Note: Descriptions are shown in the official language in which they were submitted.
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A METHOD FOR PRODUCING TAXOL AND TA~XANES FROM
EMBRYO CULTURES OF TAXUS SPECIES
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is related to a method for producing taxol and its
derivatives from somatic embryos and/or culture medium, and particularly is
related to a method :for culturing the zygote embryo or somatic embryo to
obtain
somatic poly-embryos or embryogenic calli.
2. Description of Prior Art
Taxol is an a:Lkaloid which has been isolated from the plants belonging to
the genus Taxus andl exhibits significant antitumor activity in a variety of
cancer
cell lines, including B 16 melanoma. It has been known that taxol is contained
primarily in barks of Taxus brevifolia with approximately 0.02% by gram dry
weight.
Although total synthesis of taxol has been successfully demonstrated, this
method seems to be unsuitable for commercial scale production of taxol due to
high cost requirement. In addition, semi-synthesis of taxol like compound
(for example taxotere) also was succeeded by using baccatin III or 10-deacetyl
baccatin III. Hovvever, this approach have been faced same obstacles just
mentioned above.
Therefore, tacol can only be obtained from the scarce natural resources, for
example raw materials of harvested yew. Because the content of taxol in yew
trees
is very low, 2000 to 4000 trees must cut down to yield 1 kg of taxol and
production
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of taxol from the natural sources for sufficient demands will rise significant
destruction of forest eco-system. Accordingly, there have been attempts to
produce taxol and related compounds from cells and tissue cultures of Taxes
species.
For example, USP 5,019,504 discloses a method for producing taxol or
taxol-like alkaloids by culturing bark, cambium, needle, and root tissues of
Taxes
species in a nutrient medium to produce callus or suspension cell cultures,
then
recovering taxol or taxol-like alkaloids from them. However, the above method
can produce only 1.0-3.Omg of taxol from 1 liter of cell suspension culture
medium. The above patent teaches that bark fo Yew tree can produce optimum
yield of taxol directly, and produces taxol by culturing these tissues to
induce
undifferentiated cell mass(callus) and/or by establishing cell suspension
cultures
from proliferated callus.
Still now, the above method using various parts of Yew tissues have not
commercialized partly due to low taxol yield capacity of the cells associated
or
insufficient processes developed for large scale production of target
secondary
metabolites.
W092-13961 discloses a method for producing taxol by culturing a tissue
of a plant belonging to the genus Taxes, particularly its female gametophyte
and
recovering taxol from calli or suspension cultured cells originated from it.
This
process can give 0.05% of taxol based on the dry weight of suspension culture
cells.
These methods fail to produce a sufficient amount of taxol in an industrial
scale and till now are under development to improve the taxol production
capacity.
These fact suggesting that there is a need to provide an improved method for
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increasing taxol contents in cell and/or culture medium and for screening high
taxol producing cell line to meet final goal of industrial scale production
Under these circumstances, the present inventors have made an extensive
study to develop a method for producing taxol in an industrial scale, and as a
result
thereof, we unexpectedly found for the first time that zygote embryo contains
much larger amount of taxol, based on the same dry weight, than other tissues
reported to contain taxol so far.
The present inventors have made further researches to provide a method for
increasing the mass of embryo because the size of embryo itself is too small
to
develop an industrial scale process for producing taxol. As a result thereof,
we
suprigingly found that the mass of embryo can be increased to several hundred
thousands times than the original size when the embryo is cultured onto
nutrient
media to induce a somatic embryo or embryogenic callus.
The present invention is accomplished based on the above findings.
SUMMARY OF THE INVENTION
Thus, an object of the invention is to provide a method of production of
taxol by extracting tissues of Taxus species, characterized in that said
tissue is
zygote embryo.
Other object of the invention is to provide a method of production of taxol
or its derivatives by culturing a tissue of Taxus species and recovering taxol
or its
derivatives from callus or culture medium characterized in that said tissue is
zygote embryo.
Still another object of the invention is to provide a method of production
of taxol or its derivatives, wherein it comprises the steps of:
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(a) providing living zygote embryo from Taxes species and disinfecting it;
(b) culturing said disinfected embryo onto a culture medium to produce
callus from embryo;
(c) culturing the callus obtained in (b) to produce somatic embryo from said
callus;
(d) culturing the disinfected embryo in (a) or the somatic embryo obtained
in (c) to produce embryogenic callus;
(e) liquid culturing the somatic embryo in (c) or embryogenic callus in (d);
and
(f) recovering taxol or taxol derivatives from the culture medium and from
the cells.
Yet a further object of the invention is to provide a method of producing
taxol or its derivatives comprising the steps of
(a) collecting seeds from Taxes species, disinfecting them, and producing
zygotic embryos from skid seeds;
(b) .inducing callus formation, by culturing an inoculation of said zygotic
embryo, on a solid nutrient medium, then producing a proliferated callus by
subculturing the callus on the same said solid nutrient medium further
supplemented with a compound which can prevent phenolic compounds from
inhibiting the growth of the callus;
(c) inducing the formation of a somatic embryo, by culturing said
proliferated callus on a solid nutrient medium without any plant growth
regulator;
(d) inducing the formation of embryogenic callus, by culturing said somatic
embryo on a solid medium supplemented with 1 - 4ppm of NAA and 0.5 - 2 ppm
of 6-furfurylaminopurine to induce a callus therefrom, culturing the callus on
the
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same medium supplemented with 1 -5 ppm of cytokinin to form embryoids on the
surface of the callus, and then culturing the embryoids on the same medium
supplemented with 2 -10 ppm of 2,4-D;
(e) liquid culturing said embryogenic callus; and
5 (fj recovering taxol or its derivatives from the liquid culture medium and
from the cells.
The instant invention is distinguished from the above-cited prior arts in that
it is based on the finding that the zygote embryo contains unexpectedly large
amount of taxol and further increases the mass of embryo to several hundred
thousand times through an induction of somatic embryo or embryogenic callus,
and is advantageous over the prior art because it makes it possible to produce
taxol
in an industrial scale by liquid culturing the somatic embryo or embryogenic
callus.
Other features and applications of the present invention will be easily
apparent to the skilled in the art by the following description.
DEFINITIONS
Somatic Embryogenesis(SE)
The term ' asexual embryogenesis' encompasses somatically and genetically
derived embryos. SE embryos have the same appearance as zygotic embryos. The
main features of SE embryos are that they have vascular systems and morphology
similar to zygotic embryo(torpedo, globular and heart shape in their
developmental
process). Moreover, SE embryos have an ability to produce shoot and root(i. e.
they
have regenerative capacity)
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Embryogenic Callus(EC)
Embryogenic callus is composed of normal stomatic cells and white to pale
yellow
compact callus that is opaque, often with a nodular and convoluted surface. EC
is
an early function stage of SE and the main feature of EC is that it does not
have
a differentiated morphology, as compared, for example, with zygotic or somatic
embryos. Moreover, EC does not have a perfect vascular system or regeneration
ability without special treatment.
BRIEF DESCRIPTION OF DRAWINGS
Fig 1 (A) is a HPLC chromatogram of standard taxanes.
Fig I (B~s a I-IPLC chromatogram of purified culture medium obtained from
Example 7.
Fig 2(A) is a photograph showing rat cancer cell without treatment in
Experimental Example 1.
Fig 2(B) is a photograph showing rat cancer cell treated with the extract of
Example 7 in Experimental Example 1.
Fig 3(A) is a standard curve by ELISA data.
Fig 3(B) is a curve by ELISA data carried out in Experimental Example 2.
Fig 4 is a graph showing effects of production medium on taxol production in
Example 8.
DETAILED DESCRIPTION OF THE INVENTION
The present inventors analyzed taxol contents of 24,000 Taxes cuspidata
trees planted in national forest land in Korea. Although the amounts were
significantly varied depending on the individual tree, they found that 5 g of
taxol
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was contained in 100g of dried embryos collected from superior trees. Whereas
4.Smg and 2.Omg of taxol were obtained from the same amounts of dried bark and
needle, respectively.
Therefore, the first object of the invention is to provide a method for
producing taxol wherein taxol is extracted from embryo of Taxes species. The
kind of Taxes species is not limited and any trees can be employed so long as
it
belongs to genus Taxes. Nevertheless, Taxes cuspitdata is advantageously
employed.
The extraction method for producing taxol from embryo is not particularly
limited. However, in general, extraction of taxol from embryo may be carned
out
by immersing freeze-dried powders of embryo in an alcohol, particularly
methanol.
Unfortunately, the size of zygote embryo is very small and embryo contains
more than 90% of water. Huge amount of embryos are required to produce only
small amount of taxol and the extraction of taxol directly from embryo is
disadvantageous in an economic viewpoint.
Accordingly, the present inventors had been made extensive researches to
provide a method for producing taxol in an economic way without the above-
identified problems, and as a result thereof could increase the mass of embryo
to
hundred thousands times by using a tissue culture techniques, particularly via
an
induction of somatic embryo and embryogenic callus. Therefore, the second
object of the invention is to provide a method for producing taxol or taxol
derivatives by inducing somatic embryo or embryogenic callus from the zygote
embryo of Taxes species, culturing said somatic embryo or embryogenic callus
in
a liquid media by using a shaker or bio-reactor and recovering taxol from the
culture medium and cells.
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One characteristic feature of the instant invention is that the present
invention can increase the mass of embryo from ten thousands to hundred
thousands times through an induction of somatic embryo or embryogenic callus,
whereas a simple callus induction from various tissues except embryo is
difficult
to attain such an amplification possibly due to the different capacities for
cell
division by the meristem used. Particularly, an induction of embryogenic
callus
makes it possible to increase the mass of embryo to hundred thousands times
and
thus is very important.
The induction of somatic embryo or embryogenic callus from zygote
embryo and production of taxol or its derivatives will be described
hereinafter.
In order to aseptically culture an embryo, the mature or immature seeds
collected from Taxes species during the period from August to November should
be disinfected or surface sterilized. Disinfection of surface sterilization
may be
carried out by using common techniques. For example, seeds are immersed in
70% ethanol for 30-60 seconds, washed with sterilized water two or three times
and surface sterilized with 1-3%(v/v) solution of sodium hypochlorite for 24
hours. The surface sterilized seeds are rinsed with sterilized water for more
than
5 times and embryos are detached therefrom.
The embryos may be employed for extracting taxol by following the above
described extraction techniques.
The embryos also are placed onto a solid nutrient medium to induce callus
therefrom. Induction of somatic embryo may be carned out by PEDC(Pre-
Embryogenic Determined Cell) or IEDC(Induced Embryogenic Determined Cell)
procedures.
Induction of somatic embryo using PEDC pattern is as follows:
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Embryo is cultured on a solid nutrient medium
supplemented with 1-naphthalene acetic acid(hereinafter "NAA"), kinetin and
2,4-
D for callus induction. Once callus was induced, small pieces of calli could
be
proliferated by subculturing onto the media contained proper concentrations of
macro-, micro-nutrients, and vitamins.
For the purposes of induction and proliferation of callus, common plant
tissue culture media may be employed without limitation. For example,
mBs(modified Gamborg's B,medium), Durzan, MS(Murashige & Skoog medium),
WPM(Lloyd & McCown), DKW(Driver-Kuniyuk-Walnut), GD(Gresshoff &
Doy), SH(Schenk & Hildebrandt medium) or LP(Quoirin & Lepiover) may be
employed.
It is understood that modifications may be made in these media such as
addition or deletion of various components, or alteration of proportions.
Among
others, mBs or Durzan medium is preferred. As an intermediate step for somatic
embryogenesis, the media for induction which may be the same or different from
the media for rapid growth of callus may be used. From the viewpoint that the
largest number of somatic embryos can be obtained, mBs medium is
advantageously employed.
Because most of the above mentioned media are commercially available or
well established, public may easily have access thereto. Therefore
determination
and/or optimization of suitable culture media for induction and rapid growth
of
callus are within the ability of a person skilled in the art. For example, mBs
medium, which is advantageously employed in the invention, is slightly
modified
medium of Gamborg's Bs and its composition is shown in Table 1.
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Table 1
Compound mg/1 Compound mg/1
KN03 2500 CoC12.6H20 0.025
KCl 150 NazMo04.5H20 0.25
CaCl2 150 CuS04.5Hz0 0.025
KI 1 H3B03 3
NaH2P04 300 Thiamine.HCl 2
MgS04.7H20 250 Pyridoxine.HCl 1
(NH4)ZSO4 150 Nicotinic acid 1
MnS04.4H20 10 Myo-innocitol 100
ZnS04.7H20 2 Sucrose 30000
Na2-EDTA 37.2
FeS04.7H20 27.8 pH 5.6-5.7
Besides, IEDC pattern for the production of somatic embryo may be carried
out by culturing embryo in an above-illustrated medium, to which different
plant
growth hormones are added alternatively.
For the initial culture, 2-4 ppm of 2,4-D is added. When callus is formed
from the surface of embryo and the cell mass increases 10-20 folds, subculture
was
usually followed by using the same medium having half concentration of the
auxius used.
Because most of the callus lose their growth ability due to the phenolic
compounds possibly secreted from the non-prolific cells, 0.5%(w/v) of
activated
charcoal or 1-2%(w/v) polyvinyl polypyrrolidine were incorporated. The callus
is subcultured onto a medium free of growth regulator for 2-4 months to form
somatic embryo from meri-stemic tissues of callus.
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Thus-formed somatic embryo contains large amount of taxol or taxol
derivatives(hereinafter, "taxanes") and may be employed per se to extract
taxanes.
The extraction of taxanes from somatic embryos may be carried out by using
common methods, particularly the method described hereinafter. However, the
somatic embryo is advantageously employed to induce embryogenic callus in
order
to further increase cell mass.
According to the present invention, a relatively high rate of taxanes can be
obtained by extraction of embryogenic calli as well as the above produced
somatic
embryos. The embryogenic callus may be produced by culturing the somatic
embryo obtained by PEDC or IEDC procedure described as above or the zygote
embryo. Procedures for the production of embryogenic callus is as follows: The
disinfected embryo or somatic embryo is cultured for 2-4 months in a solid
medium suitable for inducing callus, which is supplemented 1.0-4.0 ppm of NAA
and 0.5-2.0 ppm of kinetineTM(used throughout to mean 6-
Furfurylaminopuridine),
preferably with 2.0 ppm of NAA and 1.0 ppm of kinetinTM. 1-4 ppm of 2,4-D
instead of NAA plus kinetinTM may be added to the medium. The induced callus
is then cultured onto the same medium supplemented with 1-5 ppm of cytokinin
such as benzylamine purine, N-isopenthenyl aminopurine, kinetinTM or zeatin TM
for
1-2 months to form embryoid on the surface of the callus, and the embryoid is
cultured onto the same medium supplemented with 2-10 ppm of 2,4-D at about
26 °C for 2-3 months under the 16/8 hours(light/dark) condition. This
culture
enables to obtain embryogenic callus in a huge amount which is hundred
thousands times of original mass of embryos. The embryogenic callus showed a
wide range of color, for example, from yellow, brown to green.
For the induction and proliferation of embryogenic callus, any medium
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which can be employed for conventional plant cell and tissue cultures may be
used
without limitation.
The above-produced embryogenic callus contains a large amount of taxol
and may directly be employed for extraction of taxol and taxanes.
The extraction of embryogenic callus may be carried out by a common
technique, particularly the method described hereinafter.
In order to amplify the mass of the embryogenic callus in an industrial
scale, the embryogenic callus is aseptically sliced to produce single cells or
small
cell aggregates. To establish cell suspension cultures, 10%(v/v) of these
cells
were inoculated into 250m1 conic flask having SOmI of a liquid culture medium
supplemented with 2ppm of 2,4-D. Air-lift or impeller type bioreactors are
advantageously employed for this purpose in the viewpoint of continuous
culture.
The bioreactor culture may be carried out using two different culture media:
one for the growth of cells(Growth medium) and the other for the production of
taxanes(Production medium). The growth medium is preferably mBs medium
supplemented with 2-4 ppm of 2,4-D and the production medium is preferably MS
medium supplemented with 2-10 ppm of NAA.
Particularly, the production medium preferably contains elicitors in order
to increase the taxol production. The elicitors may be selected from fungal
elicitors, female gametophyte extract( 1-5 ml/1), phenylalanine(50-300 mM) and
GA3 (0.5-1.0 ppm). The examples of fungal elicitors include Cytospora abietis
ATCC No. 38688 and Penicillium minioluteum(Dierckx), NRRL 18467, which are
pulverized and subjected to a series of extraction to give extracts.
Alternatively,
it is also preferable to collect and culture fungi of pestalotiopsis genus and
to add
it's extract to the culture medium in an amount of 0.5-1.0 ml/l.
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Taxol is present in cells as well as secreted into the culture medium.
Accordingly, after the liquid culture process, taxol and taxanes can be
recovered
from the cultures or the media by using well known techniques. Separation of
cells from the culture medium may be carned out by centrifugation(for example
1000xg, 5-10 minutes) or by decantation. If the later technique is employed
for
the separation, the culture broth is allowed to stand for about 24 hours to
precipitate cells and the culture medium is decanted. The culture medium still
remained in the collected cells is removed by vacuum suction using pasteur
pipette.
Taxanes could be recovered from the somatic embryo or embryogenic
callus cells as well as the culture medium by extracting them with alcohols or
a 1:1
mixture of methanol and methylene chloride. The precipitates formed during
centrifugation may be pulverized by using a sonicator( Branson 250) and
subjected
to extraction.
According to the present invention, taxol itself as well as various taxol
derivatives are recovered. Taxol derivatives include 10-deacetyl baccatin III,
baccatin III, 10-deacetyl taxol, cephanolmannin and 7-epi-10-deacetyl
taxol(see
Fig 1 (B)). The taxol derivatives may be used as such or converted to taxol by
semi-synthesis, if necessary.
The identification of taxanes from the somatic embryo and embryogenic
callus originated from zygote embryo was accomplished by HPLC, cytotoxicity
test and ELISA using taxol and six(6) taxol derivatives supplied from NCI as
standards.
The present invention will be described in more detail by way of non-
limiting Examples.
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Example 1
Taxol was recovered from the zygote embryo of Taxes species.
Seeds were harvested from superior trees of Taxes cuspidata. The embryo
was dried in a vacuum oven of about 40 ° C for about 3 days to water
content of
less than 10% and weighed. Then, embryo was freezed with liquid nitrogen and
pulverized. Pulverized embryo was immersed in an incubator of about 28
°C
containing about 100 weight times methanol for about 7 days. The resulting
extract was filtered through a membrane filter(pore size, 0.2,um ) and
analyzed for
the content of taxol by using HPLC.
Bark and needles collected from the same tree were treated and analyzed
for the content of taxol by following the same procedure as described above.
The results are shown in Table 2.
Table 2
Tissue Taxol content in the dry tissues(100g)
Embryo Sg
Bark 4. Smg
Needle 2.Omg
Example 2
Somatic embryo was produced from the embryo by using PEDC pattern.
Seeds from selected genotype of Taxes cuspidata were surface sterilized by
immersing them into 70% ethanol and 2% sodium hypochlorite for 50 seconds and
24 hours respectively, and rinsing with sterile distilled water for 3 to 5
times after
the end of each step.
As a culture medium, mBs medium (having the ingredients and
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1S
compositions shown in the above Table 1 ) supplemented with 2.Oppm NAA,
O.Sppm kinetinTM and O.Sppm 2,4-D was employed. In vitro culture was carried
out in a growth room adjusted to 26 ° C for 8 weeks.
During the first 8 weeks of culture, the embryos enlarged and callus was
induced from surface of the most embryos. The green spots in the callus were
excised and cultured in mBs medium without any plant growth regulators for
about
3 months to induce somatic embryos.
Example 3
Somatic embryo was produced from the zygote embryo by using IEDC
pattern.
Seeds from Taxes cuspidata were sterilized by following the procedure in
Example 2 and embryos were isolated therefrom. Embryos were cultured in
Durzan medium supplemented with 2ppm 2,4-D at 23 - 28°C for 2
weeks. After
2 weeks of culture, all embryos were entirely covered with calli induced
therefrom.
After 4 weeks of culture the mass of calli reached to several ten times of the
original mass.
In order to avoid any inhibition of growth or death of callus due to phenols
secreted by itself, subculture of the callus was carried out by using the same
medium supplemented with about 1 % of polyvinyl polypyrolidone. After 8
weeks culture, when the average diameter of the calli reaches to 1 - l.Scm,
the
callus was moved to mBs medium containing no plant growth regulator, wherein
callus was grown to induce somatic embryos. Differentiation of roots was
observed in some of the embryos.
Example 4
In order to induce embryogenic callus cultures, sterilized zygote embryo in
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Example 2 and somatic embryos in Examples 3 and 4 were employed as starting
materials.
The above materials were cultured in mBs medium supplemented with
2.Oppm NAA and 1.Oppm kinetinTM at 23-28 °C for about 2.5 months to
induce
callus. After the completion of callus induction, subculture was conducted
using
the same medium with 3ppm of zeatinTM for about 6 weeks to obtain green to
yellowish embryoids.
Embryoids were cultured on the same medium containing 3ppm of 2,4-D
at 26 ° C for about 2 months under the 16/8(light/dark) hours condition
to produce
embryogenic callus which amounted to hundred thousands times of original
embryo mass. The resulting embryogenic callus differs in morphology compared
with normal callus originated from various type of Yew tissues and sometimes
produced similar appearances of zygotic embryo.
However, the surface of embryogenic callus were consisted of various color
ranging red, yellow, dark green, light brown, dark brown and black. Each
colored cell mass were sliced with brade to produce small cell aggregates,
which
were then subcultured into liquid media for 2-4 weeks. After removal of large
cell clumps from the liquid cultures, single cell and/or small cell aggregates
were
inoculated onto the plastic Petri dish having 20m1 of culture medium. The
method incorporated is liquid plating and selection of differently colored
cell lines
were conducted after 4 weeks of culture. These visual selection to obtain
different colored cell lines were routinely conducted.
Example 5
Somatic embryos obtained in Example 2 or 3, or embryogenic calli
obtained in Example 4 were dried at 25 °C and dissolved in l,ul f
methylene
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chloride. l ~l of distilled water was added and the mixtures were stirred for
10
seconds, followed by centrifugation(25,000 x g). The precipitates were dried
at
25 ° C, dissolved in SO,ul of methanol and filtered through 0.2,um
filter to obtain
extracts.
Extracts were analyzed by HPLC equipped with a reverse-phase micropore
column using a standard curve(correlation coefficient 0.999) of external
standards.
The IqPLC results showed that the peak of the standard taxanes and those of
taxane contained in the above extracts appeared at the same retention time(
14.3
minutes).
Example 6
Somatic embryo in Examples 2 and 3, and embryogenic callus in Example
4 were extracted to produce taxanes as follows:
Each O.Sg of somatic embryo in Examples 2 and 3, and embryogenic callus
in Example 4 were placed in a centrifugal tube and 2,u1 of hexene was added.
The mixtures were well mixed using a glass rod and stored at -20 ° C
for 12 hours,
followed by centrifuging at 25 °C, 25,000 x g for 20 minutes.
To precipitate were added a mixed solution of methanol:ethylene chloride
and subjected to sonication using a sonicator(Branson 250). The resulting
solutions were centrifuged at 25,000 x g and the precipitates were dired at 60
° C
to dryness to obtain extracts.
The extracts were analyzed for their taxol contents and the results were as
follows : the extract of somatic embryo of Example 2 contained 0.21-0.27mg per
g of dry cells(total taxol content was 0.5-l.2mg), the extract of somatic
embryo of
Example 3 contained 0.20-0.27mg(total taxol content was 0.5 5-1.1 mg) and the
extract of embryogenic callus of Example 4 contained 0.23-0.28mg(total taxol
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content was 0.6-l.4mg).
Example 7
For the mass production of embryogenic callus, embryogenic callus of
Example 4 was cultured using a impellar type bioreactor.
S A embryogenic callus of Example 4 was inoculated at 10% PCV(Packed
Cell Volume) onto a 250m1 Erlenmeyer flask having SOmI of liquid mBs medium
with 2ppm of 2,4-D.
Stationary phase was attained when cultured at 25-28 °C for 18 days
under aseptic
condition.
The cultures were placed in a 5 liter impeller type bioreactor containing MS
medium supplemented with 2ppm of NAA(production medium). Maintenance
of culture was carried out at 25-28°C for 30 days under an aerobic
condition.
After 30 days of culture, the culture broth was allowed to stand for 24 hours
to
precipitate cells and the cells were separated from the culture medium.
Pasteur
pipette was used to thoroughly remove culture medium from the cells.
Thus separated cells and culture medium were extracted to produce taxol
and its derivatives in the same manner described in Example 5.
The HPLC results showed that the peaks of the standard taxol and its
derivatives(Fig 1 (A)) and those of taxol and its derivatives contained in the
above
extract(Fig 1 (B)) appeared at the same retention time, indicating that these
compounds are the same.
It was calculated that the cells contained 0.09mg of taxol per g of dried
cells while the culture medium contained about 8mg of taxol/1.
Example 8
In order to examine the effect of the type of culture media on taxol
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production, the cultures of Example 7 were inoculated onto various culture
media
such as MS, mBs, WPM, DKW, Durzan, White, LP, GD, B5, DCR or SH media.
All the media tested were supplemented with 2.0 ppm of NAA and other factors
including micro-environmental conditions were equally adjusted. The period of
S culture was reduced to 20 days.
The results are shown in Fig. 4. As can be seen from Fig. 4, the
production of taxol was significantly influenced by the type of the culture
medium
and MS and mB5 gave the highest production of taxol.
Example 9
Various elicitors may be added to the production medium to increase taxol
and taxane production. The effect of elicitors on taxol production was
evaluated as follows:
( 1 ) For the present invention, an extract of Pestolotiopsis sp., which is a
fungium occurred in Taxus species, was added to the production medium as an
elicitor for taxol production.
Plant tissues such as seed and inner bark were immersed in 70% ethanol,
surface-sterilized using 15% H202 for 15 minutes and then again immersed in
70%
ethanol. Tissues were rinsed with sterile distilled water for 4 times or more
to
remove remaining agents. Thus surface-sterilized tissues were placed on media:
malt extract agar medium(malt extract 20.Og, peptone S.Og, agar lS.Og and
distilled water 1 liter), growth agar medium(glucose 40g, bacto soytonTM lOg,
sodium acetate 1 g, sodium benzoate SOmg, agar 20.Og and distilled water 1
liter)
and water medium(agar 20.Og and distilled water 1 liter) and maintained in a
thermostatically controlled growth chamber with 12/ 12(dark/light) hours of
elumination.
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When Pestalotiopsis sp. fungi were appeared within 3 days of culture, the
fungi were further cultivated on the malt extract and growth agar medium(yeast
extract 3g, bacto soyton Sg, MgS04 O.Sg, glucose Sg, sucrose lOg/L) for 4
days.
When the time of harvest, the culture broth was centrifuged to separate cells
from
5 the culture medium. The cells were dried, pulverized and extracted with
methanol to give carbohydrate fraction.
Thus obtained carbohydrate extract of Pestalotiopsis sp. was added to the
production medium to a concentration of lppm, and the culture and analysis
were
carned out by following the same procedure in Example 7. The results are shown
10 in Table 3.
(2) The procedure in Example 7 was repeated except that female
gametophyte extract 2m1/L, phenylalanine 1 OOmM or Gibberellic acid 1 ppm was
added as an elicitor to the production medium. The results are shown in Table
3.
15 Table 3
Amount of taxol
Elicitor
per liter of per 100g of
20 culture medium living cell
Fungi extract 30mg 6.3mg
Female gametophyte 25mg S.Smg
extract
Phenylalanine 21 mg 4.1 mg
Gibberellin 28mg 6.Omg
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21
Experimental Example 1
Verification of taxol from embryo cultures of Yew tree was accomplished
by cytotoxicity test using a rat cancer cell.
Cytotoxicity test using a rat cancer cell is usually carried out based on the
fact that taxol is capable of killing selectively cancer cells at the
metaphase of the
cell division. Rat cancer cells provided by Central Research and Development
Center of Pacific Corporation, in singal, Kyounggi-do, Korea were cultured in
plastic culture vessels containing an animal cell culture medium. At the early
period of culture, when cell division was occured, a drop of the extracts from
Example 7 was added to one vessel(Treatment) while no extract was added to
another vessel(Control). Then, cell division and viability of each cancer cell
were
examined. The results are shown in Fig 2(A) and Fig 2(B).
As can be seen from Fig 2(A) and 2(B), the control group showed
flourishing growth of cancer cells (Fig 2(A)) while the treatment group showed
the
death of cancer cells(Fig 2(B)). The death of cancer cells was appeared after
3
hours from the treatment and completed about 24 hours later. Therefore,
measurements of cell division or viability was not required.
Experimental Example 2
The identification of taxanes from embryo cultures of Yew trees was also
accomplished by ELISA(enzyme linked immunosorbent assay) using monoclonal
antibody as follows:
TA01 kit responding specifically to taxol and TA03 kit responding
specifically to taxanes, all of which were purchased from Hawaii Biotechnology
Group, were employed for this experiment. Taxol- and taxane-antigens were
diluted to 1 / 100 folds with PB S(phosphate buffered saline) and 100l of the
dilution
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was distributed into ELISA plate. After incubating at 25 °C for 1 hour,
the plate
was washed with TBS-T(washing buffer) solution at least four times, and SO,ul
of
PBST(phosphate buffered saline tweenTM) was added thereto. Taxol standard,
taxane standard or the extracts of Example 7 was distributed into the well in
a
consecutive three folds dilution manner. Taxol-antibody and taxane-antibody
were diluted with PB ST to 1 / 100 and 1 / 1000 folds, respectively and 50 ~l
of each
dilution was distributed. After incubating at 25 °C for 1 hour, the
plate was
washed with the washing buffer 4 times. 100,u1 of HRP(horse radish peroxidase)
diluted with PB ST to 1 /2000 folds was distributed into the well and
incubated at
25 °C for 1 hour, followed by washing with the washing buffer 4 times.
200,u1
of OPD(o-phenylenediamine) was added and incubated at 25 ° C for 1 hour
to
develop a color. Absorbance at 490nm was measured by using a ELISA reader.
The results are shown in Fig 3 (A) for the taxol standard and in Fig 3 (B )
for the
culture medium of Example 7, which demonstrated taxol activity of the embryo
cultures of Taxes species. It is understood that the foregoing detailed
description
is given merely by way of illustration and that modification and variations
may be
made therein without departing from the spirit and scope of the invention.
