KR100208656B1 - Apparatus for manufacturing taxol by online continuous process of extraction and purification using supercritical fluid - Google Patents

Abstract

The present invention relates to a method of separating taxol or a derivative thereof, which is an anticancer substance, from a target cell or a cultured target cell by a supercritical extraction process and a supercritical chromatography process on-line, extracting the taxol or a derivative thereof with high yield and then performing selective high purity purification continuously Apparatus and method. An apparatus for producing taxa of the present invention comprises: means for supplying carbon dioxide; Means for supplying a co-solvent; And a preheater for maintaining the carbon dioxide and the co-solvent introduced from the supply means of the electric carbon dioxide and the co-solvent in a supercritical state, wherein the preheater is in contact with a powder sample of the target or cultured target cells present in the supercritical milk extractor A batch extraction means for extracting taxol or a derivative thereof; A partial purification means for primarily purifying taxol or a derivative thereof by attaching the extract transferred from the electric extraction means to an adsorption column and eluting with supercritical carbon dioxide containing an auxiliary solvent; A UV detector for identifying a taxol or a derivative thereof present in the effluent eluted from the electric part refining means; Purity purifying means for purifying taxol or derivative thereof secondarily by injecting only eluted fractions of taxol or derivatives thereof identified in the electric detector into a purification column and eluting with supercritical carbon dioxide containing an auxiliary solvent; And collecting means for collecting only the fraction after confirming the taxol or its derivative present in the elution rack eluted from the electrophoresis purification means. The use of the apparatus of the present invention makes it possible to extract and purify Taxol or its derivative simultaneously in one apparatus, so that the process becomes extremely simple compared to the organic solvent extraction and purification method and a final product can be obtained without a separate distillation process . In addition, the use of organic solvents is reduced, which is highly desirable from a clean engineering standpoint.

Description

Production of Taxol by on-line continuous process of extraction and purification using supercritical fluid

The present invention relates to an apparatus and a method for producing Taxol by on-line continuous process of extraction and purification using a supercritical fluid. More specifically, the present invention relates to a method for producing a pharmaceutical composition comprising a supercritical extraction process and a supercritical chromatography process on-line to extract taxol or its derivative, which is an anticancer substance, from a target cell or a cultured oyster cell in a high yield, The present invention relates to an apparatus and method for performing continuous and simultaneous operations.

Taxol is a kind of diterpene alkaloid. It was first approved by the US FDA in October 1992 as an advanced ovarian cancer treatment, and has since been approved for breast cancer treatment and has attracted much attention until it is marketed , And many taxol-related researches are still underway. Taxol derivatives having a taxol-like structure include cephalomannine having a taxane ring, baccatin III, 10-deacetylbaccatin III, 10-diacetyl (10-deacetylcephalomannine) are known, and their content varies depending on the species of interest, but most of them are included in the attention along with taxol.

In addition, attention has been focused on the genus, Amentotaxus, Austrotaxus, Cephalotaxus, Pseudotaxus, Taxus, Torreya, Among these, the Taxus genus plants, for example, T. brevifolia, T. baccata, T. brevis, T. media, T. wallichiana, T. canadensis, and T. cuspidata are known as the most suitable raw materials for the extraction of taxol and its derivatives.

On the other hand, functional group as anticancer agent of taxol. During small cell division, taxol promotes tubulin polymerization to induce highly stable microtubules, thereby inhibiting tubulin degradation, (Horwitz et al., Nature, 277: 665-667 (1979)). Although the most widely used method for producing taxol has the anticancer effect, the natural extraction method, the total synthesis method and the semi-synthetic chemical synthesis method, which have the problems of the shortage of the taxa plant and the destruction of the ecosystem, Cell culture, and the like. At this time, extraction and purification processes are required to separate the taxol produced by these methods.

The currently used extraction and purification method of Taxol is divided into an extraction step and a high purity purification step. The solvent extraction, the evaporation, the solvent partitioning, the multiple chromatography, the recrystallization (See Journal of Liquid Chromatography, 12 (11): 2117-2132 (1989); WO / 07842). However, this method has various problems such as economical problems due to numerous processes, deterioration of taxol due to the use of toxic organic solvents, environmental pollution, and the like. To solve some of these problems, the extraction and purification method using supercritical fluid has been developed.

Supercritical fluids are fluids that are in a state of critical pressure and critical temperature, and various physical properties vary greatly around the critical point. Particularly, the change in density is closely related to the ability to dissolve various materials . When supercritical oil is used as a solvent, it has a density similar to that of a liquid, so that it has the ability to dissolve a liquid or a solid like a liquid solvent. In addition, the supercritical fluid has its solubility controlled by changes in density due to changes in pressure and temperature, and is easily separated from the solute after extraction. In addition, when supercritical oil is used in the extraction process of taxol, it has various advantages compared with the conventional organic solvent extraction. However, in the extraction of taxol using supercritical oil, the supercritical fluid has a disadvantage in that it has a very low selectivity indicating the degree to which taxol is extracted in a more homogeneous manner than other substances. Therefore, A method of increasing the selectivity by using a solvent and an adsorption column has been proposed (Korean Patent Application No. 96-19486).

However, the problem that the selectivity between supercritical carbon dioxide and taxol still used as an extraction solvent is undesirable, that is, when taxol and various organic compounds are extracted in addition to taxol in extraction using supercritical carbon dioxide, the extraction efficiency of taxol in the extract is low (W / w) of Taxol in the extract is low because of its high selectivity. This low selectivity is likewise exhibited when a wide variety of co-solvents are used. When the taxon content in the extract is low in this first extraction, the subsequent purification process becomes very complicated and the use of toxic organic solvents is inevitable for this complex process.

Supercritical chromatography began in 1962 when Klesper et al. Applied a supercritical fluid as a mobile phase to high pressure gas chromatography. The major advantages of this technology are the low viscosity of supercritical fluids, high diffusion Coefficient and good solubility, and it is also possible to change the solubility and selectivity as a control of pressure, temperature and co-solvent. Supercritical chromatography has advantages such as low amount of solvent use, low temperature extraction purification, and pollution-free process in comparison with chromatography using existing organic solvents, Is increasing.

Under these circumstances, the inventors of the present invention have focused on the simultaneous extraction of taxol and high purity purification of taxol by applying supercritical extraction and supernatant chromatography simultaneously to the extraction and purification of taxol and its derivatives, We have developed an apparatus for producing taxol by online continuous process of extraction and purification using critical fluid. By using this apparatus, an extract containing taxol or its derivative, which is extracted with high efficiency by supercritical extraction, can be directly introduced into a supercritical chromatography column and fractionated. Therefore, efficient purification can be performed, In addition to the advantages of critical-fat extraction and purification, online continuous processing is possible, and thus it is possible to improve the efficiency of taxol production, thus completing the present invention.

The main object of the present invention is to connect supercritical extraction process and supercritical chromatography process on-line to extract taxol or derivatives thereof from the target cells or cultured target cells at a high yield, and then to carry out selective high purity purification continuously And the like.

It is another object of the present invention to provide a method for producing Taxol or a derivative thereof from a target cell that has been remarked or cultured using an electric device.

FIG. 1 is a schematic view showing an apparatus for producing taxol or a derivative thereof on which a supercritical extraction process and a supercritical chromatography process of the present invention are connected on-line.

FIG. 2 shows a more preferable process flow chart of the taxa manufacturing apparatus of the present invention.

FIG. 3 is a chromatogram showing the absorbance at 228 nm of the extract obtained through the partially purified column.

FIG. 4 is a chromatogram showing the absorbance at 228 nm of the extract obtained through a high purity purification column.

DESCRIPTION OF THE REFERENCE NUMERALS

1: carbon dioxide storage container 2: auxiliary solvent storage container

3: cooling circulator 4: lamp with condenser

5: Pump 6, 7: Valve

8: Preheater 9: Mixer

10: Injector 11: Selection valve

12: valve 13: extractor

14: Partially purified column 15: High purity purification column

16: oven 17: UV detector

18: integrator 19: reverse pressure regulator

20: collectors 21, 22, 23 and 24: valves

Hereinafter, the present invention will be described more specifically.

FIG. 1 schematically shows an apparatus for manufacturing taxol or derivatives thereof connected to a supercritical extraction process and a supercritical chromatography process used in the present invention.

An apparatus for producing taxol or derivatives thereof according to the present invention by an on-line continuous process of extraction and purification comprises (i) means for supplying carbon dioxide; (Ii) means for supplying an auxiliary solvent; (Iii) a preheater for keeping the carbon dioxide and the co-solvent introduced from the supply means of the electric carbon dioxide and the co-solvent in a supercritical state, wherein the supercritical fluid is supplied to the target or the cultured target cells A batch extraction means for extracting taxol or a derivative thereof by contacting with a powder sample of the sample; (Iv) a partial purification means for firstly purifying taxol or a derivative thereof by attaching the extract transferred from the electric extraction means to an adsorption column and eluting with supercritical carbon dioxide containing a co-solvent; (V) a UV detector for identifying a taxol or a derivative thereof present in the effluent eluted in the electrical part refining means; (Vi) high-purity purification means for purifying secondarily the taxol or a derivative thereof, by injecting only the elution fraction of the taxol or its derivative identified in the electric detector into the purification column and eluting with supercritical carbon dioxide containing the auxiliary solvent; And collecting means for collecting only the fraction after confirming the taxol or its derivative present in the effluent eluted in the electrophoresis purification means (iii).

In the above, the means for supplying carbon dioxide includes a carbon dioxide storage vessel 1, a pump 4 with a cooler, and a back pressure regulator 19 for precise regulation of the pressure. The auxiliary solvent may be methanol, ethanol, dichloromethane or acetonitrile. The adsorption column is filled with silica gel, alumina or an adsorbent of an ion exchange resin. The purification column is made of silica ), Amino (amino), nitro (nitro) or glycerol, or a reversed phase filler such as C18.

FIG. 2 shows a more preferable process flow chart of the taxa manufacturing apparatus of the present invention.

The process for preparing taxol or its derivative from the target plant or cultivated plant using the above-described apparatus will be described in detail below.

[First Step: Extraction Process Using Supercritical Carbon Dioxide Containing Ethanol Coating Solvent]

The liquefied carbon dioxide supplied from the carbon dioxide storage vessel 1 is pressurized by the pump 4 to which the cooler is attached and the auxiliary solvent supplied from the auxiliary solvent storage vessel 2 is transferred through the pump 5. The carbon dioxide and the co-solvent supplied by the respective pumps are maintained at a supercritical state at a preheater 8 in the oven 16 at a temperature higher than the critical temperature, mixed in the mixer 9, And is then sent to the extractor 13 to extract the taxol. At this time, the extractor is filled with glass fiber at one end and the sample powder of the target cells or cultured target cells alternately with organic beads, and then the other end is covered with glass fiber to increase the contact area with supercritical carbon dioxide A packed bed type extractor. In addition, various methods disclosed in Korean Patent Application No. 96-19486 can be applied to increase the selectivity upon taxol extraction.

[Second Step: Partial Purification Step]

Partially purified column 14 is prepared by filling a column with a pre-selected filler either dry or wet, wherein the dry column is a method of filling a blank column with a filler that is dried, and the wet column is a high-speed liquid chromatography (High Performance Liquid Chromatography) column. At this time, the amount of the adsorbing material to be packed in the column and the size of the column are used in proportion to the amount of the extract. The efficiency of a column is high for a wet type column, but it is much easier for a dry type column in terms of manufacturing process and usage.

The extract in the first step is adsorbed to the partial purification column 14 via the selection valve 11 and eluted with supercritical carbon dioxide containing the auxiliary solvent. At this time, when the supercritical carbon dioxide is directly introduced into the partial purification column using the selection valve 11, the batch extraction proceeds in the extractor, and at the same time, the transfer extract can be adsorbed to the partial purification column 14.

[Third step: High purity purification step]

Only the fraction containing taxol when eluted in the partially purified column is introduced into the high purity purification column 15 by valve operation and the supercritical carbon dioxide is not flowed into the partially purified column using the selection valve 11, . When a partially purified extract is eluted in a high-purity purification column, taxol fractionation yields high purity taxol.

The pressure in the whole apparatus and the flow rate of the supercritical carbon dioxide, including the auxiliary solvent flowing in the column, are kept constant by using the back pressure regulator 19. In addition, the extracted taxol was confirmed using the absorbance measured at the detector 17, and the valve operation when introducing the taxol fraction in the partially purified column into the high purity purification column was as follows: the valve 23 was opened When the valve 22 and the valve 24 are closed, the extract passes through the partial purification column and then flows out to the outside through the detector and the back pressure regulator. When it is confirmed that the taxol-containing fraction is eluted from the partially purified column through the detector, the valve 21 and the valve 23 are closed and the valve 22 and the valve 24 are opened to remove the partially purified taxol- . Purified carbon dioxide flows through the high purity purification column without passing through the partial purification column by using a selection valve and then the valve 22 and the valve 24 are closed and the valve 21 and the high- When the valve 23 is opened, the eluted fractions are discharged to the outside through the detector and the back pressure regulator. At this time, if it is confirmed to be Taxol through the detected absorbance, Taxol of high purity can be obtained by fractionation.

The production of taxol using the apparatus of the present invention has the following advantages.

① The extraction and purification of Taxol or its derivatives can be carried out continuously or simultaneously.

② The process is very simple compared to the taxol production process using existing organic solvents. When the purified taxol fraction is fractionated from the high purity column, the supercritical carbon dioxide is vaporized by lowering the pressure of the fraction to lower the solubility. Taxol, the final product, can be obtained.

③ Supercritical fluids are excellent in separation speed because of low viscosity and high diffusion coefficient.

④ The use of organic solvents is reduced, which adds to the added value of clean engineering technology.

⑤ Reproducibility is very good.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be apparent to those skilled in the art that these embodiments are only for describing the present invention in more detail and that the gist of the present invention is not limited by these embodiments in accordance with the gist of the present invention.

[Example 1]

[Extraction of Taxol Using Supercritical Carbon Dioxide Containing Ethanol Coating Solvent]

The carbon dioxide and the ethanol co-solvent supplied by each pump were kept in a supercritical state and mixed to extract taxol from the attention or cultured attention cells in the extractor.

[Example 2]

[Partial purification]

The supercritical extract obtained in Example 1 was adsorbed on a caloric gel column and eluted with supercritical carbon dioxide containing ethanol co-solvent to measure the absorbance at a wavelength of UV 228 nm. FIG. 3 is a chromatogram showing the absorbance of the extract through a partially purified column. Among these, the Taxol fraction corresponding to the retention time of about 10 minutes is introduced into the high purity purification column of Example 3, so that impurities other than Taxol escape before Taxol or remain in the adsorption column to partially purify Taxol. Through this process, the concentration of taxol could be increased to 70-80%.

[Example 3]

[High purity tablet]

The taxot-containing fraction introduced into the high purity purification column in Example 2 was eluted with supercritical carbon dioxide containing ethanol co-solvent, and the absorbance at a wavelength of UV 228 nm was measured. FIG. 4 is a chromatogram showing the absorbance of the extract through a high purity purification column. As shown in FIG. 4, the extract was highly purified, and it was found that Taksol obtained in high purity can be obtained by collecting only the Taxol fraction corresponding to the retention time of about 4 minutes.

As described and demonstrated above, the present invention relates to a method and apparatus for online selection of a supercritical extraction process and a supercritical chromatography process, which extracts taxol or its derivative, which is an anticancer substance, from a target cell or a cultured target cell at a high yield, An apparatus and method for continuous and simultaneous purification are provided. Since the extraction and purification of taxol or its derivative can be simultaneously performed in one apparatus, the apparatus and method of the present invention can be reduced to five steps or less, which is extremely simple compared to the organic solvent extraction and purification method. The final product can be obtained without distillation. In addition, the use of organic solvents is reduced, which is highly desirable from a clean engineering standpoint.

Claims (1)

(I) a means for supplying carbon dioxide, comprising a carbon dioxide storage vessel (1), a pump (4) with a cooler and a back pressure regulator (19); (Ii) a means for supplying one of the co-solvents selected from the group consisting of methanol, ethanol dichloromethane and acetonitrile; (Iii) a preheater for maintaining the carbon dioxide and the co-solvent introduced from the respective carbon dioxide and co-solvent supply means in a supercritical state, the supercritical fluid containing the supercritical fluid, A batch extraction means for extracting the taxol by contacting with a powder sample; (Iv) attaching the extract transferred from the electric extraction means to an adsorption column filled with one kind of material selected from the group consisting of silicagel, alumina and ion exchange resin, and A partial purification means for first purifying taxol by eluting with supercritical carbon dioxide; And (v) a UV detector for identifying the taxol present in the effluent eluted in the electric fraction purification means; (Vi) Only the eluate fraction of Taxol identified in the electric detector is injected into a purified column filled with silica, amino, nitro or glycerol combined normal phase filler or reversed phase filler, Purity purifying means for eluting with supercritical carbon dioxide contained therein to purify the taxol secondarily; And (ⅶ) an apparatus for producing taxol by an on-line continuous process of extraction and purification, comprising collecting means for collecting only the fraction, after confirming the taxol present in the concentrate solution eluted from the high purity purification means.