JPS626675A - Method of tissue culture for duboisia leichhardtu f. muell - Google Patents

Abstract

PURPOSE:In producing a propane alkaloid useful as an agalgesic, etc., by subjecting a plant of the genus Buboisia to tissue culture, to improve yield, productivity, etc., by using a medium having a high ratio of NH4 ion to nitrate ion. CONSTITUTION:A plant such as Duboisia myoporoides R. Br, etc., belonging to the genus Duboisia, is subjected to tissue culture by using a medium comprising an inorganic component (e.g., nitrogen, phosphorus or potassium) and a carbon source (e.g., sucrose or ethanol) as essential components blended with a plant hormone, a vitamin and amino acid. In the operation, a ratio of ammonium ion/nitrate ion in the medium is adjusted to >=0.2 to carry out the tissue culture. Consequently, a tissue culture mixture containing a large amount of tropane alkaloid such as scopolamine, hyoscyamine, etc., is obtained and extracted with a proper solvent to advantageously give the tropane alkaloid.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing tropane-based alkaloids such as scopolamine and hyoscyamine by culturing plant tissues such as roots and adventitious roots of plants belonging to the genus Zboisia.

[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 in that their production is affected by the weather and the harvest season is 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 (Pl-ant Ce1l Report
ts 1.101-103 (1982)), but the scopolamine content was 20 ppm, which was lower than the content in natural plants. Furthermore, Yamada et al.
(Duboisia Leichhardtij F,
It has been found that significant amounts of scopolamine and hyoscyamine are present in adventitious roots obtained by tissue culture of Muell (Plant Cell Report).
s3.186-188 (1984)), but the amount was still not sufficient.

[Problem that the invention seeks to solve]

Therefore, when aiming for industrial production of tropane-based alkaloids using such a tissue culture method.

Furthermore, increasing productivity was an important issue.

In view of these circumstances, the present inventors researched a method for efficiently cultivating adventitious roots of plants belonging to the genus Zboisia, and as a result found the following facts.0 [Means to solve the problem] Namely, Normally, when culturing adventitious roots, a medium with a low concentration of medium components is desirable. In addition, when culturing adventitious roots of Korean carrots, it is said that it is desirable to remove ammonium ions from the medium (@ Kaisho 59-169
488. Nitto Denko). Furthermore, Yamada et al. used a medium with a low ammonium ion concentration (2 mM), such as Camborg's B5 medium, in culturing adventitious roots of Zboisia. The present inventors investigated the effects of culture medium composition on root and adventitious root growth and alkaloid content, and found that the presence of ammonium ions had a significant effect on root and adventitious root growth and alkaloid content. That is, when the concentration ratio of ammonium ions and nitrate ions is 1:5 or more, preferably 1:2 or more, compared to when no ammonium ions are present,
It was discovered that the production amount of alkaloids more than doubled.
That is, according to the method of the present invention, plants belonging to the genus Zboisia are grown in a medium in which the ratio (a/b) of ammonium ions and nitrate ions (bl) is 0.2 or more. Provided is a method for culturing a tissue of a plant belonging to the genus Zboisia, which is characterized in that a tropane-based alkaloid is produced by culturing the tissue using the present invention.

In the present invention, tissue culture is carried out using a plant belonging to the genus Zboisia, and specifically, the plant belonging to the genus Zboisia is D.
uboisia rnyoporoides RoBr
.

Duboisia Leichhardtu F, M
uell o and Du-boisia hopwood
The medium used in the present invention is a medium containing inorganic components and carbon sources as essential components, to which plant hormones and vitamins are added, and further amino acids are added as necessary. be.

Inorganic components of the medium include inorganic salts containing elements such as nitrogen, phosphorus, potassium, sodium, calcium, magnesium, sulfur, iron, manganese, zinc, boron, molybdenum, chlorine, iodine, cobalt, etc. Specifically, potassium nitrate, sodium nitrate, ammonium nitrate, ammonium chloride, potassium chloride, calcium chloride, potassium monohydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate, magnesium chloride, sodium sulfate,
Examples include compounds such as ferrous sulfate, ferric sulfate, manganese sulfate, copper sulfate, sodium molybdate, molybdenum trioxide, 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 naphthaleneacetic acid (NAA), indoleacetic acid (IAA), p-chlorophenoxyacetic acid, and 2,4-dichlorophenoxyacetic acid (
2,4-D), indolebutyric acid (IBA) and their derivatives, and benzyladenine (B
Examples include cytokinins such as A), 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 mVl or less.

The vitamins in the medium include biotin, thiamine (vitamin B1), pyridoxine (vitamin B), pyridoxal, pyridoxamine, calcium pantothenate,
Examples include ascorbic acid (vitamin C), inositol, nicotinic acid, nicotinamide, and riboplavin (vitamin Bt).

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 19/l to about 100 g of the carbon source, and about 0.1 m9 of the plant hormones.
/ l to about 100 TL9/1. About 0.1 m9/l'l to about 150 mg/l of the vitamins and about 1000 mg/l of the amino acids! It is preferable that it be used in combination.

Specifically, the medium used for the tissue culture of plants belonging to the genus Zboisia of the present invention may be a conventionally known culture medium used for tissue culture of plants, such as Murashige & Skog ('62)
oog) medium, Linsmeyer-Skoog (RM-
1965) [Linsmaier & Skoog]
tD medium, white ('63) (White)
cD medium, B of Kanhork ((xFJ, mborg)
-5 medium, Mitsui's M-9 medium.

Niche Niche Medium (Nitsch & N1tsc
Adding the carbon source and plant hormones described above to h) etc.,
Furthermore, examples include a culture medium prepared by adding the vitamins and amino acids described above as necessary, but in the present invention, among these, in particular, a culture medium prepared using H.H., Linsmeyer-Skoog, or Murashige-Skoog. Preferably, the medium is The composition of the above-mentioned conventionally known culture medium is described, for example, in "New Flora Knowledge Culture JP386-P391" by Harinai, Nakataka, 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% agar, but it is preferable to use a liquid medium in the present invention.

In the present invention, tissue culture of plants of the genus Zboisia is carried out using the above-mentioned medium, but in the present invention, the medium must satisfy the following conditions. ) and nitrate ion H (a/b) is usually 0.2 or more, preferably 0.5
Tissue culture of plants of the genus Zboisia is performed using the above-mentioned medium. A medium having such a composition ratio can be prepared by selecting any component from the above-mentioned inorganic components and selecting the amount added as appropriate. 5 Usually, for example, ammonium nitrate and potassium nitrate or components such as ammonium nitrate, ammonium chloride, and potassium chloride are mainly used to adjust the ratio (a/b) of ammonium ions () and nitrate ions () to 0.2 in the culture medium.
It is preferable to employ the +filtration method above. In the present invention, when the ratio (a/b) is usually less than 0.2, the amount of tropane alkaloid produced by tissue culture decreases.

Moreover, in this case, the production ratio of more expensive scopolamine is also reduced, which is not preferable. In the present invention, the ratio (a/b
Although the upper limit of ) is not particularly limited, it is usually preferable to keep the ratio in the range of 0.5 to 2.

The concentrations of ammonium ions @) and nitrate ions (b>) in the culture medium of the present invention can be normally set in the same concentration range (about 0.1 μM to about 100 mM) as the above-mentioned inorganic components. So ammonium ion (a)
The concentration is usually preferably in the range of 4mM to 40mM since the amount of tropane alkaloid produced will increase. On the other hand, regarding the nitrate ion (b) concentration, in the present invention, the a/b ratio is set to 0.2 or more as described above, so the nitrate ion concentration is determined to satisfy this condition.

As mentioned above, the present inventors set the concentration ratio (a/b) of ammonium ions ea) and nitrate ions (b) in the medium to 0.
．． The present invention was based on the discovery that when tissue culture of plants of the genus Zboisia is carried out at a concentration of 2 or more, the amount of tropane alkaloids produced increases significantly compared to the conventional method, and the production ratio of scopolamine also increases. Although the method of the present invention has been completed, the results are completely unexpected and surprising based on the conventional knowledge in this field. That is, in the past, for example! vF Publication No. 59-169488 states that when culturing Korean ginseng as a plant to obtain adventitious roots as cultured cells, it is desirable to remove ammonium ions, and in this case, the ratio (a/ b)
is 0.2 or less. In addition, in conventional tissue culture of plants belonging to the genus Zboisia, 1, for example, the above-mentioned Lori Plant Ce
1ls Reports 3.186-188(198
In the method shown in 4), the ammonium ion concentration is usually as low as 2mM, such as in Gamborg's B5 medium, and the ratio (a/b) of ammonium ion (b) to nitrate ion (b) is 0.2 or less. This method uses a culture medium, and in this case, the amount of tropane alkaloid produced is low. The present inventors acknowledged that conventional tissue culture in this field is in the above situation, and as a result of detailed investigation into the influence of medium composition on tissue culture of plants of the genus Zupoysia, they found that the presence of ammonium ions and the presence of ammonium ions and nitric acid. It was discovered that the ratio of ions greatly affects the growth of cultured cells obtained by tissue culture and the content of tropane-based alkaloids produced in the cells, and by studying this, the present invention was completed. This is as described above. In the tissue culture of the present invention, tissue pieces of plants belonging to the genus Zboisia are cultured using the above-mentioned medium to obtain cultured cells containing tropane alkaloids. Specific examples of the tissue pieces in this case include roots, leaves, stems, seeds, flower buds, etc., and the following tissue culture method for obtaining cultured cells containing the alkaloid. That is, usually in the present invention, callus is induced from these tissue pieces, and the cultured cells obtained by subculturing the callus are propagated and cultured using the medium of the present invention to form a large amount of culture containing tropane alkaloids. cells are obtained. In this case, in the present invention, it is particularly preferable to use roots and/or adventitious roots formed during callus induction as cultured cells, subculture them, and propagate them.

When using roots as plant tissue pieces, it is also possible to adopt a method of culturing the roots as they are to obtain cultured cells.

In the present invention, when roots and/or adventitious roots formed during callus induction are used, plant tissue pieces are used for callus induction, such as Agrobacterium.
rhizogenes) and the hair roots that emerge thereby can also be used.

Specific examples of the tropane alkaloid obtained by the method of the present invention include scopolamine, hyoscyamine, and acenal 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 tropane-based alkaloids may be used, for example, in the case of 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.

〔Effect of the invention〕

By employing the tissue culture method for plants of the genus Zboisia 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 myopo grown in our medicinal herb garden
-roides R8Br, leaves were washed and soaked in 10% antiformin solution for 10 minutes, then washed three times with sterile water, then cut into approximately 1 crn and treated with naphthaleneacetic acid and benzyladenine at 105M and 10-6M, respectively.
The cells were placed on a Linsmeyer-Skoog agar medium supplemented with the following conditions, and cultured at 25°C for 30 days. Adventitious roots generated at the same time as callus formation were cut out, transplanted into an Etch Niche liquid medium supplemented with indolebutyric acid at a concentration of 10-5M, and subcultured for 6 months. The adventitious roots thus obtained (8.1 dry weight) were cut into 1 to 2 crn,
4.6mM ammonium ion and 2mM nitrate ion
The cells were transplanted into a culture tank with a volume of 151 containing a liquid medium 101 prepared by adding indolebutyric acid to a concentration of 10 −5 M to an Etch Niche medium modified to contain 2.8 mM, and cultured with aeration for 3 weeks. Obtained adventitious root 112. F (dry weight)
After drying, add 10E of basic chloroform-methanol solution.
Extracted with. 84 1N sulfuric acid was added to this to transfer the alkaloid to the sulfuric acid layer. Furthermore, 400 m of ammonia water and 81' of chloroform were added to transfer the alkaloid to the chloroform layer, which was concentrated under reduced pressure to obtain 2.7 g of extraction residue.
A portion was analyzed using a gas chromatograph mass spectrometer.

The extraction residue was dissolved in 1N sulfuric acid '11, made weakly alkaline with sodium bicarbonate, scopolamine was separated with chloroform, and the chloroform was distilled off to obtain a syrupy base. This was neutralized with dilute hydrobromic acid, and after concentration, 440 my crystals of scopolamine hydrobromide were obtained. After adding 17um of sodium hydroxide from 41 to the extraction residue, extracting with ether,
from ether solution.

102 mp of crystals of hyoscyamine were obtained.

Table 1 shows the concentrations of ammonium ions and nitrate ions and the amount of alkaloids produced in this S combination medium.

The following columns were used for alkaloid analysis.

Power 7m: 5ilicone 0V-17 (1%) on
Chromosorb W (Mesh80-10100) 3$xH7L Glass column carrier gas: He Column temperature: 200°C Examples 2-4 and Comparative Examples 1-2 The concentrations of ammonium ions and nitrate ions in the culture medium were as shown in Table 1. The results were shown in Table 1 in the same manner as in Example 1 except that .

Claims (1)

[Claims] (1) Production of tropane-based alkaloids by tissue culturing plants belonging to the genus Zboisia using a medium in which the ratio (a/b) of ammonium ions (a) and nitrate ions (b) is 0.2 or more. Features of Zboisia tissue culture method.