JPS5982340A - Purification of abscisic acid - Google Patents

Abstract

PURPOSE:To obtain the titled acid useful as a natural-type plant hormone from a solution containing (+)-cis-trans abscisic acid, in high yield, by using a strongly basic anion exchange resin of a polystyrene-type porous adsorbent resin as an adsorbent. CONSTITUTION:A solution containing (+)-cis-trans abscisic acid such as the cultured product of an abscisic acid-producing strain (e.g., Cercospora rosicola), its cultured liquid, or their treated material, is made to contact with a strongly basic anion exchange resin or a polystyrene-type porous adsorbent resin to effect the adsorption of the abscisic acid to the resin. The adsorbed acid is eluted with an elutant containing 30-90%(v/v) of an organic solvent selected preferably from methanol, ethanol, propanol, butanol and acetone. Abscisic acid in the above solution can be purified economically, in an industrial scale by this process.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for refining (t)cis-trans abscisic acid (hereinafter abbreviated as abscisic acid). More specifically, the present invention d relates to a method for purifying (t)cis-trans type abbuidic acid using a strongly basic anion exchange resin or a polystyrene-based porous adsorption resin.

Absaicic acid is an important natural plant hormone and is expected to have a wide range of uses.

Regarding the production of abscisic acid by conventional fermentation method,
A solid culture medium and a liquid stationary light irradiation culture according to i. Ta. In addition, as a method for purifying and isolating abscisic acid from culture materials and broth,

A known method is to add an organic solvent that is hardly soluble in water, such as ethyl acetate, and extract the organic solvent layer (C) for purification, but since a large amount of organic solvent is used, this method was inconvenient. (Experientia 3315561977
Year).

Recently, the above-mentioned submerged aerated liquid culture of Cercospora rosicola was discovered, and it was revealed that more than 60μ97M of abscisic acid was accumulated in the culture solution within a culture period of 15 to 20 days, making industrialization possible. It has also been revealed that abscisic acid can be purified and isolated from a culture solution by using a method using an ion exchange resin in addition to the conventional solvent extraction (Japanese Patent Laid-Open No. 160996/1983).

However, solvent extraction has the disadvantage of using a large amount of solvent, and conventional methods using ion exchange resins have the drawback of generally poor yields.

Therefore, the present inventors investigated various methods for purifying abscisic acid from a culture solution on an industrial scale. As a result, by bringing the culture solution containing abssaic acid into contact with a strongly basic anion exchange resin or polystyrene-based porous adsorption resin, abssaic acid is adsorbed onto these resins and absorbed into the strongly basic anion exchange resin. Absaicic acid is an acid containing water-soluble organic solvents such as methanol and acetone!
The present invention was accomplished by discovering that absisic acid adsorbed to a porous polystyrene adsorption resin can be recovered in good yield by a common eluent used when using such resins. reached.

Conventionally, methods using these strongly basic anion exchange resins have been known for the purification of absisic acid [Japanese Unexamined Patent Application Publication No. 1983-1993]
-160996] It is not known that the elution rate can be significantly increased by using an eluent containing a water-soluble organic solvent, and furthermore, there is no known example of using a polystyrene-based porous adsorption resin. .

The present invention will be described in detail below.According to the present invention, by contacting a solution containing abssaicic acid with a strongly basic anion exchange resin or a polystyrene-based porous N resin, abssaicic acid is adsorbed onto the resin. , followed by elution with an eluent to purify absisic acid.・Solutions containing abscisic acid can be used to treat, for example, Cercospora
Examples include culture products, culture fluids, and processed products of abscisin-producing strains such as Rosicola. The production of absilic acid by Cercospora rosicola is described in Japanese Patent Publication No. 56-160996.

As a strong basic anion exchange resin, DOWEX 1x
2. Same 1×4. Same 1×8. Same 2 x 4゜ Same 2 x 8 (The above are Dow Chemical Company product names, Diamond 5AIOA, 5A
IIA, S A21A.

Same PA316. Same PA406. Same PA4i6゜ Same 5AI
IA, HPA-25 (all Mitsubishi Kasei product names), Amberly) IRA4°0° IRA900. Same IRA9
10 (Low, trade name of Herr & Haas), etc. are used.

As the polystyrene-based porous adsorption resin, Diamondion HP-10, Diamondion I (P-20, Diamondion HP-30, Diamondion HP-
40. HP-50, HP-21 (all Mitsubishi Kasei product names), Annoku Light XAD-2, XAD-4 (
Rohm and Haas product name) etc. are used.

When using a strong basic anion exchange resin,
A mineral acid (sulfuric acid) is used as a CO-F eluent.

Hydrochloric acid, etc.), alkalis (sodium chloride, ammonia, etc.), salts (ammonium sulfate, etc.).

methanol to an aqueous solution of sodium sulfate, etc.).

A solution prepared by adding a water-soluble organic solvent such as ethanol, gropanol, butanol, or acetate is used. As the amount of water-soluble organic solvent added increases, the elution rate of absisic acid increases.

Although it depends on the type of bath medium and the amount of absisic acid adsorbed, the usage range of water-soluble organic solvents is 30~
90 ratio (v/v) is suitable.

When using a polystyrene-based porous adsorption resin, the eluent may be methanol or ethanol, which are commonly used when using the resin.

A liquid containing an organic solvent such as globanol or acetone is used.

In the case of strongly basic anion exchange resin, mineral acid 0.1-2
When general eluents such as N, alkali 0.1-2N, and salts 0.1-2M are used, the amount of abscisic acid eluted is 10-30%, which is a low recovery rate and is not practical. The dissolution rate can be significantly increased by adding a solvent.

In the case of a polystyrene-based porous adsorption resin, an elution rate close to 100% can be obtained by elution with the above solvent.

Absaicic acid is recovered from the eluate containing abssaicic acid as follows. After concentrating the eluate under reduced pressure and removing the organic solvent, the p'f (adjusted to 2 to 3) is extracted by adding an organic solvent that is hardly soluble in water such as ethyl acetate. After separating the extract into layers, add anhydrous sulfuric acid. Dehydrate by adding sodium, decolorize by adding activated carbon if necessary, concentrate the tear fluid under reduced pressure, and crystallize absisic acid.After crystal separation, vacuum dry.
White crystals of abscisic acid can be obtained.

Examples of the present invention will be shown below.

Example 1゜i00+xA! Dispense 4 m # of potato dextrose agar medium (PH6, s) containing 2.4 p of potato dextrose prosthe (product of Difco) and 22 m of agar into large test tubes (160 x n x 16 m).
The mixture was sterilized under pressure at 0°C for 15 minutes to prepare an agar slant medium.

After the agar solidifies, Cercospora spp.
Rosicola IAM5031 was inoculated and statically cultured at 25°C for 20 days. Eight gallons of sterilized water was dispensed onto the culture on the crushed agar slant, and the box was suspended in this using a glass rod, and the resulting suspension was used as a seed culture. 15ml of this persimmon culture
Potato dextrose broth (manufactured by Difco)
2.497d! , stock (scientific name: hermitage thiolain)
ca part) extract (chop the flowers, add the same weight of water, homogenize, and drain through gauze: lachrymal fluid) 0.25 punishment/
L: Inoculate a 51-volume jar fermenter containing fermentation medium 31 with tl, pH 6.5 and incubate for 1 minute with 3 A of air aerated on the surface (0.10 odor nolea/y & hr) L, 4
The cells were cultured under agitation conditions of 0 rpm and a temperature of 25°C, and nonionic 0T221 (product of NOF Co., Ltd.) was added to a concentration of 0.5 m9/mJ for 5 days after culture, and the culture was continued. The cells were cultured for a total of J5 days. 50.5μ7Δa of abscisic acid was accumulated in the culture solution. To this culture solution, 300 f of a filter aid Radioly+1600 (manufactured by Showa Kagaku Kojosha) was added and filtrated to obtain lachrymal fluid. Diaion S A21 A (
Cl) was passed through a column of 50 g to adsorb it, washed with water, and diluted with 0.2 N
Absaicic acid was eluted with 250 g of Hα-methanol (1:1 volume ratio) solution. After the eluate was concentrated under reduced pressure, 50 vcl of ethyl acetate was added to extract absisic acid from the ethyl acetate layer. After separating the ethyl acetate layer, 2 g of anhydrous sodium sulfate was added for dehydration, the sodium sulfate was removed, and the lachrymal fluid was concentrated under reduced pressure to crystallize absisic acid. After separating the crystals, they were vacuum dried at room temperature to obtain white crystals of abscisic acid 1107717.

Example 2: Cultured in the same manner as in Example 1, 63.2μ? A tear fluid was obtained by adding 300f of Radiolite+600 to 2.61ml of a culture solution in which abscisic acid had been accumulated at a concentration of /rd.

Amberlite IR person 400 filled with this filtrate in a column
(OI()) After passing through the column to 50 vtl, washing with water was performed. Absaicic acid was then eluted with 250 IIL of a 2M NaCA!-acetone (6:4 volume ratio) solution. After concentrating the eluate under reduced pressure, hydrochloric acid was removed. In addition, the pH was adjusted to 3, and 50 cm of ethyl acetate was added thereto.After that, the mixture was purified in the same manner as in Example 1 to obtain 145 cm of white crystals of absisic acid.

Example 3: Radiolite f 600 was added to 2.61 μg of a culture solution cultured in the same manner as in Example 1 to accumulate abscisic acid at a concentration of 57.6 μg' to 300 μg. In addition, the patient passed away and obtained lachrymal fluid. This tear fluid was packed into a column called Diamond Ion I (P-2).
After passing through the column to 0.050 mg, it was washed with 0.2M NaCl. Absaicic acid was then eluted with 30% acetone water. After condensing the eluate under reduced pressure, hydrochloric acid was added to adjust the pH to 2, and 50 g of ethyl acetate was added. Thereafter, it was purified in the same manner as in Example 1, and white crystals of abscisic acid 1,30
Get mp.

Patent Applicant (LO2) Kyowa Hakko Kogyo Co., Ltd. 71 Procedural Amendment December 28, 1980 Commissioner of the Patent Office 1. Indication of the Case 1988 Patent Application No./9267 No. 2 Name of the Invention ffff method of absisic acid 3. Relationship with the case of the person making the amendment Patent applicant Postal code 100 Address 6 Otemachi-chome, Chiyoda-ku, Tokyo Address No. 1 Name
<102) Kyowa Hakko Kogyo Co., Ltd. (T5r-: 0
3-201-7211 extension 2751) Inscription of claims in the specification j lr! Claims (11←) By contacting a solution containing cis-trans-absaicic acid with a strongly basic anion exchange resin or a polystyrene-based porous adsorption resin←) cis-trans-absaicic acid is added to the resin. ←) A method for purifying cis-trans abscisic acid, which comprises adsorbing the substance and eluting the substance with an eluent.

(2) When using a strongly basic anion exchange resin, the eluent contains an organic solvent.
The method described in section.

(3) A method according to claim 2, characterized in that the organic solvent is selected from methanol, ethanol, globanol, butanol and acetone.

(4) The method according to claim 2 or 3, characterized in that the organic solvent is contained in an amount of 30 to 90% (v/v).

Claims (4)

[Claims] (1) By melting a solution containing (g) cis-trans abscisic acid with a strongly basic anion exchange resin or a porous polystyrene adsorption resin, (g) cis-;・
1. A method for purifying (t)cis-trans type abscisic acid, which comprises adsorbing lance type abscisic acid on the resin and eluting the substance with an eluent. (2) The method according to claim 1, wherein the eluent contains an organic solvent. 3. A method according to claim 2, characterized in that the organic solvent is selected from methanol, ethanol, propanol, butyl and acetone. (4) The method according to claim 2 or 3, characterized in that the organic solvent is contained in an amount of 30 to 90% (v/v).