JPS6384497A - Production of tropan based alkaloid - Google Patents

Abstract

PURPOSE:To efficiently produce a tropan based alkaloid, particularly scopolamine, hyoscyamine, etc., in a large amount, by adding amines to a culture medium in tissue cultivation of a plant capable of producing the tropan based alkaloid. CONSTITUTION:Amines, e.g. spermine, spermidine, putrescine, histamine, aminopropane, etc., are added to a culture medium containing added inorganic components, carbon sources, plant hormones, vitamins, etc., to give about 0.01mM-10mM concentration. A plant capable of producing a tropan based alkaloid, e.g. plant of the genus Duboisia, Datura, Scopolia, Hyoscyamus, etc., is subjected to tissue cultivation in the above-mentioned culture medium. Roots, leaves stems, etc., can be exemplified as the plant tissues used in the tissues cultivation, but adventitious roots obtained by previous tissue cultivation are particularly preferably used.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention produces scopolamine and The present invention relates to a method for producing a tropane alkaloid such as hyoscyamine.

[Conventional technology]

Scopolamine is used as an antispasmodic, analgesic, and parasympathetic nerve blocker, and hyoscyamine is used as a parasympathetic nerve blocker, each of which is used as a medicine. These compounds are manufactured by extracting them from natural plants, but because they are made from natural materials, there are problems such as production being affected by the weather and the harvesting period being limited. It has become. Therefore, many studies have been conducted both domestically and internationally to produce these compounds through plant tissue culture. Regarding production by callus, there is a known example of production by Yamada et al. using henbane callus (PlantCell Report
TS, 101-103 (1982)), the scopolamine content was 20 ppm, which was lower than the content in natural plants. Furthermore, Yamada et al.
uboisia Leichhardtii F, M
It has been found that significant amounts of scopolamine and hyoscyamine are present in adventitious roots obtained by tissue culture of Plant Cell (Plant Cell Reports).
3.186-188 (1984)), but the amount was still not sufficient. Therefore, 1. We investigated various culture conditions for Zboisia adventitious roots, and by setting the ratio of ammonium ions to nitrate ions in the medium to 0.2 or more and the dissolved oxygen concentration in the medium to 10 to 65 ppm, we were able to produce tropane-based alkaloids. The present applicant has each filed a patent application in 1986-14.
Although patent applications have been filed as No. 3882 and Japanese Patent Application No. 6'0-143881, it is desired to further increase the productivity from an industrial standpoint.

[Problem that the invention seeks to solve]

Therefore, when aiming at industrial production of tropane-based alkaloids using such tissue culture, it has been an important issue to further increase productivity.

In view of these circumstances, the present inventors have researched methods for efficiently cultivating tissues and cells such as adventitious roots of plants that produce tropane alkaloids, such as Zboisia, Datura, Porphyra, and Hyosus, and have found the following. I found such a fact.

[Means for Solving the Problems] In conventional tissue culture methods, when culturing cells and tissues such as callus and adventitious roots, compounds belonging to amines are not added to the medium. Therefore, the present inventors added this component to the culture medium and tried culturing, and found that the tropane alkaloid content of cultured tissues such as adventitious roots and cells obtained by culture from the tissues of plants that produce tropane alkaloids. The present invention has been completed based on the discovery that the productivity of tropane-based alkaloids is improved by promoting the growth of cells such as adventitious roots, and as a result, the productivity of tropane-based alkaloids is improved.

That is, according to the method of the present invention, a method for producing tropane alkaloids, which comprises culturing tissues of plants that produce tropane alkaloids using a medium containing amines to produce tropane alkaloids. is provided.

[Detailed description of the invention]

In the present invention, tissue culture is carried out using plants that produce tropane-based alkaloids, and specifically, Duboisia myoporoides+D
Plants of the genus Zboisia such as Uboisia 1eichhardtii, Datura tatula+ Datu
ra arboea, Daturastramoni
Plants of the genus Datura such as um, 5copolia japon
Scoboria plants such as ica, Hyoscyamus
plants of the genus Mucuna such as niger and Atropa be
Examples include plants of the family Solanaceae, such as plants of the genus Atropa such as lladonna.

In the present invention, a medium containing amines is used to produce tropane alkaloids by culturing the plant tissues. This will be explained in detail below.

The medium used in the present invention is a medium containing amines, and has inorganic components and carbon sources as essential components other than these components, and plant hormones and vitamins are added thereto. This is a medium to which amino acids are added according to the conditions.

The amines according to the present invention refer to the following amines. That is, the amines that can be used in the present invention include spermine CHJ-(C1l□)3N11- (CHz) *-N
H-(C)Iz) 3-NH2), spermidine (H
zN-(CHz) JH-(CHz) 4-NH2)
, tetramethylene diamine and its derivatives such as putrescine (HgN-(CL) t-NHz), polyalkylene polyamines such as diaminopropane CHtN-(CHtN-(CHz) *-NHz), and lower alkyls such as aminopropane, amitubukun, aminopentane, etc. The terminal position of monoamines and lower alkyl monoamines such as tyramine, tryptamine, and histamine is a hydroxyphenyl group,
Substituted lower alkyl monoamines substituted with indole or imidazole residues.

In the present invention, the usage ratio of the amines is such that the concentration of the amines in the medium is usually 0.01mM to 10mM;
It is preferably used at a concentration of 0.1mM to 5mM. In the present invention, one or two of the above amines are used.
It is okay to use a mixture of more than one species.

When the plant tissue is cultured using a medium containing amines as in the method of the present invention, the production amount of tropane alkaloids is increased compared to when a medium containing no amines is used.

In the culture medium used in the present invention, inorganic components other than the above-mentioned amines include nitrogen, phosphorus, potassium, sodium, calcium, magnesium, sulfur, iron, manganese, zinc, boron, and molybdenum. Examples include inorganic salts containing elements such as , chlorine, iodine, and cobalt; specific examples include potassium nitrate, sodium nitrate, ammonium nitrate, ammonium chloride, potassium chloride, calcium chloride, potassium monohydrogen phosphate, and 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. I can give an example.

Examples of carbon sources for 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-
Examples include auxins such as chlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), indolebutyric acid (IBM), and derivatives thereof, and cytokinins such as benzyladenine (BA), kinetin, and zeatin. In the present invention, it is usually preferable not to add cytokinins to the culture medium, but when they are added as necessary, the concentration of cytokinins is usually 10-? M(
It is preferable to use it at a low concentration of 0.02 mg/1) or less.

The vitamins in the medium include biotin, thiamine (vitamin B), pyridoxine (vitamin Bl), pyridoxal, pyridoxamine, calcium pantothenate, ascorbic acid (vitamin C), inositol, nicotinic acid, nicotinamide, and ribobravin (vitamin C). Examples include vitamin th).

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 of the inorganic components.
μM to about 100 mM, about 1 g/l to about 100 g/It of the carbon source, about 0 g/It of the plant hormones.
．． 01 μM to about 100 μM1 The above vitamins are added to about 0
．． It is preferable to use lIIIg/It to about 150 mg/L and the above aminos in O to about 1000 mg/Il.

Specifically, the medium used for the tissue culture of plants belonging to the genus Zboisia of the present invention includes a conventionally known culture medium used for tissue culture of plants, such as Murashige & Skoo ('62).
g) medium, Linsmeyer-Skoog (RM-19
65) Clinsmaier & Skoog)
medium, White medium, Gamborg 8-5 medium, Mitsui 19 medium, Nitsch N1t medium.
An example is a culture medium prepared by adding the above-mentioned carbon source and plant hormone to a culture medium of sch. Preferably, the medium is prepared using Hetch, Linsmeyer-Skoog or Murashige-Skoog medium. Regarding the composition of the above-mentioned conventionally known culture medium, for example, "
New Plant Tissue Culture JP P386-P391, Asakura Shoten, 1
It was written in 979.

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

In the tissue culture of the present invention, tissue pieces of plants that metabolize tropane alkaloids are tissue cultured using the above medium to obtain cultured cells or cultured tissues containing tropane alkaloids.

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

Specifically, the tissues of the plants used in the tissue culture of the present invention include roots, leaves, stems, seeds, flower buds, etc. of the plants, as well as those that can be used in the tissue culture of the present invention or other conventional tissue culture methods. The resulting cultured cells or tissue of the plant can be exemplified. In the present invention, among these, it is particularly preferable to use adventitious roots obtained by previously tissue culturing plant tissues and tissue culture them using the medium according to the present invention. In this case, the adventitious roots as raw materials are Adventitious roots containing a large amount of tropane alkaloids can be obtained by propagation and culture using the medium of the invention.

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

In the present invention, when using adventitious roots, plant tissue pieces are, for example, hairy root disease bacteria (e.g. Agrobacterium rh).
izogenes) and the resulting hair roots that appear can also be used (for example, the method proposed in Japanese Patent Application No. 61-89975 filed by the present applicant can also be used).

Specific examples of the tropane alkaloid obtained by the method of the present invention include scopolamine, hyoscyamine, and acetyl compounds of these compounds, and among these, scopolamine and hyoscyamine are preferred.

In the present invention, the method for separating tropane-based alkaloids from cultured cells containing them is, for example, the method used to isolate and purify tropane-based alkaloids from plants containing tropane-based alkaloids, as described in the Pharmacopoeia Act. The usual methods that have been used can be adopted.

〔Effect of the invention〕

By employing the method for producing tropane alkaloids by tissue culture of the present invention, tropane alkaloids, especially scopolamine and/or hyoscyamine, can be produced efficiently in large quantities compared to conventional methods.

〔Example〕

Hereinafter, the method of the present invention will be explained in more detail with reference to Examples.

Example 1 Duboisia mypor grown in our herb garden
oidesR,Br leaves were washed, immersed in 10% antiformin solution for 10 min, then washed 3 times with sterile water, then cut into approximately 11 pieces and treated with naphthaleneacetic acid and benzyladenine at 10-'M and 10-'M, respectively. The cells were plated on a Linsmeyer-Skoog agar medium supplemented with M and cultured at 25°C for 30 days. Adventitious roots that appeared at the same time as callus formation were cut out, and indole acetic acid was added to
The cells were transplanted into an H-H liquid medium supplemented with a concentration of 'H' and subcultured for 2 years. 10 mg (dry weight) of the adventitious roots thus obtained were placed in a 20-ml Niche Niche liquid medium containing 1 mM spermidine and 10 M indolebutyric acid.
00rn! The cells were transplanted into a large Erlenmeyer flask and cultured with shaking for 3 weeks. After drying 190 mg (dry weight) of the obtained adventitious roots, 50 ml of basic chloroform-methanol solution was added! Extracted with. 40 mf of IN sulfuric acid was added to this and the alkaloid layer was transferred to the sulfuric acid layer. Furthermore, ammonia water 2- and chloroform 40- were added to transfer the alkaloid to the chloroform layer, which was concentrated under reduced pressure and the amount of alkaloid was analyzed using a gas chromatograph. Table 1 shows the production amount of alkaloid in this case. Note that the gas chromatograph analysis was conducted under the following conditions.

Column: 5ilicone 0V-17 (1%) o
nChron+osorb W (Mesh 80
~10100) 3φ x 11I + glass column carrier gas 2N2 Column temperature -7200°C Examples 2 to 3 and Comparative Example 1 Same as Example 1 except that the diamine pretocin or diaminopropane shown in Table 1 was added to the medium. Table 1 shows the results obtained in the same manner as in Example 1 (Examples 2 to 3) and the results obtained in the same manner as in Example 1 using a medium to which no amines were added (Comparative Example 1).

Example 4 Duboisia myoporoid
After treating leaves of es R, Br) with 10% antiformin, they were plated on a Linsmeyer-Skoog (LS) agar medium containing 10-'M benzyladenine. The shoots that emerged after one month were cultured for 40 days in the same medium, and the stems of the Zboisia shoots, which had not yet become fully formed, were 1 to 2 c.
Agroba was cut into pieces of approximately
After immersing in a suspension of cterium rhizogenes HRI-1 (107 cells/-), the cells were placed on an LS agar medium containing no plant hormones. Three weeks later, adventitious roots emerged from the cut site of the shoot stem. Add this to ampicillin 0.1
The roots were treated for 2 days with an LS liquid medium containing 1.0 mg/- to obtain fungus-free, self-propagating adventitious roots infected with Zboisia.

The adventitious roots infected with Zboisia were cultured for one year in a LS liquid medium. 10 mg (dry weight) of these adventitious roots were placed in LS liquid medium 20 containing spermidine added to a concentration of 1 mM.
The cells were transplanted into a 100-ml Erlenmeyer flask containing rnl and cultured with shaking for 2 weeks. 150 mg (dry weight) of the obtained adventitious roots were dried, extracted in the same manner as in Example 1, and analyzed for alkaloid content. Table 2 shows the production amount of alkaloid in this case.

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

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

Claims (1)

[Claims] (1) A method for producing tropane alkaloids, which comprises producing tropane alkaloids by culturing the tissues of plants that produce tropane alkaloids using a medium containing amines.