JPS6331200B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention cultivates callus derived from the plant tissue of Hanakirin, a plant belonging to the genus Euphorbia, and produces sterin and red pigment chrysanthemin (cyanidin-3-3-
This invention relates to a method for co-producing glucosides. Sterine is one of the components widely contained in plants.In recent years, it has been used as a synthetic raw material for steroid hormones such as corticosteroids and pills, and it is also used as a pharmaceutical ingredient in cosmetics, surfactants, etc. It is attracting attention as a synthetic raw material for chemical products. On the other hand, dyes are often used as food coloring agents to add aesthetic appeal to foods.
Synthetic coloring agents have caused various problems as carcinogens due to their toxicity, such as mutagenicity. Therefore, from the viewpoint of safety, it is desired to use pigments derived from natural products as coloring agents for foods and the like. However, since natural cultivation is easily subject to constraints of the natural environment such as season, climate, temperature, latitude, etc., it is not possible to maintain a stable supply by collecting from natural plants. In addition, large-scale cultivation using arable land naturally competes with food production, so there is a limit to its supply, and there is also a limit to its productivity, making it extremely expensive. However, in recent years, research on plant cell culture has been progressing as a method for producing plant components. Plant cell culture grows at a much faster rate than natural plants, which grow on a yearly or monthly basis, so it is possible to produce the desired ingredients in a short period of time, and unlike natural cultivation, it is less susceptible to the effects of weather etc. It has the advantage of being able to be produced in a planned manner on an industrial scale without the need for much labor for collection. The present inventors previously succeeded in separating and collecting sterin from plant tissue culture callus of the genus Euphorbia and/or the genus Eucalyptus (see Japanese Patent Application Laid-open No. 55-26813). By culturing callus derived from Hanakirin plant tissue in a medium containing 2,4-D as a plant hormone, we succeeded in producing the red natural pigment chrysanthemin. From this point of view, the present inventors have conducted further research to enable industrially advantageous co-production of the above-mentioned sterin and the natural red pigment chrysanthemine through plant cell culture. Callus derived from tissues was treated with α-naphthalene acetic acid (NAA) at 10 ^-4 to 10 ^-7 M.
The inventors have discovered that the above-mentioned sterin and the natural red pigment chrysanthemine can be co-produced in high yield by culturing in a medium containing preferably a concentration of 10 ^-4 to ^{10 -6} M, leading to the present invention. The red pigment produced together with sterine according to the present invention is chrysanthemine (cyanidin-3-glucoside) having the following structural formula, and it is not a multi-component pigment, but only one type of chrysanthemine. The extraction and purification operation of the pigment is also easy. The cultured plant cells used in the present invention are obtained by deriving Hanakirin, a plant of the genus Euphorbia, according to a conventional method. An example will be explained below. First, after thoroughly washing the leaves of Hanakirin with deionized water, they were immersed in 70% ethyl alcohol for 5 to 10 minutes, then in a 10% bleaching powder solution for 5 to 10 minutes to sterilize the bacteria attached to the surface. Wash away residual disinfectant with sterile distilled water. Next, the sterilized leaves are cut into small pieces with a sterile scalpel to an appropriate size and treated with auxin-active substances such as 2,4-dichlorophenoxyacetic acid (2,4-D) and α-naphthaleneacetic acid (NAA). The cells are placed on a synthetic medium containing (for example, Murashige-Skoog medium). After placing on the bed, store in a bright place under constant temperature conditions of 20 to 30℃, preferably around 28℃, preferably 100 lux or more,
More preferably, the culture is carried out under light irradiation of 3,000 to 100,000 lux. After one week of such culture, a callus that produces the red pigment is formed from the leaves of Hanakirin, which is then transplanted onto a new agar medium with an appropriate composition and incubated at 20 to 30°C, preferably at 28°C. Under constant temperature before and after, in a bright place, preferably
100 lux or more, more preferably 3000 to 100000
Continue culturing under lux light irradiation. In order to obtain callus on an industrial scale, the callus may be cultured and propagated by a static culture method or a liquid culture method in the same manner as in the culture of general microorganisms. As a liquid culture method, for example, a shaking culture method in which culture is performed on a shaking culture machine, or a method in which sterile air is aerated in a closed tank made of glass, metal, etc. may be selected as appropriate depending on the purpose. Can be done. The medium used for culturing the callus according to the present invention is based on various known inorganic synthetic agar media, and contains trace amounts of organic substances such as vitamins, etc.
A carbon source, plant hormones, and various natural extracts are added. Representative examples of the inorganic synthetic agar medium include White medium, Hildebrand medium, Linsmeyer-Skoog medium, Murashige-Skoog medium, and the like. In addition, these media with improved compositions can also be used. Examples of trace organic substances such as vitamins include vitamins such as thiamine hydrochloride, pyridoxine hydrochloride, and nicotinic acid; amino acids such as glycine and asparagine; and hexahydric alcohols such as inosite and sorbitol. It may show good growth even without being added to the medium. As the carbon source, carbohydrates such as sucrose, glucose, and maltose; organic acids such as acetic acid; and alcohols such as methanol and glycerin can be used. Among them, sucrose is particularly preferred because of its high pigment productivity. The concentration used is 1-10% w/v, preferably 4-7% w/v. As a plant hormone, when inducing callus, 2,4-dichlorophenoxyacetic acid (3,4-D),
Auxin-acting substances such as β-indoleacetic acid (IAA) and α-naphthaleneacetic acid (NAA) and cytokinins such as Kinetic can be used, but from the viewpoint of chrysanthemine productivity and selectivity, , 4-D, the preferred concentration being 10 ^-4 to 10 ^-7 M, more preferably 10 ^-6 to ^{10 -7} M. However, in order to co-produce sterine and chrysanthemine in high yield according to the purpose of the present invention by culturing the induced callus, α-naphthaleneacetic acid (NAA) must be used as described above.
must be used. The concentration of NAA used is
10 ^-4 to 10 ^-7 M, preferably 10 ^-4 to 10 ^-6 M,
If this concentration is too high, callus will not grow, which is undesirable; if it is too low, callus may turn green or not grow, which is undesirable. Examples of the various natural extracts include casein hydrolyzate (0-2% w/v), coconut milk (0-25% w/v), yeast extract (0-2%
w/v), malt extract (0 to 2% w/v), etc. can be used alone or in any combination. In the method of the present invention, as described above, the callus is cultured in a culture vessel containing callus at a temperature of 100 lux or more.
It is preferably carried out by installing under light irradiation of 3,000 to 100,000 lux. As a light source, sunlight, a fluorescent lamp, an incandescent lamp, a mercury lamp, etc. can be used. If no light irradiation is performed, no dye will be produced. Sterlin and the red pigment chrysanthemine can be separated and collected from the calli thus cultured by a known method, such as a solvent extraction method. An example will be explained below. First, cells are cultured in a solvent containing an acid such as hydrochloric acid, acetic acid, or formic acid at a concentration of about 0.01 to 5% by weight, preferably water or an alcoholic solvent such as methanol or ethanol, and the culture is preferably lyophilized according to a conventional method. The cells are soaked at a temperature of -5 to 10°C to extract the produced stelline and red pigment. Next, the obtained extract is subjected to an operation such as filtration to remove the solid content, and then concentrated under reduced pressure at a temperature of 40°C or lower (too high a temperature is not preferable because the red pigment easily decomposes). Transfer this concentrated solution to a separating funnel and extract it several times with, for example, ether (hexane, heptane, petroleum ether, chloroform, methylene dichloride, ethyl acetate, etc. can also be used instead of ether) to remove the Extract the sterine. The desired red pigment chrysanthemine can be obtained by drying the residual liquid after ether extraction under reduced pressure, and further purified by cellulose thin layer chromatography, cellulose column chromatography, paper chromatography, etc. be able to. The obtained chrysanthemine can be identified by comparing it with standard chrysanthemine using paper chromatography or UV absorption spectrum, as shown in the following examples. On the other hand, the desired sterine crystals can be obtained by distilling off the ether from the ether-extracted layer and recrystallizing the residue with, for example, n-hexane. Note that instead of recrystallization, for example, column chromatography, thin layer chromatography, etc. can also be used. The obtained sterine has a melting point of about 137°C, and it was determined by comparing it with standard sterine by thin layer chromatography using various solvent systems as a developing solvent, infrared absorption spectrum, and nuclear magnetic resonance spectrum. Can be identified. As explained above, according to the method of the present invention, by culturing callus derived from the plant tissue of Hanakirin, a plant belonging to the genus Euphorbia, in a specific medium, sterine and food colorants, which are valuable as synthetic raw materials for fine chemical products, can be produced. It is possible to co-produce chrysanthemine, a natural red pigment suitable as a natural red pigment, in pure form, and moreover, it can be produced extremely efficiently compared to the case where it is produced from natural plants. Examples of the present invention will be described below, but it goes without saying that the scope of the present invention is not limited to the following examples. Example The leaves of Hanakirin were thoroughly washed with water and cut into approximately 4 cm ² pieces. Next, these sections were sterilized by immersing them in 70% ethyl alcohol for 5 minutes and then in a 10% bleaching powder solution for 10 minutes, and then immersed in sterile distilled water several times in a sterile box to wash and steam them thoroughly. Removed residual disinfectant. These leaf sections were cut into small pieces with a width of about 1 cm ² using a sterile scalpel, and the obtained small pieces of Hanakirin leaves were placed aseptically on a synthetic agar medium having the following composition. The medium was Murashige-Skoog's inorganic salt medium, 3% w/v sucrose, and 0.2% w/v malt extract.
v, 2,4-D10 ^-6 M, thiamine hydrochloride 0.1 ppm,
Pyridoxine hydrochloride 0.5ppm, nicotinic acid
0.5ppm, glycine 2ppm and inositol
Add 100ppm and adjust to PH6.0, agar 0.8%w/
A medium was used which had been sterilized in a conventional manner by adding V. Small pieces of Hanakirin leaves placed on such a medium were cultured at a culture temperature of 25°C under light irradiation of 3000 lux. After one week, a red callus appeared around the cut end of the leaf. After one month, the callus that has grown large is divided into small pieces and treated with plant hormones 2 and 4.
-D10 ^-6 M was replaced with NAA10 ^-5 M, but the callus was transplanted aseptically into a medium with the same composition as used for callus induction above, and the callus was irradiated with light at 3000 lux at a culture temperature of 25°C. The culture was carried out for a total of 6 months, repeating the same operation every 4 to 6 weeks. The cultured Hanakirin cells grown in this way were separated from the solid medium, and water was removed in a vacuum freeze dryer to obtain 10.6 g of dry callus. This dried callus was ground in a mortar and then immersed in 2% hydrochloric methanol in a cold place for 24 hours. The extract obtained after filtration is concentrated to about 50ml at a temperature below 40℃,
After transferring to a separating funnel, 100 ml of ether was added to separate the obtained sterine and chrysanthemine, and after thorough shaking, the mixture was separated from the ether layer (sterine was transferred to this ether layer). After repeating this ether extraction operation several times, the remaining liquid was further heated at 40°C.
It was concentrated to dryness under reduced pressure. This dried product was dissolved in a small amount of 0.01% methanol with hydrochloric acid, applied in a strip on cellulose TLC (20 cm x 20 cm), and developed using a developing solvent of hydrochloric acid/acetic acid/water = 5/1/5. The red band was scraped off and immersed in 2% methanol with hydrochloric acid to extract the red pigment. This extract was dried under reduced pressure at 40° C. or lower to obtain 87.5 mg of black-red powder. Paper chromatography of this dye (Toyoro Paper No. 51,
Same below) (Solvent: n-butanol/acetic acid/water =
4/1/5 upper layer, n-butanol/hydrochloric acid/water=
The Rf values of 5/1/4 upper layer, 1% hydrochloric acid and acetic acid/hydrochloric acid/water = 15/3/82) are as shown in Table 1 below. ) was in good agreement.

[Table] Also, the obtained pigment and standard chrysanthemin
When we measured the UV absorption spectrum, we found that Figure 1A
As shown in and B, the UV absorption spectrum of the red dye was in good agreement with that of standard chrysanthemin. Next, the red pigment obtained above was boiled in 20% hydrochloric acid for 5 minutes, and the aglycone obtained by hydrolysis was subjected to paper chromatography (solvent: n-butanol/acetic acid/water = 4/
1/5 upper layer, n-butanol/hydrochloric acid/water = 5/
The Rf values of 1/4 upper layer, acetic acid/hydrochloric acid/water = 5/1/5 and formic acid/hydrochloric acid/water = 2/1/2) are also shown in Table 2 below. The results were in good agreement with those obtained by decomposition.

【table】

[Table] Furthermore, the results of paper chromatography (solvent: phenol/water = 4/1 and pyridine/butanol/water = 3/6/1) of the sugar moiety of the red pigment are as shown in Table 3 below. Consistent with glucose. From these results, it can be identified that the red pigment obtained above is chrysanthemine.

[Table] On the other hand, all the ether extracts containing sterine are collected and concentrated, and the ether is distilled off to obtain an ether extract. This ether extract was dissolved in a small amount of hexane and recrystallized to obtain 970 mg of sterine. The melting point of this crystal was 136.5°C, and its infrared absorption spectrum and nuclear magnetic resonance spectrum matched well with the spectrum of authentic sterin (a mixture of sitosterol and stigmasterol). Furthermore, this crystal of chloroform/ethyl acetate =
9/1 and n-hexane/ethyl acetate = 7/3
The Rf value (see Table 4) and color development of spots generated when a silica gel G thin layer plate was sprayed with sulfuric acid using a developing solvent of matched.
From these results, it can be identified that the crystals obtained above are sterine.

[Table] Comparative example In the above example, as a plant hormone for culturing callus derived from Hanakirin leaves.
Callus was cultured in the same manner as in Example except that 2,4-D10 ^-6 M was used instead of NAA10 ^-5 M, and 9.8 g of dry callus was obtained. From this drying, chrysanthemin and sterine were separated in the same manner as in the example, and 79.4 mg of black-red powder chrysanthemin was found.
However, the amount of sterine was only 10.8 mg.

[Brief explanation of drawings]

Figure 1 is a chart of the UV absorption spectra of the red pigment obtained from callus in the example and standard chrysanthemin. Figure A shows the absorption spectrum of the red pigment of callus, and Figure B shows the absorption spectrum of standard chrysanthemin. show.

Claims (1)

[Scope of Claims] 1 Callus derived from the plant tissue of Hanakirin, a plant of the genus Euphorbia, is cultured in a medium containing α-naphthalene acetic acid (NAA) at a concentration of 10 ^-4 to ^{10 -7} M to produce sterine and red color. A method for co-producing sterine and red pigment chrysanthemine, characterized by co-producing the pigment chrysanthemine. 2. The method according to claim 1, wherein the callus is cultured under light irradiation of 100 lux or more. 3 The callus was cultured with 1 to 10% by weight of sucrose.
The method according to claim 1 or 2, which is carried out in a medium containing a concentration of .