JPH02303430A - Method for carrying out tissue culture of adventive root of tropane-based alkaloid producing plant - Google Patents

Abstract

PURPOSE:To obtain the subject adventive root useful as a medicine in high yield by culturing the adventive root in specific concentrations of nitric acid ion and sugar so as to be capable of culturing the adventive root at a high concentration when adventive root of tropane-based alkaloid producing plant is subjected to liquid culture. CONSTITUTION:A liquid culture medium or culture ingredient is fed to a liquid medium obtained by adding an adventive root of tropane-based alkaloid producing plant (e.g. Duboisia myoporoides) to a liquid medium, preferably of Nitsch & Nitsch, etc., containing a plant hormone, etc., so as to retain 10 to 30mM nitric acid and 2 to 10% sugar concentrations during culturing period and the adventive root is cultured at a high concentration (concentration of tissue-culture material at culture starting time of >=25g/l by fresh weight or concentration of the material at culture finishing time of >=200g/l in a culture tank) to provide the objective adventive root.

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 a slimming agent, analgesic, and a new parasympathetic nerve-stimulating drug, and hyoscyamine is used as a new parasympathetic nerve-stimulating drug. 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, there is a known example of production by Yamada et al. using henbane callus (Plant Ce1l).
iepor ts soil, 101. -103 (1982)
), but the scobolamine content was 20 ppm, which was lower than the content in natural plants. Also, Yamada et al.
Duboisia (Duboisia Leichhardt)
It has been found that significant amounts of scopolamine and hyoscyamine are present in adventitious roots obtained by tissue culture of Plant Ce1l Re.
ports 3.186-188 (1984)),
The amount was still not enough. Therefore,
Examining various culture conditions for Zboisia adventitious roots, the ratio of ammonium ions to nitrate ions in the medium should be 0.2 or more, and the dissolved oxygen concentration in the medium should be 10 to 65 ppa.
The present applicant has discovered that the productivity of tropane alkaloids can be improved by adding
Although a patent application has been filed for this technology, from an industrial standpoint, it is desirable to further increase its productivity.

(i! 18 to be solved by the invention) Therefore, when aiming at industrial production of tropane-based alkaloids by such tissue culture, in order to further increase productivity, the yield of the tissue fiber culture per unit volume of the culture tank is An important issue was to increase the

The yield of tissue culture (cultured cells, etc.) is determined by the nutrient base f(
Because it depends on the concentration of culture medium (medium components), the nutrient substrate is consumed during culture in the Linsmeyer and Skoog medium, White's medium, and Etch & Niche medium, which are often used in conventional culture, and tissue culture It is not possible to culture at high concentrations. In addition, as a method to increase the concentration of tissue culture, it is possible to culture the culture medium with a higher concentration of nutrient substrate than before. There are limitations to increasing tissue culture productivity by increasing nutrient substrate concentrations because of concentration insults.

In view of these circumstances, the present inventors have researched a method for efficiently cultivating adventitious roots of plants that produce tropane alkaloids, such as Zboisia, Datura, Hacilidokoro, and Hyosus, by increasing the concentration, and have found the following results. I discovered a fact.

[Means to solve the problem]

As a result, the present inventors found that during the culture period, the nitrate ion concentration in the liquid medium was 10 to 30 IIM, tJ! 1 degree to 2 to 10
By culturing the adventitious roots of plants that produce tropane alkaloids at a high concentration by performing liquid culture or supplying medium components into the culture tank so as to maintain the concentration at The present inventors have discovered that it is possible to culture the cells without causing any damage, leading to the completion of the present invention.

That is, according to the present invention, in a method of tissue culturing adventitious roots of plants that produce tropane-based alkaloids by liquid culture, the adventitious roots are cultured at a high concentration, and the nitrate ion concentration in the liquid medium is reduced to 10% throughout the culture period. -b There is provided a tissue culture method for adventitious roots of plants producing tropane-based alkaloids, which comprises supplying a liquid medium or medium components into a culture tank.

In the above, high concentration means that the concentration of the tissue culture is higher than that in conventional tissue culture. In particular, in the mm culture method of the present invention, the concentration of the tissue culture in the culture tank at the start of culture is 25g/1 expressed as fresh weight of item
higher concentration, or the concentration of tissue culture in the culture tank at the end of incubation expressed as fresh weight of tissue culture is 2
00g/7! This is particularly effective when culturing at a high concentration.

In the present invention, tissue culture is carried out using plants that produce tropane-based alkaloids.
myoporoides), Duboisia Ieichhardtii
), plants of the genus Zboisia, such as Datura
a tatura), Datura arborea
a arborea), Datura stramonium (Da
plants of the genus Datura, such as (Tura stramonium);
Scopolia japonica
plants of the genus Scopolia such as llyoscyamus niger;
For example, plants of the genus Atroba such as Atroba (belladonna) can be exemplified.

In the present invention, when producing troban alkaloids by culturing the adventitious roots of the plants, liquid culture or medium components are supplied during the culture period. This will be explained in detail below.

In the present invention, the medium components may be supplied continuously or discontinuously, and the medium may be continuously or discontinuously extracted from the culture tank during the culture period. .

In the present invention, during the culture period, the nitrate ion concentration in the culture solution is 10 to 30 IIIM, preferably 20 to 10
Provide a liquid medium or medium components containing an amount of nitric acid to maintain it at mM. Also, during the culture period, the sugar concentration in the culture solution was adjusted to 2 to 1
A liquid medium or medium component is fed containing an amount of sugar to maintain it at 0%, preferably 2-3%.

Examples of sugar sources that can be used include carbohydrates such as sig1 #N and derivatives thereof, and sig13 is preferably used. Other components in the liquid medium or medium components to be supplied are not particularly limited, and the medium components used at the start of culture may be used.

Regarding the medium components used in the present invention at the start of culture,
Inorganic components include nitrogen, phosphorus, potassium, sodium,
Examples include inorganic salts containing elements such as calcium, magnesium, sulfur, iron, manganese, zinc, boron, molybdenum, chlorine, iodine, and cobalt, specifically potassium nitrate, sodium nitrate, ammonium nitrate, ammonium chloride, and potassium chloride. , calcium chloride, phosphate l
Potassium hydrogen, potassium hydrogen phosphate, magnesium sulfate, magnesium chloride, sodium sulfate, ferrous sulfate,
Examples include compounds such as ferric sulfate, manganese sulfate, copper sulfate, sodium molybdate, molybdenum trioxide, potassium iodide, zinc sulfate, boric acid, and cobalt chloride.

As a carbon source for the medium, C! Examples include carbohydrates such as sugars, their derivatives, fatty acids and organic acids.

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 generally desirable 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 (0,021 ng/f) or less.

The vitamins in the medium include biotin, thiamine (vitamin 8), pyridoxine (vitamin B&), pyridoxal, pyridoxamine, calcium pantothenate,
Examples include ascorbic acid (vitamin C), inositol, nicotinic acid, nicotinamide, and riboflavin (vitamin aX).

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

The medium of the present invention has a nitrate ion concentration and a sugar concentration in the specified ranges, the other inorganic components in the range of about 0.1 μH to about 100 mM, and the carbon source in the range of about 1 g/g.
1 to about 10 h/l of the plant hormones #O,O
XμH to about 100μi, and the vitamins are about 0.1
1/2 to about 150 mg/lfi and 0 to about 100 mg/lfi of the above-mentioned amino acids are preferably used.

Specifically, the medium used in the tissue culture of the present invention may be a conventionally known culture medium used in plant tissue culture, such as Murashige and Skoog ('62) (
Murashige & Skoog) medium, Linsmeyer-Skoog (RM-1965) (Lins
Maier & Skoog) medium, white ('
63) (White) medium, Gamborg (
Gamborg's B-5 medium, Mitsui's M9 medium, Nitsch N1tsct+ medium, etc., the carbon source and plant hormones described above were added, and if necessary, the vitamins and amino acids described above were added. For example, in the present invention, a medium prepared using an Etch-Etchi, Linsmeyer-Skoog, or Murashige-Skoog medium is particularly preferred. Regarding the composition of the above-mentioned conventionally known culture medium, for example, see "New Plant Tissue 2 Culture J.
P386-P391, Breakfast Shoten, 1979.

In the Miho culture of the present invention, adventitious roots of plants that metabolize tropane alkaloids are tissue cultured using the above medium to obtain adventitious roots containing tropane alkaloids.

Specifically, the adventitious roots of the plant used in the tissue culture of the present invention include tissue pieces or cells such as roots, leaves, stems, seeds, flower buds, etc. of the plant used in the tissue culture of the present invention or other conventional tissues. An example of this is adventitious roots obtained by culturing in m-weave culture using the culture method.

If adventitious roots as described above are tissue cultured by the method of the present invention, they can be cultured at a high concentration, and therefore, the yield of culture per unit volume of the culture tank can be increased,
The yield of tropane alkaloids can be increased to the same level as above.

In the present invention, when using adventitious roots, plant tissue pieces may be infected with, for example, hairy root disease fungi (e.g. Agrobacterumrhumrhi).
zogenes) and the resulting hair roots can 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 alkaloids include scopolamine, hyoscyamine, and acetyl compounds of these compounds, and among these, scopolamine and 3J-styamine are preferred.

In the present invention, the method for extracting the alkaloid 1 from cultured cells containing the tropane alkaloid is as follows:
For example, conventional methods described in the Pharmacopoeia Law and the like that have been used for isolating and purifying tropane-based alkaloids from plants containing these compounds can be employed.

〔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 medicinal herb area
oidesR,Br leaves were washed, immersed in 10% antiformin solution for 10 min, then washed 3 times with sterile water, then cut into approximately ICuts, and naphthalene acetate and hensyl adenine were added to lfl'M and 10- The cells were plated on a Linsmeyer-Skoog agar medium supplemented with a concentration of 'M, and cultured at 25°C for 30 days. Adventitious roots that appeared at the same time as callus formation were cut out, and indolebutyric acid was added to
The cells were transplanted into an etch-etch liquid medium supplemented to a concentration of 0-'H, and subcultured for 2 years. 1 g (fresh weight) of the adventitious roots thus obtained was treated with indole acetic acid for 10-'H.
Etch Niche's liquid medium (nitrate ions) 8mM with a phosphate concentration of 2.5mF, sugar 3
%) to a 100 g Erlenmeyer flask containing 20 d of cellulose, and culture with shaking was started. The culture solution was sampled and the nitrate ion concentration and sugar concentration were measured.Based on these measurements, the culture solution contained the necessary nitrate ions and sugar to make the nitrate ion concentration in the culture solution 18mM and the sugar concentration 3%. 1 ml of liquid medium was added.Furthermore, similar operations were carried out on days 10, 12, 14, 17 and 188, and culture was carried out on day 211.

The obtained adventitious roots were dried and then extracted with 50 mf of a basic chloroform-methanol solution. 40 m/1N sulfuric acid was added to this, and the alkaloid layer was transferred to the sulfuric acid layer. moreover,
Ammonia water 2I+1/and chloroform 40ni/
was added to transfer the alkaloid V 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.

The sugar concentration is measured using a saccharimeter, and the nitrate ion concentration is measured using the brucine method (CJls, K 0102 (1981)).
I went there. Gas chromatograph analysis was performed under the following conditions.

Column: Si! 1cone O sea 17 (1%) o
nChromosorb W (Mesh 80-10
100) 3φX1m glass column Carrier gas: N2 Column temperature: 200°C Comparative Example 1 Table 1 shows the results of carrying out the same procedure as in Example 1 except that the addition during culturing was not performed.

Example 2 Table 1 shows the results obtained in the same manner as in Example 1 except that 1 ml of the liquid medium added in Example 1 further contained other 2.7-tinisoti medium components in addition to nitrate ions and sugar. Indicated.

Example 3 In Example 2, the sugar concentration at the start of culture was set to 5%, and the sugar concentration was maintained at 3% only when the sugar concentration became 3% or less by measuring sugar at the time of addition during culture. Table 1 shows the results of the same procedure as in Example 1, including the addition of nitrate ions, except for the addition of sugar.

Comparative Example 2 In Comparative Example], the nitrate ion concentration at the start of culture was set to 50 m
Table 1 shows the results of the same procedure as in the comparative example except that the sugar concentration was 12%.

(Margins below this page) Table 1 [Effects of the Invention] If the method for producing tropane alkaloids by tissue culture of the present invention is adopted, a large amount of tropane alkaloids, especially scopolamine and/or hyoscyamine, can be produced compared to conventional methods. can be produced efficiently.

Applicant: Mitsui Petrochemical Industries, Ltd. Agent, patent attorney Yusuke Hiraki Same patent attorney Sadaji Ishii

Claims (1)

[Scope of Claims] 1. A method for tissue culturing adventitious roots of plants that produce tropane-based alkaloids by liquid culture, in which adventitious roots are cultured at a high concentration, and the nitrate ion concentration in the liquid medium is maintained at 10 to 30 mM throughout the culture period. , and sugar concentration 2-10%
A method for culturing adventitious roots of a plant producing tropane-based alkaloids, the method comprising supplying a liquid medium or medium components into a culture tank so as to maintain the tropane-based alkaloids. 2. The concentration of tissue culture in the culture tank at the start of culture is higher than 25 g/l expressed as fresh weight of tissue culture, or the concentration of tissue culture in the culture tank at the end of culture is higher than tissue culture 2. A tissue culture method according to claim 1, characterized in that the concentration is higher than 200 g/l expressed in fresh weight of the tissue.