JPH05219975A - Production of tabersonine derivative - Google Patents

Abstract

PURPOSE:To simply obtain the subject compound useful as an intermediate for Vincristine, etc., of medicines by subjecting a cell of a plant which produces an aspidsperma type indole alkaloid to tissue culture in a culture medium to which a specific compound is added. CONSTITUTION:A cultured cell or foliage organ cultured strain of a plant which produces an aspidsperma type indole alkaloid (e.g. Catharanthus roseus var. Little Bright Eye) is subjected to tissue culture in a tissue culture medium to which 2-oxoglutamic acid is addd and then the objective compound such as 16-hydroxytabersonine is collected from the culture mixture. 2-Oxoglutaric acid is preferably added in such an amount so that the concentration of culture medium may be 5-20 mM after 9 days from the time when culture is started. Culturing temperature is preferably 23-28 deg.C. A liquid medium prepared using Murashige & Skoog medium is preferably used as the cultue medium.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a tabersonine derivative which is considered as an intermediate of biosynthesis of vincristine and vinblastine, which is important as a therapeutic drug for acute leukemia, malignant lymphoma and the like.

[0002]

BACKGROUND OF THE INVENTION Tabersonin is a perennial plant of the genus Astragalus, Amsonia e.
lliptica Roem. et. Schult) and American sturgeon
It is an aspidosperma-type indole alkaloid that is abundant in the seeds of (A. tabernaemontana Walt). Vincristine and vinblastine, which are important as therapeutic agents for acute leukemia, malignant lymphoma, etc.
Is a dimer composed of bindrin and casalanthin as constituent units, and tabersonin is considered to be a biosynthetic intermediate of bindrin. Currently, the production of vincristine or vinblastine is collected from natural or cultivated plants, and a method utilizing cultured cells of plants is not known. Furthermore, there is no case in which vindoline production was observed from callus culture of Catharanthus roseus. If the above-mentioned tabelsonin derivative such as vindoline can be synthesized from tabersonin using tissue culture, it is advantageous because it can be used as a raw material for chemical synthesis of vincristine and vinblastine.

On the other hand, as a prior art for producing a tabersonin derivative by tissue culture of Catharanthus roseus, Japanese Patent Application Laid-Open No. 2-1339.
No. 0 discloses a method for obtaining a tabersonine derivative by adding tabersonin to the culture medium of Catharanthus roseus or the culture medium of foliage organs. However, JP-A-2-1339
The taversonin derivatives described in the examples of JP-A-0 are locnericin and locnerinin, and these substances are V.DE.
Report by LUCA et al. [J. Plant Physiol. 125, 147-156 (198
6)] It is different from the substance on the biosynthetic pathway from tabersononin to bindrin. 16-hydroxy tabersonine, 16-hydroxy tabersone
-Methoxytabersonin, 16-methoxy-2,3-dihydro-3-hydroxytabersonin, 4-desacetoxybindrin and deacetylbindrin,
No examples have so far been known for producing these substances.

[0004]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for conveniently producing a tabersonon derivative by culturing plant tissue.

[0005]

Means for Solving the Problems As a result of earnest research, the present inventors have added 2-oxoglutaric acid to a cultured cell of a plant producing an aspidosperma-type indole alkaloid or a tissue culture medium of a foliage organ culture strain. Then, it was found that when the tissue culture was carried out, the taversonin derivative was produced in the culture, and the present invention was completed.

That is, the method for producing a tabersonine derivative of the present invention is carried out by adding 2-oxoglutaric acid to the tissue culture medium of a plant culture cell or a foliage organ culture strain producing an aspidosperma type indole alkaloid. It is characterized in that the tabersonsonin derivative is collected from the culture. Examples of the taversonin derivative produced by tissue culture in the production method of the present invention include 16-hydroxytabersonin, 16-methoxytabersonin and the like.

Examples of plants producing aspidosperma-type indole alkaloids used for tissue culture in the production method of the present invention include asversoidal-type indole alkaloids such as tabersonon, 16-hydroxytabersonon, 16-methoxytabersonin, and vindoline. There is no particular limitation as long as it is a plant that produces, for example, Little Bright Eye variety of Catharanthus species (Catharanthus
roseus var. Little Bright Eye), Gee-don variety (Cr
oseus G. Don), Little Delica (C. roseus cv.
Little Delicata) etc .; Amsonia te of the genus Astragalus
Examples include bernaemontana and Walt.

According to the present invention, the tissue culture of the plant is 2-
It can be carried out by a conventionally usual means except that only oxoglutaric acid is added, or ascorbic acid and / or adenosine triphosphate (hereinafter referred to as “ATP”) is further added. 2-oxoglutaric acid has a concentration in the medium of 1 to 50 mM, preferably 5 to 20 mM, and ascorbic acid has a concentration in the medium of 0.05 to 2 mM, preferably 0.1.
˜1 mM, ATP is used in an amount such that the concentration in the medium becomes 0.01 to 5 mM, preferably 0.05 to 0.5 mM.

Conventionally, since 2-oxoglutarate was not used as a medium additive in tissue culture of indole alkaloid-producing plants, it could not be easily predicted that the production of a tabersononine derivative would be recognized by the use of 2-oxoglutarate. .. The medium used in the present invention is
In addition to the 2-oxoglutarate, ascorbic acid and ATP, it contains ordinary medium components. Generally, a carbon source and an inorganic component are used as such components, and plant hormones and vitamins can be added thereto, and amino acids can be added as necessary. 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, cobalt, and the like.
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 indoleacetic acid (IAA), naphthaleneacetic acid (NAA), p-chlorophenoxyisobutyric acid, and 2,4-dichlorophenoxyacetic acid (2,4-
Auxins such as D) and cytokinins such as kinetin, zeatin and dihydrozeatin are used. Examples of vitamins include biotin and thiamine (vitamin B
₁ ), pyridoxine (vitamin B ₆ ), pantothenic acid,
Inositol, nicotinic acid, etc. are used.

As the amino acids, for example, glycine, alanine, glutamine, cysteine and the like can be added. Generally, each of the above-mentioned components has a carbon source of about 1 to about 30 g / l, an inorganic component of about 0.1 μM to 100 mM, and a plant hormone of about 0.01 to about.
Approximately 10 μM, 0.1 each of vitamins and amino acids
~ Used at a concentration of about 100 mg / l.

The medium used for the tissue culture of the present invention is a conventionally known medium used for the tissue culture of plants, for example, Murashige Skoog (1962) [Murashig
e & Skoog] medium, Rinsmeier Scoog (1965
Year) [Linsmaier Skoog] Medium, White (1954)
[White] medium, Gamborg [Gamborg] B-5 medium, Mitsui M-9 medium and the like are added with the above-mentioned plant hormones, and if necessary, the above-mentioned carbon source, vitamins, amino acids and the like are added. Although the medium prepared by the above method can be exemplified, the medium prepared by using Murashige-Skoog's medium is particularly preferable.

In the present invention, any of a liquid medium and a solid medium containing 0.1 to 1% of agar, gellan gum or the like can be used, but the liquid medium is usually preferable. In the tissue culture of the present invention, the above-mentioned plant roots, growth points, leaves, stems, seeds, pollens, anthers, sepals and the like tissue pieces or cells, or these tissue culture in a medium according to the present invention or other conventional medium It is possible to use the cultured cells or cultured tissue obtained in this way or the crown gall tissue transformed by the introduction of a plasmid. When these tissues or cells are tissue-cultured using a medium to which 2-oxoglutarate is added according to the present invention, cultured tissues or cells containing a tabersonon derivative, which could not be obtained without addition, can be obtained. From this cultured tissue or cultured cells, the taversonin derivative can be separated by extraction with an organic solvent such as methanol.

A preferred example of the tissue culture of the present invention is the following method. First, plant bodies of plants belonging to the genus Catharanthus, for example, roots, growing points, leaves, stems, after sterilizing plant pieces collected from seeds, etc., placed on solid medium of Murashige-Skoog solidified with agar, 10 ~ 35 ° C 7 ~
After about 30 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 callus is transferred to a liquid medium suitable for growth, for example, Murashige-Skoog liquid medium, and allowed to grow. The growth rate is further increased in the liquid medium. The stabilized cultured cells are added to a liquid medium containing 2-oxoglutarate according to the present invention and cultured.

The addition timing of 2-oxoglutarate, ascorbic acid and ATP in the tissue culture of the present invention is as follows.
The effect of addition can be observed on the 7th day after the start of culture, but the 9th day or later is preferable. The culture temperature in the tissue culture of the present invention is usually about 10 to about 35 ° C, particularly about 23 to 28 ° C. If the temperature is lower than about 10 ° C, the growth rate is low, and if it is higher than about 35 ° C, the growth rate is also low. Light is not always necessary to perform the tissue culture of the present invention, but the irradiation of light does not prevent the production of the tabersononin derivative.

When a liquid medium is used in the production method of the present invention, the cultured tissue or cells are separated from the medium by a method such as decantation or filtration after the culture is completed, and the desired tabelsonin derivative is separated from the medium. It can be separated by a method such as extraction with a solvent.

[0018]

The present invention will be described in more detail with reference to Examples and Comparative Examples, but the scope of the present invention is not limited to these Examples.

[0019]

Example 1 Naphthalene acetic acid and kinetin, respectively
Murashige added to have a concentration of 1 ppm and 0.1 ppm
2% in advance on Scoog's agar (1% by weight agar)
Catharanthus roseus var. Littl (Catharanthus roseus var. Littl) sterilized with antiformin solution or 70% ethanol solution
e Bright, part of anther of Madagascar)
Cultivation was performed in the dark at 0 ° C. to obtain Catharanthus roseus callus.
Next, the callus was cultivated on a rotary shaker in a Murashige-Skoog liquid medium containing the above-mentioned two components at the same concentration, and cultivated on a rotary shaker (amplitude: 25 mm, 100 rpm). Rapid and stabilized periwinkle culture cells were obtained.

1.6 g (fresh weight) of the cultured cells thus obtained was added with 1 p of each of naphthalene acetic acid and kinetin.
Transfer to a 300-mL Erlenmeyer flask containing 45 ml of Murashige-Skoog liquid medium added at a concentration of pm and 0.1 ppm, and cultivate with shaking at 25 ° C. On the 9th day of culture, 2-oxoglutarate is brought to a concentration of 10 mM. The mixture was added as above, and the mixture was further cultured for 5 days with shaking at 25 ° C.

After completion of the culture, the periwinkle culture cells were collected by filtration, air-dried at 40 ° C. overnight, and the dry weight thereof was measured to determine the growth weight of the culture cells per 1 L of the liquid medium. The alkaloid yield was measured by extracting the taversonin derivative from the obtained dry callus with methanol or the like, and quantifying it by comparison with standard taversonine using high performance liquid chromatography. The results are shown in Table 1.

[Comparative Example 1] The same operation as in Example 1 was carried out except that a medium containing no 2-oxoglutarate was used. The results are shown in Table 1.

[0023]

[Example 2] The same operation as in Example 1 was repeated except that the medium to which ascorbic acid was further added was used. The results are shown in Table 1. [Comparative Example 2] The same operation as in Example 2 was carried out except that the medium containing only ascorbic acid was used. The results are shown in Table 1.

[0024]

[Example 3] The same operation as in Example 1 was carried out except that the medium in which ATP was further added was used. The results are shown in Table 1. [Comparative Example 3] The same operation as in Example 3 was carried out except that the medium containing only ATP was used. The results are shown in Table 1.

[0025]

[Example 4] The same operation as in Example 1 was carried out except that the medium in which ascorbic acid and ATP were further added was used. The results are shown in Table 1.

[0026]

[Example 5] In Example 4, the culture was started at the addition timing of 2-oxoglutarate, ascorbic acid and ATP.
The operation was performed in the same manner as in the example except that it was the first day. The results are shown in Table 1.

[0027]

[Table 1]

[0028]

[Example 6] The same operation as in Example 1 was carried out except that the tabersonsonin derivative was extracted from the obtained dry callus using methanol or the like and then fractionated by high performance liquid chromatography. As a result of mass spectrometric analysis and TLC analysis of the separated fractions, as shown in Table 2, it was found that tabersonein was hydroxylated 16-hydroxytabersonin.

Table 2 MS 352 (M ⁺ ) TLC Rf value (diethyl ether: n-hexane = 1: 1) 0.36 Rf value (ethyl acetate: ethyl alcohol = 3: 1) 0.94 Color development with ceric ammonium sulfate Blue

[0030]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to easily synthesize a taversonin derivative by tissue culture of an aspidosperma type indole alkaloid producing plant.
Since the tabersonin derivative is considered to be an intermediate of vincristine and vinblastine, which is important as a drug, the present invention makes it possible to easily provide a raw material for the chemical synthesis of vincristine and vinblastine.

Claims (4)

[Claims] 1. 2-oxoglutaric acid is added to a tissue culture medium of a cultured cell of a plant or a foliage organ culture strain that produces an aspidosperma-type indole alkaloid, and tissue culture is performed, and a taversonin derivative is collected from the culture. A method for producing a tabersonine derivative, comprising: 2. The method according to claim 1, wherein the plant that produces the aspidosperma-type indole alkaloid is a periwinkle plant. 3. The method according to claim 1, which further comprises adding ascorbic acid and / or adenosine triphosphate to the medium and culturing. 4. The method according to claim 3, wherein the addition time of 2-oxoglutaric acid and ascorbic acid and / or adenosine triphosphate is 7 days or more after the start of culture.