KR20110012169A - Method of a micelle-based separation for pre-purification of paclitaxel - Google Patents

Abstract

PURPOSE: A micelle-based separation for pre-treating paclitaxel is provided to easily separate paclitaxel of high purity and to be used in secondary metabolite purification. CONSTITUTION: A micelle-based separation for pre-treating paclitaxel comprises: a step of adding a soluble organic solvent to Taxus genus plant, cell or cell culture medium thereof and extracting and concentrating; a step of adding non-polar organic solvent to the concentrate and extracting liquid-liquid; a step of decompressing and drying to obtain a first dry material; a step of treating the first dry material with an adsorbent and filtering to obtain a second dry material; a step of adding the second dry material to a mixture liquid of non-soluble organic solvent and cationic surfactant; a step of stirring and placing and collecting micelle(4)-formed cationic surfactant layer(3); a step of adding non-soluble organic solvent to the cationic surfactant and stirring; and a step of collecting non-soluble organic solvent layer(1) and decompressing.

Description

Method of a micelle-based separation for pre-purification of paclitaxel

The present invention relates to a micelle-based separation method for paclitaxel pretreatment from a paclitaxel-containing material, and a micelle-based separation method for paclitaxel pretreatment capable of easily separating high-purity paclitaxel in high yield by a micelle process using an adsorbent treatment and a cationic surfactant. will be.

 Paclitaxel, a diterpenoid family of cancer drugs, was discovered in the 1960s through a large-scale screening program for anticancer active substances from the National Cancer Institute of the United States, human uterine cancer, breast cancer, kaposi's sarcoma, It is known to be the most important anticancer substance for non-small cell lung cancer (NSCLC). Paclitaxel is a widely used anticancer drug approved by the FDA in 1992. Indications such as rheumatoid arthritis and Alzheimer's treatment continue to expand, and paclitaxel demand will continue to increase in the future as clinical trials involve multiple prescriptions. Paclitaxel is isolated and purified from bark of yew tree, which has a very low paclitaxel content of 0.02%, which requires high cost and causes problems of resource and ecosystem destruction. In order to overcome the above problems, a semisynthesis method of chemically bonding side chains by obtaining precursors from leaves of yew and a mass production method using plant cell culture methods obtained by culturing plant cells derived from yew trees Is being developed.

Plant cell culture method of the major production method of paclitaxel is not affected by external factors such as climate, environment, etc., because it can be stably produced in the bioreactor has the advantage that it can produce a large amount of paclitaxel of a certain quality. In order to produce paclitaxel from plant cell culture, there are several stages of separation and purification.In general, the biomass recovered from the cell culture solution is first extracted with an organic solvent, and the product is subjected to final purification through a pretreatment process. Production process. In this process, the pretreatment process in particular has a great impact on the final purification cost. Existing studies have been economically troubled by using expensive chromatography as a pretreatment process for purification or adopting a method for final purification of crude product extracted without pretreatment by high performance liquid chromatography (HPLC). As a result, there are many difficulties in scale-up.

Known techniques for the purification of paclitaxel are as follows.

International Patent Publication No. 2000/40573 discloses a low purity concentrate obtained from a liquid / liquid extraction process in a paclitaxel-containing material, first purified through a silica column, and then precipitated using an acetone / water solution. The process of obtaining paclitaxel of high purity by repeating is shown. However, in order to obtain high-purity paclitaxel, a total of three silica chromatography and two crystallization steps must be repeatedly performed. Therefore, a lot of solvent and time are required, and a precipitation method using acetone / water solution is required. It has a disadvantage in that the recovery rate drops considerably to 49-73%.

International Patent Publication No. 2000/078741 extracts natural raw materials of taxanes with an organic solvent, forms a precipitate with a base or an acid, melts them with acetone, and percolorizes them with nucleic acid to give resins and pigments. Is removed, a precipitate is formed with a base or an acid, and at least one chromatographic run and at least one crystallization with acetone and nucleic acid are disclosed. However, in general, paclitaxel is known to be decomposed into 10-diacetelpaclitaxel and bacatin III under acidic or basic conditions, making it difficult to purify paclitaxel in high yield, and there is a problem that requires a large amount of time due to a complicated process. .

The present invention is to solve the problems of the prior art as described above, paclitaxel can be easily separated in high yield paclitaxel in high yield by the micelle process using the adsorbent treatment and cationic surfactant, mass production in an efficient and economical way It is an object to provide a micelle-based separation method for pretreatment.

More specifically, by treating the adsorbent with a silica-based adsorbent before the micelle process, biomass-derived waxes and tars are effectively removed to increase the purity of the paclitaxel and the purification process and to increase the efficiency of the cationic surfactant. It is an object of the present invention to provide a micelle-based separation method for paclitaxel pretreatment that can easily separate high-purity paclitaxel in high yield by a micelle process.

The present invention has been made to achieve the above object, and provides a micelle-based separation method for paclitaxel pretreatment.

Hereinafter, the present invention will be described in more detail.

In this case, unless there is another definition in the technical terms and scientific terms used, it has the meaning that is commonly understood by those of ordinary skill in the art, unnecessarily obscure the subject matter of the present invention in the following description Description of known functions and configurations that may be omitted.

The present invention relates to a micelle-based separation method for paclitaxel pretreatment,

(a) extracting by adding a water-soluble organic solvent to a Taxus genus plant, a cell thereof, or a cell culture thereof, and then concentrating the extract;

(b) adding a non-polar organic solvent to the concentrate, followed by liquid-liquid extraction, concentration and drying under reduced pressure;

(c) drying the filtrate obtained by treating the first dried material obtained in step (b) with an adsorbent and filtering;

(d) putting the second dried product obtained in step (c) into a mixed solution of a non-aqueous organic solvent and a cationic surfactant, stirring and stabilizing to recover a cationic surfactant layer in which micelles are formed after phase separation;

(e) further adding a non-aqueous organic solvent to the recovered cationic surfactant layer of step (d), stirring and standing to recover the non-aqueous organic solvent layer after phase separation, and concentrating it under reduced pressure.

Taxus genus plant of the present invention, its cell or cell culture medium thereof refers to paclitaxel-containing material, the Taxus genus plant is Taxus brevifolia (Taxus brevifolia), Texas Taxus canadensis, Taxus cuspidata, Taxus baccata, Taxus globosa, Taxus floridana, Texas Wallichiana Taxus wallichiana), Taxus media, or Taxus chinensis, and the like.

The water-soluble organic solvent used in the step (a) is used to extract paclitaxel from biomass, specific examples of the water-soluble organic solvent used in the present invention is selected from methanol, ethanol, propanol, methylene chloride Species or mixtures of two or more species may be used, and as an organic solvent capable of recovering paclitaxel as much as possible from biomass, methanol may be preferably used. The biomass recovered from the Taxus genus plant, its cells or its cell culture in step (a) is 0.1-100% (w / v) in methanol, preferably 1-50% ( After addition to w / v), extraction and filtration by stirring for 30 minutes at room temperature may be repeated two or more times to extract the biomass-derived paclitaxel derivatives.

The non-polar organic solvent used in the step (b) serves to remove the polar impurities contained in the paclitaxel derivative through phase separation with the water-soluble organic solvent in the step (a), the specific of the non-polar organic solvent used in the present invention For example, one or two or more mixtures selected from methylene chloride, ethyl acetate, and ether may be used, and in the present invention, the polar impurities contained in the paclitaxel derivative may be most effectively removed by performing one to three liquid-liquid extractions. Can be. When the water-soluble organic solvent used for paclitaxel derivative extraction in step (a) is an alcohol, methylene chloride is generally used for liquid-liquid extraction. Methylene chloride can preferably be used with 20-30% (v / v) of the alcohol concentrate, and the use of excess methylene chloride will place a heavy burden on subsequent processes.

The adsorbent of the step (c) is preferably used a silica-based adsorbent, when using a silica-based adsorbent, it is possible to effectively remove impurities such as biomass-derived wax, tar, purity of paclitaxel and It is effective in increasing the efficiency of the purification process.

In the adsorbent treatment of step (c), the adsorbent is added in an amount of 20 to 100% (w / w), preferably 20 to 60% (w / w) of the first dry matter obtained in the step (b), and methylene One or more mixtures selected from chloride and chloroform are further added, followed by stirring at 20 to 60 ° C. for 30 minutes, followed by drying the filtrate obtained by filtration. Impurities can be effectively removed. In addition, the organic solvent used in the adsorbent treatment in step (c) should have a strong affinity for paclitaxel and a weak affinity for impurities such as tar and wax components.

The water-insoluble organic solvent of step (d) and (e) is dichloromethane, carbon tetrachloride, chloroform, 1,2-dichloroethane, di-ethyl ether (di-ethyl ether), methyl-t-butyl ether, di-iso-propyl ether (di-iso-propyl ether), one or a mixture of two or more selected from benzene The non-aqueous organic solvent used in step (d) may be used for efficient removal of nonpolar impurities through phase separation from the cationic surfactant layer, and the non-aqueous organic solvent used in step (e) may be used. The purpose is to recover paclitaxel contained in the micelles. Most preferably, a mixture of methyl-t-butyl ether and hexane may be used as the water-insoluble organic solvent in step (d), and most preferably as the water-insoluble organic solvent in step (e). Preferably methyl-t-butyl ether can be used.

Step (d) of the present invention is a method of removing impurities by micelle formation and phase separation using a cationic surfactant in a micelle based separation method. Cationic surfactants used for micelle formation include one or two or more selected from N-cetylpridinium chloride, hexadecyltrimethylammonium chloride, dodecyltrimethylammonium chloride Mixtures may be used, most preferably hexadecyltrimethylammonium chloride. As shown in FIG. 1, the cationic surfactant is composed of (5) (hydrophilic) and hydrophobic (6) (hydrophobic), and the hydrophobic tail group surrounds paclitaxel (7) that is insoluble in water. Externally, there is a hydrophilic head group and can be dissolved in an aqueous solution. In the micelle process of step (d), the water-insoluble organic solvent layer (1) is phase separated (2) from the cationic surfactant layer (3) including paclitaxel to effectively remove impurities from the cationic surfactant layer containing paclitaxel. Can be. In addition, the cationic surfactant used in the micelle process of the present invention is preferably used in an aqueous solution, 1 to 20% (w / v), most preferably 5 to 15% (w / v) of the aqueous solution In the case of using the aqueous solution of the cationic surfactant in the above range, it is effective to easily separate high purity paclitaxel in high yield.

The micelle-based separation method for paclitaxel pretreatment according to the present invention can be easily separated in high yield with high yield of paclitaxel by a micelle process using an adsorbent treatment and a cationic surfactant, and can be mass-produced in an efficient and economical manner.

More specifically, it is possible to effectively remove components such as waxes and tars derived from biomass by treating the adsorbent with a silica-based adsorbent before the micelle process, and purifying and purifying paclitaxel by performing a micelle process using a cationic surfactant. It is effective in increasing the efficiency of the process and easily separating high purity paclitaxel in high yield.

Hereinafter, the present invention will be described in more detail with reference to Examples, which are intended only for understanding the constitution and effects of the present invention, and are not intended to limit the scope of the present invention.

reagent

In the present invention, as a cationic surfactant, N-cetylpridinium chloride (N-cetylpridinium chloride, 99%, Acros, USA), hexadecyltrimethylammonium chloride (99%, Acros, USA), dodecyltrimethylammonium chloride ( dodecyltrimethylammonium chloride, 98%, Acros, USA) and sodium dodecyl sulfate (99%, Acros, USA), sodium dioctyl sulfosuccinate (98%) as anionic surfactant , Acros, USA), Tween 80 (Samchun Pure Chemical Co., Korea), polyethylene glycol 6000 (99%, Samchun Pure Chemical Co., Korea), Triton X- 100 (Sigma-Aldrich, USA) was used respectively.

Test Example 1 Analysis Method of Paclitaxel

The content of paclitaxel was analyzed using HPLC (Waters), and all the assay samples were taken three times to determine the content as an average value after analysis. The column used for analysis was Capcell Pak C18 UG 120 (250 mm × 4.6 mm, Shiseido, Japan), the column temperature was 40 ° C, the mobile phase was acetonitrile / water (65/35 ~ 35/65, v / v gradient), 1.0 mL / min, sample injection amount was 20 μL, and a UV (227 nm) detector was used. Paclitaxel standard was used as Sigma-Aldrich product (purity: 95%).

Preparation Example 1 Plant Cell Culture

Plant cell cultures used in this experiment were cultured using cell lines obtained from the leaves of Taxus chinensis. Decanter centrifuge (Westfalia, CA 150 Clarifying Decanter) was used to recover plant cells from the culture, and high-speed centrifuge (α-Lavel, BTRX 205 GD-35 CDEFP) was used to recover plant cell debris. . In the present invention, the collected plant cells and the cell pieces are referred to as biomass.

[Examples 1 to 12 and Comparative Examples 1 to 20] Purification of paclitaxel using an adsorbent treatment and a micelle process

The biomass recovered from the cell culture was added to methanol at 1% (w / v), extracted and filtered at room temperature for 30 minutes, and the reaction was repeated four times by adding fresh methanol to the biomass. Extraction was carried out. The extract filtrate was collected and concentrated in a concentrator (CCA-1100, EYELA, Japan) (30% of the stock), methylene chloride was added to the concentrate (25% of the concentrate) and liquid-liquid extraction was performed for 30 minutes at room temperature. Repeated three times to remove the polar impurities, concentrated under reduced pressure and dried. To the first dried product obtained after drying the liquid-liquid extract (paclitaxel purity: 8%), 50% (w / w) of sylopute (Fuji Silysia Chemical Ltd., Japan) as an adsorbent and methylene chloride were added (25% of the concentrate). Stir at 40 ° C. for 30 minutes and then filter. The filtrate was dried at 30 ° C. under reduced pressure and used in a micelle process. After drying the filtrate, the second dried product (paclitaxel purity: 9.8%) was dissolved in 25 mL of MtBE (methyl-t-butyl-ether), 12.5 mL of an aqueous surfactant solution and 25 mL of hexane were added thereto, followed by stirring at room temperature. After stirring for 30 minutes, the mixture was suspended to recover the surfactant layer in which micelles were formed after phase separation, and four repeated experiments were performed under the same conditions. Paclitaxel was recovered to the MtBE layer by putting MtBE back into the surfactant layer recovered through four repeated experiments to decompose micelles from the surfactant layer, and four repeated experiments were performed in the same manner. After concentrating the recovered MtBE layer under reduced pressure, the concentrate was dried in a vacuum oven (UP-2000, EYELA, Japan) for 24 hours and analyzed by HPLC.

The principle and schematic diagram of the micelle-based separation process for paclitaxel pretreatment are shown in FIG. 1, and the paclitaxel purity before and after adsorbent treatment of the present invention is shown in FIG. 2. As shown in Table 1, Examples 1 to 12 and Comparative Examples 1 to 20 were carried out by varying the type and concentration of the surfactant.

TABLE 1

The purity and yield of paclitaxel according to the tablets of Examples 1 to 12 and Comparative Examples 1 to 20 are shown in FIGS. 3 to 5.

Figure 3 shows the yield (a) and purity (b) of paclitaxel according to the concentration of the cationic surfactant (CPC, CTMAC, LTMAC), the yield of paclitaxel when the concentration of the cationic surfactant is 5% (w / v) And it was confirmed that the purity showed the maximum value. In particular, the highest yield (~ 99%) was obtained at 5% (w / v) of CTMAC. In the case of purity, all three types of surfactants showed a similar tendency of 21% at concentrations above 5% (w / v). In addition, the time required for phase separation was about 1 hour in the case of CPC and LTMAC, whereas it took about 30 minutes in the case of CTMAC.

Figure 4 shows the yield (a) and purity (b) of paclitaxel according to the concentration of anionic surfactants (SDS, AOT), in the case of AOT among anionic surfactants at an concentration of 1% (w / v) or more It is difficult to utilize the activator in the micelle process because it is not dissolved in water, and in the case of SDS, the yield and purity increase with increasing surfactant concentration, but it is confirmed that there is almost no change at 5% (w / v) or more. In addition, the time required for phase separation was about 1.5 hours, and the time required for phase separation was higher than that of the cationic surfactant.

In the micelle process, as the concentration of the surfactant increases, paclitaxel is wrapped inside the micelle to move well into the aqueous phase, but it is difficult to recover paclitaxel from the aqueous phase after the micelle formation and phase separation. do.

Figure 5 shows the yield (a) and purity (b) of paclitaxel according to the concentration of non-ionic surfactant (Tween-80, PEG-6000, TX-100), as shown in Figure 5 yield and purity Not only does it increase by more than 30% and 10%, but the surfactant is not dissolved in water at concentrations of 1% (w / v) and higher, and it takes a long time (2 to 4 hours) to separate the phases, making it difficult to use in the micelle process. .

1 shows the principle and schematic of a micelle based separation process for paclitaxel pretreatment.

Description of the main parts of the drawing

1: water-insoluble organic solvent layer 5: hydrophilic

2: phase separation 6: hydrophobic

3: cationic surfactant layer 7: paclitaxel

4: micelle

Figure 2 shows the paclitaxel purity before (a) and after (b) treatment of the adsorbent, Figure 3 shows the yield (a) and purity (b) of paclitaxel according to the type and concentration of the cationic surfactant, Figure 4 Shows the yield (a) and purity (b) of paclitaxel according to the type and concentration of anionic surfactant, Figure 5 shows the yield (a) and purity (b) of paclitaxel according to the type and concentration of nonionic surfactant It is shown.

Claims (8)

(a) extracting by adding a water-soluble organic solvent to a Taxus genus plant, a cell thereof, or a cell culture thereof, and then concentrating the extract; (b) adding a non-polar organic solvent to the concentrate, followed by liquid-liquid extraction, concentration and drying under reduced pressure; (c) drying the filtrate obtained by treating the first dried material obtained in step (b) with an adsorbent and filtering; (d) putting the second dried product obtained in step (c) into a mixed solution of a non-aqueous organic solvent and a cationic surfactant, stirring and stabilizing to recover a cationic surfactant layer in which micelles are formed after phase separation; (e) further adding a non-aqueous organic solvent to the recovered cationic surfactant layer of step (d), stirring and standing to recover the non-aqueous organic solvent layer after phase separation, and concentrating it under reduced pressure; A micelle based separation method for paclitaxel pretreatment comprising a. The method of claim 1, The water-soluble organic solvent of step (a) is one or a mixture of two or more selected from methanol, ethanol, propanol, methylene chloride, and the nonpolar organic solvent of step (b) is selected from methylene chloride, ethyl acetate, ether Species or mixtures of two or more species, and the water-insoluble organic solvents of steps (d) and (e) are dichloromethane, carbon tetrachloride, chloroform, 1,2-dichloroethane (1,2). 1 selected from -dichloroethane, di-ethyl ether, methylt-butyl ether, di-iso-propyl ether, hexane, benzene Micell based separation method for paclitaxel pretreatment, characterized in that the species or a mixture of two or more species. 3. The method of claim 2, The water-insoluble organic solvent of step (d) is a mixture of methyl-t-butyl ether and hexane, and the water-insoluble organic solvent of step (e) is methyl-t-butyl ether (methylt-butyl micelle-based separation method for paclitaxel pretreatment, characterized in that ether). The method of claim 1, The adsorbent of step (c) is a micelle-based separation method for paclitaxel pretreatment, characterized in that using a silica-based adsorbent. The method of claim 4, wherein In the adsorbent treatment of step (c), the adsorbent is added at 20 to 100% (w / w) of the first dried material obtained in the step (b), and one or two or more mixtures selected from methylene chloride and chloroform are added. The micelle-based separation method for paclitaxel pretreatment, further comprising stirring. The method of claim 1, The cationic surfactant of step (d) is one or two selected from N-cetylpridinium chloride (N-cetylpridinium chloride), hexadecyltrimethylammonium chloride, dodecyltrimethylammonium chloride The micelle-based separation method for paclitaxel pretreatment, characterized in that a mixture of the above. The method of claim 6, The cationic surfactant is a micelle-based separation method for paclitaxel pretreatment, characterized in that used in the aqueous solution. The method of claim 7, wherein The cationic surfactant is a micelle-based separation method for paclitaxel pretreatment, characterized in that used in 1 ~ 20% (w / v) of the aqueous solution.

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