JPH02215397A - Production of panaxsaponin by tissue culture of panax japonicus - Google Patents

Abstract

PURPOSE:To accomplish stable mass production of panaxsaponin in high efficiency in a short period by making a culture of callus produced by culture of the tissue of Panax Japonicus in an organic medium containing a nitrogen source such as NH and a carbon source such as glucose. CONSTITUTION:A plant body such as the root of Panax Japonicus is cut, sterilized with ethyl alcohol, and inoculated into a Murashige.Skoog medium containing 0.01-5ppm of an auxin (e.g. indoleacetic acid) followed by culture in a dark place at 20-25 deg.C for 10-30 days to produce callus. An organic culture medium is prepared by formulating a nitrogen source such as NH4NO3 so as to be 2.8-55.6mM (0.05-1.0g/l) of NH and 11.3-80.6mM (0.7-5.0g/l) of NO and a carbon source so as to be 3-7 wt.% of sucrose and/or 3-5wt.% of glucose. Thence, the above callus is inoculated into the organic medium and put to culture in a dark place at 20-25 deg.C. The resultant callus is dried under an air stream, ground and extracted with e.g. ca.50% ethanol for ca.24hr, thus obtaining a liquid extract. This liquid extract is purified by e.g. chromatography to produce the objective panaxsaponin.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for efficiently producing chikusetsusaponin by tissue culturing callus obtained from horse chestnut ginseng.

(Prior art) Panax ginseng (Panax Japonicus C)
,^0Meyer) is a type of medicinal ginseng, and its roots and stems are called the herbal medicine ``chikusetsu ginseng'' and are known to have medicinal effects such as improving stomach health, expectoration, and fever reduction. The medicinal components of "Ginseng" are mainly saponins, and saponins Ib, TV, rVa, V, and m are known. Among these, it has been reported that tikusetusaponin■ has an anti-stress ulcer effect (Pharmaceutical Journal 10
7(2), 135-139 (1987)).

Chikusetsu saponin is currently obtained from "Chikusetsu ginseng", which is obtained by cultivating horse chestnut ginseng. However, horse chestnut ginseng is only cultivated in specific regions (Nagano Prefecture, Fukushima Prefecture, Tohoku region, etc.), and there is a limit to the amount of labor available to nine people. Moreover, since it is cultivated under natural conditions, the season, weather,
It is easily influenced by soil quality etc. Furthermore, several years of cultivation are required before the roots and stems can be used as herbal medicines. As described above, it is not possible to stably obtain large amounts of Chiksetsusaponin due to constraints on the cultivation of the raw material, horse chestnut ginseng.

(Problem to be solved by the invention) The present invention solves the above-mentioned conventional problems.

The purpose of this invention is to provide a method for efficiently producing chiksetsusaponin from horse chestnut ginseng. Another object of the present invention is to provide a method for stably obtaining a large amount of saponin without being affected by natural conditions such as season, weather, and soil quality. Still another object of the present invention is to provide a method for efficiently producing Chikusetsaponin by tissue culture of Panax ginseng.

(Means for Solving the Problems) The method for producing chikusetsusaponin of the present invention is... (a)
) Panax Japonicus C
, 80 Meyer) tissue to induce callus; (b) the obtained callus is grown in an organic medium containing NH,+ and NOs- as a nitrogen source and sucrose and/or glucose as a carbon source; and (C) the step of extracting chikusetsusaponin from the callus, thereby achieving the above object.

To produce Chikusetsu saponin by the method of the present invention, first, the roots of horse chestnut, one stem, one leaf, and one seed of the plant are cut, sterilized with alcohol, etc., and callus is induced by a normal plant tissue culture method. .

Culture conditions do not need to be particularly special. As a medium,
Murashige-Skoog's medium, White's medium, Linsmeyer-Skoog's medium, Gauslett's medium, Heller's medium, Gamborg's medium, and modified media thereof, which are commonly used for plant tissue culture, can be used. In particular, Murashige-Skoog's medium is preferably used. In addition, to further promote callus induction, plant hormones (auxins, cytokinins, etc.) may be added as necessary.
is added. One example is auxin.

2,4-dichlorophenoxyacetic acid (2,4-D),
Indole acetic acid (IAA), indole 3-butyric acid (I
BA).

Examples include naphthalene acetic acid (NAA). There are 2 cytokinins, such as kinetin, benzyladenine (B
^) etc. Usually, auxins are 0.01 to 5p
pm ratio, cytokinins are 0.1 to 10 ppm
is added to the medium at a ratio of Usually 15-35 in the dark
When cultured at ℃, a callus is formed on the cut surface of the tissue after 10 to 30 days. This callus is transferred to an appropriate solid medium such as an agar medium and grown to an appropriate amount. Furthermore, if necessary, this callus is transferred to a solid medium or a liquid medium and culture is continued. The same medium as above can be used for this medium, and Murashige-Skoog's medium is particularly preferably used. It is preferable to culture in the dark, and the culture temperature is 20°C.
~25°C is used.

The callus thus obtained is transferred to an organic medium containing specific nitrogen and carbon sources. This medium contains a specific nitrogen source and carbon source shown below. 9 Conventional tissue culture media are sufficient, except that they contain specific nitrogen and carbon sources. for example,
The above callus induction or growth medium is used. As the nitrogen source, NH4NO3, (N)14)2SO
, etc. (dissociates to produce NH,+)
, and NaN.

Nitrate salts such as 803 (which dissociates to produce NO3-) are used. It is preferable to use a combination of an ammonium salt and a nitrate, and a combination of NH4NO3 and KNO is particularly preferable. KNO3 contains potassium, a metal element necessary for plant growth.

It is also thought to be effective for callus proliferation. The concentration of these nitrogen sources in the medium is 2.8-55.6mM (ammonium salt as ammonium ion (NH,+)).
0.05~1.0g/l), preferably 5.0~
41.1mM (0.09-0.74g/1), and nitrate is 11.3mM as nitrate ion (N(13-)
~80.6mM (0.7-5.0g/1).

Preferably 14.5-64.5mM (0.9-4.0
g/1).

The specific carbon source mentioned above is sucrose.

and glucose are used alone or in combination. What are the concentrations of these carbon sources in the medium?

For sucrose it is 3-7% by weight, preferably 3-4% by weight, and for glucose it is 3-5% by weight. If the content of the nitrogen source and carbon source is outside the above range, the production amount of saponin will decrease.

Plant hormones such as auxin and cytokinin may be added to the medium as necessary for the purpose of callus growth. The culture is preferably carried out in the dark at a culture temperature of 20 to 25°C, similar to the callus induction and proliferation described above.

The callus thus cultured is dried under appropriate conditions, such as ventilation drying, and if necessary, pulverized, and an appropriate extraction solvent is added thereto for extraction. For example, 50
% ethanol for 24 hours.

By allowing the mixture to stand at room temperature, an extract containing Chikusetsusaponin can be obtained. By evaporating the extract to dryness under relatively mild conditions, an extract containing Chikusetsaponin can be obtained. If necessary, this extract is subjected to various chromatography methods known to those skilled in the art to obtain purified saponin.

The presence of saponin in the extract can be confirmed, for example, by silica gel thin layer chromatography.

(Example) The invention is illustrated by the following examples.

Example 1 2.4- as auxin in Murashige-Skoog's medium
An agar medium for callus induction was prepared by adding D and kinetin at tppm and agar at 0.9%, and 50 ml of the culture medium was dispensed into 100 ml Erlenmeyer flasks and sterilized by autoclaving.

On this agar medium, small pieces of roots of horsetail ginseng sterilized with primary sodium hypochlorite were placed aseptically and cultured in the dark at 25°C for 4 weeks to induce callus. the obtained callus.

IBA2 as auxin in Murashige-Skoog medium
ppm and BA O,lppm, and agar 0
．． The cells were transplanted onto a growth agar medium supplemented with 9% and cultured at 25°C for 4 weeks. This callus was further transferred to a liquid growth medium having the same composition as the growth medium described above but not containing agar, and cultured at 25° C. for 21 days to allow growth.

Next, in order to cause the callus to produce saponin, 3% by weight of sucrose as a carbon source and 5.5% by weight as a nitrogen source were added to Murashige-Skoog's medium. OmM N8
4NO- (5.0mM as ammonium ion (0,
09g/])) and NO0[1 as nitrate ion
An organic medium supplemented with 4.5mM (0.9g/1)] was prepared.

Separately, the concentration of KNO3 was 21.0 mM (1.3 g/l) as nitrate ion, 29. OmM(1,
Three types of media were prepared: 8g/l) and 60.0mM (3.7g/l). After sterilizing flasks containing 100 ml of each medium in an autoclave, the above-mentioned callus was inoculated at a fresh weight of 4 Og per flask. By culturing in the dark at 25°C for 3 weeks, the fresh weight of each
4g, 17g, 16g and 17g of callus were obtained. - Dry this callus at 60°C for 24 hours to obtain dry callus, each with a dry weight of 0.89g.
, 0.10 g, 0.98 g and 0.1 g. When converted to the organic medium 11, the dry weight is 8.9 g/l and 10.1 g/l, respectively.
9.88/l right and 10. It becomes Og/1.

The relationship between NO3− concentration of the medium and callus dry weight [dry weight per 11 medium (g/l)] is shown in Figure 1 with a black circle (・).
Indicated by show. From FIG. 1, it can be seen that callus proliferates favorably by culturing using the medium of the present invention. This dried callus was extracted with 50% ethanol, and the extract was evaporated to dryness. This was dissolved in methanol to form a solution and treated with Sep Pak (Waters) to give 19.0 mg of each dry callus Ig, 21.
Saponin fractions of 2 mg, 20.4 mg and 22.1 mg were obtained. This fraction was then treated with CHCl3:M
The upper layer of eOH:H20=65:35:10 was subjected to silica gel thin layer chromatography using a developing solvent. Thiksetsaponin was confirmed by spraying 10% sulfuric acid on the developed thin layer plate, heating it at 105°C for 10 minutes, and scanning the absorption at a wavelength of 520 nm using a chromatographic scanner (Chem,
Pharm, Buli.

11.1546 (1963)).

Comparative Example 1 Nitrate ion concentration in organic medium was 9.7mM (0,6
Callus was cultured in the same manner as in Example 1, except that the concentration was (g/l). The results are shown in FIG. 1 along with the results of Example 1 by black angle of view marks (■). From Figure 1, when this medium is used, the callus is 4.18/I L as a dry matter.
It can be seen that callus growth is not promoted if the concentration of nitrate ions is too low.

Example 2 Of the auxin in the agar medium for callus induction in Example 1, 2.4-D was changed to NAA, BA in the growth medium was changed to kinetin, and ammonium ion was added as a nitrogen source in the organic medium. 1 degree is 41.1mM (0.74g
/I ), and the concentration of nitrate ions is 16.1 mR1 (1
,0g/l), 40.3mM (2,5g/l)
, 50.0mM (3,Ig/l) or 60.0mM
M (3.7g/1) was excluded.

Calli of horse chestnut ginseng was cultured in the same manner as in Example 1, and dried callus was obtained. The results of measuring this dry weight are shown in FIG. 1 as white circles (◯) together with the results of Example 1. 1st
From the figure, it can be seen that callus proliferates favorably by culturing using the above medium. 15mg, 17Jmg, 2 from each 1g of dry callus, respectively.
Crude saponin fractions of 1.7 mg and 22.7 mg were obtained, and the presence of saponin was confirmed in the same manner as in Example 1.

Comparative Example 2 Ammonium ion in organic medium was 72.2mM (
1,3 g/l) and nitrate ion concentration to 16.1 mM (1,0 g/l) and 40 JmM, respectively.
(2,5g/I) or 59.7mM (3,7
Callus was cultured in the same manner as in Example 2, except that the concentration was changed to (g/1). The results are shown in Example 1. The results of Comparative Example 1 and Example 2 are shown in Figure 1 with a white square mark (△
)show. From Figure 1.

Using this medium, the callus will be 5.0g/dry matter.
Only 1 was obtained, indicating that callus growth deteriorates when the concentration of ammonium ions is too high.

Comparative Example 3 Ammonium ion concentration in organic medium was OmM (Og/
l), and the nitrate ion concentration was 4°68mM, respectively.
(0,29g/1), 9.35mM (0,
58g/1).

19.4mM (1,20g/1) or 37.
Except that it was 6mM (2,33g/1).
Callus was cultured according to Example 2. The result is
Example 1. The results of Comparative Example 1, Example 2, and Comparative Example 2 are shown in FIG. 1 by black triangles (mm). From Figure 1, when this medium is used, the callus yields 4.0 g/1 as dry matter.
It can be seen that if the ammonium ion concentration is too low, the growth of callus deteriorates.

Example 3 Using the same callus induction medium and growth medium as in Example 1, ginseng callus was cultured.

This callus was treated with the same nitrogen source as in Example 1 [ammonium ion 5mM (0,09g/1) and nitrate ion 20.9mM (1,3g/i)] and 1% sucrose as a carbon source. , 3% sucrose, 5% sucrose, 7% sucrose, 1% glucose.

The cells were cultured in seven types of organic media containing 3% glucose or 5% glucose (Modified Murashige-Skoog medium). The calli obtained using these media were made into dry calli in the same manner as in Example 1, and the dry weight was measured. The results are shown in FIG. 2 by white circles (O rainbow course) and white squares (△nigel course).

Furthermore, the amount of crude saponin obtained from 1 g of dried callus in the same manner as in Example 1 is shown in Figure 3 with white circles (○:
Indicated by a rainbow mark and a square mark (△nigel course). From Figures 2 and 3, it can be seen that 3-7% sucrose or 3-7% sucrose or
It can be seen that by culturing using an organic medium containing 5% glucose, callus proliferates favorably, and at the same time, chikusetsusaponin is produced with high efficiency.

Comparative Example 4 Horse chestnut ginseng callus was cultured in the same manner as in Example 3, except that 1, 3 or 5% fructose was used as the carbon source in the organic medium, and dried callus was obtained. The weight (g/l) of this dry callus.

The results of Example 3 are shown in FIG. 2 with white angle of view marks (b). Furthermore, the amount of crude saponin obtained from 1 g of these dried calluses in the same manner as in Example 3.

The results of Example 3 are shown in FIG. 3 along with the white angle of view (b).

From Figures 2 and 3, when fructose is used as a carbon source in the organic medium, the production of callus and saponin is less than half that of when sucrose or glucose is used, resulting in improved productivity. It turns out that it gets worse.

(Effects of the Invention) According to the present invention, by tissue culturing the callus obtained from horse chestnut ginseng using a specific medium,
A method for efficiently producing saponin is provided. According to the method of the present invention, it is possible to stably obtain a large amount of saponin in a relatively short period of time without being affected by natural conditions such as season, weather, and soil quality. The saponin obtained in this way can be used in a wide range of fields such as pharmaceutical raw materials and health food additives.

[Brief explanation of the drawing]

Figure 1 is a graph showing changes in the yield of horse chestnut ginseng callus when the ammonium ion concentration and nitrate ion concentration of the specific organic medium used in the method of the present invention are changed; A graph showing the change in callus yield when the type and concentration of carbon source in the organic medium was changed; and Figure 3 shows the change in the yield of callus when the type and concentration of carbon source in the organic medium was changed. It is a graph showing changes in the amount of. that's all

Claims (1)

[Claims] 1. (a) Panax ginseng (Panax japonicu)
sC. A. Meyer) tissue to induce callus; (b) the obtained callus is treated with NH as a nitrogen source;
_4^+ and NO_3^-, and a step of culturing in an organic medium containing sucrose and/or glucose as a carbon source; and (c) a step of extracting chikusetsusaponin from the callus. Manufacturing method. 2. NH_4^+ in the medium is 2.8-55.6mM (
0.05-1.0 g/l), and NO_3^
- is 11.3-80.6mM (0.7-5.0g/l)
The manufacturing method according to claim 1, wherein the content is in a proportion of . 3. The production method according to claim 1, wherein the medium contains sucrose in a proportion of 3 to 7% by weight and/or glucose in a proportion of 3 to 5% by weight.