KR20150086805A - Method for purifying paclitaxel and pharmaceutical composition contains paclitaxel purified with this - Google Patents

Abstract

The present invention relates to a paclitaxel purification method and a pharmaceutical composition which contains the paclitaxel obtained through using the method. More specifically, the method for extracting the paclitaxel from Taxus genus biomass can significantly increase the extraction efficiency compared to the conventional extraction method and obtain high-purity paclitaxel.

Description

TECHNICAL FIELD The present invention relates to a method for purifying paclitaxel and a pharmaceutical composition comprising paclitaxel purified by the method.

The present invention relates to a method for purifying paclitaxel and a pharmaceutical composition comprising paclitaxel purified by the method. More particularly, the present invention relates to a method for extracting paclitaxel from a taxa biomass, And a pharmaceutical composition comprising paclitaxel purified by the method and a method for purifying paclitaxel capable of obtaining paclitaxel of high purity.

Paclitaxel is an anticancer substance found in the epidermis of a tree of interest and has been approved by the US Food and Drug Administration (FDA) for the treatment of ovarian cancer, breast cancer, Kaposi's sarcoma, and non-small cell lung cancer (NSCLC) It is currently the most widely used anticancer drug currently, and currently holds the largest market share among anticancer drugs, accounting for 22% of the major anticancer drug market. Rheumatoid arthritis, and Alzheimer's disease. In addition, clinical trials are being conducted on multiple prescriptions for various treatment methods, and the demand for paclitaxel is expected to continue to increase in the future.

There are three main methods of producing paclitaxel. First, it is difficult to continuously supply the raw materials by direct extraction from the tree of interest, and there are many difficulties in extraction and purification, and it is not suitable for protecting the tree as an environmental protection water. Second, it is a semi-synthetic method to obtain the precursor (baccatin Ⅲ, 10-deacetylbaccatin Ⅲ, 10-deacetyl paclitaxel, etc.) from the leaf of the tree to chemically bond the side chain. This method also has the same problems as the direct extraction because the precursor must be obtained directly from the tree of interest. Third, it is a method to induce callus from a tree of interest and cultivate plant cells in a main bioreactor through a seed culture. The third method has the advantage of mass production of paclitaxel of constant quality because it can be produced stably in the bioreactor without being influenced by external factors such as climate and environment.

The separation and purification of the anticancer paclitaxel from plant cell cultures results in a high purity (> 98%) product through several stages of extraction and purification. In general, the separation and purification processes consist of a step of extracting paclitaxel from the raw material biomass with an organic solvent, and a step of producing a product through a pre-treatment and final purification. In this process, in particular, the pre- .

On the other hand, in the patent 10-0474228 (published on May 15, 2000), paclitaxel was isolated from a mixture obtained by reacting a mixture of paclitaxel and cephalomannine with an aliphatic alcohol or an amine in the presence of N-halosuccinimide And a method for separating paclitaxel.

The above patent and conventional paclitaxel separation and / or extraction methods can be used as a pretreatment process for paclitaxel purification, in which raw paclitaxel extracted with or without high-throughput chromatography is subjected to high performance liquid chromatography (HPLC) chromatography was used for the final purification.

However, the above conventional paclitaxel separation and / or purification method can not be applied to the case of using paclitaxel extract of low purity as a sample of HPLC, in view of economical efficiency such as using a large amount of organic solvent, shortening the lifetime of the packed resin in the column, There are many problems, and there were also many difficulties in scale-up and mass production.

In addition, the paclitaxel extraction method using an organic solvent, which is one of the conventional paclitaxel extraction methods, has a purity of 0.5% or less of paclitaxel extracted and a very low purity of about 10% even after a simple pretreatment.

SUMMARY OF THE INVENTION Accordingly, the present invention has been made to solve the above-mentioned problems. Accordingly, it is an object of the present invention to provide a method for extracting paclitaxel from Taxus biomass, which comprises extracting paclitaxel with paclitaxel And a pharmaceutical composition comprising paclitaxel purified by the method.

The present invention relates to a method for producing an extract of Taxus genus biomass comprising paclitaxel by an extraction method of at least one of organic solvent extraction, vacuum extraction, liquid-liquid extraction and hexane precipitation extraction; Mixing the extract and the water-soluble organic solvent to prepare a mixture; Adding water to the mixture to perform fractional precipitation; And stirring after fractional precipitation to obtain paclitaxel; , And the amount of water added is a method of purifying paclitaxel by mixing the water-soluble organic solvent and water in a volume ratio of 50:50 to 20:80.

According to one preferred embodiment of the present invention, the water-soluble organic solvent is C ₁ To C ₄ alcohols and methylene chloride.

According to another preferred embodiment of the present invention, the stirring may be performed at 10 to 35 ° C for 5 minutes to 2 hours at 100 to 300 rpm.

According to another preferred embodiment of the present invention, it may further include storing at 0 to 35 ° C for 10 minutes to 2 hours after the stirring.

According to another preferred embodiment of the present invention, the storage may be performed at 0 to 10 ° C for 10 minutes to 2 hours.

According to another preferred embodiment of the present invention, the added amount of water may be mixed in a volume ratio of water-soluble organic solvent and water of 47: 53 to 35: 65.

According to another preferred embodiment of the present invention, the paclitaxel purification method may have an extraction yield of 40% or more.

According to another preferred embodiment of the present invention, the paclitaxel purification method can obtain paclitaxel having a purity of 1.5 to 4 times higher than the paclitaxel purity of the extract.

The present invention also provides a pharmaceutical composition for an anticancer agent comprising paclitaxel purified by the above-described purification method.

Further, the present invention provides a pharmaceutical composition for the treatment of rheumatoid arthritis comprising paclitaxel purified by the above-described purification method.

In addition, the present invention provides a pharmaceutical composition for treating Alzheimer's disease comprising paclitaxel purified by the above-described purification method.

There is an effect of providing a method for purifying paclitaxel capable of obtaining paclitaxel having a significantly higher extraction efficiency and purity than the conventional paclitaxel extraction method, and a pharmaceutical composition comprising paclitaxel purified through the method.

1 is a paclitaxel purity of Examples 2 to 4 measured in Experimental Example 1. Fig.
2 is paclitaxel purity of Examples 5 to 10 measured in Experimental Example 2. Fig.
Fig. 3 shows the yields of paclitaxel extraction in Examples 5 to 10 measured in Experimental Example 2. Fig.
4 shows the paclitaxel extraction yields of Examples 11 to 12 and Comparative Examples 1 and 2 measured in Experimental Example 3. FIG.
5 shows the paclitaxel extraction yields of Example 2, Examples 13 to 16, Comparative Example 1 and Comparative Examples 3 to 4 measured in Experimental Example 4.
6 is paclitaxel purity of Examples 11 to 12 and Examples 17 to 24 measured in Experimental Example 5;
7 shows the paclitaxel extraction yields of Examples 11 to 12 and Examples 17 to 24 measured in Experimental Example 5.
8 shows the yield of paclitaxel extraction in Example 4 and Comparative Example 5 measured in Experimental Example 6.

Hereinafter, the present invention will be described in detail.

As described above, the conventional paclitaxel separation and / or purification method uses a low purity paclitaxel extract as a sample of high performance liquid chromatography (HPLC), using a large amount of an organic solvent, there have been many problems in terms of economics such as shortening the lifetime of the resin and decreasing the throughput, and there have been many difficulties in scale-up and mass production. In addition, the paclitaxel extraction method using an organic solvent, which is one of the conventional paclitaxel extraction methods, has a purity of 0.5% or less of paclitaxel extracted and a very low purity of about 10% even after a simple pretreatment.

Accordingly, the present invention relates to a method for producing an extract of Taxus genus comprising paclitaxel, which comprises extracting the biomass with at least one extraction method selected from the group consisting of organic solvent extraction, vacuum extraction, liquid-liquid extraction and hexane precipitation extraction; Mixing the extract and the water-soluble organic solvent to prepare a mixture; Adding water to the mixture to perform fractional precipitation; And stirring after fractional precipitation to obtain paclitaxel; And a method for purifying paclitaxel in which water is mixed with the water-soluble organic solvent and water at a volume ratio of 50: 50 to 20: 80 to the mixture. Accordingly, there is an effect of providing a method for purifying paclitaxel which can obtain extraction efficiency and paclitaxel of high purity which is significantly higher than that of the conventional paclitaxel extraction method, and a pharmaceutical composition containing paclitaxel purified by the method.

The present invention relates to a method for producing an extract of Taxus genus biomass comprising paclitaxel by an extraction method of at least one of organic solvent extraction, vacuum extraction, liquid-liquid extraction and hexane precipitation extraction; Mixing the extract and the water-soluble organic solvent to prepare a mixture; Adding water to the mixture to perform fractional precipitation; And stirring after fractional precipitation to obtain paclitaxel; ≪ RTI ID = 0.0 > paclitaxel < / RTI >

First, the step of producing an extract from the Taxus genus biomass containing paclitaxel is described.

The taxa biomass including paclitaxel may include one or more of the group consisting of a plant belonging to the genus Taxus, a cell thereof, and a cell culture solution thereof.

In addition, the plant in the taxus is Taxus brevifolia , Taxus canadensis , Taxus cuspidata , Taxus baccata , Taxus globosa , Taxus floridana , Taxus wallichiana , Taxus media ) or Taxus chinensis ), and the like, but the present invention is not limited thereto.

The method for preparing the extract from the taxus biomass is not particularly limited as long as it is a method for carrying out the extraction to extract the components contained in the biomass. For example, extraction can be carried out by one or more extraction methods selected from organic solvent extraction, vacuum extraction, liquid-liquid extraction and hexane precipitation extraction.

Specifically, the organic solvent for the organic solvent extraction is not particularly limited as long as it is usually used for extracting an active ingredient from a plant, but preferably includes at least one kind selected from the group consisting of C ₁ to C ₄ alcohols and water .

Further, the conditions for extracting the organic solvent are not particularly limited as long as they can be used in a method for extracting an active ingredient from a plant, but they can be preferably carried out at 20 to 45 ° C for 30 minutes to 2 hours.

If the temperature is lower than 20 캜, the extraction efficiency of the paclitaxel may be decreased due to the low temperature. If the temperature is higher than 45 캜, the paclitaxel decomposes due to the high temperature Lt; / RTI >

If the treatment time is less than 30 minutes, the extraction efficiency of the paclitaxel may be lowered because the paclitaxel in the biomass is not extracted. If the treatment time exceeds 2 hours, May occur.

In addition, the solvent extraction can be repeated one to several times, preferably two times or more, more preferably three to five times.

The liquid-liquid extraction is not particularly limited as long as it is a method for removing polar compounds contained in paclitaxel by using phase separation between a non-polar organic solvent and a polar organic solvent.

For example, the nonpolar organic solvent may include at least one member selected from the group consisting of methylene chloride, ethyl acetate, and ether. In addition, the polar organic solvent may include at least one selected from the group consisting of C ₁ to C ₄ alcohols and water.

In addition, the liquid-liquid extraction can be repeated one to several times, preferably two times or more, more preferably three to five times.

Further, when the polar organic solvent is a C ₁ -C ₄ alcohol, it is preferable to use methylene chloride as the nonpolar organic solvent, and methylene chloride is preferably used at 20 to 30% (v / v) of the alcohol concentrate And the use of excess methylene chloride will result in a heavy burden on subsequent processes.

Next, the extract and the water-soluble organic solvent are mixed to prepare a mixture.

The mixing is not particularly limited as long as it is a method for mixing solid and liquid. For example, stirring may be performed using a stirrer.

In addition, the mixing is not particularly limited as far as it is a mixing amount capable of uniformly dispersing the extract in the water-soluble organic solvent, but preferably 1 to 5 parts by weight of the extract may be mixed with 100 parts by weight of the water-soluble organic solvent.

If less than 1 part by weight of the extract is mixed with 100 parts by weight of the water-soluble organic solvent, the extraction yield may be decreased and economic problems may be caused due to excessive use of the solvent. When the extract is mixed, the paclitaxel extract may have a problem of lowering the purity thereof.

The water-soluble organic solvent is not particularly limited as long as it is an organic solvent ordinarily used for extracting an active ingredient from a plant. Preferably, the water-soluble organic solvent may include at least one selected from the group consisting of C ₁ to C ₄ alcohols and methylene chloride , More preferably methanol.

Next, water is added to the mixture to perform fractional precipitation.

The water may be added in a volume ratio of water-soluble organic solvent to water of 50:50 to 20:80, more preferably 47: 53 to 35:65 in volume ratio of water-soluble organic solvent to water.

As shown in Experimental Example 6 and FIG. 8, it was found that when methanol was used as a water-soluble organic solvent, paclitaxel could be extracted at about 11% higher yield than in the case of using acetone.

Then, after the fractional precipitation, the paclitaxel is obtained by stirring.

The stirring is not particularly limited as long as it is a condition used for extracting components from a plant, but it can be stirred preferably at 15 to 26 ° C for 5 minutes to 2 hours at 100 to 300 rpm.

If stirring is conducted at a temperature lower than 15 ° C, the extraction efficiency of paclitaxel may decrease due to the low temperature. If stirring is carried out at a temperature exceeding 26 ° C, the extraction efficiency of paclitaxel decreases due to the high temperature May occur.

If stirring is carried out for less than 5 minutes, the paclitaxel extraction yield may be decreased due to poor precipitation of paclitaxel. If stirring is performed for more than 2 hours, the operation efficiency is lowered due to excessive operation time There may be a problem.

If stirring at a speed of less than 100 rpm, the paclitaxel extraction time may be increased due to the slow paclitaxel precipitation, which is an advantage that can be obtained by stirring, and when stirred at a speed exceeding 300 rpm, The mixing efficiency may be lowered due to mixing.

In addition, according to one embodiment of the present invention, paclitaxel may be further obtained by storing the mixture at 0 to 35 ° C for 10 minutes to 2 hours after the stirring, preferably at 0 to 10 ° C For 10 minutes to 2 hours to obtain paclitaxel.

If stored at a temperature below 0 ° C, excessive energy may be used relative to sedimentation efficiency, and storage at a temperature exceeding 35 ° C may result in degradation of paclitaxel extraction yield and degradation of paclitaxel.

If stored for less than 10 minutes, the precipitation of the paclitaxel is not easy and the extraction yield may decrease. If the storage time is more than 2 hours, the efficiency of operation due to excessive operation time may decrease May occur.

As described above, the paclitaxel purification method has an extraction yield of 40% or more, and paclitaxel having a purity of 1.5 to 4 times higher than the paclitaxel purity of the extracted extract can be obtained. This is significantly higher than the paclitaxel purity of 10% or less that can be obtained by the conventional paclitaxel purification method.

In addition, the present invention provides a pharmaceutical composition comprising paclitaxel purified by the method for purifying paclitaxel as described above. The pharmaceutical composition is not particularly limited as long as it can usually include paclitaxel, but it may preferably be an anti-cancer agent, a rheumatoid arthritis treatment agent, or an Alzheimer's treatment agent.

The anticancer agent is a generic term for a chemotherapeutic agent used for the treatment of malignant tumors. Most anticancer drugs interfere with various metabolic pathways of cancer cells and inhibit the synthesis of hexane or exhibit anticancer activity. Since it is difficult to inhibit only the tumor cells without damaging the normal cells, the mechanism of action has been studied by a number of companies with various different medicines. It is the most important field of application of biotechnology related to medicine. Currently available anticancer drugs are roughly classified into three types, depending on the mechanism of action, such as revitalizing the immune system, antagonism of metabolism, and antibiotics directly killing tumor cells.

The pharmaceutical composition containing paclitaxel purified by the purification method according to the present invention may be administered orally or parenterally in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, May be formulated in the form of sterile injectable solutions.

More specifically, when formulating the composition, it can be prepared using a diluent or an excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, and the like.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Examples of liquid formulations for oral use include suspensions, solutions, emulsions and syrups. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like have.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The paclitaxel according to the present invention may vary depending on the age, sex and body weight of the patient, but is generally administered in an amount of 0.01 to 500 mg / kg, preferably 0.1 to 100 mg / kg, can do. Also, the dosage of paclitaxel may be increased or decreased depending on route of administration, degree of disease, sex, weight, age, and the like. Accordingly, the dosage is not limited in any way to the scope of the present invention.

Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are intended to further illustrate the present invention, and the scope of the present invention is not limited to these examples.

[ Example ]

Preparation Example  One. Biomass  Ready

Biomass was obtained by culturing a cell line obtained from leaves of Taxus chinensis .

Specifically, the suspension cells obtained from Taxus sp. Leaves were cultured by stirring at 150 rpm in darkness condition at 24 캜. The suspension cells were cultured in a modified Gemborg's B5 medium, 30 g / l sucrose, 10 쨉 naphthalene acetic acid, 0.2 쨉 m 6-benzylamino purine, 1 g / l casein hydrolyzate and 1 g / l 2- (N-morpholino) ethane sulfonic acid.

Cell culture was changed to new medium every 2 weeks. To prolong production and culture, 1 ~ 2% maltose was added on days 7 and 21, and 4 ㎛ of silver nitrate (AgNO ₃ ) Was added. The cultured cells were harvested from the culture medium using a decanter (using CA150 Claritying decanter from Westpalia) and a high-speed centrifuge (using BTPX205GD-35CDEEP from Alpha-LaSalle) to recover cell pieces and cell pieces. The recovered plant cells and cell fragments were combined and used as biomass.

Example  One.

The ratio of the biomass to methanol obtained in Preparation Example 1 was added in a ratio of 1/1 (w / v), and the mixture was subjected to methanol extraction for 30 minutes at 25 ° C, followed by filtration to prepare an extract. Then, methanol was added to the biomass extracted with primary methanol 1/1 (w / v), and the same procedure was repeated four times to prepare an extract.

The extract was concentrated under reduced pressure to 30% of the stock solution using a rotary evaporator (CCA-1100, EYELA, Japan), and methylene chloride was added at 25% by volume of the concentrate. After stirring at 25 ° C for 30 minutes, - Liquid extraction was performed. After removing the upper methanol layer containing polar impurities, the lower methylene chloride layer was collected and concentrated under reduced pressure using a rotary evaporator (CCA-1100, EYELA, Japan) to prepare a liquid-liquid extract The filtrate was filtered with a filter paper (150 mm, Whatman) and dried to prepare a crude extract.

Then, 50 l of methylene chloride was added to 10 g of the crude extract to remove tar and wax components derived from the plant cell, and the adsorbent sylopute (purchased from Fuji Silysia Chemical Ltd., Japan) was added to 100% w / w) and reacted in a 40 ° C thermostat bath (PS-1000, EYELA, Japan) for 30 minutes, followed by filtration. The filtrate obtained by the filtration was dried at 30 ° C under a reduced pressure to obtain a dried sample, and the dried sample was used in the hexane precipitation process.

In the hexane precipitation process, the dried sample was dissolved in methylene chloride and dropped in hexane to induce precipitation to remove nonpolar impurities (methylene chloride / hexane = 1/10, v / v). After hexane precipitation, the extract was filtered and dried in a vacuum oven (UP-2000, EYELA, Japan) at 35 ° C for 24 hours.

A vacuum dried extract of 17.6%, 21.3%, 27.6%, 32.6% or 42.0% purity was then used as the paclitaxel sample.

Example  2. Paclitaxel  refine

The vacuum dried extract having a purity of 21.3% prepared in Example 1 and methanol were mixed at a ratio of 0.5: 99.5 (w / v), stirred at 180 rpm until a ratio of methanol to water was 50: 50 by volume at 25 ° C, Was added dropwise to prepare a mixture for paclitaxel immersion. After that, the mixture for paclitaxel immersion was stirred at 180 rpm for 10 minutes at 25 ° C, filtered and dried in a vacuum oven at 35 ° C for 24 hours to obtain paclitaxel.

Example  3.

Paclitaxel was prepared in the same manner as in Example 2 except that water was added until the ratio of methanol to water was 40:60 by volume.

Example  4.

A paclitaxel was prepared in the same manner as in Example 2, except that water was added until the ratio of methanol to water was 30:70 by volume.

Experimental Example  1. Refined Paclitaxel  And extraction efficiency analysis

For the purity analysis of paclitaxel purified in Examples 2 to 4, a HPLC system (SCL-10AVP, Shimadzu, Japan) and Capcell Pak C ₁₈ UG 120 (250 mm x 4.6 mm, Shiseido, Japan) Respectively. The mobile phase was acetonitrile / water (65/35 to 35/65, v / v, gradient mode), and the flow rate of the mobile phase was 1.0 ml / min and the sample injection volume was 20 μl. The purity of paclitaxel was then analyzed using a UV detector at 227 nm. Sigma-Aldrich product (purity: 95%) was used as a paclitaxel standard substance. The purity of the measured paclitaxel is shown in Fig.

In addition, the extraction efficiency of paclitaxel was calculated based on the following equation (1). The measured paclitaxel extraction efficiency is shown in Fig.

As can be seen in FIG. 1, the paclitaxel extraction yield increased with increasing water content (as the methanol / water ratio decreased). The paclitaxel extraction yields were 42%, 84.3%, and 92% when the ratio of methanol to water (v / v) was 50:50, 40:60, and 30:70, respectively. Particularly, it was confirmed that the extraction yield of Example 3 in which the ratio of methanol to water was 40:60 was much larger than that of Example 2 in which the ratio of methanol and water was 50:50.

It was possible to obtain paclitaxel at a high yield by simply changing the mixture ratio of methanol and water without storing at low temperature (0 to 4 ° C) after fractional precipitation. In particular, Example 4 was able to obtain a paclitaxel extraction yield of about 92%.

Furthermore, the purity of paclitaxel was 60.2%, 63.6% and 57.7% when the ratio of methanol to water (v / v) was 50:50, 40:60 and 30:70, respectively.

Thus, the paclitaxel purification method of the present invention was able to recover the paclitaxel precipitate immediately after storage at 25 ° C, regardless of the ratio of methanol to water, without storing at an additional low temperature (0 to 4 ° C).

Example  5.

Each of the paclitaxel was purified in the same manner as in Example 2, except that each of the paclitaxel extracts obtained in Example 1, which had a purity of 17.6%, 32.6% or 42.0%, was used.

Example  6 to 10.

Each of paclitaxel was purified in the same manner as in Example 5, except that the extraction conditions as described in Table 1 below were used.

division The mixing volume ratio (v / v) of methanol and water Example 6 45:55 Example 7 40:60 Example 8 35:65 Example 9 30:70 Example 10 20:80

Experimental Example  2.

HPLC analysis and extraction yields of paclitaxel obtained in Examples 5 to 10 were analyzed in the same manner as in Experimental Example 1. The results of paclitaxel purity analysis and paclitaxel extraction are shown in FIG. 2 and FIG. 3, respectively.

As can be seen in FIG. 2, the paclitaxel purity obtained tended to decrease slightly as the amount of water to be mixed with methanol increased. It was also found that the purity of paclitaxel obtained increases with increasing purity of paclitaxel before fractional precipitation at the same methanol / water mixing volume ratio.

As can be seen in FIG. 3, the extraction yield of paclitaxel tended to increase with increasing water to methanol regardless of the purity of paclitaxel prior to fractional precipitation. It was also found that the higher the purity of paclitaxel before the fractional precipitation at the same methanol / water mixing volume ratio, the greater the extraction yield of paclitaxel.

Therefore, even when the mixing ratio of methanol / water (v / v) is adjusted, paclitaxel of high purity can be obtained without further storage at a low temperature (0 to 4 ° C) after fractional precipitation in various samples (crude extract purity: 17.6 to 42.0% It was confirmed that it can be recovered at a high yield.

Example  11.

The paclitaxel was purified in the same manner as in Example 2, except that the vacuum-dried extract prepared in Example 1 had a purity of 27.6%.

Example  12.

The paclitaxel was purified by the same method as in Example 11, except that the mixture for the paclitaxel immersion mixture was stirred and then stored at 25 캜 for 30 minutes and then filtered.

Comparative Example  1-2.

Paclitaxel was purified in the same manner as in Example 12, except that the extraction conditions described in Table 2 were used.

division Stirring condition Storage conditions. Comparative Example 1 Do not stir. Store at 25 ° C for 10 minutes. Comparative Example 2 Do not stir. Store at 25 ° C for 40 minutes.

Experimental Example  3.

HPLC analysis and extraction yields of paclitaxel obtained in Examples 11 to 12 and Comparative Examples 1 and 2 were analyzed in the same manner as in Experimental Example 1, and the yield of paclitaxel extraction was shown in FIG.

As can be seen in Fig. 4, the extraction yields of paclitaxel in Examples 11 to 12, when they were added to both the vacuum-dried extract and methanol and water for 10 minutes, were 50.0% and 77.8%, respectively. On the other hand, the extraction yields of paclitaxel in Comparative Examples 1 and 2, which were obtained by adding water to both the vacuum dried extract and methanol without stirring, were 11.6% and 36.6%, respectively.

From this result, it was confirmed that adding water to the vacuum dried extract and methanol mixture and stirring for 10 minutes is an important factor for improving paclitaxel extraction yield. It was also confirmed that the paclitaxel extraction yields of Examples 11 and 12 were more favorable for paclitaxel purification after the stirring (storage) and filtration.

Example  13.

The paclitaxel was purified by the same method as in Example 2, except that the mixture was kept at 4 캜 for 30 minutes and filtered after stirring the mixture for paclitaxel immersion.

Example  14-16 and Comparative Example  3 to 4.

Paclitaxel was purified in the same manner as in Example 13, except that the extraction conditions described in Table 3 were used.

division Stirring condition Storage conditions. Example 14 Stir at 180 rpm for 10 minutes at 25 ° C. Store at 4 ° C for 1 hour. Example 15 Stir at 180 rpm for 40 min at 25 < 0 > C. Do not archive. Example 16 Stir at 180 rpm for 70 min at 25 < 0 > C. Do not archive. Comparative Example 3 Do not stir. Store at 25 ° C for 10 minutes and at 4 ° C for 30 minutes. Comparative Example 4 Do not stir. Store at 25 ° C for 10 minutes and at 4 ° C for 1 hour.

Experimental Example  4.

HPLC analysis and extraction yields of paclitaxel obtained in Example 2, Examples 13 to 16, Comparative Example 1 and Comparative Examples 3 to 4 were analyzed in the same manner as in Experimental Example 1, and the yield of paclitaxel extraction was shown in FIG.

As can be seen in FIG. 5, the paclitaxel extraction yields of Example 2 and Examples 13 to 14, which were stored at low temperature (4 ° C) after stirring for 10 minutes after the addition of water, were 42%, 82.5%, or 84.7% The paclitaxel extraction yields of Examples 15 to 16, which were stirred for 40 minutes or 70 minutes, were 71.3% or 81%, respectively. The paclitaxel extraction yields of Comparative Examples 3 and 4, which were stored at low temperature Were 33.6% or 48.92%, respectively.

In addition, paclitaxel extraction yields were much higher than those of Comparative Examples 3 and 4, which were stored at low temperature (4 ° C) without stirring, in Example 2 and Examples 13 to 16, which were stirred for 10 minutes.

Particularly, Example 15, in which water was added to the mixture and stirred at 25 ° C for 40 minutes, showed a high paclitaxel extraction yield (as compared with Example 14 (84.7%) which was stored at low temperature (4 ° C) for one hour after stirring for 10 minutes 81%).

As a result, the paclitaxel extraction yield (~ 81%) can be obtained without stirring at a low temperature, compared with the conventional method in which the protein was stored at a low temperature for a long time. Water was added to the vacuum- Time agitation was very important for improving paclitaxel extraction yield.

Example  17-24.

Paclitaxel was purified in the same manner as in Example 11, except that the extraction conditions described in Table 4 were used.

division Stirring condition Storage conditions. Example 17 Stir at 180 rpm for 10 minutes at 25 < 0 > C. Store at 25 ° C for 1 hour. Example 18 Stir at 180 rpm for 10 minutes at 25 < 0 > C. Store at 25 ° C for 2 hours. Example 19 Stir at 180 rpm for 10 minutes at 25 < 0 > C. Store at 4 ° C for 30 minutes. Example 20 Stir at 180 rpm for 10 minutes at 25 < 0 > C. Store at 4 ° C for 1 hour. Example 21 Stir at 180 rpm for 10 minutes at 25 < 0 > C. Store at 4 ° C for 2 hours. Example 22 Stir at 180 rpm for 40 minutes at 25 < 0 > C. Do not archive. Example 23 Stir at 180 rpm for 70 minutes at 25 < 0 > C. Do not archive. Example 24 Stir at 180 rpm for 130 minutes at 25 < 0 > C. Do not archive.

Experimental Example  5.

The paclitaxel obtained in Examples 11 to 12 and Examples 17 to 24 was analyzed by HPLC and the extraction yield by the same method as in Experimental Example 1. The paclitaxel purity analysis result is shown in Fig. 6, and the paclitaxel extraction yield is shown in Fig.

As can be seen in FIG. 6, the paclitaxel purity of Examples 11 to 12 and Examples 17 to 18 stored at 25 ° C. after stirring was 64%, 67%, 64.7%, and 64.3%, respectively. ) The paclitaxel purity of the stored Example 11 and 19 to 21 were 64%, 63.7%, 62.1% and 65.2%, respectively. The paclitaxel purity of Example 11 and Examples 22 to 24, which were stirred for 10 minutes or more, were 64%, 66%, 62.4% and 66.8%, respectively.

As can be seen in FIG. 7, the paclitaxel extraction yield rapidly increased between 0 and 30 minutes after storage, and thereafter, the paclitaxel extraction yield was not increased.

Specifically, the extraction yield of paclitaxel according to the settling time (0 minutes, 30 minutes, 1 hour, 2 hours) was 50.6%, 77.8% for Examples 11 to 12 and Examples 17 to 18, respectively, , 78.2% and 82.1%, respectively. In Examples 11 and 19 to 21, which were stored at a low temperature (4 ° C) after stirring, 50.6% were 84.3%, 88.4% and 89.6%. The paclitaxel extraction yields of Examples 22 to 24, which were stirred for 10 minutes or more, were 83.2%, 86.9%, and 87.1%, respectively.

In particular, when water was added to a vacuum-dried extract and a methanol mixture, storage at a low temperature (4 ° C) or stirring at 25 ° C for 40 minutes or more in comparison with storage at 25 ° C in a fractional precipitation time (30 minutes to 2 hours) Of the total.

As compared with the conventional method in which all the water was added and stored at a low temperature (0 to 4 ° C) immediately after the addition of water in the conventional fractionation precipitation, water was added to the dry extract and the organic solvent mixture, and then the paclitaxel of the present invention The purification method was confirmed to be a very economical method because paclitaxel with high purity can be fractionally precipitated at a high yield.

Comparative Example  5.

The paclitaxel was purified in the same manner as in Example 4, except that acetone was used instead of methanol.

Experimental Example  6.

HPLC analysis and extraction yields of paclitaxel obtained in Example 4 and Comparative Example 5 were analyzed in the same manner as in Experimental Example 1, and the yield of paclitaxel extraction was shown in FIG.

As can be seen from Fig. 8, the yield of paclitaxel extraction obtained after stirring was 81% in Comparative Example 5 using mass spectrometry and 92% in Example 4 using methanol. Therefore, it was found that the use of methanol rather than acetone was more effective in increasing the paclitaxel extraction yield.

As can be seen from the Examples, Comparative Examples and Experimental Examples, it was confirmed that adding water to the dry extract and the organic solvent mixture during the fractional precipitation and stirring the mixture was very effective in increasing the paclitaxel extraction yield. These results suggest that the addition of water to the dry extract and the organic solvent mixture in the conventional fractional precipitation results in a high concentration of water at 25 ° C after the addition of water as compared with a method in which water is added and stored at low temperature (0 to 4 ° C) Paclitaxel of high purity can be recovered in a short time at a high yield. As a result, it was found that the paclitaxel purification method of the present invention is more suitable for paclitaxel purification than the conventional fractionation method, and is a more economical method.

Claims (11)

Preparing an extract of Taxus genus biomass containing paclitaxel by one or more extraction methods selected from the group consisting of organic solvent extraction, reduced pressure extraction, liquid-liquid extraction, and hexane precipitation extraction;
Mixing the extract and the water-soluble organic solvent to prepare a mixture;
Adding water to the mixture to perform fractional precipitation; And
Stirring after the fractional precipitation to obtain paclitaxel; Lt; / RTI >
Wherein the water is added in a volume ratio of water-soluble organic solvent to water in a ratio of 50:50 to 20:80. The method according to claim 1, wherein the water-soluble organic solvent is C ₁ ~ Method of purification of paclitaxel, characterized in that it comprises at least one of the group consisting of alcohols and methylene chloride of the C _4. The method for purifying paclitaxel according to claim 1, wherein the agitation is carried out at 10 to 35 DEG C for 5 minutes to 2 hours at 100 to 300 rpm. The method for purifying paclitaxel according to claim 1, further comprising storing the mixture at 0 to 35 ° C for 10 minutes to 2 hours after the stirring. 5. The method of claim 4, wherein the storage is performed at 0 to 10 DEG C for 10 minutes to 2 hours. The method of claim 1, wherein the paclitaxel is purified by the method of claim 1, wherein the paclitaxel is paclitaxel having a purity of 1.5 to 4 times higher than the paclitaxel purity of the extract. [Claim 3] The method according to claim 1, wherein the paclitaxel extraction method has a paclitaxel extraction yield of 40% or more. The method for purifying paclitaxel according to claim 1, wherein the water is added in a volume ratio of water-soluble organic solvent to water of 47: 53 to 35: 65. 9. A pharmaceutical composition for treating Alzheimer's disease comprising paclitaxel purified by the purification method of any one of claims 1 to 8. 9. A pharmaceutical composition for an anticancer drug, which comprises paclitaxel purified by the purification method of any one of claims 1 to 8. A pharmaceutical composition for the treatment of rheumatoid arthritis, which comprises paclitaxel purified by the purification method of any one of claims 1 to 8.

Images