JP3144947B2 - Method for producing taxane-type diterpene - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

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 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, a plant of the genus Taxus. , Have complex ester groups associated with activity. It has been reported that taxol is present in all parts of the yew tree plant, and its content is highest in bark.
Currently, taxol is produced from natural or cultivated plants, but yew plants are 20 c
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 if the bark is peeled off. If taxol or a taxane-type diterpene such as baccatin III, which is a precursor of taxol, can be synthesized using tissue culture, a large amount of taxol can be easily obtained without cutting down trees. It is.

A method for producing taxol using cultured cells of a plant has been described in detail in the text
s brevifolia NUTT) A method for producing taxol using cultured cells has been patented (US Patent: 5019504). And
Although taxol production is described as 1-3 mg / l, it is insufficient for industrial production.

On the other hand, as a prior art of the taxol production method, baccatin III (bac
catin III) is a patent from Holton et al. (US Paten
t: 5015744]. Since the tissue culture method does not require a large amount of plant tissue, it is further advantageous for taxol production.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for producing taxane-type diterpenes easily and in large quantities by plant tissue culture.

[0006]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that when tissue culture is performed by adding lithium ions to the culture cells of cultured cells or cultured tissues of taxane-type diterpene-producing plants, The present inventors have found that the productivity of taxane-type diterpenes in the product is improved, and completed the present invention.

That is, the present invention is characterized in that a cultured cell or tissue of a plant that produces a taxane-type diterpene is tissue-cultured in a tissue culture medium containing lithium ions, and the taxane-type diterpene is recovered from the tissue culture. This is a method for producing a taxane-type diterpene.

Hereinafter, the present invention will be described in detail. The taxane-type diterpene to be subjected to the production method of the present invention,
There is no particular limitation as long as it is a diterpene having a taxane skeleton, and examples thereof include taxol, baccatin III, cephalomannine, and 10-deacetylbaccatin.

Examples of plants producing taxane-type diterpenes used for tissue culture in the method of the present invention include, for example, yew (Taxus baccata LINN) and yew (T. cu.
spidata SIEB.et ZUCC), Caraboku (T. cuspidata SIE)
B.et ZUCC var.nana REHDER), Taiheiyoichii (T.
brevifolia NUTT), Canada yew (T. canadiensis MAR)
And Yew genus plants such as SH).

[0010] The tissue culture of the plant can be performed by a conventionally known method except that lithium ions are present in the tissue culture medium.

Examples of the mode in which lithium ions are present in the tissue culture medium include adding lithium chloride, lithium acetate, lithium bromide, lithium oxalate, and the like to the tissue culture medium. It is necessary that the concentration of lithium ions be 0.01 mM or more in the culture medium.
It is preferable to adjust the concentration in the range of mM to 50 mM.

[0012] Heretofore, there have been no reports on the use of lithium ions as an additive to a culture medium in tissue culture of taxane-type diterpene-producing plants, and the use of such lithium ions unexpectedly increases the amount of taxane-type diterpene produced. I found out.

The medium used in the present invention contains ordinary medium components in addition to the lithium ions. In general, an inorganic component and a carbon source are used as such components, and plant hormones and vitamins can be added thereto, and further, 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, and a mixture of two or more of these sugar sources at an appropriate ratio can be used.

Examples of the inorganic components include phosphorus, nitrogen, potassium, calcium, magnesium, sulfur, iron, manganese, zinc, boron, copper, molybdenum, chlorine, sodium, iodine, cobalt and the like. 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, and cobalt chloride.

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

The vitamins include, for example, biotin, thiamine (vitamin B1), pyridoxine (vitamin B
6), pantothenic acid, inositol, nicotinic acid and the like are used.

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

As the medium used for the tissue culture of the present invention, conventionally known mediums used for tissue culture of plants, for example, Murashige Skoog (1962) [Murashige
&Skoog's medium, Rinsmeier Skoog (1965)
[Linsmaier Skoog] Medium, Woody Plant
Medium (1981) Medium of Woody Plant Medium,
A medium prepared by adding the above-mentioned plant hormones to B-5 medium of Gamborg, M-9 medium of Mitsui, etc., and further adding the above-mentioned carbon source, vitamins, amino acids and the like as needed. Among them, a medium prepared using a Woody Plant Medium medium is particularly preferable.

In the present invention, any of a liquid medium and a solid medium usually containing 0.1 to 1% of agar, gellan gum or the like can be used, but a liquid medium is usually preferable. In the tissue culture of the present invention, the roots, growth points, leaves, stems, seeds, pollens, anthers, sepals and other tissue fragments or cells of the above-mentioned plants, or these are cultured in the above-mentioned medium or other conventional medium. The resulting cultured cells or cultured tissues can be used. The cultured tissue includes shoots and adventitious roots obtained by differentiating from the tissue or callus.

When these tissues or cells are cultured in a medium containing lithium ions according to the present invention,
A cultured tissue or cultured cell with high productivity of the taxane-type diterpene can be obtained as compared with the case without addition. The taxane-type diterpene can be separated from the cultured tissue or cells by extraction with an organic solvent such as methanol.

A preferred example of the tissue culture of the present invention includes the following method. First, a plant of a plant belonging to the genus Taxus, for example, roots, growth points, leaves, stems, after sterilization treatment of plant pieces collected from seeds and the like, placed on a solid medium of woody plant medium solidified with gellan gum,
After about 14 to 60 days have passed at 10 to 35 ° C, a portion of the tissue piece is callusized. When the callus thus obtained is subcultured, the growth rate gradually increases and a stabilized callus is obtained. Here, the term "stabilized callus" refers to a callus having a property that a portion 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. In the culture until a stabilized callus is obtained, it is preferable that lithium ions are not present in the medium from the viewpoint of preventing cell death.

The stabilized callus is transferred to a liquid medium suitable for growth, for example, Woody Plant Medium, and grown. The growth rate is further increased in the liquid medium. In the present invention, the stabilized callus or cells constituting the callus are cultured in a solid medium or a liquid medium containing lithium ions.

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

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

[0025]

The present invention will be described more specifically below with reference to examples and comparative examples. However, the technical scope of the present invention is not limited by these examples. (Example 1) Sterilize 2% antiformin solution or 70% ethanol solution or the like in advance in a woody plant medium solid medium (gellan gum 0.25% by weight) to which naphthalene acetic acid was added to a concentration of 10 ^-5 M. A part of the treated stem of Taxus baccata (Taxus baccata LINN) was placed on a bed and allowed to stand still at 25 ° C. in a dark place to obtain calliphorus callus. Next, 0.1 g of this callus (fresh weight) was transferred to a φ18 mm well containing 1 ml of a liquid medium of Woody Plant Medium to which the above-mentioned components were added at the same concentration, and cultivated in a rotary shaker (amplitude: 25 mm, 120 rpm). 28
Planting was continued every day to increase the growth rate of the callus.

The woody cells obtained by adding 0.1 g (fresh weight) of the cultured cells thus obtained to the above components at the same concentration, and further adding lithium chloride so that the final concentration of lithium ions becomes 0.01 mM. The medium was transferred to a φ18 mm well containing 1 ml of a liquid medium of Plant Medium and cultured with shaking at 25 ° C. for 28 days.

After completion of the culture, the cultured cells of the yew tree were collected by filtration, freeze-dried, and the dry weight 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 dried callus using methanol or the like, and compared with standard taxol, cephalomannine, and baccatin III by high performance liquid chromatography to determine the taxane-type diterpene yield. Table 1 shows the results.

(Comparative Example 1) The procedure of Example 1 was repeated, except that a medium to which lithium ions were not added was used. Table 1 shows the results. (Example 2) The same operation as in Example 1 was carried out except that the final concentration of the added lithium ion was 0.1 mM. Table 1 shows the results. (Example 3) The same operation as in Example 1 was performed except that the final concentration of the added lithium ion was 1 mM. Table 1 shows the results. (Example 4) The procedure of Example 1 was repeated, except that the final concentration of the added lithium ion was 10 mM. Table 1 shows the results. Example 5 The same operation as in Example 1 was performed except that the final concentration of the added lithium ion was 50 mM. Table 1 shows the results.

[0029]

[Table 1]

[0030]

According to the present invention, a large amount of taxol can be easily obtained by tissue culture of a taxane-type diterpene-producing plant.

Claims (2)

(57) [Claims] 1. Yew producing taxane type diterpene
A stable callus of a genus plant is tissue-cultured in a tissue culture medium containing 0.01 to 50 mM lithium ion, and a taxane-type diterpene is recovered from the obtained tissue culture. For producing a taxane-type diterpene. 2. The method according to claim 1, wherein the yew plant is yew .