ ~ ~ ~ ~ pCT/U~92/030&8
r,,:.:.:
- 13 -
a
which process preserves taxane content and increases
the accessibility of taxanes to extraction. This
process comprises providing intact clippings wherein a
substantial amount of foliage is attached to the stems
of taxane-containing plant matter, drying the intact
clippings to reduce the volatile content of the
clippings to produce substantially dry clippings, and
recovering the substantially dry clippings.
Preferably, the process of preparing the plant material
is conducted at a temperature of less than about 70°C
and is not conducted under unobstructed direct
sunlight.
The plant materials, which can be used as a
source for taxanes, prepared according to the method of
this invention which method comprises providing intact
clippings, drying the intact clippings to reduce the
volatile components to produce substantially dry
clippings and recovering the substantially dry
clippings are also a part of this invention.
This invention also relates to a process for
obtaining a arcade taxane mixture from taxane-containing
plants. The process comprises an initial solvent
treatment comprising the steps of:
A. extracting taxanes from plant matter by
contacting the plant matter with an organic solvent and
obtaining a substantially solvent-free first residue
3~
rich in taxanes; .
21~8'~~
Yd0 92/18492 ?CTlU~92/03088 ,..~:.~
;~; : .w,~
- 14 -
B. partitioning the first residue between
an organic solvent capable of dissolving the taxanes
and an aqueous solvent which does not dissolve
significant quantities of taxanes to produce an organic
phase and an aqueous phase;
C. separating the organic phase from the
aqueous phase and recovering the organic phase
substantially free of the aqueous phase; and
D. forming a second residue from the second
organic phase; and
subjecting said second residue to a finishing
treatment.
Taxanes present in the second residue are
further purified through a finishing treatment selected
from either treatment E or F. Finishing treatment E
a 20
comprises the steps of:
dissolving the second residue in a solvent
which allows for the reversible attachment of the
taxanes to a solid support,
introducing a solid support to the solvent
comprising the dissolved second residue and allowing
the taxanes of the second residue to attach to said
30 solid support,
separating said solid support from the
solvent;
35 '
CA 02108265 2003-09-22
- 15 -
sequentially contacting the solid support
with attached taxanes with a series of solvents which
solvents have differing taxane eluting properties, and
eluting a taxane-rich fraction from the solid
support as the crude taxane mixture.
Finishing treatment F comprises partitioning
the second residue between an aqueous mixture and a
nonpolar nonmiscible organic solvent, wherein the
aqueous mixture comprises a single phase of water and a
polar organic solvent miscible with water which polar
organic solvent is present in the aqueous mixture in an
amount sufficient to allow the taxanes present in the
second residue to enter the aqueous mixture, and
recovering the crude taxane mixture from the aqueous
mixture.
In addition to the process of preparing a
crude taxane mixture, this invention also includes the
crude taxane mixture compositions prepared according to
the method disclosed herein.
This invention also provides a process for
separating two or more taxanes including taxo~ and
cephalomannine comprising the steps of:
providing a mixture comprising two or more
taxanes in a solvent suitable for loading onto a normal
phase chromatography column,
* trade-mark
CA 02108265 2003-09-22
- 16 -
loading the taxane comprising mixture onto a
normal phase chromatography column packed with a solid
support in a solvent suitable as a mobile phase, and
separating the taxanes by eluting the normal
phase chromatography column with a mobile phase having
a sufficient polarity to separately elute taxol and
cephalomannine.
Additionally, the present invention provides
a process for separating taxol from cephalomannine
using normal phase chromatography wherein a mixture of
ethyl acetate and methylene chloride is used as the
mobile phase.
A process for obtaining taxanes from taxane-
containing plant matter and separating taxanes is also
a part of this invention. This process comprises:
providing intact clippings wherein a
substantial amount of foliage is attached to the stems
of taxane-containing plant matter, drying the intact
clippings to reduce its volatile content to produce
substantially dry clippings and recovering said
substantially dry clippings,
extracting taxanes from said substantially
dry clippings according to the following steps
comprising:
A. contacting the plant matter with an
organic solvent and obtaining a substantially solvent-
free first residue rich in taxanes:
* trade-mark
.,WO 92/18492 ? - ~ ~ ~ PCl'/U~92/03~88
- 17 -
B. partitioning the first residue between
an organic solvent capable of dissolving the taxanes
and an aqueous solvent which does not dissolve
significant quantities of taxanes to produce an organic
phase and an aqueous phase;
C. separating the organic phase from the
aqueous phase and recovering the organic phase
substantially free of the aqueous phase:
D. forming a second residue from the second
organic phase;
subjecting said organic phase to a finishing
treatment selected from either
E. dissolving the second residue in a
solvent which allows fox the reversible attachment of
the taxanes unto a solid support,
introducing a solid support to the solvent
comprising the dissolved second residue and allowing
the taxanes of the second residue to attach to said
solid support,
separating said solid support from the
solvent,
sequentially contacting the solid support
with attached taxanes with a series of solvents which
solvents have differing taxane eluting properties, and
eluting a taxane-rich fraction from the solid
support as the crude taxane mixture, or
CA 02108265 2003-09-22
18
F. partitioning the second residue between
an aqueous mixture and a nonpolar nonmiscible organic
solvent, wherein the aqueous mixture comprises a single
phase of water and a polar organic solvent miscible
with water which polar organic solvent is present in
said mixture in an amount sufficient to allow the
taxanes present in the second residue to enter the
aqueous mixture, and
recovering the crude taxane mixture from the
second aqueous mixture,
providing the crude taxane mixture obtained
from either treatments E or F, said crude taxane
mixture comprising two or more taxanes, in a solvent
suitable for loading onto a normal phase chromatography
column:
loading the taxane comprising mixture onto a
normal phase chromatography column packed with a solid
support in a solvent suitable as a mobile phase:
separating the taxanes~by eluting the normal
phase chromatography column with a mobile phase having
a sufficient polarity to separately elute taxol and
cephalomannine.
Accordingly, it is an object of this
invention to provide a consistent and renewable source
of taxanes and method for efficiently recovering
taxanes.
* trade-mark
WO 92/18492 ~ ~ ~ ~ ? ~ J Pf'TlU592103088
~C;r~a;.s.
r;.,
_ 19
It is a further object of this invention to
provide a method for the isolation of taxanes from
plant matter that reduces the need for multiple
elutions on a variety of normal phase chromatography
packings.
It is an additional object of this invention
to provide a method for the isolation of taxanes which
method avoids or minimizes the use of certain
chlorinated hydrocarbons such as methylene chloride and
chloroform.
It is also an object of this invention to
provide a method for the isolation of taxanes from
plant matter that produces taxanes in high yields.
It is another object of this invention to
provide a method for the drying of taxane-containing
plant matter that minimizes loss of taxanes. Another
object of this invention is to provide substantially
dry plant materials which can be used as a source for
taxanes.
It is a further object of this invention to
provide a method for the isolation of taxanes from
plant matter that permits the use of simple agitation
to remove the organic components from the plant matter.
It is also an object of this invention to
provide a method for the isolation of taxanes from
plant matter that eliminates the need for filtration
steps.
~~.~8~~
iW0 92/18492 FCT/LJS92/03088
,:.. _;:
- 20 --
a
It is an additional object of this invention
to provide a method for the isolation of taxanes from
plant matter that does not reguire the use of aqueous
elution media as found in reverse phase chromatography.
It is a further object of this invention to
provide a method for the isolation of taxanes from
plant matter that prevents the epimerization of C-7
stereocenter.
It is an additional object of this invention
to provide a method for the isolation of taxanes from
plant matter in which all taxanes possess substantially
distinct retention times in normal phase
chromatography.
It is another object of this invention to
provide a method for obtaining a crude taxane mixture.
It is a further object of this invention to
provide a composition comprising a crude taxane
mixture.
DETAILED DESCRIPTTON OF THE INVENTION
Any plant matter which contains taxanes is
useful in the methods of the present invention.'
Furthermore, the plant matter used may be of pure
genetic origin, mixed genetic or hybrid origin, or
unknown genetic origin. Plant matter harvested from
plants growing in the wild may be used as a source for
taxanes. Examples include T. brevifolia, T. baccata,
CA 02108265 2003-09-22
- 21 -
T. cuspidata, and T. wallachiana. Preferably, plant
matter from cultivars of plants belonging to the genus
Taxus* is used as a source for taxanes and in particular
certain varieties or species of cultivated ornamental
S
Taxus~ As used herein, "cultivars" means an assemblage
of cultivated plants which is clearly distinguished by
any characters (morphological, physiological,
cytological, chemical, or others), and which when
reproduced (sexually or asexually), retains its
distinguishing characters. Hortus Third: A Concise
Dictionary of Plants Cultivated in the United States,
MacMillan Publishing Co., Inc., 1976.
Surprisingly, it has been discovered that
several varieties of ornamental a s cultivars have
been identified with taxol* content in the fresh leaves
comparable to or higher than that of the dried stem
bark of ~ brevifolia. Among the identified varieties
are: Ti X media "Henryi', ,~ X a 'a 'Runyan', T.
c~3_spidata, ~, X media 'Halloran' , ~ X Media
.Hatfield', ~ X ed a 'Hicksii', ~ X a a 'Nigra',
~ X Media 'Tauntonii', ~ X media 'Dark Green
Spreader' and cultivated ~ cuspidata 'Hrevifolia',
cusDidata 'Spreader'. ether varieties with taxol*
content in the dried leaves comparable to that of the
dried stem bark of ~ brevifolia include ~ X me_ dia
'Wardii', ~ X media 'Hrownii', and T. X m_gdia
'Densiformis'.
* trade-mark
CA 02108265 2003-09-22
22
" Preferred cultivars may be selected from -the
group consisting of ~ X media 'Densiformis', ~ X
media 'Hicksii', ~ X is 'Dark Green Spreader', T. X
medi 'Runyan', ~ X med'a Brownii', T. X me_ did
'Wardii', ~ X med'a 'Halloran', T. X me '
did Hatfield ,
T. X em did 'Nigra', ~ X med'a 'Tauntonii', T.
L0
cuspidata 'Brevifolia', and ~ cuspidata. Most
preferably, however, are cultivars whose leaves have a
higher taxane content than the dried stem bark of
brevifolia are used as a source for taxanes. Most
preferred cultivars may be selected from the group
15 consisting of T. cuspidata: ~ X me 'a 'Halloran': T,= X
media 'Hatfield'; ~ X media 'Nigra'; T. X med
'Tauntonii': ~ X edi 'Dark Green Spreader'; and ~ X
e~ did 'Hicksii'. The cultivars stated above are
20 available from commercial nurseries.
The plant matter used in the methods of this
invention may be any portion of the cultivar that
contains taxanes and may comprise the leaves, stems,
25 branches, bark, roots or mixtures thereof. In the
preferred embodiment of this invention, leaves are
dried on the small stems as intact clippings prior to
stripping in order to preserve their taxol content. As
used herein, "intact clippings" is meant to include any
clipping in which a substantial amount of the original
foliage or leaves remains attached to the stems. A
clipping is also referred to by those in the art as a
* trade mark
CA 02108265 2003-09-22
- 23 -
"trimming". In addition, the taxol content of the
small stems carrying the leaves is found to be
approximately 60-70% of that of the leaves.
The plant matter to be extracted according to
the methods of this invention is used in either fresh
or dried form. As used herein, "fresh" is meant to
include plant matter that retains substantially all of
its volatile content. Preferably, the plant matter is
dried at a temperature of. less than about 70~C under
either fully lighted, shaded or dark conditions such
that the drying is not conducted~under unobstructed
direct sunlight. As used herein, "fully lighted" is
meant to include any condition in wh~ch ambient visible
light, such as sunlight or that found in a greenhouse
or artificially lighted building is not substantially
blocked, reflected, or otherwise prevented from
reaching the plant matter. More preferably, the plant
matter is dried at a temperature of between about 65'C
and about 20'C under shaded or dark conditions. Dry
air or low humidity are preferred conditions for
drying. Preferably the relative humidity is not above
about 80%: more preferably not above about 50% relative
humidity. As used herein, "shaded conditions" is meant
to include an environment where between about 40% and
about 70% of external unobstructed visible sunlight is
blocked, reflected, or otherwise prevented from
reaching the plant matter that is being dried.
* trade-mark
CA 02108265 2003-09-22
- 24 -
'' Alternatively, exposure of plant matter to light which
light is absorbed by the plant and converted to heat,
causing the temperature of the plant to rise above
about 70°C should be avoided. Preferably, at least a
25% reduction in the weight of the plant matter is
obtained during the drying step. In a more preferred
embodiment, the plant matter is dried until between
about a 40% and about a 70% weight loss is obtained.
while not wishing to be bound by theory, it
is believed that drying the plant matter under the
conditions of this invention substantially preserves
15~ the taxane content of the plant matter by limiting
their decomposition. In addition, it is also be~~eved,
while not wishing to be bound by any theory, that the
method of drying the plant matter according to the
present invention increases the accessibility of taxol~
and other taxanes to extraction.
Those of skill in the art will recognize that
there is a relationship between the temperature of
ZS drying and the duration of drying. For example, drying
at a relatively higher temperature will require a
shorter period of time than drying at a relatively
lower temperature. Any~method of drying the plant
matter to a weight loss as described above, while
leaving the taxane content of the plant matter
substantially unaffected, is withi.~.~. the scope of this
* trade mark
. W~ 9Z/18492 ~ -~ ~ ~ ~ ~ '~ PCfI(JS92/03088
.:,~:
- 25 -
invention. Such methods may include freeze-drying and
S
microwave drying.
While the pressure at which the plant matter
is dried is not deemed to be critical, it is understood
that the use of decreased atmospheric pressure can
promote drying at lower temperatures or shorter periods
of time. The practitioner may therefore make a
selection among various temperatures and atmospheric
pressures of drying without departing from the scope of
this invention.
Plant matter, prepared according to the
process of this invention, comprising substantially dry
intact clippings provide a plant material also a part
of this invention which can be used as a source for
taxanes. Following the drying step, the clippings are
recovered for subsequent use as a source of taxanes.
While the plant matter to be extracted may be
used, in substantially intact form, it is preferably
manipulated to increase the surface area of the plant
matter and thereby increase the rate of taxane
extraction. More preferably, the plant matter is cut,
crushed or manipulated to form a powder. Most
preferably, the plant matter is ground either by hand
or by mechanical means. Suitable methods of grinding
include the use of a blender, ball mill grinder, Wiley
Mill or Fritz Mill. Other grinding apparati and
',~r~.~~~.~5n~~~
WO 92/18492 PCT/US92/03088 ,,f~..
t, ::::
- 26 -
methods may be used without departing from the scope
and spirit of this invention.
Preferably, the plant matter is ground to a
particle size of between about 40-80 mesh. More
S
preferably, the plant matter is ground to a particle
size of about 60 mesh.
To extract the taxanes, the fresh or dried
plant matter to be extracted, in any physical form
described above, is subjected to one or more initial
solvent treatments comprising contacting the plant
matter with an organic solvent to form an extract from
which a substantially solvent-free first residue rich
in taxanes may be obtained. Multiple extractions of.
the plant matter with the organic solvent may enhance
taxane extraction. Preferably, the weight: volume ratio
of plant matter to organic solvent ranges from about
1:8 to 1:12. A more preferred weight:volume ratio is
about 1:10. A weight:volume.ratio of plant matter to
total volume of organic solvent used to extract the
plant matter ranges from about 1:10 to.about 1:150. A
more preferred weight:volume ratio of plant matter to
total volume of organic solvent is about 1:40.
Preferably, contact of the plant matter with
the organic solvent is promoted by percolation or
soxhlet extraction. More preferably, contact is
promoted by shaking. Most preferably, contact is
accomplished by agitating the plant. matter as it soaks
CA 02108265 2002-10-25
r WG~,92/18492 PCT/US92/03088
- 27 -
in the organic solvent. In this step, substantially
all of the extractable organic matter, including
taxanes, is removed from the plant matter and carried
into the organic solvent.
The organic solvent which is contacted with the plant
matter is preferably one commonly used with extracting
organic chemicals from plant matter. 1?referred organic
solvents include chloroform, carbon tetrachloride, ethanol,
acetone, ethyl acetate, methylene chloride, methanol, methyl
ethyl ketone, methyl isobutyl ketone, methyl t-butyl ether,
or mixtures thereof. Preferably the organic solvent is
ethanol. ~~he~ore, it has been found
that between about three to ~'~out six vo7.umes of
between about 40 ml and about 200 ml of t:he organic
solvent, applied sequentially over a period of about 8
hours to about 48 hours, are sufficient t:o remove
essentially 100% of all taxanes from about 10 grams of
plant matter. Also, it has been found that between
about three to about six valumes of between 2 liters
and about 5 liters of the organic solvent, applied
. sequentially over a period of about 8 hours to about 48
hours are sufficient to remove essentially 100% of all
taxanes from about 100og of plant matter..
In a separate preferred embodiment, the plant
matter is-optionally first defatted with hexane,
hexanes, pentane, petroleum ether, isooctane, or
mixtures thereof, followed by contacting the plant
~~.5~~5
WO 92/y8492 PC1'/US92/03088 _~;,
_ 28 _
" matter with the organic solvent. For example, it has
been found that the use of two volumes of appraximately
100 ml hexane is sufficient to defat about l0 grams
graund, dried plant matter over a period of between
about eight hours and about two days.
In a more preferred embodiment the plant
material is first defatted with hexane then extracted
by shaking with the organic solvent, ethanol, acetone
ar ethyl acetate, for a period of 24 hours, changing
the solvent four times during this period.
Those of skill in the art will recognize that
the multiple uses of relatively smaller volumes of
solvent is more effective in extracting materials than
the single use of a larger solvent volume. Thus, the
practitioner may depart from the solvent volumes used
in each of the steps involving extracting,
partitioning, or otherwise removing organic materials
described herein, as well as the number of iterations
of extraction, without departing from the scope of the
invention.
Hydrophilic moieties extracted from the pJ.ant
matter by the organic solvent are then separated from
the taxanes and removed by partitioning the extracted
plant matter obtained from the organic solvent between
a suitable pair of organic and aqueous solvents such
that the taxanes remain in an organic solvent and the
hydrophilic moieties are removed in an aqueous solvent.
~~~;<-::WO 92>18492 c~ ~ ~ . ~ PC~C/US921030R~
- 29 -
Organic and aqueous solvents which are immiscible with
one another and may be separated from each other
following partitioning to the extent that the purity of
one phase is not substantially contaminated by the
other, are suitable, provided that the taxanes have
sufficient solubility in the organic solvent to remain
in the organic solvent. Furthermore, the suitable
aqueous solvents should not dissolve significant
quantities of taxanes. Accordingly, in this and any
subsequent step involving the partitioning of the
taxanes between two different phases or their further
purification, any solvent from a previous step
remaining with the taxanes being purified, which
solvent interferes with the action of solvents in a
subsequent step is removed. For example, a solvent
2p from one step which interferes with the partitioning of
an organic and an aqueous solvent in a subsequent step
by increasing the miscibility of the organic and
aqueous solvents, is removed prior to contacting the
taxanes with the organic and aqueous solvents to be
partitioned.
Preferably, following extraction of the plant
matter with the organic solvent, the extract is
3p
evaporated to dryness forming a first residue, with or
without heating to promote the rate of solvent
evaporation. If heating is used, the temperatures
which promote epimerization of the taxanes should b,e
2~.~a?~~
WO 92/18492 9~CT/IJS92/Oa088
- 30 -
a
avoided. Preferably, temperatures should not exceed
about 35°C to about 40°C.
Once the solvent has been evaporated, the
material remaining as the first residue is dissolved in
an organic solvent such as methylene chloride or, more
preferably, ethyl acetate to form a second extract. A
weight: volume ratio of first residue to organic solvent
of about 1 to about 10 to 20 is preferred. For
example, one gram of residue would be dissolved in
about 10 to 20 ml of organic solvent. The second
extract is washed by partitioning with an aqueous
solvent component which does not dissolve significant
- quantities of taxanes to remove any hydrophilic
moieties that may remain in the second extract. Water,
similar polar solvents or mixtures thereof are suitable
as the aqueous solvent. Preferably the aqueous solvent
is water. A volume of aqueous solvent of about one
half of that of the organic solvent is generally
sufficient to wash the second extract. The aqueous
solvent is then separated from the second extract. The
aqueous solvent may be discarded.
The second extract, contains the taxanes.
The second extract is optionally treated with a drying
agent to remove water found in the extract, followed by
removal of the drying agent and solvent evaporation to
form a second residua. Preferred drying agents are
anhydrous magnesium sulphate, 4A molecular sieves,
CA 02108265 2002-10-25
.a "
WO 92/18492 PC?/US92/03088
- 31 -
calcium chloride or mixtures thereof. A more preferred
drying agent is anhydrous sodium sulphate. The
practitioner may find it advantageous to add.a small
volume of an organic solvent to the drying agent before
combining the taxane containing second extract and the
drying agent. Adding an organic solvent to the drying
agent tends to prevent taxanes from adhering to the
drying agent, and thereby improves yields. The drying
1'0
agent may be removed by gravity filtration, vacuum
filtration, or by decanting the solvent from the drying
agent. Another method to remove residual water from
the second extract is to cool the second extract to a
temperature at which the water in tha second extract is
in the frozen state without freezing the organic
solvent. The liquid nonaqueous material could then be
~0 decanted separating it from the aqueous material.
Further purification of the taxanes is
accomplished according to the invention by subjecting
the second residue to a finishing treatment.
According to the method of one finishing treatment, the
second residue is dissolved in a solvent, e.g., ethanol,
which allows for the reversible attachment of the taxanes
onto a solid support. A solid support is then introduced
into the solvent comprising the dissolved second residue
and components from the second residue including the
taxanes are allowed to attach to the solid support.
Following separation of the solid support from the
CA 02108265 2003-09-22
32
solvent, preferably by evaporating the solvent so as to
leave a residue on the celite* which residue forms a
coating comprising the taxanes, the solid support is
sequentially eluted with a series of solvents Which
results in the elution of a taxane rich fraction. An
example of a suitable solid support is celite.
Preferably the celite is coated with components of the
second residue which have been dissolved in an organic
solvent comprising a mixture of methanol and ethyl
acetate by evaporating the solvent from the mixture of
solvent, second residue and celite. Preferably the
-methanol -and---ethyl--acetate-wa~rewpresent in a volume- ,
volume ratio of 1:3.
The coated celite is preferably eluted first with
--hexane ---which- does not----sigra'if-icantly elute the taxanes but
elutes other less polar molecules. A crude taxane mixture of
the present invention is then eluted from the celite preferably
with ethyl acetate, methylene chloride or another similarly
polar solvent, which is more polar than the first solvent. -
Another finishing step to further purify
the taxanes present in the second residue comprises
partitioning the second residue between an aqueous
mixture and a nonpolar nonmiscible organic solvent.
.
preferably, the second residue is first dissolved in
the aqueous mixture which is then combined with the
nonpolar nonmiscible organic solvent. Preferred
nonpolar nonmiscible organic solvents are such solvents
* trade~rcark
,:a::,~'~ 92/18492 ~ '~ ~ .~ ~) f FC?/1J~92f03088
N . ~. .. r,1 ~~~ ~'
- ~3 -
as hexane, pentane, petroleum, ether, heptane, iso-
octane, and mixtures thereof. preferably, the nonpolar
nonmiscible organic solvent is hexane. A preferred
ratio of nonpolar nonmiscible organic solvent to
aqueous mixture is 2 to 1. The aqueous mixture is a
single phase aqueous mixture comprising water and a
polar organic solvent miscible with water. The polar
organic solvent is present in the aqueous mixture in an
amount sufficient to allow the taxanes present in the
second residue to enter into the aqueous mixture.
Preferably, the aqueous mixture comprises more than
abcsut 50% polar organic solvent and the balance water.
More preferably, the aqueous mixture comprises about 9
parts polar organic solvent and about 1 part water. In
a preferred embodiment, the polar organic solvent is
acetonitrile. In a more preferred embodiment, the
polar organic solvent is methanol..
The aqueous mixture comprising the crude
taxane mixture of the present invention is separated
from the nonpolar nonmiscible organic solvent. The
aqueous mixture may be evaporated to remove the polar
organic component and the crude taxane mixture may be
combined with an organic solvent to further purify the
taxanes. Because the aqueous mixture is predominantly
acetonitrile or methanol in composition, it is
preferably evaporated directly to form a third residue
comprising the crude taxane mixture. Heat, vacuum and
CA 02108265 2003-09-22
- 34 -
combinations thereof may be used to promote
evaporation, provided that the temperature of the
aqueous mixture does not exceed about 40'C. The direct
evaporation of the aqueous mixture avoids the
possibility that any taxanes might be lost in
unnecessary extraction steps.
Any of the foregoing initial solvent
treatment steps or finishing treatment steps of the
process to obtain the crude taxane mixture may be
repeated one or more times. For example, any of the
foregoing steps may be repeated at least one, two,
three or four times.
Specific taxanes may be purified from the
crude taxane mixtures of this invention by any commonly
used purification protocols such as crystallization or
trituration. Preferred taxanes which may be present in
the crude taxane mixture and may be further purified
according to the methods of this invention include
taxol, cephalomannine, desacetylcephalomannine,
baccatin III, 10-desacetyl baccatin III and 10-
desacetyltaxol. Crystallization may be performed by
allowing crystal formation from a substantially
saturated solution of the crude taxane mixture.
Alternatively, crystals may be grown by adding a seed
crystal of the desired taxane product to a
substantially saturated solution of crude taxanes.
Ideally, a single solvent is used as the
*trade-mark
WO 92/18~b92 ~ ~ J P~I'/US92/03088
~'i2,r ~1
4 ~ i ~.e)
i..:-v.r; ':
- 35 -
crystallization solvent, although the use of solvent
mixtures is contemplated by this invention. The
temperature of the crystallization solvent or solvents
is not critical so long as' substantially pure taxanes
result.
According to the present invention, specific
taxanes may also be purified from the crude taxane
mixture using a series of normal phase chromatography
columns. The specific taxanes are eluted by passing
solvent mixtures having progressively increased
elutrophic power through the columns. Generally, in
normal phase column chromatography, the mobile phase
comprises a two component system. The first component
is a nonpolar organic solvent which controls the rate
of compound elution. The second component is a polar
organic solvent which elutes the compounds to be
separated. Increasing amounts of the second component
in the mobile phase generally decrease the contact time
of the eluting compounds with the column packing.
Higher concentrations of the second component therefore
have the effect of decreasing the elution time.
The separation of taxanes may be improved by
using a mobile phase that follows a gradient of
increasing elutropic power, although isocratic elution
is also contemplated by this invention. Preferred
normal phase column chromatography packing materials
for one or more of the chromatography columns may be
~~a~.~y,~~'J:~
i3~0 92118492 PCT/ iJS92J03088
,,....
- 36 -
selected from the group consisting of silica gel,
florisil, alumina, and celite. A specifically
preferred normal phase column chromatography packing
material is silica gel 60, 230-400 mesh, manufactured
by E. Merck, and available from Brinkman Instrument
Co., Westbury, N.Y.
To prepare the crude taxane mixture, third
residue, for running on a chromatography column, the
third residue may be triturated (digested) repeatedly
(1-5 times) with an organic solvent which dissolves the
taxanes. Such an organic solvent may be selected from
the group consisting of ether, methylene chloride,
methanol, chloroform, ethyl acetate and acetone. The
preferred organic solvent is methylene chloride. The
third residue dissolved in the organic solvent is
combined with a solid support such as diatomaceous
earth or celite and evaporated to dryness, thereby
adhering the taxanes to the solid support.
The taxane-containing solid support is then
loaded directly onto the top of a packed normal phase
chromatography column.
' Once the solid support has been placed
directly on top of the packed normal phase
chromatography column, a mobile phase capable of
separating the taxanes from one another and from other
plant components is passed through the chromatography
column. The mobile phase comprises (1) a nonpolar
~e~o 9ai~~n~2 ~crius9ze~o~og8
,.. ~.;
"... . ;
° 37 -
" component selected from the group consisting of hexane,
petroleum ether, iso-octane, and solvents having
similar polarities, and (2) a polar component selected
from acetone, ethyl acetate, ether, methyl t-butyl
ether, chloroform, and solvents having similar
polarities. Preferably, the mobile phase comprises
hexane and acetone.
In a preferred embodiment, the mobile phase
comprises initially between about 60% to 85a hexane and
between about 15% to 40p acetone and follows a gradient
of increasing elutropic power. The mobile phase can
have any final composition so long as effective
separation of taxanes is accomplished. Most
preferably, however, the mobile phase has an initial
cancentration of about 75% hexane and about 25%
acetone, and a final concentration of about 0% hexane
and about 100% acetone. In the case of a
hexane/acetone mixture, it is the hexane which controls
the rate of elution and the acetone which elutes the
taxanes. The desired taxanes are eluted from fractions
comprising about 60% hexane and about 40o acetone, and
about 0% hexane and about 100% acetone. Alternatively,
any other series of solvent mixtures having polarities
similar to those of the hexane/acetone mixtures
described above are suitable to elute the taxane
molecules from the first normal phase chromatography
Column.
CA 02108265 2003-09-22
- 38 -
Preferably, the normal phase chromatography
column is run under pressure. For a 5 X 16 cm column
containing 160g silica gel 60 the flow rate is
preferably about 100 ml/5 min.
The presence of taxanes in the fractions
collected from the columns may be detected by thin
*.
layer chromatography ("TLC") using silica gel G W254
(Machery Nagel, Duren) using 5% methanolJchloroform as
a developing system and p=anisaldehyde/
sulfuric acid as a visualizing reagent. Taxo~'and
cephalomannine appear as a bluish-grey spot with an Rf
value of 0.62 in ,the fractions eluted with
hexane/acetone ratios of 60:40 and 55:45. Baccatin III
appears as a blue spot with an Rf value of 0.55 in the
fractions eluted with hexane/acetone 50:50 and 45:55.
The taxane 10-desacetyl baccatin III appears as a
purplish spot with an Rf value of 0.28 in the fraction
eluted with hexane/acetone 0:100.
The hexane/acetone solvent from the taxane
rich fractions is evaporated to dryness to form a
taxane-rich product.,
Further purification of the taxanes taxol and
cephalomannine may be achieved by loading the taxane-
rich product comprising taxol and cephalomannine onto
another normal phase chromatography column comprising
silica gel 60*or similar material packed with about 1%
methanol and 99% methylene chloride. The elutropic
* trade-marks
CA 02108265 2003-09-22
- 39 -
power of the mobile phase is increased by increasing
the concentration of methanol in increments of about
0.5% methanol, until the mobile phase has a composition
of about 2.5% methanol, 97.5% methylene chloride. The
mobile phase passing through a 2 cm X 40 cm column
preferably has a flow rate of about 8 ml/min. The
taxanes, taxol* and cephalomannine, are collected from
fractions eluted with about 2.5% methanol and 97.5%
methylene chloride. Alternatively, any other series of
solvent mixtures having polarities similar to that of
the methanol/methylene chloride solvent mixtures is
15 suitable to elute the taxanes from the second normal
*
phase chromatography column. The taxol-cephalomannine
rich fractions from the methanol/methylene chloride
column are evaporated to dryness to form another
taxane-rich product.
Surprisingly, taxol may be separated from
cephalomannine according to the method of the present
invention by providing a mixture comprising two or more
taxanes in a solvent suitable for loading onto a normal
phase chromatography column. The taxane comprising
mixture is then loaded onto a normal phase
chromatography column packed with a solid support in a
solvent suitable as a mobile phase. The taxanes,
including taxol*and cephalomannine are then separately
eluted with a mobile~phase having a sufficient polarity
to separately elute taxol and cephalomannine.
*trade-mark
CA 02108265 2003-09-22
- 40 -
preferably, a mixture of ethyl acetate and metHylene
chloride is used as the mobile phase. According to a
preferred embodiment of the present invention, the
second taxane rich product is dissolved in a mixture of
about 20% ethyl acetate and 80% methylene chloride and
loaded on a normal phase chromatography column packed
With silica gel 60, or similar solid support as
14
described above, in the presence of a mobile phase
comprising about 20% ethyl acetate and 80% methylene
chloride. The elutropic power of the mobile phase is
increased by increasing the concentration of ethyl
acetate in increments of about 5% ethyl acetate,. until
. the mobile phase has a composition of about 50% ethyl
acetate, 50% methylene chloride. A flow rate of about
8 ml/min is~preferred for a 1 cm X 32 cm column.
Substantially, pure taxol is collected from fractions
eluted with about 45% ethyl acetate, 55% methylene
chloride. Substantially, pure cephalomannine is
collected from fractions eluted with a mobile phase
comprising about 50% ethyl acetate and 50% methylene
chloride. Alternatively, any other series of solvent
mixtures having polarities similar to those of the
ethyl acetate/methylene chloride mixtures described
above are suitable to elute the taxane molecules, taxol
and cephalomannine, from the normal phase
chromatography column.
* trade-rrerk
CA 02108265 2003-09-22
- 41 -
Depending on the degree of separation of
taxanes present in the crude taxane mixture achieved as
a result of the initial normal phase chromatography
step, additional chromatography of the taxanes using
ethyl acetate and methylene chloride as the mobile
phase, surprisingly, may separate and purify the
t
taxanes, including the separation of taxol from
cephalomannine, obviating the need for intermediate
chromatography using methanol-methylene chloride.
Any of the foregoing chromatography steps may
be repeated one or more times, for example one, two,
three or four times, to further purify or separate the
taxanes at a particular~step.
Although column chromatography is a preferred
method, any other chromatographic configuration, such
as radial normal phase chromatography, as performed on
a chromatotron, are useful in the methods of the
present invention. Also, other suitable detection
means may be used to identify taxanes without departing
from the scope of the invention.
EXAMPLES:
Example 1: Quantification of taxol content of
different varieties of cultivated ornamental Taxus.
The fresh leaves from clippings of various
cultivated varieties of ornamental Taxus were analyzed
*trade-mark
CA 02108265 2003-09-22
- 42 -
for their taxol content following the procedure
outlined below:
Extraction
a. Leaves which had been stripped from
clippings were well mixed and weighed.
IO
b. Ten gram samples of each variety of
cultivated ornamental Taxus were blended in a blender
with 1Q0 ml 95% ethanol, and the mixture transferred
quantitatively into a 250 ml Erlenmeyer flask. The
leaves and ethanol were allowed to extract by
percolation for 24 hours, and then filtered with
15 rinsing (1 X.25 ml). The percolation process was
repeated three additional times.
c. The filtrates were combined (500m1) and
evaporated to dryness under vacuum at a temperature not
20 exceeding 40'C. The weight of the ethanol extract, or
first residue, was recorded.
P~_eparation of the ethanol extract for HPLC analysis
25 a, Approximately 100 mg of the extracts
were weighed into screwcap vials. The caps were lined
with a piece of aluminum foil to avoid contact of the
extract with the cover-lining during partitioning.
b. The extracts were partitioned between
water (1 ml) and methylene chloride (2 ml x 5) or until
the methylene chloride became colorless. The combined
methylene chloride washes were pipetted into 25 ml
*trade-mark
?:~ ~~?~5
,:~?oW~ 92/18497. PCT/US92/!~3088
- 43 -
Erlenmeyer flasks and evaporated to dryness to produce
a second residue. The weight of the second residue was
recorded.
c. The second residue was dissolved in
ethyl acetate (5 ml) and methanol (2 ml) with
sonication. Celite (200 mg to 650 mg) was introduced
and allowed to be coated with the extracted material.
The solvent was evaporated to dryness in vacuo to
produce a celite material coated with the second
residue.
d. The residue-coated celite was
~antitatively transferred into a petri-dish. The dish
was left under a hood until the last traces of solvent
had been removed (10 minutes). The final product was
triturated until a free flowing, uniform powder was
obtained. The resulting powder was packed into a
Pasteur pipet that served as a column.
e. The column was eluted with hexane (7 ml,
or until' the eluent was colorless), followed by
methylene chloride (5-6 ml).
f. The methylene chloride eluent was
evaporated in vaeuo, and the weight of the remaining
residue was recorded.
g. The remaining residue was dissolved with
sonication in 1 ml methanol (HPLC grade). The solution
was withdrawn in a 1 ml syringe and filtered through a
~'~ 92/18492 PCT/IJS92/03088
- 44 -
Millex filtering unit (0.45~C) into a Wheaton vial, and
capped.
HPLC conditions
Column: ~. Bondapak C-18 (10~C, Waters Associates)
Mobile Phase: methanol/water (65:35)
Flow rate: 1.2 ml/minute
Detection: U.V. at 227 nm
AUFS: 0.1
Amount in~Lected: 10,1.
Results of these analyses are shown in Table 1.
20
30
CA 02108265 2003-09-22
- 45 -
*
Table 1 Taxol Content in fresh leaves of certain Taxus Cultivars
Cultivar % Ethanol %Taxol~
Extractives Average
+ S.D.'
~. X media 'Henryi' 17.63 0.00272
~ X media 'Densiformis' 18,93 0.00342
~ X media 'Hicksii' 15.43 0.01042
~ X media 'Dark Green Spreader' 15.32 O,
~ X m i 'Runyan' 17.04 0.00572
~ X m~ia 'Brownii' 19.55 0.00272
T X m is ' Wardii' 14. 89 0.00522
T cu~idata 'Brevifolia' 14.100.36 0.00970.0037
C.V.=3896
~ X m i 'Brownii' 16.07~0.93 0.00465~0.00096
C.V. =20.696
1 S ~ ~s 14.7310.46 0.0141 ~0.00226
C.V.=1696
~ X media 'Densiformis' 13.3 f 1.36 0.00594~0.00039
C.V. =6.596
~,, X media 'Halloran' 14.79~0.18 0.01183~0.00057
C.V.=4.896
~ X media 'Hatfield' 12.83~0.29 0.0121 ~0.00042
C.V.=3.5%
~ X media 'Hicksii' 15.67~0.29 0.0152~0.0023
C.V.=15%
2 5 ~ X ~ ~Nigra'4 15.1310.23 0.0291 ~0.0057
C.V.=19.6%
T baccata 'Repandens' 12.83 f 0.57 0.00178~0.00074
C.V.=41.5%
~ n=3, except for ~ X media 'Hicksii', 'Fairview',
C.V. - coefficient of variation.
'Wardii' and 'Spreader' where n=4.
Single values based on single determinations.
Synonyms = 'Tautoni', ~ cusgidata 'Tauntonii'
Some authorities treat as ~ baccata 'Nigra'
Vegetatively propagated unnamed cultivar of
cuspidata.
*trade-mark
CA 02108265 2003-09-22
- 46 -
T. X m is 'Tauntonii'3 16.47~0.92 0.0198~0.00245
C.V.=129
T X m is 'Hicksii' 19.71 ~ 1.01 0.0093+0.0013
C.V.=14%
S
T. X m is 'Fairview' 19.25~0.38 0.0013~0.0004
C.V.~30.7%
T. X m is 'VVardii' 18.97~0.45 0.0084~0.0036
C.V.=43%
1 Q T a i ata 'Spreader' 19.52 ~0.49 0.0032 ~0.00036
C.V.=llY6
Example 2: Effect on Taxol recovery of stripDincr
leaves from stems before drvin~ leaves.
The fresh leaves (100g) from clippings which
15 were representative samples from large collections (3-6
lbs.) of certain cultivars were analyzed for their
taxol content using the method described under Example
1. Table 2 shows the percent taxol extracted from
fresh leaves compared to that extracted from dry
20 leaves. The calculated taxol~content is the % taxol*
expected in the dry plant material based on the % taxol
content determined for the fresh material and the %
moisture of the plant material. Percent Taxol~
(calculated) - % Taxol fresh /[1-(% moisture/100)].
25 The % moisture is determined from the difference in
weight of the plant material before and after drying.
A ratio of found dry % taxol content to
calculated dry % taxol content was then calculated
(F/C). According to this calculation, a number less
30 than 1.00 indicates a loss of taxol in the drying
procedure (e.g., 0.90 indicates a 10% loss), and a
number greater than 1.00 indicates that the drying
method yields a quantity of taxol greater than is found
by extracting fresh clippings (e.g., 1.20 indicates a
35 20% gain).
*trade-mark
CA 02108265 2003-09-22
47
" 2(a) Fresh clippings of the same cultivars
were separated into leaves and small stems. A portion
(log) of the leaves of each cultivar were extracted and
analyzed as described under Example 1. Results are
shown in Table 2(a). Another portion of the leaves of
each cultivar was allowed to dry at room temperature
until na further weight loss was observed. A sample of
the dried leaves was ground in a Wiley Mill; and a log
portion was extracted and analyzed as described under
Example 1. The results are shown in Table 2(a).
2(b) Alternatively, clippings of ~ X media
'Dark Green Spreader' were dried intact (i.e., without
separating the leaves from their stems) at room
temperature until no further weight loss was observed.
The dried leaves were then stripped from their stems,
ground in a Wiley Mill* and a lOg portiow was extracted
and analyzed as described under Example 1. The results
are shown in Table 2(b).
The data of Table 2(b) were obtained by
analysis of 10 replicates of a large sample of plant
material. The 131% retention in taxol content obtained
according to the method in 2(b) compared with a mean .
33% retention in taxol content obtained according to
the method in 2(a) is reproducible. Surprisingly,
drying according to the method of retaining the
clippings intact enhances the recovery of taxol . ..
compared to ethanol extractions of leaves dried after
they are stripped from their stems. An almost 4-fold
increase.in taxol retention occurs by drying the .
clippings intact as compared to drying after stripping
the leaves.
* trade-mark
CA 02108265 2003-09-22
48-
f~fN 1 tD P m e'1
\ f'1a f'7N N f'fN Y1
.
V. O O O O O O O r-1
!~
_ D
O
d O
O
*
~~ O rf 0~r1 P N 1 O r
O U ~ aoo ~D n i ~D
x .a o o N N ,.~1 0 ..nl
0 0 0 0 0 0 0 0
~
x~- 0 0 0 0 0 1 0 0 0
ao
d o
N
A O
~
1 ~ o -~ w .. ~ ~, a .c~ a N o '
v7 O N f'fCv 1~ f'1iC1N f'1N
~ C
.. 0l x O O O O O O O O f"1
,~ O
o O o o O o O o O
7
O x~- 0 0 0 0 0 o O o O
U
N N
d
x > _
~o
E O y
C U
t
-~r L it O vt1n O ~rO t~ t~
~ it
m x e n 1. o o~o n ~c
Iw .~ 47 .-~N rl N ~1N .1 n-i
d
U
O
1"~
O
N
O N N .~n a t aDaD n a~
> -~ W D vDv0 vo tn~t1111v0v0
'O
4l U1
-~ N
L O
O O
tl!
'O dl * O
~0 Ip "~ CO IHV ~D P 1 P N m
o O O 1
x O O . O O O
~ ~ o 0 -tt0 0 0 0 0
0
E ~
r 0 0 0 0 0 1 0 0 0
a. d
c w rA
-~~ ar
c o .N
c ~
c r ~ ~c o m n o w o eo
.. y 1J
V Li7 1~ ~ If11t1 P ~ 01
X
rr ta7 .~ .i.i .i .a.r.~ .i
0
x
t0 N !r 1r
E-~ d d
p fl
E ..a ' H
W0 -.. Wo
~ w ~ ~ ~ ~
N -..i .. p~ t O -.iC~
~ 1 ~
r
~ ~ ~ N ~ C H
4J ~. l. N ~C = 'p.!L b
r i
-a ~ t G V fr t ~ O L it cC
3 S ~ -~ a~ a~..,~ ~ A s~ A to N
~ ' = o x v a f..m 3 c
v ~ ~
CA 02108265 2003-09-22
- 49 -
Example 3: Determination of taxol content of fresh
stems and stripped dried stems.
* ,
The percent taxol content of fresh stems
(100g) from clippings of certain * ultivars was analyzed
and compared to the percent taxol content of stems
(lOg) which were dried and ground as described under
Example 2(a). The fresh and ground stems were
extracted and analyzed as described under * xample 1.
Results are shown in Table 3. The % taxol calculated
was determined as described under Example 2. Also, the
ratio of found dry % taxol content to calculated dry %
taxol~content (F/C) was determined as described under
Example 2.
20
30
*trade-mark
CA 02108265 2003-09-22
- 50 -
N i 1 I
vt'1 ar N v e~ 1 ~ 11
U v O r O ~D 1 1
O .-~ O O O i 1 O
'D
d
~o
O 1H If1riW D 1 47N
3
x wo v ~nn m t ~Io
A o o .~N o 1 0 ..,
.-,
E. o 0 0 0 0 1 0 0
A
a V . 1
1 x-- 0 0 0 0 0 1 0 0
0
a~
al a
a
w o
. o ov r o00 ~ o l in
to
o c x N a o .-m n co~ .r
~
a o A o o .~0 0 0 ~ o
c
O sr ~ E 0 0 0 0 o o ~ o
~
~
b > D x O O O O O O 1 O
1
t N
a ~
N V
p1c = o N iwo N o~.-an
y
e
1S ~x r~ a
.~ ...,..~ ~, .,.~..,
w~
c x
oc ~w
y ..,
c b o
V w
* -i ~
U ~
uw D O~co O ~t.aao
~ u~
2 0 H s
a
ae
o '
o ..~
o 0 o N o e~ 1 0,
a .e x n N ~oo ~ ~ o v
a A o 0 0 ., 0 1 0 0
o H o 0 0 0 0 1 0 0
w 1
25 or x o 0 0 0 0 1 0 0
as
d
~
>
~ N n It10t
G
J ~ N v0 ~
l .
rl rl~ ~ O N O
wy
x
xw
30
w
cd -
E v ~ H
'C
fr O -..~Ir c -.i
81
r0 ...1w .~p~ C 0 ~ ~ I
it
>
-- ~ N ~ ~C >. C 3 '0'd
a W
cd
U O
CJ C!1 iC 7 A . yOH
- i
O r D ft E m 3
v . _ . _ _ _ _ _
_
35
CA 02108265 2003-09-22
- 51 -
" Exam_ple 4: ra) Determination of taxol content of
leaves of non-stripped barn-dried clippings.
Fresh intact clippings (stems + leaves). of
T.X media 'Densiformis' (Table 4(A)) and T_.X media.
'Dark Green Spreader' (Table 4(B)) were placed on
aluminum wire cloth (18 x 16 inch, 0.010 gauge window
screen) attached to a wooden frame. The frames were
placed in a drying barn on supports 30" above the
floor. The plant material was allowed to dry in the'
dark without additional heat. A fan provided air
movement: The drying apparatus used is a "Roanoke"*
style model 7,5-I-G Bulk Curing Barri (Gregory
Manufacturing Company, Inc., Lewistown, Woodville, NC
27849). The temperature ranged between 30'C at 8 a.m.
to 40'C at 3:30 p.m. Drying lasted for 2 days. The
moisture of the leaves prior to drying was determined
to be 64.5% (~, X ed a 'Densiformis') and 66% (~ X
_ media 'Dark Green Spreader') based on the difference in
weight of the leaves before and after drying.
Immediately before analysis, the dried leaves (10 gms)
were recovered and stripped from their stems, ground
using a Wiley mill; and extracted by percolation with
ethanol, acetone or ethyl acetate. Analysis was
carried out as described under Example 1.
The taxol content of the dried leaves was
compared with fresh leaves of the same variety which
were processed immediately after stripping by blending
with the extraction solvent. Results are shown in
Table 4. Calculated dry % taxol content was determined
according to the method described under Example 2. A
ratio of found dry % taxol content to calculated dry
taxol~ content (F/C) was then calculated according to
the method described under Example *.
jbl Determination of taxol content of leaves
of non-stripped a~reenhouse-dried clippincts.
* trade-marks
CA 02108265 2003-09-22
- 52 -
Fresh intact clippings (stems + leaves) of
T. X media 'Densiformis' and T_. X media 'Dark Green
Spreader' were placed on aluminum wire cloth (18 x 16
inch, 0.010 gauge window screen) attached to a wooden
frame. The frames-were placed on supports 30" above
the floor. The plant material was allowed to dry in a
polyethylene covered greenhouse (length 60', width 20'
and height 9') approximately 55% shaded (using a shade
fabric) and ventilated with two Arvin air coolers, fan
only, without adding water to the cooling pads. The-
temperature ranged between 15.5'C at 8 a.m. to 44'C at
3:30 p.m. Drying lasted for 6 days. The moisture of
the leaves prior to drying was determined to be 62%
X media 'Densiformis') and 66% (T. X media 'Dark Green
Spreader') based on the difference in weight of the
leaves before a-nd after drying. The dried leaves were
recovered and processed as in Example 4(a), and
analyzed as described under Example 1. Results are
compared with fresh leaves of the same cultivar and are
shown in Table 4.
(c) Determination of taxol* content of leaves
of non-stripped cli~ping~s dried outdoors under shaded
conditions.
Fresh intact clippings (stems & leaves) of T.
X media 'Densiformis' and T. X media 'Dark Green
Spreader' were placed on aluminum wire cloth (18 x 16 -
inch, 0.010 gauge window screen) attached to a wooden
frame. The frames were placed under a shade structure
out of doors on supports 30" above the ground. The
plant material was allowed to dry under a structure
, constructed by placing'a shade fabric over a 2" x 6"
frame, approximately 7 feet above the ground. The.
shade fabric reduced ambient sunlight by about 80%. No
fan was used. The temperature ranged between an
average 22'C at 8:00 a.m. to an average 36'C at 3:00
* grade-mark
CA 02108265 2003-09-22
- 53 -
p.m. Drying lasted for 10 days. The moisture of the
leaves prior to drying was determined to be 63.3% (T. X
media 'Densiformis') and 64% (T. X media 'Dark Green
Spreader') based on the difference in weight of the
leaves before and after drying. The dried leaves were
recovered and processed as in Example 4(a), and
analyzed as described under Example 1. Results are
compared with fresh leaves of the same cultivar and are
shown in Table 4.
(d) Determination of taxol' content of leaves
of nonstripped clippings dried indoors at room
temperature.
Fresh intact clippings (stems & leaves) of T.
X media 'Densiformis' and T. X media 'Dark green
Spreader' were placed on aluminum wire cloth (18 x 16
inch 0.010 gauge window screen) attached to a wooden
frame and dried according to the procedure described
under Example 2(b). The moisture of the leaves prior
to drying was determined to be 63.3% (T. X media
'Densiformis') and 65% (~ X ed'a 'Dark Green
Spreader') based on the difference in weight of the
leaves before and after drying. The leaves were
recovered and processed according to the procedure
described under Example 4(a), and analyzed according to
the procedure described under Example 1. Results are
compared with fresh leaves of the same cultivar and are
shown in Table 4.
35
*trade-mark
CA 02108265 2003-09-22
- 54 -
N -
t
W
E
N
1 1 1 1 1 1 1
1 t 1 m rf o P 0~ O ~D i i o~ 1 1
U 1 1 1 CD v O O n O co 1 1 h 1 1
\ 1 1 1 . . . . . . . l 1 .
W ~ ls.1 1 1 O ~ ~ ~ ~ ~ O 1 1 O 1 I
N
H
G:
Z
5c ~
o
N o
~,
W ~ >. ..v e~ oD ~ O vD ~D w
U
~
~C O D O o O O O O O O
er 11 If
ono oar oao on o~ or. 0 0
o 'v o o~ o o o~ o~n or. orr
. .
W 411 1 1 Il1 . n . . . 1 1 . 1 I
nl . ,.i a-1
i~1 1 1 O O O O O O O 1 1 O 1 1
r1 ~ ~ ~ e1 a!1 ~ .~~
l01 1 1 +I +t +1 +I +1 tl tl 1 1 +1 1 1
o ~ ~ 1 I I o c ,r In N a o I 1 o 1 1
n a a a a a n a
a ~ ~ 1 1 I 0 Il1 m 11'11C1 l"w D I 1 ~0 1 I
V7 ' V .~ o o -~ o a
O
Hz E o> o> o> o> o> o> o> p>
lO ~
W U O o o O O O O d
V V U V V U V V
~> x
3 E1
~
N ~ ICf
W
W W r Il1v n i N fV v u1 1t1
~
..a o o e~ v ., er o o .+ rr
m ro x rr rr ~ w
WN cry or o on o~c o o~ ol~ o~ orr ol oln
a 0 o o o o o o o o o o
.c .v a n .
+I n ~D n Ilf o ef ef . . . 1 1 . 1 1
rr . O ..a
O O O O 11'1O O O O O I 1 O 1 1
15 N N ri r1 ~ ~ ff tf! 1 .-1
+~ +i +I +I O +I +1 +1 tl +I t 1 +I I 1
~~
~ III on on o~n +In lna u~a Na ern o0 1 1 o,u I I
ty ~p p 115 fV n a to a1 vD t~ P a I I N I 1
E.,
L C O O O ~ O O ~ O O ~ 1
WW
d = O> O> O> O> O> O> O> O> O> O> O>
' ~ ou ou oti oc ou ou ov oc~ oti oti oti
i:.~ i
0
, .. .
,
Hr
x
Hw
20~
Z
*a
a
oW
R ~
x ,~.~ d ~ .+ al R .~ al .-~ ar
z
E C O C O C O C O G O C
w 41C O ~ C O ..a C O ~ t O .-rC O -~
> 1p y >. ~O i> >, IC i~ ?, i0 id >,~0 J~ >.
W a t C1 .C t 41 ~ ~ 41 .C t al .CZ C t
U
O i~ V il ~ TJ i~ it U i~ ~ O a~~ U iJ
V~W rC W W iC W W it W W 4 iviW 4 W
a
~.
25~
1
w
x
'~ ~ o
z m
o t ~ w
. d a~
~ ' A
u -~ a o d~ 1
>.~ o~
C a E
d
'C r 'p
3
0
r ~ o c
~ : A ~'
,.
o ~ m ~o t~n~ ~c
~
a .
CA 02108265 2003-09-22
- 55 -
- .
1 I 1 1 I 1 1
O I 1 1 P O N ~D r n n 1 I .-~ I 1
, V 1 I 1 In r .-1 ~ r1 co e'~ 1 I Ov 1 1
O \ I 1 1 . . . . . . . 1 1 1 1
,S 1 1 I .-~ .~ rl .1 .-1O .~ 1 1 O 1 1
H
W
C Ir1 r In
0J >~ ~f' CO N fa ~ r1
L
Qt G .1 O O .-1 O O .d O
H O O O O O O O O
~ ~ I' 1r II Ir In
C~ 'fl O O O o O O O O
r ~c aD r e~ O~ C,
d 1 1 I . . I 1 1 1
. rr
.~C~ I 1 1 O O O O O O O 1 1 O 1 I
0~ n r 0~ Ir'1ac aD m
la 10 I t 1 +1 +I +1 +I +1 +1 +1 i 1 +1 1 1
l0 ~ I 1 I ~D n r ao In ~o v 1 1 .-i t 1
p p p p p p p p
O O 1 i I .m 1 W c1 n r a I 1 1~1 1 I
U N .~ ,.~ N ,.~.,.~N N
. . . . .
~~ O O O O O O O O
9 ,7 ;7 ~ ~ ~ ~ ~
IO ~ i
. J O O O O O O O o
V V V V V V V V
~
ci
X
In o, N O In N c. ao .-i r O
N .-1 ~i !'1 r .'t f'1 O rl N N
~ II
O O O O O O O O O O O
In I1 ~ u1 x rI rr rr
O O O O O O O O O O O
rr w ~ aD a~ ~ ~D u1 r
. . . . . . O . . . 1 1 . I 1
~p rl Q . . ~ . . .
O O O O O O O O O O 1 1 O 1 1
N c N G7 n .-1 .-t Iflr CD CO
+I +I tl +I +1 +1 +1 +1 +I +1 I 1 +1 1 I
O ~0 O O N C~ m N aD ~D 1 I O 1 1
II 11 II II a a n II p II 11
~ O ~ ~ ~ ~ ~ r ~O a N 1 1 n 1 1
+1 C O O O n .a ,.~ N ..a.~ t-f N
. . . .
O O O O O 0 O O O O O O
> > > > 9 ~ '~ > > >
_
. Q' c oci ou ou oc~ ociov oci ociov oci cv
r
L
G
_ u
*
2 .~ , w ~o
0 0 ~o
x y ~ y ~ it
,o ev nr ar a O
a ~ <
o a ., a~ ~ ..~ d .~ al .~ d
~~ c o c o c o c ~ o c o
,~ .. ..
ar c o .~ c o . c o
> R ~r >. ~C ii >. 10 J~ >, 10 y >,A ~r>.
L G7 L L 01 L L d7 L L d L t GIL
O y U ~ ~ U ~ ~ U s~ y U s~a~ U
V7 W iC W W ~C W W it W W 4 W W 4 W
25
I
d
H
y
m A
m
1 ~ H
d d
~ ph A
A
d CL H
~ d
L O G
N O L 1 E-~
N
3 ~ ~ d w c
0 ~ m ~
.. I ,
l
~ H L O
~ H N 1r
t9 OG
O D
Ca. ~D
~~~~~~7~
WO 92/18492 PCT/iJS92/03088
- 56 -
Example 5: Solvent of extraction.
Fresh clippings of T. X media 'Hicksii' were
stripped into leaves and, small stems which were then
left to dry at room temperature under conditions as
described under Example 2(a). For analysis, a portion
of dried leaves (10g) was ground and then extracted by
percolation (4 X 100 ml) with one of several organic
solvents utilizing the procedure of Example 1. The
moisture content of the fresh leaves was 630. Analysis
was carried out as described under Example 1. Results
are shown in Table 5.
20
30
CA 02108265 2003-09-22
J
...
n
x
a r O t~1 .w C
r W
D r1 W a N n
w ~ x
p ..i ..r .~ .i .-i
O O O O O O
O O O O O O
>
.-
~ H
ma
0a
mm
o a
H a '
00
w o 0 0 0 0 0
U O~ m to N pt a
N o a, r
~
2 a ~ N .-, ..~ .,
0
X
~
> C
O
G O
dx
w a ...
CI E~ ~ N
w
v v
C .~ d
w O O 'C
C -~ C ..i
y A
.~ ~ t o
va
o w or r
w y ~ U
w x ar d
W w 'a 'D 41
..r -.~ C
d
s r z
m U U .I~ i0
E d
.O d d ~
~ 0
H ~i.~ 1 C1 0 rr
0 7
O O C
d >, ?. .r
a~
> ~ = ~ d t t .
a , . Cr
.. v
i ~ ~ a
v
~
~~.~R',~5
WO 92/18f92 ' PCT/US92/03058 ,r,,~:>~,
- 58 -
Example 6> Method of extraction.
Three methods of extraction were compared
using two replicates in.each instance. Leaves of T. X
media °Hicksii" were stripped from fresh clippings and
allowed to dry at room temperature under conditions as
described under Example 2(a). The dried leaves (10
gms) were then ground and extracted using acetone by
percolation, under soxhlet conditions or by shaking
(soaking with agitation). Extraction by percolation
was performed according to the procedure described
under Example 1. Extraction under soxhlet conditions
was carried out using one portion (100 ml) of solvent
for 12 hours, followed by exchanges with fresh solvent
at four hour intervals; a total of 400 ml of solvent
was used for each 10g sample of plant material over the
24-hour period of extraction: Extraction by shaking
(soaking with agitation) was carried out using the same
schedule of solvent exchange as described under soxhlet
conditions. The extracts were then processed and
analyzed as described under Example 1. Extracts
obtained from the percolation method of extraction were
combined and analyzed; extracts obtained from the other
two methods of extraction were analyzed at each step of
the extraction process. Results are shown in Table 6.
30
CA 02108265 2003-09-22
59
c G
a~
o
..,
.,
w ~ n ~ o
U
.
r
O
.r, o n N
w
w N ...1
O
O >
U
~, +1 +, +.
,
v~ '~' '
o
m .~ c9 0 < u,
o m o vo 0
O ~ O N O
> +1 O ~ O ' O
O O O
O O O
1a
0
1 0 '~ ~ ~' p 1D f !'1 f'1 \D
~
V
rl n iCf 1Cf 10 O N
'i
11
a ..i ~ x .., ..~ ., ..m -, N
3
O O O O O O
v E" o o 0 0 0 0
f"'
~'
..i
..i
.Ix
w.
b
1 1 ~ ~' m
tl.,~ 1 1 O O N .a
y d
~ ~ ~ O O O O
~ 1 1
G GO O O O O
O 1C
Q1 e~ 1 1 oD e'f r 01
O ~"~ ef c~I
x ~ G, ~ ~ O O O O
y A * 3 ~ 1 1 0 0 0 0
~r
E"' 1 1 c
~ v~ o 0 0
>a x s~
w
N
?C E 1 1
2 0 ~ Q' a ~ ~ o o 0 0
w
tn 1 1 O O O O
o~
a
'o ., 1 1 .1 0 In ~c
oa N n ~o N
1 1
s ev Q, .., ..~ ..
v ~ ~ 0 0 0 0
*'
xo cn 1 1 0 0 0 0
o er
r"
H ~ ~ w -1 N ~ N ~ N
w Lr Ip
0 'w C
o*..~
vl
o
o sK
H
~, w V .
n ao O o O In
11.1 ~ ~ N f"1 1f 41 01 1D
3 0 o ~ 3 .~ ., ~ .; .~ ~,
~ w
~w
ao
.~
o
a o
>m c
w a o . o ,~a
w
3 5 m L v c se b
o
.r . !.. . . ~d
e x to
it
it
tl d x a O t
a ~ w o. In v:
E
~~.~?~,~5
~'O 92/18492 ~ Pt'T/LJS92/03088 ,~
- 60 -
Example 7: Partitionina of the first residue between
water and methylene chloride.
Fresh clippings of ~'. X media °Nigra~ were
dried in a bulk tobacco curing barn according to the
method described under Example 4(a). A portion of the
clippings were ground using a Wiley Mill, then
extracted by percolation using a solvent selected from
among ethanol, acetone and ethyl acetate and using the
procedure described under Example 1. Another portion
of clippings was separated into leaves and stems, and
the two portions were ground separately using a Wiley
Mill. Each ground portion was then extracted by
percolation using a solvent selected from among
ethanol, acetone and ethyl acetate and using the
procedure described under Example 1.
(a) Partitioning° of the first residue of the
ethanol extract between water and methylene chloride
A known weight of the residue of the ethanol
extract was partitioned between water (1 ml) and
methylene chloride (2 ml x 5). The methylene chloride
phase was then prepared and analyzed by HPLC according
to the method described under Example 1. The results
are shown in Table 7a.
(b) Partitioning' of the first residue of the
acetone extract between water and methylene chloride
A known weight of the residue of the acetone
extract was partitioned between water (1 ml) and
methylene chloride (2 ml x 5). The methylene chloride
phase was then prepared and analyzed by HPLC according
to the method described under Example 1. The results
are shown in Table 7b.
(c) Partitioning of the first residue of the
ethyl acetate extract between water and meth~lene
chloride.
~.,,.WO 92/18492 ~ ,~_ ~ ~ ~ ~ ',~~ PC I'/US921J3~88
- 61 -
V '
A known weight of the first residue of the
ethyl acetate extract was partitioned between water (1
ml) and methylene chloride (2 ml x 5). The methylene
chloride phase was then prepared and analyzed by HPZC
according to the method described under Example 1. The
results are shown in Table 7c.
15
25
35
CA 02108265 2003-09-22
_ 62
J
p
U
d
it
c
sJ
ar
tn
a ,r ..
.,
z cr v~
1~
_ 3 = ~ n
O
. .~ 0~ o ~o eoenO 1 ~ . H m
l
b O P1 ~ N t9~ .1 C N N N N O O
O O O O O O ~ O O O O O O
~
d Ew o 0 0 0 0 o Ew o o 0 0 0 0
c
O -
..;
X
y
N v0O 010~ ,~ O 4141 P1t'1If1
~
U V t Ip N m n N o V e-ve~.r letN vo
.-. t
~
N N . . . . . ~,
~T N
LT
m a 3 ' o,o. v.n ~ v ~ ~'~' c' o,
w ' 3
~
- . . ~
l .i.,
..,
~" ~ m
o~ Irl.r N InO G c o~ n ea n n In
~ ~ w o n N n ~ x ~ eoIn Into 0
o
c x In .O p,
'O tr t
.
o C C ~ ~ m ~ m o~N N ~ d a~ n a Inn n
E A
B
w .", O s 3 ., .r...1.r.-I.-IU x ~ n n n .~ .r
'- Z
~
E ~ V y
m
y CO i~.~i O ao o~.r ofowo Ix ~ n lc1~ o n o~
= d
..., ~ y . a i r, o N o~ t, o ao< voao r'
fl V N N
~ --
. ,,, x ." . . . . . ~, N
to ~e ~
p~ v~
S t ao N ~ n w .-IC Z N n IW n .i a
E t
E
V p, n n e-Ie~N n p U O vD1e1lefa a
... G.
v
U
w Q d
L ta7 a O 0~ ~DN ~O ~C c 1e1O O saa ~D
!'1r1m O~~ n O N N 1D 10a a
d 1 S ~ . . . . 1 y
~ ~ 0 0
~
y o A o 0 0 0 0 o d 0 0 0 0
3 ~
~
., y c 0 0 0 0 0 o N ~ o 0 0 0 0 0
2 ~ c
0 E
~ m s ..l.~..l.~..~~., c ..~..l.~ .,., ..
.c - c
--
>
c
N
E
c
y y o .p Inae ..le,n O co o~O N In In
'p
C ,~ N n la W D f y If1u1.1 1e1u1 In
y .1 ~
O C7 n n e~ ~ r ~ d ~ ~lO O n n
.. d
~
d
Lt~7 N N N N r1ri ~C ~ ~ ~ ~ D O
? ~
w
N
w
O
'"'
~
o
d A
c
1~r .~ rl fV.1 fV.aH1 ~ rl N ~ N rl N
E as ~
t
O
pv C
..i 1~ ID ..l
o
y O r n. m o or o. m
x n
~ ~
wy '~ ~ v H t y
~
ww ,
.,. - , a v~
mo
r
H
d
r
d
A
E
CA 02108265 2003-09-22
- 63 -
w
v~
~3 ~
m a~ ~ ~ O
o
N NN N ~1v-1
O OO O O O
E"''~ O OO O O O
1~
N
p V t n emn ao o~N
~
NN
y ~ ~o nN o ~on
x A
E
V U 3 ri .~N N rlr-1
'~
fp
is
y
x
w
c a~ nv m ~om
_
d N nn a N 0t
Z S ~ x ~
~
my n aev N N
E
y .~ r erv a n n
3
'
d
U
...1,~ ..rOrr 1~1v01f1
d
r, v0~ .~ O~n
t0
C'
a~ ~
'
c~ u ~ ~~ ~. ~
o o
2~ n '
N rO N ~ N
1
.i
~. O Nn n .mn
W 4 O ~ 0 O
E
. ~ .. ~ .
r .~ a
-i nao c, .-~n
4.-~ o nv m n n
O of ao n~o ~o v ,r
E.
...r
wa 0 00 0 0 0
.a
C
~ rt Nrl N ~1N
as
N
30 ~ =
m
> a
.,. ip ...r d N
y
U t
f l~R
3S
'y1'~ 92/18492 PCT/US92/03088 .fr~a,
-- 64 -
V
Example 8: Partitioning of the first residue between
water and ethy_1 acetate followed by second solvent
partitioning between hexane and an aqueous mixture.
Fresh intact~clippings of T. X media 'Nigra'
were dried in a bulk'~'tobacco curing barn according to
the method described under Example 4(a). A portion of
the clippings was ground using a Wiley Mill, then
extracted by percolation using a solvent selected from
among ethanol, acetone and ethyl acetate and using the
procedure described under Example 1. Another portion
of clippings was separated into leaves and stems, and
the two portions were ground separately using a Wiley
Mill. Each ground portion was then extracted by.
percolation using a solvent selected from among
ethanol, acetone and ethyl acetate and using the '
procedure described under Example 1.
(a) Partitioning of the first residue of the
ethanol extract between water and et~l acetate
followed by second solvent partitioning between hexane
and an aqueous mixture (methanol: water).
A known weight of the residue of the ethanol
extract was partitioned between water (1 ml) and ethyl
acetate (2 ml x 6). The ethyl acetate phase was
evaporated and the second residue was then partitioned
between two parts hexane and one part methanol: water
(9:1). The methanol:water phase was evaporated to
dryness in vacuo. The residue was dissolved with
sonication in 1 ml methanol (HPLC grade) and the
' solution was withdrawn in a 1 ml syringe and filtered
through a Millex filtering unit (o.45~c) into a Wheaton
vial, and capped. HPLC analysis was then conducted as
described. under Example 1. Results are shown in Table
sa.
(b) Partitioning of the first residue of the
acetone extract between water and ethyl acetate
.,~9~~ 92/18492 ~ ~ ~ ~ ~ ~ ~ PC'~'/US92/030~8
- 65 -
followed by second solvent partitioning between hexane
and an agueous mixture (methanol: water).
A known weight of the residue of the acetone
extract was partitioned between water (1 ml) and ethyl
acetate (2 ml x 6). The ethyl acetate phase was
evaporated and the second residue was then partitioned
between two parts hexane and one part methanol: water
(9:1). The methanol:water phase was evaporated to
dryness in vacuo. The residue was dissolved with
sonication in 1 ml methanol (HPLC grade) and. the
solution was withdrawn in a 1 ml syringe and filtered
through a Millex filtering unit (0.45~C) into a Wheaton
vial, and capped. HPLC analysis was then conducted as
described under Example 1. Results are shown in Table
sb.
(c) Partitioning of the first residue of the
ethyl acetate extract between water and ethyl acetate
followed by second solvent partitioning_"between hexane
and an aqueous mixture (methanol: water).
A known weight of the residue of the ethyl
acetate extract was partitioned between water (1 ml)
and ethyl acetate (2 ml x 6). The ethyl acetate phase
was evaporated and the second residue was then
partitioned between two parts hexane and one part
methanol:water (9:1). The methanol:water phase was
evaporated to dryness in vacuo. The residue was
dissolved with sonication in 1 ml methanol (HPLC grade)
and the solution was withdrawn in a 1 ml syringe and
filtered through a Millex filter unit (0.45~a) into a
Wheaton vial, and capped. HPLC analysis was then
conducted as described under Example 1. Results are
shown in Table 8c.
Comparison of Tables 7 and 8 indicates that
purification by partitioning produces comparable yields
CA 02108265 2003-09-22
- 66 -
of taxol*when compared to the more expensive (in
materials and labor) method of purifying using celite.
~ ..
15
20.
30
3 5 * trad~rk
CA 02108265 2003-09-22
- 67 -
c
0 2s
~d
a >'a
U 'O
-.1
I
w C
~ a
w
wo
0
a
o c " 'P
o~ v~
~ +~
~ r
I 0~o n r .~o -~ I a o~r
3 = n
O e~n eI n .-1.a O N N N N O O
O O O O O O ~ O O O O O O
~
O
Ew o 0 0 0 0 o Ew o 0 0 o c o
~~ '
00 ~ ~
c E
o
.,., ..,
\ f'!O 1~1e~ N Il1 \ f .r ~p
L C Q! Q1
-1 ~ O CD 01 CD~ O -i~D O N ofO~
y
O : :
a
w .aosr ..;r e m aou; o wi cv~o
t t
E
y! ~ P7N N f'1N N Z f'1t'1rf~f !'1f'1
On CL
' '
'~
Z ,. O v~ O v,
S .~
..aE ..~ E
N d v v
V d V ~
p > N a C ..~O n .r eft~a C O~In eDv u1~
fW d
~ ~ ~ N ~ eD r n r ~ 'Wt'1r O~
~
a Qt ~ x
.1 C X C1 N O O .-iCC!~X 01 ~-1N N W 0 ~l1
t t
p w = .a.i.r .i W Z e~n N N .~rr
G. Cr
E -
a~ .a
o
y a O
,
A E N E
.
W V V
2 ~ v
0
N % ... cW c,vo~-,~-,o ... v c ..~r,.m eo.~
W
O ~ a O~ O O .~'C O 1~1~D n O C~O
A ~
a
a. I E N o a n ccr'I E c,o vc.-yrin
t t
m v .r~ v r,n w vo,..vpw nn
c. cs.
1r ~ N
w
O
w ~ r1
..1 d
1r
~
'o
d ~
a
7 it ?~ raO O !~ m O C aDu'1a O~ N ..r
N O O O N r y ~-1ICIIHN vDn
2 d U =
S -~
!", O O O O O O O 41 O O O O O O
d a
Z
t it j o ~ O ~ O O 4 ~ o ~ o O o
- ~ ~
f r . . .. ,.. . ., ....
. -1 i .I .I.~ -1r r i
a~
a
w c
o nr
w
vd
i
y y,, .i ~
~
w r.1N r1 N ~I!V~C ~ N ~1N ~iN
i
w C
~
30 W O ~
C~ a
!T C C' t
> a ~ > a
n
.
-. a -. d ~. a ~ ~, b
= ~ a ~ ~ ~ a
-
E
Fr .~. ~l '~ !, J .)
VIVO 92/18492 PCT/US92/O~n88
- s8 -
..a r.b voo,N
3
ro 0 0 0 0 0 0
~
Hro 0 4 0 0 0 0
1~
o
U
M ~
ro
W 01b' 6f1rd01a"1
.C
~ cha a v v sr
x a.
W
b
a
C CON ~Dt0ST10
Q1
x a;~ o~r O
~
Cl I'~N r-1riIDh
.C
r1 ~ P1~V'eff7N N
~
dJ
W LT
s vv ~ ~r r,n m r,
O N o r r ~rr
ro
H .-Wc o ~ocon
r
W COc0 01r I~h
~ ,
~ P O t0N srsf
U o a~ w e own
~S f O O
O
ft p O d o
E o o 0 o O o
C
W rle-~r!.-W e-1
~ -~
sQ,' r4N P9N 'iN
~
I
s s
S.~ N -X11 i
p
9 O. ~
U ~
V N
WO 92/18492 "s~'' - ~ '~-~' ~ J ~'CT/US92/03~88
n
69 _
-
Examt~le 9: Elution of the eelitecolumn with ethyl
acetate instead of meth~lene chloride.
Fresh clippings of T. X media 'Hicksii' were
separated into leaves and stems and allowed to dry at
room temperature until no further weight loss was
observed. The dried leaves were then ground using a
Wiley Mill and extracted by percolation with acetone.
Analysis was carried out in duplicate using 10g leaves.
The acetone extract was treated and analyzed as
described under Example 7(b), except that ethyl acetate
was substituted for methyl~ne chloride. Results are
shown in Table 9.
20
30
CA 02108265 2003-09-22
J
70
n
p
)
N >
0 ~ .
5 ~' o v
r
a
~ o
y
~ ce
. .r i o o
u ,
~u ~
. ~"~ 0 0
d
~
'~
10 ' ~ v 0 0
d
t~
~ ro
ar y
V w
~' ~E =
~"
O~N
t ii O
1. ~.~ a,ry d o
3 ~
Cl 3 4) ~ N
iJ U
i I
d ~ 0
I5 3~0
~a
c ~ .
.
N Cl -~r O C t .~. ~ N
C ?, i0 Vl p~ ~ 01
~.1 ?~ ~ L ~ ~ tot0
..1
N N
R t c
it -.r
l.i p,
W
20 x c
Gl .i W
Nt
* N
O
O > ~0
CI b " o'
m..~ c t ~r A ,
E
'
m-.~ ~ y dt
U .~ 3 Cr CJ
G. "'
tC C! C R
U O
C M
R c iJ
V
C ~.1
tr'"r ~'~ ~
~
H
c ii t ..
, ~
y~ p,
'~
~w a ~
r =W m
~ .
~ .
y ..~ w C
-~ it O
~ D
~ a s~
30 td
:
W ~ n
N N
iJ O QJ ~ ~ rt r1
ci w t
'
y ~. w
w c c
w ~p O .,~ .sd
ca
N
N
3 5 ~, ,, ~ N
c
?~_~~>~~
,~,;,,, WO 92118492 PCl'I U~92/030$v
_ m _
V
Example 10~ (a) Defatting of the plant material prior
to extraction by percolation using acetone.
A sample of leaves of T. X media ~Hicksii' was
dried and ground using the method described under
Example 9. Dried, ground leaves (10g) were defatted
with hexane by percolation for 2 days, changing the
solvent every 24 hours (2 x 100 ml). The combined
hexane extracts were evaporated to dryness (residue
average 200 mg which represents 21% of the total hexane
and acetone extractives). The marc (remaining residue)
was transferred quantitatively into a 250 ml Erlenmeyer
flask and extracted with acetone by percolation,
changing the solvent every 24 hours for 4 days (4 x 100
ml). The combined acetone extracts were evaporated to
dryness. A known weight of the acetone residue was '
partitioned between water (1 ml) and methylene chloride
(2 ml x 5). The methylene chloride phase was
evaporated to dryness in vacuo and the residue
reconstituted in methanol (1 ml). The methanolic
solution was filtered through a Millex filtering unit,
diluted 1:1 with methanol and used for HPLC analysis
according to the method described under Example 1.
Results are shown in Table 10.
(b) Defattina of the plant material~rior to
extraction
by soakinqlwith agitation (shaking~,~ using
acetone.
Dried, ground leaves (log) as in (a) were
defatted with hexane by soaking with agitation for 8
hours, changing the solvent every 4 hours (loo ml x 2).
The average hexane extractives was 194 mg or 12% of the
total hexane and acetone extractives. The marc was
transferred quantitatively into a 250 ml Erlenmeyer
flask and extracted with acetone (4 x 100 ml) by
soaking with agitation over a 24 hour-period, changing
WC~ 92/1492 PC'~'/L1S92/03088
t - 72 -
the solvent Zit~l2, 16, 20, and 24 hours. The extracts,
collected at each point, were combined, evaporated to
dryness in vacuo and a known portion of the resulting
residue was partitioned between water (1 ml) and
methylene chloride (2 ml x 5). The methylene chloride
phase was evaporated to dryness and the residue
reconstituted in methanol (1 ml). The methanolic
solution was filtered through a Millex filtering unit,
diluted 1:1 with methanol and used for HPLC analysis.
Results are shown in Table 10.
(c) Defatting of the plant material prior to
extraction
by soxhlet usincr acetone.
Dried, ground leaves (lOg) as in (a) were
defatted with hexane by soxhlet extraction for eight
hours, changing the solvent every four hours (100 ml x
2). The average hexane extractives was 297 mg or 260
of the total hexane and acetone extractives. The mare,
in the soxhlet, was further extracted with acetone (4 x
100 ml) according to the method described under Example
6. Extraction, partitioning and analysis are as
described under Example 10(b) and the results are shown
in Table 10.
30
CA 02108265 2003-09-22
- 73
n
o v m vo
~ off o o
vi r.
0 0 ow o.r
x +i . .
~ ,a
t0 O O O U1
.~1 r1
E~ C) +1 +1 tl
cr .n nr ~r
4 ~ ~o
. H .. .
.
~ o> o> o>
.. . ..
~t o o o a
U V
0
m
w f,,
* w
oy rH Leo e~r.~
p 'p .. W o~ .. ev
pv n
x .. .r .. .r
.a e~
00 00 00
"'' E' ~ O O C O
O O
O~
U
rr *r
X Q1 0
l
N
i C ~
:
c ~
a d ~ y C m e~ o
Ew v t~
U ~
_ ~, O ~0 N N C7 If1
S 1~ C' ~ .-t
~ ~ J3 t d' '
~
1 V f R 41
a 10 f
P1
: i
c
.
c
o.co
~~ U
~
0
pi
,, . vD v N
x w O
r p
.
0 ~ N
. v l1 H m
. m ~
V N ~ d '~ N N ~ O
E 0~ ~ r
1.1 ~
y~ N i9 ~ O O ~
'O d O 3 ~ ~ O .H
C ~ Q''
..r
~ aJ 'C
w
O C~
2 0 '~ w
y
w
.p
o .~
w .-~ U e-s ae t~
n er
v
w L ~ L ~
~
G9 'C 0v ; m CO
0~
'~
'~
x p~ O O O
O
~. ~ v
O
C1
2 5 '" '
ar c
A c
'y o o ~ c
x a a
y ~
i ~ w H .i ~ H
.~ .i
=
..
m
3 ~ ?"
rl ~1 ~ 1V
~s N N
10
C
4
r C
U t
35 ~s~.~'ar c
rr ~ O ~ t b
C
?
d t O
a sn N
2~i~°26~
1W0 92/18492 P~CT/US92/030$$ t.,
74
,.
Example 11: partitionina of the acetone extract of the
defatted plant material between water and ethyl
acetate.
A sample of leaves of T. X media 'Hicksii' was
dried and ground using the method described under
Example 9. Dried, ground leaves (lOg) were defatted
with hexane and extracted by percolation using acetone
using the method described under Example 10(a). A
known weight of the acetone residue was partitioned
between water (~. ml) and ethyl acetate (2 ml x 5). The
ethyl acetate phase was evaporated to dryness in vacuo
and the residue reconstituted in methanol (1 ml). The
methanolic solution was filtered, diluted and analyzed
as described under Example l0(a). Results are shown in
Table 11.
25
35
,~=,,,WO 92/1$492 i~ '~ ~ S~ ~ ~ ~ PCT/US92/030$$
- 75 -
a ''
~ O~
O
~'
O
f0 C~
~ H ro
..
, ~,
~ to
d
O ~ O
~ "~
i
. O
0
1 d
d~ W N
~o~
a .u ro
.G a~ ..,
~ . ea
~r.-a eo
,
~ 3
~
3 B o~ 0
0
w a~ w H ~ ~
o
o y ~i
~ N
5 a
ro
1
~o
W
~,~~
~ a ~ ~ v Sri
N ~ .o
oc~
-.~ ro
.u c
t~ ar ~a
ro s~
xvx
~
~ o
a~ ~ ro w ~
.. ao
~
ro H o ~ r
, cs. ~
m
~ ~21~ u~o~
~ 6
~p v o0
~w
,o ~a
o v
r, c ~,
w~N~
O
~ ~ U ~.
a
~ ~ ~ O~
C b.WT O~
U w . ~ V
i x
N o
O
.~ ~ a~ x ~, ~
w
.~ .N .~ U
1~ ?~ d~ -..1
w U
CI 'O
'ty A1 C LT
f0ro C
U
WO -.
i
C
I
~,~E ~3~~ ~;
a
~a ~ ~ d
x
w
~
s
~
m~,wH
N
' r-1 .r..l
r1 N
~ '-1
'~' N
QI n"
~
'3 5
~ ~c
WO 92/18 92 PCT/US92/03D88 ,..r;>~.,
t: .
-~ 76 _
U
Example 12: Determination of taxane levels in T. X
media 'Niqra!usinqasolid phase finishincx treatment.
Fresh leaves of T. X media 'Nigra' (Rhode Island
Nurseries, Newport, Rhode Island) were stripped from
their stems, and the following procedures were carried
out using three replicates. The stripped fresh leaves
(1o gms) were placed in a blaring blender along with 100
ml of 95~ ethanol to macerate the leaves. The leaves
were ground for 2 minutes.
The ground leaves arid the ethanol were
quantitatively transferred to a 250 ml Erlenmeyer flask
and the leaves were allowed to soak for 24 hours. The
ethanol was filtered with rinsing (25 ml ethanol) and
the ground leaves were returned to the Erlenmeyer
flask, to which another 100 ml volume of 95% ethanol
was added. This procedure was repeated three more
times until a 500 ml volume of ethanol extract was
obtained.
The ethanol extract was evaporated to dryness in
vacuo at a temperature not exceeding 40°C to leave a
residue ("first residue"). The weight of this residue
was 1.47 g ~ 0.059 (C.V'. = 4%).
About l00 mg of the first residue was
transferred into a 4 ml screwcap vial. The vial cap
was lined with aluminum foil to avoid contact of the
vial cap with the residue during subsequent
partitioning steps.
The first residue was partitioned between. about
1 ml of water and about five 2 ml volumes of methylene
chloride. The methylene chloride was then transferred
to a 25 ml Erlenmeyer flask and the solvent was
evaporated in vacuo to form a second residue.
The residue from the methylene chloride layer
was then dissolved in about 5 ml of ethyl acetate and
about a ml of methanol. To assist in the dissolution
.~.,;..~0 92/18492 ~ '~ ~ ~ ~ JPC.'T/US92/030~3~
,.~._,~t...,=.~'7~
- 77 -
a '
of the residue the flask was sonicated. When the
residue was completely dissolved, about 650 mg of
celite was added. The residue was adhered to the
celite by allowing the solvent to evaporate under
reduced pressure to produce a coated celite material.
The coated celite was transferred to a petri
dish, then triturated until a free-flowing powder was
obtained. A Pasteur pipette was then packed with the
triturated powder.
Hexane was washed through the column until the
eluent appeared colorless (7 ml). Methylene chloride
(5-6 ml) was then used to elute the taxanes. The
methylene chloride was then evaporated in vacuo to
produce the crude taxane mixture. The weight of this
1$ mixture was 39.1 mg ~ 7.43 (C.V. = 19%).
The crude taxanes were dissolved in 1 ml HPLC
grade methanol. Sonication was used to assist in the
dissolution of the crude taxanes. The solution was
filtered through a Millex filtering unit (0.45,) and
collected in a flask.
The identity and amount of specific taxanes in
'the solution was determined using HPLC analysis from a
10 g sample. A ~C Hondapak C-18 (10~C) column, obtained
from Waters Associates was used fox the HPLC analysis.
A mixture of methanol (650) and water (350) at a
flow rate of 1.2 ml per min was used as the mobile
phase. Taxanes were detected with a U.V. detector set
at a wavelength of 227 nm.
The retention times and percent compositian of
the solution is shown below in Table 12.
3S
CA 02108265 2003-09-22
- 78 -
J
Table 12. Rt Values and Percent Composition of
Specific Taxanes Isolated from T. X media 'Nigra'
Rt Value
Taxane (min.) _$
Composition
Taxol~ 11.5 0.291
cephalomannine 10.5
baccatin III 4.7 -
10-desacetyltaxol - -
10-desacetylcephalomannine - -
10-desacetyl baccatin III 4.2 -
14
example 13. Percent Taxol obtained from Barn-Dried
leaves extracted accordincLto the method of Example 12.
Fresh intact clippings of T. X e~n,d,~a 'Nigra~
15 leaves, still attached to their stems were placed on an
aluminum wire cloth (18 in by 16 in, 0.10 gauge window
screen) attached to a wooden frame inside a "Roanoak"
style drying barn, model 7.5-1-G Bulk Curing Barn
obtained from Gregory Manufacturing Company, Inc., of
20 Woodville, North Carolina. The frames were placed on
supports about~30 in. above floor level. The plant
matter was dried in a ventilated barn in the dark.
During the drying step, the temperature
varied betweew about 30'C at 8 a.m. to about 40'C at
25 3:30 p.m. Drying was continued for two days. The
clippings were analyzed for taxane content following
the procedure described in Example 12. The following
taxanes were detected: taxol; cephalomannine: baccatin
III. 10-desacetyl taxol: 10-desacetylcephalomannine;
30 and 10-desacetyl baccatin III. The percent taxol
present in the composition was 0.034.
*trade-mark
* trade-maxk
CA 02108265 2003-09-22
- 79 -
.
Example 14. Isolation of taxol from the leaves of T. X
media 'Dark Green S,~reader' using a solid phase
finish~n4 treatment.
Fresh clippings of T. X med'a 'Dark Green
Spreader' were separated into leaves and small stems
and then allowed to dry at room temperature according
to the method described under Example 2(a). The dried
leaves (500g, showing a taxol content of 0.0074% as
determined by the method described under Example .1)
were ground in a Wiley Mill to a mesh size of about 60
and extracted by percolation with 95% ethanol, changing
the solvent every 24 hours (1.5L,x 5). The combined
ethanol extracts were evaporated on a Buchi rotary
evaporator under reduced pressure at a temperature not
exceeding about 40'C.
The ethanol residue (112 g, residue A) was
partitioned between water (about 500 ml) and the
following volumes of methylene chloride: one volume of
about 1 liter, and three volumes of about 500 ml each.
The methylene chloride layers were separated, combined
and dried over anhydrous sodium sulphate. The drying
agent was removed from the methylene chloride using
vacuum filtration. The methylene chloride solvent was
evaporated using a Buchi rotary evaporator under
reduced pressure to produce a second residue (27.88,
residue H).
The second residue (27.8g) was dissolved in
an ethyl acetate/methanol mixture (3:1, 600 ml) and the
resulting solution divided into two 300 ml portions.
Each portion was uniformly coated onto about 2008
celite 545 (Fisher) as described under Example 1.
The celite coated with the second residue,
Residue B was packed into two columns (5 x 32 cm each)
and each washed successively, under pressure, with
hexane (2.2L) followed by methylene chloride (1.2L).
*trade mark
CA 02108265 2003-09-22
- 80 -
v
Evaporation of the hexane and methylene chloride washes
iI vacuo yielded combined residues weighing 19.258
(Residue C) and 6.358 (Residue D), respecti~rely. .
Residue D comprising the crude taxane mixture
(6.358) was dissolved in 250 ml ethyl acetate and
adsorbed onto celite (508) according to the method
described under Example 1. The resulting material was
applied onto a silica gel 60 flash column (1608, 5 x 16
cm, 230-400 mesh, E. Merck, 1 column volume=500 ml)
packed in hexane/acetone (?5:25). The polarity of the
mobile phase was gradually increased to hexane/acetone
(55:45) by increasing the percentage of acetone in 5%
increments. Fractions we're collected, under pressure,
at a flow rate of 100m1/5-minutes. The column was
eluted with one column volume of hexane/acetone (?5:25)
collected as one 500 ml-fraction, followed~by two '
column.volumes of hexane/acetone (?0:30) collected as 4
fractions 250 ml-each , two column volumes of
hexane/acetone (65:35) as 4 fractions 250m1-each, one
column volume of hexane/acetone (60:40) as 2 fractions
250m1-each, one column volume of hexane/acetone (55:45)'
as 10 fractions 50m1-each. The collected fractions
were examined by thin-layer chromatography on precoated
silica gel G W2~~(Machinery Nagel; Duren). Fractions
eluted with hexane/acetone (60:40, 500 ml) and
hexane/acetone (55:45, 150 ml) were combined and
evaporated to dryness 'fir vacuo (Fraction E, 0.4278).
In this fraction, 8 components were identified by thin
layer chromatography using 5% methanol in chloroform as
a developing system and p-anisa~ldehyde/sulfuric acid as
a- visualizing agent. The taxol-cephalomannine mixture
appeared as a bluish-grey spot, with an Rf value of
0.62.
Fraction E was further fractionated on a
silica gel 60 flash column. The material (42? mg) was
* trade-mzrks
CA 02108265 2003-09-22
- 81 -
dissolved in 1% methanol/methylene chloride (2 ml) and
applied onto a silica~gel 60 column (55g, 2 x 40cm,
230-400 mesh, E. Merck, 1 column volume=120 ml), packed
in 1% methanol/methylene chloride. The column was
eluted, at low pressure to provide a flow rate of 8
ml/min., successively with 1% methanol/methylene
chloride (2 column volumes as 20 fractions 12 ml-each),
1.5% methanol/methylene chloride (1 column volume as l0
fractions 12 ml-each), 2% methanol/methylene chloride
(1 column volume as 10 fractions 12 ml-each) and 2.5%
methanol/methylene chloride (3 column volumes as 74
fractions 5 ml-each). Fractions were monitored by thin
layer chromatography using the same developing and
visualizing reagent systems described above. The
taxol*cephalomannine mixture was identified in
fractions 78-86 eluted with 2.5% methanol/methylene
chloride. These were combined and evaporated tc
dryness in-vacuo to give a residue (40.35 mg, Fraction
F). This fraction when examined by HPLC indicated the
presence of taxol together with cephalomannine.
Taxol* was separated from cephalomannine by
dissolving Fraction F (40.35 mg) in 1 ml of a solvent
mixture of 20% ethyl acetate in methylene chloride and
applying the solubilized Fraction F onto a silica gel
60 flash column (lOg, 1x32cm, 230-400 mesh E. Merck, 1
column volume = 21 ml) packed in the 20% ethyl
acetate/methylene chloride solvent mixture. The column
was eluted, at low pressure to provide a flow rate of 8
ml/min., successively with 20% ethyl acetate/methylene
chloride (2 column volumes as 4 fractions 11 ml-each),
25% ethyl acetate/methylene chloride tl column volume
as 2 fractions 10 ml-each), 30% ethyl acetate/methylene
chloride (1 column volume as 5 fractions 4 ml-each),
35% ethyl acetate/methylene chloride (2 column volumes
as 9 fractions 4.5 mI each), 40% ethyl
*trade-mark
CA 02108265 2003-09-22
- 82 -
V
acetate/methylene chloride (1 column volume as 7
fractions 3 ml-each), and 45% ethyl acetate/methylene
chloride (2 column volumes, as 20 fractions 2 ml-each).
The fractions were monitored by thin-layer
chromatography on silica gel G Wz~' using ethyl
acetate/methylene chloride (1:1) as developing solvent
and p-anisaldehyde/sulfuric acid as visualizing
reagent. Taxoh'(26mg) was obtained in a pure form, as
determined by HPLC analysis, in fraction's 35-50 eluted
with 45% ethyl acetate in methylene chloride.
Example 15: Isolation of taxol~from the leaves of T. X
media 'Niara~ using a partitioning finishing treatment
ste .
Intact clippings of ~ X media 'Nigra~ were.
barn-dried as described in Example 4(a). The leaves
obtained from the barn-dried intact clippings (500g,
showing a taxol content of 0.043% dry weight as
determined by the method of Example 1) were ground in a
Wiley Mill to a mesh size of about 60 and treated by
percolation with 95% ethanol (8L). The combined
ethanol extracts were evaporated to dryness on a Buchi
rotary evaporator under reduced pressure at a
temperature not exceeding 40'C.
The ethanol residue (134.4g, first residue,
residue A) was partitioned between water (about 600 ml)
and the following volumes of ethyl acetate: two
volumes of about 1.2 L, and three volumes of about 600
ml each. The ethyl acetate layers-were separated,
combined and dried over anhydrous sodium sulfate. The
drying agent was removed from the ethyl acetate using
vacuum filtration. The ethyl acetate solvent was
evaporated using a Buchi rotary evaporator under
reduced pressure to produce a second residue (44g,
3 5 Residue 8) .
'* trade-marks
CA 02108265 2003-09-22
- 83 -
Residue B was partitioned between a biphasic
mixture of about 1 part water, 9 parts methanol (500
ml), and two volumes of hexane (1000 ml).
Subsequently, each phase was washed with the opposite
solvent (100 ml x 5) and the washings combined with
their respective phases. The combined hexane layers
were evaporate to dryness to yield a reside (17.7g,
residue C) and the combined water/methanol layers were
evaporated, 'fir vacuo, to produce a residue of a crude
i0 taxane mixture (3l.lg, residue D).
Residue D (3l.lg) was washed with sonication
using methylene chloride (300 ml x 4). The methylene
chloride washings were combined, and evaporated 'fir.
vacuo to yield a methylene chloride soluble portion of
Residue D (1?.3g, Residue E, showing a taxol content of
167mg as determined by the method of Example (1)). The
weight of the methylene chloride insoluble portion of
Residue D was 13.8g and showed a taxol content of 20mg
as determined by the method of Example 1.
Residue E (17.3g) was dissolved in 250 ml
ethyl acetate and adsorbed onto celite (30g). The
resulting material was applied onto a silica gel 60
flash column (315g, 5 x 32 cm, 230-400 mesh, E. Merck,
1 column volume=600 m1) packed in hexane/acetone
(70:30). The polarity of mobile phase was gradually
increased to hexane/acetone (45:55) by increasing the ,
acetone content in 5% increments and then the column
washed with 100% acetone. Fractions were collected,
under pressure, at a flow rate of 100 ml/5-minutes.
The column was eluted with two column volumes of
hexane/acetone (70:30) collected as eight fractions:
300 ml, 100 ml, 125 ml, 200 ml, 160 ml, 190 ml and 210
ml, followed by one column volume of hexane/acetone
(65:35) collected as 2 fractions: 175 ml and 300 ml,
one column volume of hexane/acetone (60:40) as one
~ trade-mark
CA 02108265 2003-09-22
- 84 -
fraction 500 ml-fraction, one column volume of
hexane/acetone (50:50) as ? fractions 100 ml-each, one
column volume of hexane/acetone (45:55) as 6 fractions:
100 ml-each, and 3 column volumes of 100% acetone as 4
fractions: 450 ml-each. The collected fractions were
examined by thin-layer chromatography on precoated
silica gel G W25' (Machery Nagel, Duren) using 5%
methanol in chloroform as a developing system and p-
anisaldehyde/sulfuric acid as a visualizing reagent.
Fractions eluted with hexane/acetone (55:45,
100 ml) and hexane/acetone (50:50, 450-ml) were
combined and evaporated to dryness ~ vacuo (Fraction
F, 1.61g).
In this fraction, 8 components were
identified using 5% methanol in chloroform as a
developing system and p-anisaldehyde/sulfuric acid as a
visualizing reagent. Taxol*cephalomannine mixture'
appeared as a bluish-gray spot, with a Rf value of 0.62.
While we have hereinbefore described a number
of embodiments of this invention, it is apparent that
the basic constructions can be altered to provide other
embodiments which utilize the methods of this
invention. Therefore, it will be appreciated that the
scope of this invention is defined by the claims
appended hereto rather than by the specific embodiments
which have been presented hereinbefore by way of
example.
35
* trade-mark
