JPH08198863A - Production of taxane-type diterpene - Google Patents

Abstract

PURPOSE: To easily obtain in large quantities the subject compound including taxol useful as a medicine for ovary cancer, breast cancer, etc., by treating a taxane-type diterpene-productive plant, before or after its harvest, with a taxane-type diterpene production promoter. CONSTITUTION: This compound is obtained by (A) treating a taxane-type diterpene-productive plant either grown naturally or cultivated (pref. Taxus baccata LINN) with a taxane-type diterpene production promoter, a jasmone compound of formula I (R<1a> , R<1b> , R<1c> , R<1d> , R<1e> and R<1f> are each H, OH, a 1-6C alkyl, etc.; R<2> , R<3> , R<4> , R<5> and R<6a> are each H or a 1-6C alkyl; R<6b> is OH, etc.; R<7> is OH, etc.; (n) is an integer of 1-7) (e.g. a compound of formula II), and after a lapse of specified period, harvesting part or the whole of the treated plant followed by extraction from the harvest and then purification; or (B) harvesting part or the whole of the above plant followed by treating it with the above promoter, and after storage for a specified period, by extraction from the treated harvest and then purification.

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]

2. Description of the Related Art 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 belongs to the Taxus genus Taxus. , Have 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 bark, but also in bark, its content is several hundred ppm per dry weight.
And low. Taxol is currently collected from natural or cultivated plants, but Taxus is 20 cm above the ground.
It is a slow-growing plant that takes 10 years or more to grow to the height of the plant, and the plant body withers when the bark is peeled off, so it is difficult to obtain a large amount of taxol.

At present, as an alternative method to the extraction from the bark, a method of chemically converting 10-deacetylbaccatin III obtained from leaves of Taxus baccata LINN into taxol (US Pat. No. 5,015,744). However, 10-deacetylbaccatin III in the leaf has been investigated.
Since the content is as low as 0.1%, further improvement of the content of 10-deacetylbaccatin III is desired for industrially carrying out this method. From such a situation, the present inventors have conducted diligent studies in order to increase the content of taxane-type diterpene in the natural or cultivated plant, and as a result, treated the plant with a taxane-type diterpene production promoter. By doing so, I found that the purpose was achieved.

[0004]

An object of the present invention is to obtain taxane-type diterpene and other useful taxane-type diterpenes from natural or cultivated taxane-type diterpene-producing plants. It is to provide a way to increase.

[0005]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that a taxane-type diterpene-producing plant is treated with a taxane-type diterpene production promoter before or after its harvest. Found that the content of taxane-type diterpenes in the plant was significantly increased,
The present invention has been completed.

That is, in obtaining the compound from a plant that produces a taxane-type diterpene, the following steps are performed: (1) The plant that naturally grows or is cultivated is treated with a taxane-type diterpene production promoter, After a period of time
The whole or a part of the treated plant is harvested, and the compound is extracted and purified from the obtained harvest (2) The whole or a part of the plant that naturally grows or is cultivated. After harvesting, the compound is treated with a taxane-type diterpene production promoter, stored for a certain period of time, and then extracted or purified of the compound from the treated harvest. It is a manufacturing method.

Hereinafter, the present invention will be described in detail. 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-epicepharomanin, baccatin VI, taxakifine and its analogs, taxane 1a and its analogs Body, xylosyl cephalomannine, xylosyl taxol, and baccatin III
And compounds in which various hydrocarbon groups are bonded to C-13 of 10-deacetylbaccatin III. Among these compounds, taxol, baccatin III and 10-deacetylbaccatin III are exemplified as particularly preferable target compounds.

Examples of plants producing taxane-type diterpenes used in the production method of the present invention include, for example, Taxus baccata LINN and Taxus (T. cuspidata SIE).
B.etZUCC), Carabok (T. cuspidata SIEB.et ZUCC v
ar. nana REHDER), T. brevifolia
NUTT), Canadian yew (T. canadiensis MARSH), Chinese yew (T. chinensis), Indian yew (T. wallichiana),
Taxus plants such as T. media can be mentioned. In the present invention, among these, Taihei Yew, Yew Yew and T.media are particularly preferable.

The present invention can be applied to natural or cultivated plants of the aforementioned plant species. In addition, when the plant is cultivated, according to the present invention, cultivation can be carried out by a conventionally known method except that the plant is treated with a taxane-type diterpene production promoter. Examples of the taxane-type diterpene production promoter used in the present invention include jasmonic acids, coronatine, fatty acids, and derivatives thereof. Examples of the jasmonic acids include those represented by the general formula (I):

[0010]

[Chemical 7]

[In the formula, 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 an alkoxy group having 1 to 6 carbon atoms. Representation; R
² , R ³ , R ⁴ , R ⁵ and R ^6a each represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; C ¹ -C ² -C ³
Side chain consisting of -C ⁴ -C ⁵ -C ⁶ may contain one or more double bonds; R ^6b is hydroxyl or -O
Represents a carbohydrate residue; R ⁷ is a hydroxyl group, OM (where
M is an alkali metal atom, an alkaline earth metal atom or NH
Represents ₄ . ), NR ^8a R ^8b (wherein R ^8a and R ^8b are
Each independently represents 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 (wherein 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; The ring may form a double bond between adjacent ring member carbon atoms. ] The compound shown by the general formula (II):

[0012]

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[In the formula, 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 an alkoxy group having 1 to 6 carbon atoms. Representation; R
² , R ³ , R ⁴ , R ⁵ and R ⁶ each represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; C ¹ -C ² -C ³
Side chain consisting of -C ⁴ -C ⁵ -C ⁶ may contain one or more double bonds; R ⁷ is a hydroxyl group, OM
(Here, M represents an alkali metal atom, an alkaline earth metal atom, or NH _4. ), NR ^8a R ^8b (wherein R ^8a and R ^8b are each independently a hydrogen atom, a carbon number of 1 to 6). An acyl group, an alkyl group having 1 to 6 carbon atoms or an amino acid residue), OR ⁹ (wherein R ⁹ represents an alkyl group having 1 to 6 carbon atoms or a carbohydrate residue) or 1 to carbon atoms 6 represents an alkyl group; n represents an integer of 1 to 7; the 5-membered ring may form a double bond between adjacent ring member carbon atoms. ] And the compound represented by the general formula (III):

[0014]

[Chemical 9]

[In the formula, 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 an alkoxy group having 1 to 6 carbon atoms. Representation; R
² , R ³ , R ⁴ , R ⁵ and R ⁶ each represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; C ¹ -C ² -C ³
Side chain consisting of -C ⁴ -C ⁵ -C ⁶ may contain one or more double bonds; R ⁷ is a hydroxyl group, OM
(Here, M represents an alkali metal atom, an alkaline earth metal atom, or NH _4. ), NR ^8a R ^8b (wherein R ^8a and R ^8b are each independently a hydrogen atom, a carbon number of 1 to 6). An acyl group, an alkyl group having 1 to 6 carbon atoms or an amino acid residue), OR ⁹ (wherein R ⁹ represents an alkyl group having 1 to 6 carbon atoms or a carbohydrate residue) or 1 to carbon atoms 6 represents an alkyl group; n represents an integer of 1 to 7; the 5-membered ring may form a double bond between adjacent ring member carbon atoms. ] The compound shown by these is mentioned.

In the above general formulas (I), (II) and (III),
And R^1a, R^1b, R^1c, R^1d, R ^1e, R^1f, R², R
³, R^Four, R^Five, R⁶, R^6a, R⁷, R^8a, R^8bOr R
⁹Examples of the alkyl group having 1 to 6 carbon atoms represented by
For example, methyl group, ethyl group, n-propyl group, isopropyl
Group, n-butyl group, isobutyl group, sec-butyl group, t-
Butyl group, n-pentyl group, n-hexyl group
It

In the above formulas (I), (II) and (III),
And R^1a, R^1b, R^1c, R^1d, R ^1eOr R^1fRepresented by
Examples of the alkoxy group having 1 to 6 carbon atoms include
Si group, ethoxy group, n-propoxy group, isopropoxy
Group, n-butoxy group, isobutoxy group, sec-butoxy group
Group, t-butoxy group, n-pentyloxy group, n-hexyl
Examples include a siloxy group. R⁷Is OM
And an alkali metal atom or alkaline earth represented by M
Examples of the metal atom include sodium, potassium and calcium.
Cium is mentioned.

In the case where R ⁷ is NR ^8a R ^8b , R
The acyl group having 1 to 6 carbon atoms represented by ^8a or R ^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. Can be mentioned.

In the case where R ⁷ is NR ^8a R ^8b , R
Examples of the amino acid residue represented by ^8a or R ^8b include an isoleucyl group, a tyrosyl group and a tryptophyll group.
In the case where R ⁷ is OR ⁹ , a carbohydrate residue represented by R ⁹ and R ^6b in the general formula (I) is —O—
When it is a carbohydrate residue, examples of the carbohydrate residue include a glucopyranosyl group. In the compounds represented by the general formulas (I), (II) and (III), the 5-membered ring may form a double bond between adjacent ring member carbon atoms. Specific examples of the compound represented by the general formula (I) include the compounds shown below. (Compound A)

[0020]

[Chemical 10]

(Compound B)

[0022]

[Chemical 11]

(Compound C)

[0024]

[Chemical 12]

(Compound D)

[0026]

[Chemical 13]

Specific examples of the compound represented by the general formula (II) include the compounds shown below. (Compound E)

[0028]

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(Compound F)

[0030]

[Chemical 15]

(Compound G)

[0032]

Embedded image

(Compound H)

[0034]

[Chemical 17]

Specific examples of the compound represented by the general formula (III) include the compounds shown below. (Compound I) R ^1a , R ^1b , R ^1c , R ^1d , R ^1e , R ^1f , R
^{2, R 3, R 4,} R 5, R 6: H C 3 and double bond formation between the C ⁴ R ^7: -OH or -OCH ₃ n: 1~3 (Compound J) R ^1a, R ^1b , R ^1c , R ^1d , R ^1e , R ^1f , R
^{2, R 3, R 4,} R 5, R 6: H R 7: -OH n: In the compound represented by one Formula (III), R ^1a, R
^A compound in which ^1b , R ^1c , R ^1d , R ^1e or R ^1f is a hydroxyl group,
Alternatively, specific examples of the compound in which a double bond is formed between adjacent ring member carbon atoms in the 5-membered ring include the compounds shown below. (Compound K)

[0036]

Embedded image

(Compound L)

[0038]

[Chemical 19]

(Compound M)

[0040]

Embedded image

(Compound N)

[0042]

[Chemical 21]

The compound represented by the above general formula (I), (II) or (III)
Preferred compounds include^1a, R^1b, R
^1c, R^1d, R^1e, R^1f, R², R³, R^Four, R^FiveAnd R
⁶Is a hydrogen atom, and R⁷Is a hydroxyl group or a methoxy group
C¹-C²-C³-C^Four-C^Five-C⁶Side chain consisting of
Does not contain a double bond, or C¹And C², C ²
And C³Or C³And C^FourCompounds containing double bonds between
You can The general formulas (I) and (I
Various types of jasmonic acids represented by I) or (III) are used.
There are body isomers (cis trans isomer, optical isomer)
However, even if each isomer is used alone,
May be used in.

Among the above jasmonic acids, R ^1a , R ^1b , and R ^1b in the above general formulas (I), (II) and (III)
R ^1c , R ^1d , R ^1e , R ^1f , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are hydrogen atoms, R ⁷ is a hydroxyl group or a methoxy group, n is 1 and C ³ Especially preferred are the compounds containing a double bond between C and C ⁴ , which are tuberonic acid or methyl tuberonate, cucurbic acid or methyl cucurbate, and jasmonic acid or methyl jasmonate.

These jasmonic acids are prepared synthetically or by extraction from plants (H. Yamane et a
l. Agric. Biol. Chem., 44 , 2857-2864 (1980)). Among the jasmonic acids, the compound represented by the formula (I) or (III) is more stable than the unstable cis type because the α-position of the 5-membered ring carbonyl group causes epimerization by acid, alkali or heat. It is easy to become a transformer type. In equilibrium experiments with natural or synthetic jasmonic acid, the trans form was 90
%, Cis type is present at 10%. Generally, the cis type is considered to have stronger activity, but the jasmonic acids used in the present invention include all stereoisomeric compounds represented by the above formula (I) or (III) and a mixture thereof. . The coronatine that can be used as the taxane-type diterpene production promoter is represented by the general formula (IV):

[0046]

[Chemical formula 22]

Or general formula (V):

[0048]

[Chemical formula 23]

[In the formula, R ¹⁰ is a hydroxyl group, OR ¹¹ (wherein R ¹¹ represents an alkyl group having 1 to 6 carbon atoms or a carbohydrate residue), OM ¹ (where M ¹ is Represents an alkali metal atom, an alkaline earth metal atom or NH ₄ ) or NR ^12a R ^12b (wherein R ^12a and R ^12b are each independently a hydrogen atom, an acyl group having 1 to 6 carbon atoms, or 1 carbon atom).
~ 6 alkyl group, amino acid residue or general formula (VI):

[0050]

[Chemical formula 24]

(Where R¹³Is a hydrogen atom, hydroxyl group, charcoal
Alkyl groups having 1 to 6 primes and alkoxy groups having 1 to 6 carbons
Or the following formula-CO-R¹⁶ (In the formula, R¹⁶Is a hydroxyl group, OM²(Where M²Is
Lucari metal atom, alkaline earth metal atom or NH_FourThe table
You. ), NR^17aR^17b(Where R^17aAnd R
^17bAre each independently a hydrogen atom or an acyl group having 1 to 6 carbon atoms.
Group, alkyl group having 1 to 6 carbon atoms or amino acid residue
You. ) Or OR¹⁸(Where R¹⁸Is a carbon number of 1 to 6
It represents a rukyl group or a carbohydrate residue. ) Represents. )
Represents a group represented by R ^14a, R^14b, R^15aAnd R^15bIs
Independently hydrogen atom, hydroxyl group, C1-6 al
It represents a kill group or an alkoxy group having 1 to 6 carbon atoms. )
Represents a group represented by ) Represents; R^19a, R^19b, R^20a,
R^20b, R^{twenty one}, R^{twenty two}, R^23a, R^23b, R^24a,
R^{twenty four b}, R^25a, R^25b, R²⁶And R²⁸Are each German
Vertically hydrogen atom, hydroxyl group, alkyl group having 1 to 6 carbon atoms, or
Represents an alkoxy group having 1 to 6 carbon atoms; R²⁷Is the hydrogen source
Child, alkyl group having 1 to 6 carbon atoms or carbohydrate residue
A 5-membered ring and a 6-membered ring in the formula are
A double bond may be formed. ] And other compounds
You can

In the above general formulas (IV), (V) and (VI), R ¹¹ , R ^12a , R ^12b , R ¹³ , R ^14a , R ^14b ,
R ^15a , R ^15b , R ^17a , R ^17b , R ¹⁸ , R ^19a , R
^19b , R ^20a , R ^20b , R ²¹ , R ²² , R ^23a , R ^23b ,
The ^{R 24a, R 24b, R 25a} , R 25 b, an alkyl group having 1 to 6 carbon atoms represented by R ^26, or R ^27, for example a methyl group, an ethyl group, n- propyl group, an isopropyl radical, n
-Butyl group, isobutyl group, sec-butyl group, t-butyl group, n-pentyl group, neopentyl group, t-pentyl group, n-hexyl group, isohexyl group and the like.

[0053] In the general formula (IV), (V) and ^{(VI), R 13, R} 14a, R 14b, R 15 a, R 15b,
R ^19a , R ^19b , R ^20a , R ^20b , R ²¹ , R ²² ,
Examples of the alkoxy group having 1 to 6 carbon atoms represented by R ^23a , R ^23b , R ^24a , R ^24b , R ^25a , R ^25b , or R ²⁶ include a methoxy group, an ethoxy group, an n-propoxy group, and isopropoxy group. Group, n-butoxy group, isobutoxy group, sec-butoxy group, t-butoxy group, n-pentyloxy group, neopentyloxy group, t-pentyloxy group, n-hexyloxy group, isohexyloxy group and the like. To be

When R ¹⁰ or R ¹⁶ is OM ¹ or OM ² , examples of the alkali metal atom or the alkaline earth metal atom represented by M ¹ or M ² include sodium, potassium and calcium. To be R ¹⁰ or R
^{In the case where 16} is NR ^12a NR ^12b or NR ^17a R ^17b , the acyl group having 1 to 6 carbon atoms represented by R ^12a , R ^12b , R ^17a or R ^17b may be straight chain or branched chain. Well, for example, formyl group, acetyl group, propionyl group, butyryl group, valeryl group, hexanoyl group, acryloyl group and the like can be mentioned.

R ¹⁰ or R ¹⁶ is NR ^12a NR ^12b or N
In the case of R ^17a R ^17b , R ^12a , R ^12b , R
Examples of the amino acid residue represented by ^17a or R ^17b include isoleucyl group, valyl group, glutamyl group, lysyl group and the like. When R ¹⁰ or R ¹⁶ is OR ¹¹ or OR ¹⁸ , examples of the carbohydrate residue represented by R ¹¹ or R ¹⁸ include a glucopyranosyl group.
In the general formula (V), examples of the carbohydrate residue represented by R ²⁷ include a glucopyranosyl group.
Preferred compounds of coronatines include the following formula (VIII):

[0056]

[Chemical 25]

Coronatine represented by: or the following formula (IX):

[0058]

[Chemical formula 26]

The coronafasic acid represented by

When the taxane-type diterpene production promoter is a fatty acid and / or its derivative, the main chain has 10 carbon atoms.
-22, especially even fatty acids are preferred. These fatty acids may be saturated or unsaturated fatty acids containing one or more double bonds in the carbon chain. In addition, one or two of the hydrogen atoms bonded to the carbon chain
One or more of them may be substituted with a hydrocarbon group having 1 to 6 carbon atoms, a hydroxyl group or an amino group. Further, the double bond contained in the unsaturated fatty acid may be cis type, trans type, or a mixture of both, but a fatty acid containing a cis type double bond is preferable.

Specific examples of the above fatty acids include capric acid, decenoic acid, lauric acid, dodecenoic acid, myristic acid, myristooleic acid, palmitic acid, palmitooleic acid, stearic acid, oleic acid, vaccenic acid. , Linear fatty acids such as linoleic acid, α-linolenic acid, γ-linolenic acid, tetraoctadecenoic acid, arachidic acid, arachidonic acid, eicosatetraenoic acid, eicosapentaenoic acid, behenic acid, docosahexaenoic acid, ricinoleic acid, etc. Examples thereof include branched fatty acids such as hydroxy fatty acid and 14-methylpalmitic acid. Of these, oleic acid, linoleic acid, linolenic acid and arachidonic acid are preferable, and α-linolenic acid is particularly preferable.

Among the substituents, examples of the hydrocarbon group having 1 to 6 carbon atoms include methyl group, ethyl group, propyl group, cyclopropyl group, butyl group, isobutyl group, pentyl group and hexyl group. Examples of the amino group include an amino group, a monomethylamino group and a dimethylamino group. In place of the fatty acid of the general formula ^{(VII): 29 -COR 30 (} VII) [ wherein R, R ²⁹ -CO represents an atomic group derived from the fatty acid; R ³⁰ is OR ³¹ (wherein , R ³¹ represents an alkyl group having 1 to 6 carbon atoms or a carbohydrate residue), OM ³ (wherein M ³ represents an alkali metal atom, an alkaline earth metal atom or NH ₄ ), or NR ^32a R ^32b (where R
^32a and R ^32b are each independently a hydrogen atom and a carbon number of 1.
Represents an alkyl group of ~ 6, an amino acid residue. ) Represents. ]
You may use the fatty acid derivative shown by.

In the above general formula (VII), R ³¹ , R ^32a
Or, as the alkyl group having 1 to 6 carbon atoms represented by R ^32b , for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, t-butyl group , N-pentyl group, isopentyl group,
Examples include neopentyl group, t-pentyl group, n-hexyl group and isohexyl group. When R ³⁰ is OR ³¹ , the carbohydrate residue represented by R ³¹ includes, for example, a glucopyranosyl group. When R ³⁰ is OM ³ , the alkali metal atom or alkaline earth metal atom represented by M ³ includes, for example, sodium, potassium, calcium and the like. R ³⁰ is NR ^32a R
^In the case of ^32b , examples of the amino acid residue represented by R ^32a or R ^32b include a glycyl group, a leucyl group, an isoleucyl group, a glutamyl group, a lysyl group, a phenylalanyl group, a tyrosyl group and a tryptophyll group. To be

In the present invention, each of the taxane-type diterpene production promoters may be used alone or in any combination of two or more. The treatment of the taxane-type diterpene production promoter may be carried out only once, or may be carried out in plural times. Furthermore, when the said process is implemented in multiple times, the process amount of the said production promoter can be changed suitably.

As a method for treating a target plant with a taxane-type diterpene production accelerator, when the accelerator is water-soluble, the aqueous solution is used as it is, or when the accelerator is insoluble in water. Examples of the method include a method of directly spraying an aqueous solvent or emulsion containing the above-mentioned accelerator onto a plant. here,
Examples of the water solvent include a solvent that is miscible with water or an aqueous solution containing a surfactant, and the accelerator is dissolved in the solution to form a uniform solution. In addition, as an emulsion, the accelerator is once dissolved in a solvent immiscible with a surfactant and / or water,
The thing which emulsified this in the aqueous solution is illustrated.

Examples of the water-miscible solvent which can be used as the water solvent include methanol and / or ethanol, and examples of the water-immiscible solvent which can be used in the emulsion are isopropyl alcohol, xylene, toluene,
Examples thereof include methylnaphthalene, cyclohexanone, isophorone, benzyl alcohol, N, N-dimethylformamide, N-methyl-2-pyrrolidone, diacetone alcohol, methyl isobutyl ketone and hexanol. Further, as the surfactant used in the water solvent and the emulsion, polyoxyethylene alkyl ether,
Nonionic surfactants such as polyoxyethylene alkylphenol ether, polyoxyethylene alkyl ester, polyoxyethylene alkylamine, sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, glycerin fatty acid ester, fatty acid salt, alkyl sulfate, alkylbenzene sulfone Phonate,
Anionic surfactants such as alkylnaphthalene sulphonate, dialkyl sulphosuccinate, alkyl phosphate, polyoxyethylene alkyl sulphate, cationic surfactants such as tetraalkylammonium, amphoteric ions such as fatialkyl betaine Examples of the surface active agent include:

When the above-mentioned taxane-type diterpene production promoter is volatile, a plant for treating the promoter raw material, a solution containing the raw material, or a support impregnated with the solution containing the raw material. It is also possible to place the treatment near the body and carry out the treatment by means of the vapor of the accelerator emitted from them. Further, the treatment for promoting the production of taxane-type diterpene in the present invention is carried out by plant science (Plant Scienc
e) It can also be carried out by injecting the treatment liquid containing the above-mentioned taxane-type diterpene production promoter into the trunk of the plant by a syringe or the like, as disclosed in 101: 115-124. .

There is no particular limitation on the timing of treatment with the taxane-type diterpene production promoter according to the present invention, but it is preferable to treat the taxane-type diterpene production promoter during a period of active metabolism in the plant. Specifically, from spring to autumn, just before the time when new stems and leaves grow and expand, that is, from the time when the dormancy of the shoots ends, the growth and expansion of the stems and leaves end, and the plant becomes dormant. It is desirable to perform the processing before entering. For example, in the case of a plant that grows or is cultivated naturally in a temperate to subarctic climatic zone such as north of central Japan, the period from March to November is an appropriate period for treatment.

When the target plant is treated with the taxane-type diterpene production promoter, the promoter acts on plant cells, and although there are differences depending on various conditions such as soil conditions, season / climate, and plant age, The content of taxane-type diterpenes in the leaves, stems, bark, roots, etc. of the plant begins to increase within a few weeks from the day. Therefore, the harvest of each organ containing a taxane-type diterpene, the treated taxane-type diterpene production promoter sufficiently exerts the effect, it is preferable to perform at a time when the content of the taxane-type diterpene in the organ reaches the maximum, soil Although it is difficult to specify a suitable harvest time depending on various conditions such as conditions, seasons / climate, and age of plants,
Usually, a period from several weeks after the treatment to several months after the treatment can be exemplified as an appropriate harvesting period. In addition, depending on the region, it is possible to extend the period from the treatment to the harvest to 10 months or more.

Furthermore, the taxane-type diterpene production promoter can be used as a post-harvesting agent after harvesting the whole plant or a part thereof. Phytochemistry Volume 37 No. 2 405-4
On page 08 (1994), it was reported that the contents of taxol and cephalomannine contained in the organs of T. media during the storage period after harvest of leaves and branches were increased. Thus, by carrying out the above-mentioned production promoter treatment during the period in which the plant is stored, it is possible to positively increase the taxane-type diterpene content.

As the post-harvest treatment method, the treatment method for the plant body before harvest as exemplified above can be partially modified and used. As a preferable treatment method, the harvested plant body is exemplified above. , An aqueous solution containing a taxane-type diterpene production accelerator, a method of immersing in a water solvent or emulsion for a certain time, an aqueous solution containing the taxane-type diterpene production accelerator, a method of spraying an aqueous solvent or emulsion to the harvested plant is exemplified. Further, when the production accelerator is volatile, a method of sending air containing the volatile production accelerator to the storage of the harvested product and sealing it for a certain period of time is exemplified. The harvested product thus treated can be used as an extraction material for taxane-type diterpenes after being stored for a certain period of time. The storage temperature at this time is 4 to 30.
The temperature and the storage period may be, for example, several weeks to several months.

The concentration of the taxane-type diterpene production-promoting agent in the treatment liquid that is sprayed on the target plant individual or immersed or sprayed on the harvested plant is
The concentration can be exemplified as 0.001 to 1000 μM, and the concentration range of 0.01 to 100 μM is particularly preferable. ,Also,
The treatment amount of the treatment liquid can be arbitrarily selected as long as no phytotoxicity is observed in the target plant, but if the target plant has a size of about 50 cm to 1 m, it is 1 to 1 per individual. 1000 ml can be illustrated. However,
The effect of the taxane-type diterpene production promoter according to the present invention is not limited to only the treated part, but is transmitted through the plant tissue and reaches the untreated part. Therefore, the treatment amount is less than 1 ml, and uniform treatment is possible. If you can't, you can still get the effect.

The taxane-type diterpene-containing plant organ obtained as described above can be extracted with an organic solvent such as methanol to extract the taxane-type diterpene. The extract thus obtained can be purified to high-purity taxol by known solvent extraction, column chromatography, preparative high performance liquid chromatography and the like.

The following method can be mentioned as a preferred example of the production method of the present invention. First, seedlings are obtained by germinating the seeds of a taxane-type diterpene-producing plant belonging to the genus Taxus or by cutting young shoots of the plant. Further grow the seedlings obtained in this way, to plants that have reached several tens of centimeters to several meters, during the period during which the metabolism of the plant from spring to autumn is active, the taxane-type diterpene production promoter exemplified above is added. Treat and continue growing for weeks to months. A part of the leaves and bark of the treated plant is collected, the content of taxane-type diterpenes is measured over time, and after the content is increased, leaves, branches, bark, roots, etc. are harvested,
Taxane-type diterpenes are separated and purified from these organs according to a conventional method.

[0075]

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] The yew (Taxus baccata LINN) obtained by cutting was planted on the open field and cultivated in the summer with shading to protect it from light. In late May, the third year after planting, individuals with a tree height of 50 cm to 1 m were prepared by dissolving methyl jasmonate in ethanol and diluting with distilled water to obtain a final concentration of methyl jasmonate of 1 to 100 μM (0.5.
% Ethanol solution) was uniformly sprayed at 100 ml per individual. Three months after this treatment, in late August, young leaves and stems developed in the year were harvested and dried under reduced pressure at room temperature for 48 hours. After crushing the obtained dried leaves and stems, the taxane-type diterpenes were extracted with methanol and partially purified according to a conventional method, and then the taxane was compared with the standard product by high performance liquid chromatography to compare the peak area. The type diterpene content was measured. The results are shown in Table 1.

[Example 2] The same procedure as in Example 1 was repeated except that the taxane-type diterpene production promoter was coronatine and the final concentration of coronatine in the treatment solution was 0.01 to 1 µM. did. The results are shown in Table 1.

[Example 3] In Example 1, the taxane-type diterpene production promoter was α-linolenic acid, and the final concentration of α-linolenic acid in the treatment liquid was 1 to 100 µM.
It was carried out in the same manner as in Example 1 except that the treatment liquid was ultrasonicated prior to spraying in order to promote emulsification in the treatment liquid. The results are shown in Table 1.

[Comparative Example 1] In Example 1, except that the treatment liquid to be sprayed was a 0.5% ethanol aqueous solution.
It carried out like Example 1. The results are shown in Table 1.

[Example 4] Taxus media'Hicksii 'obtained by germination of seeds was planted in the open field and cultivated in the summer in the dark with a gauze shade. 5th year from planting
At the end of the month, individuals whose tree height has reached 20-40 cm are dissolved in ethanol with methyl jasmonate and diluted with distilled water to give a final concentration of methyl jasmonate of 1-100 μM (0.
1% of the solution adjusted to 5% ethanol aqueous solution)
30 ml was uniformly sprayed per individual. Three months after this treatment, young leaves and stems developed in the year were harvested in late August, and the taxane-type diterpene content was measured in the same manner as in Example 1 below. Table 2 shows the results.

[Example 5] The procedure of Example 4 was repeated, except that the taxane-type diterpene production promoter was coronatine and the final concentration of coronatine in the treatment solution was 0.01 to 1 µM. did. Table 2 shows the results.

[Example 6] In Example 4, the taxane-type diterpene production promoter was α-linolenic acid, and the final concentration of α-linolenic acid in the treatment liquid was 1 to 100 µM.
It was carried out in the same manner as in Example 4, except that the treatment liquid was sonicated prior to spraying in order to promote emulsification in the treatment liquid. Table 2 shows the results.

[Comparative Example 2] In Example 4, except that the treatment liquid to be sprayed was a 0.5% aqueous ethanol solution.
It carried out like Example 4. Table 2 shows the results.

[Example 7] Taxus baccata LINN was cultivated in the same manner as in Example 1, and from the plant whose plant height reached 50 cm to 1 m at the end of August, the third year after planting, 10 cm from the tip. At that point, I cut a young shoot. 100g of this cutting and 100μ of methyl jasmonate
With 50 ml of 0.5% aqueous ethanol solution containing M,
It was placed in a closed container and kept at 25 ° C. in the dark. On the 7th day after starting the treatment, remove the methyl jasmonate solution from the container,
The inside of the container was replaced with air, and the container was kept at 25 ° C. in the dark for 14 days. The thus obtained cutting was dried under reduced pressure at room temperature for 48 hours, and the taxane-type diterpene content was measured in the same manner as in Example 1. The results are shown in Table 3.

Comparative Example 3 The procedure of Example 7 was repeated, except that the methyl jasmonate solution was replaced with a 0.5% aqueous ethanol solution. The results are shown in Table 3.

Example 8 In Example 7, instead of sealing the cutting with the methyl jasmonate solution, it was immersed in a 1 μM coronatine solution (0.5% ethanol aqueous solution) for 6 hours, and then at 21 ° C. for 21 days. It carried out like Example 7 except holding in the dark place. The results are shown in Table 3.

[Comparative Example 4] The procedure of Example 8 was repeated, except that a 0.5% aqueous ethanol solution was used instead of the coronatine solution. The results are shown in Table 3.

[Ninth Embodiment] Taxus in the same manner as in the fourth embodiment.
By cultivating media'Hicksii ', young shoots were cut 10 cm from the tip of individuals whose tree height reached 20-40 cm in late August, the third year after planting. 0.5 g containing 30 g of this cutting and 100 μM of methyl jasmonate
A 50% aqueous solution of ethanol was placed in a closed container and kept at 25 ° C. in the dark. Seven days after the start of the treatment, the methyl jasmonate solution was removed from the container, the inside of the container was replaced with air, and the container was kept at 25 ° C. in the dark for another 14 days.
The thus obtained cutting was dried under reduced pressure at room temperature for 48 hours, and the taxane-type diterpene content was measured in the same manner as in Example 1. The results are shown in Table 4.

Comparative Example 5 The procedure of Example 9 was repeated, except that a 0.5% aqueous ethanol solution was used instead of the methyl jasmonate solution. The results are shown in Table 4.

Example 10 In Example 9, instead of sealing the cutting with the methyl jasmonate solution, it was immersed in a 1 μM coronatine solution (0.5% aqueous ethanol solution) for 6 hours, and then at 21 ° C. for 21 days. The same procedure as in Example 9 was carried out except that the sample was kept in the dark. The results are shown in Table 4.

[Comparative Example 6] The procedure of Example 10 was repeated, except that a 0.5% aqueous ethanol solution was used instead of the coronatine solution. The results are shown in Table 4.

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[Table 1]

[0093]

[Table 2]

[0094]

[Table 3]

[0095]

[Table 4]

[0096]

INDUSTRIAL APPLICABILITY According to the present invention, a taxane-type diterpene-producing plant or a harvest thereof is treated with a taxane-type diterpene production promoter, whereby the content of the taxane-type diterpene contained in the plant or the harvest is increased. As a result, taxane type diterpenes can be easily obtained in large quantities.

Claims (17)

[Claims] 1. In obtaining the compound from a plant that produces a taxane-type diterpene, the following steps are carried out: (1) treating the plant that naturally grows or is cultivated with a taxane-type diterpene production promoter; After a period of time
The whole or a part of the treated plant is harvested, and the compound is extracted and purified from the obtained harvest (2) The whole or a part of the plant that naturally grows or is cultivated. After harvesting, the compound is treated with a taxane-type diterpene production promoter, stored for a certain period of time, and then extracted or purified of the compound from the treated harvest. Production method. 2. The method according to claim 1, wherein the taxane-type diterpene production promoter is jasmonic acid. 3. Jasmonic acids are represented by the general formula (I): [In the formula, R ^1a , R ^1b , R ^1c , R ^1d , R ^1e and R ^1f each represent a hydrogen atom, a hydroxyl group, an alkyl group having 1 to 6 carbon atoms or an alkoxy group having 1 to 6 carbon atoms; ² , R ³ , R
⁴ , R ⁵ and R ^6a are each a hydrogen atom or a carbon number of 1 to
It represents an alkyl ^{group; C 1 -C 2 -C 3 -C} 4 -C 5
Side chain consisting of -C ⁶ may contain one or more double bonds; R ^6b represents hydroxyl group or -O- carbohydrate residue; R ⁷ is a hydroxyl group, OM (wherein, M represents an alkali metal atom, an alkaline earth metal atom or NH _4. ), NR ^8a R ^8b (wherein R ^8a and R ^8b are each independently a hydrogen atom, an acyl group having 1 to 6 carbon atoms, a carbon atom). Number 1
~ 6 alkyl groups or amino acid residues. ), OR ⁹
(Wherein 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; A double bond may be formed between adjacent ring member carbon atoms. ] The method of Claim 2 which is a compound shown by these. 4. Jasmonic acids are represented by the general formula (II): [In the formula, R ^1a , R ^1b , R ^1c , R ^1d , R ^1e and R ^1f each represent a hydrogen atom, a hydroxyl group, an alkyl group having 1 to 6 carbon atoms or an alkoxy group having 1 to 6 carbon atoms; ² , R ³ , R
⁴ , R ⁵ and R ⁶ are each a hydrogen atom or a carbon number of 1 to
It represents an alkyl ^{group; C 1 -C 2 -C 3 -C} 4 -C 5
Side chain consisting of -C ⁶ has one or more may contain a double bond; R ⁷ is a hydroxyl group, OM (wherein, M is an alkali metal atom, an alkaline earth metal atom or 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 (wherein 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; May form a double bond between adjacent ring member carbon atoms. ] The method of Claim 2 which is a compound shown by these. 5. Jasmonic acids are represented by the general formula (III): [In the formula, R ^1a , R ^1b , R ^1c , R ^1d , R ^1e and R ^1f each represent a hydrogen atom, a hydroxyl group, an alkyl group having 1 to 6 carbon atoms or an alkoxy group having 1 to 6 carbon atoms; ² , R ³ , R
⁴ , R ⁵ and R ⁶ are each a hydrogen atom or a carbon number of 1 to
It represents an alkyl ^{group; C 1 -C 2 -C 3 -C} 4 -C 5
Side chain consisting of -C ⁶ has one or more may contain a double bond; R ⁷ is a hydroxyl group, OM (wherein, M is an alkali metal atom, an alkaline earth metal atom or 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 (wherein 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; May form a double bond between adjacent ring member carbon atoms. ] The method of Claim 2 which is a compound shown by these. 6. A taxane-type diterpene production promoter
General formula (IV):Or general formula (V):[Wherein, R^TenIs a hydroxyl group, OR¹¹(Where R¹¹Is charcoal
It represents an alkyl group having a prime number of 1 to 6 or a carbohydrate residue. ),
OM¹(Where M¹Is an alkali metal atom, alkali
Earth metal atom or NH_FourRepresents ) Or NR^12aR^12b
(Where R^12aAnd R^12bAre independent hydrogen
Child, acyl group having 1 to 6 carbon atoms, alkyl having 1 to 6 carbon atoms
Group, amino acid residue or general formula (VI):(Where R¹³Is a hydrogen atom, a hydroxyl group, a carbon number of 1 to 6
Alkyl group, alkoxy group having 1 to 6 carbon atoms or the following formula-CO-R¹⁶ (In the formula, R¹⁶Is a hydroxyl group, OM²(Where M²Is
Lucari metal atom, alkaline earth metal atom or NH_FourThe table
You. ), NR^17aR^17b(Where R^17aAnd R
^17bAre each independently a hydrogen atom or an acyl group having 1 to 6 carbon atoms.
Group, alkyl group having 1 to 6 carbon atoms or amino acid residue
You. ) Or OR¹⁸(Where R¹⁸Is a carbon number of 1 to 6
It represents a rukyl group or a carbohydrate residue. ) Represents. )
Represents a group represented by R ^14a, R^14b, R^15aAnd R^15bIs
Independently hydrogen atom, hydroxyl group, C1-6 al
It represents a kill group or an alkoxy group having 1 to 6 carbon atoms. )
Represents a group represented by ) Represents; R^19a, R^19b, R^20a,
R^20b, R^{twenty one}, R^{twenty two}, R^23a, R^23b, R^24a,
R^{twenty four b}, R^25a, R^25b, R²⁶And R²⁸Are each German
Vertically hydrogen atom, hydroxyl group, alkyl group having 1 to 6 carbon atoms, or
Represents an alkoxy group having 1 to 6 carbon atoms; R²⁷Is the hydrogen source
Child, alkyl group having 1 to 6 carbon atoms or carbohydrate residue
A 5-membered ring and a 6-membered ring in the formula are
A double bond may be formed. ] It is a compound shown by
The method of claim 1. 7. The method according to claim 1, wherein the taxane-type diterpene production promoter is a fatty acid and / or a derivative thereof. 8. The method according to claim 7, wherein the fatty acid is a straight chain saturated fatty acid or a straight chain unsaturated fatty acid having a main chain having 10 to 22 carbon atoms. 9. The fatty acid has at least one selected from a hydrocarbon group having 1 to 6 carbon atoms, a hydroxyl group and an amino group having a main chain having 10 to 22 carbon atoms as a substituent. The method according to claim 7, which is a fatty acid having one or more fatty acids. 10. A fatty acid whose main chain has 10 to 22 carbon atoms.
The method according to claim 7, which is an unsaturated fatty acid containing one or more cis double bonds in the main chain. 11. The method according to claim 7, wherein the fatty acid is at least one selected from oleic acid, linoleic acid, linolenic acid and arachidonic acid. 12. The fatty acid derivative has the general formula (VII): R ²⁹ —COR ³⁰ (VII) [wherein, R ²⁹ —CO represents an atomic group derived from the fatty acid according to claims 8 to 11; R ³⁰ Is OR ³¹ (wherein R ³¹ represents an alkyl group having 1 to 6 carbon atoms or a carbohydrate residue), OM ³ (wherein M ³ represents an alkali metal atom, an alkaline earth metal atom or NH ₄ ). .) Or NR ^32a
R ^32b (wherein R ^32a and R ^32b each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or an amino acid residue). ] The method of Claim 7 which is a fatty acid derivative shown by these. 13. The method according to claim 1, wherein the taxane-type diterpene production promoter is at least one selected from the compound group according to claims 2 to 12. 14. The method according to claim 1, wherein the taxane-type diterpene-producing plant is a Taxus plant. 15. The Taxus is Taxus (Taxus)
baccata LINN). 16. The method according to claim 14, wherein the Taxus plant is Taxus media. 17. The taxane-type diterpene is taxol,
10-deacetyltaxol, 7-epitaxol, baccatin III, 10-deacetylbaccatin III, 7-epibaccatin III, cephalomannine, 10-deacetylcephalomanin, 7-epicephalomanine, baccatin VI,
The method according to claim 1, wherein the method is at least one selected from taxagifin, taxane 1a, xylosyl cephalomannine, and xylosyl taxol.