JPH08140690A - Production of taxane-type diterpene - Google Patents

Abstract

PURPOSE: To provide a method for easily producing taxane-type diterpenes used for therapeutic agents, etc., for ovary cancer, breast cancer, lung cancer, etc., by tissue culturing the cultured cell or cultured tissue of a plant producing a taxane-type diterpene in a medium containing cyclic polysaccharides, and recovering the products from the cultured substances. CONSTITUTION: This method for producing a taxane-type diterpene comprises tissue culturing the cultured cell and/or cultured tissue of a plant (e.g. Taxus breviforia NUTT) producing the taxane-type diterpene (e.g. Taxol) in a culture medium containing cyclic polysaccharides (e.g. α-cyclodextrin) in the presence of a jasmonic acid expressed by the formula R<0> is a group of the formula (CH2 )n COR<7> [R<7> is hydroxy, OM (M is an alkali metal atom, an alkaline earth metal atom or NH4 ), an alkoxy, etc.; (n) is an integer of 1-7]; R<1a> -R<1f> are each H, OH, a 1-6C alkyl or a 1-6C alkoxy; R<2> to R<5> and R<6a> are each H or a 1-6C alkyl; R<6b> is OH, -O-carbohydrate residue, etc.} and recovering the products from the tissue cultured substance to obtain the taxane-type diterpene used for the therapeutic agents, etc., for the ovary cancer, breast cancer, lung cancer, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a taxane-type diterpene containing taxol which is useful as a therapeutic agent for ovarian cancer, breast cancer, lung cancer and the like.

[0002]

BACKGROUND OF THE INVENTION Taxol, which is useful as a therapeutic agent for ovarian cancer, breast cancer, lung cancer, etc., is a taxane-type diterpene isolated and identified from Taxus brevifolia NUTT, which is a Taxus genus plant of the Taxus family. Yes, with complex ester groups associated with activity. It has been reported that taxol is present in every part of the yew plant, and its content is highest in the bark.
Currently, taxol is produced from natural or cultivated plants, but Taxus plants are 20c above ground.
It is a slow-growing plant that takes more than 10 years to grow to a height of m, and it is difficult to easily obtain a large amount of taxol because the tree will die when the bark is peeled off. If taxane or a taxane-type precursor, taxane-type diterpene such as baccatin III, can be synthesized using tissue culture, a large amount of taxol can be easily obtained without cutting trees, which is advantageous. Is.

[0003] For the taxol production method using the cultured cells of the plant up to now, see
s brevifolia NUTT) Taxol production by cultured cells has been patented in the United States [US Patent: 5019504], and the taxol production amount is described as 1 to 3 mg / l, but it is insufficient for industrial production. Is. Moreover, the productivity of taxol in cell culture is unstable, and cells with high productivity can be obtained primarily by selection, but it is difficult to maintain its content by subculturing [ERM Wickremesine et a
l., World Congress on Cell and Tissue Culture (199
2)].

On the other hand, as a prior art of the taxol production method, baccatin III (bacc) which is a taxol biosynthetic precursor is used.
A semi-synthesis method from atin III) is disclosed in US patent to Holton et al. [US Patent: 5015744]. The use of the plant tissue culture method enables the production of semi-synthetic raw materials and is advantageous for taxol production.

[0005]

An object of the present invention is to provide a simple method for producing a taxane-type diterpene by culturing plant tissue.

[0006]

The taxane-type diterpenes are poorly soluble in water and have low stability in an aqueous solution. If it is possible to improve the solubility of taxane-type diterpenes in water and stabilize them, it is considered possible to improve productivity. We have
As a result of diligent research, it was found that the taxane-type diterpene productivity in the culture is improved when the tissue culture is carried out by adding the cyclic polysaccharide to the tissue culture medium of the cultured cells or cultured tissue of the taxane-type diterpene-producing plant. The present invention has been completed.

That is, according to the present invention, the taxane-type diterpene is recovered from a tissue culture medium or a cultured tissue of a plant producing a taxane-type diterpene, in a tissue culture medium containing a cyclic polysaccharide, and recovering the taxane-type diterpene from the obtained tissue culture. It is a characteristic method for producing a taxane-type diterpene.

The present invention will be described in detail below. The taxane-type diterpene to be the subject of the production method of the present invention,
There is no particular limitation as long as it is a diterpene having a taxane skeleton, for example, taxol, 10-deacetyltaxol,
7-epitaxol, baccatin III, 10-deacetylbaccatin III, 7-epibaccatin III, cephalomannine, 10-deacetylcephalomanin, 7-epicephalomanin, taxakifine and its analogs, taxane 1a and its analogs, Examples include xylosyl cephalomannine and xylosyl taxol.

The taxane-type diterpene-producing plants used for tissue culture in the method of the present invention include, for example, Taxus baccata LINN and Taxus (T. cu).
spidata SIEB.et ZUCC), Karaboku (T. cuspidata SIE
B.et ZUCC var.nana REHDER), Taihei Yew (T.
brevifolia NUTT), Canadian yew (T. canadiensis MAR
SH), Chinese yew (T. chinensis), T. media, etc.

The tissue culture of the plant can be carried out by a conventionally known method except that the cyclic polysaccharide is added according to the present invention.

The cyclic polysaccharides used in the present invention include
Cyclodextrins, cyclofructans, and their derivatives can be illustrated. Cyclic polysaccharides have a cavity inside due to their cyclic structure, the cavity opening and outside are hydrophilic, and the inside of the ring shows a property that is difficult to adapt to water. is there. Utilizing this property, it has many uses such as changing a poorly water-soluble substance to water-soluble, stabilizing unstable substances, retaining volatile substances such as perfumes, and suppressing peculiar odor. Is used for foods such as freeze-dried black tea and hams and sausages for suppressing specific odors. Cyclodextrin is
It is a substance in which 6 to 8 glucose are bound in a donut shape and is synthesized from starch by the action of cyclodextrin synthase produced by a special microorganism such as Bacillus macerans . Further, cyclofructan is a fructose is bound by six to eight donut-like substance, Ba
It is synthesized from inulin by the action of cyclofructan synthase produced by special microorganisms such as cillus circulans . As cyclodextrin or a derivative thereof which is the object of the present invention, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, branched cyclodextrin thereof, partially methylated cyclodextrin and the like can be used. Examples of branched cyclodextrins include glycosyl-α
-Cyclodextrin, maltosyl-α-cyclodextrin, maltotriosyl-α-cyclodextrin, glycosyl-β-cyclodextrin, glycosyl-γ-cyclodextrin, galactosyl-α-
Examples thereof include compounds in which a sugar is attached to a ring on a branch such as cyclodextrin. As the cyclofructan or a derivative thereof, any of compounds having 6 to 8 fructose and a β2-1 flaxside bond, branched cyclofructans thereof, partially methylated cyclofructans and the like can be used.

The cyclic polysaccharide has a concentration of 0.
It is preferably from 01 to 50 mM, and especially from 0.1 to 3
It is more preferable for the method of the present invention to adjust to the range of 0 mM.

Heretofore, no example has been reported in which tissue culture was carried out in the presence of a cyclic polysaccharide as a medium additive in tissue culture of a taxane-type diterpene-producing plant, and furthermore, leakage of the taxane-type diterpene to the medium was thereby caused. Accelerated and increased production was unexpected.

The medium used for the tissue culture of the present invention is a conventionally known medium used for tissue culture of plants, for example, Murashige Skoog (1962) [Murashige
& Skoog] 's medium, Rinsmeier Scoog (1965)
[Linsmaier Skoog] medium, Woody plant
Medium (1981) [Woody Plant Medium] medium,
Examples include Gamborg's B-5 medium and Mitsui's M-9 medium. Plant hormones are added to these media, and if necessary, carbon sources, inorganic components, vitamins,
Amino acids and the like can also be added.

As the carbon source, disaccharides such as sucrose, maltose and lactose, monosaccharides such as glucose, fructose and galactose, starch, or a mixture of two or more of these sugar sources in an appropriate ratio can be used.

Examples of the inorganic component include phosphorus, nitrogen, potassium, calcium, magnesium, sulfur, iron, manganese, zinc, boron, copper, molybdenum, chlorine, sodium, iodine and cobalt. These components are, for example, Potassium nitrate, sodium nitrate, calcium nitrate,
Potassium chloride, potassium monohydrogen phosphate, potassium dihydrogen phosphate, calcium chloride, magnesium sulfate, sodium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, zinc sulfate, boric acid, copper sulfate, sodium molybdate, It can be added as a compound such as molybdenum trioxide, potassium iodide, or cobalt chloride.

Examples of plant hormones include auxins such as indoleacetic acid (IAA), naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin,
Cytokinins such as zeatin and dihydrozeatin are used.

Examples of vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B).
6), pantothenic acid, inositol, nicotinic acid, etc. are used.

As the amino acids, for example, glycine, phenylalanine, leucine, glutamine, cysteine and the like can be added. Generally, each of the above components has an inorganic content of about 0.
It is used at a concentration of 1 μM to 100 mM, a carbon source of about 1 to about 30 g / l, plant hormones of about 0.01 to about 10 μM, and vitamins and amino acids of about 0.1 to about 100 mg / l, respectively.

In the present invention, either a liquid medium or a solid medium containing 0.1% to 1% of agar or gellan gum can be used, but the liquid medium is usually preferable. In the tissue culture of the present invention, the root of the plant, the growing point, leaves, stems, seeds,
Tissue pieces or cells such as pollen, anthers and sepals, or cultured cells obtained by tissue-culturing these with the above-mentioned medium or other conventional medium can be used. The present invention also provides Agrobacterium tumefaciens or A. rhizogen.
It can also be used for tumor cells and / or hairy roots obtained by infecting plant tissue with es .

When these tissues or cells are tissue-cultured by using the medium to which the cyclic polysaccharide is added according to the present invention, a highly-produced taxane-type diterpene-producing culture tissue or cultured cells can be obtained as compared with the case of no addition.

As a method of enhancing the effect of the present invention, Japanese Patent Application Nos. 6-36156, 6-104211, 6-10421.
A combination with a method of culturing in the presence of jasmonic acid and its derivative, which is disclosed in the specification of No. 2,6-104213 as a production promoting substance of a taxane compound, can be mentioned. As the jasmonic acids, for example, general formula (I):

[0023]

[Chemical 4]

[Wherein R ⁰ is the following formula:-(CH ₂ ) _n --CO--R ⁷ {wherein R ⁷ is a hydroxyl group, OM (where M is an alkali metal atom, an alkaline earth metal atom or Represents NH ₄ ),
NR ^8a R ^8b (wherein R ^8a and R ^8b each independently represent a hydrogen atom, an acyl group having 1 to 6 carbon atoms, an alkyl group having 1 to 6 carbon atoms, or an amino acid residue), OR ⁹ (Here, R ⁹ represents an alkyl group having 1 to 6 carbon atoms or a carbohydrate residue.) Or an alkyl group having 1 to 6 carbon atoms, n
Represents an integer of 1 to 7. } Represents a group represented by; R ^1a ,
R ^1b , R ^1c , R ^1d , R ^1e and R ^1f are each a hydrogen atom, a hydroxyl group, an alkyl group having 1 to 6 carbon atoms, or 1 carbon atom.
Represents a 6 alkoxy ^{group; R 2, R 3, R} 4, R 5 and R
^6a represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms respectively; the side chain composed of C ¹ -C ² -C ³ -C ⁴ -C ⁵ -C ⁶ has one or more double bonds. May include a bond;
R ^6b represents a hydroxyl group or an —O-carbohydrate residue;
The member ring may form a double bond between adjacent ring member carbon atoms. ] The compound shown by these, or general formula (II):

[0025]

Embedded image

[Wherein R ⁰ is the following formula:-(CH ₂ ) _n -CO-R ⁷ {wherein R ⁷ is a hydroxyl group, OM (where M is an alkali metal atom, an alkaline earth metal atom or Represents NH ₄ ),
NR ^8a R ^8b (wherein R ^8a and R ^8b each independently represent a hydrogen atom, an acyl group having 1 to 6 carbon atoms, an alkyl group having 1 to 6 carbon atoms, or an amino acid residue), OR ⁹ (Here, R ⁹ represents an alkyl group having 1 to 6 carbon atoms or a carbohydrate residue.) Or an alkyl group having 1 to 6 carbon atoms, n
Represents an integer of 1 to 7. } Represents a group represented by; R ^1a ,
R ^1b , R ^1c , R ^1d , R ^1e and R ^1f are each a hydrogen atom, a hydroxyl group, an alkyl group having 1 to 6 carbon atoms, or 1 carbon atom.
Represents a 6 alkoxy ^{group; R 2, R 3, R} 4, R 5 and R
⁶ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms respectively; the side chain composed of C ¹ -C ² -C ³ -C ⁴ -C ⁵ -C ⁶ has one or two or more double chains. May include a bond;
The 5-membered ring may form a double bond between adjacent ring member carbon atoms. ] The compound shown by these, or general formula (III):

[0027]

[Chemical 6]

[Wherein R ⁰ is the following formula:-(CH ₂ ) _n --CO--R ⁷ {wherein R ⁷ is a hydroxyl group, OM (where M is an alkali metal atom, an alkaline earth metal atom or Represents NH ₄ ),
NR ^8a R ^8b (wherein R ^8a and R ^8b each independently represent a hydrogen atom, an acyl group having 1 to 6 carbon atoms, an alkyl group having 1 to 6 carbon atoms, or an amino acid residue), OR ⁹ (Here, R ⁹ represents an alkyl group having 1 to 6 carbon atoms or a carbohydrate residue.) Or an alkyl group having 1 to 6 carbon atoms, n
Represents an integer of 1 to 7. } Represents a group represented by; R ^1a ,
R ^1b , R ^1c , R ^1d , R ^1e and R ^1f are each a hydrogen atom, a hydroxyl group, an alkyl group having 1 to 6 carbon atoms, or 1 carbon atom.
Represents a 6 alkoxy ^{group; R 2, R 3, R} 4, R 5 and R
⁶ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms respectively; the side chain composed of C ¹ -C ² -C ³ -C ⁴ -C ⁵ -C ⁶ has one or two or more double chains. May include a bond;
The 5-membered ring may form a double bond between adjacent ring member carbon atoms. ] The compound etc. which are shown are mentioned.

In the above general formulas (I), (II) and (III),
And R⁰, R^1a, R^1b, R^1c, R^1d, R^1e, R^1f,
R², R³, R^Four, R^Five, R⁶, R^6a, R⁷, R^8a, R^8b,or
Is R⁹As the alkyl group having 1 to 6 carbon atoms represented by,
For example, methyl group, ethyl group, n-propyl group, isopropyl group
Pill group, n-butyl group, isobutyl group, sec-butyl group,
t-butyl group, n-pentyl group, isopentyl group, neo
Pentyl group, t-pentyl group, n-hexyl group, isohexyl group
A xyl group etc. are mentioned. The above general formulas (I), (II) and
And in (III), R^1a, R^1b, R^1c, R^1d, R ^1eOr
R^1fThe alkoxy group having 1 to 6 carbon atoms represented by
For example, methoxy group, ethoxy group, n-propoxy group, a
Sopropoxy group, n-butoxy group, isobutoxy group, se
c-butoxy group, t-butoxy group, n-pentyloxy
Group, isopentyloxy group, neopentyloxy group, t
-Pentyloxy group, n-hexyloxy group, isohexyl
Examples thereof include a siloxy group.

When R ⁷ is OM, examples of the alkali metal atom or alkaline earth metal atom represented by M include sodium, potassium and calcium. In the case where R ⁷ is NR ^8a R ^8b , R ^8a , R
The acyl group having 1 to 6 carbon atoms represented by ^8b may be linear or branched, and examples thereof include formyl group, acetyl group, propionyl group, butyryl group, valeryl group, hexanoyl group and acryloyl group. .

When R ⁷ is NR ^8a R ^8b , R
^8a, as the amino acid residue represented by R ^8b, for example isoleucyl group, tyrosyl group, and tryptophyl groups. When R ⁷ is OR ⁹ , the carbohydrate residue represented by R ⁹ includes, for example, a glucopyranosyl group. In the general formula (I), examples of the carbohydrate residue when R ^6b is an —O-carbohydrate residue include a glucopyranosyl group.

The compounds represented by the general formula (I), (II) or (III) are preferably those in which R ^{0 is-} (C
H _{2) n} COOH or - (CH ₂₎ a ^{_{n COOCH 3, R 0, R}} 1a, R 1b, R 1c, R 1d, R 1e, R 1f, R 2,
R ³ , R ⁴ , R ⁵ , R ^6a , and R ⁶ each represent a hydrogen atom or contain a double bond between C ¹ and C ² , C ³ and C ⁴ , or C ³ and C ^4. Examples of the compound are:

Preferred specific examples of the jasmonic acids represented by the general formula (I) include the compounds shown below. (Compound A)

[0034]

[Chemical 7]

(Compound B)

[0036]

Embedded image

(Compound C)

[0038]

[Chemical 9]

(Compound D)

[0040]

[Chemical 10]

Preferred specific examples of the jasmonic acids represented by the general formula (II) include the compounds shown below. (Compound E)

[0042]

[Chemical 11]

(Compound F)

[0044]

[Chemical 12]

(Compound G)

[0046]

[Chemical 13]

(Compound H)

[0048]

Embedded image

Preferred specific examples of the jasmonic acids represented by the general formula (III) include the compounds shown below. (Compound I) R ⁰ :-(CH ₂ ) _n COOH or-(C
H ₂ ) _n COOCH ₃ (n = 1 to 3) R ^1a , R ^1b , R ^1c , R ^1d , R ^1e , R ^1f , R ² , R ³ , R ⁴ , R
^5, R ^6: between H C ³ -C ^4: double bond (Compound _{^{J) R 0: -CH 2 COOH}} R 1a, R 1b, R 1c, R 1d, R 1e, R 1f, R 2, R 3 , R ⁴ , R
⁵ , R ⁶ : H Further, in the compound represented by the general formula (III), the 5-membered ring may be further substituted with a hydroxyl group, and a double bond is formed between adjacent ring member carbon atoms. Good.

Specific examples of the compound in which the 5-membered ring is further substituted with a hydroxyl group, or the compound in which a double bond is formed between adjacent ring member carbon atoms include the compounds shown below. (Compound K)

[0051]

[Chemical 15]

(Compound L)

[0053]

Embedded image

(Compound M)

[0055]

[Chemical 17]

(Compound N)

[0057]

Embedded image

The jasmonic acids include various stereoisomers.
(Cis trans isomer, optical isomer)
Use each isomer alone or in the form of a mixture
Good. All of the above jasmonic acids are taxane-type dithenes.
It has the effect of improving the productivity of rupen,
R in formulas (I), (II), and (III)⁰Is -CH₂CO
OH or -CH₂COOCH₃And R^1a, R^1b,
R^1c, R^1d, R^1e, R^1f, R ², R³, R^Four, R^Five, R⁶,
R^6aIs a hydrogen atom, and C³And C^FourForming a double bond between
Compound, jasmonic acid or jasmonic acid
Chill, tuberonic acid or methyl tuberonate, and cucurbit
Acid or methyl cucurbitate has an effect on productivity improvement
Is particularly preferable in terms of size.

These jasmonic acids are prepared synthetically or by extraction from plants (H. Yamane et a
L., Agric. Biol. Chem., 44 , 2857-2864 (1980)). On the other hand, jasmonic acid is a plant hormone-like substance that induces various reactions involved in growth promotion, tissue maturation, and expression of disease resistance. Volume 4, pages 523-531 (1990)
It is described in.

Therefore, the jasmonic acids can be produced by the cultured cells or tissue to be used, in addition to being added from the outside of the culture system. As a method of promoting the production of this endogenous jasmonic acid by the cultured cells or the cultured tissue, it is possible to exemplify the addition of the culture of the microorganism or its extract, the heat-treated product or the plant extract to the medium. MJ Mueller et al., Proc. Natl. Aca
d. Sci. USA, 90 (16), 7490-7494 (1993),
A method of adding the mold cell wall fraction can be exemplified. Furthermore, it is also possible to increase the production amount of endogenous jasmonic acid by mechanically or partially damaging the cultured cells or tissue to be used with ultraviolet rays, heat, etc., specifically, RACleeman et al., Pro
c. Natl. Acad. Sci. USA, 89 (11), 4938-4941 (198
The method for mechanically destroying a part of cells described in 9) can be exemplified.

Since jasmonic acids are poorly soluble in water, they are usually dissolved in an organic solvent such as ethanol or methanol or a surfactant, and then added to the medium. The free form of jasmonic acid may be used as it is, or may be neutralized with an alkali to form a salt. The α-position of the 5-membered ring carbonyl group of jasmonic acids causes epimerization by an acid, an alkali, or heat, and thus tends to be a stable trans type rather than an unstable cis type. In equilibrium experiments with natural or synthetic jasmonic acid, 90% trans type and cis type
Exists in 10% condition. Generally, the cis type is more active, but the jasmonic acids that can be used in the present invention include all stereoisomeric compounds represented by the above formulas (I) (II) (III) and their stereoisomeric compounds. Including mixtures.

Jasmonic acids have a concentration of 0.
It is necessary to adjust the concentration to 01 to 1000 μM, and it is particularly preferable to adjust the concentration of jasmonic acids to the range of 0.1 to 500 μM. It is effective to add the jasmonic acids during the growth phase or stationary phase of the cultured cells, and among these, it is particularly preferable to add the jasmonic acids during the transition from the growth phase to the stationary phase. The timing of the treatment for increasing the production of endogenous jasmonic acids is also the same. For example, when cells are transplanted every 21 days, the 7th to 16th days are the appropriate period of the treatment for adding jasmonic acids or increasing the production amount of endogenous jasmonic acids. The addition of the jasmonic acids and the treatment for increasing the production amount of the endogenous jasmonic acids may be performed once or may be performed in multiple times.

Further, the present invention controls the oxygen concentration in the gas phase in the incubator, which is disclosed in Japanese Patent Application No. 6-146826, under the condition that the oxygen concentration in the incubator is lower than the oxygen concentration in the atmosphere from the beginning of the culture. It is used in combination with a method of culturing or controlling the concentration of dissolved oxygen in a fluid medium in contact with tissues and / or cells at a temperature lower than the saturated dissolved oxygen concentration at the temperature from the initial stage of culture. be able to.

Here, the early stage of culture refers to the start of culture or the 7th day after the start of culture, and the oxygen concentration in the gas phase in the incubator or the dissolution in a fluid medium contacting tissues and / or cells. It is preferable to control the oxygen concentration from the start of culture. The period of control may be controlled to the conditions throughout the entire culture period, or may be controlled only for a part of the entire culture period, and is not particularly limited, but during the entire culture period. It is preferable to control for at least 3 days.

The oxygen concentration in the gas phase in the incubator is 4 to
It is necessary to control to 15%, and it is particularly preferable to control to 6 to 12%. Further, the dissolved oxygen concentration in the fluid medium is 1 of the saturated dissolved oxygen concentration value at that temperature.
To 75%, especially 10 to
It is preferable to control to 75%.

The present invention is also directed to Japanese Patent Application No. 5-284893.
No. 6-104213, the method of dividing cells into a plurality of layers according to the difference in specific gravity and culturing the cells contained in at least one layer can be used together. As a method for separating cells by specific gravity, generally known is a method in which a density gradient is created using a medium for centrifugation, the cells are overlaid, and then the cells are centrifuged.

Ficoll and Percol are used as the medium for centrifugation.
l (both manufactured by Pharmacia LKB Biotechnology), sucrose, cesium chloride and the like are used. There is no particular limitation on the number of layers forming the density gradient. The specific gravity difference between the layers is not particularly limited, and the specific gravity differences may be the same or different. Therefore, the definition of the density gradient includes the case where the gradient changes continuously (the number of layers forming the density gradient is infinite, and the difference in specific gravity between layers is close to 0).

By thus forming a density gradient and superimposing the cells on one another and centrifuging, the cells can be divided into a plurality of layers according to the difference in specific gravity. The specific gravity of the layer to be created is
It is usually in the range of 1.00 to 1.20 g / ml, preferably 1.03 to 1.11 g / ml. As the layer to be cultured, at least one layer may be selected, or all layers may be selected and cultured.

When a plurality of layers are selected and cultured, the plurality of layers can be individually cultured, but two or more layers of the selected plurality of layers are mixed and cultured. You can also Cultured cells with high taxane-type diterpene-producing ability are usually obtained by culturing cells contained in a layer having a specific gravity of 1.07 or less, but may vary depending on the cells to be cultivated and the conditions of the culture, and are not necessarily limited to this range. It is not something that will be done. Further, it is recognized that the cells in the layer having a high specific gravity tend to have a higher taxane-type diterpene content simply by fractionating the cells by the difference in the specific gravity. Therefore, in order to more reliably obtain the taxane-type diterpene high-producing cultured cells, after culturing the cells of all the fractionated layers for a certain period of time, the taxane-type diterpene concentration contained in the cells of each layer is measured, and Among these, it is desirable to select a layer containing highly taxane-type diterpene-producing cells.

Also, by preparing a centrifuge medium having a specific gravity of 1.07 g / ml and centrifuging by the above-mentioned method, the cultured cells are divided into a plurality of layers due to the difference in specific gravity. Can be divided.

The present invention also relates to Japanese Patent Application No. 6-201150.
And the method of culturing in the presence of at least one selected from compounds containing heavy metals, complex ions containing heavy metal ions, and heavy metal ions described in the specification. Here, as the heavy metal, it is preferable to use a copper group represented by silver, an iron group represented by cobalt, and a compound containing the heavy metal, a complex ion containing the heavy metal, or a form of the metal ion. It is preferable to use. Further, the present invention may be used in combination with the method of culturing in the presence of amines described in Japanese Patent Application No. 6-201151. Here, as amines,
Polyamines, specifically putrescine, spermidine, spermine, ethylenediamine, N, N-diethyl-1,3
It is preferable to use at least one selected from the group consisting of propanediamine, diethylenetriamine, and salts of these compounds.

It is also possible to combine the method according to the present invention with some or all of the methods described in the above-mentioned prior patents.

The taxane-type diterpene can be separated from the cultured tissue or cultured cells obtained as described above by extraction with an organic solvent such as methanol. The following method is mentioned as a preferable example of the tissue culture of the present invention.

First, plant pieces of plants belonging to the genus Taxus, for example, plant pieces collected from roots, growing points, leaves, stems, seeds, etc. are sterilized and then placed on Woody Plant Medium solid medium solidified with gellan gum. Place it on the floor and at 10-35 ° C for 14-
After about 60 days, a part of the tissue piece is turned into callus. When the callus thus obtained is subcultured, the growth rate gradually increases and a stable callus is obtained. The term "stabilized callus" as used herein refers to one in which a part of the callus does not differentiate into shoots or roots during culture and retains the state of the callus and has a uniform cell growth rate.

The stabilized callus is transferred to a liquid medium suitable for growth, for example, a liquid medium of Woody Plant Medium and allowed to grow. The growth rate is further increased in the liquid medium. In the present invention, the stabilized callus or cells constituting the callus is cultured in a solid medium or liquid medium containing cyclic polysaccharide. The cyclic polysaccharide may be added to the medium from the beginning of the culture, or may be added to the medium during the culture, and the addition timing is not particularly limited.

The culture temperature in the tissue culture of the present invention is preferably about 10 to about 35 ° C., particularly about 23 to 28 ° C., because the growth rate is high. The culture period is 14 ~
42 days is preferred.

When a liquid medium is used in the culture method of the present invention, the cultured cells are separated from the medium by a method such as decantation or filtration after the completion of the culture, and the desired taxane-type diterpene is isolated from the cultured cells and / or the medium. Can be separated by a method such as extraction with an organic solvent.

[0078]

EXAMPLES The present invention will be described in more detail below with reference to examples and comparative examples, but the scope of the present invention is not limited to these examples.

Example 1 Woody plant medium solid medium (gellan gum 0.25% by weight) supplemented with naphthalene acetic acid to a concentration of 10 ⁻⁵ M was prepared in advance.
A part of the stem of Taxus baccata LINN, which had been sterilized with a 2% antiformin solution or a 70% ethanol solution, was placed on a bed and cultivated at 25 ° C. in the dark to obtain Taxus callus. Next, 0.1g of this callus (fresh weight)
Was transferred to a φ18 mm well containing 1 ml of liquid Woody Plant Medium to which the above components were added at the same concentration, and swirling culture (amplitude 25 mm, 120 rpm) on a rotary shaker.
Then, it was planted every 28 days to accelerate the growth rate of the callus.

1 g (fresh weight) of the cultured cells thus obtained was transferred to an Erlenmeyer flask containing 20 ml of Liquid Woody Plant Medium to which the above components were added at the same concentration, and cultured by shaking at 25 ° C. for 14 days. On the 14th day of culture, β-cyclodextrin was added to a final concentration of 0.01 mM,
It was further cultured for 7 days.

After the completion of the culture, the Taxus vulgaris cultured cells were collected by filtration, freeze-dried, and the dry weight thereof was measured to determine the growth weight of the cultured cells per liter of the liquid medium. The taxane-type diterpene was extracted from the obtained dry callus with methanol and the like, and the taxane-type diterpene yield was measured by comparative quantification with standard products taxol, cephalomannine and baccatin III using high performance liquid chromatography. Table 1 shows the results.

[Comparative Example 1] The same operation as in Example 1 was carried out except that β-cyclodextrin was not added. Table 1 shows the results.

[Example 2] The same operation as in Example 1 was carried out except that the final concentration of β-cyclodextrin added was 0.1 mM. Table 1 shows the results.

Example 3 The procedure of Example 1 was repeated, except that the final concentration of β-cyclodextrin added was 1 mM. Table 1 shows the results.

Example 4 The same operation as in Example 1 was carried out except that the final concentration of β-cyclodextrin added was 10 mM. Table 1 shows the results.

Example 5 The same procedure as in Example 1 was repeated except that 6-O-α-D-glucosyl-β-cyclodextrin was added in place of β-cyclodextrin so that the final concentration was 50 mM. It operated like the example. Table 1 shows the results.

[Examples 6 to 10] In Examples 1 to 5, jasmonic acid methyl ester was added at a final concentration of
The same operation as in the example was carried out except that 100 μM was added. The results are shown in Table 2.

Comparative Example 2 The same operation as in Example 1 was carried out except that jasmonic acid methyl ester was further added so that the final concentration thereof was 100 μM. The results are shown in Table 2.

Example 11 The same procedure as in Example 9 was carried out except that α-cyclodextrin was added in place of β-cyclodextrin so that the final concentration was 10 mM. Table 3 shows the results.

Example 12 The same operation as in Example 9 was carried out except that γ-cyclodextrin was added in place of β-cyclodextrin so that the final concentration was 10 mM. Table 3 shows the results.

Example 13 In Example 9, instead of β-cyclodextrin, cyclofructan (a compound having seven fructose bound thereto) was used at a final concentration of 10 mM.
The same operation as in the example was carried out except that the addition was performed so that Table 3 shows the results.

[0092]

[Table 1]

[0093]

[Table 2]

[0094]

[Table 3]

[0095]

According to the present invention, a large amount of taxane-type diterpenes can be easily obtained by tissue-culturing a taxane-type diterpene-producing plant using a tissue culture medium containing a cyclic polysaccharide.

Claims (11)

[Claims] 1. A taxane-type diterpene is recovered from a tissue culture medium of a plant that produces a taxane-type diterpene by culturing the tissue and / or the cultured tissue in a tissue culture medium containing a cyclic polysaccharide. And a method for producing a taxane-type diterpene. 2. The taxane-type diterpene is taxol,
10-deacetyltaxol, 7-epitaxol, baccatin III, 10-deacetylbaccatin III, 7-epibaccatin III, cephalomannine, 10-deacetylcephalomanin, 7-epicephalomanine, taxakifine and its analogs, taxane The method for producing a taxane-type diterpene according to claim 1, which is at least one selected from the group consisting of 1a and its analogs, xylosyl cephalomannine, and xylosyl taxol. 3. The method for producing a taxane-type diterpene according to claim 1, wherein the cyclic polysaccharide is α-cyclodextrin. 4. The method for producing a taxane-type diterpene according to claim 1, wherein the cyclic polysaccharide is β-cyclodextrin. 5. The method for producing a taxane-type diterpene according to claim 1, wherein the cyclic polysaccharide is γ-cyclodextrin. 6. The method for producing a taxane-type diterpene according to claim 1, wherein the cyclic polysaccharide is cyclofructan. 7. The method for producing a taxane-type diterpene according to claim 1, wherein the plant producing the taxane-type diterpene is a Taxus genus plant. 8. The concentration of cyclic polysaccharide in the tissue culture medium is 0.
It is 01-50 mM, The manufacturing method of the taxane-type diterpene of Claim 1 characterized by the above-mentioned. 9. A taxane-type diterpene-producing plant tissue and / or cell is prepared from the following general formula (I): [Wherein, R ⁰ is the following formula: — (CH ₂ ) _n —CO—R ⁷ {wherein R ⁷ is a hydroxyl group, OM (where M is an alkali metal atom, an alkaline earth metal atom or NH ₄ ) Represents),
NR ^8a R ^8b (wherein R ^8a and R ^8b each independently represent a hydrogen atom, an acyl group having 1 to 6 carbon atoms, an alkyl group having 1 to 6 carbon atoms, or an amino acid residue), OR ⁹ (Here, R ⁹ represents an alkyl group having 1 to 6 carbon atoms or a carbohydrate residue.) Or an alkyl group having 1 to 6 carbon atoms, n
Represents an integer of 1 to 7. } Represents a group represented by; R ^1a ,
R ^1b , R ^1c , R ^1d , R ^1e and R ^1f are each a hydrogen atom, a hydroxyl group, a C 1-6 alkyl group, or a C 1
Represents an alkoxy group of 6; R ² , R ³ , R ⁴ , R ⁵ and R ^6a
Each represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; the side chain composed of C ¹ -C ² -C ³ -C ⁴ -C ⁵ -C ⁶ is
It may contain one or more double bonds; R ^6b
Represents a hydroxyl group or an -O-carbohydrate residue; the 5-membered ring may form a double bond between adjacent ring member carbon atoms. ] It culture | cultivates in presence of jasmonic acid shown by these, The manufacturing method of the taxane-type diterpene of Claim 1 characterized by the above-mentioned. 10. A taxane-type diterpene-producing plant tissue and / or cell is prepared from the following general formula (II): [Wherein, R ⁰ is the following formula: — (CH ₂ ) _n —CO—R ⁷ {wherein R ⁷ is a hydroxyl group, OM (where M is an alkali metal atom, an alkaline earth metal atom or NH ₄ ) Represents),
NR ^8a R ^8b (wherein R ^8a and R ^8b each independently represent a hydrogen atom, an acyl group having 1 to 6 carbon atoms, an alkyl group having 1 to 6 carbon atoms, or an amino acid residue), OR ⁹ (Here, R ⁹ represents an alkyl group having 1 to 6 carbon atoms or a carbohydrate residue.) Or an alkyl group having 1 to 6 carbon atoms, n
Represents an integer of 1 to 7. } Represents a group represented by; R ^1a ,
R ^1b , R ^1c , R ^1d , R ^1e and R ^1f are each a hydrogen atom, a hydroxyl group, an alkyl group having 1 to 6 carbon atoms, or 1 carbon atom.
Represents a 6 alkoxy ^{group; R 2, R 3, R} 4, R 5 and R
⁶ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms respectively; the side chain composed of C ¹ -C ² -C ³ -C ⁴ -C ⁵ -C ⁶ has one or two or more double chains. May include a bond;
The 5-membered ring may form a double bond between adjacent ring member carbon atoms. ] It culture | cultivates in presence of jasmonic acid shown by these, The manufacturing method of the taxane-type diterpene of Claim 1 characterized by the above-mentioned. 11. A plant tissue and / or cell producing a taxane-type diterpene is prepared by the following general formula (III): [Wherein, R ⁰ is the following formula: — (CH ₂ ) _n —CO—R ⁷ {wherein R ⁷ is a hydroxyl group, OM (where M is an alkali metal atom, an alkaline earth metal atom or NH ₄ ) Represents),
NR ^8a R ^8b (wherein R ^8a and R ^8b each independently represent a hydrogen atom, an acyl group having 1 to 6 carbon atoms, an alkyl group having 1 to 6 carbon atoms, or an amino acid residue), OR ⁹ (Here, R ⁹ represents an alkyl group having 1 to 6 carbon atoms or a carbohydrate residue.) Or an alkyl group having 1 to 6 carbon atoms, n
Represents an integer of 1 to 7. } Represents a group represented by; R ^1a ,
R ^1b , R ^1c , R ^1d , R ^1e and R ^1f are each a hydrogen atom, a hydroxyl group, an alkyl group having 1 to 6 carbon atoms, or 1 carbon atom.
Represents a 6 alkoxy ^{group; R 2, R 3, R} 4, R 5 and R
⁶ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms respectively; the side chain composed of C ¹ -C ² -C ³ -C ⁴ -C ⁵ -C ⁶ has one or two or more double chains. May include a bond;
The 5-membered ring may form a double bond between adjacent ring member carbon atoms. ] It culture | cultivates in presence of jasmonic acid shown by these, The manufacturing method of the taxane-type diterpene of Claim 1 characterized by the above-mentioned.