JPH0655150B2 - Method for producing tropane alkaloids - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention is directed to scopolamine and scopolamine by tissue-culturing a tissue of a plant that metabolizes and produces tropane-based alkaloids such as Zubosia, Datura, Hasiridokoro, and Hyosus. And / or a method for producing tropane-based alkaloids such as hyoscyamine.

[Conventional technology]

Scopolamine is an antispasmodic agent, an analgesic agent, and a parasympathetic nerve palliative or abstinent drug, and hyoscyamine is a parasympathetic nerve nerfant or abolition drug. These compounds are produced by being extracted from natural plants, but since natural products are used as raw materials, their production is affected by the weather, and the harvest time is limited. I'm running. Therefore, a lot of researches for producing these compounds by plant tissue culture have been conducted both in Japan and abroad. In callus production, an example of production of chickpea callus by Yamada et al. Is known [Plant Cell Reports 1 , 101-10].
3 (1982)], the scopolamine content was 20 ppm, which was lower than that in natural plants. Yamada et al. Also found that adventitious roots obtained by tissue culture of Duboisia Leichhardtii F. Muell contained significant amounts of scopolamine and hyoscyamine (Pl.
Ant Cell Reports 3 , 186-188 (1984)], but the amount was not sufficient. Therefore, we examined various culture conditions of Zboiscia adventitious roots and improved the productivity of tropane alkaloids by setting the ratio of ammonium ion to nitrate ion in the medium to 0.2 or more and the dissolved oxygen concentration in the medium to 10 to 65 ppm. The present applicant has filed patent applications as Japanese Patent Application No. 60-143882 and Japanese Patent Application No. 60-143881 respectively, but from an industrial point of view, it is desired to further increase the productivity. .

[Problems to be solved by the invention]

Therefore, when aiming at industrial production of tropane-type alkaloids by such tissue culture, it was an important issue to further increase productivity.

In view of such circumstances, the present inventors have found that plants that produce tropane-type alkaloids such as Zubosia, Datsura, Hasiridokoro, and Hyosuka ([Natural Drug Encyclopedia] (edited by Takuo Okuda, published by Hirokawa Shoten, April 1986). 15th) 303
As a result of research on a method for efficiently culturing tissues and cells such as adventitious roots, the following facts were found.

[Means for solving problems]

In the case of culturing cells and tissues such as callus and adventitious roots in the conventional tissue culture method, amines such as tetramethylenediamine, polyalkylenepolyamine and lower alkylmonoamine are not added to the medium. Therefore, the present inventors tried to culture by adding this component to the medium, and cultured tissue such as adventitious roots obtained by culturing from the tissue of a plant that produces tropane-based alkaloids, tropane-based alkaloids of cells. The inventors have found that the content is improved, or the growth of tissues and cells such as adventitious roots is promoted, and as a result, the productivity of tropane-based alkaloids is improved, and the present invention has been completed.

That is, according to the method of the present invention, a tropane-type alkaloid characterized by producing a tropane-type alkaloid by tissue-culturing a tissue of a plant that produces a tropane-type alkaloid using a medium containing a predetermined amine. A production method is provided.

[Specific Description of the Invention]

In the present invention, the tissue culture is performed using a plant that produces a tropane-type alkaloid. Specific examples of the applicable plant include Duboisia myoporoides, Duboisia leichhardtii.
Zubosia plants such as Datura tatula, Datura arboe
a, Datura stramonium, etc., Scopolia jap
Examples thereof include plants of the genus Scoporia such as onica, plants of the genus Hyoscus such as Hyoscyamus niger, and plants of the family Solanaceae such as Atropa belladonna.

In the present invention, a medium containing amines is used for culturing the tissue of the plant to produce a tropane-type alkaloid. This will be described in detail below.

The medium used in the present invention is a medium containing amines, in which an inorganic component and a carbon source are essential components other than the above components, and plant hormones and vitamins are added to the medium, and further necessary. It is a medium to which amino acids are added according to.

The amines according to the present invention refer to the amines shown below. That is, the amines that can be used in the present invention include spermine [H ₂ N- (CH ₂ ) ₃ NH- (CH ₂ ) _4- NH- (CH ₂ ) _3- NH ₂ ], spermidine [H ₂ N- (CH ₂ ) ₃ NH- (CH ₂ ) _4- NH ₂ ], putrescine (H ₂ N- (CH
₂ ) _4- NH ₂ ] and other tetramethylenediamine and its derivatives, diaminopropane [H ₂ N- (CH ₂ ) ₃ -NH ₂ ] and other polyalkylene polyamines, and lower alkyls such as aminopropane, aminobutane and aminopentane. Substituted lower alkyl monoamines in which the terminal positions of monoamines and lower alkyl monoamines such as tyramine, tryptamine, histamine and the like are substituted with a hydroxyphenyl group, an indole residue or an imidazole residue.

In the present invention, as the use ratio of the amines, the concentration of the amines in the medium is usually 0.01 mM to 10 mM, preferably
It is used at a concentration of 0.1 mM to 5 mM. In the present invention, the amines may be used alone or in combination of two or more. When the tissue of the plant is cultured using the medium containing the amines as in the method of the present invention, the production amount of tropane-based alkaloids is increased as compared with the case where the medium containing no amines is used.

In the medium used in the present invention, for the medium components other than the amines described above, the inorganic components include nitrogen, phosphorus, potassium, sodium, calcium, magnesium, sulfur, iron, manganese, zinc, boron and molybdenum. Examples thereof include inorganic salts containing elements such as chlorine, iodine, and cobalt. Specifically, potassium nitrate, sodium nitrate, ammonium nitrate, ammonium chloride, potassium chloride, calcium chloride, potassium monohydrogen phosphate, potassium dihydrogen phosphate. , Magnesium sulfate, magnesium chloride, sodium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, copper sulfate, sodium molybdate, molybdenum trioxide, potassium iodide, zinc sulfate, boric acid, cobalt chloride, etc. It can be illustrated.

Examples of the carbon source of the medium include carbohydrates such as sucrose and derivatives thereof, organic acids such as fatty acids, and primary alcohols such as ethanol.

Examples of plant hormones in the medium include naphthalene acetic acid (NAA), indole acetic acid (IAA), p-chlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), and indolebutyric acid ( Examples thereof include auxins such as IBA) and their derivatives, and cytokinins such as benzyladenine (BA), kinetin and zeatin. In the present invention, it is generally desirable not to add cytokinins to the medium, but when added as necessary, cytokinins should be used at a low concentration of usually 10 ⁻⁷ M (0.02 mg /) or less. preferable.

The vitamins in the medium include biotin, thiamine (vitamin B ₁ ), pyridoxine (vitamin B ₆ ), pyridoxal, pyridoxamine, calcium pantothenate, ascorbic acid (vitamin C), inositol, nicotinic acid, nicotinic acid amide and ribobrabin ( vitamin
B ₂ ) etc. can be illustrated.

Examples of amino acids in the medium include glycine, alanine, glutamic acid, cysteine, phenylalanine and lysine.

The medium of the present invention usually contains about 0.1 μM of a pre-inorganic component.
To about 100 mM, about 1 g / to about 100 g of the carbon source
/, About 0.01μM to about 100μ of the plant hormones
It is preferable to use M, the vitamins in an amount of about 0.1 mg / to about 150 mg / and the amino acids in an amount of 0 to about 1000 mg /.

Specifically as the medium used for tissue culture of the Zboisia plant of the present invention, a medium used for tissue culture of conventionally known plants, for example, Murashige Skoog ('62) [Murashige & Skoog ] Medium, Rinsmeier Skoog (RM-1965) [Linsmaier & Skoog]
Medium, white ('63) [White] medium, Gamborg [Gamborg's B-5 medium, Mitsui's M-9 medium, Nitsch Nitsch's medium, etc., with the above-mentioned carbon source and plant hormones added. However, it is possible to exemplify a medium prepared by adding the above-mentioned vitamins and amino acids, if necessary, but in the present invention, among them, especially Nichi-Nichi, Rinsmeier-Skoog or Murashige-Skoog's medium is used. The medium prepared is preferred. Regarding the composition of the conventionally known medium described above, for example, Takeuchi, Nakajima, and Furuya “New Plant Tissue Culture” P386 to P391, Asakura Shoten, 1979.
Listed in the year.

The medium that can be used in the present invention is a liquid medium or a solid medium containing 0.5 to 1% of agar, gelatin or the like, but it is preferable to use a liquid medium in the present invention.

In the tissue culture of the present invention, a tissue piece of a plant that metabolizes and produces tropane-type alkaloids is tissue-cultured using the above-mentioned medium to obtain cultured cells or tissue containing tropane-type alkaloids.

In the present invention, adventitious roots are particularly preferable as the cultured tissue.

Specific examples of the tissue of the plant used for the tissue culture of the present invention include the root, leaf, stem, seed and flower bud of the plant, as well as the tissue culture according to the present invention or other conventional tissue culture methods. The culture range or culture tissue of the plant thus obtained can be exemplified. In the present invention, of these, it is particularly preferable to use adventitious roots obtained by tissue-cultivating plant tissue in advance, and perform tissue culture using the medium of the present invention. Are grown and cultured using the medium of the present invention to obtain adventitious roots containing a large amount of tropane-type alkaloids.

In the present invention, callus is usually induced from the above-mentioned tissue pieces or cells, and cultured cells or cultured tissues obtained by subculturing the callus are grown and cultured using the medium of the present invention to contain a large amount of tropane-type alkaloids. Culture to
In particular, it is preferable to use a tissue culture method such as obtaining adventitious roots.

In the present invention, when an adventitious root is used, it is also possible to infect a plant tissue piece with, for example, a hairy root pathogen (for example, Agrobacterium rhizogenes), and use a hair root that appears by this (for example, Japanese Patent Application No. 61-89975 relating to the present applicant). You can also use the method proposed in.

Specific examples of the tropane-based alkaloids obtained by the method of the present invention include scopolamine, hyoscyamine and acetyl compounds of these compounds, and among them, scopolamine and hyoscyamine are preferable.

In the present invention, the method for separating the alkaloids from the cultured cells containing the tropane-based alkaloids is used, for example, in the case of isolating and purifying these compounds from plants containing the tropane-based alkaloids described in, for example, the Pharmacy Method. The usual methods that have been used can be adopted.

〔The invention's effect〕

When the method for producing tropane-based alkaloids by tissue culture of the present invention is adopted, tropane-based alkaloids, particularly scopolamine and / or hyoscyamine, can be efficiently produced in large amounts as compared with the conventional method.

〔Example〕

Hereinafter, the method of the present invention will be described more specifically with reference to Examples.

Example 1 Leaves of Duboisia myporoides R.Br cultivated in our herb garden were washed, immersed in a 10% antiformin solution for 10 minutes, washed with sterilized water three times, and then cut into about 1 cm, and naphthalene acetic acid. And benzyladenine at 10 ^-5 M and 1 respectively
The plate is placed on a Rinsmeier-Skoog agar medium added to 0 ^-6 M and cultured at 25 ° C for 30 days. Adventitious roots generated at the same time as callus formation were cut out and indole butyric acid was removed.
The cells were transplanted to a liquid medium of Nichi-Nitsu, which was added to 10 ^-5 M, and subcultured for 2 years. A 100 ml Erlenmeyer flask containing 20 ml of the liquid medium of Nichi-Nitsu, to which 10 mg (dry weight) of adventitious root thus obtained was added so that spermidine was added to 1 mM and indolebutyric acid was added to 10 ^-5 M Then, the cells were cultured for 3 weeks with shaking. 190 mg (dry weight) of the obtained adventitious root was dried and then extracted with 50 ml of a basic chloroform-methanol solution. To this, 40 ml of 1N sulfuric acid was added to transfer the alkaloid layer to the sulfuric acid layer. further,
2 ml of ammonia water and 40 ml of chloroform were added to transfer the alkaloid to the chloroform layer, which was concentrated under reduced pressure.
The amount of alkaloids was analyzed by gas chromatography. Table 1 shows the amount of calcaloid produced in this case. The gas chromatograph analysis was performed under the following conditions.

Column: Silicone 0V-17 (1%) on Chromosorb W (Mes
h 80-100) 3 mm × 1 mm glass column Carrier gas: N ₂ Column temperature: 200 ° C. Examples 2-3 and Comparative Example 1 As Example 1 except that diamine pretocin or diaminopropane shown in Table 1 was added to the medium. Table 1 shows the results obtained in the same manner (Examples 2 to 3) and the results obtained in the same manner as Example 1 (Comparative Example 1) using a medium to which amines were not added.

Example 4 Zuboisia (Duboisia myoporoides R. Br) leaves were treated with 10% antiformin and then benzyladenine 10 ^-5 M
The plate was placed on an agar medium containing Rinsmeier Scoog (LS). One month later, the stems of Zubosia shoots that have not yet been lignified obtained by subculturing the shoots in the same medium for 40 days were cut to about 1 to 2 cm, and the suspensions of Agrobacterium rhizogenes HRI-1 that had been shake-cultured for 24 hours were suspended. Suspension (10 ⁷ / m
After soaking in l), it was placed on LS agar medium containing no plant hormone. After 3 weeks, adventitious roots were generated from the cutting sites of shoot stems. This was treated for 2 days in an LS liquid medium containing 0.1 mg / ml of ampicillin to obtain self-propagating Zboiscia-infected adventitious roots containing no bacterium. The adventitious roots infected with Zubosia were subcultured in LS liquid medium for 1 year. 20 mg of this adventitious root (dry weight) LS liquid medium containing spermidine added to 1 mM
The mixture was transferred to a 100 ml Erlenmeyer flask containing 100 ml and shake-cultured for 2 weeks. 150 mg (dry weight) of the obtained adventitious root is dried,
Extraction was performed in the same manner as in Example 1 and the amount of alkaloids was analyzed. The production amount of alkaloids in this case is shown in Table 2.

Example 5 Table 2 shows the results obtained in the same manner as in Example 4 except that a medium containing putrescine as the amines was used.

Comparative Example 2 Table 2 shows the results obtained in the same manner as in Example 4 except that spermidine was not added.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12R 1:91)

Claims (1)

[Claims] 1. A plant tissue producing a tropane-type alkaloid is prepared by using tetramethylenediamine, a derivative of tetramethylenediamine, polyalkylenepolyamines, lower alkylmonoamines and lower alkylmonoamines having a hydroxyphenyl group and an indole residue. Of tropane-based alkaloids characterized by producing tropane-based alkaloids by tissue culture using a medium containing at least one selected from the group consisting of substituted lower alkyl monoamines substituted with a group or an imidazole residue. Production method.