JP2000236894A - Radicicol manufacturing method - Google Patents

Abstract

(57) [Problem] To provide a method capable of producing high-purity radicicol easily and inexpensively on an industrial scale. SOLUTION: This is a method for producing or purifying radicicol, comprising the following steps: (1) a step of adding a base to a culture solution in which radicicol-producing bacteria are cultured to adjust the culture solution to pH 7.0 or more; 2) a step of adsorbing the base addition salt of radicicol contained in the culture solution to the synthetic adsorbent and then eluting the base addition salt with an organic solvent or a water-containing organic solvent; and (3) contained in the eluate Converting the base addition salt into radicicol in a free form by a neutralization treatment.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing radicicol having a physiological activity such as an antifungal activity. More specifically, the present invention relates to a method for easily producing high-purity radicicol from a culture solution of radicicol-producing bacteria.

[0002]

2. Description of the Related Art Radicicol is a physiologically active substance represented by the following formula (I) and has an antifungal activity [Nat.
171 ), 344 (1953)], an anticancer effect (JP-B-43-8718), an angiogenesis inhibitory effect (JP-A-6-279279), an immunosuppressive effect (JP-A-6-279279).
It is known to have a pharmacological action such as that described in US Pat.

Embedded image

Conventionally, radicicol has been known as monosporium ( M
onosporium ) by a microbiological production method using radicicol- producing bacteria and the like. As a method of separating and purifying radicicol from the culture solution, a radicicol is extracted by adding an organic solvent such as ethyl acetate to a microbial culture solution or microbial cells, and then a normal phase system using a carrier such as silica gel or A method is known in which a target substance is separated by a combination of reversed-phase column chromatography and an eluate is concentrated to obtain a crude crystal (Japanese Patent Publication No. 43-8718).
No.). However, this method has a problem that a large amount of organic solvent is required for extraction because radicicol has low solubility in an organic solvent mixed with water. Another problem is that a large amount of silica gel and an organic solvent must be used in purification by silica gel column chromatography.

As another purification method, the culture supernatant is adjusted to pH 2 with hydrochloric acid or the like, and the desired product is extracted with an organic solvent such as methylene chloride. The extract is dissolved in benzene-acetone and subjected to silica gel column chromatography. And a method of crystallizing the target substance from the eluate [Journal of Natural Products, 42 , 374 (1)].
979)]. However, this method has many steps,
In addition, since this method has the same problem as the method described in JP-B-43-8718 in that a large amount of silica gel and an organic solvent are required in order to use silica gel column chromatography, this method is also industrial. This is not a satisfactory method.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for efficiently producing or purifying radicicol. More specifically, it is an object of the present invention to provide a method capable of producing high-purity radicicol easily and inexpensively on an industrial scale. The present inventors have conducted intensive studies in order to solve the above problems, as a result of adjusting the culture solution of radicicol-producing bacteria to basic, and separating radicicol from the culture solution as a water-soluble base addition salt. It has been found that high-purity radicicol can be easily and efficiently produced without using a large amount of an organic solvent. The present invention has been completed based on the above findings.

That is, the present invention relates to a method for producing or purifying radicicol represented by the following formula (I),

Embedded image The following steps: (1) a step of adjusting the pH of the culture solution to 7.0 or higher by adding a base to the culture solution in which the radicicol-producing bacteria have been cultured; (2) removing a base addition salt of radicicol contained in the culture solution A step of eluting the base addition salt with an organic solvent or a water-containing organic solvent after being adsorbed to the synthetic adsorbent; and (3) neutralizing the base addition salt contained in the eluate into free form radicicol. Providing a method comprising the step of converting.

According to a preferred embodiment of the present invention, the above method of adjusting the culture solution to pH 7.5 to 10, more preferably pH 8 to 9, the above method using an alkali metal hydroxide, preferably sodium hydroxide, as a base. The synthetic adsorbent is a non-ionic porous resin, preferably Diaion SP
The above-mentioned method, wherein the radicicol-producing bacterium belongs to the genus Penicillium , more preferably Penicillium luteo-aurantium ( Penicillium).
luteo-aurantium ); and the above method using an organic solvent, preferably methanol, for elution.

[0008] From another viewpoint, there is provided a method for producing radicicol, comprising a step of separating radicicol as a base addition salt from a culture solution in which radicicol-producing bacteria are cultured. According to a preferred embodiment of this method, a base is added to the culture solution to at least pH 7.0, preferably pH 7.5 to 1
The above method of converting to a base addition salt by adjusting the pH to 0, more preferably pH 8 to 9; the above method of using an alkali metal addition salt, preferably a sodium salt as the base addition salt; a synthetic adsorbent, preferably a nonionic porous material Resin,
More preferably, the above method of separating a base addition salt using Diaion SP207; a radicicol-producing bacterium belonging to the genus Penicillium , more preferably a penicillium luteo-aurantium ( Penicillium l.).
uteo-aurantium ); and a method for separating a base addition salt with a synthetic adsorbent, wherein the organic adsorbent is preferably used to elute the base addition salt from the synthetic adsorbent. .

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION As a radicicol-producing bacterium,
Any microorganism may be used as long as it has radicicol-producing ability, and its type is not particularly limited. For example, Monosporium bonorden
den ) [Nature, 171 , 344 (195)
3): IFO8176 etc.], HumicolagriseaTraaen (JP-B-43-871)
No. 8), penicillium luteo-aurantium ( P
enicillium luteo-aurantium ) [Journal of Natural Product
s), 42 , 374 (1979): NRRL 3130,
NRRL 3131 etc.], Cylindrocarponradicicola [Transactions the British Mycological Society (Trans. Br. Mycol. Soc.), 49 , 563 (19)
66)] and the like, among which those belonging to the genus Penicillium are preferred. The type of nutrient medium used for culturing radicicol-producing bacteria is not particularly limited, and any medium commonly used for culturing the producing bacteria can be used. For example, a nutrient medium or the like used for culturing ordinary filamentous fungi can be used.

The method of culturing radicicol-producing bacteria is not particularly limited, but liquid culture, particularly deep stirring culture, is suitable. The culturing temperature is preferably about 20 to 40 ° C, more preferably about 25 to 30 ° C, and preferably pH 3 to 9,
More preferably, it is desirable to carry out the culture in the vicinity of a weakly acidic pH of 3 to 7. When the concentration of radicicol in the culture solution reaches a maximum, the culture is stopped, and a culture solution used for purification can be obtained. The concentration of radicicol contained in the culture solution is not particularly limited, but is preferably 0.1 to 2.0 g / L, and more preferably 0.5 to 1.0 g / L.

In the method of the present invention, a base is added to the culture solution obtained as described above, radicicol contained in the culture solution is converted into a base addition salt, and then the base addition salt is converted from the culture solution. It is characterized by including a separating step. The type of base added to the culture solution is not particularly limited, and any base may be used as long as it can produce a water-soluble base addition salt of radicicol. Examples of the base include alkali metal hydroxides such as lithium hydroxide, sodium hydroxide, and potassium hydroxide; alkaline earth metal hydroxides such as magnesium hydroxide, calcium hydroxide, and barium hydroxide; and aluminum hydroxide. Ammonia; alkali metal carbonates such as sodium carbonate and potassium carbonate; alkaline earth metal carbonates such as magnesium carbonate; alkali metal bicarbonates such as sodium hydrogen carbonate and potassium hydrogen carbonate; dimethyl Organic amines such as amine, trimethylamine, diethylamine, triethylamine, pyridine, piperidine and piperazine can be mentioned. These may be used in combination of two or more.
Of these, alkali metal hydroxides are preferred.

A base is added to the culture solution, and the culture solution is adjusted to pH7.
By adjusting the pH to 0 or more, preferably pH 7.5 to 10, more preferably pH 8 to 9, radicicol in the culture solution can be converted to a base addition salt. The base may be added as a solid, but may also be added as an aqueous solution having an appropriate concentration. The culture solution immediately after adding the base may be used in the next separation step. For example, a culture solution adjusted to be basic by adding a required amount of base is preferably 10%.
After stirring at 〜40 ° C., more preferably 20-30 ° C., preferably 10-60 minutes, more preferably 15-30 minutes, it is desirable to separate the base addition salt. When this treatment is performed, radicicol that has been insolubilized in the culture solution is converted into a water-soluble base addition salt, and the recovery rate may be increased in some cases.

The method of separating the base addition salt of radicicol produced in the culture solution is not particularly limited. For example, the separation can be carried out efficiently by using a synthetic adsorbent. In general, the base addition salt can be adsorbed to the synthetic adsorbent by filling the column with the synthetic adsorbent and then passing the culture solution through the column. The base addition salt can also be adsorbed to the synthetic adsorbent by a batch method in which a synthetic adsorbent is directly added to the culture solution and stirring is performed. When the base addition salt is adsorbed on the synthetic adsorbent, the culture solution in a state in which the microbial cells are suspended may be used as it is, but generally, the culture solution after separating the microbial cells is used. It is desirable. Separation of microbial cells can be performed by a usual separation operation, for example, filtration, centrifugation, membrane separation, etc., preferably filtration. Filtration is
For example, it can be performed with a nutsche, a filter press, a basket separator or the like.

The kind of the synthetic adsorbent is not particularly limited, and can adsorb the radicicol base addition salt in the culture solution adjusted to basicity, and can remove the base addition salt adsorbed by an appropriate eluent. Any substance that can be eluted may be used. For example, aromatic HP series, aromatic SP800 series, aromatic modified SP200 series, acrylic HPMG series (manufactured by Mitsubishi Chemical Corporation: Diaion Ion Exchange Resin Synthetic Adsorbent Manual 12th edition Mitsubishi Chemical Corporation) Edited by Ion Exchange Resin Department, published on September 10, 1997), aromatic XAD-2, XAD-4, acrylic XAD-7,
A nonionic porous resin such as XAD-8 (above, manufactured by Rohm and Haas Co.) can be used (here, the nonionic porous resin does not have a functional group such as an ion exchange group, and is a van der Waal). -Means a resin which adsorbs various organic substances by the loose force). Preferably Diaion SP2
Resin obtained by copolymerizing a vinyl monomer containing styrene and divinylbenzene, such as 07 (manufactured by Mitsubishi Chemical Corporation) can be used.

After washing the synthetic adsorbent adsorbing the base addition salt of radicicol, the adsorbed base addition salt can be eluted using an organic solvent or a water-containing organic solvent as an eluent. In this case, the organic solvent is preferably a water-soluble organic solvent. The water-soluble organic solvent means an organic solvent that dissolves in water at room temperature in an amount of 5% by volume or more. For washing, an aqueous solution of the above-mentioned base can be used in addition to water, and the concentration of the base is preferably about 0.01 to 0.05 normal, more preferably about 0.015 to 0.02 normal. For example, it is preferable to perform cleaning using an aqueous solution of an alkali metal hydroxide having the above concentration. Washing can be carried out by appropriately combining water and an aqueous base solution, or two or more aqueous base solutions or two or more aqueous base solutions having appropriate concentrations may be appropriately combined. As the eluent, a water-soluble organic solvent is preferable, and two or more kinds of organic solvents may be mixed and used, or a water-containing organic solvent to which an appropriate amount of water is added may be used as the eluent. Examples of the organic solvent include acetone, methanol, ethanol, propanol, and the like, and preferably, methanol can be used.

After the eluate containing the base addition salt of radicicol is subjected to a neutralization treatment, radicicol in a free form can be isolated by an appropriate separation method. The neutralization treatment is not particularly limited. For example, an acid such as hydrochloric acid or sulfuric acid is added to the above eluate to obtain a pH of 7.0 or less, preferably pH 5 or less.
To 7, more preferably to pH 6 to 6.5. By concentrating or cooling the solution after the treatment under normal pressure or reduced pressure, radicicol in a free form can be precipitated. When concentrating under reduced pressure,
Preferably 700 to 760 mmHg, more preferably 7
The solution is preferably concentrated under a reduced pressure of about 20 to 760 mmHg, preferably at a temperature of about 30 to 80 ° C., more preferably about 40 to 50 ° C., until the concentration becomes higher than the saturated concentration of radicicol. The temperature at which radicicol is precipitated from the obtained concentrate is preferably 20 to 30 ° C,
More preferably, it is 20 to 25 ° C. After concentrating and neutralizing the solution after neutralization, radicicol can be precipitated from the organic solvent by dissolving the residue in an organic solvent and, if necessary, concentrating the solution under normal pressure or reduced pressure. As the organic solvent to be added, a water-soluble organic solvent is preferable, and for example, methanol, ethanol, acetone and the like can be used, and methanol is more preferable. In addition, a seed crystal or the like may be appropriately used to precipitate a crystal.

The precipitated radicicol can be isolated by ordinary operations such as filtration and drying, and if necessary, the obtained crystals are recrystallized from methanol, ethanol, acetone or the like to improve the purity. Can be done. Incidentally, radicicol represented by the above formula has an asymmetric carbon, there are stereoisomers such as optically active isomers and diastereoisomers, the method of the present invention, all stereoisomers and It can be used to produce the mixture.

According to the method of the present invention, radicicol of high purity can be produced or purified without requiring a large amount of an organic solvent or purification by silica gel column chromatography. Further, the method of the present invention can be carried out using only a water-soluble organic solvent as an organic solvent, in which case, as in the conventional method, ethyl acetate, benzene, chloroform, and water-insoluble organic solvents such as toluene. It is possible to produce or purify radicicol of high purity without using a solvent.

[0019]

EXAMPLES The present invention will be described more specifically with reference to the following examples, but the scope of the present invention is not limited to the following examples. Example 1 10 L of a culture solution obtained in the same manner as in Reference Example 1 was adjusted to pH 8.5 with a 10 N aqueous solution of sodium hydroxide, followed by filtration with Nutsche to obtain a filtrate from which microbial cells were removed. 7 L of the filtrate contained 4.2 g of radicicol. The obtained filtrate is applied to a column (400 mL) filled with a synthetic adsorbent (Diaion SP207 manufactured by Mitsubishi Chemical Corporation) at a flow rate SV [eluate amount (weight) per hour per synthetic adsorbent (weight)] 3. After passing through the column, the column was sequentially washed with 1.2 L of a 0.02 N aqueous sodium hydroxide solution and 1.2 L of water. Next, elution was performed with 1.6 L of 100% methanol. The column eluate containing the sodium salt of radicicol was adjusted to pH 6.5 with concentrated hydrochloric acid and converted to free form radicicol. The obtained reaction solution was concentrated to 25 mL, the concentrated solution was allowed to stand at 25 ° C. for 24 hours, and then filtered to obtain crude radicicol crystals with a purity of 95% with a recovery rate of 75% (3. 2g). The crude crystals of radicicol were subjected to a recrystallization operation using methanol to give a purity of 98%.
Was obtained at a recovery rate of 90% (2.8 g in terms of pure product).

Example 2 The same procedure as in Example 1 was repeated, except that the neutralized reaction solution containing radicicol was concentrated to dryness, the residue was dissolved in 1 L of methanol, and further concentrated to 25 mL to obtain a concentrated solution. Was manufactured. As a result, radicicol crude crystals having a purity of 95% were obtained at a recovery rate of 75% (3.2 g in terms of pure product).

Example 3 Radicicol was produced in the same manner as in Example 1 except that the extract containing the microbial cells was passed directly through the column without filtration. As a result, 95% pure radicicol crude crystals
It was obtained with a recovery of 0% (2.9 g in terms of pure product). The crude crystals of radicicol were subjected to a recrystallization operation using methanol to obtain radicicol having a purity of 98% with a recovery rate of 90% (2.6 g in terms of pure product).

Example 4 10 L of the culture solution obtained in the same manner as in Reference Example 2 was adjusted to pH 8.5 with a 10 N aqueous solution of sodium hydroxide, and then filtered with a Nutsche to obtain a filtrate from which microbial cells were removed. 7 L of the filtrate contained 4.2 g of radicicol. After passing the obtained filtrate through a column (400 mL capacity) packed with a synthetic adsorbent (Diaion SP207 manufactured by Mitsubishi Chemical Corporation) at a flow rate of SV3, 1.2 L of a 0.02 N aqueous sodium hydroxide solution, and Washed sequentially with 1.2 L of water. Next, elution was performed with 1.6 L of 100% methanol. The column eluate containing the sodium salt of radicicol was adjusted to pH 6.5 with concentrated hydrochloric acid and converted to free form radicicol. The obtained reaction solution was concentrated to 25 mL, and the concentrated solution was allowed to stand at 25 ° C. for 24 hours, and then filtered to obtain crude radicicol with a purity of 95% with a recovery of 75% (3. 2g). The crude crystals of radicicol were subjected to a recrystallization operation with methanol to obtain radicicol with a purity of 98% at a recovery rate of 90% (2.
8g).

Example 5 The neutralized reaction solution containing radicicol was concentrated to dryness,
Radicicol was produced in the same manner as in Example 4, except that the concentrate was dissolved in L of methanol and further concentrated to 25 mL to obtain a concentrated solution. As a result, radicicol crude crystals having a purity of 95% were obtained with a recovery rate of 75% (3.2 in pure product conversion).
g).

Example 6 Radicicol was produced in the same manner as in Example 4 except that the extract containing the microbial cells was passed directly through the column without filtration.
As a result, the radicicol crude crystals having a purity of 95% were reduced to 70%.
(2.9 g in terms of pure product). The crude crystals of radicicol were subjected to a recrystallization operation using methanol to obtain radicicol having a purity of 98% with a recovery rate of 90% (2.6 g in terms of pure product).

[0025] Reference Example 1 Penicillium as an inoculum Ruteo - aurantium (Pen
icillium luteo-aurantium ). The strain was transferred to a seed medium (glucose 10) in a 2 L Erlenmeyer flask.
g / L, peptone 5 g / L, dried yeast 5 g / L, vegetable juice 200 g / L, trimagnesium phosphate 0.5 g /
L, adjusted to pH 6.0 before sterilization) Inoculate 300 mL,
The cells were cultured at 30 ° C. for 48 hours with shaking (220 rpm). 1.8 L of this seed culture was added to the main fermentation medium (sucrose 70 g / 30 g) in a 30 L stainless steel jar fermenter.
L, corn steep liquor 30 g / L, magnesium sulfate 0.5 g / L, monopotassium dihydrogen phosphate 0.5 g / L
L, trimagnesium phosphate 0.5g / L, pH before sterilization
(Adjusted to 7.0) 18 L was inoculated, and aeration and agitation culture (rotation speed: 270 rpm, aeration rate: 18 L / min) was performed at 25 ° C. for 5 days. 10 L of the culture was adjusted to pH 8.5 with 10 N sodium hydroxide, and then filtered through Nutsche to obtain a filtrate from which microbial cells had been removed. 7 L of the filtrate contained 4.2 g of radicicol.

Reference Example 2 A culture solution containing radicicol was obtained in the same manner as in Reference Example 1 by using a bacterium belonging to the genus Penicillium as the inoculum. 10 L of the culture was adjusted to pH 8.5 with a 10 N aqueous sodium hydroxide solution, and then filtered through Nutsche to obtain a filtrate from which microbial cells had been removed. 7 L of the filtrate contained 4.2 g of radicicol.

[0027]

Industrial Applicability According to the present invention, a method for producing or purifying radicicol suitable for an industrial scale, in which radicicol of high purity can be easily obtained at a high recovery rate without using a large amount of an organic solvent. A method is provided.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat (Reference) C12R 1:80) (72) Inventor Tetsuo Nishimura 1-1-1, Kyowa-cho, Hofu-shi, Yamaguchi Kyowa Hakko Kogyo Co., Ltd. F-term (reference) in the company's R & D center

Claims (11)

[Claims] 1. A method for producing radicicol represented by the following formula (I): The following steps: (1) a step of adjusting the pH of the culture solution to 7.0 or higher by adding a base to the culture solution in which the radicicol-producing bacteria have been cultured; (2) removing a base addition salt of radicicol contained in the culture solution A step of eluting the base addition salt with an organic solvent or a water-containing organic solvent after being adsorbed to the synthetic adsorbent; and (3) neutralizing the base addition salt contained in the eluate into free form radicicol. A method comprising the step of converting. 2. The method according to claim 1, wherein the pH of the culture solution is adjusted to 7.5 to 10. 3. The culture solution is adjusted to pH 8-9.
The method described in. 4. The method according to claim 1, wherein an alkali metal hydroxide is used as the base. 5. The method according to claim 1, wherein the synthetic adsorbent is a nonionic porous resin. 6. A method for producing radicicol, comprising a step of separating radicicol as a base addition salt from a culture solution in which radicicol-producing bacteria have been cultured. 7. The method according to claim 6, comprising a step of adding a base to the culture solution to adjust the pH to 7.0 or more to convert the culture solution into a base addition salt. 8. A culture solution containing a base added at pH 7.5-10.
The method according to claim 7, which comprises a step of converting the compound to a base addition salt by adjusting the pH of the solution. 9. The method according to claim 6, wherein an alkali metal addition salt is used as the base addition salt. 10. The method according to claim 6, wherein the base addition salt is separated by a synthetic adsorbent. 11. The method according to claim 10, wherein the synthetic adsorbent is a non-ionic porous resin.