KR20220135587A - Extraction method of paclitaxel using gas bubble - Google Patents

Abstract

The present invention relates to a method for extracting paclitaxel using gas bubbles. More specifically, the present invention provides a method for extracting paclitaxel which can efficiently recover paclitaxel by introducing a gas flow rate in a specific range to biomass containing paclitaxel to break up the surface of the biomass.

Description

Extraction method of paclitaxel using gas bubble

The present invention relates to a method for extracting paclitaxel using gas bubbles, and more particularly, by introducing a gas flow rate in a specific range into biomass containing paclitaxel and crushing the surface of the biomass, paclitaxel can be efficiently recovered. A method for extracting paclitaxel is provided.

Paclitaxel is a diterpenoid anticancer substance found in the epidermis of yew tree, and is currently the most used anticancer 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. The demand for paclitaxel is expected to continue to increase with the continued development of indications (alzheimer's disease, rheumatoid arthritis) and treatment methods.

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

Kim et al. Korean J. Biotechnol. According to Bioeng ., 15, 346-351 (2000), the recovery process generally involves first 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 investigating the extraction tendency using propyl alcohol, chloroform, diethyl ether, acetone, methanol, etc.), it was found that methanol was 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 major 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 is a disadvantage that it takes a lot of organic solvent and time 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, recently, extraction using ultrasound has been reported as a very effective method in that it can improve the extraction efficiency of target components by disrupting 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); Ming, G. et al., Food Chem ., 175 , 181-188 (2015)).

However, although the operation time was shortened by reducing the number of extractions through microwaves or ultrasonic waves, additional equipment and costs are incurred for a high recovery rate, and the process is complicated, dangerous, cumbersome, and consumes a lot of energy. There is a limit 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 reduces equipment and energy, as well as process convenience and process feasibility, is still is in need.

Under this background, the present inventors have attempted to dramatically improve a method for recovering paclitaxel from biomass by extraction using gas bubbles. That is, it was attempted to improve the recovery rate of biomass-derived paclitaxel by optimizing the gas flow rate and operating time, which are major process variables in the extraction process introducing gas bubbles. In addition, it solves the problems of using many organic solvents and long extraction times, which were limitations of the existing solvent extraction methods (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)) It was intended to dramatically improve the complexity of the process, the risk of the device, and the high cost due to energy consumption, which were the limitations of the process.

The present invention has been devised to solve the above problems, and provides an economical and simplified process for extracting paclitaxel while dramatically increasing the recovery rate of paclitaxel, an anticancer substance, from biomass through an extraction method using gas bubbles. there is 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 (biomass) 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 of step a) may include at least one selected from the group consisting of Taxus plants, its cells, its cell debris and its cell culture solution. .

According to another preferred embodiment of the present invention, the organic solvent of 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 the 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 1.00L/min to 15.00L/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 a lot of organic solvent 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 apparatus, which were the limitations of the extraction process using microwave or ultrasonic waves. And it is possible to remarkably improve the high cost problem due to the consumption of a lot of energy. Therefore, the paclitaxel extraction method of the present invention is considered to be very useful for commercial mass production of anticancer substance paclitaxel from biomass derived from plants of the genus Taxus .

1 is a schematic diagram showing a process of extracting paclitaxel from biomass, (A) shows a conventional extraction method, which is a conventional solvent extraction method, (B) shows an extraction method using gas bubbles.
2 is a graph showing the change in yield of paclitaxel from biomass according to the conventional extraction and gas flow rate (1.15, 4.52 and 9.41 L/min, respectively) at room temperature.
3 is a scanning electron micrograph of a biomass sample dried by solvent extraction using a different method, (A) before extraction, (B) is a conventional conventional solvent extraction method (one-time extraction), (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's a photo.

As described above, the conventional solvent extraction method requires four or more extraction steps to recover most of the paclitaxel from biomass. Extraction using microwaves or ultrasound shortens the number of extractions, but for high recovery, additional equipment and costs are incurred, 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 that. Therefore, the development of a high-efficiency biomass extraction process that minimizes and/or simplifies the equipment required to reduce the number of extractions, and at the same time reduces equipment and energy, as well as process convenience and process feasibility, is still is in need.

Accordingly, the present invention comprises the steps of: a) obtaining a biomass derived from a taxus genus plant containing paclitaxel; b) mixing the biomass with an organic solvent; and c) injecting gas bubbles into the mixture of step b) to extract paclitaxel. The paclitaxel extraction method of the present invention solves the problems of using a lot of organic solvent 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 the device, and a lot of energy consumption, which were the limitations of the extraction process using microwave or ultrasound can dramatically improve the high cost problem caused by

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

In the paclitaxel extraction method of the present invention, the biomass of step a) may include one or more selected from the group consisting of plants of the genus Taxus, cells thereof, cell debris thereof, and cell cultures thereof.

In addition, the taxus genus plants are Taxus brevifolia ( Taxus brevifolia ), Taxus canadensis ( Taxus canadensis ), Taxus cuspidata ( Taxus cuspidata ), Taxus baccata ( Taxus baccata ), Taxus glow Bossa ( Taxus globosa ), Taxus floridana ( Taxus floridana ), Taxus wallichiana ( Taxus wallichiana ), Taxus media ( Taxus media ) or Taxus chinensis ( Taxus chinensis ) and the like 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 plant cell culture medium.

Next, step b) of mixing the biomass with an 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 normally used to extract the active ingredient from a plant, but preferably at least one selected from the group consisting of C ₁ to C ₄ alcohol and water. may include

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

In addition, the biomass and organic solvent of step b) may be mixed in a ratio of 1:1 to 1:6 (w/v), but is not limited thereto, and can be used to extract active ingredients from plants in general. If there is a ratio, it is not particularly limited. In one embodiment of the present invention, the biomass and the organic solvent were uniformly 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 bubble in step c) may be injected at a constant gas flow rate regardless of the purity of paclitaxel in the sample. Preferably, the gas bubbles will be 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, most preferably 5.00 L/min to 10.00 L/min. can

If the gas bubble is 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 when the gas bubble is injected at a gas flow rate exceeding 15.00 L/min Excessive evaporation of the extraction solvent may be caused, which may cause a problem in that extraction efficiency is reduced.

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 carried out for 6 minutes to 15 minutes, more preferably 8 minutes to 12 minutes, and most preferably 8 minutes to 10 minutes.

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

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

The diameter of the injected gas bubble may be 0.5 to 5.0 mm, but as long as the diameter of the bubble can be formed, it may be used without limitation. For example, it is also possible to perform extraction by injecting microbubbles 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 was 58%, whereas the gas flow rates during extraction were 1.15, 4.52, and 9.41 L/min, respectively. The recovery rates when introduced were 61, 66 and 75%. The recovery rate of paclitaxel increased as the gas flow rate and operating time 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 shortened the operation time compared to the conventional optimized solvent extraction method, and the extraction efficiency according to the reduction in operation time was significantly increased. .

In addition, in another specific embodiment of the present invention, the surface of the biomass was observed after extraction using a solvent extraction method or gas bubbles using a scanning electron microscope (MIRA LMH; Tescan, Czech Republic). In the case of the biomass before extraction, the surface of the biomass shows a very smooth shape as shown in FIG. showed shape.

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

The present inventors have proposed a method for remarkably shortening the fractional precipitation time by injecting gas bubbles in the fractional precipitation step during purification of paclitaxel, but this improves the mass transfer of solute by injecting gas bubbles, and the gas bubbles themselves are nuclear As a principle of promoting the precipitation of paclitaxel by acting as the center of formation (the role of a seed in precipitation), it shortens the operation 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 a lot of organic solvent and long extraction time, which were limitations of the existing solvent extraction method, and at the same time, the complexity of the process, which was a limitation of the extraction process using microwave or ultrasonic, and the The high cost problem caused by risk and high energy consumption can be remarkably improved.

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, more preferably 10 to 30°C.

In the separation and purification of paclitaxel, the conventional fractional precipitation method was performed at a low temperature of 0 to 4 ° C.

However, in the present invention, after adding an organic solvent in a certain ratio to the biomass, in a 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, it does not require a separate low-temperature storage for fractional precipitation, so it is suitable for mass production process.

Hereinafter, the present invention will be described in more detail through examples. However, since the present invention can have various changes and can have various forms, the specific examples and descriptions described below are only for helping the understanding of the present invention, and the present invention is limited to specific disclosed forms it's not meant to be 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 originating from Taxus chinensis were cultured by stirring at 150 rpm in dark conditions at 24°C. Suspension cells were prepared using modified Gamborg's B5 medium, 30 g/L sucrose, 10 mM naphthalene acetic acid, 0.2 μM 6-benzylaminopurine, 1 g Cultured in /L casein hydrolysate, 1 g/L 2-(N-morpholino)ethanesulfonic acid (2-(N-morpholino)ethanesulfonic acid). Cell culture was replaced with a fresh medium every 2 weeks, and in order to extend the culture time, 1-2% (w/v) maltose was added on the 7th and 21st days, and inducer As an elicitor, 4 mM AgNO ₃ was added at the beginning of the culture [7,19]. Plant cells and cell debris were recovered from the culture medium after plant cell culture using a decanter (Westfalia, CA150 Claritying Decanter) and a high-speed centrifuge (α-Laval, BTPX205GD-35CDEEP). The recovered plant cells and cell fragments were combined and referred to as biomass.

Gas bubble-assisted extraction

Most paclitaxel produced by plant cell culture is contained in biomass (plant cells), and Kim et al., Korean J. Biotechnol. According to Bioeng ., 15 , 346-351 (2000), as a result of investigating extraction efficiency using various organic solvents, methanol was found to be the most effective. For the optimization of major 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 the gas at room temperature was The effect of gas flow rate and operating 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 gas generating unit (each 1.15, 4.52 and 9.41 L/min) on the extraction efficiency were investigated. In addition, the operation time (1, 2, 4, 6, 8, 10 and 12 minutes, respectively) was changed, and extraction was performed once under stirring (~500 rpm). After extraction, it was filtered under reduced pressure with filter paper (185 mm, ADVANTEC) to recover the paclitaxel extraction filtrate, concentrated using a concentrator (CCA-1100, EYELA, Japan), and then dried under complete vacuum (40°C, overnight (overnight), 760 mmHg) did. Purity and yield of paclitaxel were measured from dried crude paclitaxel through HPLC analysis.

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

A conventional extraction method and a 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 , in the case of the conventional solvent extraction method without gas bubble introduction, the recovery rate of paclitaxel is 58%, whereas when the gas flow rate during extraction is introduced at 1.15, 4.52 and 9.41 L/min, respectively, the recovery rate is 61, 66 and 75%. The recovery rate of paclitaxel increased as the gas flow rate and operating time increased. According to these results, it is judged that the extraction efficiency is improved because the gas bubbles break the cell wall and the paclitaxel contained in the cell is effectively extracted.

Investigation of the morphology 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 is 1 mg, and the form of the dried biomass was investigated at an acceleration voltage of 10-15 kV.

In the case of biomass before extraction, as shown in Fig. 3(A), the surface of the biomass showed a very smooth shape, and in the case of conventional extraction (one-time extraction) using methanol, a wrinkled shape as shown in Fig. 3(B) was obtained. seemed On the other hand, in the case of extraction (one-time extraction) using gas bubbles, the biomass surface was very rough and shrunken (crushed) due to strong impact as shown in FIGS. 3(C), 3(D) and 3(E). shape, and as the gas flow rate increased, the surface showed a more corrugated shape. That is, a sufficiently shrunken (crushed) surface was formed so that all of the paclitaxel contained in the cell could be extracted, and as a result, the extraction efficiency was effectively improved. From these results, it was confirmed again that paclitaxel could 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 problem of using a lot of organic solvent and long extraction time, which were the limitations of the traditional extraction process, was solved, and at the same time, the complexity of the process, the risk of the device, and a lot of energy consumption, which were the limitations of the extraction process using microwave or ultrasound, were solved. It was possible to dramatically improve the high cost problem caused by

Claims (8)

A method for extracting paclitaxel comprising the following steps a) to c):
a) obtaining a biomass derived from a taxus genus plant containing paclitaxel;
b) mixing the biomass with an organic solvent; and
c) extracting paclitaxel by injecting gas bubbles into the mixture of step b). According to claim 1, wherein the biomass of step a) is Paclitaxel extraction, characterized in that it comprises at least one selected from the group consisting of Taxus plants, its cells, its cell debris, and its cell culture solution. Way. The method according to claim 1, wherein the organic solvent in step b) comprises at least one selected from the group consisting of C ₁ to C ₄ alcohol and water. The method of claim 1, wherein the biomass and the organic solvent in step b) are mixed in a ratio of 1:1 to 1:6 (w/v). The method of claim 1, wherein the extraction using gas bubbles in step c) is performed once. The method according to claim 1, wherein the gas flow rate is 1.00 L/min to 15.00 L/min when the gas bubble is injected in step c). The method of claim 1, wherein the extraction using gas bubbles in step c) is performed for 15 minutes or less. The method of claim 1, wherein the extraction using gas bubbles in step c) is performed at 0 to 30°C.

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