JPH01281078A - Novel callus induced cell and production thereof - Google Patents

Abstract

PURPOSE:To obtain a callus induced cell capable of producing a large amount of lignans by transplanting a callus cell derived from a tissue fragment of a plant belonging to the genus Schizandra into a medium containing a specific plant growth regulator and cultivating. CONSTITUTION:A callus cell derived from a tissue fragment of a plant belonging to the genus Schizandra is transplanted into a medium containing at least one plant growth regulator selected from a group consisting of indoleacetic acid, 2,4-dichlorophenoxyacetic acid, alpha-naphthaleneacetic acid and indolebutyric acid or at least one plant growth regulator selected from the group and at least one plant growth regulator selected a group consisting of kinetin, zeatin, dihydrozeatin, benzyladenine and isopentenyladenine and cultivated. Consequently, a callus derivative capable of producing a large amount of lignans having improving action on liver disorder and antitumor action is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to novel callus-inducing cells that produce lignans useful as pharmaceuticals, and a method for producing the callus-inducing cells.

[Prior Art and Problems] Chosengomin, which is a plant belonging to the genus Schizandra, has been used in the formulation of sho-seitoba, seisho-ekki-to, etc. since ancient times. Furthermore, its constituent lignans [
Schizandrin A to D, deoxyschizandrin
n).

gomisin A = D, F - H
, J., pregomisin, etc.] is well known to be a useful substance as a pharmaceutical having liver damage-improving effect and anti-ulcer effect. An industrial synthesis method for these lignans has not yet been reported, and in order to supply them as pharmaceuticals, the method of extracting and purifying them from natural plants of the genus Schizandra can be cited. However, this method has the following reasons: ■ The amount of lignans contained in the plant is very small and the content is unstable, ■ It takes a long time to cultivate the plant, and ■ The amount of cultivation is small. The method is short and good enough for mass production.

Therefore, one possible method for mass production is to tissue culture large quantities of callus cells derived from tissue pieces of Schizandra plants and extract lignans from the cells. There is no report that lignans were contained in callus cells. Also,
Although it is possible to obtain plants in a short period of time by tissue culture, it is difficult to say that this method is suitable for producing large amounts of lignans.

[Means for Solving the Problems] The present inventors cultivated a large amount of callus cells derived from tissue pieces of Schizandra plants, and established a method for extracting dananes from the cells. As a result of intensive studies to find callus cells that have the ability to produce lignans, we have obtained a new callus-inducing cell that has the ability to produce lignans.

That is, the present invention provides callus cells induced from tissue pieces of plants of the genus Schizandra, treated with at least one plant growth regulator selected from indoleacetic acid, 2,4-dichlorophenoxyacetic acid, α-naphthaleneacetic acid, and indolebutyric acid, or indoleacetic acid. , 2.4-dichlorophenoxyacetic acid, α-naphthaleneacetic acid, and indolebutyric acid, and at least one plant selected from kinetin, zeatin, dihydrozeatin, benzyladenine, and isopentenyladenine. New callus-induced cells obtained by transplanting and culturing in a medium containing a growth regulator, and callus cells induced from tissue pieces of Schizandra plants, were treated with indole acetic acid, 2
．． 4-Zine [10 At least one plant growth regulator selected from phenoxyacetic acid, α-naphthaleneacetic acid, and indolebutyric acid, or at least one selected from indoleacetic acid, 2,4-dichlorophenoxyacetic acid, α-naphthaleneacetic acid, and indolebutyric acid and at least one plant growth regulator selected from kinetin, zeatin, dihydro [1] zeatin, benzyladenine and isopentenyl adenine, and cultured to obtain new callus-inducing cells. This is a novel method for producing callus-induced cells.

[Example] The present invention will be explained in detail below.

Examples of plants of the genus Schizandra in the present invention include plants of the genus Schizandra of the family Elegantaceae, and Schizandra chinensis.

In order to induce callus cells as raw materials from tissue pieces of Schizandra plants, first, callus cells from tissues such as polar parts, petioles, stems, roots, inflorescences, fruits, etc. of Schizandra plants, such as leaf tissues, are Tissue pieces cut to about 3 to 7 R11, root tissues cut to about 5 to +OXX, fruit tissues cut to about 2 to 4 pieces, etc. are sterilized using the commonly used general sterilization method ( For example, after sterilization using an ethanol aqueous solution, hypochlorous acid aqueous solution, sterilized water, etc.), leave it in a culture medium and store it under aerobic conditions at a temperature of 20~
Examples include a method of culturing at 30°C, preferably 25°C, for 4 to 8 weeks. At this time, it is okay to shine light on it.

As a medium, use a solid medium that is based on various known inorganic synthetic media, to which sugars, plant growth regulators, vitamins, natural substances, etc. are added, and solidified by adding agar or gellan gum, etc. I can do it.

Typical synthetic media include White's medium, Murashige & Skoog
Koog's medium (hereinafter referred to as MS medium), Linsmeier & Sko
og) medium, Nitsch medium, Nitsch & N1tsch medium, Gamborg et al. medium, Fle
ller) medium, and other mediums based on these mediums,
Examples include Shino, etc. whose composition has been changed.

Specific examples of inorganic components of the inorganic synthetic medium include copper, nitrogen,
Phosphorus, potassium, calcium, magnesium, sulfur,
Iron, manganese, zinc, boron, molybdenum, chlorine, sodium, iodine, Kobal I, etc. More specifically, potassium nitrate, sodium nitrate, calcium nitrate, ammonium nitrate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, ethylenediaminetetraacetic acid Sodium, potassium chloride, calcium chloride, magnesium sulfate, sodium sulfate, ammonium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, zinc sulfate, boric acid, copper sulfate, sodium molybdate, molybdenum trioxide, potassium iodide, Examples include cobalt chloride.

Specific examples of sugars include carbohydrates such as sucrose, inositol, and glucose, and derivatives thereof.

A specific example of a plant growth regulator is indole acetic acid (
IAA), α-naphthaleneacetic acid (NAA).

Auxins such as 2,4-dichlorophenoxyacetic acid (2,4-D) and indolebutyric acid (IBA), kinetin, zeatin, dihydrozeadin, benzyladenine (I
3A), cytokinins such as isopentenyl adenine, and the like.

Specific examples of vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B8), pantothenic acid, ascorbic acid (vitamin C), nicotinic acid, etc., and specific examples of amino acids include glycine, alanine, etc. , glutamine, cysteine, etc.

Specific examples of natural substances include casein hydrolyzate, coconut milk, yeast extract, etc. In addition to the above-mentioned specific examples, substances necessary for culture may be added as appropriate.

Moreover, a stable strain can be obtained by subculturing the callus cells obtained in this way two to three times under the culture conditions used for the above-mentioned callus induction.

Below, specific examples of the production of callus cells induced from tissue pieces of plants of the genus Schizandra, which are the raw materials for the method for producing novel callus-inducing cells of plants of the genus Schizandra of the present invention, will be shown.

Specific Example 1 The collected leaves of Schistemrum were lightly washed, rinsed thoroughly with running water, and washed for 15 minutes in tap water with a few drops of a surfactant (household synthetic detergent) added while stirring to avoid damage. This operation was repeated twice and further rinsed under running water for 1 hour. It was immersed in 70% ethanol for 1 minute, then sterilized with a 2% aqueous sodium hypochlorite solution for 7 minutes, and then washed three times with sterile water. Sterilize this to approximately S XX
After cutting each sample, MS medium (2,4-D 0 , 5
ppm, kinetin 0, 5 ppIll, agar 08
% larvae) and cultured in the dark at 25°C. After 1 week, small pieces enlarged, and after 3 weeks, callus cells were obtained (
Induction success rate 75°0%).

Specific Example 2 The roots of the collected Schisandra japonica were thoroughly cleaned of mud, rinsed thoroughly with running water, and washed for 15 minutes in tap water with a few drops of a surfactant (household synthetic detergent) added while stirring. This operation was repeated three times, and further rinsed under running water for 2 hours. 7
Immersed in 0% ethanol for 1 minute, then sterilized with 2% sodium hypochlorite aqueous solution for 10 minutes, and then sterilized with sterilized water for 3 minutes.
Washed twice. After cutting this aseptically into about 5 categories,
The cells were placed on MS medium (containing 2,4-D 0 , 1 ppm, benzyladenine 0, 3 ppm, and 0.8% agar) and cultured in the dark at 25°C. After one week, the small pieces enlarged. Callus cells were obtained after 6 weeks (induction success rate 6
9.5%).

Specific Example 3 The seeds of the collected Schistemrum were taken out, washed, rinsed thoroughly with running water, and washed for 15 minutes with stirring in tap water with a few drops of a surfactant (household synthetic detergent) added. .

This operation was repeated three times, and the product was further rinsed under running water for 1 hour. It was immersed in 70% ethanol for 1 minute, then sterilized with a 2% aqueous sodium hypochlorite solution for 7 minutes, and then washed three times with sterile water. After dividing this into two aseptically, MS medium (2,4-Do, 5 ppm, benzyladenine 0
, 5 ppm, containing 0.8% agar) and cultured at 25°C in the dark. After one week, small pieces enlarged, and 4
Callus cells were obtained after a week (induction success rate 80.0%).

The callus cells obtained in the same way were treated with indole acetic acid,
At least one plant growth regulator selected from 2.4-dichlorophenoxyacetic acid, α-naphthaleneacetic acid and indolebutyric acid, or at least one selected from indoleacetic acid, 2.4-dichlorophenoxyacetic acid, α-naphthaleneacetic acid and indolebutyric acid and at least one plant growth regulator selected from kinetin, zeatin, dihydrozeatin, benzyladenine, and isopentenyladenine, under aerobic conditions, at a temperature of 20 to 30°C. Culture is preferably carried out at 25°C for 2 to 8 weeks. At this time, light irradiation is not necessarily required, but light may be irradiated depending on the case.

The medium is a liquid medium based on various known inorganic synthetic media, to which inorganic components, sugars, vitamins, natural substances, etc. are added, or agar or gellan gum, etc. is added to these liquid mediums and solidified. A solid medium containing at least one plant growth regulator selected from indoleacetic acid, 2,4-dichlorophenoxyacetic acid, α-naphthaleneacetic acid, and indolebutyric acid, or indoleacetic acid, 2,4-dichlorophenoxyacetic acid,
Using a medium containing at least one plant growth regulator selected from α-naphthaleneacetic acid and indolebutyric acid and at least one plant growth regulator selected from kinetin, zeatin, dihydrozeatin, benzyladenine, and isopentenyladenine. I can do it.

Typical synthetic media include White's medium, MS medium, Linsmeyer-Skoog's medium, Niche's medium, Niche-Hetch's medium, Gamborg et al.'s medium, Heller's medium, and others. , those with changed compositions, etc.

Specific examples of inorganic components of the inorganic synthetic medium include copper, nitrogen,
Phosphorus, potassium, calcium, magnesium, sulfur,
Iron, manganese, zinc, boron, molybdenum, chlorine, sodium, iodine, cobalt, etc. More specifically, potassium nitrate, sodium nitrate, calcium nitrate, ammonium nitrate, potassium dihydrogen phosphate, sodium trihydrogen phosphate, ethylenenoaminetetraacetic acid sodium, potassium chloride,
Calcium chloride, magnesium sulfate, sodium sulfate,
Examples include ammonium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, zinc sulfate, boric acid, copper sulfate, sodium molybdate, molybdenum trioxide, potassium iodide, cobalt chloride, and the like.

Specific examples of sugars include carbohydrates such as sucrose, inotol, and glucose, and derivatives thereof.

Specific examples of vitamins include biotin, thiamine (vitamin B1), pyridoquinone (vitamin B),
Examples include pantothenic acid, ascorbic acid (vitamin C), nicotinic acid, etc., and specific examples of amino acids include glycine, alanine, glutamine, cysteine, etc.

Specific examples of natural substances include casein hydrolyzate, coconut milk, yeast extract, etc. In addition to the above-mentioned specific examples, substances necessary for culture may be added as appropriate.

A plant growth regulating substance is added to the medium containing the above components, and indole acetic acid, 2.4
- When adding at least one plant growth regulator selected from dichlorophenoxyacetic acid, α-naphthaleneacetic acid and indolebutyric acid, the concentration should be between 0.1 and 50%.
×l Q-IIM, especially 0.5~l Ox l O-”
It is preferable to set it to M.

Also, at least one plant growth regulator selected from indoleacetic acid, 2,4-dichlorophenoxyacetic acid, α-naphthaleneacetic acid, and indolebutyric acid, and at least one plant growth regulator selected from kinetin, zeatin, dihydrozeatin, benzyladenine, and isopentenyladenine. When adding a plant growth regulator, the concentration of at least one plant growth regulator selected from indoleacetic acid, 2,4-dichlorophenoxyacetic acid, α-naphthaleneacetic acid, and indolebutyric acid is 0.1 to 50×.
l O-"M, especially from 0.5 to 10 x 10-"M,
The concentration of at least one plant growth regulator selected from kinetin, zeatin, dihydrozeatin, benzyladenine and isopentenyladenine is 0.01 to 1.
It is preferable to set it to 0XIO-'M, especially 0.1 to 5XIO-'M.

[Effects of the Invention] According to the present invention, lignans that can be used in pharmaceuticals can be obtained in large quantities and at low cost in a short period of time.

[Examples] Hereinafter, the present invention will be specifically explained with reference to production examples, but the present invention is not limited thereto in any way.

Examples 1 to 6 The calli obtained in Examples 1 to 3 were cut into small pieces, each of which was placed in a medium with the conditions shown below, and cultured in the dark at 25°C for 4 weeks or more to obtain callus-induced cells. Ta. These callus-induced cells contained lignans as shown below.

Claims (2)

[Claims] (1) Callus cells derived from tissue pieces of plants of the genus Schizandra are treated with at least one plant growth regulator selected from indoleacetic acid, 2,4-dichlorophenoxyacetic acid, α-naphthaleneacetic acid, and indolebutyric acid, or indoleacetic acid, 2 , 4-dichlorophenoxyacetic acid, α-naphthaleneacetic acid, and indolebutyric acid; and at least one plant growth regulator selected from kinetin, zeatin, dihydrozeatin, benzyladenine, and isopentenyladenine. Novel callus-induced cells obtained by transplanting and culturing in a medium containing (2) Callus cells derived from tissue pieces of Schizandra plants are treated with at least one plant growth regulator selected from indoleacetic acid, 2,4-dichlorophenoxyacetic acid, α-naphthaleneacetic acid, and indolebutyric acid, or indoleacetic acid, 2 , 4-dichlorophenoxyacetic acid, α-naphthaleneacetic acid, and indolebutyric acid; and at least one plant growth regulator selected from kinetin, zeatin, dihydrozeatin, benzyladenine, and isopentenyladenine. 1. A method for producing a new callus-inducing cell, which comprises transplanting and culturing it in a medium containing the same, to obtain a new callus-inducing cell.