KR101491915B1 - Increase material for fractional precipitation and purification method using thereof - Google Patents

Abstract

The present invention relates to a paclitaxel purification method, and more particularly, to a paclitaxel purification method which is used in a fractional precipitation method in a high performance liquid chromatography (HPLC) preprocessing process, comprising mass purification of paclitaxel, Paclitaxel, and paclitaxel. The present invention relates to a paclitaxel fraction-precipitating surface-area-increasing substance and a paclitaxel purification method using the same.

Description

TECHNICAL FIELD [0001] The present invention relates to a paclitaxel fractionated precipitation surface area increasing substance and a paclitaxel purification method using the same,

The present invention relates to a paclitaxel purification method, and more particularly, to a paclitaxel purification method which is used in a fractional precipitation method in a high performance liquid chromatography (HPLC) preprocessing process, comprising mass purification of paclitaxel, Paclitaxel, and paclitaxel. The present invention relates to a paclitaxel fraction-precipitating surface-area-increasing substance and a paclitaxel purification method using the same.

Paclitaxel is an anticancer substance found in the epidermis of a tree of interest and has been approved by the US Food and Drug Administration (FDA) for the treatment of ovarian cancer, breast cancer, Kaposi's sarcoma, and non-small cell lung cancer (NSCLC) It is currently the most widely used anticancer drug currently, and currently holds the largest market share among anticancer drugs, accounting for 22% of the major anticancer drug market. Rheumatoid arthritis, and Alzheimer's disease. In addition, clinical trials are being conducted on multiple prescriptions for various treatment methods, and the demand for paclitaxel is expected to continue to increase in the future.

There are three main methods of producing paclitaxel. First, it is difficult to continuously supply the raw materials by direct extraction from the tree of interest, and there are many difficulties in extraction and purification, and it is not suitable for protecting the tree as an environmental protection water. Second, it is a semi-synthetic method to obtain the precursor (baccatin Ⅲ, 10-deacetylbaccatin Ⅲ, 10-deacetyl paclitaxel, etc.) from the leaf of the tree to chemically bond the side chain. However, this method also has the same problems as direct extraction because the precursor must be obtained directly from the tree of interest. Third, it is a method to induce callus from a tree of interest and cultivate plant cells in a main bioreactor through a seed culture. The third method has the advantage of mass production of paclitaxel of constant quality because it can be produced stably in the bioreactor without being affected by external factors such as climate and environment.

Separation and purification of the anticancer paclitaxel from plant cell cultures will result in a product of high purity (> 98% purity) through several steps of extraction and purification. In general, the separation and purification processes consist of a step of extracting paclitaxel from the raw material biomass with an organic solvent, and a step of producing a product through a pre-treatment and final purification. In this process, in particular, the pre- . Previous studies have shown that crude paclitaxel, which has been subjected to expensive chromatography as a pretreatment process for purification, or that has been extracted without pretreatment, is directly purified by high performance liquid chromatography (HPLC) There are many problems in terms of economics, and there are many difficulties in scale-up and mass production. Generally, when paclitaxel is extracted from biomass using an organic solvent, the purity is less than 0.5%, and even after a simple pretreatment, the purity is as low as about 10%. Final purification of these samples directly by HPLC is not economical and is not suitable for mass production such as using a large amount of organic solvent, shortening the lifetime of the packed resin packed in the column, reducing the throughput. Therefore, the purity of the sample should be increased as much as possible (at least 50%) through the pretreatment process to reduce the cost of final purification, especially HPLC purification.

Fractional precipitation has been developed and used in the pre-processing of conventional HPLC. The fractionation is carried out by recovering the plant cells from the plant cell culture solution, recovering the recovered plant cells by solvent extraction, and then subjecting the cells to adsorption and hexane precipitation. The purity of the sample is about 15% Paclitaxel of high purity was obtained simply. However, disadvantages of fractional precipitation are that it takes a lot of time (more than 3 days) to precipitate and it is difficult to apply to mass production process.

Therefore, there is a need for a paclitaxel purification method capable of obtaining paclitaxel with high purity (60% or more) in a shorter time than conventional HPLC pretreatment processes.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in order to solve the above problems, and it is an object of the present invention to provide a purification method capable of purifying paclitaxel raw material to obtain paclitaxel of high purity The purpose of this is to provide.

The present invention relates to a method for producing an extract from a mixture comprising a Taxus genus plant-derived biomass and a water-soluble organic solvent; Mixing the extract with a nonpolar organic solvent, and then extracting the liquid to obtain a crude extract; Mixing the crude extract, the nonpolar organic solvent and the adsorbent, and then filtering to prepare a filtrate; Adding a mixed solution containing the filtrate and the nonpolar organic solvent to a non-aqueous organic solvent to produce a precipitate; Preparing a reaction solution containing the precipitate, the water-soluble organic solvent and the surface-increasing substance; And 6) storing the reaction solution at 1 to 10 ° C to obtain a paclitaxel precipitate; ≪ RTI ID = 0.0 > paclitaxel < / RTI >

According to a preferred embodiment of the present invention, the biomass derived from the plant belonging to the Taxus family can be obtained from a plant belonging to the Taxus family, a cell thereof or a cell culture derived from the cell.

According to another preferred embodiment of the present invention, the water-soluble organic solvent may be at least one selected from the group consisting of C ₁ to C ₅ alcohols and water.

According to another preferred embodiment of the present invention, the nonpolar organic solvent may be at least one selected from the group consisting of methylene chloride, ethyl acetate and ether.

According to another preferred embodiment of the present invention, the water-insoluble organic solvent is selected from the group consisting of dichloromethane, carbon tetrachloride, chloroform, 1,2-dichloroethane, A group consisting of di-ethyl ether, methyl t-butyl ether, di-iso-propyl ether, hexane and benzene May be used.

According to another preferred embodiment of the present invention, the surface-increasing material is selected from the group consisting of porous silica and porous alumina; Or porous alumina.

According to another preferred embodiment of the present invention, the reaction solution of step 6 is stored for 1 to 12 hours, and the yield of paclitaxel may be 60% or more.

According to another preferred embodiment of the present invention, at least one of the adsorbent activated carbon, active clay, sylopute and silica (SiO ₂ ) may be used.

According to another preferred embodiment of the present invention, the precipitation in step 6 may be performed for 5 to 18 hours.

The present invention also relates to a surface-increasing agent comprising porous silica and porous alumina; Or porosity, wherein the surface enhancer is added to a mixture comprising paclitaxel, methanol and water to provide a paclitaxel fractionated sedimentation enhancer having a zeta potential of 0 to 40 mV do.

According to a preferred embodiment of the present invention, the surface area increasing body may have an average surface area of 200 to 290 m 2 / g.

According to another preferred embodiment of the present invention, the surface enhancer may include a porous silica: porous alumina in a weight ratio of 0.01 to 30:70 to 99.99.

The present invention can provide a purification method capable of obtaining paclitaxel of high purity (60% or more) in a shorter time than the conventional HPLC preprocessing process of paclitaxel. Thus, the purification method has an effect of being applicable to the mass production process of paclitaxel.

Further, the paclitaxel purified by the purification method of the present invention has paclitaxel having a very small crystal particle size of 100 μm or less and high pharmacological activity, thus providing paclitaxel having high utilization as a raw material drug.

Further, it is possible to provide a paclitaxel purification method which can be very effectively applied to the drying of the product (paclitaxel) after purification (in particular, residual solvent and moisture removal).

1 is a graph showing the yield of paclitaxel measured in Experimental Example 2. Fig.
2 is a purity graph of paclitaxel measured in Experimental Example 2. Fig.
3 is a graph showing the zeta potential value in Experimental Example 3 and the yield of paclitaxel after 6 hours of fractional precipitation.
4 is a result image of a paclitaxel precipitate observed by an electron microscope according to the fractional precipitation time in Experimental Example 4. Fig.
FIG. 5 is a graph showing the correlation between the mean diameter of the paclitaxel precipitate observed by an electron microscope and the settling time by fraction in Experimental Example 4. FIG.
FIG. 6 shows the relationship between the particle size of the paclitaxel precipitate measured in the same fractionation time (18 hours) of Experimental Example 4 and the zeta potency measured in Experimental Example 3. FIG.

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

As described above, the conventional fractional precipitation used in the pretreatment process of HPLC has a lot of time (3 days or more) for the precipitation, which is difficult to be applied to the mass production process. Therefore, there is a need for a paclitaxel purification method capable of obtaining paclitaxel with high purity (60% or more) in a shorter time than conventional HPLC pretreatment processes.

Accordingly, the present invention relates to porous silica and porous alumina; Or porous alumina, and a purification method using the surface-increasing agent, and a method for purifying the above-described surface-increasing agent. Thus, it is possible to obtain paclitaxel with high purity (60% or more) in a shorter time than the conventional HPLC preprocessing process of paclitaxel. In addition, the purification method has an effect that can be applied to a mass production process of paclitaxel.

The paclitaxel fractionation settling surface area increasing substance of the present invention may be porous silica and porous alumina; Or porous alumina.

When the surfactant is added to a solvent, the surfactant is added to facilitate the precipitation of the paclitaxel due to its increased surface area. The surfactant can be used to easily measure the zeta potential because the surfactant floats in the solvent. , Which can also control the zeta potential. Also, the surface increasing agent may be porous alumina; Or porous silica and porous alumina.

In addition, when the surface enhancer is added to a mixture containing paclitaxel, methanol and water, the zeta potential may be controlled to 0 to 40 mV, but is not limited thereto.

Furthermore, the porous alumina and the porous silica which can be used for the surface increasing agent are not particularly limited as long as they can be usually purchased and / or synthesized.

In addition, the surface enhancer may comprise a porous silica-alumina mixture; Or porous alumina can be used, and those prepared by a spray pyrolysis process can be used.

The spray pyrolysis process comprises the steps of preparing a spray solution by adding a precursor for forming pores to a solution of a silica precursor and an aluminum precursor or an aluminum precursor dissolved therein; Moving the spraying solution to a reaction part of a spray pyrolysis reactor including a drying part and a reaction part through a carrier gas after dropletization; And collecting the particles passing through the reaction part.

The drying unit may be divided into two zones, the temperature of the drying unit may be 200 to 400 ° C, and the temperature difference between one zone and two zones may be 40 to 60 ° C.

The temperature of the reaction part may be 550 to 650 ° C.

Further, the drying part and the reaction part may be those having an average diameter of 50 to 60 mm and a length of 1,000 to 1,500 mm.

The carrier gas is not particularly limited as long as it is capable of transporting a solution liquidized by a spray pyrolysis reactor, but it may be air.

The particles collected by the above-described method may further be calcined at 500 to 600 ° C for 3 to 5 hours.

The particles subjected to the calcination step may have an average surface area of 200 to 290 m ² / g, more preferably 220 to 260 m ² / g.

The pore volume average of the particles subjected to the sintering step may be 0.5 to 0.7 cm ³ / g, the pore average diameter may be 6.5 to 9.0 nm, and more preferably, the average pore diameter may be 7.0 - 9.0 nm.

If the surface enhancer is a porous silica-alumina mixture, the total concentration of the silica precursor and the aluminum precursor is 0.1 to 0.3 M, the solution containing the silica precursor 0.01 to 30 weight ratio and the aluminum precursor 70 to 99.99 weight ratio The surface-increasing agent can be produced through the spray pyrolysis process as described above.

If the surface area increasing material is porous alumina, a solution containing 0.2 M of an aluminum precursor may be used to prepare a spray pyrolysis process as described above.

Further, the present invention provides a method for purifying paclitaxel using the surface enhancer produced as described above.

The paclitaxel purification method of the present invention comprises the steps of: 1) preparing an extract from a mixture comprising Taxus genus plant-derived biomass and a water-soluble organic solvent; Mixing the extract with a nonpolar organic solvent, and then extracting the liquid to obtain a crude extract; Mixing the crude extract, the nonpolar organic solvent and the adsorbent, and then filtering to prepare a filtrate; Adding a mixed solution containing the filtrate and the nonpolar organic solvent to a non-aqueous organic solvent to produce a precipitate; Preparing a reaction solution containing the precipitate, the water-soluble organic solvent and the surface-increasing substance; And 6) storing the reaction solution at 1 to 10 ° C to obtain a paclitaxel precipitate; .

First, an extract is prepared from a mixture comprising plant-derived biomass and a water-soluble organic solvent in a taxus genus as a first step.

The plant belonging to the Taxus family is not particularly limited as long as it is a plant belonging to the Taxus family in the usual plant classification. For example, Taxus breather non polyamic (Taxus brevifolia), Taxus Kana den sheath (Taxus canadensis , Taxus cuspidata , Taxus baccata , Taxus globosa , Taxus floridana , Taxus wallichiana , Taxus media ) or Taxus chinensis ) can be used.

In addition, the biomass derived from the plant in the taxus is not particularly limited as long as it is a biomass derived from a plant belonging to the Taxus family, a cell thereof or a cell culture thereof.

Further, the water-soluble organic solvent is used for extracting paclitaxel from biomass, and is not particularly limited as long as it is usually used for extracting paclitaxel from biomass of a plant. Preferably, the organic solvent is a C ₁ to C ₅ alcohol and water And methanol, and more preferably, methanol may be used.

In addition, the extract may contain 70,000 to 90,000 parts by weight of a water-soluble organic solvent per 100 parts by weight of the biomass, but the present invention is not limited thereto.

In addition, the method for extracting the extract from the mixture is not particularly limited as long as it is a method for extracting the extract from a plant biomass. For example, it can be extracted by stirring at 20 to 28 ° C for 20 to 40 minutes. The extraction can be carried out once, preferably 2 to 4 times.

On the other hand, when the extraction liquid is mixed with a non-polar organic solvent without being concentrated, liquid-liquid extraction may proceed, and the efficiency of liquid-liquid extraction may be lowered due to the residual water. It may be more preferable to concentrate and use the extract.

Therefore, in one embodiment of the present invention, the extract can be used by concentrating. The concentration is not particularly limited as long as it is a method used for concentrating a liquid, but preferably a vacuum concentration method or a vacuum concentration method can be used, and more preferably, a vacuum concentration method can be used.

The reduced-pressure concentration can concentrate the extract to 20-50% of the stock solution.

The concentration under reduced pressure is not particularly limited as long as it is a condition commonly used for the concentration of the liquid under reduced pressure, but preferably 700 to 800 mmHg and 35 to 50 ° C.

Next, in step 2, a crude extract is prepared by mixing the extract and the nonpolar organic solvent, followed by liquid-liquid extraction.

The non-polar organic solvent serves to remove polar impurities contained in the starting paclitaxel through phase separation with a water-soluble organic solvent. The nonpolar organic solvent is not particularly limited as long as it is a nonpolar organic solvent used in liquid-liquid extraction, Chloride, ethyl acetate, and ether. More preferably, it may further include methylene chloride.

The extract may be used in an amount of 300 to 500 parts by weight based on 100 parts by weight of the nonpolar organic solvent.

If less than 300 parts by weight of the extract is mixed with 100 parts by weight of the nonpolar organic solvent, a large burden may be caused to remove the nonpolar organic solvent in the subsequent step. If the extract is mixed in an amount of more than 500 parts by weight based on 100 parts by weight of the nonpolar organic solvent, the extraction efficiency of the substance to be extracted may be lowered.

The liquid-liquid extraction can be performed one or more times, preferably two to four times.

Further, the crude extract is present in a non-polar organic solvent which is a lower layer of a water-soluble organic solvent in liquid-liquid extraction, and can be obtained by recovering the non-polar organic solvent layer.

The crude extract is not particularly limited as long as it is a method for obtaining an extract from an organic solvent. Preferably, the non-polar organic solvent can be obtained by concentration and / or drying at 20 to 28 ° C and 700 to 800 mmHg .

Next, in Step 3, the crude extract, the nonpolar organic solvent and the adsorbent are mixed and then filtered to prepare a filtrate.

The nonpolar organic solvent serves to remove tar and wax components from the crude extract and is not particularly limited as long as it is a nonpolar organic solvent used for removing plant-derived tar and wax components from plant cells. Preferably, the nonpolar organic solvent is methylene chloride , Ethyl acetate, and an ether. More preferably, it may further include methylene chloride.

The adsorbent is not particularly limited as long as it is usually used for removing plant-derived tar and wax components, but is preferably activated carbon, active clay, sylopute and silica (SiO ₂ ) May be included.

Further, the crude extract, the nonpolar organic solvent, and the adsorbent may be mixed with 240,000 to 800,000 parts by weight of a nonpolar organic solvent and 30 to 70 parts by weight of an adsorbent based on 100 parts by weight of the crude extract.

On the other hand, when the filtrate is mixed with a non-polar organic solvent without being dried and then added to a non-aqueous organic solvent, precipitation efficiency may be lowered due to the influence of the residual solvent, It may be more preferable to dry the filtrate than to carry out the step 4 without drying.

Therefore, in one embodiment of the present invention, the filtrate may be dried and used. The drying is not particularly limited as long as it is generally used for drying the liquid to prepare a dry sample. Preferably, the drying may be performed by a reduced-pressure drying method or a vacuum drying method, and more preferably, a reduced-pressure drying method may be used.

In addition, the reduced-pressure drying is not particularly limited as long as it is a condition used for producing a sample under reduced pressure by drying under reduced pressure, but it can be performed preferably at 700 to 800 mmHg and at 35 to 50 ° C for 20 to 50 minutes.

Next, in step 4, a mixed solution containing the filtrate and the nonpolar organic solvent is added to the water-insoluble organic solvent to prepare a precipitate.

The nonpolar organic solvent serves to remove the nonpolar impurities from the filtrate. The nonpolar organic solvent is not particularly limited as long as it is a nonpolar organic solvent used for removing nonpolar impurities from a plant-derived extract. Preferably, methylene chloride, ethyl acetate, Ether, and the like. More preferably, it may further include methylene chloride.

The water-insoluble organic solvent may be at least one selected from the group consisting of dichloromethane, carbon tetrachloride, chloroform, 1,2-dichloroethane, di-ethyl ether, And may include at least one member selected from the group consisting of methyl t-butyl ether, di-iso-propyl ether, hexane, and benzene. More preferably, it may further comprise a nucleic acid.

The mixing of the filtrate and the nonpolar organic solvent may include 100 to 1,000 parts by weight of the nonpolar organic solvent with respect to 100 parts by weight of the filtrate.

The mixed solution to be added to the water-insoluble organic solvent may be added at a ratio of water-insoluble organic solvent: mixed solution = 100: 5 to 20 (v / v).

On the other hand, when the precipitate is mixed with a water-soluble organic solvent without drying, the precipitation efficiency may be lowered due to the residual solvent, so that the precipitate is dried It may be more preferable to use it.

Thus, in one embodiment of the present invention, the precipitate may be dried and used. The drying is not particularly limited as long as it is a method used for drying a sample. Preferably, vacuum drying or vacuum drying may be used, more preferably vacuum drying.

In addition, the vacuum drying is not particularly limited as long as it is a condition used for drying the sample under vacuum, but it may preferably be performed using a vacuum dryer for 18 to 30 hours.

Next, Step 5 is to prepare a reaction solution containing the precipitate, the water-soluble organic solvent and the surface-increasing substance.

The surface-increasing substance is added to a solvent to increase the surface area thereof and to facilitate the precipitation of paclitaxel. In addition, when a surface-increasing substance having a floating property in a solvent is used, measurement of the zeta potential can be very easy, and thereby the zeta potential can be controlled.

If an ion exchange resin or glass bead is used as the surface increasing material, it is difficult to measure the zeta potential because it can not float in a solvent, and the surface increasing material is not uniformly dispersed in the precipitation solution, have.

Therefore, preferably, the surface-increasing agent of the present invention produced by the above-mentioned method can be used, more preferably porous silica and porous alumina; Or porous alumina; may be used.

In addition, the water-soluble organic solvent is used for dissolving a precipitate to separate paclitaxel having a purity of 60% or more. Usually, the organic solvent is not particularly limited as long as it is an organic solvent used in fractional precipitation, but preferably a C ₁ to C ₅ alcohol And water, and more preferably, methanol and / or water.

If methanol and water are used as the water-soluble organic solvent, water containing 25 to 40 parts by weight of water per 20 to 50 parts by weight of water, preferably 100 parts by weight of methanol may be used per 100 parts by weight of methanol.

On the other hand, when methanol and water are used as the water-soluble organic solvent in the preparation of the reaction solution and the precipitate is mixed with methanol and water at once, the precipitate may contain a large amount of impurities and the precipitation efficiency (purity) may be lowered It is more preferable that the precipitate is first dissolved in one kind of the water-soluble organic solvent rather than the precipitate is mixed in one water-soluble organic solvent without dissolving it first.

Therefore, in one embodiment of the present invention, the precipitate can be used by dissolving it in one kind of a water-soluble organic solvent. The water-soluble organic solvent may be methanol or water, but it is preferable to add water to the solution after dissolving the precipitate in methanol to prepare a dissolving solution.

The mixing ratio of the methanol and the precipitate is not particularly limited, but it is preferable to prepare a solution containing the precipitate 1: methanol 150 to 300 (w / v).

Furthermore, the mixing ratio of the dissolving liquid and water is not particularly limited, but preferably water can be added until the concentration of the dissolving liquid becomes 55 to 70%.

Next, in step 6, the reaction solution is stored at 1 to 10 DEG C to obtain a paclitaxel precipitate.

The reaction solution can be stored at 1 to 10 ° C, preferably at 3 to 8 ° C, more preferably at 4 to 7 ° C.

The storage is carried out in order to induce the precipitation reaction of paclitaxel and can be carried out for 6 to 18 hours which is much shorter than 3 days, which is the time required for conventional fractionation.

The paclitaxel precipitate obtained above has an average diameter of 10 to 100 mu m and can exhibit high purity of 70 to 85%.

Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are intended to further illustrate the present invention, and the scope of the present invention is not limited to these examples.

[ Example ]

Preparation Example . Biomass  Ready

The biomass used in this example was prepared using a plant cell culture solution. The plant cell culture solution contained Taxus After the induction the callus (callus) from the leaves of chinensis) to give a cell line (cell line) to give a cell suspension to the culture and suspended. The suspension cells were cultured in a darkness condition at 24 ° C with stirring at 150 rpm. The cultured suspension cells were cultured in a modified Gemborg's B5 medium, 30 g / l sucrose, 10 쨉 naphthalene acetic acid (NAA), 0.2 쨉 6-benzylamino (BA) purine, 1 g / l casein hydrolysate and 1 g / l 2- (N-morpholino) ethanesulfonic acid (MES). The cultivation was carried out every two weeks with a new medium, and 1.5% maltose was added on the 7th day and the 21st day to prolong production and culture, and as an elicitor, It was added to a 4 ㎛ silver nitrate (AgNO _3).

Plant cells and cell debris were recovered from the culture after decantation using a decanter (CA150 Claritying Decanter from Westpalia) and a high-speed centrifuge (using BTPX205GD-35CDEEP from Alpha-LaSalle). Hereinafter, recovered plant cells and cell fragments were used as biomass.

Example  1. Surface area Increase body  Produce.

Aluminum nitrate (Al (NO ₃ ) ₃ .9H ₂ O, manufactured by Aldrich) was used as the aluminum precursor, and tetraethyl orthosilicate (TEOS, 97% Were used. (Ethylene glycol) -block-poly (ethylene glycol) -block-poly (ethylene glycol) -block-poly (ethylene glycol) ); Product of Aldrich, product name: P123) was used.

For the preparation of the surface area increasing agent, the silica precursor was dissolved in water, the aluminum precursor was dissolved, and P123 was added to prepare spray solutions 1 and 2. Also, the total concentration of the aluminum precursor and silica was fixed at 0.2 M, and the molar ratio of aluminum P123 was fixed at 0.07. The molar ratio of the aluminum precursor to the silica precursor was changed to 100: 0 and 80: 20, and the molar ratio is shown in Table 1 below.

division Molar ratio (%) Aluminum precursor Silica precursor The surface-increasing agent 1 (spray solution 1) 100 0 The surface-increasing agent 2 (spray solution 2) 80 20

The spray solutions 1 and 2 were dropletized using an ultrasonic droplet generating apparatus (aerosol generator, six oscillators, 1.7 MHz, Dongrim Engineering), and then transferred into the reactor through a carrier gas. The carrier gas used was air and the flow rate was fixed at 10 L / min. The reactor of the drying part and the reaction part of the reactor was a quartz tube having a length of 1200 mm and a diameter of 55 mm. The drying unit was divided into two zones, each temperature was fixed at 300 ° C and 350 ° C, and the temperature of the reaction part was fixed at 600 ° C.

The particles passing through the reaction part were collected using a Teflon filter. In order to remove the organic substances remaining in the collected particles, they were removed by firing in an electric furnace. The temperature of the electric furnace was fired in an air atmosphere at 550 캜 (temperature raising rate: 1 캜 / min) for 4 hours to obtain surface enhancers 1 to 2.

Example  2. Paclitaxel  Fractional sedimentation

Biomass: methanol was mixed with 1: 1 (w / v) to mix the biomass and methanol prepared in the preparation example, followed by extraction at 25 ° C for 30 minutes to obtain an extract. The extracted extract was filtered, and fresh methanol was added to the filtered biomass, and extraction was repeated four times in the same manner as above. The filtrate was collected and added to a concentrator (CCA-1100, EYELA, Japan) and concentrated to 30% of the filtrate added at 40 ° C and under reduced pressure to prepare a concentrate. Thereafter, the concentrate and methylene chloride were mixed. The concentrate: methylene chloride was mixed at 1: 4 (v / v), and liquid-liquid extraction was performed for 30 minutes at 25 ° C for 3 times Lt; / RTI >

The polar impurities produced by the liquid-liquid extraction were removed from the upper methanol layer, and the paclitaxel was collected from the methylene chloride layer, concentrated under reduced pressure at 25 ° C, and dried to obtain crude extract.

In order to remove the plant-derived tar and wax components from the crude extract, crude extract and methylene chloride were mixed, and a crude extract solution was prepared by mixing crude extract: methylene chloride 1: 5 (v / w). Then, 50% (w / w) of the adsorbent sylopute (Fuji Silyv / a Chemical Ltd., Japan) was added to the crude extract solution and the mixture was reacted at 40 ° C for 30 minutes with stirring After filtration, a filtrate solution was obtained. The filtrate was dried at 30 ° C and under reduced pressure to prepare a dried sample.

The dried sample was dissolved in methylene chloride and then dropped on hexane to induce precipitation to remove non-polar impurities (methylene chloride / hexane = 1/10, v / v). Next, the precipitate from which the non-polar impurities were removed was filtered to recover the paclitaxel precipitate, and the recovered precipitate was vacuum dried (UP-2000, EYELA, Japan) for 24 hours.

For the fractional precipitation, the vacuum dried precipitate was dissolved in methanol (pure paclitaxel basis: 0.5%, w / v) and the precipitate was distilled under stirring (180 rpm) until the concentration of dissolved methanol was 61.5% water) was dropped one by one to prepare a reaction solution. The reaction solution was then added to the precipitate.

To increase the surface area per working volume (S / V, 1007.589 mm &^lt; ^{-1 >} ), the surface increasing agent 1 prepared in Example 1 was added to the precipitate to which the reaction solution was added, 1. The surface area was fixed at 1007.589 mm ^-1 and the yield and purity of paclitaxel were determined according to the fractionation time (6 hours, 12 hours, 18 hours, and 24 hours) in order to avoid the effect of surface area increase.

The precipitated solution 1 was stored in a low-temperature (4 to 7 ° C) temperature and humidity chamber (KCL-2000W, EYELA, Japan) to obtain paclitaxel precipitate 1. The paclitaxel precipitate 1 was filtered and dried in a vacuum oven (UP-2000, EYELA, Japan) at 35 ° C for 24 hours to prepare dry paclitaxel precipitate 1.

Example  3.

The dried paclitaxel precipitate was prepared in the same manner as in Example 2 except that the surface area increasing agent 2 prepared in Example 1 was added to the precipitate to which the reaction solution was added to prepare precipitate solution 2 from which dry paclitaxel precipitate 2 ≪ / RTI >

Comparative Example  One.

A dried paclitaxel precipitate was prepared in the same manner as in Example 2, except that the surface-increasing agent was not added to the precipitate to which the reaction solution was added.

Comparative Example  2 to 4

The surface-increasing agents 3 to 5 were prepared in the same manner as in Example 1 except that the molar ratio of the aluminum precursor to the silica precursor was changed to 50:50, 20:80, and 0: 100, and the molar ratios were shown in Table 2 below .

division Molar ratio (%) Aluminum precursor Silica precursor The surface-increasing agent 3 (spray solution 3) 50 50 The surface-increasing agent 4 (spray solution 4) 20 80 Surface area increasing body 5 (spray solution 5) 0 100

The dried paclitaxel precipitate was prepared in the same manner as in Example 2 except that the surface-increasing agent 3, the surface-increasing agent 4 or the surface-increasing agent 5 prepared above was added to the precipitate to which the reaction solution had been added, , Precipitation solution 4 or precipitation solution 5, respectively, from which dry precipitated paclitaxel 3, dried paclitaxel precipitate 4 or dried paclitaxel precipitate 5 was obtained.

Experimental Example  1. Surface area Increasing sieve  Property Analysis

Nitrogen adsorption / desorption isotherms of the surface increasing agents 1 to 2 prepared in Example 1 and the surface increasing agents 3 to 5 prepared in Comparative Examples 2 to 4 were pretreated at 200 ° C and then ASAP 2000 of Micrometrics Co. Lt; -1 > C. The surface area was calculated using adsorption data and the Brunauer-Emmett-Teller (BET) method at a relative pressure range of 0.06 to 0.3. The pore volume was calculated from the adsorption volume at a relative pressure of 0.995 and the pore size was calculated from the desorption isotherm using the Barrett-Joyner-Halenda (BJH) method in the range of 17-3000 Å. The calculated results are shown in Table 3.

division Average surface area (m 2 / g) Pore volume (cm < 3 > / g) Average diameter of pores (nm) Surface area increasing body 1 239 0.65 8.6 Surface area increasing body 2 253 0.50 7.1 Surface area increasing body 3 302 0.58 6.9 Surface area increasing body 4 411 0.62 6.0 Surface area increasing body 5 375 0.99 9.4

As can be seen in Table 3, the surface area increasing substances 1 to 5 were found to have no particular difference in the average surface area, the volume of the pores and the average diameter of the pores.

Experimental Example  2. Paclitaxel  Content analysis

For the analysis of paclitaxel contents, high-performance liquid chromatography of dried paclitaxel precipitates 1 to 5 and comparative dry paclitaxel precipitates prepared in Examples 2 to 3 and Comparative Examples 2 to 4 was carried out.

The high-performance liquid chromatography (HPLC) system was a HPLC system manufactured by Waters Corporation and a Capell Pak C18 (250 × 4.6 mm, Shiseido, Japan) column. The mobile phase was fed with a mixed solution of acetonitrile and distilled water (65/35 to 35/65, v / v, gradient mode) at a flow rate of 1.0 ml / min. The amount of sample injection was 20 μl and was detected by UV at 227 nm. A standard quantitative curve was used for HPLC analysis, and a standard sample was a Sigma-Aldrich product (purity: 95%). The measured paclitaxel yield is shown in Fig. 1, and the paclitaxel purity measured is shown in Fig.

As shown in Fig. 1, when the surface-increasing agent was added (Examples 2 to 3 and Comparative Examples 2 to 4), the yield was lower than that of the paclitaxel purification method (Comparative Example 1) 3 times more than the others.

In addition, when one of the surface enhancers 1 to 5 was used for increasing the surface area, the yield of paclitaxel increased with the precipitation time and the time required for fractional precipitation could be shortened.

Further, when the surface-increasing agent 1 or 2 was used (Example 2 or 3), the precipitation yield reached a steady state when fractional precipitation was carried out for 12 hours. However, when the surface-increasing agents 3 to 5 were used (Comparative Examples 2 to 4), when the fractional precipitation was carried out for 18 hours, the precipitation yield reached a steady state, and the fractional precipitation had to proceed for a longer time than the embodiment of the present invention .

In addition, the yield when the surface-increasing agent 1 or 2 was added at 12 hours of fractional precipitation (Example 2 or 3) was 76% at maximum, which was 3.3 times higher than 23% of Comparative Example 1 .

As can be seen in FIG. 2, the paclitaxel purity tended to be about 80% at the maximum, regardless of the change in the zeta potential depending on the surface-increasing agent.

Therefore, FIGS. 1 and 2 show that the addition of the surface enhancer 1 during the fractional precipitation is the most effective as the surface enhancer because it can obtain paclitaxel with high purity (81%) at the highest yield (76%). Further, in the case where the surface-increasing substance is added to the case where the surface-increasing substance is not added, the time required for fractional precipitation is drastically shortened to 18 hours (Comparative Examples 1 to 4) to 6 hours (Examples 2 to 3) Respectively.

Experimental Example  3. Zeta Potential and Paclitaxel  Analysis of association of manufacturing yield

The change of the zeta potential value was measured according to the mixing ratio of the silica precursor and the alumina precursor. The zeta potential values were measured by measuring changes in the zeta potential value between the surface-increasing agent and the reaction solution of the precipitating solutions 1 to 5 prepared in Examples 2 to 3 and Comparative Examples 2 to 4.

The zeta potentials of the above precipitating solutions 1 to 5 were measured using ELS-Z (Photal, Japan). The measured zeta potentials were verified using ELS-Z software, and the zeta potential values of the surface-increasing substances suspended in the solutions 1 to 5 were confirmed. The surface-increasing agents 1 to 5 can effectively float the precipitating solution, and the measurement of the zeta potential is very easy. The measured zeta potentials are shown in Table 4 below.

division The added surface enhancement substance Zeta potential (mV) Precipitation solution 1 Surface area increasing body 1 35.41 Precipitation solution 2 Surface area increasing body 2 8.16 Precipitation solution 3 Surface area increasing body 3 -23.08 Precipitation solution 4 Surface area increasing body 4 -23.95 Precipitation solution 5 Surface area increasing body 5 -21.57

The zeta potential values shown in Table 4 and the yield of paclitaxel after 6 hours of fractionation were plotted in FIG. 3 in order to examine the relationship between the zeta potential value and the yield according to the mixing ratio of the silica precursor and the alumina precursor.

As can be seen in FIG. 3, it was found that the zeta potential value increased as the alumina addition ratio increased, and that the yield of paclitaxel produced increased proportionally as the zeta potential value increased.

Therefore, when performing fractional precipitation for the same time, it was found that the addition of the surface area increasing substance 1 is most effective as the surface area increasing substance because paclitaxel can be produced with the highest yield (about 68%). Also, it was confirmed that the surface area increasing substance 2 prepared by adding silica and alumina at 20: 80 also improved the production yield of paclitaxel about twice as much as the surface area increasing substances 3 to 5.

Experimental Example  4. Analysis of sediment type and size

The paclitaxel precipitates 1 to 5 prepared in Examples 2 to 3 and Comparative Examples 2 to 4 were analyzed with an electron microscope (SV-35 Video Microscope System, Some Tech., Inc., Japan) to analyze the shape and size of the precipitate obtained through fractional precipitation. Korea).

The electron microscope observed paclitaxel precipitates at high magnification (x100). The observed paclitaxel precipitate was visualized in IT-Plus software (Some Tech., Korea), and the shape and size of the paclitaxel precipitate were confirmed. The results of the paclitaxel precipitate observed with an electron microscope according to the fractional precipitation time are shown in FIG. 5 shows a correlation graph between the mean diameter of the paclitaxel precipitate observed by the above electron microscope and the settling time by fraction.

As can be seen in Fig. 4, the paclitaxel precipitate grows in branches around the nucleus and has a needle shape as a whole. However, as the zeta potential value increases to positive (+) value, a thinner needle type precipitate is formed, and as the negative value increases, it becomes thicker needle shape.

It was also found that the morphology of paclitaxel precipitates grows with increasing number of branches around the nucleus as the fractional precipitation time (6 ~ 12 hours) elapses. After 12 hours of fractional precipitation, the number of precipitates increases, And it became small.

As can be seen from FIG. 5, in the case of the same fractionation precipitation time, when the surface-increasing substance was added (Examples 2 to 3 and Comparative Examples 2 to 4) as compared with Comparative Example 1 in which the surface-increasing substance was not added, the size of the paclitaxel precipitate Which was significantly decreased.

Therefore, it was found that when performing fractional precipitation for the same time, addition of the surface-increasing agent 1 is most preferable for producing a paclitaxel precipitate having a small size.

In addition, the relationship between the particle size of the paclitaxel precipitate measured in the same fractionation time (18 hours) and the zeta potency measured in Experimental Example 3 is shown in FIG.

As can be seen in FIG. 6, the particle size of the paclitaxel precipitate was related to the zeta potential value of the solution depending on the addition of the surface area increasing agent. As the zeta potential value of the fractional precipitating solution increased, the particle size of the paclitaxel precipitate Of the total amount of water.

Generally, in the case of active pharmaceutical ingredient (API), it is aimed to increase the utilization rate by reducing the crystal size of the drug in the crystallization process. As the size of the crystal grains becomes smaller, the dissolution rate, uniformity of drug dispersion, and bioavailability of the formulation can be improved. In addition, the smaller the crystal grain size, the better the removal of residual water and residual solvent in the drying step after purification. In this respect, the particle size reduction of paclitaxel by the surface-increasing agent in the fractional precipitation process of the present invention may be useful in terms of utilization of pharmaceuticals.

Claims (12)

Taxus genus Plant-derived biomass and a water-soluble organic solvent to prepare an extract from the mixture;
Mixing the extract with a nonpolar organic solvent, and then extracting the liquid to obtain a crude extract;
Mixing the crude extract, the nonpolar organic solvent and the adsorbent, and then filtering to prepare a filtrate;
Adding a mixed solution containing the filtrate and the nonpolar organic solvent to a non-aqueous organic solvent to produce a precipitate;
Preparing a reaction solution containing the precipitate, the water-soluble organic solvent and the surface-increasing substance; And
Storing the reaction solution at 1 to 10 DEG C to obtain a paclitaxel precipitate;
Wherein the surface area increasing material comprises porous silica and porous alumina in a weight ratio of 0.01 to 20:80 to 99.99, or the surface area increasing material comprises only porous alumina,
Wherein the surface area increasing material has an average surface area of 220 to 260 m < 2 > / g, an average pore volume of the surface area increasing material is 0.5 to 0.7 cm < ³ > / g, an average diameter of the pores of the surface increasing material is 6.5 to 9.0 nm,
The yield of paclitaxel was 55% to 69% when the reaction solution of step 6 was fractionally precipitated for 6 hours, and the yield of paclitaxel was 70% to 85% when the reaction solution of step 6 was fractionally precipitated for 12 hours ≪ / RTI > The paclitaxel purification method according to claim 1, wherein the biomass derived from the plant belonging to the Taxus family is a biomass derived from a plant belonging to the Taxus family, a cell thereof or a cell culture thereof. The paclitaxel purification method according to claim 1, wherein the water-soluble organic solvent is at least _{one selected} from the group consisting of C ₁ to C ₅ alcohols and water. The paclitaxel purification method according to claim 1, wherein the nonpolar organic solvent is at least one member selected from the group consisting of methylene chloride, ethyl acetate and ether. The method of claim 1, wherein the water-insoluble organic solvent is selected from the group consisting of dichloromethane, carbon tetrachloride, chloroform, 1,2-dichloroethane, di-ethyl a paclitaxel tablet having at least one of the group consisting of ether, methyl t-butyl ether, di-iso-propyl ether, hexane and benzene, Way. delete The paclitaxel purification method according to claim 1, wherein the adsorbent is at least one selected from the group consisting of activated carbon, active clay, sylopute and silica (SiO ₂ ). The method of claim 1, wherein the reaction solution of step 6 is stored for 6 to 18 hours. delete A surface-increasing agent comprising porous silica and porous alumina in a weight ratio of 0.01 to 20: 80 to 99.99; Or a porous alumina;
Wherein the surface area increasing material has an average surface area of 220 to 260 m < 2 > / g, an average pore volume of the surface increasing material is 0.5 to 0.7 cm < ³ > / g, an average diameter of the pores of the surface increasing material is 6.5 to 9.0 nm
Wherein the surface enhancer is added to a mixture comprising paclitaxel, methanol and water to provide a paclitaxel fractionation settling surface area enhancement with a zeta potential of 0 to 40 mV. delete delete

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