KR20020071633A - A purification method of pravastatin precursor compactin and derivatives thereof using HP20 resin and isopropyl alcohol - Google Patents

Abstract

PURPOSE: Provided is a purification method of a pravastatin precursor, compactin, and its derivatives by absorption chromatography with HP20 resin as controlling the concentration of isopropyl alcohol, thereby purifying them time and cost effectively. CONSTITUTION: The purification method of a pravastatin precursor, compactin, and its derivatives comprises the steps of: fermenting a strain producing a pravastatin precursor, compactin, and its derivatives to obtain a fermented solution; subjecting the fermented solution to absorption chromatography with HP20 resin using isopropyl alcohol as absorbing solution; and recovering a pravastatin precursor, compactin, and its derivatives using isopropyl alcohol as an eluent.

Description

A purification method of pravastatin precursor compactin and derivatives conjugate using HP20 resin and isopropyl alcohol}

The present invention relates to a method for concentrating and purifying pravastatin precursor comptintin and derivatives thereof, and more particularly, by concentrating and purifying pravastatin precursor compactin and its derivatives by adsorption chromatography using HP20 resin and isopropyl alcohol. It is purified.

Pravastatin precursor compactin is disclosed in US Pat. Nos. 3,983,140 and 1,453,425 as substances capable of lowering cholesterol concentrations. It is a substance that is effective in heart disease such as myocardial infarction or arteriosclerosis caused by hyperlipidemia, especially hyperlipidemia by lowering cholesterol concentration.

Strains used in the preparation method of such pravastatin precursors include penicillium brevicompactin, Penicillmyces sp., Trichoderma longibriatum, Tricorderma pseudo Trichoderma psudokoning, Hyphomyces chrisopomus and Penicillium citrinum (Japanese Patent Publication No. 4-349034). Pravastatin precursors can be prepared by such strains or chemical transformations. In addition, Korean Patent No. 0186758 discloses a method of preparing pravastatin precursor compactin and its derivatives by aerobic culturing of Gliocladium sp. YJ-9515 (KCTC 02528BP).

Korean Patent No. 100212482 discloses a method for preparing pravastatin sodium using Streptomyces exfoliatus YJ-118, and No. 0186706 to Streptomyces roseochromogenes subsp. A method of preparing pravastatin sodium by adding and culturing pravastatin precursor to a culture medium of KTCC 12385 is described.

The present invention provides a method for purifying pravastatin precursor compactin and its derivatives. Korean Patent No. 0186706 describes a method for purifying a culture medium cultured with Streptomyces roseochromogenes subspecies KCCM 1285 cells to obtain pravastatin sodium salt. Stirring for about time and centrifuging to remove the cells; Centrifuging the supernatant by adsorption column chromatography using HP20 resin, and then washing again; Pravastatin sodium was obtained through a complex process of several steps, such as eluting with an eluent.

The present invention provides a method for purifying pravastatin precursor compactin consisting of a minimal number of steps of simply passing the culture solution through HP20 resin and eluting again this several step process.

The present invention purifies pravastatin precursor compactin prepared by culturing Gliocladium sp. And Penicilium sp. By affinity chromatography using HP20 resin and isopropyl alcohol. When isopropyl alcohol is used in the adsorption solution and the elution solution, it is characterized in that the method is adjusted to be 0-20% in the adsorption solution and 60% or more in the elution solution.

Production strains used in the present invention glyocladium genus and penicillium genus can be used a known fungal culture method, but for the production and seed culture medium for passage of strain preservation medium, several stages of seed culture and medium production for the present culture , Agitation cultivation according to this, and a culture technique method capable of producing the target product in a large amount can be used. Glyclodium strains generally grow at temperatures of 10-36 ° C. Of these, the optimum temperature for the production of pravastatin precursor is 23-28 ° C. and the most suitable temperature is 25 ° C. The growable pH is 4.0-8.0 and the optimal pH is 5.0-6.0 [Korean Patent No. 0186758].

When culturing the strain, the carbon source of the culture medium may be glucose, glycerol, maltose, and as the nitrogen source, Corn stip liquor, tryptone, soybean peptone, skim soybean meal, yeast extract and sodium nitrate Salts may include magnesium sulfate, ammonium sulfate and monophosphate. It can be cultured under aerobic conditions in such medium.

Culture conditions and culture conditions of the strain for obtaining the pravastatin precursor used in the present invention is as described in Korean Patent No. 0186758 (Youngjin Pharm.).

The present invention utilizes isopropyl alcohol as an adsorption solution and an elution solution for the pravastatin precursor cultured from the strain, etc. without further centrifugation or filtration, and by adjusting the concentration thereof, by using the adsorption to HP20 resin Purification of pravastatin precursor compactin and derivatives thereof in high yield.

Example 1 Influence of Isopropyl Alcohol Concentration When Using Strains of Glycodium

The filtrate fermented with the genus Gliocladium strain was 0.5 g / l of Compectin and its derivatives, and the alcohol content was adjusted to the concentration shown in Table 1 below, and 20 L of this solution was adsorbed onto 1000 ml of HP20.

Isopropyl alcohol concentration in adsorption liquid (%) 0 10 20 30 40 50 Content of compactin and its derivatives (g) 0.0 0.0 0.0 1.2 8.0 9.1 Adsorption rate (%) 100 100 100 88 20 9

The adsorption rate was 80% or more when the alcohol content was in the range of 0-30%, but the adsorption rate was 20% or less at a concentration of 40% or more.

Example 2: Effect of Isopropyl Alcohol Concentration When Using Penicillium Strain

The filtrate fermented with the genus Penicillium strain to adjust the content of the compactin and its derivatives to 1.0 g / l, 10 L of the solution adjusted to the alcohol content to the concentration as shown in Table 2 is adsorbed to 1000 ml HP20.

Isopropyl alcohol concentration in adsorption liquid (%) 0 10 20 30 40 50 Content of compactin and its derivatives (g) 0.0 0.0 0.0 1.2 8.1 9.2 Adsorption rate (%) 100 100 100 98 19 8

The adsorption rate was 80% or more in the range of 0-30% alcohol content, but less than 20% in the concentration of 40% or more.

Example 3

Pravastatin-producing strains are cultured using Gliocladium sp. And Penicillmyces sp. The culture solution is filtered, and water is added to the filtrate so that the content of pravastatin compactin and its derivatives is 0.5 g / l and the alcohol content is 10%. After diluting 20 L (10 g total) of this culture to 1000 ml of HP20 column, eluting with 5 L of eluent having different alcohol concentration, the content and purity of the compactin and its derivatives in the eluted solution were measured. The chromaticity was converted into 100 by the total chromaticity in the adsorption liquid before adsorption. Chromaticity was measured at OD660 nm.

Alcohol content in eluent (%) 0 10 20 30 40 50 60 80 100 Content of compactin and derivatives thereof in eluted solution (g) 0.0 0.0 0.0 1.1 6.5 8.9 9.1 8.9 9.2 Elution rate (%) 0 0 0 11 65 89 91 89 92 Chromaticity (%) 10 10 10 13 21 26 25 27 24

Purifying or concentrating pravastatin precursor compactin and its derivatives, the elution rate of the compactin and its derivatives in the eluent was 11% or less in the range of 0-30% alcohol, but 65% in the 40% alcohol content. Above 60%, above 90%. The color of brown decreased by more than 80% of undiluted solution. The eluent volume was concentrated to 1/4 of the adsorption liquid.

Example 4 Content of Isopropyl Alcohol in Eluent and Content of Compectin and Its Derivatives after Elution

1000 ml of HP20 column was mixed with 5 liters (10 g total) of liquid containing 2 g / L of compactin and its derivatives and 0% of alcohol content by adding water and alcohol to a solution containing the culture of penicillium strain. After adsorbing to eluent, the eluent was eluted with 5 L of eluent, and the content and chromaticity of the compactin and its derivatives were measured. The chromaticity was converted into 100 as the total chromaticity in the adsorption liquid before adsorption.

Isopropyl Alcohol Content in Eluent (%) 0 10 20 30 40 50 60 80 100 Content of compactin and derivatives thereof in eluted solution (g) 0 0 0 1.2 6.8 8.2 9.2 9.1 9.3 Elution rate (%) 0 0 0 12 68 82 92 91 93 Chromaticity (%) 9 11 10 15 21 23 20 25 27

In case of purification or concentration of compactin and its derivatives, the elution rate of the compactin and its derivatives in the eluent was less than 12% in the range of 0-30% alcohol, but 68% in the 40% alcohol content and 60%. In the above, it was 90% or more. The color of brown decreased by more than 75% of the stock solution. The eluted liquid has more than 50% alcohol content, which has a lower boiling point than water, which is effective for concentrating at low temperatures and saving energy.

According to the present invention, when purifying the pravastatin precursor compactin and its derivatives by adjusting the isopropyl alcohol concentration by adsorption chromatography with HP20 resin, the purification process is minimized, thereby reducing the cost and time.

Claims (1)

Fermenting the production strain of pravastatin precursor compactin and its derivatives to obtain a fermentation broth; Adsorption chromatography of the fermentation broth using HP20 resin using isopropyl alcohol as an adsorption solution; And In the method for purifying the complex and the derivatives of the pravastatin precursor comprising the step of recovering the pravastatin precursor compactin and derivatives thereof using isopropyl alcohol as eluent, Adsorbing the isopropyl alcohol content in the adsorption solution in the range of 0-30% and allowing the isopropyl alcohol content in the elution solution to be at least 60%.