KR102558908B1 - Fractional precipitation method performed by introducing negative pressure cavitation in purifying paclitaxel - Google Patents

Abstract

The present invention relates to a fractional precipitation method performed by introducing negative pressure cavitation in the purification of paclitaxel, and more particularly, to a fractional precipitation method capable of obtaining paclitaxel in high yield in a short time through a simplified and shorter process than the conventional fractional precipitation by introducing negative pressure cavitation under specific conditions to a sample containing raw paclitaxel, and a method for purifying paclitaxel using the same.

Description

Fractional precipitation method performed by introducing negative pressure cavitation in purifying paclitaxel}

The present invention relates to a fractional precipitation method performed by introducing negative pressure cavitation in the purification of paclitaxel, and more particularly, to a fractional precipitation method capable of obtaining paclitaxel in high yield in a short time through a simplified and shorter process than the conventional fractional precipitation by introducing negative pressure cavitation under specific conditions to a sample containing raw paclitaxel, and a method for purifying paclitaxel using the same.

Paclitaxel (C ₄₇ H ₅₁ NO ₁₄ ) is a diterpenoid alkaloid found in the epidermis of the yew tree. This molecule induces apoptosis in tumor cells by inhibiting depolymerization by enhancing microtubule stability. Paclitaxel, which has a unique pharmacological effect, is widely used in the treatment of ovarian cancer, breast cancer, head and neck cancer, kaposi's sarcoma, and non-small cell lung cancer, and the demand for paclitaxel is expected to continue to increase as indications for paclitaxel expand and prescription methods are developed. The main production methods of paclitaxel include direct extraction from yew, semi-synthesis using precursors, and plant cell culture using callus derived from plants. Among them, plant cell culture has the advantage of stably producing paclitaxel of constant quality in a bioreactor without being affected by external factors (climate, environment).

In plant cell culture, paclitaxel is mostly accumulated in biomass (plant cells), and paclitaxel is produced through several steps of extraction, pretreatment, and final purification. In particular, the pretreatment process has a great influence on the final purification cost. 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.

Fractional precipitation is a simple and efficient pretreatment method using the difference in solubility of paclitaxel. In 2000, the fractional precipitation method of plant cell-derived paclitaxel was first reported [Kim et al., "Fractional precipitation for paclitaxel pre-purification from plant cell cultures of Taxus chinensis ," Biotechnol. Lett. 22 1753-1756 (2000)], and then optimizing the fractionation conditions (solvent, temperature, sample characteristics) to obtain high purity paclitaxel in high yield [Kim et al., "A novel prepurification for paclitaxel from plant cell cultures," Process Biochem. 37 679-682 (2002); "Optimal temperature control in fractional precipitation for paclitaxel pre-purification," Process Biochem. 41 276-280 (2006)]. However, since fractional precipitation is operated at low temperatures for a long time, there is a limit to its application to the mass production process of paclitaxel in terms of economic feasibility. In order to improve these problems, studies to improve precipitation efficiency by increasing the surface area per working volume (S / V) of the precipitator using glass beads or ion exchange resins have been attempted [Kim et al., "Fractional precipitation for paclitaxel pre-purification from plant cell cultures of Taxus chinensis ," Biotechnol. Lett. 22 1753-1756 (2000); Kim et al., "A novel prepurification for paclitaxel from plant cell cultures," Process Biochem. 37 679-682 (2002); Kim et al., "Optimal temperature control in fractional precipitation for paclitaxel pre-purification," Process Biochem. 41 276-280 (2006)]. Furthermore, studies were also conducted to improve precipitation efficiency by adjusting sample purity and content during precipitation [Kim et al., "Evaluation of the effect of crude extract purity and pure paclitaxel content on the increased surface area fractional precipitation process for the purification of paclitaxel," Process Biochem. 47 2388-2397 (2012)]. Although the long precipitation time has been reduced to some extent through these studies, it is still insufficient for application to the mass production process of paclitaxel. In 2014, the precipitation time was shortened by adjusting the polarity of the precipitation solution (methanol-distilled water mixture), but the purity of paclitaxel rather decreased due to the excessive addition of distilled water [Kim et al., "Improved fractional precipitation method for purification of paclitaxel," Process Biochem. 49 1370-1376 (2014)]. Recently, the existing long precipitation time has been dramatically improved by fractional precipitation using ultrasound [Kim et al., "Ultrasound-assisted fractional precipitation of paclitaxel from Taxus chinensis cell cultures," Process Biochem., 87 , 238-243 (2019)]. That is, by introducing ultrasound into the precipitation process, the precipitation time could be shortened by increasing the nucleation rate. This result is attributed to ultrasonic cavitation, and it is explained that cavitation bubbles eventually become unstable and collapse during the process of compression and expansion, resulting in microjets of the precipitated solution, intense local heating, and high-pressure shock waves [Schueller BS et al., "Ultrasound Enhanced Adsorption and Desorption of Phenol on Activated Carbon and Polymeric Resin," Ind. Eng. Chem. Res. 40 4912-4918 (2001)]. In the case of ultrasonic fractional precipitation, operation time can be reduced, but additional equipment and energy costs may occur and the process may be complicated. Therefore, there is still a need for a new fractional precipitation technology that ensures not only process efficiency but also process convenience and feasibility. Therefore, the present invention conceived an idea from the cavitation-based fractional precipitation technique to develop a new green negative pressure cavitation fractional precipitation process for efficient recovery of paclitaxel from biomass.

The present invention has been devised to solve the above-described problems, and an object of the present invention is to provide a fractional precipitation method and a method for purifying paclitaxel using the same, which not only dramatically shortens the fractional precipitation time through the introduction of negative pressure cavitation when purifying paclitaxel, but also improves the dissolution rate, uniformity of drug dispersion, and oral bioavailability of the drug when formulated by reducing the average particle size of paclitaxel.

In order to solve the above problems, the present invention provides a fractional precipitation method performed by introducing negative pressure cavitation during paclitaxel purification.

According to a preferred embodiment of the present invention, the fractional precipitation method may be to mix a sample containing paclitaxel with a water-soluble organic solvent, add water, and then introduce negative pressure cavitation.

According to another preferred embodiment of the present invention, the water-soluble organic solvent may include one selected from the group consisting of C ₁ to C ₄ alcohol and methylene chloride.

According to another preferred embodiment of the present invention, the water may be added until the volume ratio of the water-soluble organic solvent and water is 50:50 to 20:80.

According to another preferred embodiment of the present invention, the introduction of negative pressure cavitation may generate cavitation bubbles by applying a negative pressure of -260 mmHg to -50 mmHg.

According to another preferred embodiment of the present invention, the negative pressure cavitation introduction may be performed for 1 minute to 10 minutes.

According to another preferred embodiment of the present invention, the fractional precipitation method may be performed at room temperature.

According to another preferred embodiment of the present invention, the average particle size of paclitaxel after fractional precipitation by introducing the negative pressure cavitation can be reduced to 10 μm to 20 μm.

In another aspect, the present invention provides a method for purifying paclitaxel comprising the following steps a) to e):

a) obtaining biomass from a cell culture medium of a Taxus genus plant;

b) performing organic solvent extraction;

c) performing liquid-liquid extraction;

d) treating the adsorbent; and

e) performing the fractional precipitation method described above.

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.

The fractional precipitation method using negative pressure cavitation and the method for purifying paclitaxel including the method of the present invention dramatically shorten the precipitation time compared to the conventional fractional precipitation method without introducing negative pressure cavitation, and significantly increase the yield of paclitaxel compared to the same operation time. In addition, it does not require low-temperature storage for fractional precipitation, and it is suitable for the process for mass production of paclitaxel because it can perform faster and more effective precipitation with less capital investment while efficiently utilizing energy. In addition, by reducing the average particle size of paclitaxel, there is an advantage in improving the dissolution rate, uniformity of drug dispersion, and oral bioavailability of the drug during formulation.

FIG. 1(A) is a schematic diagram showing a traditional fractional precipitation method without introducing negative pressure during purification of paclitaxel, and FIG. 1(B) is a schematic diagram showing a fractional precipitation method using negative pressure cavitation during purification of paclitaxel.
Figure 2 is a graph showing the effect of atmospheric pressure (0 mmHg) and negative pressure (-50 mmHg, -100 mmHg, -150 mmHg and -200 mmHg) on the yield of paclitaxel.

As described above, in the past, various attempts have been made to improve precipitation efficiency by optimizing conditions such as solvent, temperature, and sample characteristics during fractional precipitation, increasing the inner surface area of the precipitator, adjusting the polarity of the precipitation solution, or ultrasonic treatment.

Therefore, in the present invention, a solution to the above problems was sought by developing a new green negative pressure cavitation fractionation precipitation process for efficient recovery of paclitaxel from biomass and optimizing the main process parameters (negative pressure and operating time). The fractional precipitation method employing negative pressure cavitation according to the present invention can obtain high-purity paclitaxel in high yield in a short time through a shorter and simplified process than conventional fractional precipitation, and does not require separate low-temperature storage, so it is suitable for a process for mass production of paclitaxel.

Accordingly, a first aspect of the present invention relates to a fractional precipitation method performed by introducing negative pressure cavitation when purifying paclitaxel.

Conventionally, the method for extracting and purifying paclitaxel from biomass consists of solvent extraction, liquid-liquid extraction, adsorbent treatment, hexane precipitation, and fractional precipitation. After solvent extraction, liquid-liquid extraction, adsorbent treatment, hexane precipitation, and fractional precipitation using biomass, the purity of the sample is improved to 0.5-0.7%, 6-9%, 9-10%, 21-27%, and 46-61%, respectively. A large amount of impurities were removed through this pretreatment process, and the purity of crude paclitaxel was greatly increased, making it suitable for the final HPLC process. However, since the fractional precipitation takes a lot of time (~ 3 days) for precipitation and the precipitation is performed at low temperature (0-4 ° C), there is a limit to its application to the mass production process of paclitaxel in terms of economic feasibility.

The present inventors have confirmed that the precipitation time can be drastically reduced when negative pressure cavitation is introduced into the fractional precipitation method in the purification of paclitaxel.

Accordingly, the fractional precipitation method of the present invention is performed by mixing a raw sample containing paclitaxel with a water-soluble organic solvent, adding water, and then introducing negative pressure cavitation.

In the fractional precipitation method of the present invention, the dissolution of the sample in the water-soluble organic solvent is not particularly limited as long as the sample is dissolved in the water-soluble organic solvent in an amount capable of uniformly dispersing the sample, but preferably, the pure paclitaxel may be dissolved in the water-soluble organic solvent so that the content is 0.1% to 1% (w / v).

The water-soluble organic solvent is not particularly limited as long as it is an organic solvent commonly used to extract active ingredients from plants, but preferably includes at least one of the group consisting of C ₁ to C ₄ alcohol and methylene chloride, and more preferably methanol.

Next, after dissolving the sample in a water-soluble organic solvent, fractional precipitation is performed by adding water until the volume ratio of the water-soluble organic solvent and water is 50:50 to 20:80 (v/v). At this time, distilled water may be used as the water, and it is preferable to add it dropwise while stirring.

The stirring speed may be 100 to 400 rpm. If stirring at a speed of less than 100 rpm, mass transfer in the precipitate solution is lowered, and the paclitaxel precipitation speed, which is an advantage obtained by stirring, may increase the paclitaxel precipitation time. In the case of stirring at a speed of more than 400 rpm, excessive mixing may cause a problem of lowering energy efficiency as well as operational efficiency.

The amount of water added may be mixed until the volume ratio of the water-soluble organic solvent and water is 50:50 to 20:80, and more preferably, the volume ratio of the water-soluble organic solvent and water is 47:53 to 35:65.

When the water addition is complete, negative pressure cavitation is introduced.

In the present invention, the term "negative pressure" refers to a pressure lower than atmospheric pressure (760 mmHg). Therefore, in the fractional precipitation method of the present invention, the negative pressure refers to a pressure lower than the atmospheric pressure of 760 mmHg, and includes, for example, a pressure of 500 mmHg to 710 mmHg, but is not limited thereto.

In the fractional precipitation method of the present invention, the introduction of negative pressure cavitation may be performed by generating cavitation bubbles by applying a negative pressure in the above-mentioned range.

In the present invention, the term "negative pressure cavitation" is cavitation caused by negative pressure, which is introduced into a liquid-solid system to increase turbulence, collision, and mass transfer between the extraction solvent and the matrix.

In a specific embodiment of the present invention, in order to evaluate the effect of negative pressure on fractional precipitation during purification of paclitaxel, negative pressure conditions were varied to 0 mmHg, -50 mmHg, -100 mmHg, -150 mmHg, and -200 mmHg, and the recovery rate of paclitaxel according to operating time was evaluated under each condition. At this time, the negative pressure conditions of 0 mmHg, -50 mmHg, -100 mmHg, -150 mmHg and -200 mmHg mean that the fractional precipitation was performed under atmospheric pressure conditions of 760 mmHg, 710 mmHg, 660 mmHg, 610 mmHg and 560 mmHg, respectively. As confirmed in FIG. 2, in the control group where traditional discernment precipitation was performed without the introduction of negative pressure, it showed a low paclitaxel yield of 69.5% despite the 20 -minute operation time.However, a short operation time of -50 mmHg to -200 mmHg is applied to discern, 74.4% to 98.6% even in short operation time of 1 minute to 10 minutes. Very high paclitaxel yield was shown. In particular, when fractional precipitation was performed by introducing a negative pressure of -200 mmHg, it was possible to recover most of the paclitaxel (> 97%) in a very short operation time of 1 minute, and thus the fractionation yield and fractionation operation time were significantly improved compared to the control group.

Therefore, in the fractional precipitation method of the present invention, the introduction of negative pressure cavitation may be performed by generating cavitation bubbles by applying a pressure condition of 500 mmHg to 710 mmHg, that is, a negative pressure of -260 mmHg to -50 mmHg. Preferably, the negative pressure cavitation introduction may be performed by generating cavitation bubbles by applying a pressure condition of 500 mmHg to 680 mmHg, that is, a negative pressure of -260 mmHg to -80 mmHg. If the fractional precipitation is performed at a pressure of less than 500 mmHg, the operation may become difficult due to evaporation of the precipitation solution, and when the fractional precipitation is performed at a pressure exceeding 710 mmHg, the effect of increasing the recovery rate of paclitaxel may be insignificant compared to when the fractional precipitation is performed at atmospheric pressure. Therefore, in order to maximize the efficiency of fractional precipitation when purifying paclitaxel, it is preferable to perform fractional precipitation at a negative pressure within the above range.

At this time, the introduction of the negative pressure cavitation may be performed for 1 minute to 10 minutes. When a negative pressure of -100 mmHg or more is introduced, that is, when fractional precipitation is performed at a pressure of 660 mmHg or less, paclitaxel can be obtained with a high recovery rate of >97% simply by introducing negative pressure cavitation for 5 minutes or less.

However, when the fractional precipitation using negative pressure cavitation is performed for less than 1 minute, paclitaxel may not be sufficiently recovered, and when it is performed for more than 10 minutes, the effect of increasing the recovery rate of paclitaxel over time is insignificant, resulting in an unnecessary increase in operating time.

In another specific embodiment of the present invention, the operating time of the fractional sedimentation method introducing negative pressure cavitation according to the present invention and the conventional fractional sedimentation method without introducing negative pressure cavitation were compared. As confirmed in Table 1, the fractional precipitation method using negative pressure cavitation according to the present invention can obtain paclitaxel at a high recovery rate even though the operation time is significantly reduced compared to various conventional fractional precipitation methods, and thus can be effectively applied to the mass production process of paclitaxel.

The fractional precipitation method of the present invention can be performed by applying negative pressure into the solvent using only a commercially available vacuum controller and vacuum pump without a specially designed negative pressure cavitation device.

The fractional precipitation method using negative pressure cavitation of the present invention minimizes and/or simplifies the device, thereby dramatically improving the complexity of the process, the risk of the device, and the high cost due to high energy consumption, which were limitations of the fractional precipitation using ultrasonic waves.

The fractional precipitation method of the present invention may be performed at room temperature. In the case of paclitaxel purification, the conventional fractional precipitation method is performed at a low temperature of 0 ° C to 4 ° C, so there is a limit to its application to the mass production process of paclitaxel in terms of economic feasibility.

However, since the fractional precipitation method performed in the purification of paclitaxel of the present invention is performed by introducing negative pressure cavitation to a sample containing raw paclitaxel at room temperature, it does not require low-temperature storage for fractional precipitation, so it is suitable for application to a mass production process.

In the present invention, the term "room temperature" includes a temperature range of 0 °C to 30 °C, preferably 5 °C to 30 °C, and more preferably 10 °C to 30 °C, but is not limited thereto.

In the paclitaxel purification method of the present invention, the average particle size of paclitaxel after fractional precipitation by introducing negative pressure cavitation can be reduced to 10 μm to 20 μm.

As confirmed in Table 2, the average particle size was measured at 52.529 μm when conventional fractionation precipitation without introduction of negative pressure was performed, whereas the average particle size was 15.868 μm at 1 to 10 minutes of precipitation when negative pressure was introduced. The average particle size was reduced by 3.3 times when negative pressure was introduced. Through this, by reducing the average particle size of paclitaxel, it is possible to improve the drug dissolution rate, uniformity of drug dispersion, and oral bioavailability during formulation.

A second aspect of the present invention relates to a method for purifying paclitaxel using the aforementioned fractional precipitation method.

The paclitaxel purification method of the present invention includes the steps of a) obtaining biomass from a cell culture medium of a taxus genus plant before performing the fractional precipitation method; b) performing organic solvent extraction; c) performing liquid-liquid extraction; and d) treating the adsorbent.

In the method for purifying paclitaxel of the present invention, the biomass obtained from the cell culture solution of a taxus genus plant 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 solution.

In addition, the Taxus brevifolia, Taxus canadensis, Taxus cuspidata, Taxus baccata , Taxus globosa , Taxus floridana , Taxus wallichiana , Taxus media ( Taxus media ) or Taxus chinensis ( Taxus chinensis ), etc. may be included, but is not limited thereto.

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.

In the method for purifying paclitaxel of the present invention, the organic solvent extraction in step b) is not particularly limited as long as an organic solvent commonly used for extracting an active ingredient from a plant is used, but preferably contains a C ₁ to C ₄ alcohol.

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 may be preferably used as an organic solvent capable of recovering as much paclitaxel from biomass as possible.

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

In addition, the solvent extraction may be repeated once to several times, preferably twice or more, more preferably three to five times, and then the obtained extract is concentrated under reduced pressure and then dried. It can be used in the liquid-liquid extraction step.

In the paclitaxel purification method of the present invention, the liquid-liquid extraction in step c) is not particularly limited as long as it is a method for removing polar impurities contained in paclitaxel by using a phase separation of a non-polar organic solvent and a polar organic solvent.

For example, the non-polar organic solvent may include one or more compounds 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.

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

Furthermore, if the polar organic solvent is a C ₁ to C ₄ alcohol, it is preferable to use methylene chloride as the non-polar organic solvent, and methylene chloride is preferably used in an amount of 20 to 30% (v/v) of the alcohol concentrate, and excessive use of methylene chloride causes a lot of burden in subsequent processes.

In the paclitaxel purification method of the present invention, it is preferable to use a silica-based adsorbent to remove impurities such as tar in the adsorbent treatment in step d). In the case of using the silica-based adsorbent, impurities such as biomass-derived wax and tar can be effectively removed, and it is effective in increasing the purity of paclitaxel and the efficiency of the purification process.

In addition, the adsorbent in step d) may be treated so that the ratio of the raw paclitaxel extract obtained in step c) to the adsorbent is 1:0.5 to 1:2.5 (w/w). The adsorbent may be treated at 20 °C to 60 °C for 30 minutes, then filtered with a filter paper and dried under reduced pressure.

In the paclitaxel purification method of the present invention, after treating the adsorbent, the hexane precipitation step may be omitted and the fractional precipitation step may be performed. Conventionally, a method for extracting and purifying paclitaxel from biomass is composed of solvent extraction, liquid-liquid extraction, adsorbent treatment, hexane precipitation, and fractional precipitation. However, in the case of performing fractional precipitation by introducing negative pressure cavitation during paclitaxel purification, paclitaxel having a purity similar to that of the conventional purification method can be obtained in a high yield in a short time even if the hexane precipitation step is omitted.

Therefore, the paclitaxel purification method of the present invention omits the hexane precipitation step from the conventional solvent extraction, liquid-liquid extraction, adsorbent treatment, and hexane precipitation steps, thereby simplifying the complexity of the process and can be very usefully used for mass production of paclitaxel.

Hereinafter, the present invention will be described in more detail through examples. However, the present invention can have various changes and various forms, and the specific embodiments and descriptions described below are only intended to aid understanding of the present invention, and are not intended to limit the present invention to specific disclosed forms. 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

Suspension cells derived from Taxus chinensis were cultured by stirring at 150 rpm in a modified Gamborg's B5 medium at 24 ° C. under dark condition [Kim et al., "Ultrasound-assisted fractional precipitation of paclitaxel from Taxus chinensis cell cultures," Process Biochem., 87 , 238-24 3 (2019); Kim et al., "Adsorption kinetics, mechanism, isotherm, and thermodynamic analysis of paclitaxel from extracts of Taxus chinensis cell cultures onto Sylopute," Biotechnol. Bioproc. Eng ., 24 , 513-521 (2019)]. After cell culture, plant cells (biomass) were recovered from the culture medium using a decanter (Westfalia, CA150 Claritying Decanter) and a high-speed centrifuge (α-Laval, BTPX205GD-35CDEEP).

Fractional precipitation using negative pressure cavitation and analysis of paclitaxel content

2-1. Sample preparation for differential acupuncture points

After extracting by mixing the biomass recovered in Example 1 with methanol at a ratio of 1:1 (w/v), liquid-liquid extraction was performed by adding methylene chloride (25% of the extract). Paclitaxel was recovered as a lower methylene chloride layer through phase separation, and concentrated and dried. The dried crude extract was treated with an adsorbent (Sylopute) and filtered. The filtrate was dried under reduced pressure at 30 °C and purified through a silica-gel 60N (Timely, Japan) column (eluent: 1.5% (v/v) methanol dissolved in dichloromethane). The sample (purity: 20.47%) obtained through this was used for fractional precipitation using negative pressure cavitation.

2-2. Negative pressure cavitation fractional precipitation

A schematic diagram of the traditional fractionation precipitation without negative pressure and the negative pressure cavitation fractionation precipitation process is shown in FIG. 1 . For fractional precipitation, crude paclitaxel (purity: 20.47%) obtained in Example 2-1 was dissolved in methanol (pure paclitaxel content dissolved in methanol: 0.5%, w/v), and distilled water was added dropwise under stirring (335 rpm) until the methanol:water ratio (v/v) reached 61.5:38.5 at room temperature. To investigate the effect of negative pressure, negative pressure was applied into the precipitation solution using a vacuum controller unit (EYELA NVC-3000, Japan) and a diaphragm vacuum pump (EYELA NVP-1000, Japan). The effect of varying the negative pressure (0 mmHg, -50 mmHg, -100 mmHg, -150 mmHg or -200 mmHg) on the precipitation efficiency of paclitaxel at a precipitation temperature of 5 °C was investigated. In addition, the operation time (1 minute, 5 minutes, 10 minutes or 20 minutes) was changed to confirm the precipitation time, paclitaxel yield and purity. After precipitation, the precipitate was filtered under reduced pressure with a filter paper (185 mm, ADVANTEC) and dried in a vacuum oven (UP-2000, EYELA, Japan) at 40 °C for 24 hours. The paclitaxel content of the dried precipitate was analyzed by HPLC.

2-3. Analysis of paclitaxel content

To analyze the content of paclitaxel in each sample obtained in Examples 2-1 and 2-2, a HPLC (high performance liquid chromatography) system (SCL-10AVP, Shimadzu, Japan) and a Capsule Pak C ₁₈ (Capcell Pak C ₁₈ ; 250 × 4.6 mm, Shiseido, Japan) column was used. The mobile phase was a mixed solution of distilled water and acetonitrile (65/35 to 35/65, 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. A standard paclitaxel sample (purity: 97%) was purchased from Sigma-Aldrich and used.

The results of fractional precipitation according to negative pressure changes (0 mmHg, -50 mmHg, -100 mmHg, -150 mmHg, -200 mmHg) are shown in FIG. 2 . As confirmed in FIG. 2 , the yield of paclitaxel was 74.4% to 98.6% at negative pressures of -50 mmHg to -200 mmHg. As the negative pressure increased, the yield of paclitaxel increased. On the other hand, in the traditional fractionated precipitation (control group), the yield of paclitaxel at 20 minutes of fractionated precipitation was 69.5%, which was very low compared to the fractionated precipitation using negative pressure. In addition, the time required for fractional precipitation at negative pressures of -50 mmHg to -200 mmHg was 1 to 10 minutes, respectively. In particular, when precipitating at negative pressure -200 mmHg, most paclitaxel could be recovered (>97%) in a short operation time (1 minute). Through this, it was confirmed that the fractional precipitation yield and the time required for fractional precipitation were significantly improved compared to the control group. The purity of paclitaxel in the precipitate according to the negative pressure was about 34 to 41%, and there was little difference in purity (data not shown). These results were found to be similar to the results of research on fractional precipitation using conventional polymer materials [Kim et al., "Decreasing particle size of paclitaxel using polymer in fractional precipitation process , " Korean Chem. Eng. Res. 54 278-283 (2016)].

2-4. Comparison of settling time between conventional fractionation and fractionation using negative pressure cavitation

Table 1 compares the results of previous studies on the fractional precipitation of paclitaxel with those of this study. Fractional precipitation using glass beads or ion exchange resin as surface area-increasing materials [1. Kim et al., "Improvement of fractional precipitation process for pre-purification of paclitaxel," Process Biochem. , 44 736-741 (2009); 2. Kim et al., "Effect of reactor type on the purification efficiency of paclitaxel in the increased surface area fractional precipitation process," Sep. Purif. Technol. 99 14-19 (2012)] and traditional fractional precipitation without using surface area increasing materials [3. Kim et al., "Fractional precipitation for paclitaxel pre-purification from plant cell cultures of Taxus chinensis ," Biotechnol. Lett. 22 1753-1756 (2000); 4. Kim et al., "A novel prepurification for paclitaxel from plant cell cultures," Process Biochem. 37 679-682 (2002); 5. Kim et al., "Optimal temperature control in fractional precipitation for paclitaxel pre-purification," Process Biochem. 41 276-280 (2006)], the settling times were -12 hours and -72 hours, respectively. Fractionated precipitation by increasing the ratio of distilled water in the precipitation solution (methanol-distilled water mixture) [6. Kim et al., "Improved fractional precipitation method for purification of paclitaxel," Process Biochem. 49 1370-1376 (2014)], the settling time was ~0.5 h. In addition, ultrasonic cavitation fractionated precipitation [7. In Kim et al., "Ultrasound-assisted fractional precipitation of paclitaxel from Taxus chinensis cell cultures," Process Biochem., 87 , 238-243 (2019)], the precipitation time was 0.08 to 0.50 hours. The settling time of fractional precipitation using negative pressure cavitation designed in the present invention was 0.02 to 0.16 hours. That is, by introducing a negative pressure, the operation time of the fractionation was drastically shortened, and the long precipitation time, which was a problem in the existing fractionation, could be remarkably shortened.

Comparison of fractionation time according to whether or not negative pressure cavitation was introduced during fractional precipitation of paclitaxel document number Fractional precipitation method Methanol:distilled water ratio (v/v) storage temperature
(℃) surface area increasing material fractionation settling time
(hr) One Add surface area increaser 61.5:38.5 low temperature
(0-4) glass bead ~12 2 Add surface area increaser 61.5:38.5 low temperature
(0-4) ion exchange resin 6-12 3,4,5 3. Optimization of fractional sedimentation conditions
4. Adsorbent treatment
(activated clay)
5. Temperature control 61.5:38.5 low temperature
(0-4) doesn't exist ~72 6 Methanol: distilled water
rate change 50:50-20:80 low temperature
(0-4) doesn't exist ~0.5 7 ultrasonic cavitation 61.5:38.5 low temperature
(5) doesn't exist 0.08-0.50 original invention negative pressure cavitation 61.5:38.5 low temperature
(5) doesn't exist 0.02-0.16

These results suggest that, as in ultrasonic cavitation fractionation, the negative pressure cavitation bubbles themselves act as sites for heterogeneous nucleation, lowering the free energy barrier for nucleation and accelerating sedimentation. In addition, it is believed that this is because homogeneous nucleation is promoted due to shock waves and microjets generated by the collapse of negative pressure cavitation bubbles.

Confirmation of the characteristics of fractionated sedimentation using negative pressure cavitation

3-1. Sediment size measurement

In order to investigate the characteristics of the fractionation sedimentation more quantitatively, first, the particle size of the precipitate was measured in the traditional fractionation (control group) without negative pressure and the fractionation using negative pressure cavitation.

Specifically, in order to examine the shape and size of the precipitate obtained in the fractional precipitation of Example 2-2, an electron microscope (SV-35 Video Microscope System, Some Tech, Korea) was used [Kim et al., "Kinetic and thermodynamic characteristics of fractional precipitation of (+)-dihydromyricetin," Process Biochem. 53 224-231 (2017)]. The precipitate was observed at high magnification (x200), and the size of the precipitate was measured from video images using the IT-Plus System (Some Tech, Korea).

3-2. Estimation of diffusion coefficient

In order to investigate the characteristics of fractionation more quantitatively, secondly, the viscosity of the precipitation solution and the diffusion coefficient of paclitaxel were measured in the conventional fractionation (control) without negative pressure and the fractionation using negative pressure cavitation.

The diffusion coefficient of the paclitaxel molecule (B) diffusing into the fractionation precipitation solvent (A) was calculated from the Stokes-Einstein equation and can be expressed as Equation 1.

[Equation 1]

In Equation 1, k is the Boltzmann constant (Boltzmann constant, )ego, is the paclitaxel molecular radius, is the dynamic viscosity of the solution, and T is the absolute temperature of the solution. The viscosity of the solution was measured using a viscometer (Viscolite 700, Hydromotion, UK).

Values of kinetic parameters for fractional precipitation of paclitaxel at different negative pressures Fractionated sedimentation type average particle size
(μm) Viscosity (g/cm s) diffusion coefficient, ( ) control group* 52.529 1.2 6.348 Using negative pressure cavitation bubbles
(-200 mmHg) 15.868 0.9 28.020

*No introduction of negative pressure (atmospheric pressure 760 mmHg)

As confirmed in Table 2, the viscosities of the control and fractionation solutions with negative pressure were measured as 1.2 and 0.9 g/cm·s, respectively. The average particle size was measured to be 52.529 μm in fractionation sedimentation without introducing negative pressure, whereas the average particle size was measured to be 15.868 μm in 1 to 10 minutes of sedimentation when negative pressure was introduced, indicating a 3.3-fold decrease in average particle size when negative pressure was introduced. In general, reducing the particle size of drug substances has the advantage of improving dissolution rate, uniformity of drug dispersion, oral bioavailability, etc. during formulation. To investigate the diffusion behavior according to the fractional precipitation method, the diffusion coefficient of paclitaxel was calculated using the Stokes-Einstein equation. As a result of calculating (diffusion coefficient), as shown in Table 2, in the case of traditional fractionated sedimentation (control group) without negative pressure value is 6.348 On the other hand, in the case of fractional precipitation using negative pressure cavitation value is , and the diffusion coefficient with the introduction of negative pressure It was found that the value increased by 4.4 times. In fractional precipitation, the increase in diffusion coefficient has a significant effect on homogeneous nucleation. This is believed to be due to intense local heating (hotspots), high-pressure shock waves and microjets, which are cavitational phenomena caused by the collapse of negative pressure cavitation bubbles.

3-3. Kinetic model

In general, the generation process of sediment particles is divided into nucleation and growth stages. The Johnson-Mehl-Avrami-Komolgorov (JMAK) equation, which is mainly used in crystallization or precipitation processes, deals with the kinetics of isothermal phase-transformation in the nucleation and particle growth processes and is expressed as Equation 2.

[Equation 2]

Equation 3 can be obtained by linearizing Equation 2.

[Equation 3]

Using Equation 3 big was plotted, and n and k were obtained from the slope and intercept of the straight line, respectively.

where X is the precipitate fraction, t is time, k is the rate constant, and n is the Avrami index describing the structure of the crystal and the nature of crystal nucleation.

To confirm the validity of the kinetic model (JMAK model) in the fractional precipitation process of paclitaxel, the coefficient of determination (coefficient of determination, ) was used. The closer the coefficient of determination is to 1, the smaller the error between the experimental value and the calculated value.

The experimental data of FIG. 2 was applied to the JMAK equation to kinetically analyze the precipitation pattern of paclitaxel according to the precipitation time.

Based on Equation 2 to apply the JMAK equation to the fractional precipitation versus log t was plotted (data not shown). Table 3 shows the JMAK exponent (n) and rate constant (k) calculated from the slope and intercept of the straight line along with the coefficient of determination (r ² ). The n values were 0.2166 (control) and 0.0406 to 0.2407 (-50 to -200 mmHg). The k values (min ^-1 ) were 0.6140 (control) and 1.3183 to 3.7120 (-50 to -200 mmHg). In the case of fractional precipitation using negative pressure (-50 to -200 mmHg) compared to the control group, the rate constants increased by 2.147 to 6.046 times, respectively. Arrhenius equation [Kim et al., "Kinetic and thermodynamic characteristics of microwave-assisted extraction for the recovery of paclitaxel from Taxus chinensis ," Process Biochem. 76 187-193 (2019)] as a result of calculating the activation energy change (ΔE _a =ΔE _{a, negative pressure} -ΔE _{a, control} ) according to negative pressure (-50 ~ -200 mmHg), the ΔE _a value was -1767 ~ -4161 J / mol. That is, when fractional precipitation was performed using negative pressure, there was an effect of reducing activation energy. From these results, it was possible to reduce the activation energy (minimum energy required for the reaction to proceed) by introducing negative pressure into the fractionation precipitation, thereby increasing the precipitation rate. As a result of the kinetic analysis, it was found that the JMAK model had a high r ² value (>0.828) and was suitable for the fractional precipitation process of paclitaxel.

Effect of Negative Pressure Cavitation Bubbles on Average Particle Size and Diffusivity Sound pressure (mmHg) n k ΔE _a (J/mol)
(E _a , _{negative pressure} -E _a , _control ) r ² control group 0.2166 0.6140 - 0.926 -50 0.1601 1.3183 -1767 0.890 -100 0.2407 1.8395 -2537 0.890 -150 0.1738 2.3763 -3129 0.936 -200 0.0406 3.7120 -4161 0.828

Claims (10)

As a fractional precipitation method performed by introducing negative pressure cavitation in the purification of paclitaxel,
A fractional precipitation method is performed by mixing a sample containing paclitaxel with methanol, adding water, and then applying negative pressure of -260 mmHg to -100 mmHg to generate cavitation bubbles, introducing negative pressure cavitation for 1 to 5 minutes,
After performing the fractional precipitation method, more than 97% of paclitaxel is recovered. delete delete The fractional precipitation method according to claim 1, wherein the water is added until the volume ratio of methanol to water is 50:50 to 20:80. delete delete The fractional precipitation method according to claim 1, wherein the fractional precipitation method is performed at room temperature. The fractionated precipitation method according to claim 1, wherein the average particle size of paclitaxel after the fractional precipitation is performed by introducing negative pressure cavitation is reduced to 10 μm to 20 μm. Paclitaxel purification method comprising the following steps a) to e):
a) obtaining biomass from a cell culture medium of a Taxus genus plant;
b) performing organic solvent extraction;
c) performing liquid-liquid extraction;
d) treating the adsorbent; and
e) performing the fractional precipitation method of any one of claims 1, 4, 7 and 8. 10. The method of claim 9, 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.