KR20170049353A - Extraction method of high purity cordycepin - Google Patents

Abstract

The present invention relates to an extraction method of high purity cordycepin and, more specifically, to an extraction method of high purity cordycepin, which extracts vegetable worms using an extraction solvent, and then can rapidly extract high purity cordycepin from the vegetable worms through performing a porous gel column chromatography and silica gel column chromatography.

Description

{Extraction method of high purity cordycepin}

The present invention relates to a method for extracting high purity cordycepin. More specifically, the present invention relates to a method for rapidly extracting high purity cordycepin from Cordyceps.

It is known that there are about 80 kinds of mushrooms in Korea (Mycologia, 50, 169 ~ 222, 1985) and 80 species in Korea (Kyo -Hak publishing co. Seoul, 13-18, 1996). Among them, Cordyceps sinensis ( Cordyceps sp.) And Cordyceps sp. ( Cordyceps sp.) Have been reported to be used as medicinal herbs from ancient times such as tuberculosis, asthma, drug addiction detoxification, B., 5, 253-260, 1940, Icons of Medicinal Fungi from China Science Press, China, p.575, 1989; USDA Agricultural Reserch Service, 1990). Typical cordyceps used for medicinal use are mainly Cordyceps sinensis), Cody sepseu Millie other leases (Cordyceps militaris), Soi to Cody Rhodes sepseu operational Ogle (Cordyceps ophioglossoides), Cody sepseu small barley Blow (Cordyceps sobolifera), Cody sepseu beret brother Beria (Cordyceps beauveria) or coordinating sepseu bassi Ana (Cordyceps bassiana ).

Cordycepin, which is the most important component of the Cordyceps, has been studied by Cunningham et al. In 1950, and many studies have been conducted on the pharmacological activity. Codisepin inhibits the synthesis of m-RNA and is known to have antibacterial, antifungal, immunological and anticancer effects (Nature, 166, 949-954, 1950; Cancer Res., 21, 216-220, 1961; Biochem. Biophys., Acta., 80, 640-647, 1964).

In recent years, studies on the inhibitory action of platelet aggregation of cordesiepin have been conducted. As an example of this, Korean Patent No. 464876 discloses an antithrombotic composition containing cordycepin of Cordyceps. Further, as a method for separating and purifying the codistin component, Korean Patent Laid-Open No. 2001-0054264 discloses codistepin for anticancer drug isolated from Cordyceps and its preparation method. However, a method for rapidly extracting cordycepsin of high purity from Cordyceps has yet to be disclosed.

A problem to be solved by the present invention is to provide a novel extraction method for obtaining high purity cordycepin in a short time.

In order to solve the above-described problems, the present invention provides a method for producing a Chinese cabbage, comprising: (s1) extracting a Chinese cabbage extract from Cordyceps; (s2) passing the Cordyceps cristatus extract through a porous gel column chromatography to obtain a cordycepin fraction; And (s3) purifying the above-mentioned cordierite fraction through silica gel column chromatography and then recrystallizing to obtain codistepin.

The present inventors have made intensive researches to overcome the problems of the prior art. As a result, the present inventors have extracted the Cordygotic Herb Extract using a specific extraction solvent and purified it by porous gel column chromatography and silica gel column chromatography, Codispin can be rapidly extracted. Thus, the present invention has been completed.

In the present invention, the extraction solvent in step (s1) may be water, an organic solvent or a mixture thereof. Preferably, the extraction solvent in step (s1) may be an organic solvent, and the organic solvent may be selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, and ethyl acetate. More preferably, the organic solvent may be ethanol, methanol or a mixture thereof. In this case, the content of cordycepin in the extract can be remarkably increased.

In the present invention, the extraction in step (s1) may be carried out by pulverizing Cordyceps, and extracting the extractant by shaking for 12 to 36 hours, preferably 16 to 32 hours, more preferably 20 to 28 hours. If the incubation time is less than 12 hours, the extract is not sufficiently extracted. If the incubation time is more than 36 hours, it takes too much time, which is uneconomical. After left to shake, the Cordyceps cristata extract is filtered using a filter paper, concentrated under reduced pressure, and then the extracting solvent is removed to obtain a purified Cordyceps cristatus extract.

Preferably, the porous gel column chromatography in the step (s2) may use a porous adsorbent as a filler, more preferably a hydrophobic gel such as styrene and divinyl benzene. Even more preferably, the copolymer of styrene and divinylbenzene may have a particle size of 0.250-850 탆 and a pore radius of 38-600 Å. Most preferably, the filler may be DIAION HP-20, MCI GEL CHP20P or Sepabeads SP850.

The DIAION HP-20 is a porous synthetic adsorbent that is a copolymer of styrene and divinylbenzene, and has a particle size of 0.250-850 μm and a pore radius of 200-300 Å. DIAION HP-20 has a large number of pores and high adsorption capacity due to high specific surface area. Due to its relatively large pore size, it is suitable for adsorption of large molecules (MW 1,500 or more) and is easy to elute the adsorbed material. DIAION HP-20 is hydrophobic on the surface of the particles, so that hydrophobic groups in organic molecules can be adsorbed when the organic material is adsorbed on the synthetic adsorbent.

The MCI GEL CHP20P is a porous synthetic adsorbent that is a copolymer of styrene and divinylbenzene. Its particle size is 75-150 탆 and its pore radius is 400-600 Å.

The Sepabeads SP850 is also a porous synthetic adsorbent that is a copolymer of styrene and divinylbenzene, has a particle size of 250-850 탆 and an average pore radius of 38 Å.

In the present invention, the step (s2) may be performed by adsorbing the Cordygotic Herb Extract on a column, followed by flowing ethanol or methanol, preferably ethanol, to obtain an eluate to obtain a cordysepine fraction. Preferably, the step (s2) may be performed by adsorbing the Cordyceps mellifera extract to a column, washing the column with water, and then eluting with ethanol or methanol, preferably ethanol. The ethanol may be used at a concentration of 10 to 50%, preferably 15 to 45%, more preferably 20 to 40%. In the case of using an ethanol solution as compared to other solvents, cordycepin is eluted at one time and no further elution step is required, so that the cordycepin extract can be rapidly obtained.

Preferably, the silica gel which can be used in the step (s3) may be a silica gel commonly used in the technical field of the present invention. For example, the silica gel may be obtained from Merck & / RTI > 107734 can be used, and the result of separation is the same as in the case of using other silica gel, but it is advantageous in terms of time. Merck no. 109385 can be used for 230-400 mesh ASTM column chromatography with silica gel with a particle size of 0.040-0.063 mm. Merck no. 107734 can be used for 70-230 mesh ASTM column chromatography with silica gel with a particle size of 0.063 to 0.200 mm.

In the present invention, the step (s3) may be carried out by adding dichloromethane / methanol / water in an amount of 5 to 9 / 0.5 to 3 / 0.3 to 1, preferably 6 to 8 / 0.7 to 2.5 / 0.4 to 0.6 (dichloromethane / methanol / water ) In a volume ratio of 1: 1. When the solvent is mixed in the volume ratio as described above, it is possible to more accurately separate the fraction of cordysepin than to use other solvents, thereby extracting the higher purity cordycepin.

After fractionation by silica gel column chromatography in step (s3), fractions containing codistepin can be identified by thin film chromatography (TLC). High purity cordycepin can be finally obtained through a recrystallization process in which a small fraction containing cordycepin is concentrated 2 to 4 times under reduced pressure, left at a low temperature, and filtered. Codisepin extracted by the above extraction method may have a high purity of 98% or more.

The present invention also provides a high purity cordycepin having a purity of 98% or more extracted by the above extraction method.

In the method of extracting cordycepin of the present invention, it is possible to rapidly extract cordycepsin of high purity from the Cordyceps sinensis extract through porous gel column chromatography and silica gel column chromatography by extracting cordyceps using an extraction solvent.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

Reference Example  1: Cordisceptin ( Cordycepin ) Material information

[Chemical Formula 1]

Material name: Cordycepin

CAS No .: 73-03-0

Molecular formula: C ₁₀ H ₁₃ N ₅ O ₃

Molecular Weight: 251.25

Example  1: Preparation of Cordyceps sinensis extract

50 g of Cordyceps was pulverized, and 1 liter of ethanol was added thereto, followed by shaking for one day. The extract was filtered using a filter paper, concentrated under reduced pressure, and then ethanol was removed to obtain an extract of Cordyceps militaris. The same amount of Cordyceps was pulverized and 1 liter of distilled water was added thereto, followed by stirring at 60 ° C for 3 hours. The extract was filtered using a filter paper and concentrated under reduced pressure to prepare a water extract of Cordyceps mellifera. The cordycepin content of the ethanol extracts and water extracts of the Chinese caterpillar fungus was measured by high performance liquid chromatography (HPLC). The results are shown in Table 1 below.

sample Codispein content (%) Cordyceps juice extract 0.95% Ethanol extract of Cordyceps 3.82%

As can be seen from the results of Table 1, it can be seen that when ethanol was used, the content of cordycepin was significantly increased about four times as compared with the method of extracting cordyceps using a conventional water as a solvent.

Example  2: step of obtaining a cordycepin fraction by porous gel chromatography

Porous gel column chromatography was carried out using the caterpillar powder ethanol extract prepared in Example 1.

The column was filled with DIAION HP-20 filler and washed several times with distilled water. Approximately 5 g of the ethanol extract of Cordyceps was dissolved in distilled water and adsorbed on a DIAION HP-20 column. After washing the column with about 300 ml of distilled water, 20% ethanol was flown to obtain a 20% ethanol eluate. Then, 40% ethanol was added to the eluate to obtain a 40% ethanol eluate.

The same amount of ethanol extract was dissolved in distilled water and adsorbed on a DIAION HP-20 column. After washing the column with about 300 ml of distilled water, 20% methanol was flowed to obtain a 20% methanol eluate, and 40% methanol was further flowed to obtain a 40% methanol eluate and each was concentrated under reduced pressure.

The content of cordycepin in the 20%, 40% ethanol fraction and 20% and 40% methanol fraction of the codisepin produced was measured by HPLC and the results are shown in Table 2 below.

sample Codispein content (%) yield 20% ethanol fraction 39.6 9.2% 40% ethanol fraction ND ^* 0.9% 20% methanol fraction 40.8 5.9% 40% methanol fraction 38.3 3.1%

^* ND: Not Detect

As can be seen from the results of Table 2 above, it can be seen that by performing the porous gel chromatography, a fraction containing a higher content of cordycepin can be obtained, which is faster than the known methods. There was no significant difference in the content and total yield when ethanol solution and methanol solution were used, but it was confirmed that when eluting with ethanol solution, cordisepin was eluted at one time. That is, when the ethanol solution is used, the separation step and the volume of the eluate can be reduced, so that the separation can be performed more rapidly.

Example  3: silica gel column  Step of obtaining high purity cordycepin by chromatography and recrystallization

Silica gel column chromatography was performed to obtain high purity cordycepin from a fraction containing 40% of cordycepin.

The silica gel (Merck No. 109385) was subjected to silica gel column chromatography by changing the composition of CH ₂ Cl ₂ / MeOH / H ₂ O (7/1 / 0.5 to 7/2 / 0.5) . The fractions containing codistepin were identified by thin film chromatography (TLC), and the small fraction 3 was concentrated under reduced pressure, left at a low temperature, and then filtered to obtain highly purified codistepin. The obtained cordine sebine content was measured by HPLC and the results are shown in Table 3 below.

sample Codispein content (%) Products manufactured by Codisepin 98.2 Codispefin standard (sigma) 100

As can be seen from the results of Table 3, when the content of the codispefin standard product purchased from Sigma was compared, it was confirmed that codisepin having a purity of 98% or more can be obtained through the extraction method of the present invention.

Claims (10)

(s1) extracting the Chinese cabbage extract from Cordyceps;
(s2) passing the Cordyceps cristatus extract through a porous gel column chromatography to obtain a cordycepin fraction; And
(s3) purifying the above-mentioned cordierite fraction through silica gel column chromatography and then recrystallizing to obtain cordycepin. The method of claim 1, wherein the step (s1) comprises extracting water with an extraction solvent using an organic solvent or a mixture thereof. [3] The method of claim 2, wherein the step (s1) comprises using an organic solvent such as methanol, ethanol or a mixture thereof as an extraction solvent. The method according to claim 1, wherein the extraction of step (s1) is performed by pulverizing Cordyceps, and extracting the extract by shaking for 12 to 36 hours in an extraction solvent. The method according to claim 1, wherein DIAION HP-20, MCI GEL CHP20P or Sepabeads SP850 is used as a filler in the porous gel column chromatography of step (s2). The method according to claim 1, wherein the step (s2) comprises adsorbing the Cordygotic Herb Extract on a column, followed by flowing ethanol or methanol to obtain an eluate. [7] The method of claim 6, wherein the step (s2) comprises adsorbing the Cordygotic Herb Extract on a column, followed by flowing ethanol to obtain an eluate. The method according to claim 1, wherein the step (s3) is performed using a solvent mixture of dichloromethane / methanol / water in a volume ratio of 5 to 9 / 0.5 to 3 / 0.3 to 1 (dichloromethane / methanol / water) Wherein the step of extracting the cordycepsin is carried out by a method comprising the steps of: The method according to claim 1, wherein the purity of cordycepin extracted by the extraction method is 98% or more. A high purity cordycepin having a purity of 98% or more extracted by the extraction method of claim 1.