KR19980078646A - Extraction of desorption from yew leaves - Google Patents

Abstract

The present invention relates to a method for extracting Taxol from the leaves of Taxus cuspidata, comprising the steps of: (a) extracting and concentrating the leaves of attention with methanol; and (b) a mixed solution of chloroform and distilled water. Concentrating the chloroform soluble portion after partitioning to (c) separating the concentrate obtained in (b) by column chromatography using strong anion exchange resin, (d) the active fraction obtained in (c) Purifying by normal phase column chromatography, (e) purifying the active fraction obtained in (d) by reverse phase column chromatography, and (f) purifying the active fraction obtained in (e) by a semi-prep high pressure liquid. A method comprising purification by means of chromatography (Semi-Prep HPLC), according to the method of the present invention, provides a very simple, efficient and economical separation of taxol from leaves of interest. can do.

Description

How to Extract Taxol from Yew Leaves

The present invention relates to a method of extracting Taxol from the leaves of Taxus cuspidata.

Taxol (TAXOL: paclitaxel) obtained from Taxus spp. Is an extremely complex diterpene-based taxane compound with more than 20 asymmetric centers. It is a known anticancer substance. Taxol research began in the 1960s with a large screening program of anticancer actives conducted by the US National Cancer Institute on 35,000 plant species, with extracts from the Pacificus bark (Taxus brevifolia) bark in various tumors, including leukemia and lung cancer. After being reported to exhibit strong activity against, the active material was isolated in 1969 by Wall et al., And in 1971 the structure of Taxol as the body of the active material was revealed (Eric K. Rowinsky et al., J. National Can.Inst., 82, 11247 (1990)).

Unlike conventional anticancer drugs, Colchicine or Vinca alkaloid, which decompose microtubules and show anticancer activity, Taxol promotes microtubule assembly and the next step, the decomposition process. Schiff et al. (Schiff, PB, J. Fant and SB Horwitz, Nature, 277, 665 (1979)) indicate that they have a specific mechanism of action that inhibits ovaries. It is mainly used for the treatment of cancer and breast cancer. According to the current NCI clinical trials, Taxol is known to treat 30% of ovarian cancer, 50% breast cancer, and 20% lung cancer among terminal cancer patients who cannot be treated with conventional anticancer drugs (David, G.). , et al., J. Nat. Prod., 53, 1 (1990)).

As such, the demand for taxol as an anticancer agent is rapidly increasing, but there is a lot of difficulties in the treatment of authorized ovarian cancer and breast cancer as well as various clinical experiments such as lung cancer and skin cancer due to the shortage of supply.

At present, the supply of taxol is dependent on about 0.02% of the native bark on the earth, but the content of the taxol is low and the plant is killed when the bark is removed. Supply is limited.

Taxol is a complex compound with a lot of subsidiary carbon, which was reported in 1994 by several research groups on total synthesis, but it seems difficult to produce economically by chemical synthesis. In addition, the cell culture of attention, which grows very slowly in nature, has many problems, such as slow growth of the cells and various browning of the cells, such as easily occurring cells (US Pat. No. 5,019,504). Therefore, efforts have been made to develop a method of directly extracting Taxol using leaves that can be obtained year-round without damaging the trees instead of the bark of yew.

Methods of separating Taxol from leaves of yew are reported in Korean Patent Publication Nos. 95-10903, 95-25034 and 97-1339. They insist on the method of extracting Taxol from the yew leaves, but in reality, it is a level that examines the Taxol content of the yew leaves and is not actually a method for separating or producing Taxol. In particular, there are no solutions to chlorophyll-based substances that have a decisive effect on the separability and economics in the actual production of Taxol from yew leaves. Without this solution, simple content investigation experiments may be possible, but the actual production is impossible. I don't recognize it at all.

The process of extracting and separating Taxol from leaves differs from the process of extracting and separating the bark of yeast, which is important for the separation of silica column chromatography, which is important for the separation of Taxol. It also acts as an obstacle and causes a fatal damage to the filling, which shortens the life of the filling, thereby increasing the production cost. Therefore, a technique for effectively treating chlorophyll-based materials in the process of separating Taxol from the leaves of attention should be developed.

It is an object of the present invention to provide an effective method for the separation and purification of Taxol from leaves of interest.

1 is a ¹ H-NMR spectrum of Taxol obtained by the method of Example 1. FIG.

2 is a ¹³ C-NMR spectrum of Taxol obtained by the method of Example 1. FIG.

3 is a result of analyzing the molecular weight of Taxol obtained by the method of Example 1 by ESI / MS.

4 shows the results of HPLC analysis of taxol obtained by the method of Example 1. FIG.

Fig. 5 is a chromatogram analyzed by HPLC of a sample of yew leaves extracted before treatment with an ion exchange resin.

Fig. 6 is a chromatogram analyzed by HPLC of a sample of yew leaves extracted after treatment with an ion exchange resin.

Taxol separation and purification method of the present invention

(a) extracting the leaves of yew with methanol and concentrating,

(b) distributing the concentrate obtained in (a) into a mixed solution of chloroform and distilled water, and then concentrating the chloroform soluble portion;

(c) separating the concentrate obtained in (b) by column chromatography using strong anion exchange resin,

(d) purifying the active fraction obtained in (c) by normal phase column chromatography,

(e) purifying the active fraction obtained in (d) by reverse phase column chromatography, and

(f) purifying the active fraction obtained in (e) by semi-prep high pressure liquid chromatography (Semi-Prep HPLC).

In step (a), 5 to 7 L of methanol is added per kg of dried yew leaves, followed by stirring at room temperature, preferably 20-40 ° C. for 1 to 5 hours, preferably 3 hours or more. By repeatedly extracting Taxol and related substances, the extract is concentrated under reduced pressure. Subsequently, in step (b), the concentrate is stirred and distributed together with the mixed solution of chloroform and distilled water, and then the distilled water layer containing polar impurities is removed, the chloroform layer is separated and concentrated under reduced pressure.

In step (c), the concentrate obtained in step (b) is purified by strong anion exchange resin column chromatography to remove chlorophyll pigments. At this time, as strong anion exchange resin, Amberlite IRA-416 (Rohm and Haas, USA), Permutit ESB (Permutit AG, Germany), and Dowex 2- as strong base anion exchange resin. x8 (Dow Chemical Co., USA) can be used and Amberlite IRA-416 is preferred. As the developing solvent, methanol at a concentration of 30 to 70%, preferably 40 to 60% can be used.

Then, in step (d), the active fractions obtained in step (c) are collected, concentrated and purified by column chromatography. In this case, as the filler, silica gel 60 (63-200 μm), silica gel 40 (63-200 μm) and silica gel 60 (200-500 μm) can be used, and silica gel 60 (63-200 μm) is preferably used. . As a developing solvent, a mixture of benzene: acetone, n-hexane: ethyl acetate, n-hexane: acetone can be used in various ways, and the mixing ratio thereof is 7: 1 (v / v) to 1: 1 (v / v). Various adjustments can be made within the range of. Preferably, the benzene: acetone mixed solvent is 7: 1 (v / v) to 1: 1 (v / v) and the n-hexane: ethyl acetate mixed solvent is 5: 1 (v / v) to 1: 1 (v / v) and n-hexane: acetone mixed solvents are used with varying ratios from 3: 1 (v / v) to 1: 1 (v / v).

The active fractions, including eluted taxol, are combined, concentrated and purified by reverse phase column chromatography in step (e). At this time, as a reverse phase filler, Lichroprep RP-18 (40-63 μm, Merck, USA), Lycroprep RP-2 (25-40 μm) or Lycroprep RP-8 (40-63) Μm), and it is preferred to use Lycroprep RP-18 (40-63 μm). Anhydrous methanol, methylene chloride or acetonitrile can be used as a developing solvent, It is preferable to use anhydrous methanol.

The eluted active fractions are concentrated and purified by high pressure liquid chromatography in the last step (f). At this time, SG 120 (15-30 micrometers, Shiseido, Japan) or AG 120 (15-30 micrometers) can be used as a filler, and it is preferable to use SG 120 (15-30 micrometers). As the developing solvent, it is preferable to use methanol at a concentration of 50 to 65%, preferably 55 to 60%.

The Taxol-containing fraction purified according to the above method is crystallized by recrystallization or dried in a lyophilizer to obtain Taxol as a white powder.

According to the method of the present invention, 80 to 100 mg of taxol can be obtained with about 98% or more purity from 1 kg of yew leaves.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and do not limit the present invention.

Example 1 Purification of Taxol from Yew Leaves

10 kg of dried Jejudo yew leaves were put in a 100 L extraction tank, 30 L of anhydrous methanol was added thereto, and stirred at room temperature for 3 hours to extract Taxol and related substances. This process was repeated twice. The methanol extract was concentrated in a 20 L vacuum concentrator, dissolved in 10 L chloroform, stirred with 10 L in distilled water, and the layers separated. The distilled water layer containing the polar impurity was removed, and the chloroform layer was concentrated under reduced pressure again and added to a glass column of 25 cm x 60 cm length filled with Amberlite IRA-416 ion exchange resin, and 300 ml of 40% methanol as the developing solvent. Ion exchange chromatography was performed at a flow rate of / min. The eluted samples were collected in 1 ml fractions and chlorophyll pigments were removed by taking only fractions 1-20.

The soluble portion fraction from which chlorophyll and the like was removed was concentrated and added to a glass column of 15 cm diameter x 50 cm length filled with silica gel 60 (70-230 mesh, manufactured by Merck), and a mixed solvent of benzene: acetone 7: was developed as a developing solvent. Primary separation purification was performed by passing through a column at a flow rate of 150 ml / min, changing from 1 (v / v) to 1: 1 (v / v). At this time, the eluted samples were collected in 500 ml fractions and fractions 63 to 75 were taken as active fractions. The active fractions were concentrated and added to a glass column of 3.1 cm in diameter and 25 cm in length filled with Lichroprep RP-18 (40-63 μm, available from Merck), and 50 mL / methanol as a developing solvent. Secondary separation purification was performed using the flow rate of minutes. The eluted samples were collected in 300 ml fractions and fractions 20 to 25 were taken as active fractions.

The active fractions were concentrated and separated and purified using the final step, Semi-Prep HPLC. At this time, the HPLC system was used by connecting the DC-1200 Fraction Collector, manufactured by Ayela, Japan, to SCL-8A HPLC, manufactured by Shimadzu, Japan, and the column having a diameter of 2 cm x length 20 A cm filled octadecyl silane (ODS) column (SG120 column, Shiseido, Japan) was used. As a developing solvent, 60% methanol was used at a flow rate of 20 ml / min. Eluted samples were collected in 20 ml fractions and fractions 32 to 37 were taken as active fractions.

The active fractions were lyophilized to afford 900 mg of 98.5% Taxol in the form of a white powder.

Example 2 Purification of Taxol from Yew Leaves

The chloroform layer obtained through methanol extraction and chloroform: distilled water distribution was concentrated under reduced pressure again from 10 kg of dried dried leaf of yew as in Example 1, except that 50% methanol was used as a developing solvent. In the same manner, the chlorophyll pigments were removed by ion exchange chromatography.

Except that the soluble portion from which chlorophyll and the like was removed was concentrated, except that the n-hexane: ethyl acetate mixed solvent was changed from 5: 1 (v / v) to 1: 1 (v / v) as a developing solvent. Primary separation and purification in the same manner as in 1, and secondary separation and purification in the same manner as in Example 1.

The active fractions were concentrated and semi-prep HPLC was carried out in the same manner as in Example 1 except that 55% methanol was used as the developing solvent.

The eluted active fraction was concentrated to about ½ and crystallized in a 0 ° C. cold room to yield 880 mg of 99.1% Taxol in the form of a white powder.

Example 3 Purification of Taxol from Yew Leaves

The chloroform layer obtained through methanol extraction and chloroform: distilled water distribution from 10 kg of the dried leaf of the yew leaves pulverized as in Example 1 was concentrated under reduced pressure, and then the same as in Example 1 except that 60% methanol was used as a developing solvent. Chlorophyll pigments were removed by ion exchange chromatography.

Concentrate the soluble part from which chlorophyll and the like has been removed, and then use it as a developing solvent except changing n-hexane: ethyl acetate mixed solvent from 3: 1 (v / v) to 1: 1 (v / v). Primary separation and purification in the same manner as in Example 1, and secondary separation and purification in the same manner as in Example 1.

The active fractions were concentrated and then subjected to Semi-Prep HPLC and crystallization as in Example 2 to give 960 mg of 98.7% Taxol in the form of white powder.

Example 4 Identification of Taxol by Instrumental Analysis

In order to confirm that the Taxol obtained by the method of the present invention is equivalent to a known Taxol, various instrumental analyzes were performed as follows using the Taxol obtained in Example 1.

1) NMR

¹ H and ¹³ C-NMR spectra were obtained at 600 MHz and the results are shown in FIGS. 1 and 2, respectively.

2) mass spectrometry

The results of molecular weight analysis by ESI / MS are shown in FIG. 3. As can be seen in FIG. 3, it was confirmed that the molecular weight of Taxol separated by the method of the present invention is 853 since [M + H] ⁺ peak is observed at m / z 854.3.

3) HPLC / UV Spectrum

The Taxol obtained in Example 1 was vacuum dried at 40 ° C. for 5 hours, and then HPLC was performed under the following conditions using a sample diluted with methanol, and the results are shown in FIG. 4:

Column: Kurosil-G, 6μ

250 x 3.2 mm

Mobile phase: A (10 mM ammonium acetate, pH 4.0): B (CH ₃ CN) = 55: 45

Isocratic elution.

Flow rate: 0.6 ml / min

Detection: UV, 228 nm

The results of the instrumental analysis were compared with those of commercially available standard Taxol (Sigma, USA), and it was confirmed that the Taxol separated by the method of the present invention is equivalent to commercial Taxol.

Example 5 Identification of Suitable Ion Exchange Resins

In the method of the present invention, the following experiment was conducted to identify the type of ion exchange resin capable of removing pigments such as chlorophyll most effectively in ion exchange chromatography.

Amberlite IR-120 (Rohm and Haas, USA) and Permutit RS (Permutit AG, Germany) were used as sulphonated polystyrene strong-acid cation exchanger, and carboxylic acid- Amberlite IRA-50 and Fermutite C as type weak acid cation exchanger, Amberlite IRA-416 and Fermutite ESB as strong anion exchange resin, and Amberlite IR-45 and Fermuti as weak anion exchange resin. E was used. Each ion exchange resin was packed into a column, washed and filled with 100% purified water, and then a vacuum concentrate of the chloroform layer obtained through methanol extraction and chloroform: distilled water partition was loaded from the dried leaf pulverized in Example 1. Methanol was added to the column at 5% intervals from 10% to 70% as the developing solvent, and the eluate was confirmed by HPLC.

As a result, the strong positive ion exchange resin, weak positive ion exchange resin, and weak anion exchange resin were not only good in taxol but also most colored colored impurities, which resulted in poor purification results. However, in the case of strong anion exchange resins, even when developed with 70% methanol, most colored impurities do not escape due to binding to the ion exchange resins, thereby solving the problem of pigment removal. When the concentration of methanol was 40 to 60% as a developing solvent, taxol was best escaped but colored impurities were not released.

Example 6 Check the Effect of Strong Anion Exchange Resin

In order to confirm the purification efficiency when using the strong anion exchange resin found to be the most effective in Example 5, the following experiment was carried out.

The vacuum concentrate from the dried leaf of yew leaves pulverized in Example 1 and the chloroform layer obtained through the distillation of chloroform: distilled water were concentrated and the eluate of this concentrate was purified by strong anion exchange chromatography as in Example 5, respectively. -G (Curosil-G, 6μ, 250 x 3.2 mm, manufactured by Phenomex), and 10 mM ammonium acetate (pH 4.0) and CH ₃ CN 50:50 mixed solvent as a developing solvent at a flow rate of 1.0 ml / min. Passed through and detected at UV 228 nm.

The results are shown in FIGS. 5 and 6, FIG. 5 is an HPLC chromatogram before treatment with an ion exchange resin, and FIG. 6 is an HPLC chromatogram after treatment with an ion exchange resin. Comparing the two HPLC chromatograms shows the effect of ion exchange resin treatment. From the starting point of the chromatogram to the 4-minute residence time peaks are the colored substances of the chlorophyll lineage. It can be seen that after the ion exchange resin treatment, the peaks in this area have been significantly reduced, and the relative ratio of Taxol in the 9.6-component range has more than doubled.

The method of the present invention greatly improves the degree of separation in silica gel column chromatography by effectively removing impurities such as chlorophyll by using an inexpensive ion exchange resin in the process of separating and purifying Taxol from the leaves of yew, thereby increasing productivity by substantially increasing the column throughput. In addition to significantly improving the efficiency of the column filler, it is very economical to reduce the production cost in mass production by extending the life of the column filler. In addition, by treating the yew leaf extract with an ion exchange resin, treatment with n-hexane, which is a pre-treatment step of yew leaf extraction, may be omitted, which is much simpler and more efficient than conventional separation and purification methods.

Claims (13)

In the method of extracting Taxol from the leaves of Taxus cuspidata, (a) extracting the leaves of attention from methanol and concentrating; (b) distributing the concentrate obtained in (a) into a mixed solution of chloroform and distilled water Then concentrating the chloroform soluble portion, (c) separating the concentrate obtained in (b) by column chromatography using strong anion exchange resin, and (d) the active fraction obtained in (c) is purified by column chromatography Purifying by chromatography, (e) purifying the active fraction obtained in (d) by reverse phase column chromatography, and (f) purifying the active fraction obtained in (e) by semi-prep high pressure liquid chromatography ( Purifying by Semi-Prep HPLC). The method of claim 1, Process using anhydrous methanol in step (a). The method of claim 1, Amberlite IRA-416 (Rohm and Haas, USA), Permutit ESB (Permutit AG, Germany) and Doexx 2-x8 as strong anion exchange resins in step (c). Dow Chemical Co., USA). The method of claim 1, Process using the concentration of 40 to 60% methanol as the developing solvent in step (c). The method of claim 1, Method of using a silica gel column in step (d). The method of claim 5, Silica gel 60 (63-200 μm), Silica gel 40 (63-200 μm) and Silica gel 60 (200-500 μm) are used as fillers for the silica gel column. The method of claim 1, Method of using a benzene: acetone mixed solvent from 7: 1 (v / v) to 1: 1 (v / v) to the developing solvent in step (d). The method of claim 1, Method of using a hexane: ethyl acetate mixed solvent from 5: 1 (v / v) to 1: 1 (v / v) as the developing solvent in step (d). The method of claim 1, Method of using a hexane: acetone mixed solvent from 31 (v / v) to 1: 1 (v / v) as the developing solvent in step (d). The method of claim 1, Lichroprep RP-18 (40-63 μm, Merck, USA), Lycroprep RP-2 (25-40 μm) or Lycroprep RP- as the filler of the reversed phase column in step (e). 8 (40-63 μm). The method of claim 1, Method of using anhydrous methanol as the developing solvent in the step (e). The method of claim 1, Using an octadecyl silane (ODS) column in step (f). The method of claim 1, Process using 50 to 65% methanol as the developing solvent in step (f).