JPH036139B2 - - Google Patents

Description

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

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

For the production of abscisic acid by conventional fermentation methods, Cercospora rosicola
Solid culture and liquid stationary light irradiation culture using A. rosicola) are known, but the amount of abscisic acid produced is small and the culture period requires a long period of 30 to 40 days, which is difficult from an industrial perspective. In addition, a known method for purifying and isolating abscisic acid from culture materials and broth is to add an organic solvent that is not soluble in water, such as ethyl acetate, and extract it into the organic solvent layer for purification.
Since a large amount of organic solvent is used, it is still difficult from an industrial perspective. (Experientia 33 1556 1977
Year).

Recently, the above-mentioned aerated agitation liquid probe culture of Cercospora rosicola was discovered, and it was revealed that more than 60 μg/ml of abscisic acid was accumulated in the culture solution after 15 to 20 days of culture, making industrialization possible. . Furthermore, it has become clear 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 conventionally known solvent extraction (Japanese Patent Application Laid-Open No. 160996/1983).

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

Therefore, the present inventors investigated various methods for purifying abscisic acid from a culture solution on an industrial scale. As a result, by contacting a culture solution containing absisic acid with a strongly basic anion exchange resin or a polystyrene-based porous adsorption resin, abscisic acid is adsorbed on these resins, and abscisin adsorbed on the strongly basic anion exchange resin is absorbed. The acid is treated with an acid or alkali or salt containing a water-soluble organic solvent such as methanol or acetone, and the abscisic acid adsorbed on a polystyrene-based porous adsorption resin is treated with a general eluent used when using the resin. They found that it can be recovered in good yield and completed the present invention.

Conventionally, methods using strong basic anion exchange resins have been known for the purification of abscisic acid, but the elution rate can be significantly increased if an eluent containing a water-soluble organic solvent is used [JP-A-56-160996]. It is not known that this is possible, and furthermore, there are no known examples of using polystyrene-based porous adsorption resins.

The present invention will be explained in detail below.

According to the present invention, abscisic acid is adsorbed onto the resin by contacting a solution containing abscisic acid with a strongly basic anion exchange resin or a porous polystyrene adsorption resin, and then eluted with an eluent. Abscisic acid can be purified.

Examples of solutions containing abscisic acid include cultures of abscisic acid-producing strains such as Cercospora rosicola, culture solutions, and processed products thereof. The production of abscisic acid by Cercospora rosicola is described in JP-A-160996. The structural formula of abscisic acid is It is.

Strongly basic anion exchange resins include DOWEX 1x2, DOWEX 1x4, DOWEX 1x8, DOWEX 2x4, and DOWEX 2x8 (all Dow Chemical company product names, DOWEX SA10A, DIAION SA11A, DOWEX SA21A, PA316, same
PA408, PA416, HPA-10, HPA-25
(The above are Mitsubishi Kasei product names), Amberlight
IRA400, IRA900, IRA910 (Rohm and Haas product name), etc. are used.

Diaion HP-10, Diamondion HP-20, Diamondion HP-30, Diamondion HP-30,
HP-40, HP50, HP-21 (all Mitsubishi Chemical product names), Amberlite XAD-2, XAD-
4 (Rohm and Haas Company product name), etc. are used.

When using a strongly basic anion exchange resin, the eluent should be an aqueous solution of mineral acid (sulfuric acid, hydrochloric acid, etc.), alkali (potassible soda, potash, ammonia, etc.), or salt (ammonium sulfate, sodium sulfate, etc.). methanol, ethanol, propanol,
A solution containing a water-soluble organic solvent such as butanol or acetone is used. As the amount of water-soluble organic solvent added increases, the elution rate of abscisic acid increases. Although it depends on the type of organic solvent and the amount of abscisic acid adsorbed, the range of use of the water-soluble organic solvent is preferably 30 to 90% (v/v).

When using a polystyrene-based porous adsorption resin, a liquid containing an organic solvent such as methanol, ethanol, propanol, or acetone, which is commonly used when using the resin, is used as the eluent.

For strongly basic anion exchange resins, mineral acid 0.1
When using general eluents such as ~2N, alkali 0.1~2N, and salts 0.1~2M, the amount of abscisic acid eluted is 10~30%, which is low and impractical; however, adding a water-soluble organic solvent By this, the dissolution rate can be significantly increased.

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

Recovery of abscisic acid from the eluate containing abscisic acid is performed as follows. After the eluate is concentrated under reduced pressure to remove the organic solvent, the pH is adjusted to 2 to 3, and an organic solvent that is hardly soluble in water, such as ethyl acetate, is added for extraction. After separating the extract into layers, it is dehydrated by adding anhydrous sodium sulfate, and if necessary, activated carbon is added to decolorize it, and the liquid is concentrated under reduced pressure to crystallize abscisic acid. After crystal separation, white crystals of abscisic acid can be obtained by vacuum drying.

Examples of the present invention will be shown below.

Example 1 Dispense 14 ml of potato dextrose agar medium (PH6.5) containing 2.4 g of potato dextrose broth (Difco product) and 2 g of agar per 100 ml into a wide test tube (160 mm x 16 mm) and heat at 120°C. The mixture was sterilized under pressure for 15 minutes to prepare an agar slant medium. After the agar had solidified, Cercospora rosicola IAM5031 was inoculated over the entire surface of the agar medium and cultured stationary at 25°C for 20 days. 8 ml of sterilized water was dispensed into the culture on the obtained agar slant, and the bacterial cells were suspended therein using a glass rod, and the resulting suspension was used as a seed culture. Add 15 ml of this seed culture to potato dextrose broth (manufactured by Difco) 2.4 g/dl, stock (scientific name:
Mathiolaincana) extract (chopped flowers, added the same weight of water, homogenized, and filtered through gauze) was inoculated into a 5-volume jar fermenter containing 0.25 ml/dl of fermentation medium 3 at pH 6.5. , surface ventilation of 3 air per minute (0.10Kg moles/
hr) and cultured under stirring conditions of 400 rpm and temperature conditions of 25°C, and on the 5th day of culture, nonionic OT221
(Nippon Oil & Fats Co., Ltd. product) was added at a concentration of 0.5 mg/ml, and the culture was continued for a total of 15 days. Abscisic acid accumulated in the culture solution at 50.5 μg/ml. Add radiolite #600 as a superfluid to this culture solution.
(Showa Chemical Industry Co., Ltd. product) 300g was added and filtered to obtain a liquid. This liquid was passed through 50 ml of Diaion SA21A (Cl) packed in a column to adsorb it, then washed with water, and 0.2N HCl-methanol (1:1 volume ratio)
Abscisic acid was eluted with 250 ml of solution. After the eluate was concentrated under reduced pressure, 50 ml of ethyl acetate was added to extract abscisic 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 separated, and the liquid was concentrated under reduced pressure to crystallize abscisic acid. After separating the crystals, they were dried under vacuum at room temperature to obtain 110 mg of white crystals of abscisic acid.

Example 2 300 g of Radiolite #600 was added to culture solution 2.6, which was cultured in the same manner as in Example 1 and accumulated abscisic acid at a concentration of 63.2 μg/ml, to obtain a filtrate. Amberlite packed with this liquid in a column
After passing through the column into 50 ml of IRA400 (OH), washing with water was performed. Next, 2M NaCl-acetone (6:4 volume ratio)
Abscisic acid was eluted with 250 ml of solution. After concentrating the eluate under reduced pressure, add hydrochloric acid to adjust the pH to 3.
50ml of ethyl acetate was added. After that, it was purified in the same manner as in Example 1, and white crystals of abscisic acid 145
I got mg.

Example 3 300 g of Radiolite #600 was added to culture solution 2.6, which was cultured in the same manner as in Example 1 and accumulated abscisic acid at a concentration of 57.6 μg/ml, and filtered to obtain a solution. Diaion packed with this liquid in a column
After passing through the column into 50 ml of HP-20, it was washed with 0.2M NaCl. Abscisic acid was then eluted with 30% acetone water. After the eluate was concentrated under reduced pressure, hydrochloric acid was added to adjust the pH to 2, and 50 ml of ethyl acetate was added. Thereafter, the product was purified in the same manner as in Example 1 to obtain 130 mg of white crystals of abscisic acid.

Claims (1)

[Claims] 1. By contacting a solution containing (+) cis-trans abscisic acid with a polystyrene-based porous adsorption resin, the (+) cis-trans abscisic acid is adsorbed onto the resin, and the substance is Either by elution with an eluent or by contacting a solution containing (+) cis-trans abscisic acid with a strongly basic anion exchange resin, (+) cis-trans abscisic acid is adsorbed onto the resin, and methanol A method for purifying (+) cis-trans type abscisic acid, which comprises eluting the substance using an eluent containing a water-soluble organic solvent selected from , ethanol, propanol, butanol, and acetone. 2. Claim 1, characterized in that an eluent containing 30 to 90% (v/v) of an organic solvent is used.
The method described in section.