JPH0249777B2 - SHIRIKAGERUNOSAISEIHO - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for regenerating silica gel, and more particularly to a method for regenerating silica gel used for purification of coenzyme Q. Coenzyme Q is involved in the electron transport system in vivo, and is used as a therapeutic agent for diseases such as heart failure. This coenzyme Q is produced by methods such as synthesis, fermentation, and extraction from natural products, but as it is a pharmaceutical, it is required to be highly pure. Column chromatography using silica gel as an adsorbent is used as a purification method for this purpose, but due to the separation of coenzyme Q and impurities in column chromatography, the regeneration of silica gel is insufficient. Therefore, it was difficult to reuse it. For these reasons, column chromatography using silica gel has been problematic as an industrial purification method in terms of workability and the amount of silica gel used.

The present inventors investigated various silica gel regeneration methods in silica gel column chromatography, and found that impurities that had been adsorbed on the silica gel were removed by adsorption, elution, washing the silica gel with a solvent, and then drying the silica gel in the column. Coenzyme Q can be removed by column chromatography using regenerated silica gel obtained by this method.
The silica gel can be well separated from impurities, making it possible to use the silica gel repeatedly and reducing the amount of silica gel used. Furthermore, it has been found that this regeneration method is applied to the silica gel that is still filled in the column container, so that operations such as filling and taking out the silica gel can be omitted, and the present invention has been completed.

That is, the present invention purifies coenzyme Q by adsorbing coenzyme Q to silica gel using column chromatography using silica gel as an adsorbent and eluting it with an organic solvent for elution. In a method for regenerating silica gel after cleaning silica gel with a cleaning organic solvent, the silica gel is brought into contact with a nonpolar organic solvent while the silica gel after cleaning is held in the column (hereinafter referred to as the first solvent treatment). Alternatively, without contacting the column, the organic solvent remaining in the column is discharged, the silica gel is sufficiently dried, and then the silica gel is brought into contact with a non-polar organic solvent (hereinafter referred to as the second solvent treatment). This is a method for regenerating silica gel in a closed system.

The starting materials to be purified in the present invention are synthesized,
This is crude coenzyme Q that contains impurities such as various lipids that are mixed in when coenzyme Q is produced by methods such as fermentation and extraction from natural products. Coenzyme Q in the present invention may be any of coenzymes _Q1 to _Q12 , or may be a mixture of two or more thereof.

The silica gel used in the present invention is not particularly limited, but for practical purposes, for example, silica gel (trade name Wako Gel C-200, manufactured by Wako Pure Chemical Industries, Ltd.), silica gel (trade name Wako Gel C-300, manufactured by Wako Pure Chemical Industries, Ltd.), silica gel (Product name: Art 7734, manufactured by Merck & Co.),
Koyoto silica gel (product name ART 9385, manufactured by Merck & Co., Ltd.), silica gel (product name KT-3063, manufactured by Fujigel), and silica gel (product name 4B, manufactured by Fujigel)
Commercially available products such as are preferably used.

Adsorption with silica gel, elution, and washing are performed by conventional methods. In practice, the elution organic solvent used in the elution treatment is usually a single solvent such as benzene and toluene, or n-hexane,
n-pentane, isooctane, cyclohexane,
A mixed solvent whose polarity is adjusted by mixing a nonpolar solvent such as benzene and petroleum ether with a polar solvent such as chloroform, ethyl ether, isopropyl ether, ethyl acetate, acetone, methanol, and ethanol is used.

In order to remove impurities adsorbed on the silica gel after elution, silica gel is washed using, for example, chloroform, ethyl ether,
Isopropyl ether, benzene, ethyl acetate,
It is carried out using acetone, methanol, ethanol, n-hexane, and n-heptane, each alone or in a mixture thereof. Note that the cleaning solvent used for cleaning must have greater polarity than the elution organic solvent used for elution. The amount of solvent used for cleaning is not particularly limited and may be as long as it is necessary to remove impurities adsorbed on silica gel, but in general, silica gel 1
Approximately 2 to 15 per kg is preferred.

In the present invention, the first solvent treatment can be omitted, but is preferably carried out in order to improve the separation ability of the regenerated silica gel.

The following nonpolar organic solvents are used in each of the first solvent treatment and the second solvent treatment of the present invention. i.e. n-hexane, n-heptane, n-
Non-polar organic solvents such as pentane, isooctane, cyclohexane, petroleum ether, benzene and toluene are used.

The amount of solvent required for the first solvent treatment is not particularly limited, but in practice, it is generally preferred that the amount of solvent be about 30 or less per kg of silica gel. The first solvent treatment is performed at room temperature or above room temperature and below the boiling point of the solvent. Furthermore, heating within this temperature range is not prohibited.
In the first solvent treatment, it is preferable to apply pressure so that the solvent flows smoothly through the gaps between the silica gel particles, and in practical terms, the pressure is set at 2 kg/cm ² or less.

Following the wash or first solvent treatment, the solvent in the column is forced out of the column. This solvent may be discharged naturally by weight, or may be forcibly discharged by pressurization or suction. In order to shorten the discharge time and/or reduce the burden of drying, it is preferable to force the discharge. Moreover, this discharge is preferably performed in an inert gas atmosphere.
Practically, it is preferable to extrude using pressurized inert gas, and the pressure at this time is usually 2 kg/cm ² or less.

After the solvent in the column is discharged, the silica gel is dried in order to substantially completely remove the solvent and water adhering to the silica gel and remaining in the gaps between the silica gel particles. There are no particular restrictions on these drying conditions. That is, the drying temperature may be a temperature that does not cause deterioration of the silica gel, and may be room temperature to normal temperature, or may be heated. In practice, the temperature is usually about room temperature to about 100°C. The pressure is also not particularly limited, but it may be any pressure that the column container can withstand, such as reduced pressure, normal pressure, or increased pressure. It is preferable to dry while passing an inert gas as described above and suctioning from the bottom of the column. When drying under heating, heating may be applied from outside the column container, or heated inert gas may be passed through the column.

Furthermore, it is preferable that the same solvent be used in the first and second solvent treatments.

There is no particular limit to the amount of solvent used in the second solvent treatment, but in practice it is approximately 2
~10 is preferred, and the inflow of the solvent in this treatment is preferably carried out under pressure. The pressure at this time is 5
Kg/cm ² or less is preferable. Further, the temperature at this time may be room temperature to room temperature. Impurities and moisture have been removed from the silica gel in the column thus restored to the adsorption separation ability of new silica gel. The silica gel thus regenerated according to the present invention can be reused as is for adsorption.

The silica gel used to purify coenzyme Q was previously thought to be unable to be reused due to insufficient regeneration, but the present invention restores the adsorption separation ability of silica gel and makes it possible to reuse it. Eliminates the need to replace silica gel and reduces the amount of silica gel used.
Therefore, the present invention has great industrial significance since it is improved in terms of cost and operation.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 Protaminobacter ruber NC, a methanol-assimilating bacterium, was grown in a synthetic medium containing methanol.
IB 2879 was cultured at 28° C. with air aeration and mechanical stirring. After the culture was completed, the cells were collected using a centrifuge and dried using a spray dryer to obtain 18 kg of dried bacterial cells.

60 kg of acetone was added to 18 kg of dried bacterial cells, and the mixture was stirred and extracted at 30°C for 1 hour, and the bacterial cells were separated by filtration. This operation was repeated three times, and the three extracts were combined and concentrated under reduced pressure to dryness. 1000 ml of n-hexane was added thereto to dissolve and remove insoluble impurities. to this
Add 200ml of methanol solution containing 5% of 28wt% ammonia water, stir for 20 minutes, stand still, and
-The hexane layer and the ammonia/methanol layer were separated, and the ammonia/methanol layer was removed. Then, 200 ml of a methanol solution containing 5 vol% water was added to the hexane layer, stirred for 20 minutes, and then allowed to stand still.
-Separate the hexane layer and water/methanol layer,
The water/methanol layer was removed. The n-hexane layer was concentrated under reduced pressure and dried, and dissolved in 150 ml of acetone to remove insoluble matter. 150 ml of methanol was added to this liquid, and the mixture was placed in a -10°C deep freezer and allowed to stand overnight to precipitate crystals, which were filtered to obtain 20 g of crude crystals (purity 79.8%) containing coenzyme Q ₁₀ .

Silica gel (trade name: Wako Gel C) suspended in n-hexane was placed in a column container with a diameter of 26 mm and a length of 300 mm.
-300, manufactured by Wako Pure Chemical Industries), and charged 5 g of the above-mentioned crude crystals dissolved in n-hexane to the top of the column.
- When the mixture with hexane was flowed down at a flow rate of 200 ml/hr, the coenzyme
Effluent containing Q ₁₀ began to flow out of the column.
A 500 ml portion was collected as a fraction containing pure coenzyme Q ₁₀ , and the solvent was removed from it to obtain 3.55 g of pure coenzyme Q ₁₀ . This corresponds to about 89% of coenzyme Q ₁₀ in the crude crystals.

The silica gel column was washed with 300 ml of a 1:1 mixed solvent (vol) of acetone:n-hexane to remove impurities. Then add 0.5 to the column
Kg/cm ² of nitrogen gas was passed through the column for 60 minutes to discharge the solvent in the column. Thereafter, vacuum drying was performed for one day while heating the column to 80°C. After that 2Kg/
300 ml of n-hexane pressurized at cm ² was flowed and treated with a solvent at room temperature (corresponding to the second solvent treatment) to regenerate the silica gel in the column.

A column packed with regenerated silica gel was charged with 5 g of the above crude crystals dissolved in n-hexane at the top of the column, and the acetone content was 1 vol%.
A mixture of acetone and n-hexane at a flow rate of 200
The flow rate was ml/hr. Collect 500 ml as a pure coenzyme Q ₁₀ -containing fraction, remove the solvent from it, and obtain 3.51
g of pure coenzyme Q ₁₀ was obtained. This corresponds to about 88% of coenzyme Q ₁₀ in the crude crystals.

Example 2 Example 1 except that the silica gel after adsorption and elution was washed with a 1:1 mixed solvent (vol) of acetone and n-hexane, and then 500 ml of n-hexane was poured into the sample and the first solvent treatment was carried out at room temperature. I went in the same way. Pure coenzyme when using this regenerated silica gel column
_Q10 The amount obtained was 3.5g. This corresponds to about 90% of the coenzyme Q ₁₀ in the crude crystals.

Comparative example 1 Acetone on silica gel column after adsorption and elution:
The procedure was carried out in the same manner as in Example 1, except that after washing by flowing 300 ml of a 1:1 mixed solvent (vol) of n-hexane to remove impurities, the column was regenerated by flowing 500 ml of hexane without discharging or drying. Ivy. When this regenerated silica gel column was reused, the amount of pure coenzyme _Q10 obtained was 2.2 g. This corresponds to about 55% of the crude crystalline coenzyme _Q10 .

Example 3 Isodecarenol and 2,3-dimethoxy-5-
2500 ml of acetonitrile was added as an extractant to 50 g of a reaction solution containing coenzyme Q ₁₀ obtained by reacting with methylhydroquinone, and extraction was performed at 30°C for 10 minutes to obtain an extract, which was filtered. . This operation was repeated five times, and the resulting extracts were combined, concentrated under reduced pressure, and dried to obtain a solid. Add 200ml of methyl ethyl ketone to this solid, stir and dissolve at 20°C, filter, put this filtrate in a -10°C freezer, leave for 2 hours, and after confirming that the temperature of the liquid has reached -10°C, the purity is 99.4. % of powdered coenzyme Q ₁₀ was added thereto, and the mixture was allowed to stand for an additional 5 days to precipitate crystals.
When the precipitated crystals were filtered and dried under vacuum, 20 g of crude crystals (purity 71.0%) containing coenzyme Q ₁₀ were obtained.
I got it.

A column container with a diameter of 26 mm and a length of 300 mm was filled with 50 g of silica gel (product name: Art 7734, manufactured by Merck & Co., Ltd.) suspended in n-heptane.
5 g of the above crude crystals dissolved in heptane were charged to the top of the column, and a mixture of ethyl acetate and n-heptane with an ethyl acetate content of 5 vol% was added at a flow rate of 180 ml/heptane.
When the column was allowed to flow down at hr, an effluent containing coenzyme Q ₁₀ began to flow out of the column approximately 2 hours and 10 minutes after the start of flow. 400 ml was collected as a fraction containing pure coenzyme Q ₁₀ , and the solvent was removed from it to obtain 3.27 g of pure coenzyme Q ₁₀ . This is about 92 coenzyme Q ₁₀ in the crude crystals.
%.

This silica gel was washed with 200 ml of ethyl acetate to remove impurities, and then nitrogen gas of 0.5 Kg/cm ² was passed through the column for 60 minutes to discharge the solvent in the column. The column was then vacuum dried for one day while being heated to 85°C. Thereafter, 400 ml of n-heptane pressurized at 2 kg/cm ² was flowed, and solvent treatment was performed at room temperature (corresponding to the second solvent treatment) to regenerate the silica gel in the column.

Into a column filled with regenerated silica gel, 5 g of the above crude crystals dissolved in n-heptane were charged to the top of the column, and the ethyl acetate content was 5 vol.
% ethyl acetate and n-heptane at a flow rate of
The flow rate was 180ml/hr. Collect 400 ml of pure coenzyme Q into ₁₀ portions, remove the solvent, and reduce to 3.25 ml.
g of pure coenzyme Q ₁₀ was obtained. This corresponds to about 92% of coenzyme Q ₁₀ in the crude crystals.

Example 4 The same procedure as in Example 3 was carried out, except that the silica gel was washed with ethyl acetate after adsorption and elution, and then 300 ml of n-heptane was poured into the gel for the first solvent treatment. When this regenerated silica gel column was used, the amount of pure coenzyme Q ₁₀ obtained was 3.23 g. This corresponds to about 91% of coenzyme Q ₁₀ in the crude crystals.

Comparative Example 2 After adsorption and elution, the silica gel column was washed with 200 ml of ethyl acetate to remove impurities.
Add n-heptane to the column without draining and drying.
The same procedure as in Example 3 was carried out, except that 1500 ml was flowed and the column was regenerated. When this regenerated silica gel was used, the amount of pure coenzyme _Q10 obtained was 2.5 g.
This corresponds to about 70% of coenzyme Q ₁₀ in the crude crystals.

Example 5 The procedure was carried out in the same manner as in Example 3, except that following washing with ethyl acetate, the solvent in the column was allowed to drain naturally by gravity by leaving the valve at the bottom of the column open for one day. Substantially the same results as Example 3 were obtained.

In addition, in the above-mentioned Examples and Comparative Examples, unless otherwise specified, each treatment was performed at room temperature.

Claims (1)

[Claims] 1 Purify coenzyme Q by adsorbing coenzyme Q onto silica gel using column chromatography using silica gel as an adsorbent, eluating it with an organic solvent for elution, and then using an organic solvent for washing that is more polar than the organic solvent for elution. In the method for regenerating silica gel after washing the silica gel with silica gel, the organic solvent remaining in the column is discharged while the silica gel after washing is retained in the column, the silica gel is sufficiently dried, and then the silica gel is A method for regenerating silica gel in a closed system characterized by contact with a nonpolar organic solvent.