KR0150339B1 - A method for isolating taxol from taxus genus plant by fractional precipitation - Google Patents

Abstract

The present invention relates to a method for purifying Taxol from Taxus genus plants. More specifically, the present invention provides a high recovery rate by a simple process of pretreatment of the taxus plant, solvent extraction and fractional precipitation, to separate crude taxol, and to purify crude taxol by fractional precipitation. The present invention relates to a method for economically purifying high purity taxol. The method for purifying Taxol from the Taxus plant according to the present invention is a starting material of the Taxus plant sample, which is pulverized and powdered dried leaves or skins of the yeast, or the tissue cells of the yeast are cultured in a fermenter and the plant cells Using a cake-like sample of the plant cells obtained by the recovery, mixing and filtering an aqueous sodium chloride solution to a sample of the plant of the genus Taxus, adding methanol to stir and stirring, vacuum filtration and concentrating the filtrate under reduced pressure; Extracted by adding methylene chloride to the concentrate obtained in the previous step, followed by extracting with purified water, removing the water layer, and adding a mixed solvent of methyl-t-butyl ether, hexane, and cetylpyridinium chloride to the concentrated solution obtained by concentrating under reduced pressure. Removing and adding methyl-t-butyl ether to the water layer again and extracting to remove the water layer and concentrate under reduced pressure; Adding the concentrate obtained in the above step to hexane to recover the precipitate precipitate by centrifugation and drying to extract the crude taxol; And adding methanol to the regulator taxol obtained from the electrical process, dissolving the regulator taxol, adding purified water, leaving it for 1 to 2 days, and filtering and vacuum drying the filtrate.

Description

Method for Purifying Taxol from Taxus Plants by Fractional Precipitation

The present invention relates to a method for purifying Taxol from Taxus genus plants. More specifically, the present invention provides a high recovery rate by a simple process of pretreatment of the taxus plant, solvent extraction and fractional precipitation, to separate crude taxol, and to purify crude taxol by fractional precipitation. The present invention relates to a method for economically purifying high purity taxol.

Generally, taxane-based compounds have potent anti-tumor effects. Taxol is the first known compound with a taxane ring. Taxol is known to have significant effects on the treatment of leukemia and cancer and has antitumor effects. Recently, according to a clinical study conducted by the National Cancer Institute (NCI), about 30% of patients with ovarian cancer, It has been reported to be effective in treating about 50% of breast cancer patients and about 20% of lung cancer patients. In addition, baccatin III, 10-deacetylbaccatin III, 10-deacetyltaxol, cepharomanmannine and diacetylcepa having structures similar to taxol. Romancene (deacetylcephalomannine) is a derivative of taxol, some of which have important precursor and anti-tumor effects used for semisynthesis.

The structure of each of these taxane compounds is shown below.

On the other hand, as a method of manufacturing such a taxol, the total synthesis method, the semisynthesis method, the separation method, etc. are known.

However, as can be seen from the complex structure of Taxol, the total synthesis method, which is a method of synthesizing Taxol using available starting materials, was not economically available and had a very low yield, so it was limited in practical use in industry.

On the other hand, the semisynthetic method of synthesizing taxol using taxol precursors such as 10-diacetylbaccatin III requires a process of separating and purifying taxol precursors from plants and a multi-step process of converting purified taxol precursors into taxol. Falling had the problem.

Therefore, the separation method has been widely used in the Taxol manufacturing method due to the above problems, this separation method is a method of separating and purifying Taxol directly from the plant, it has the advantage that can be produced Taxol with efficacy as an anti-tumor treatment with economics Various attempts have been made to effectively separate high-purity taxols, and related arts are disclosed in various documents as follows.

WO 94/12268 discloses a method for separating and purifying taxol of high purity by purifying a solution containing a taxane solute using a semipermeable membrane and a reverse osmosis apparatus.

However, the above-described separation and purification method requires a high level of skill in operating the device because it uses a complex semipermeable membrane and a reverse osmosis device, and has a problem in that it is not possible to separate and purify taxol economically because an expensive device is used.

EP 553,780, on the other hand, dried and pulverized Taxus plants, extracted with methanol, extracted and concentrated in vacuo, diluted with water, diluted with cyclohexane, solvent extracted with methylene chloride and evaporated to evaporate the solvent. A method of separating the taxol and precursors by obtaining a crude taxol of purity and then purifying the crude taxol by silica gel column chromatography is disclosed.

In addition, WO 92/18492 further finely grinds plants, extracts and concentrates by adding methanol, concentrates, adds methylene chloride or ethyl acetate, and partitions to add water and washes to obtain low-purity crude taxol, which is crude taxol. To purify the silica gel into a normal phase liquid column.

WO 92/07842 discloses a method for pulverizing a plant, adding ethanol, chloroform and methanol to extract a low purity crude taxol, and then purifying the crude taxol using reverse phase liquid chromatography. .

WO 94/13827 discloses finely divided crude taxols by pulverizing the plants, adding a mixed extractant of ethanol, methanol and acetone, adding activated carbon or charcoal, extracting and purifying the crude taxol, and then converting the crude taxols into a silica gel normal liquid column. A method of purifying is disclosed.

In addition, Japanese Patent Application Laid-open No. Hei 6-157329 discloses a method for extracting a low-purity crude taxol by pulverizing a plant and extracting the solvent by adding ethyl acetate, ether, acetonitrile and acetone.

However, the conventional separation and purification method described above is mainly a method for purifying low-purity regulator taxol using chromatography, and the method described above is a terpenoid compound, lipid, which is structurally similar to taxol in the prepared taxol. Because it contains a large amount of complex mixtures such as (lipid), chlorophyll and phenol, it is impossible to obtain high-purity taxol even by multi-step elution using multiple columns when performing chromatography. He had the problem of burdening the poem column.

In addition, in the conventional separation and purification method, since the purity of the extracted crude taxol is low, the solubility of the sample in the solvent decreases when performing chromatography, and the recovery and purification efficiency of the taxol is poor and the yield is low. In order to obtain a crystalline taxol, an additional crystallization process is required, and thus, the conventional taxol separation and purification method has a limitation in practical use in industry.

As a result, an object of the present invention is to provide a method for purifying crystalline taxol of high purity from a plant sample of the taxus plant in an economical and high yield by a fractional precipitation process using a solubility difference of taxol.

Hereinafter, the purification method of the taxol according to the present invention will be described in more detail for each process.

In the present invention, the plant sample of the genus Taxus as a starting material for obtaining taxol is pulverized and powdered dried leaves or bark of the yeast, or the tissue cells of the yeast are cultured in a fermenter and the plant cells are recovered. A cake-like sample of plant cells obtained by using the same is used, and any substance obtained by the electric method can be efficiently used in the purification method of the taxol according to the present invention. In the present invention, the plants of the genus Taxus brevifolia (Taxus brevifolia), Taxus canadensis (Taxus canadensis), Taxus cuspidata (Taxus cuspidata), Taxus bacata (Taxus baccata), Taxus globosa, Taxus floridana, Taxus wallichiana, Taxus media, and Taxus chinensis are possible.

[Step 1]

Primary solvent extraction

An aqueous sodium chloride solution was added to the cake-like sample of the taxus plant, mixed, filtered, methanol was added, stirred for 10 to 30 minutes, vacuum filtered, and the filtrate was concentrated under reduced pressure at 30 to 40 ° C. At this time, the amount of the sodium chloride solution is preferably 50 to 200% (v / w) to the cake-like sample, the amount of methanol is preferably 50 to 200% (v / w) to the cake-like material, methanol The addition and vacuum filtration operation can be repeated several times as necessary. In addition, it is preferable to maintain the pressure at 15 to 40 in.Hg at the time of concentration under reduced pressure, and the concentration under reduced pressure is preferably performed until the volume of the concentrate to the cake material is 30 to 100% (v / w). On the other hand, when the concentration of the filtrate under reduced pressure is maintained at 40 ° C. or higher, epimerization of the taxol and taxol derivatives is promoted and high purity taxol cannot be obtained.

[Step 2]

Second solvent extraction

Methylene chloride was added to the concentrated solution obtained under reduced pressure, and the purified water was mixed and extracted to remove the water-soluble impurities present in the extract. The water layer was concentrated and concentrated under reduced pressure at 20 to 40 ° C. A mixed solvent of t-butyl ether, hexane, and cetylpyridinium chloride was added and extracted to remove the organic layer, and methyl-t-butyl ether was added to the water layer and extracted to remove the water layer. And concentrated under reduced pressure at 20 to 40 ℃. In this case, in order to remove the water-soluble impurities present in the water layer, purified water is added to the methyl-t-butyl ether before concentration under reduced pressure, mixed and extracted, and then a step of removing the water layer may be further included. In addition, the amount of methylene chloride added is preferably 20 to 100% (v / v) with respect to the concentrate obtained in the first step, the amount of purified water is preferably 5 to 20% (v / v), and the mixed solvent It is preferable that the addition amount is 50 to 100% (v / v). At this time, in order to effectively extract taxol and taxol derivatives from the concentrate, it is required to adjust the polarity of the solvent. For this purpose, the mixing ratio of each solvent in the aforementioned mixed solvent is methyl-t-butyl ether: hexane: cetylpyridinium chloride. It is preferable to maintain 2: 2: 1 by volume ratio. In addition, the amount of methyl t-butyl ether added to the water layer from which the organic layer is removed is preferably 40 to 60% (v / v) based on the concentrate obtained in the first step. In addition, the addition operation of each organic solvent and purified water in the process can be repeated as many times as necessary, the pressure is preferably maintained at 15 to 30 in.Hg when concentrated under reduced pressure, the concentrated under reduced pressure for the concentrate obtained in the first step Preferably, the final concentrate is made to have a volume of 0.5 to 5% (v / v).

[Step 3]

Precipitation and Centrifugation

The concentrate obtained from the above process is added to hexane to recover the precipitate precipitate by centrifugation and then dried to give crude taxol. In this case, in order to increase the purity of the adjuster taxol, centrifugation and methylene chloride are added to and dissolved in the dried precipitate, and then added to hexane to precipitate and centrifuged to recover the precipitate, followed by drying. Moreover, it is preferable that the addition amount of hexane is 300 to 500% (v / v) with respect to a concentrate.

In addition, in the extraction process of the regulator taxol, the addition of a solvent, vacuum filtration and precipitation may be repeated several times as necessary to improve the purity of the regulator taxol.

[Step 4]

Fractional precipitation

Methanol was added to the crude taxol obtained from the electrical process to dissolve the crude taxol, purified water was added, and the mixture was left at low temperature of 0 to 8 ° C. for 1 to 2 days while being filtered at low speed to uniformly precipitate crystals of the taxol and filtered. Was vacuum dried at 30 to 40 ° C. for 1 to 3 hours to obtain taxol crystals of the present invention. At this time, in order to increase the purity of the taxol may be further included a step of removing the water-soluble impurities dissolved in the filtrate by adding 10 to 60% aqueous methanol solution to the filtrate. In addition, the methanol addition amount is preferably 800 to 1,600% (v / w) with respect to the adjuster taxol, the addition amount of purified water is preferably 300 to 1,200% (v / w) with respect to the adjuster taxol, and the addition of purified water is small. It is preferable to add by dividing. In addition, the process can be repeated several times as necessary. According to the fractional precipitation of this process, taxol having a purity of about 70% or more from the crude taxol can be purified at a recovery rate of 90% or more by a single fractionation, and taxol having a purity of about 85% or more by two fractional precipitations. By, three times of fractional precipitation, crystalline taxol having a high purity of about 98% or more can be purified with a recovery rate of about 90% or more.

Quantitative Analysis

In order to measure the purity and recovery of taxol obtained from the electrical process, high-performance liquid chromatography (HPLC) was used to quantitatively analyze the conditions in Table 1 below.

According to the aforementioned taxol separation and purification method of the present invention, a crystalline taxol having a purity of about 70 to 99% or more can be obtained from the taxus plant by a simple process with a high yield of 90% or more.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1

Purification of Taxol from Samples Obtained from Dried Leaves or Peel of Yew

200 g of 10% sodium chloride aqueous solution was added to 200 g of cake-like sample obtained by pulverizing and pulverizing the dried leaf or bark of the powder, followed by filtration. Then, 200 ml of methanol was added and stirred for 20 minutes, followed by vacuum filtration. Each time was added three times and stirred for 20 minutes, and vacuum filtered. The filtrate was concentrated under reduced pressure at a pressure of 25 in.Hg and a temperature of 40.degree. C. to give a concentrated solution to 100 ml. Then, 20 ml of methylene chloride was added three times and extracted with 10 ml of purified water. The water layer was removed and 15 in. Hg was removed. It concentrated under reduced pressure at the pressure of and the temperature of 30 degreeC. A mixed solvent consisting of 25 ml of methyl-t-butyl ether, 25 ml of hexane and 12.5 ml of cetylpyridinium chloride in the concentrate was added and extracted to remove the organic layer, and 12.5 ml of methyl-t-butyl ether was added to the water layer again. The mixture was extracted and extracted to remove the water layer, and concentrated under reduced pressure to 1 ml at a pressure of 20 in.Hg and a temperature of 30 ° C. The concentrate was added to 4 ml of hexane, the precipitate precipitate was collected by centrifugation, and then dried to obtain crude taxol having a purity of 30%. 12.5 ml of methanol was added to 1 g of the crude taxol, and the crude taxol was dissolved. After stirring, 4.2 ml of purified water was added in small portions. The mixture was left at 4 ° C for 2 days with stirring, filtered using a filter having a diameter of 0.45 µm, and the filtrate was filtered. Vacuum drying at 2 ° C. for 2 hours yielded taxol crystals of the present invention. The obtained taxol was quantitatively analyzed by high performance liquid chromatography, and the purity of the taxol was found to be 73% and the yield was 98%.

Example 2

Extraction from Samples Obtained by Tissue Culture of Attention

Taxol crystals were obtained in the same manner as in Example 1, except that the tissue cells of interest were cultured in a fermenter and a cake-like sample obtained by collecting plant cells was used. Quantitative analysis of the obtained taxol using high performance liquid chromatography revealed that the taxol had a purity of 75% and a yield of 98%.

[Examples 3 to 4]

Take 200 mg of Taxol obtained in Examples 1 to 2, add 2.5 ml of methanol, add 1.5 ml of purified water in small portions while stirring, and leave it at 4 ° C for 1 day with stirring to use a filter having a diameter of 0.45 µm. After filtration and washing with 20% aqueous methanol solution was added to the filtrate, and vacuum-dried at 35 ℃ 2 hours to obtain the taxol crystal of the present invention. The obtained taxol was quantified using high performance liquid chromatography, and the purity of the taxol was found to be 85% and 88%, respectively, and the yields were 95% and 97%, respectively.

[Examples 5 to 6]

To 100 mg of Taxol obtained in Examples 3 to 4, 1.25 ml of methanol was added thereto, and 0.42 ml of purified water was added in small portions while stirring. The mixture was left to stand at 4 ° C. for 1 day while stirring, using a filter having a diameter of 0.45 µm. After filtration and washing with 20% aqueous methanol solution was added to the filtrate, and vacuum-dried at 35 ℃ 2 hours to obtain the taxol crystal of the present invention. The obtained taxol was quantified using high performance liquid chromatography, and the purity of the taxol was found to be 98% and 99%, respectively, and the yields were 90% and 91%, respectively.

As described and demonstrated in detail above, the crystalline taxol having a high purity of about 70% to 98% or more from the Taxus plant by the purification method of the present invention is economically and about 90% by a fractional precipitation process using a solubility difference of Taxol. It was confirmed that separation and purification were possible at the above high yield.

Claims (3)

(Iii) a mixture of aqueous sodium chloride solution to the sample of Taxus genus) plants, filtered, stirred for 10 to 30 minutes by adding methanol, followed by vacuum filtration and concentration of the filtrate under reduced pressure at 30 to 40 ° C; (Ii) Methylene chloride was added to the concentrate obtained in the above step, followed by extraction with purified water, followed by extraction. The aqueous layer was removed and concentrated under reduced pressure at 20 to 40 ° C. to methyl-t-butyl ether, hexane and cetylpyridinium chloride. The mixed solvent of 2: 2: 1 (v / v / v) was added and extracted to remove the organic layer, and methyl-t-butyl ether was added to the water layer, followed by extraction to remove the water layer, and again to methyl-t-butyl ether. Extracting by mixing purified water and removing the water layer to concentrate under reduced pressure at 20 to 40 ℃; (Iii) The concentrate obtained in the above process was added to hexane to recover the precipitate precipitated by centrifugation, dried, methylene chloride was added again, dissolved, added to hexane, precipitated and centrifuged, and the precipitate was dried to extract crude taxol. Process of doing; And (iii) 800 to 1,600% (v / w) of methanol was added to the adjuster taxol obtained in the electrical process to dissolve the adjuster taxol, and purified water was added to the adjuster taxol 300 to 1,200% (v / w). w) was added and left for 1 to 2 days at 0 to 8 ℃ and filtered, and then the filtrate was washed by adding 10 to 60% aqueous methanol solution to the filtrate, vacuum drying for 1 to 3 hours at 30 to 40 ℃ A method for purifying taxol from a taxus plant comprising a step of making. The Taxus plant according to claim 1, wherein the sample of the Taxus plant is a sample obtained by grinding and pulverizing dried dried leaves or bark of Yew, or a sample obtained by culturing tissue cells of Yew. Method for Purifying Taxol According to claim 1 or 2, Taxus plants include Taxus brevifolia, Taxus canadensis, Taxus cuspidata, Taxus bacata (Taxus baccata), Taxus globosa, Taxus floridana, Taxus wallichiana, Taxus media and Taxus chinensis. A method for purifying Taxol from Taxus plants, characterized in that it is selected from the group consisting of: