JPH01273597A - Production of tropan based alkaloid - Google Patents

Abstract

PURPOSE:To efficiently produce a tropan based alkaloid in a large amount by subjecting tissues or cells of a specific plant to tissue culture using a culture medium containing an adenosyl compound or cyclic adenosylphosphoric acid. CONSTITUTION:Tissues or cells of a plant, such as Duboisia.myoporoides or Datura.tatula, capable of producing a tropan based alkaloid are subjected to tissue culture using a culture medium containing an adenosyl compound, such as 3'-deoxyadenosine, or a cyclic adenosyphosphoric acid, such as adenosine 3',5'-cyclic phosphate, an inorganic component and carbon source to improve the productivity of the tropan based alkaloid, such as scopolamine and/or hyoscyamine. Furthermore, the content of the above-mentioned adenosyl compound in the culture medium is preferably 10<-2>-6mM.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention is directed to culturing the tissues or cells of plants that metabolize tropane-based alkaloids, such as Zboisia, Datura, Hashiridokoro, Hiyosu, etc., using a specific medium. The present invention relates to a method for producing tropane alkaloids such as scopolamine and/or hyoscyamine.

(Prior Art) Scopolamine is used as an antispasmodic, analgesic, and parasympathetic nerve blocker, and hyoscyamine is used as a parasympathetic nerve blocker. These compounds are manufactured by extracting them from natural plants, but because they are made from natural materials, there are problems such as their production being affected by the weather and the harvesting period being limited. It has become. Therefore, numerous 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 (Plant Ce1lR
eports, 101-103 (1982)),
The scopolamine content was 20 ppm, which was lower than the content in natural plants. Furthermore, Yamada et al.
We found that significant amounts of scopolamine and hyoscyamine were present in the adventitious roots obtained by tissue culture of Plant Ce1l Re.
ports 3.186-188 (1984)),
The amount was still not enough.

Therefore, we investigated various culture conditions for Zboisia adventitious roots.
The ratio of ammonium ions and nitrate ions in the medium was set to 0.2.
The present applicant has discovered that the productivity of tropane alkaloids can be improved by adjusting the concentration of dissolved oxygen in the medium to 10 to 65 ppm, and the present applicant has discovered that the productivity of tropane-based alkaloids can be improved by adjusting the concentration of dissolved oxygen in the medium to 10 to 65 ppm, and the present applicant has discovered that the productivity of tropane-based alkaloids can be improved by adjusting the concentration of dissolved oxygen in the medium to 10 to 65 ppm. 6674
Although a patent application has been filed for this technology, from an industrial standpoint, it is desirable to further increase its productivity.

[Problem to be solved by the invention]

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

(Means for Solving the Problems) In view of the above-mentioned circumstances, the present inventors have devised a method to efficiently collect Mi fibers and cells of adventitious roots of plants that produce tropane alkaloids, such as Zboisia, Datura, Hashiridokoro, and Hiyosu. As a result of research into culturing methods, the following facts were discovered.

That is, when tissues or cells of the plant are cultured using a medium containing an adenosyl compound or cyclic adenosyl phosphate at a specific concentration, the resulting cultured cells or cultured tissues obtained by culturing adventitious roots, etc. The present inventors have completed the present invention by discovering that the content of tropane alkaloids is increased, or the growth of cultured tissues such as cultured cells and adventitious roots is promoted, resulting in improved productivity of tropane alkaloids.

That is, according to the present invention, tropane alkaloids are produced by culturing the tissues or cells of plants that produce tropane alkaloids using a medium containing an adenosyl compound or cyclic adenosyl phosphate. A method for producing a tropane alkaloid is provided.

In the present invention, tissue culture is carried out using plants that produce tropane-based alkaloids.
myoporoides), Duboisia 1eichhardtii
) and other plants of the genus Zboisia, Datur
a tatula) + Datu arborea
ra arborea), Datura stramonium (D
plants of the genus Datura such as Atura stramonium, Scopolia japonica (Scopolia japonica),
plants of the genus Scopolia such as llyoscyamus ntger, plants of the genus Mucuna such as llyoscyamus ntger, and plants of the genus Atropa belladonna (Atropa belladonna).
Examples include plants of the Solanaceae family, such as plants of the genus Atropa, such as Atropa (belladonna).

In the present invention, a medium containing an adenosyl compound or cyclic adenosyl phosphate is used when culturing the plant tissue to produce a tropane alkaloid. This will be explained in detail below.

The medium used in the present invention is a medium containing an adenosyl compound as an essential component, and has inorganic components and a carbon source as essential components other than this component, to which plant hormones and vitamins are added. It is.

The adenosyl compound according to the present invention has the general formula (1) [wherein R1 represents 11 or a lower alkyl group, R2 represents H, 0
11 or a phosphoric acid group, R3 is OH, a phosphoric acid group, or R
'-CHCHzCHz-3-(R' is II or -COO
Hl) Shown. ] and its derivatives, specifically adenosine, 3'-deoxyadenosine,
Adenosine 3'-monophosphate, adenosine 5-monophosphate,
Examples include adenosyl phosphates such as adenosine 5'-diphosphate and adenosine 5'-triphosphate, and S-adenosyl compounds such as S-adenosylmethionine, S-adenosylhomocystine, and S-adenosylmethylthiopropylamine. I can do it.

The cyclic adenosyl phosphoric acids according to the present invention are cyclic adenosyl phosphoric acids represented by the general formula (n) I B [wherein R1 is the same as in the general formula (1)] and derivatives thereof. A specific example is adenosine 3', 5'-cyclic phosphate.

In the present invention, the carboxyl group and phosphate group in these compounds may form a salt with the same metal ion as the inorganic component described below constituting the medium, and this salt can also be used as a medium component in the present invention. .

In the present invention, the ratio of the adenosyl compound contained in the medium is usually in the range of IQ-'mM to 10mM, but within this range, the range of Ill'mM to 6mM is particularly preferable.Adenosyl compound in the medium If the content of the component is outside this range, the amount of tropane alkaloid produced will not improve significantly even if tissue culture is performed using such a medium. It is preferable to perform tissue culture using a culture medium.

When using cyclic adenosyl phosphate in the medium, the usage rate is usually 0.01 μM to 100 μM,
Among these, 0.1 μM to 10 μM is preferable.

In the medium used in the present invention, other medium components other than the adenosyl compound and cyclic adenosyl phosphate described above include nitrogen, phosphorus, potassium, sodium, calcium, magnesium, sulfur, iron, Manganese, zinc, boron, molybdenum, chlorine,
Examples include inorganic salts containing elements such as iodine and cobalt, specifically potassium nitrate, sodium nitrate, ammonium nitrate, ammonium chloride, potassium chloride, calcium chloride, potassium monohydrogen phosphate, potassium dihydrogen phosphate, and magnesium sulfate. Examples include compounds such as magnesium chloride, sodium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, copper sulfate, sodium molybdate, molybdenum dioxide, potassium iodide, zinc sulfate, boric acid, and cobalt chloride.

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 naphthaleneacetic acid (NAA), indoleacetic acid (IA^), p-chlorophenoxyacetic acid, 2.4-dichlorophenoxyacetic acid (2,4-D), and indolebutyric acid ( Examples include auxins such as IB8) and derivatives thereof, and cytokinins such as benzyladenine (BA), kinetin, and zeatin. In the present invention, it is generally preferable not to add cytokinins to the culture medium, but when 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/j2) or less.

The vitamins in the medium include biotin, thiamine (vitamin AT), pyridoxine (vitamin B6), pyridoxal, pyridoxamine, calcium pantothenate,
Examples include ascorbic acid (vitamin C), inositol, nicotinic acid, nicotinamide, and riboflavin (vitamin BX).

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

The medium of the present invention usually contains about 0.1 of the inorganic components.
μP to about 100mM, and about 1g/ff of the carbon source
i or about 100g//! , about 0 of the plant hormones
, O1 μn to about 100 μN, and the vitamins are about 0
．． 1 mg/e to about 150 mg/I;! It is desirable that the amino acids be used in an amount of 0 to about 100 mg/i2.

Specifically, the medium used for the tissue culture of plants belonging to the genus Zboisia of the present invention includes a conventionally known medium used for tissue culture of plants, such as Murashige & Skoo ('62).
g) medium, Linsmeyer-Skoog (R1'l-
1965) (Linsmaier & Skoog
) medium, White ('63) (White medium, Gamborg B-5 medium, Mitsui M-9 medium, Nitsch N
An example is a medium prepared by adding the above-mentioned carbon source and plant hormone to a medium of 1tsch), and further adding the above-mentioned vitamins, amino acids, etc. as necessary. Preferably, the medium is prepared using Hetch, Linsmeyer-Skoog or Murashige-Skoog medium. The composition of the above-mentioned conventionally known culture medium is described, for example, in "New Plant Tissue Culture JP 386-P391," written by Tenshi, Septum, and Furuya, Shokusho Shoten, 1979.

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.

Specifically, the tissue or cells of the plant used in the tissue culture of the present invention include plant tissue pieces or cells such as roots, leaves, stems, seeds, flower buds, etc. of the plant, as well as tissue culture or other cells related to the present invention. Examples include cultured cells or tissue of the plant obtained by the conventional tissue culture method. In the present invention, it is particularly preferable to use adventitious roots obtained by culturing plant tissues in advance and tissue culture them using the medium according to the present invention. Adventitious roots containing a large amount of tropane alkaloids can be obtained by propagation and culture using the medium of the present invention.

In addition, in the present invention, callus is induced from the tissue pieces or cells described above, and the cultured cells or tissue obtained by subculturing the callus are grown and cultured using the medium of the present invention to produce a large amount of tropane-based alkaloids. It is also preferred to use methods of tissue culture to obtain cultures containing, in particular adventitious roots.

In the present invention, when using adventitious roots, plant tissue pieces are infected with hairy root disease fungi (e.g., eight grobacterium um).
tropane obtained by the method of the present invention. Specific examples of the alkaloids include scopolamine, hyoscyamine, and acetyl compounds of these compounds, and among these, scopolamine and hyoscyamine are preferred.

In the present invention, methods for separating tropane-based alkaloids from cultured cells or cultured tissues include, for example, the method of isolating and purifying these compounds from plants containing tropane-based alkaloids, as described in the Pharmacopoeia Act, etc. Any conventional method that has been used can be used.

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

Examples 1, 2, 3.4 and 5 Duboisia mypor grown in our herb garden
oiclesR,Br leaves were washed, immersed in 10% antiformin solution for 10 min, then washed three times with sterile water, then cut into approximately 1 (m) and treated with naphthaleneacetic acid and benzyladenine at 10-'M and Place on Linsmeyer-Skoog agar medium supplemented with 10-6M and culture at 25°C for 30 days.Adventitious roots that have developed at the same time as callus are excised, and indolebutyric acid is added at 10-6M.
-''M, and subcultured for 2 years. 10cm (dry weight) of the adventitious roots thus obtained were treated with indole acetic acid for 10cm.
The S-adenosylmethionine concentration was 0.0 on the 7th day after the culture was started.
S-adenosylmethionine was added to the liquid medium at concentrations of 3mM, 0.1mM, 0.3n+M, 1,0mM, and 5.0mM, and cultured with shaking for an additional 14 days. After drying the obtained adventitious roots, 50ml of basic chloroform-methanol solution was added. Extracted with. This has 40m1 of IN.
Sulfuric acid was added to transfer the alkaloid layer to the sulfuric acid layer. Further, 2 d of aqueous ammonia and 40 m/ml of chloroform were added to transfer the alkaloids to the chloroform layer, which was concentrated under reduced pressure and the amount of alkaloids 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
nChromosorb H (Mesh 80-100
) 3 cylinders φ x 1m glass force ram carrier gas 2N2 Column temperature: 200°C Comparative Example 1 The same procedure as in Example 1 was used except that a medium without S-adenosylmethionine was used. The results are shown in Table 1. Ta.

Example 6.7 The same procedure as in Example 1 was performed except that 0.11 and 1.0 mM of adenosine 5'--phosphate was used instead of S-adenosylmethionine. The results are shown in Table 1. Ta.

Comparative Example 2 Table 1 shows the results of the same procedure as in Example 1 except that 10.0 mM of adenosine 5'-monophosphate was used instead of S-adenosylmethionine.

Examples 8, 9.10 In Example 1, adenosine 3', 5'-cyclic phosphate was added at 0.1 μM and 1 in place of S-adenosylmethionine.
．． Table 1 shows the results obtained in the same manner as in the example except that 0 μm gate and 10.0 μm gate were used.

(Margins below this page) [Effects of the invention] If the method for producing tropane alkaloids by tissue culture of the present invention is adopted, tropane alkaloids, especially scopolamine and/or hyoscyamine, can be produced in large quantities more efficiently than in conventional methods. Can be produced well.

Applicant: New Manufacturing Technology Research Association for Chemicals Utilizing Biofunctions Agent: Yusuke Hiragi, Attorney: Sadatsugu Ishii

Claims (3)

[Claims] (1) Tropane-based alkaloids are produced by culturing the tissues or cells of plants that produce tropane-based alkaloids using a medium containing an adenosyl compound or cyclic adenosyl phosphate. Production method. (2) Adenosyl compound at 10^-^2mM to 6mM
The method for producing tropane alkaloids according to claim 1, characterized in that a medium containing M is used. (3) The method for producing tropane-based alkaloids according to claim 1, characterized in that a medium containing 0.1 μM to 10 μn of cyclic adenosyl phosphate is used.