KR102541768B1 - Extraction method of paclitaxel using gas bubble - Google Patents

Abstract

The present invention relates to a method for extracting paclitaxel using gas bubbles, and more particularly, to a method for efficiently recovering paclitaxel by introducing a gas flow rate in a specific range to biomass containing paclitaxel to break up the surface of the biomass. A paclitaxel extraction method is provided.

Description

Extraction method of paclitaxel using gas bubble {Extraction method of paclitaxel using gas bubble}

The present invention relates to a method for extracting paclitaxel using gas bubbles, and more particularly, to a method for efficiently recovering paclitaxel by introducing a gas flow rate in a specific range to biomass containing paclitaxel to break up the surface of the biomass. A paclitaxel extraction method is provided.

Paclitaxel is a diterpenoid-based anti-cancer substance found in the epidermis of the yew tree and is currently the most widely used anti-cancer drug. It has obtained U.S. Food and Drug Adnimistration (FDA) approval for the treatment of ovarian cancer, breast cancer, head and neck cancer, Kaposi's sarcoma, and non-small cell lung cancer. With continued development of indications (Alzheimer's disease treatment, rheumatoid arthritis) and treatment methods, the demand for paclitaxel is expected to continue to increase.

The main production methods of paclitaxel include a direct extraction method from a yew tree, a semi-synthetic method by obtaining a precursor, and a method obtained by culturing plant cells by inducing callus from a plant. Among these, the plant cell culture method is very advantageous for mass production because it can stably produce paclitaxel of constant quality in a bioreactor without being affected by external factors (climate, environment) compared to other methods. Paclitaxel produced by plant cell culture is mostly contained in biomass (plant cells), and paclitaxel production consists of several stages of extraction and purification processes. From an economic point of view, it is very important to first recover paclitaxel contained in biomass in high yield.

Kim et al., Korean J. Biotechnol. According to Bioeng ., 15, 346-351 (2000), it is common for the recovery process to first perform extraction with an organic solvent (conventional solvent extraction), and various types of organic solvents (methylene chloride, methyl-t-butyl ether, ethanol, iso As a result of examining the extraction tendency using propyl alcohol, chloroform, diethyl ether, acetone, methanol, etc.), it was found that methanol was the most effective for biomass extraction by obtaining the highest recovery rate with the smallest amount. In addition, it was attempted to increase the recovery rate of paclitaxel from biomass by optimizing the main process variables (methanol concentration, biomass/methanol mixing ratio, extraction time, number of extractions, etc.).

However, in the case of the conventional solvent extraction method, there was a disadvantage in that a lot of organic solvents and time were required due to low extraction efficiency. Kim et al., Korean J. Biotechnol. Bioeng ., 23 , 281-284 (2008) reported a method for recovering paclitaxel from biomass by extraction using microwaves. In addition, it has recently been reported that extraction using ultrasound is a very effective method in that it can improve the extraction efficiency of the target component by breaking the cell wall (Davoud, S et al., Ind. Crop. Prod., 55 , 163-172 (2014); Chunying, L. et al., J. Sep. Sci., 38 , 291-300 (2015); Pstsaporn, P. et al., Sep. Purif. Technol ., 144 , 37-45 (2015). Ana, C, S. et al. , Trends Food Sci. Technol., 21 , 323-331 (2010);

However, although the operation time is shortened by reducing the number of extractions through microwave or ultrasonic waves, additional equipment and costs are incurred for high recovery rate, as well as the process is complicated, dangerous, and cumbersome, and a lot of energy is consumed, making industrial use difficult. There are limits to its application to mass production processes. As a result, the development of a high-efficiency biomass extraction process that minimizes or simplifies the equipment required to shorten the number of extractions, and at the same time saves equipment and saves energy, as well as process convenience and process feasibility, is still needed. It is necessary.

Under this background, the present inventors have attempted to dramatically improve the method for recovering paclitaxel from biomass by extraction using gas bubbles. That is, an attempt was made to improve the recovery rate of biomass-derived paclitaxel by optimizing the gas flow rate and operation time, which are major process variables in the extraction process using gas bubbles. In addition, it solves the problem of using many organic solvents and long extraction time, which were limitations of the existing solvent extraction method (Kim et al. , Korean J. Biotechnol. Bioeng ., 15, 346-351 (2000)), and at the same time using microwave or ultrasonic waves. Extraction ( Korean J. Biotechnol. Bioeng ., 23 , 281-284(2008)) The limitations of the process, such as the complexity of the process, the risk of equipment, and the high cost due to high energy consumption, were drastically improved.

The present invention has been made to solve the above problems, and dramatically increases the recovery rate of paclitaxel, an anticancer substance, from biomass through an extraction method using gas bubbles. has a purpose to

In order to solve the above problems, the present invention provides a paclitaxel extraction method comprising the following steps a) to c): a) Taxus genus plant-derived biomass containing paclitaxel obtaining; b) mixing the biomass with an organic solvent; and c) extracting paclitaxel by injecting gas bubbles into the mixture of step b).

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, the extraction using gas bubbles in step c) may be performed once.

According to another preferred embodiment of the present invention, the gas flow rate during the gas bubble injection in step c) may be 1.00 L/min to 15.00 L/min.

According to another preferred embodiment of the present invention, the extraction using gas bubbles in step c) may be performed for 15 minutes or less.

According to another preferred embodiment of the present invention, the extraction using gas bubbles in step c) may be performed at 0 to 30°C.

The extraction method of paclitaxel using gas bubbles of the present invention solves the problems of using many organic solvents and long extraction time, which were the limitations of the existing solvent extraction method, and at the same time, the complexity of the process and the risk of the device, which were the limitations of the extraction process using microwave or ultrasonic waves. And it is possible to dramatically improve the high cost problem caused by a lot of energy consumption. Therefore, it is believed that the paclitaxel extraction method of the present invention can be very usefully used for commercial mass production of paclitaxel, an anticancer substance, from biomass derived from plants of the genus Taxus .

Figure 1 is a schematic diagram showing a process for extracting paclitaxel from biomass, (A) shows a traditional extraction method, which is a conventional solvent extraction method, and (B) shows an extraction method using gas bubbles.
Figure 2 is a graph showing the change in the yield of paclitaxel from biomass according to conventional extraction and gas flow rates (1.15, 4.52 and 9.41 L/min, respectively) at room temperature.
Figure 3 shows scanning electron micrographs of biomass samples dried by solvent extraction using different methods, (A) before extraction, (B) the conventional traditional solvent extraction method (extraction once), (C ) Scanning electron microscope for extraction method using gas bubbles (1.15 L/min), (D) extraction method using gas bubbles (4.52 L/min) and (E) extraction method using gas bubbles (9.14 L/min) It is a picture.

As described above, the conventional solvent extraction method requires 4 or more extraction processes to recover most of the paclitaxel from biomass, and thus has a disadvantage in that it requires a lot of organic solvents and time for high paclitaxel recovery. Extraction using microwaves or ultrasonic waves shortens the number of extractions, but additional equipment and costs are incurred for high recovery rate, and the process is complicated, dangerous, cumbersome, and consumes a lot of energy, so it is applied to industrial mass production processes. There was a limit to doing it. Therefore, the development of a high-efficiency biomass extraction process that minimizes and/or simplifies the equipment required to shorten the number of extractions, saves equipment and saves energy, as well as process convenience and process feasibility is still in progress. It is necessary.

Accordingly, the present invention comprises the steps of a) obtaining taxus genus plant-derived biomass containing paclitaxel; b) mixing the biomass with an organic solvent; and c) extracting paclitaxel by injecting gas bubbles into the mixture of step b). The paclitaxel extraction method of the present invention solves the problems of using many organic solvents and long extraction time, which were the limitations of the existing solvent extraction process, and at the same time, the complexity of the process or the risk of equipment and high energy consumption, which were the limitations of the extraction process using microwave or ultrasonic waves. The high cost problem caused by can be dramatically improved.

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

In the paclitaxel extraction method 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.

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 mixing the biomass with the organic solvent will be described.

In the paclitaxel extraction method of the present invention, the organic solvent is not particularly limited as long as it is commonly used to extract active ingredients from plants, but is preferably at least one selected from the group consisting of C ₁ to C ₄ alcohol and water. can include

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

In addition, the biomass and organic solvent in step b) may be mixed in a ratio of 1:1 to 1:6 (w/v), but is not limited thereto, and can be usually used to extract active ingredients from plants. If there is a ratio, it is not particularly limited. In one embodiment of the present invention, biomass and organic solvent were constantly mixed at a ratio of 1:2 (w/v) for optimal extraction efficiency.

Next, step c) of extracting paclitaxel by injecting gas bubbles into the mixture of step b) will be described.

In the paclitaxel extraction method of the present invention, the gas bubbles in step c) may be injected at a constant gas flow rate regardless of the purity of paclitaxel in the sample. Preferably, gas bubbles are injected at a gas flow rate of 1.00 L/min to 15.00 L/min, more preferably 3.00 L/min to 13.00 L/min, and most preferably 5.00 L/min to 10.00 L/min. can

If gas bubbles are injected at a gas flow rate of 1.00 L/min or less, the effect of increasing the recovery rate of paclitaxel may be insignificant compared to the conventional solvent extraction method, and gas bubbles are injected at a gas flow rate exceeding 15.00 L/min. Excessive evaporation of the extraction solvent may cause a problem of lowering extraction efficiency.

In the paclitaxel extraction method of the present invention, the extraction using gas bubbles in step c) may be performed for 15 minutes or less. Preferably, it may be performed for 6 minutes to 15 minutes, more preferably 8 minutes to 12 minutes, and most preferably 8 minutes to 10 minutes.

If the extraction using gas bubbles is performed for 6 minutes or less, paclitaxel may not be sufficiently recovered from the biomass, and if it is performed for more than 15 minutes, the effect of increasing the recovery rate of paclitaxel over time is insignificant, resulting in unnecessary operation time. can cause an increase in

Gas bubbles may be injected using a bubbler. For example, gas bubbles may be injected by connecting an air hose, tube, or pipe to the oxygen outlet of the bubble generator, but is not limited thereto.

The injected gas bubble may have a diameter of 0.5 to 5.0 mm, but any diameter at which bubbles can be formed may be used without limitation. For example, it is also possible to perform extraction by injecting fine bubbles in the range of 0.5 mm or less.

In the paclitaxel extraction method of the present invention, the extraction using gas bubbles in step c) may be performed once.

In a specific embodiment of the present invention, stirring (~ 500 rpm), extraction was performed once, and the recovery rate of paclitaxel was calculated by Equation 1 below.

[Equation 1]

As shown in FIG. 2, in the case of the conventional solvent extraction method, which is a conventional solvent extraction method without introducing gas bubbles, the recovery rate of paclitaxel is 58%, while the gas flow rate during extraction is 1.15, 4.52, and 9.41 L/min, respectively. Recovery rates when introduced were 61, 66 and 75%. As the gas flow rate and operation time increased, the recovery rate of paclitaxel increased.

As a result, the optimal extraction method using gas bubbles was performed for 8 to 12 minutes at a gas flow rate of 9.41 L/min, and it is possible to recover about 75% of paclitaxel with only one extraction under optimal conditions. did

Through these results, it was confirmed that the paclitaxel extraction method using gas bubbles of the present invention significantly shortens the operation time compared to the conventional optimized solvent extraction method, and dramatically increases the extraction efficiency according to the reduction of the operation time. .

In addition, in another specific embodiment of the present invention, the biomass surface was observed after extraction using a solvent extraction method or gas bubble using a scanning electron microscope (MIRA LMH; Tescan, Czech Republic). In the case of biomass before extraction, the biomass surface is very smooth as shown in FIG. 3(A), and in the case of the traditional solvent extraction method using methanol (one-time extraction), as shown in FIG. showed shape.

On the other hand, in the case of the extraction method using gas bubbles (extraction once), the biomass surface is very rough and shriveled (crushed) form due to strong impact, as shown in FIGS. 3 (C), 3 (D) and 3 (E). , and the expression became more wrinkled as the gas flow rate increased. That is, a sufficiently shriveled (crushed) surface was formed so that paclitaxel contained in the cells could be effectively extracted, and as a result, the extraction efficiency was dramatically improved. From these results, it was confirmed that paclitaxel can be effectively extracted/recovered from biomass by extraction using gas bubbles.

The present inventors have proposed a method of drastically shortening the fractionation precipitation time by injecting gas bubbles in the fractionation precipitation step during the purification of paclitaxel, but this improves solute mass transfer by injecting gas bubbles, and the gas bubbles themselves are nuclei The principle of accelerating the precipitation of paclitaxel by acting as the center of formation (serving as a seed in precipitation), which shortens the operating time required for the fractional precipitation step (Kim et al. Process Biochemistry 99 (2020) 316-323 ), It is completely different from the biomass crushing effect confirmed in the present invention.

The optimized paclitaxel extraction method using gas bubbles of the present invention solves the problems of using many organic solvents and long extraction time, which were the limitations of the existing solvent extraction method, and at the same time, the complexity of the process, which was the limitation of the extraction process using microwave or ultrasound, It is possible to drastically improve the high cost problem caused by the risk and high energy consumption.

In the paclitaxel extraction method of the present invention, the extraction using gas bubbles in step c) may be performed at 0 to 30°C, preferably 5 to 30°C, and more preferably 10 to 30°C.

In the separation and purification of paclitaxel, the conventional fractional precipitation method is performed at a low temperature of 0 to 4 ° C., so there is a limit to its application to the mass production process of paclitaxel in terms of economic feasibility.

However, in the present invention, after adding a certain ratio of organic solvent to biomass, the temperature range including room temperature (eg, 5 to 30 ° C.) at a gas flow rate of 1.00 L/min to 15.00 L/min for 15 minutes Since the recovery rate of paclitaxel can be remarkably improved only by injecting gas bubbles during the following process, it is suitable for application to a mass production process because it does not require low-temperature storage for fractional precipitation.

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. 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.

Gas bubble-assisted extraction

Paclitaxel produced by plant cell culture is mostly contained in biomass (plant cells), Kim et al., Korean J. Biotechnol. According to Bioeng ., 15 , 346-351 (2000), as a result of investigating the extraction efficiency using various organic solvents, it was found that methanol was most effective. In order to optimize the main process parameters, the optimal biomass/methanol ratio (1:2, w/v) suggested by Kim et al., Process Biochem ., 39 , 1985-1991 (2004) was kept constant and gas The effect of gas flow rate and operation time was investigated.

Specifically, by injecting gas bubbles into the extraction solution using a gassing unit (SH-A2, Amazonpet, Korea) (a hole having a diameter of 0.5 mm), the gas flow rate through the gassing unit (each 1.15, 4.52 and 9.41 L/min) on the extraction efficiency was investigated. In addition, the operation time (1, 2, 4, 6, 8, 10 and 12 minutes, respectively) was varied and extracted once under stirring (~500 rpm). After extraction, the paclitaxel extraction filtrate was recovered by filtration under reduced pressure with a filter paper (185 mm, ADVANTEC), concentrated using a concentrator (CCA-1100, EYELA, Japan) and completely vacuum dried (40 ° C, overnight, 760 mmHg) did The dried crude paclitaxel (crude paclitaxel) was measured for paclitaxel purity and yield through HPLC analysis.

For paclitaxel content analysis, an HPLC system (SCL-10AVP, Shimadzu, Japan) and a Capsule Pak C ₁₈ (Capcell Pak C ₁₈ ; 250 Х 4.6 mm, Shiseido, Japan) column were used. The mobile phase was flowed with a mixed solution of distilled water and acetonitrile (65/35 to 35/65, v/v, gradient solvent composition method) at a flow rate of 1.0 ml/min. The sample injection amount was 20 μl and was detected by UV at 227 nm [9]. For HPLC analysis, a standard quantification curve was used, and a standard sample from Sigma-Aldrich (purity >97%) was used.

The traditional extraction method and the biomass extraction method using gas bubbles are shown in FIG. 1 . In addition, the yield of paclitaxel was calculated by Equation 1 below.

[Equation 1]

As shown in FIG. 2, the recovery rate of paclitaxel is 58% in the case of the conventional solvent extraction method without gas bubble introduction, whereas the recovery rate is 61, 66 and 75%. As the gas flow rate and operation time increased, the recovery rate of paclitaxel increased. These results indicate that the gas bubbles disrupted the cell walls and effectively extracted paclitaxel contained in the cells, resulting in improved extraction efficiency.

Morphological investigation of biomass through scanning electron microscopy (SEM) analysis

A scanning electron microscope (MIRA LMH; Tescan, Czech Republic) was used to observe the biomass surface. The amount of analysis was 1 mg and the shape of the dried biomass was investigated at an accelerating voltage of 10-15 kV.

In the case of biomass before extraction, as shown in FIG. 3(A), the biomass surface showed a very smooth shape, and in the case of traditional extraction using methanol (extraction once), as shown in FIG. seemed On the other hand, in the case of extraction using gas bubbles (extraction once), as shown in FIGS. 3 (C), 3 (D), and 3 (E), the biomass surface is very rough and shriveled (crushed) due to strong impact. As the gas flow rate increased, the surface showed a more wrinkled shape. That is, a sufficiently shriveled (crushed) surface was formed so that all of the paclitaxel contained in the cells could be extracted, and as a result, the extraction efficiency was effectively improved. From these results, it was confirmed again that paclitaxel can be effectively extracted/recovered from biomass by extraction using gas bubbles. In addition, it was found that there was little difference in the purity of paclitaxel (~4.5%) obtained from biomass extraction with or without gas bubbles (data not shown).

As a result, through this study, the problems of using many organic solvents and long extraction time, which were the limitations of the traditional extraction process, were solved, and at the same time, the complexity of the process, the risk of equipment and high energy consumption, which were the limitations of the extraction process using microwave or ultrasonic waves, were solved. It was possible to dramatically improve the high cost problem caused by

Claims (8)

Paclitaxel extraction method comprising the following steps a) to c):
a) obtaining taxus genus plant-derived biomass containing paclitaxel;
b) mixing the biomass with one organic solvent selected from the group consisting of a C ₁ to C ₄ alcohol and a mixed solvent of a C ₁ to C ₄ alcohol and water; and
c) extracting paclitaxel by injecting gas bubbles into the mixture of step b). 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. method. delete The method of extracting 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 extracting paclitaxel according to claim 1, wherein the extraction using gas bubbles in step c) is performed once. The method of extracting paclitaxel according to claim 1, wherein the gas flow rate during the gas bubble injection in step c) is 1.00 L/min to 15.00 L/min. The method of extracting paclitaxel according to claim 1, wherein the extraction using gas bubbles in step c) is performed for 15 minutes or less. The method of extracting paclitaxel according to claim 1, wherein the extraction using gas bubbles in step c) is performed at 0 to 30°C.

Images