KR102572099B1 - Pre-treatment method of paclitaxel extract through a tandem water and hexane washing and purification method of paclitaxel using the same - Google Patents

Abstract

The present invention relates to a method for pre-treating a paclitaxel extract through sequential washing with water and hexane and a method for purifying paclitaxel using the same, and more particularly, to a method for extracting a biomass containing paclitaxel with an organic solvent and sequentially extracting the paclitaxel with water and A pretreatment method capable of effectively purifying paclitaxel by performing hexane washing is provided.

Description

Pre-treatment method of paclitaxel extract through a tandem water and hexane washing and purification method of paclitaxel using the same}

The present invention relates to a method for pre-treating a paclitaxel extract through sequential washing with water and hexane and a method for purifying paclitaxel using the same, and more particularly, to a method for extracting a biomass containing paclitaxel with an organic solvent and sequentially extracting the paclitaxel with water and A pretreatment method capable of effectively purifying paclitaxel by performing hexane washing is provided.

Paclitaxel is a diterpenoid anticancer substance found in the epidermis of the yew tree, and its chemical structure and anticancer mechanism were identified by Wani et al. in 1971 and Schiff et al. in 1979. It is the most important anticancer drug that has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of Kaposi's sarcoma and non-small cell lung cancer (NSCLC).

The main production methods of paclitaxel include direct extraction from yew trees, and chemically synthesizing side chains by obtaining precursors (baccatin III, 10-diacetyl baccatin III, 10-diacetyl paclitaxel, etc.) from the leaves of yew trees. There is a semi-synthetic method of combining, a method of inducing callus from a yew tree and culturing plant cells in a main culture medium after seed culture.

Separation/purification of paclitaxel by plant cell culture produces a product of high purity (>98%) through several steps of extraction and purification process. In general, in the separation and purification process, paclitaxel is first extracted with an organic solvent from biomass (plant cells containing paclitaxel) as a raw material, followed by pre-purification and final purification. consists of a process for producing In particular, the pre-purification process greatly affects the final purification cost (Kim et al., Process Biochemistry, 87: 238-243 (2019)).

According to the prior art, expensive chromatography is used as a pretreatment process, or raw paclitaxel extracted without pretreatment is immediately finally purified by HPLC (high performance liquid chromatography), resulting in many problems in terms of economy. In addition, there were many difficulties in scale-up and industrial mass production (Kim et al., Process Biochemistry, 59: 216-222 (2017)). In general, when paclitaxel is extracted from biomass using an organic solvent, the purity is 5% or less, and the purity is very low, 10% or less even after a simple pretreatment process. Final purification of these samples directly by HPLC is quite uneconomical, such as the use of a large amount of organic solvent, shortened lifespan of the raw material (resin) packed in the column, and reduced throughput, and is not suitable for mass production. No (Kim et al., Process Biochemistry, 51: 1738-1743 (2016)). Therefore, the purity of the sample should be increased as much as possible through the pretreatment process to reduce the cost of final purification, especially purification using HPLC. Therefore, there is an urgent need to develop a simple and economical pretreatment process capable of obtaining high-purity paclitaxel in high yield.

In the prior art (Kim et al. Process Biochemistry, 87: 238-243 (2019); Kim et al. Process Biochemistry, 59: 216-222 (2017); and Kim et al. Process Biochemistry, 39, 1985-1991 (2004)) According to this, the separation/purification process for mass production of paclitaxel consists of liquid-liquid extraction, adsorbent treatment, hexane precipitation, and fractional precipitation. The purity of the extract obtained by extracting biomass using an organic solvent (methanol) is usually about 0.5-0.7%, which is subjected to subsequent separation/purification processes such as liquid-liquid extraction, adsorbent treatment, hexane precipitation and fractional precipitation. After this, the purity of the sample is improved to 6-9%, 9-10%, 21-27%, and 46-61%, respectively (Kim et al., Process Biochemistry, 51: 1738-1743 (2016)). Through this pretreatment process, a large amount of impurities are removed and the purity of crude paclitaxel is greatly increased, making it suitable for final purification using HPLC. However, it is still difficult to efficiently mass-produce paclitaxel because a complex process consisting of several steps takes a lot of time and requires the use of a large amount of organic solvent, and there is a limit to reducing the production cost of paclitaxel. Therefore, the present invention tried to develop a new pretreatment process for the purification of paclitaxel through a simple and convenient sequential washing method of water and hexane.

The present invention has been made to solve the above problems, and provides a paclitaxel pretreatment method using a sequential washing method of water and hexane, in which the pretreatment efficiency is dramatically improved compared to the conventional paclitaxel pretreatment process.

More specifically, the optimization of the pretreatment method is achieved through sequential washing with water and hexane, thereby improving the efficiency of the pretreatment method, such as increasing the purity and yield of paclitaxel and reducing operating time, as well as reducing the steps of the pretreatment method. The purpose is to drastically shorten it.

Furthermore, another object of the present invention is to provide a method for purifying paclitaxel using the above-described pretreatment method for paclitaxel.

In order to solve the above problems, the present invention provides a method for pre-treatment of paclitaxel comprising the following steps a) to d): a) taxus genus plant-derived biomass containing paclitaxel Obtaining; b) extracting the biomass with an organic solvent to obtain a crude extract of paclitaxel; c) adding water to the paclitaxel raw extract, stirring and washing, and filtering to obtain a cake; and d) adding hexane to the filtrate followed by stirring and washing.

According to a preferred embodiment of the present invention, the biomass in step a) may include at least one selected from the group consisting of a taxus genus plant, its cells, its cell debris, and its cell culture medium. .

According to another preferred embodiment of the present invention, the organic solvent in step b) may include at least one selected from the group consisting of C ₁ to C ₄ alcohol and water.

According to another preferred embodiment of the present invention, the biomass and organic solvent in step b) may be mixed in a ratio of 1:1 to 1:6 (w/v).

According to another preferred embodiment of the present invention, step c) is performed at 15 ° C to 35 ° C for 5 to 20 minutes. can be done

According to another preferred embodiment of the present invention, in step c), water may be added so that the ratio of the raw extract to water is 1:5 to 1:60 (w/v).

According to another preferred embodiment of the present invention, step d) may be performed at 15 ° C to 35 ° C for 10 to 20 minutes.

According to another preferred embodiment of the present invention, in step d), hexane may be added so that the ratio of the filtrate to hexane is 1:150 to 1:170 (w/v).

According to another preferred embodiment of the present invention, the step of drying the filtrate may not be included between steps c) and d).

The present invention also provides a method for purifying paclitaxel including the above-described pretreatment method for paclitaxel.

The paclitaxel pretreatment method using sequential washing of water and hexane of the present invention provides a shortened and simplified paclitaxel pretreatment process compared to the conventional pretreatment process for separation and purification of paclitaxel, and solvent extraction, which is a conventional paclitaxel separation and purification method , liquid-liquid extraction, adsorbent treatment, and hexane precipitation can be performed sequentially to obtain paclitaxel with higher purity and yield than paclitaxel samples obtained, so there is a remarkable effect of obtaining high-purity paclitaxel in high yield in a short time. there is. In addition, since all of the paclitaxel pretreatment methods of the present invention can be performed at room temperature, high cost due to energy consumption for low temperature storage can be improved. Therefore, the paclitaxel pretreatment method of the present invention is very suitable for application to mass production of paclitaxel, and through this, the cost reduction effect of paclitaxel can be induced.

Figure 1 (A) shows the HPLC chromatogram results for the raw extract obtained from biomass extraction using methanol, and Figure 1 (B) shows the HPLC chromatogram results for the sample (cake) obtained after washing with water. 1(C) shows the HPLC chromatogram results of the filtrate after washing with water.
Figure 2 shows various samples (cake) during hexane washing: hexane ratios (1:80, 1:100, 1:120, 1:140, 1:160 and 1:180 (w / v), respectively) and operation time It is a graph showing the purity of paclitaxel.
FIG. 3(A) shows the HPLC chromatogram result of the sample (cake) obtained after washing with hexane, and FIG. 3(B) shows the HPLC chromatogram result of the filtrate after washing with hexane.
FIG. 4 shows the HPLC chromatogram results of the filtrate obtained by immediately washing with hexane and filtering the sample (cake) obtained through filtration after washing with water without drying.
Figure 5 is a schematic diagram showing a sequential washing method for pre-purification of paclitaxel from biomass.

As described above, the conventional pretreatment process for the purification of paclitaxel uses expensive chromatography, or when the extracted raw extract is directly purified by the HPLC process, a large amount of organic solvent is used, raw materials packed in a column ( It is very uneconomical and not suitable for mass production due to shortened resin) life span and reduced throughput. In addition, there is a difficulty in efficiently producing paclitaxel due to the complexity of the process and the consumption of a lot of pretreatment process time according to the conventional multi-step pretreatment process, which makes it difficult to reduce the cost of paclitaxel.

Accordingly, in the present invention, a) obtaining taxus genus plant-derived biomass containing paclitaxel; b) extracting the biomass with an organic solvent to obtain a crude extract of paclitaxel; c) adding water to the paclitaxel raw extract, stirring and washing, and filtering to obtain a cake; and d) adding hexane to the filtrate (cake), followed by stirring and washing. Through this, it is possible to obtain high purity paclitaxel in a high yield in a short time through a process that is shorter and simpler than the conventional paclitaxel pretreatment process, thereby significantly improving the efficiency of the pretreatment process (increasing the purity and yield of paclitaxel and shortening operation time). Therefore, there is an advantage that it is very suitable for mass production of paclitaxel.

First, step a) of obtaining biomass derived from a Taxus genus plant containing paclitaxel will be described.

In the paclitaxel pretreatment method of the present invention, the biomass derived from a taxus genus plant containing paclitaxel is at least one selected from the group consisting of a taxus genus plant, its cells, its cell debris, and its cell culture solution. can include

In addition, the Taxus genus plants are Taxus brevifolia , Taxus canadensis, Taxus cuspidata , Taxus baccata , Taxus Glo Bossa ( Taxus globosa ), Taxus Floridana ( Taxus floridana ), Taxus wallichiana ( Taxus wallichiana ), Taxus media ( Taxus media ) or Taxus chinensis ( Taxus chinensis ), etc. may be included, but are limited thereto It is not.

The step of obtaining the biomass is not particularly limited as long as it is a method of obtaining biomass from a conventional plant cell culture medium.

Next, step b) of extracting the biomass with an organic solvent to obtain a crude paclitaxel extract will be described.

In the paclitaxel pretreatment method of the present invention, the organic solvent in step b) is not particularly limited as long as it is commonly used to extract active ingredients from plants, but is preferably selected from the group consisting of C ₁ to C ₄ alcohol and water. One or more may be included.

As a specific example of the organic solvent, one or a mixture of two or more selected from methanol, ethanol, propanol, and methylene chloride may be used, and methanol is preferably used as an organic solvent capable of recovering as much paclitaxel from biomass as possible. can

Furthermore, the organic solvent extraction conditions are not particularly limited as long as they are conditions that can be used in the method of extracting active ingredients from plants, but preferably can be performed at 20 ° C to 45 ° C for 30 minutes to 2 hours.

If the treatment is performed at a temperature of less than 20 ° C, the extraction efficiency of paclitaxel may decrease due to the low temperature. can happen

If the treatment takes less than 30 minutes, all of the paclitaxel in the biomass is not extracted, which may cause a problem of lowering the extraction efficiency of paclitaxel, and if the treatment takes more than 2 hours, an economic problem due to excessive operating time. may occur.

In the paclitaxel pretreatment method of the present invention, the biomass and organic solvent in the above step may be mixed in a ratio of 1:1 to 1:6 (w/v), but is not limited thereto, Any ratio that can be used for extraction is not particularly limited. In one embodiment of the present invention, biomass and organic solvent were constantly mixed at a 1:1 (w/v) ratio for optimal extraction efficiency.

In addition, the solvent extraction may be repeated one to several times, preferably two or more times, more preferably three to five times, and then the obtained extract is concentrated under reduced pressure and then dried. and can be used in the water washing step.

Next, step c), which is a step of adding water to the paclitaxel raw extract, stirring and washing, and filtering to obtain a cake, will be described.

In the paclitaxel pretreatment method of the present invention, step c) is performed at 15° C. to 35° C. for 5 to 60 minutes. It may be performed, preferably at 20 ° C to 30 ° C for 5 to 30 minutes, more preferably at 20 ° C to 30 ° C for 5 to 20 minutes.

If carried out at a temperature of less than 15 ° C, economic problems may occur due to low temperature, and when treated at a temperature exceeding 35 ° C, a problem of decomposition of paclitaxel due to high temperature may occur.

In addition, when performed for less than 5 minutes, a problem may occur in which paclitaxel is not sufficiently washed, and when performed for more than 60 minutes, a problem in operation efficiency may occur due to excessive operation time.

In the paclitaxel pretreatment method of the present invention, in step c), water may be added so that the ratio of the raw extract to water is 1:5 to 1:60 (w/v), preferably 1:25 to 1: 45 (w / v), more preferably 1:35 to 1:45 (w / v), water may be added, and then the filtrate (cake) obtained by filtering after stirring and washing is hexane washing step can be used for

In a specific embodiment of the present invention, distilled water is added to a sample (purity: 4.5%) obtained from biomass extraction using methanol so that the sample: water ratio is 1:10 to 1:60 (w / v) After that, washing with water was performed for 10 minutes under stirring, and then HPLC was performed on the obtained sample to confirm the purity of paclitaxel. As a result, the highest purity and yield of paclitaxel could be obtained when the sample:water ratio was 1:40 (w/v), and the purity at this time was ~12.2%, as shown in FIG. 1(B), and the yield was was identified as ~99.9%. In addition, as a result of recovering the filtrate after washing with water and performing HPLC, a large amount of polar impurities were detected as shown in FIG. It was confirmed that only .

Finally, step d), which is a step of sequentially stirring and washing after adding hexane to the filtrate (cake) obtained after washing with water, will be described.

In the paclitaxel pretreatment method of the present invention, step d) may be performed at 15°C to 35°C for 10 to 20 minutes, preferably at 20°C to 35°C for 10 to 20 minutes, more preferably It may be performed at 20°C to 30°C for 10 to 20 minutes.

If carried out at a temperature of less than 15 ° C, economic problems may occur due to low temperature, and when treated at a temperature exceeding 35 ° C, a problem of decomposition of paclitaxel due to high temperature may occur.

In addition, when carried out for less than 10 minutes, paclitaxel may not precipitate sufficiently, and when carried out for more than 20 minutes, the effect of increasing the purity and yield of paclitaxel due to the increase in operating time hardly appears, resulting in operational efficiency. Falling issues can occur.

In the paclitaxel pretreatment method of the present invention, in step d), hexane may be added so that the ratio of the filtrate (cake) to hexane is 1:150 to 1:170 (w/v), more preferably 1 :155 to 1:165 (w/v), hexane may be added, followed by stirring and washing, followed by filtration, and the resulting filtrate (cake) may be used in an additional purification process.

In a specific embodiment of the present invention, hexane is added to the dried sample after washing with water so that the sample:hexane ratio is 1:80 to 1:180 (w / v), and then hexane washing is performed for 10 to 40 minutes under stirring. After that, HPLC was performed on the obtained sample to confirm the purity of paclitaxel. As a result, as confirmed in FIG. 2, when the sample:hexane ratio was 1:160 (w/v) or more and the operating time was 20 minutes or more, almost no increase in purity was observed. Therefore, it was confirmed that the sample:hexane ratio was 1:160 (w/v) and the treatment time was 20 minutes as the optimal hexane washing conditions. As a result of performing HPLC on the sample obtained through filtration after hexane washing under the above optimal hexane treatment conditions, the purity of paclitaxel was confirmed to be ~30.2% and the yield was ~99.9%, as shown in FIG. 3(A). .

In addition, as a result of performing HPLC by recovering the filtrate after washing with hexane under the above optimal conditions, a large amount of non-polar impurities were detected as shown in FIG. It was confirmed that only contained non-polar impurities were effectively removed.

In the paclitaxel pretreatment method of the present invention, a step of drying the filtrate (precipitate, cake) is not included between steps c) and d).

In a specific embodiment of the present invention, in order to investigate the possibility of a sequential washing process of water and hexane, the filtrate (cake) obtained through filtration after water washing was directly used for hexane washing without drying, and then for the obtained sample HPLC was performed to confirm the purity of paclitaxel. At this time, the optimal conditions identified above were applied to the water washing and hexane washing conditions, respectively. As a result, the purity of paclitaxel in the sample was almost similar to the purity of paclitaxel (30.2%) obtained from the result of washing the sample washed with water at ~30% and then sequentially washing with hexane.

Through these results, the paclitaxel pretreatment method of the present invention can obtain paclitaxel with higher purity and yield than the paclitaxel sample obtained by sequentially performing solvent extraction, liquid-liquid extraction, adsorbent treatment, and hexane precipitation, and thus more simplified It was confirmed that high-purity paclitaxel could be obtained in high yield in a short time through the process. In addition, since all of the paclitaxel pretreatment methods of the present invention can be performed at room temperature, high cost due to energy consumption for low temperature storage can be improved.

Furthermore, the present invention provides a paclitaxel purification method including the above-described paclitaxel pretreatment method.

The paclitaxel purification method of the present invention is performed in a simplified process by extracting biomass with an organic solvent and sequentially washing with water and hexane instead of the conventional solvent extraction, liquid-liquid extraction, adsorbent treatment, and hexane precipitation steps, thereby reducing the complexity of the process. It can be very useful for mass production of paclitaxel by simplifying.

Hereinafter, the present invention will be described in more detail through examples. However, the present invention can be made with various changes and can have various forms, and the specific embodiments and descriptions described below are only intended to help the understanding of the present invention, and the present invention is limited to specific disclosure forms. It's not something I want to do. It should be understood that the scope of the present invention includes all modifications, equivalents and substitutes included in the spirit and scope of the present invention.

biomass preparation

The plant cell culture medium used in this example was cultured using a cell line obtained from the leaves of Taxus chinensis . Suspension cells derived from Taxus chinensis were cultured at 24° C. under darkness conditions with agitation at 150 rpm. Suspension cells were prepared in modified Gamborg's B5 medium, 30 g/L sucrose, 10 mM naphthalene acetic acid, 0.2 μM 6-benzylaminopurine, 1 g /L casein hydrolysate, 1 g/L 2-(N-morpholino) ethanesulfonic acid (2-(N-morpholino) ethanesulfonic acid). The cell culture was changed to a new medium every 2 weeks, and 1 to 2% (w/v) maltose was added on the 7th and 21st days to extend the culture time, and the inducer As an elicitor, 4 mM AgNO ₃ was added at the beginning of the culture [7,19]. After plant cell culture, plant cells and cell debris were recovered from the culture medium using a decanter (Westfalia, CA150 Claritying Decanter) and a high-speed centrifuge (α-Laval, BTPX205GD-35CDEEP). The collected plant cells and cell fragments were collectively referred to as biomass.

Pretreatment of paclitaxel samples by water washing process and analysis of paclitaxel content

2-1. Sample preparation for water washing process

The biomass recovered from the plant cell culture medium of Example 1 was dissolved in methanol (biomass/methanol ratio = 1:1, w/v), extracted at room temperature for 30 minutes, filtered, and new methanol was added to the biomass. Extraction was repeated 4 times in the same way. The extract was concentrated under reduced pressure in a rotary evaporator (CCA-1100, EYELA, Japan), and then completely dried at 40 °C for 24 hours using a vacuum oven (UP-2000; EYELA). The purity of the dried sample was 4.5%, and it was used in the water washing process.

2-2. water washing process

After adding distilled water to the sample (purity: 4.5%) obtained from biomass extraction using methanol in Example 2-1, water washing was performed by stirring (~ 300 rpm). Water washing was performed at room temperature, the sample/water ratio was set at 1:10-1:60 (w/v), and the treatment time was 10 minutes. The precipitate obtained after water pretreatment was filtered (Whatman Grade 5, 2.5 mm, particle retention, 150 mm diameter), and the cake obtained was used for hexane washing. As a result of HPLC analysis, the purity of the sample was 11.2%.

2-3. Analysis of paclitaxel content

To analyze the content of paclitaxel in each sample obtained in Examples 2-1 and 2-2, HPLC (high performance liquid chromatography) system (SCL-10AVP, Shimadzu, Japan) and Capsule Pak C ₁₈ (Capcell Pak C ₁₈ ; 250 × 4.6 mm, Shiseido, Japan) column was used (Kim et al., Process Biochemistry, 99, 316-323; and Kim et al., Process Biochemistry, 87: 238-243). The mobile phase was an acetonitrile-distilled water mixed solution (35/65-65/35, v/v, gradient solvent composition method) and the flow rate was 1.0 mL/min. The sample injection amount was 20 μL and was detected by UV at 227 nm. Standard paclitaxel samples (purity: 95%) were purchased from Sigma-Aldrich and used.

As a result of drying the sample obtained from the biomass extraction using methanol in Example 2-1 and performing HPLC, the purity of paclitaxel was confirmed to be 4.5%, as shown in FIG. 1(A).

In Example 2-2, the sample: water ratio was 1:10 to 1:60 (w / v), washed with water for 10 minutes, and then dried and HPLC was performed on the cake obtained through filtration. Figure 1 As confirmed in (B), when the sample:water ratio was 1:40 (w/v), the highest paclitaxel purity (~12.2%) and yield (~99.9%) were exhibited.

In addition, as a result of confirming the chromatogram obtained by HPLC analysis by recovering the filtrate after washing with water, a large amount of polar impurities were detected as shown in FIG. It was found to be very effective in removing Furthermore, there was almost no paclitaxel in the filtrate, and through this, it was found that most of the paclitaxel could be recovered with the sample (cake) obtained through filtration after washing.

Pretreatment of paclitaxel samples by hexane washing process and analysis of paclitaxel content

3-1. Hexane washing process

After adding hexane to the sample (purity: 11.2%) obtained through water washing in Example 2-2, hexane washing was performed by stirring (~ 300 rpm). Hexane washing was carried out at room temperature, and in order to investigate the optimal hexane addition amount, the sample:hexane ratio was 1:80, 1:100, 1:120, 1:140, 1:160 and 1:180 (w/v). each changed. In addition, the treatment time was changed to 10, 20, 30 and 40 minutes, respectively, to investigate the optimal treatment time. After washing with hexane, a sample (cake) was obtained through filtration (Whatman Grade 5, 2.5 mm, particle size, 150 mm diameter), and dried to calculate the purity and yield of paclitaxel through HPLC analysis.

3-2. Analysis of paclitaxel content

HPLC was performed in the same manner as in Example 2-3 to analyze the content of paclitaxel.

As confirmed in FIG. 2, when the sample:hexane ratio was 1:160 (w/v) or more, the purity was hardly increased, and the purity was hardly increased after 20 minutes of treatment. Therefore, it was confirmed that the sample:hexane ratio was 1:160 (w/v) and the treatment time was 20 minutes as the optimal hexane washing conditions. As a result of performing HPLC on the sample (cake) obtained through filtration after hexane washing under the above optimal hexane treatment conditions, as shown in FIG. 3(A), the purity of paclitaxel was confirmed to be ~30.2%, and the yield was ~99.9 % confirmed.

In addition, as a result of confirming the chromatogram obtained by HPLC analysis after recovering the filtrate after washing with hexane under the above optimal conditions, a large amount of non-polar impurities were detected in the filtrate, as shown in FIG. It was found to be very effective in removing non-polar impurities contained in this sample. Furthermore, almost no paclitaxel was present in the filtrate, and through this, it was found that most of the paclitaxel could be recovered with the sample (cake) obtained through filtration after washing.

Pretreatment of Paclitaxel Samples by Sequential Washing Process with Water and Hexane and Analysis of Paclitaxel Content

4-1. Sequential washing process of water and hexane

In order to investigate the possibility of a sequential washing process of water and hexane, the sample (cake) obtained through filtration after water washing was directly used for hexane washing without drying. This has the advantage of shortening the operating time by omitting the drying process after washing with water.

Specifically, the sample (purity: 4.5%) obtained from the biomass extraction using methanol of Example 2-1 was subjected to the optimal condition of the water washing process: water: sample ratio of 1:40 (w / v) for 10 minutes Water washing was performed during water washing, and hexane washing was sequentially performed using a sample (cake) obtained through filtration (Whatman Grade 5, 2.5 mm, particle size, 150 mm diameter) after washing with water. Similarly, hexane washing was performed for 20 minutes with a sample:hexane ratio of 1:160 (w/v), which is the optimal condition for the hexane washing process, and then filtration (Whatman Grade 5, 2.5 mm, particle size, 150 mm diameter) The sample (cake) obtained through was dried.

4-2. Analysis of paclitaxel content

HPLC was performed in the same manner as in Example 2-3 to analyze the content of paclitaxel.

As a result, as shown in FIG. 4 , the purity of paclitaxel was almost similar to the purity of paclitaxel (30.2%) obtained from the result of sequentially washing with hexane after drying the sample washed with water at about 30%. As a result, the process time could be shortened by immediately performing hexane washing without drying the sample (cake) obtained after filtering the water-washed sample.

A schematic diagram of the “sequential washing process of water and hexane” designed in the present invention is shown in FIG. 5.

Claims (10)

A method for pre-treatment of paclitaxel comprising the following steps a) to d):
a) obtaining taxus genus plant-derived biomass containing paclitaxel;
b) extracting the biomass with an organic solvent to obtain a crude extract of paclitaxel;
c) adding water so that the ratio of the raw paclitaxel extract to water is 1:5 to 1:60 (w/v), stirring and washing, and filtering to obtain a cake; and
d) adding hexane so that the ratio of the cake to hexane is 1:150 to 1:170 (w/v), followed by stirring and washing at 15° C. to 35° C. for 10 to 20 minutes. The method of claim 1, wherein the biomass in step a) comprises at least one species selected from the group consisting of a taxus genus plant, its cells, its cell debris, and its cell culture medium. preprocessing method. The pretreatment method for paclitaxel according to claim 1, wherein the organic solvent in step b) includes at least one selected from the group consisting of C ₁ to C ₄ alcohol and water. The pretreatment method for paclitaxel according to claim 1, wherein the biomass and the organic solvent in step b) are mixed in a ratio of 1:1 to 1:6 (w/v). The method of claim 1, wherein step c) is performed at 15° C. to 35° C. for 5 minutes to 60 minutes. A method for pre-treatment of paclitaxel, characterized in that performing. delete delete delete The paclitaxel pretreatment method according to claim 1, wherein a step of drying the filtrate is not included between steps c) and d). A method for purifying paclitaxel, comprising the method for pre-treating paclitaxel according to any one of claims 1 to 5 and 9.