KR100466734B1 - A extracting and refining method of taxol by using extracting solvent from fungi culture medium containing taxol - Google Patents

Abstract

The fungal culture named Pestalotia Heterocornis , which produces Taxol, is extracted and concentrated using an organic solvent such as dichloromethane, ethyl acetate, chloroform, and the concentrate is partitioned with purified water. Purification with cyclohexane: ethyl ether allows for simple preparation of taxols in remarkably high yields.

Description

Extraction and purification of Taxol by solvent extraction from fungal cultures containing Taxol {A EXTRACTING AND REFINING METHOD OF TAXOL BY USING EXTRACTING SOLVENT FROM FUNGI CULTURE MEDIUM CONTAINING TAXOL}

[Industrial use]

The present invention relates to a method for effectively extracting and purifying Taxol from a fungal culture containing Taxol, and more particularly, to Pestalotia heterocornis obtained from Taxus (Taxus) yew tree or the soil in which the tree grows. Pestalotia Heterocornis ) relates to a method for effectively extracting and purifying Taxol from a fungal culture called Pestalotia Heterocornis . On June 18, 1997, the fungus was deposited with the Korea Institute of Science and Technology Biotechnology Research Institute located at 52, Eeun-dong, Yuseong-gu, Daejeon, Korea, and received accession number KCTC 0340BP.

[Private Technology]

Taxol is a substance having excellent anticancer activity, and is a terpenoid-based compound contained in bark of yew as a substance known for treating leukemia and anticancer agent. same.

[Formula 1]

Taxol is a compound approved by the US Food and Drug Administration as a leukemia therapeutic agent and an anticancer agent, and is particularly a compound having excellent anticancer activity against breast cancer and ovarian cancer. The use of the above-mentioned Taxol structure, leukemia therapeutics and anticancer agents can be used in the journal of the American Chemical Society, May 1971, Vol. 93, No. 9, pages 2325-2327. In addition, the above-mentioned document also describes the alcohol extraction method of Taxol.

As mentioned above, Taxol can be extracted from the bark of the yew tree, but the yield obtained therefrom is extremely low, about 100 mg or less per kilogram of bark, and there is a risk of environmental destruction, and the production of the yew tree There is a problem that the supply is not desired because it depends entirely on growth.

In order to solve the above problems, U.S. Patent No. 4,924,011 discloses a chemical method for preparing taxol, but the chemical method requires a complicated process and thus has a problem of low economic efficiency.

Recently, in International Patent Application PCT / US 93/03416, University of Montana, plant pathologist Andrea Stierle et al., A fungus producing taxol from inner bark of yew trees. A method for separating Taxomyces andreanae is disclosed . However, the taxol produced by the fungus is only a small amount of 1-2 micrograms per liter. In addition, after extraction of the culture with dichloromethane, column chromatography using silica gel and prep-liquid chromatography (prep-LC) extraction and purification process is not economical.

The present invention has been made to solve the problems of the prior art as described above, the object of the present invention is to obtain a Taxol genus (Taxus) tree or obtained from the soil in which the tree grows, which can be easily produced in a significantly high yield The present invention provides a method for effectively extracting and purifying Taxol from a culture of fungi called Pestalotia Heterocornis , which produces Taxol.

In order to achieve the object of the present invention as described above, the present invention provides a method for extracting and purifying Taxol from a fungal culture named Pestalotia Heterocornis producing Taxol.

Here, it is preferable that the fungal culture further comprises using an organic solvent selected from the group consisting of dichloromethane, ethyl acetate, chloroform, and one or more thereof. In addition, the organic solvent layer from which the fungal cultures were extracted is concentrated to prepare a concentrate, the prepared concentrate is dissolved in chloroform, and the dissolved concentrate is added with 0.5 to 4 times the water of the glofoform. It is preferable to further include the step of, and further comprising the step of adding 1 to 5 times the cyclohexane: ethyl ether mixed in a ratio of 1: 1 to 1: 3 of the organic solvent layer purified with water. Do.

EXAMPLE

Representative Example

The fungus named Pestalotia Heterocornis is incubated in a fermentor, and then the culture is completely ground using a waring blender and extracted using an organic solvent such as dichloromethane, ethyl acetate, chloroform, and the like. At this time, the amount of the organic solvent used is preferably used in an amount of 1 to 3 times the volume of the mycelial culture. After repeating this operation 2 to 3 times, the organic solvent layer formed is concentrated under reduced pressure at a temperature of less than 35 ° C.

The concentrate is dissolved in 5 to 20 times the weight of the concentrate in chloroform, the insoluble suspended solids are filtered, and purified water is added to 0.5 to 4 times the amount of chloroform to separate the layers 2-3 times. The water layer is discarded and the chloroform layer is concentrated under reduced pressure at a temperature of less than 35 ° C. The concentrate was dissolved in 5 to 20 times the weight of the concentrate in ethanol: butanol = 1: 1, the insoluble suspended solids were filtered out, and cyclohexane: ethyl ether = 1: 1 to 1: 3 was added to ethanol: butanol = 1: 1. Add 5 to 5 times to separate layers 2-3 times. The cyclohexane layer was discarded and the ethanol layer was vacuum dried at 30 to 45 ° C.

The dry matter can be analyzed by the following method.

The thin layer chromatography is used to identify Taxol standards as comparative materials. In this case, a silica gel plate is used as the stationary phase, and chloroform / acetonitrile = 7: 3 (v / v) and hexane / ethyl acetate = 1: 3 (v / v) are used as the mobile phase. The Taxol standard and the dry matter are then dried on TLC to prepare a vanillin-sulphate spray and spray and confirm at a particular wavelength to determine whether the compound produced is Taxol.

In order to confirm Taxol more reliably, the dried material should be dissolved in methanol of high performance liquid chromatography class, and analyzed by high performance liquid chromatography, and then the retention time and the peak area should be checked in comparison with the standard and the peak. As a result of the check, when the peak appears at the same retention time as the Taxol standard, it can be determined whether the dry matter is Taxol.

Preferred Embodiment

A preferred embodiment of the present invention is described. However, the following examples are merely examples of the present invention showing the constitution and effects of the present invention, and the present invention is not limited to the following examples.

Example 1

Take 100 liters of the culture, which is called Pestalotia Heterocornis , and culture it for 3 weeks in a fermenter. = 1: 1 was added and vigorously stirred to separate the water-soluble and non-aqueous materials, and an organic solvent layer was taken. The separation operation of the water-soluble and water-insoluble material was further repeated twice. The organic solvent layer was collected and concentrated under reduced pressure at less than 35 ° C. to obtain 10 g of concentrate.

Example 2

The concentrate was dissolved in 100 ml of chloroform and the insoluble suspended solids were filtered and 400 ml of purified water was added to separate the layers. This operation was repeated once more. The water layer was discarded and the chloroform layer was concentrated under reduced pressure at a temperature of less than 35 ° C. The concentrated solution was dissolved in 100 ml of ethanol: butanol = 1: 1, the insoluble suspended solids were filtered off, and the layers were separated by adding 400 ml of cyclohexane: ethyl ether = 1: 2. This operation was repeated once more. The cyclohexane: ethyl ether layer was discarded and the ethanol: butanol layer was vacuum dried at 35 ° C.

Example 3

In order to determine whether or not the dried product prepared in the above example was Taxol was operated as follows.

Using thin layer chromatography, Taxol standard was identified as a comparative material. At this time, a silica gel 5 × 10 cm plate was used as the stationary phase, and chloroform / acetonitrile = 7: 3 (v / v) and hexane / ethyl acetate = 1: 3 (v / v) were used as the mobile phase. The Taxol standard and the dried product were dissolved in chloroform and developed in TLC, and then vanillin-sulfuric acid spray liquid was prepared and sprayed. Therefore, it could be determined that the dry matter was Taxol.

More accurate analysis was attempted using high performance liquid chromatography. As a result of comparing the peaks of the Taxol standard with the peaks of the dried material using a MicroBondapak column (μBondapak, C18), the peaks of Taxol appear at 14 minutes and 30 seconds, which is the same retention time. It was confirmed that the amount was 3.85 mg.

As described above, by extracting and purifying the culture solution obtained by culturing the fungus named Pestalotia Heterocornis according to the present invention by the above method, Taxol can be easily obtained in a remarkably high yield.

1 is a graph of High Performance Liquid Chromatography (HPLC) analysis of Taxol prepared according to the method of the present invention,

2 is a graph of HPLC analysis of Taxol standard.

Claims (3)

Treating dichloroethane: ethyl acetate with a Taxol-producing strain culture medium to obtain an organic solvent layer; Concentrating the organic solvent layer and then adding chloroform to remove suspended matter; Adding purified water to the mixture from which the float is removed to obtain a chloroform layer; Concentrating the chloroform layer under reduced pressure and adding ethanol: butanol (1: 1); Removing the suspension of the mixture and then adding a cyclohexane: ethyl ether (1: 1) solution and Obtaining an ethanol layer of the mixture; Taxol extraction method from taxol-producing strain comprising a. The method of claim 1, wherein the Taxol producing strain is Pestalotia Heterocornis . The method of claim 1 wherein the method is Treating Taxol-producing strain culture pulverized with 1 to 3 times dichloroethane: ethyl acetate to obtain an organic solvent layer; Concentrating the organic solvent layer and then adding chloroform 5 to 20 times the weight of the concentrate to remove suspended matter; Adding 0.5 to 4 times purified water of chloroform to the mixture from which the float is removed to obtain a chloroform layer; Concentrating the chloroform layer under reduced pressure and adding an ethanol: butanol (1: 1) solution of 5-20 times the weight of the concentrate; Removing the suspended solids of the mixture and adding a 1-5 times cyclohexane: ethyl ether (1: 1-1: 3) solution to the ethanol: butanol (1: 1) solution and Obtaining an ethanol layer of the mixture; Method for extracting Taxol from a taxa produced strain containing.

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