JPS5923798B2 - Manufacturing method of glycyrrhizin - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing glycyrrhizin (C4□H62016), and particularly to a method for obtaining glycyrrhizin by dramatically increasing the production amount of glycyrrhizin by culturing licorice callus and simultaneously causing organ differentiation.

Licorice callus (an amorphous mass of cells that has lost the organ-forming ability of the plant, with an external appearance resembling the fused tissue of the plant, produced when small pieces of the licorice plant are tissue cultured in a medium) Regarding the component of ), Furuya et al.
Licorice callus was induced from the licorice root using the medium of E and the medium of Murashige & Skoog in the absence or presence of light, with the addition of hormones and yeast extract, and its components were obtained. (TetrahedronL) is not mentioned.
etters27 2567-2579 (1971),
Collection of Abstracts from the 96th Annual Meeting of the Pharmaceutical Society of Japan, Volume 245 (1976)).

Furthermore, in Japanese Patent Publication No. 50-16440, small pieces of licorice plants are cultured in a medium containing auxin and cytokinin to generate callus, and the callus is cultured aerobically in a liquid medium to disperse cells in the liquid. It has been described that licorice extract for tobacco flavoring can be obtained by growing licorice into a suspension and then superconcentrating the culture solution, but it has been shown that this licorice extract contains only a trace amount of glycyrrhizin. ing.

As a result of research on the production of glycyrrhizin by culturing licorice calli, the present inventor found that almost no glycyrrhizin was produced by culturing small pieces of licorice plants alone, and that glycyrrhizin production increased rapidly due to organ differentiation. We have obtained the knowledge that a hormonal balance between auxin and cytokinin in the culture medium is necessary for the improvement of human organ differentiation and the organ differentiation of crabs, and based on this finding, we have completed the present invention.

That is, in the present invention, licorice callus is cultured in a nutrient medium containing 10-6 mol/l or less of auxin and 10-4 to 10-6 mol/l of cytokinin, simultaneously differentiated into organs, and glycyrrhizin is extracted from the organodifferentiated culture. This is a method for producing glycyrrhizin, which is characterized by collecting glycyrrhizin.

The present invention will be explained in detail below.

First, in the present invention, the plant body of licorice, such as leaves, stems, roots,
Alternatively, the seeds are sterilized and cultured in a nutrient medium to generate callus, and the licorice callus is used.

As a nutrient medium for generating this licorice callus,
For example, Whyte's medium, Marashige
&Skoog's medium, Gautheret's medium, etc. are used.

In this case, these media contain auxin 10-6 to 10-
Preferably, the cytokinin is present at a concentration of 4 molar or even less than 10-6 molar.

Examples of auxins include 2,4-dichlorophenoxyacetic acid, indoleacetic acid, naphthaleneacetic acid, indolebutyric acid, etc. Examples of cytokinins include adenine, kinetin, benzyladenine, zeatin, zeatin riboside, isopentynyladenosine, etc. , and natural cytokinin.

The above-mentioned licorice plants are cultured at 15-30° C. for about 15-30 days to generate callus, which is then subcultured for 5-6 generations.

The medium and temperature conditions for subculture may be the same as those for callus generation, but the auxin concentration should be 10-6 to 10
It is preferable to keep the concentration lower than -4 molar and the same as that during organ differentiation.

Next, the licorice callus generated in this manner is cultured in a nutrient medium containing auxin and cytokinin for organ differentiation.

In this case, the nutrient medium must always contain auxin at a concentration of 104 molar or less, preferably 10-6 to 10-9 molar, and cytokinin at a concentration of 10'' to 104 molar.

In addition, as a nutrient medium, the above-mentioned Why ite medium,
Murashige &Skoog's medium, Gauthe
ret media and their modified media, as well as other nutrient media used for plant tissue culture, including inorganic components, sugars (e.g., glucose, sucrose, fructose, etc.), organic acids, carbon sources such as alcohols, and further as necessary. Depending on the situation, a nutritional culture medium containing vitamins, amino acids, yeast extract, peptone, etc. can be used.

Callus can be cultured by static culture or shaking culture.

Light irradiation during culture is not particularly limited, and culture may be performed under light irradiation or in the dark.

When the callus is cultured in the above medium at 15 to 30°C for about 30 days, the development of shoots, roots, and stems is observed.

In order to collect glycyrrhizin from the culture obtained in this way, glycyrrhizin is extracted using a conventional method for collecting glycyrrhizin, for example, using a solvent such as water, water-ethanol, or water-acetone, and the extract is poured into an anion exchange resin column. to adsorb glycyrrhizin.

Next, the column is washed with water, and a 50-thialcohol aqueous solution containing 1% ammonia is passed through an anion exchange resin column to remove glycyrrhizine as an ammonium salt.

An alcoholic aqueous solution of glycyrrhizin ammonium salt is desalted by passing through a cation exchange resin to obtain an alcoholic aqueous solution of glycyrrhizin.

Next, this solution is concentrated under reduced pressure to precipitate glycyrrhizin crystals.

Glycyrrhizine can then be obtained by recrystallizing it from alcohol containing a small amount of water.

In order to clarify that the production amount of glycyrrhizin is dramatically improved by the present invention, Examples and Comparative Examples will be shown and specifically explained, but the present invention is not limited by these Examples. .

Example 1 10.7m9/A of naphthaleneacetic acid was added to the medium shown in Table 1 (hereinafter referred to as medium A). (5×10 −5 molar concentration) and 1 og/13 agar were added and sterilized in a conventional manner to prepare a medium.

Add licorice strips to this medium with 70% ethanol aqueous solution.
．． After sterilizing with an aqueous sodium hypochlorite solution (5), the specimens were thoroughly washed with sterile water, placed on a bed, and kept at 25° C. in a dark place.

After 21 days, white callus was generated from the cut surface of the strip, so the strip with callus was transplanted to a medium with the same composition, and
Subculture was performed.

This callus was inoculated into medium 11, which was prepared by adding 0.0221 m9/l (10-7 molar concentration) of 2,4-dichlorophenoxyacetic acid and 2.15 μ/# kinetin (10-' molar concentration) to medium A. Shaking culture was performed at 25°C for 30 days.

Thus, 80 cultures in which the development of roots, stems, leaves, etc. are observed.
g was obtained.

Extraction was carried out by adding 240 g of water to this culture, and this process was repeated.

The extract was passed through an anion exchange resin column to adsorb glycyrrhizine, and the column was washed with water.

Next, a 50% aqueous ethanol solution containing 1st group ammonia was passed through the column to desorb glycyrrhizin as an ammonium salt.

This ethanol aqueous solution was desalted by passing through a cation exchange resin column to obtain an ethanol aqueous solution of glycyrrhizin.

This solution was concentrated under reduced pressure to precipitate glycyrrhizin, and then recrystallized from ethanol with a water content of 5 parts.

The yield of glycyrrhizin was 0.28 g (7% based on dry matter).

Example 2 Naphthalene acetic acid 10.7 m9/l (5X10
-5 molar concentration), kinetin 0.2Tn9/l (9,
3XIO-7 molar concentration) and 10 g/l of agar were added and sterilized in a conventional manner to prepare a medium.

Licorice strips were sterilized and washed in the same manner as in Example 1, then placed on this medium, and kept at 25° C. in the dark.

After 15 days, a white callus was generated from the cut surface of the strip, and this was subcultured for 6 generations using a medium with the same composition.

This callus was mixed into medium A with 0.0175 m9/l of indoleacetic acid (10-7 molar concentration) and 2.12 m9/l of benzyladenine (10-'-El concentration).
was inoculated into medium 11 supplemented with , and cultured with shaking at 25° C. for 31 days.

Culture 10 in which the development of roots, leaves, stems, etc. is thus observed
0 g was obtained.

Extraction from this culture as described in Example 1,
Crystallization and recrystallization were performed to obtain 0.35 g of glycyrrhizin.

Example 3 The subcultured callus obtained in Example 2 was mixed with naphthaleneacetic acid 0.225 mI//l (10-mol/l/concentration) and kinetin 2.15/7 (10-'mol/l) in medium A. The cells were inoculated into medium 11 supplemented with the following concentrations: 1 to 10%, and cultured with shaking at 25°C for 35 days.

Thus, the culture 86 in which the development of roots, leaves, stems, etc. is observed.
g was obtained.

Extraction from this culture as described in Example 1,
Crystallization and recrystallization were performed to obtain 0.30 g of glycyrrhizin.

Example 4 The subcultured callus obtained in Example 2 was treated with medium A ninaphthalene acetic acid 0.212 m9/It (10-7 molar concentration).
and Penzyladeni710.71n9/l (5X 10
-5 molar concentration) and agar 10 m9/l, and static culture was performed at 25° C. for 30 days while irradiating with a fluorescent lamp.

Thus, there were 7 cultures in which the development of roots, leaves, stems, etc. was observed.
7g was obtained.

Extraction from this culture as described in Example 1,
Crystallization and recrystallization were performed to obtain 0.27 g of glycyrrhizin.

Example 5 Add 2,4-dichlorophenoxyacetic acid 2/l・(
9.05 x 10-molar concentration), kinetin 0.2 rIu
? / l (9,3X 10-7 mo/I/concentration) and agar 1
0 g/l was added and sterilized in a conventional manner to prepare a culture medium.

Licorice strips were sterilized and washed in the same manner as in Example 1, placed on this medium, and kept at 25° C. in a dark place.

After 15 days, a white callus developed from the cut surface of the strip, and this was added to medium A with 0.0% of 2,4-dichlorophenoxyacetic acid.
The cells were placed in a medium containing 2 m9/A' (9,05X 10-7 molar concentration), kinetin 0.02/7 (9,3XIO4 molar concentration), and 10 g/l agar, and subcultured for 6 generations.

This callus was placed in medium A containing 2,4-dichlorophenoxyacetic acid 0.21n9/Al' (9,05X 10-7 molar concentration), kinetin 2/A'(9,3X10''6 molar concentration) and agar log. Place the plate on a medium containing 3.
Culture was performed for 0 days.

Thus, 10 nutrients that are recognized to develop in roots, leaves, stems, etc.
0 g was obtained.

Extraction from this culture as described in Example 1,
Crystallization and recrystallization were performed to obtain glycyrrhizin o, 3i.

Comparative Example 1 The subcultured callus obtained in Example 5 was mixed with indole acetic acid 2.0 m/l (1, I X 10-' molar concentration) and kinetin 0.2 m9/l (9,3 X 10-' molar concentration) in medium A. -7
The cells were inoculated into a medium supplemented with molarity) and cultured with shaking at 25°C for 30 days.

No development of organs such as leaves or stems was observed in the culture.

And the culture is 100 f! /13.

Extraction from this culture as described in Example 1,
Crystallize and recrystallize to obtain glycyrrhizin 0.15.9
(3.5% of dry matter) was obtained.

Comparative Example 2 The subcultured callus obtained in Example 5 was mixed with indole acetic acid 2.0 μ/l (1, I x 10−5 molar concentration L) in medium A and Tokaine=-F-7215 m9/l (10 '
The cells were inoculated into a medium supplemented with molarity) and cultured at 25°C for 30 days.

No development of organs such as leaves or stems was observed in the culture.

and the culture was 90 g/l.

Extraction, crystallization, and recrystallization were performed from this culture in the same manner as described in Example 1 to obtain glycyrrhizin 0.14.9.

Comparative Example 3 The subcultured callus obtained in Example 5 was mixed with indole acetic acid 0.01751n9/11 (10' molar concentration) and kinetin 0.2 rIu//l (9,3 x 10-1) in medium A.
7 molar concentration) and cultured at 25° C. for 30 days.

No development of organs such as leaves or stems was observed in the culture.

and the culture was 40 g/it.

Extraction from this culture as described in Example 1,
Crystallize and recrystallize to give glycyrrhizin 0.06. ?
I got it.

Comparative Example 4 The subcultured callus obtained in Example 5 was added to medium A with 0.01751 n9/l of indole acetic acid (10-7 molar concentration) and 0.2159/l of kinetin (10-3 molar concentration). The cells were inoculated into a culture medium prepared in this way, and cultured at 25°C for 30 days.

In this case, a culture of 5 g/l was obtained in which only a small portion of leaves, stems, etc. were observed to develop, but the culture died.

Extraction from this culture as described in Example 1,
Crystallization and recrystallization were performed to obtain 0.01 g of glycyrrhizin.

As is clear from the results of Comparative Examples 1 to 4 described above, when licorice callus is cultured in a nutrient medium that does not contain auxin and cytokinin in the concentration range as in the present invention, the yield of glycyrrhizin is lower than that obtained in the present invention. The yield of glycyrrhizin is extremely small compared to that of glycyrrhizin.

Claims (1)

[Claims] 1. Licorice callus is cultured in a nutrient medium containing auxin 10' mol/II or less and cytokinin 10" to 10-6 mol/l, and at the same time, organodifferentiated, and glycyrrhizin is collected from this organodifferentiated culture. A method for producing glycyrrhizin.