KR101756529B1

KR101756529B1 - Pre-treatment method of paclitaxel extract using water

Info

Publication number: KR101756529B1
Application number: KR1020150113511A
Authority: KR
Inventors: 김진현; 이충기
Original assignee: 공주대학교 산학협력단
Priority date: 2015-08-11
Filing date: 2015-08-11
Publication date: 2017-07-11
Also published as: KR20170019291A

Abstract

The present invention relates to a pretreatment process for paclitaxel containing water, and more particularly, to a process for the treatment of paclitaxel in a short period of time in a biomass recovered from a plant cell culture of a taxus genus plant, Of paclitaxel in a high yield, and a method of pretreating paclitaxel using water.

Description

[0001] PRE-TREATMENT METHOD OF PACLITAXEL EXTRACT USING WATER [0002]

Paclitaxel is a diterpenoid anticancer substance found in the epidermis of the yew tree and its chemical structure and anticancer mechanism have been revealed by Wani et al. (1971) and Schiff et al. (1979). Paclitaxel 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 an anticancer agent being used.

The main production methods of paclitaxel include a direct extraction method directly extracted from the tree of interest, a semi-synthetic method in which the side chains are chemically bonded by obtaining precursors (baccatin Ⅲ, 10-deacetylbaccatin Ⅲ, 10-deacetylpaclitaxel, etc.) And plant cell culture, which is a method of inducing callus and culturing plant cells in a main culture medium after seed culture. Among these methods, the plant cell culture method can be stably produced in the bioreactor without being influenced by external factors such as climate and environment, so that the paclitaxel of a certain quality can be mass produced and coped with the increase in demand sufficiently. Many separation and purification steps are required to obtain high purity paclitaxel from plant cell cultures. In general, paclitaxel is first extracted from the biomass with a solvent and subjected to pre-treatment and final purification to obtain high-paclitaxel.

However, in the prior art, crude extract was purified by HPLC (High Performance Liquid Chromatography) using expensive chromatography as a pretreatment process for purification. Particularly when HPLC is used for final purification of low purity samples without pretreatment, it is very uneconomical due to the use of a large amount of organic solvent, the shortening of the life of the packed resin in the column and the reduction in throughput, It is not suitable for the process.

Therefore, a pretreatment method that can easily and economically obtain paclitaxel of high purity at a high yield is desperately needed. In 2002, Kim et al. Prepared a novel prepurification for paclitaxel from plant cell cultures. Process Biochem 2002; 37: 679-82; Pyo et al. A large-scale purification of paclitaxel from cell cultures of Taxus chinensis. Process Biochem 2004; 39: 1985-91; And Kim's Paclitaxel: Recovery and purification in the commercialization step. Korean J. Biotechnol. Pre-treatment processes for the mass production of paclitaxel have been developed by Bioeng 2006; 21 (1): 1-10, which consists of solvent extraction, liquid-liquid extraction, adsorbent treatment, hexane precipitation and fractional precipitation 1).

The purity of the samples were 0.5-0.7%, 6-9%, 9-10%, 21-27%, 46-24%, respectively by the solvent extraction, liquid-liquid extraction, adsorbent treatment, hexane precipitation and fractional precipitation using biomass. 61%. Through this pretreatment process, a large amount of impurities were removed, and the purity of the crude paclitaxel was greatly increased, making it suitable for the final HPLC process. However, the increase of purity in the adsorbent treatment process was insignificant (<4%) in the pretreatment process, and the preprocessing process of many stages consumed a lot of time for the pretreatment process, and the use of a large amount of solvent, complicated process, There is a difficulty in mass production of paclitaxel and it is difficult to reduce the cost of paclitaxel.

Accordingly, the present invention has been made to solve the above-mentioned problems, and provides a pretreatment process of paclitaxel including a pretreatment method of paclitaxel using water, wherein the pretreatment efficiency is drastically improved as compared with the conventional paclitaxel pretreatment process.

More specifically, the water pre-treatment process is optimized and the efficiency of the pretreatment process such as paclitaxel purity and yield increase and shortening of the operation time is improved by searching various paths of the subsequent pre-treatment process after the water pretreatment But also to shorten the pretreatment process remarkably.

In order to solve the above-mentioned problems, the present invention provides a pretreatment process for paclitaxel comprising the following steps a) to c): a) extracting a biomass from a cell culture of a Taxus genus plant comprising paclitaxel ; b) extracting the biomass with an organic solvent to obtain a paclitaxel crude extract; And c) adding water to the obtained crude extract to perform a pretreatment.

According to a preferred embodiment of the present invention, in the pretreatment step, the step c) may be carried out at 15 to 35 ° C for 5 to 60 minutes.

According to another preferred embodiment of the present invention, in the pretreatment step, the amount of water added in step c) may be 1: 5 to 1:60 (w / v).

According to another preferred embodiment of the present invention, in the pretreatment step, the ratio of the crude extract to water may be 1:35 to 1:45.

According to another preferred embodiment of the present invention, in the pretreatment step, c ') liquid-liquid extraction step may further be included between step b) and step c) or step c).

According to a preferred embodiment of the present invention, the pre-treatment step may further include the following steps after performing the pre-treatment step in the order of steps a), b) and c): d) Processing; And e) performing fractional precipitation.

According to another preferred embodiment of the present invention, in the preprocessing step, the order of steps a), b), c ') and c) or steps a), b), c) and c' , The method may further comprise the following steps: d) treating the adsorbent; And e) performing fractional precipitation.

According to another preferred embodiment of the present invention, in the pretreatment step, the fractional precipitation of step e) may include the following steps e-1) to e-3): e-1) d) Dissolving the obtained paclitaxel crude extract in a water-soluble organic solvent; e-2) water is added with stirring until the volume ratio of the water-soluble organic solvent and water is 50:50 to 20:80 to perform fractional precipitation; And e-3) followed by fractional precipitation to obtain paclitaxel.

According to another preferred embodiment of the present invention, in the pretreatment step, between the step d) and the step e), the hexane precipitation step is not included.

According to another preferred embodiment of the present invention, the paclitaxel purity is 5 to 13% and the paclitaxel yield is 90 to 100% after the steps a), b) and c) Lt; / RTI >

According to another preferred embodiment of the present invention, in the pretreatment step, the paclitaxel purity after the steps a) to e) is omitted and the paclitaxel purity is 50 to 55% 95%. &Lt; / RTI >

According to another preferred embodiment of the present invention, in the pre-processing step, The paclitaxel purity after the step (b) and (c) or between the step c) and step (d) after step c) is 50 to 60% and the yield of paclitaxel is 80 to 95 %. &Lt; / RTI >

The pretreatment step of paclitaxel including the pretreatment method using water of the present invention not only provides a preprocessing step of paclitaxel which is much shorter than the conventional pretreatment step for separation and purification of paclitaxel, , There is a remarkable effect that paclitaxel of high purity can be obtained in a high yield in a short time.

Specifically, if the pretreatment step of the conventional paclitaxel such as liquid-liquid extraction and hexane precipitation is omitted, if the pretreatment step of the paclitaxel including the pretreatment method using the water of the present invention is carried out, the paclitaxel still having a high purity can be obtained at a high yield . As a result, the operating time is remarkably reduced, the amount of the solvent used is reduced, and the complicated process is simplified. Thus, the paclitaxel pretreatment process of the present invention is well suited for mass production of paclitaxel, thereby reducing the cost of paclitaxel.

1 is a schematic diagram showing a process for purifying paclitaxel from biomass in the prior art.
Figure 2 is a graph showing the effect of crude extract / water ratio on purity of paclitaxel during water pretreatment.
Figure 3 is a graph showing the effect of crude extract / water ratio on the yield of paclitaxel during water pretreatment.
4 is a graph showing the chromatogram of the water supernatant.
Fig. 5 is a schematic diagram showing a subsequent pretreatment step (A) involving liquid-liquid extraction after water pretreatment and a subsequent pretreatment step (B) without liquid-liquid extraction.
6 is a chromatogram showing the purity of paclitaxel after performing the biomass extraction step.
Figure 7 is a chromatogram showing the purity of paclitaxel after the water pretreatment step.
Figure 8 is a chromatogram showing the purity of paclitaxel after performing the liquid-liquid extraction step.
9 is a chromatogram showing the purity of paclitaxel after the adsorbent treatment step has been carried out through the liquid-liquid extraction step.
10 is a chromatogram showing the purity of paclitaxel after performing an improved fractional precipitation through a liquid-liquid extraction step and an adsorbent treatment step.
11 is a graph comparing the paclitaxel purity (right graph) after biomass extraction, water pretreatment, liquid-liquid extraction, paclitaxel purity (left graph) and biomass extraction, liquid-liquid extraction, water pretreatment and adsorbent treatment.
Figure 12 is a chromatogram showing the purity of paclitaxel after the adsorbent treatment step without the liquid-liquid extraction step.
Figure 13 is a chromatogram showing the purity of paclitaxel after adsorbent treatment and improved fractional precipitation steps without the liquid-liquid extraction step.

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

As described above, the conventional pretreatment process for purification of paclitaxel requires a large amount of organic solvent when using expensive chromatography or ultrafiltration of the extracted crude extract directly by HPLC, shortening the lifetime of the resin, reducing throughput, etc., it is very uneconomical and not suitable for mass production. In addition, due to the many pre-processing steps and the complexity of the process in the conventional pretreatment processes, it has been difficult to efficiently produce paclitaxel, which is a cause of difficulty in reducing the cost of paclitaxel.

Accordingly, the present invention provides a method for producing a taxane, comprising: a) obtaining a biomass from a cell culture of a Taxus genus plant comprising paclitaxel; b) organic solvent extraction of the biomass to obtain a crude extract of paclitaxel; And c) adding water to the obtained crude extract to perform pretreatment. Thus, a solution to the above-mentioned problem has been sought by providing a pretreatment process for paclitaxel. Thus, paclitaxel with a high purity can be obtained in a short time at a high yield through a remarkably shortened process than the conventional paclitaxel pretreatment process, so that the efficiency of the pretreatment process (paclitaxel purity, yield and operation time) can be remarkably improved, It is very suitable for mass production of the < RTI ID = 0.0 >

First, the step of obtaining biomass from a cell culture of a Taxus genus plant containing paclitaxel is described.

The biomass obtained from the cell culture solution of the Taxus plant containing the paclitaxel may include at least one selected from the group consisting of a plant belonging to the Taxus family, a cell thereof, a cell debris 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), Florida TAXUS I (Taxus floridana), TAXUS May Reach Ana (Taxus wallichiana , Taxus media , or Taxus chinensis ), and the like, but the present invention is not limited thereto.

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

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

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

Specific examples of the organic solvent include methanol, ethanol, propanol, methylene chloride, and mixtures of two or more thereof. An organic solvent capable of recovering paclitaxel as much as possible from the biomass is preferably methanol .

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 may be repeated one to several times, preferably two or more times, more preferably three to five times, and then the resulting extract is concentrated under reduced pressure and then dried And can be used in the water pretreatment step or the liquid-liquid extraction step.

Next, the step of performing preprocessing by adding water to the paclitaxel crude extract obtained after organic solvent extraction will be described.

The water pretreatment may be carried out at 15 to 35 ° C for 5 to 60 minutes, preferably at 20 to 30 ° C for 5 to 30 minutes, more preferably at 25 to 30 ° C for 5 to 15 minutes .

If it is carried out at a temperature lower than 15 캜, economical problems due to low temperature may occur, and when the temperature is exceeded 35 캜, paclitaxel may be decomposed due to high temperature.

If the treatment is carried out for less than 5 minutes, a problem that the paclitaxel is not sufficiently precipitated may occur. If the treatment is performed for more than 60 minutes, the operation efficiency may be lowered due to excessive operation time.

The water pre-treatment may be performed at a ratio of 1: 5 to 1:60 (w / v), preferably 1:25 to 1:45 (w / v) More preferably 1:35 to 1:45 (w / v), and then the resulting precipitate can be filtered through a filter paper and then dried to be used in a liquid-liquid extraction step or an adsorbent treatment step .

Alternatively, the paclitaxel pretreatment process of the present invention may further comprise a c) liquid-liquid extraction step between step b) and step c) or step c).

Specifically, the step c ') may be selectively added between steps b) and c) or between the steps d) and adsorbent treatment steps to be performed in step c) and subsequent steps, But those skilled in the art will generally be able to carry out the present invention.

The paclitaxel pretreatment process of the present invention may be carried out in the order of steps a), b) and c), and then d) treating the adsorbent; And e) performing a fractional precipitation.

Further, The paclitaxel pretreatment step may be carried out in the order of steps a), b), c ') and c) or steps a), b), c) and c'), and then d) treating the adsorbent; And e) performing a fractional precipitation.

Specifically, when the silica-based adsorbent is used, it is preferable to use a silica-based adsorbent as the adsorbent in the step d). The impurities such as biomass-derived wax and tar can be effectively removed, and the purity and purification of paclitaxel To increase the efficiency of the system.

Also, the adsorbent of step d) may be treated so that the ratio of the crude extract / adsorbent obtained in step c) or step c ') is 1: 0.5 to 1: 2.5 (w / w).

The adsorbent can be treated at 20 to 60 ° C for 30 minutes, and then filtered with a filter paper and dried under reduced pressure.

The above c ') liquid-liquid extraction is not particularly limited so long as it is a method for removing polar impurities contained in paclitaxel using the phase separation between a nonpolar organic solvent and a polar organic solvent.

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

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, if the polar organic solvent is a C ₁ -C ₄ alcohol, it is preferred to use methylene chloride as the nonpolar organic solvent, and methylene chloride may be preferably used at 20 to 30% (v / v) of the alcohol concentrate , And the use of excess methylene chloride places a heavy burden on the subsequent process.

The fractional precipitation of step e) may include the following steps e-1) to e-3): dissolving the paclitaxel crude extract obtained after step d) in a water-soluble organic solvent; e-2) water is added with stirring until the volume ratio of the water-soluble organic solvent and water is 50:50 to 20:80 to perform fractional precipitation; And e-3) followed by fractional precipitation to obtain paclitaxel.

In step e-1), the crude extract is dissolved in the water-soluble organic solvent, but it is not particularly limited as long as the water-soluble organic solvent is a mixture of water and a solvent capable of uniformly dispersing the crude extract. Preferably, 1 to 5 parts by weight of crude extract can be dissolved.

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, the step of performing fractional precipitation by adding water with stirring until the volume ratio of the water-soluble organic solvent and water becomes 50:50 to 20:80, will be described.

The amount of the water added may be in the range of 50:50 to 20:80 by volume of the water-soluble organic solvent and water, more preferably 47:53 to 35:65 by volume of the water-soluble organic solvent and water.

Next, in the step of obtaining paclitaxel by stirring after the fractional precipitation of e-3), stirring conditions are not particularly limited so long as they are conditions used for component precipitation in plants, but preferably 5 to 2 Lt; RTI ID = 0.0 > 100-300 < / RTI >

If stirring is carried out at a temperature lower than 15 ° C, economical problems may arise in the precipitation of paclitaxel due to the low temperature. If stirring is carried out at a temperature exceeding 26 ° C, the precipitation efficiency of paclitaxel may decrease due to the high temperature have.

If stirring is carried out for less than 5 minutes, the precipitation of paclitaxel may not be carried out well and the paclitaxel precipitate yield may be decreased. If stirring is performed for more than 2 hours, the operation efficiency is lowered There may be a problem.

When stirring at a speed of less than 100 rpm, the paclitaxel precipitation time may be increased due to a slow paclitaxel precipitation speed, which is an advantage obtained by stirring, and when agitation is performed at a speed exceeding 300 rpm, excessive mixing There is a possibility that the operation efficiency is lowered.

In addition, the paclitaxel pretreatment process of the present invention may be characterized in that it does not include a hexane precipitation step between steps d) and e).

The purity and yield of paclitaxel after the pretreatment of paclitaxel comprising steps a), b) and c) are 5-13% and 90-100%, preferably 10-13% and 95-100% .

Specifically, since the pretreatment method using water does not dissolve paclitaxel in water, unlike the other steps of the pretreatment process, even if only the water is treated for about 10 minutes without using an organic solvent, even if the polar impurities are effectively removed Paclitaxel of high purity (~ 12.1%) can be obtained in a high yield (~ 99.4%) as shown in Table 1 and Table 1.

The paclitaxel purity when the c ') liquid-liquid extraction step is omitted and the step up to step e) is performed may be 50 to 55%, and the paclitaxel yield may be 80 to 95%.

Specifically, even when the conventional liquid-liquid extraction step and the hexane precipitation step are omitted and paclitaxel is pretreated, paclitaxel having a purity of 50.0% can be obtained at a high yield (~ 87.9%) in a short time (10 minutes) ). &Lt; / RTI > From these results, it has been confirmed that the liquid-liquid extraction and hexane precipitation processes can be omitted in the conventional pretreatment process, and as a result, the paclitaxel process can be simplified and the paclitaxel- Pretreatment process is very suitable.

The paclitaxel purity when the c) liquid-liquid extraction step is carried out between step b) and step c) or step c) and step d) to step e) is 50 to 60% The yield may be 80-95%.

Specifically, as shown in FIG. 11, the steps a) -b) -c ') - c) -d) and the steps a) -b) -c) -c') - d) d), paclitaxel with high purity of 38% and 36.8%, respectively, can be obtained irrespective of the order of step c ').

Further, even when the pretreatment including the liquid-liquid extraction step is omitted, since the conventional hexane precipitation step is omitted, the process is still shortened compared to the conventional paclitaxel pretreatment process, and as shown in FIG. 10 and Table 1, After performing the precipitation, paclitaxel having a purity of 56.5% can be obtained in a short time (10 minutes) at a high yield (~ 86.2%).

The present invention will now be described more specifically with reference to the following examples. However, the following examples should not be construed as limiting the scope of the present invention, but should be construed to facilitate understanding of the present invention.

Biomass Ready

The plant cell culture used in the present invention was cultured using a cell line obtained from leaves of Taxus chinensis , a taxa species. Suspension cells originating from Taxus cinnensis were cultured by stirring at 150 rpm at 24 캜 in dark condition. Suspension cells were cultured in a modified Gamborg's B5 medium, 30 g / l sucrose, 10 μM naphthaleneacetic acid, 0.2 μM 6-benzylaminopurine, 1 g / l casein hydrolyzate, 1 g / (N-morpholino) ethanesulfonic acid). Cell culture was changed to fresh medium every 2 weeks. To prolong production and culture, 1-2% maltose was added on days 7 and 21, and 4 μM AgNO ₃ was added as an inducing factor at the beginning of culture . After plant cell culture, plant cells and cell debris were recovered from the culture using a decanter (Westfalia, CA150 Claritying Decanter) and a high-speed centrifuge (α-Laval, BTPX205GD-35CDEEP). The recovered plant cells and cell debris were combined and used as biomass.

1) Sample preparation for pretreatment

In Example 1, the ratio of the biomass recovered from the plant cell culture liquid to the methanol was 1/1 (w / v), and the extraction was performed at room temperature for 30 minutes. Then, fresh methanol was added to the biomass The same procedure was repeated four times. The extract was concentrated under reduced pressure on a rotary evaporator (CCA-1100, EYELA, Japan) and completely dried at 35 ° C for 24 hours using a vacuum oven (UP-2000; EYELA) The crude extract had a purity of 2.54%.

2) Water pretreatment method

Water was added to the dried crude extract (purity: 2.54%) obtained from the biomass extraction to perform pre-treatment. Pretreatment was carried out at room temperature and the treatment time was 10 minutes. The ratio of crude extract / water was varied to 1: 5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60 (w / v) . The precipitate obtained after pretreatment of water was filtered using a filter paper (Whatman Grade 5, 2.5 mm, particle capture, 150 mm diameter) and dried to be used for liquid-liquid extraction or adsorbent treatment process.

3) liquid-liquid extraction

The crude extract (purity: 12.1%) obtained by the water pretreatment was dissolved in methanol (crude extract / methanol ratio, 1:80 (w / v)) and methylene chloride was added (25% of methanol) Stirred for 30 minutes then stagnated to induce phase separation. After removing the upper methanol layer containing the polar impurities, the lower methylene chloride layer was recovered and the liquid-liquid extraction was repeated three times. Each recovered methylene chloride layer was concentrated / dried under reduced pressure using a concentrator (CCA-1100, EYELA, Japan).

4) Adsorbent treatment

The dried crude extract (purity: 12.1%) obtained by water pretreatment and the dried crude extract (purity: 28.6%) obtained by liquid-liquid extraction were dissolved in methylene chloride at a ratio of 20% (v / w) Experiments were performed by adding sylopute (Fuji Silysia Chemical Ltd., Japan). The adsorbent treatment temperature was 40 ° C, the adsorbent treatment time was 30 minutes, and the ratio of crude extract / silo paste was 1: 1 (w / w). After the adsorbent treatment, the filtrate was filtered using filter paper (Whatman Grade 4, 20-25 mm, particle capture, 150 mm diameter) and the filtrate was dried under reduced pressure at 30 ° C.

5) Improved fractional precipitation

Two kinds of dried crude extracts (purity: 30.0% and 36.8%) obtained through adsorbent treatment were dissolved in methanol so that the pure paclitaxel content in methanol was 0.5% (w / v), and the methanol / water ratio (v / v) was 20:80 under stirring (180 rpm). After addition of distilled water, the solution was stirred for 10 minutes. After precipitation, the precipitate was filtered and dried in a vacuum oven (UP-2000, EYELA, Japan) at 35 ° C for 24 hours.

6) Paclitaxel analysis

The HPLC system (SCL-10AVP; Shimadzu, Japan) and Capell Pak C ₁₈ (250 × 4.6 mm, Shiseido, Japan) columns were used for paclitaxel content analysis. The mobile phase was flowed with a mixed solution of acetonitrile and distilled water (65/35 to 35/65, v / v, gradient mode) at a flow rate of 1.0 ml / min. Sample injection volume was 20 μl and detected by UV at 227 nm. A standard quantitative curve was used for HPLC analysis, and a standard sample was a Sigma-Aldrich product (purity: 98.7%).

Water pretreatment effect

Water was added to the crude extract (purity: 2.54%) in the same manner as in Example 2-2) to examine the pretreatment effect of water. The crude extract / water ratio was changed to 1: 5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60 (w / v) And yield were investigated.

2, the purity of paclitaxel was higher when the crude extract / water ratio was 1: 5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60 (w / v) , 7.2, 9.2, 10.2, 10.5, 12.1, 11.1, and 11.8%, respectively. The paclitaxel purity increased until the crude extract / water ratio reached 1:40 (w / v) and thereafter remained almost unchanged. That is, as the volume of water increased, paclitaxel purity increased gradually and then tended to be almost unchanged after 1:40 (w / v).

As shown in FIG. 3, the yield of paclitaxel was 1: 5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60 (w / v) 99.99, 99.6, 99.6, 99.6, 99.4, 99.3, 99.1%, respectively, and most of the paclitaxel was recovered (> 99%) in all ranges.

Therefore, the optimum water ratio for water pretreatment was 1:40 (w / v). After the optimized water pretreatment, the filtrate was recovered and the chromatogram was confirmed. As shown in FIG. 4, it was found that there was a large amount of polar impurities and almost no paclitaxel.

Since the water pretreatment method first introduced in the present invention utilizes the property that paclitaxel, which is an anticancer agent, does not dissolve in water, unlike other methods of the pretreatment process, the pretreatment with water alone for 10 minutes, Paclitaxel of high purity (~ 12.1%) was obtained in high yield (~ 99.4%) by simple, convenient and effective removal of polar impurities.

Effect of water pretreatment on subsequent processes when liquid-liquid extraction is included

As shown in the results of Example 3 above, the purity of paclitaxel could be improved at a short time (10 minutes) at room temperature by using a simple water pretreatment. To further investigate the effect of the water pre-treatment on the subsequent process, the subsequent process liquid-liquid extraction, adsorbent treatment and improved fractionation precipitation were performed. In this case, the efficiency of the process was examined by dividing the liquid-liquid extraction (FIG. 5A) and the liquid-liquid extraction (FIG. 5B) before the adsorbent treatment.

First, in order to investigate the influence of the water pretreatment on the subsequent process when liquid-liquid extraction was included (Fig. 5 (A)), biomass extraction (Fig. 6, purity: 2.5%) and drying (Fig. 7, purity: 12.1%) were sequentially carried out in the order of liquid-liquid extraction, adsorbent treatment and improved fractional precipitation. The crude extract (purity: 12.1%) of Example 3 was dissolved in methanol (crude extract / methanol ratio, 1:80 (w / v)) and methylene chloride was added (25% As a result of the liquid extraction, paclitaxel having a purity of 28.6% was obtained as shown in FIG. 8, and polar impurities were removed in particular.

The crude extract (purity: 28.6%) obtained by liquid-liquid extraction was dissolved in methylene chloride, and adsorbent treatment was carried out at 40 ° C for 30 minutes by adding a commercial adsorbent sylvestool (extract / silofut ratio, 1/1, w / w) As a result, paclitaxel having a purity of 36.8% was obtained as shown in FIG. When water pretreatment was introduced, paclitaxel with very high purity (~ 36.8%) could be obtained by removing tar and wax components as well as a large amount of polar impurities in the subsequent liquid - liquid extraction and adsorbent treatment steps.

As shown in FIG. 10, paclitaxel having a purity of 56.5% was obtained in a short time (10 minutes) as a result of performing an improved fractional precipitation using a sample (purity: 36.8%) obtained through adsorbent treatment. It has been reported in the technical field of the present invention that it is possible to reduce the cost in the final HPLC process by increasing the purity (> 50%) of the possible samples in the pretreatment process. Therefore, it was possible to obtain samples with high purity (purity: 56.5%) which can be used for the final HPLC process even after the subsequent liquid - liquid extraction, adsorbent treatment and improved fractionation precipitation using a simple water pretreatment.

In addition, pretreatment was carried out in the order of extraction of biomass, liquid-liquid extraction, water pretreatment and adsorbent treatment, and the effect of pretreatment of liquid-liquid extraction was examined. The results are shown in FIG.

As shown in FIG. 11, the paclitaxel purity after performing the adsorbent treatment step including liquid-liquid extraction between the water pretreatment step and the adsorbent treatment step was 36.8% (left graph in FIG. 11) The paclitaxel purity after performing the adsorbent treatment step including the liquid-liquid extraction between the water pretreatment steps was 38% (the right graph in Fig. 11). Therefore, paclitaxel with high purity can still be obtained by performing the steps of biomass extraction, liquid-liquid extraction, water pretreatment, and adsorbent treatment steps. Therefore, even after performing the fractional precipitation, It is expected that a sample of high purity can be obtained.

As a result, regardless of the order of liquid-liquid extraction, paclitaxel with high purity (~ 38%) was obtained at the adsorbent treatment stage by the introduction of water pretreatment and dramatically simplified the pre- .

Effect of water pretreatment on subsequent processes when liquid-liquid extraction is omitted

(Fig. 6, purity: 2.5%) and a pre-treated water obtained by the pretreatment of water to investigate the effect of water pretreatment on the subsequent process in the absence of liquid-liquid extraction (Fig. 5 The extract (FIG. 7, purity: 12.1%) was sequentially carried out in the order of adsorbent treatment and improved fractional precipitation.

The crude extract after the water pretreatment in Example 3 was dissolved in methylene chloride, adsorbent treatment was carried out at 40 占 폚 for 30 minutes by adding a commercial adsorbent siloxuter (extract / sulphurous ratio, 1/1, w / w) Paclitaxel with a purity of 30.0% was obtained as shown in Fig. Even if there is no liquid-liquid extraction process that mainly removes polar impurities, if the adsorbent treatment is performed after the pretreatment of water, the purities are further improved by removing not only the dark color, tar and wax components but also a large amount of polar impurities from the sample (12.1% %).

Therefore, when the adsorbent treatment is performed after the pretreatment of water, paclitaxel having a very high purity (~ 30.0%) can be obtained in the conventional process as compared with a purity of 10% or less after adsorbent treatment.

As shown in FIG. 13, paclitaxel with a purity of 50.0% was obtained in a short time (10 minutes) as a result of performing an improved fractional precipitation using a sample (purity: 30.0%) obtained by treating with an adsorbent. These results demonstrate that paclitaxel with high purity (> 50%) required for pretreatment by adsorbent treatment and improved fractional precipitation (no need for liquid-liquid extraction and hexane precipitation in conventional separation / purification processes) .

From these results, it is confirmed that the liquid-liquid extraction process can be eliminated in the pretreatment process, which makes it very suitable for the mass production of paclitaxel by reducing the amount of solvent by simplifying (simplifying) the process and shortening the processing time of the subsequent process .

Evaluation of efficiency of water pretreatment

In order to investigate the effect of the water pretreatment on the subsequent process in more detail, the pre-purification process of the paclitaxel reported in the prior art document [1-8] and the pre- purification process are shown in Table 1 below. All of the biomass used in the three processes in Table 1 are the same at 1 kg.

In the prior art processes of Table 1 above, the numbers mentioned under biomass extraction, liquid-liquid extraction, adsorbent treatment, hexane precipitation and fractional precipitation each refer to different prior art documents, The prior art documents are as follows:

[1] Kim JH, Kang IS, Choi HK, Hong SS, Lee HS. A novel prepurification for paclitaxel from plant cell cultures, Process Biochem 2002; 37: 679-82;

[2] Pyo SH, Park HB, Song BK, Han BH, Kim JH. A large-scale purification of paclitaxel from cell cultures of Taxus chinensis , Process Biochem 2004; 39: 1985-91;

[3] Gamborg OL, Miller RA, Ojima K. Nutrient requirements of suspension cultures of soybean root cells, Exp Cell Res 1968; 50: 151-8;

[4] Kim JH. Optimization of liquid-liquid extraction conditions for paclitaxel separation from plant cell cultures, KSBBJ 2009; 24: 212-215;

[5] Choi HK, Son SJ, Na GH, Hong SS, Park YS, Song JY. Mass production of paclitaxel by plant cell culture, Korean J Plant Biotechnol 2002; 29: 59-62;

[6] Lee CG, Kim JH. Improved fractional precipitation method for purification of paclitaxel, Process Biochem 2014; 49: 1370-76;

[7] Jeon KY, Kim JH. Improvement of fractional precipitation process for pre-purification of paclitaxel, Process Biochem 2009; 44: 736-41; And

[8] Pyo SH, Choi HJ. An improved high-performance liquid chromatography process for the large-scale production of paclitaxel, Sep Purif Technol 2011; 76: 378-84.

As shown in FIG. 1, the pretreatment process of the prior art documents was performed in order of liquid-liquid extraction, adsorbent treatment, hexane precipitation and fractional precipitation without extracting water after extraction of biomass. The overall yield of the sample ) And operating hours were 46.5 ~ 61.0%, 69.3 ~ 75.4% and 18.2 ~ 76.2 hours, respectively.

On the other hand, when the liquid-liquid extraction is included as in Example 4 (FIG. 5A), the liquid-liquid extraction, the adsorbent treatment and the improved fractionation precipitation were performed after the biomass extraction and the water pretreatment, The final order, overall yield and operating time were 56.5%, 86.2% and 4.4 hours, respectively.

5 (B)), only the biomass extraction and pretreatment of water, followed by the treatment with the adsorbent and the improved fractional precipitation were performed. As a result, Yield and operating time were 50.0%, 87.9% and 2.9 hours, respectively.

From these results, we could obtain a high purity crude extract from the biomass extract in a high yield from the biomass extract in a high yield. In comparison with the conventional pretreatment for paclitaxel separation / purification, the purity, yield and operating time It was found to be effective.

Particularly in the process of treating the adsorbent, not only the dark color, tar and wax components but also a large amount of polar impurities are removed, so that paclitaxel having high purity can be obtained compared with the conventional process of treating the adsorbent without water pretreatment. From the above results, it can be concluded that pretreatment by introducing water pretreatment can dramatically shorten the existing pretreatment process and can also provide a high purity (> 50%) sample suitable for final HPLC purification in a short operation time and high yield .

Claims

A pretreatment method of paclitaxel extract comprising the steps of a) to e)
a) obtaining a biomass from a cell culture of a Taxus genus plant comprising paclitaxel;
b) organic solvent extraction of the biomass to obtain a crude extract of paclitaxel;
c) adding water to the crude extract so that the ratio of crude extract to water is 1: 5 ~ 1: 60 (w / v);
d) treating the adsorbent; And
e) Performing fractional precipitation.

The method according to claim 1, wherein the step c) is carried out at 15 to 35 DEG C for 5 to 60 minutes Wherein the paclitaxel extract is administered to a patient in need thereof.

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The method according to claim 1, wherein the ratio of the crude extract to water is 1:35 to 1:45 (w / v).

The method according to claim 1, further comprising the step of c) liquid-liquid extraction between step b) and step c) or step c) and step d).

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6. The method according to claim 1 or 5, wherein the fractional precipitation of step e) comprises the following steps e-1) to e-3)
e-1) dissolving the paclitaxel crude extract obtained in step d) in a water-soluble organic solvent;
e-2) water is added with stirring until the volume ratio of the water-soluble organic solvent and water is 50:50 to 20:80 to perform fractional precipitation; And
e-3) stirring after fractional precipitation to obtain paclitaxel.

6. The method according to any one of claims 1 to 5, further comprising, between step d) and step e), no step of hexane precipitation.

The method according to claim 1, wherein the paclitaxel purity after the step c) is 5 to 13% and the paclitaxel yield is 90 to 100%.

The method according to claim 1, wherein the paclitaxel purity is 50 to 55% and the paclitaxel yield is 80 to 95% after step e).

[Claim 6] The method according to claim 5, wherein the paclitaxel purity is 50 to 60% and the paclitaxel yield is 80 to 95% after step e).