JPH01218595A - Production of tropan-based alkaloid - Google Patents

Abstract

PURPOSE:To improve industrial productivity of tropan-based alkaloid efficiently and in a large amount, by subjecting tissue of plant to produce a tropon-based alkaloid to tissue culture in a medium containing a phenylalanine derivative. CONSTITUTION:A medium such as Murashige & Skoog medium, etc., is incorporated with >=1.0muM phenylalanine derivative (e.g., 4-methyl-phenylalanine), an amino acid derivative shown by formula I [R<1> is lower alkyl or halogen; R<2> is H or group shown by formula II; R<3> is OH, NH2 or OM (N is metallic ion)], 0.1muM-100mM inorganic component such as Ca, 1-100g/l carbon source such as sucrose, 0.01-100muM plant hormone to give a medium for tissue culture. Then, the medium is inoculated with a plant tissue such as Duboisia myoporoides to produce a tropon-based alkaloid, which is multiplied and cultivated to give a culture mixture or an adventitious root containing the alkaloid. Then the adventitious root is dried and extracted to isolate a tropon-based alkaloid such as scopolamine.

Description

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

[Conventional technology]

Among the tropane-based alkaloids, scopolamine is used as an antispasmodic, analgesic, and a new parasympathetic nerve suppressant, and hyoscyamine is used as a new parasympathetic nerve suppressant.

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, many studies have been conducted both domestically and internationally to produce these compounds through plant tissue culture. Regarding production by callus, an example of production by Yamada et al. using henbane callus is known (Plant Ce1
l Reports, 101-103 (1982)
), but the scopolamine content was 20 ppm, which was lower than the content in natural plants. Also, Yamada et al.
Duboisia Le-ichhard
It has been found that significant amounts of scopolamine and hyoscyamine are present in adventitious roots obtained by tissue culture of Plant Ce1lR).
eports i, 186-188 (1984)), but the amount was still not sufficient. Therefore, 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 higher and the dissolved oxygen concentration in the medium to 10 to 65 ppm, we improved the productivity of tropane-based alkaloids. The present applicant has each filed a patent application in 1986.
Although patent applications have been filed as No. 143882 and Japanese Patent Application No. 143881/1988, it is desired to further increase the productivity from an industrial standpoint.

[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.

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 to solve the problem]

In other words, when tissues of plants that produce tropane-based alkaloids are cultured using a medium containing specific amino acids at specific concentrations, tropane-based alkaloids are produced in cultured cells or cultured tissue obtained by culturing adventitious roots. The inventors have now completed the present invention by discovering that the alkaloid content is increased or the growth of cultured tissue fibers such as cultured cells and adventitious roots is promoted, resulting in improved productivity of tropane alkaloids.

That is, according to the present invention, the tissue of a plant that produces tropane alkaloids is treated with a phenylalanine derivative in 1.
0. Provided is a method for producing tropane alkaloids, which comprises producing tropane alkaloids by culturing tissue using a medium containing at least uM.

[Detailed description of the invention]

In the present invention, tissue culture is carried out using plants that produce tropane-based alkaloids.
5yoporoides) + Duboisia 1eichhardti 4 (Duboisia 1eichhardti)
i) and other plants of the genus Zboisia, Datu
ra tatuJa) + Datura arborea (Dat
ura arborea) + plants of the genus Datura such as Datura strawonius, Scopolia japonica (Scopolia j
aponica), plants of the genus Mucuna such as Iyoscyan+us niger, and Atropa belladonna (^trop).
Examples include plants of the Solanaceae family, such as plants of the genus Atropa such as A belladonna).

In the present invention, a medium containing 1.0 .mu.h or more of a phenylalanine derivative is used to produce tropane alkaloids by culturing the aforementioned plant tissues. This will be explained in detail below.

The medium used in the present invention is a medium containing a phenylalanine derivative as an essential component, and other components other than this component include an inorganic component and a carbon source, to which plant hormones and vitamins are added, Furthermore, it is a medium to which amino acids other than the phenylalanine derivative according to the present invention, such as glycine, glutamic acid, and lysine, may be added as necessary.

The phenylalanine derivative according to the present invention has the general formula (1) % formula % (1) [In the formula, RI represents a lower alkyl group or a halogen atom, and 9t is H or R-C- (R is H or a lower alkyl group)
and R3 is OL NiF! 0 again? f (M represents a metal ion), n represents an integer of 0 to 3,]
It is a derivative of the amino acid represented by 4, specifically 4
- Alkylphenylalanine such as methyl-phenylalanine, halogenated phenylalanine such as 4-chlorophenylalanine, 4-fluorophenylalanine, 4-fluorof.

Examples include amide derivatives such as enylalanine amide.
In the present invention, the carboxyl group in these compounds may form a salt with the same metal ion as the below-mentioned inorganic component constituting the medium, and this salt can also be used as a medium component in the present invention.

In the present invention, the proportion of the above-mentioned phenylalanine derivative contained in the medium is 1.0 μM or more.

Among these ranges, a range of 2 μM to 30 μM is particularly preferred. The upper limit of phenylalanine derivative concentration is usually 1n+M
It is. The content of phenylalanine derivatives in the medium is 1
．． If the concentration is less than 0μ, the amount of tropane alkaloid produced will not improve significantly even if tissue culture is performed using such a medium. Therefore, in the present invention, a medium with the content of this component within the above range is used. Tissue culture is performed.

In the medium used in the present invention, other medium components other than the above-mentioned phenylalanine derivatives include nitrogen, phosphorus, potassium, sodium, calcium, magnesium, sulfur, iron, manganese, zinc, boron, 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, potassium dihydrogen phosphate, Magnesium sulfate, magnesium chloride, sodium sulfate, ferrous sulfate, ferric sulfate
Iron, manganese sulfate, copper sulfate, sodium molybdate,
Examples include compounds such as 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 the plant hormones in the medium include naphthalene acetic acid (NA^), indole vinegar fi (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 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 ■/l) or less.

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

The medium of the present invention usually contains about 0.1 of the inorganic components.
μ gate to about 1100 II, and about 1 g/l to about 100 g// of said carbon source! , about 0.0 of the plant hormones
1 μm to about 100 μi, and about 0.1 μm of the above vitamins.
■/l or about 150B//! It is desirable to use it together.

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) (Linsmaier & Skoog)
medium, white medium ('63),
Gamborg's B-5 medium, Mitsui's M
-9 medium, Nitsch N1tsc
An example of a culture medium prepared by adding the phenylalanine derivative according to the present invention, the above-mentioned carbon source and plant hormone to the medium etc. of h), and further adding the above-mentioned vitamins etc. as necessary; Among these, media prepared using H-H, Linsmeyer-Skoog, or Murashige-Skoog are particularly preferred.

Regarding the composition of the above-mentioned conventionally known culture medium, for example, see [New Plant Tissue Culture J P38] by Tenshi, Nakajima, and Furuya.
6-P391, Asakura 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.

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-described tissue pieces or cells, and cultured cells or cultured MAm obtained by subculturing the callus are grown and cultured using the above-mentioned medium of the present invention to obtain a large amount of tropane-based alkaloids. Preference is given to using methods of tissue culture, such as obtaining cultures containing, in particular adventitious roots.

In the present invention, when using adventitious roots, plant tissue pieces may be treated with hairy root disease bacteria (e.g., Agrobacteria+rh).
zogenes) and the resulting hair roots can be used (for example, the method proposed in Japanese Unexamined Patent Publication No. 62-248429 filed by the present applicant can also be used). Specific examples of the alkaloid 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 a method that can be used, for example, when isolating and purifying 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.

〔Example〕

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

Example 1.2.3.4 Dabofsia myopa grown in our herb garden
ro+desR,Br leaves were washed, immersed in 10% antiformin solution for 10 minutes, then washed three times with sterile water, cut into approximately 1 cm pieces, and treated with naphthaleneacetic acid and benzyladenine at 10-'M and 1, respectively. 〇-Place on Linsmeyer-Skoog agar medium added so as to form a snout, and culture at 25°C for 30 days. Cut out the adventitious roots that occurred at the same time as callus formation, and add 10% indolebutyric acid.
The cells were transplanted into a liquid medium of H.H. and subcultured for 2 years. Culture was started by transplanting 10 cm (dry weight) of the adventitious roots thus obtained into a 100-volume Erlenmeyer flask containing 20-cm of Etch Niche liquid medium supplemented with indolebutyric acid to a concentration of 10-'H. sometimes 4
- Fluorophenylalanine concentrations are 1 μM and 3 μ', respectively.
4-Fluorophenylalanine was added to the above liquid medium to give M, 10 μM and 30 μM, and cultured with shaking for an additional 3 weeks. The obtained adventitious roots were dried and then extracted with 50% of a basic chloroform-methanol solution. 40@l 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 td 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 QV-17 (1%) o
nChroa+osorb W (Mesh 8ON1
00) 3ms φ Indicated.

Comparative Example 2 Table 1 shows the results of carrying out the same procedure as in Example 1 except that the 4-fluoro-phenylalanine concentration was changed to 0.05 μM.

(Margins below main text) Example 5.6.7 Duboisia myoporoid
After treating the leaves of s R, Br) with 10% antiformin, they were placed on a Linsmeyer-Skoog (LS) agar medium containing 10-'M benzyladenine. The shoots that emerged after 1 month were cultured for 40 days in the same medium, and the stems of the Zboisia shoots, which had not yet matured, were cultured at 1 to 2 CI.
Agrobacterium rhizogenes cut into about 11 pieces and cultured with shaking for 24 hours
After being immersed in a suspension of HRI-1 (10 7 cells/ml), the cells were placed on an LS agar medium containing no plant hormones. After 3 weeks, adventitious roots developed from the cut site of the stem of the surface streak. This was treated with an LS liquid medium containing 0.1 mg/mI of ampicillin for 2 days to obtain bacteria-free, self-propagating adventitious roots infected with Zboisia. This Zboisia-infected adventitious root was grown in LS liquid medium.
It was subcultured for a year. This adventitious root 10■ (dry weight 4-
Fluorophenylalanine 4 degrees is 3 μM each, 10
μA, LS liquid medium 20 added to 30 μ-
The cells were transplanted into a 100 m Erlenmeyer flask containing mZ and cultured with shaking for 3 weeks. 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.

Comparative Example 1 Table 2 shows the results of the same procedure as in Example 5 except that 4-fluorophenylalanine was not added.

Comparative Example 2 Table 2 shows the results of the same procedure as in Example 5 except that the 4-fluorophenylalanine concentration was changed to 1 μm.
It was shown to.

(Margin below this page) Examples 8, 9.10 10 cm of adventitious roots obtained in the same manner as in Example 1 were added so that the 4-chlorophenylalanine concentrations were 3 μN, 10 μN, and 30 #M, respectively. Also, indolebutyric acid 10
-Etch Niche liquid medium 2 added so that jM
011 into a 1 ooId volume Erlenmeyer flask.

After culturing for 3 weeks, the cells were treated in the same manner as in Example 1, and the amount of alkaloid was analyzed. Table 3 shows the alkaloid production amount in this case.

Comparative Example 1 Table 3 shows the results of the same procedure as in Example 1 except that 4-chlorophenylalanine was not added in the sample.

Comparative Example 2 In Example 8, the 4-chlorophenylalanine concentration was 0.
．． Table 3 shows the results obtained in the same manner as in the example except that 5IIM was used. - (Blank below next summer) [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 compared to conventional methods. Efficiency (can be produced.) - Applicant: New Manufacturing Technology Research Association for Chemicals Utilizing Biofunctions, Patent Attorney: Yusuke Hiraki

Claims (1)

[Claims] (1) A method for producing tropane alkaloids, which comprises culturing the tissue of a plant that produces tropane alkaloids using a medium containing 1.0 μM or more of a phenylalanine derivative to produce tropane alkaloids.