KR20210053636A - How to prepare physiologically acceptable salts and nuts thereof - Google Patents

Abstract

The present invention relates to a method for preparing nuts made by extracting ergosterol epoxide from shiitake mushrooms and physiologically acceptable salts thereof, and more specifically, to a method for preparing nuts made by extracting ergosterol epoxide from shiitake mushrooms and physiologically acceptable salts thereof which can prepare a large amount of ergosterol epoxide in high yield by going through processes of extraction, concentration treatment, fractionation and purification with an organic solvent based on findings that the residues of shiitake mushrooms after an anticancer polysaccharide extraction process and the lower alcohol-soluble parts of shiitake mushrooms that are discarded in a pretreatment process for the extraction of anticancer polysaccharides have ergosterol epoxide in large amount which is widely used as raw materials for pharmaceuticals, etc., because of anti-cancer effect, whitening effect and anti-inflammatory activity thereof.

Description

TECHNICAL FIELD How to prepare physiologically acceptable salts and nuts thereof TECHNICAL FIELD

The present invention relates to a method for preparing nuts and physiologically acceptable salts thereof made by extracting ergosterol epoxide from shiitake mushrooms, and more particularly, to a method for preparing a shiitake mushroom residue and an anticancer polysaccharide after an anticancer polysaccharide extraction process. It was discovered that a large amount of ergosterol epoxide, which is widely used as a raw material for pharmaceuticals, is present in the lower alcohol-soluble part of shiitake mushrooms discarded in the pretreatment process for the extraction of mushrooms because of its anticancer effect, whitening effect and anti-inflammatory activity. It relates to a method for producing physiologically acceptable salts and nuts thereof, capable of producing a large amount of ergosterol epoxide in high yield by undergoing extraction, concentration treatment, fractionation, and purification by means of.

Shiitake (Lentinus edodes) is a mushroom belonging to the Basidiomycete Pleurotus oyster family, and unlike plants, it cannot assimilate carbon by chloroplasts, so it is parasitic on endemic trees and old trees. Since ancient times, shiitake has been called by various names such as Hyangsim, Margo, and oak mushrooms. In China, it is called changu and in Japan it is called Shiitake. When expressing altitude in English, oak mushrooms are used, or Shiitake, the Japanese pronunciation of altitude, is used.

The origin of shiitake mushrooms is estimated to be China, and since ancient times, it has been eaten in Eastern countries such as China, Korea, and Japan for its unique taste and aroma. It contains a nucleic acid seasoning called guanylic acid, which gives it a unique fragrance and umami. Lidademin lowers cholesterol and contains a large amount of vitamins B1, B2 and D. It is a health food containing an anticancer component called Lentinan. It has lower blood cholesterol, anticancer action, lower blood pressure, antiviral action, etc., and is recognized as a relatively high-grade health supplement or health food as well as a function as a food for survival, and consumption is increasing year by year as income increases.

Ergosterol epoxide is a kind of ergosterol derivative, which is a kind of fungal sterol, and its physiological activity is reported to have an immune function to suppress allergic diseases in humans [Kreisel et al., 1990, Zentralbl Mikrobiol 145; 393-396], antioxidant activity [Kim Sang-wook et al., 1999, Bull Korean Chem Soc 20; 819-823], inhibitory effect on the proliferation of influenza virus [Lindequist et al., 1989, Pharmazie 44; 579-580], reported that ergosterol epoxide isolated from chlorella strongly inhibited the carcinogenic effect of TPA [Yasukawa et al., 1996, Biol Pharm Bull 19; 573-576].

Accordingly, the inventors of the present invention observed that the extract of shiitake mushrooms exhibited anticancer activity as a result of research and efforts to find a substance having anticancer activity for shiitake mushrooms, and through the separation and identification of fractions and active ingredients according to the activity, It was revealed that the anticancer activity was based on ergosterol epoxide, and the ergosterol epoxide was found in a large amount in the soluble portion of lower alcohol such as methanol and ethanol, which was discarded during the purification of the remaining fractions and immune-enhancing polysaccharides after removing the immune enhancing polysaccharide extracts The present invention was completed by developing a technology capable of separating and purifying high-purity ergosterol epoxide with high added value as a raw material for pharmaceuticals and the like by discovering that it exists and using it.

Accordingly, the present invention provides ergosterol epoxide and physiologically acceptable salts thereof in the lower alcohol-soluble site of shiitake mushrooms discarded in the pretreatment process for the extraction of anticancer polysaccharides and residues of shiitake mushrooms after the anticancer polysaccharide extraction process. Its purpose is to provide a method of separation.

As described above, the method of separating ergosterol epoxide from shiitake mushrooms of the present invention is the residue of shiitake mushrooms after the extraction process of anticancer polysaccharides and the lower alcohol-soluble part of shiitake mushrooms discarded in the pretreatment process for extraction of anticancer polysaccharides. It has anti-cancer effect, whitening effect and anti-inflammatory activity, so it is discovered that a large amount of ergosterol epoxide, which is widely used as a raw material for pharmaceuticals, is present, and it is extracted, fractionated, and purified using the same to obtain a large amount of ergosterol epoxide. Can be effectively separated and purified.

The present invention is an effective method for obtaining high-purity ergosterol epoxides and physiologically acceptable salts thereof in large quantities, and since it can be obtained from the lower alcohol soluble portion obtained from the isolation process of immune-enhancing polysaccharides of shiitake mushrooms, it is very low. Useful materials can be obtained at cost.

1A is a proton NMR spectrum of ergosterol epoxide isolated from shiitake mushroom powder,
1b is a carbon NMR spectrum of ergosterol epoxide isolated from shiitake mushroom powder,
2 is an exploded view showing a process of separating ergosterol epoxide from shiitake mushrooms by a method according to the present invention.

The present invention is a first step of obtaining an organic solvent extract by extracting the anticancer polysaccharide extraction residue of shiitake mushrooms or shiitake mushrooms with an organic solvent; A second step of concentrating the organic solvent extract and fractionating with hexane and water to obtain a hexane layer extract; A third step of dissolving the hexane layer extract in a mixed solvent of hexane and ethyl acetate and then performing silica gel column chromatography to collect active fractions; A fourth step of dissolving the active fraction in a mixed solvent of hexane and ether and then performing silica gel column chromatography to collect the active fraction; And a fifth step of recrystallizing the active fraction of the fourth step using petroleum ether and purifying it using high performance liquid chromatography (HPLC) to obtain a compound represented by the following Formula 1 or a physiologically acceptable salt thereof. It features a method of preparing an ergosterol epoxide or a physiologically acceptable salt thereof.

Formula 1

The present invention will be described in more detail as follows.

The present invention has anticancer effect, whitening effect, and anti-inflammatory activity in the residue of shiitake mushrooms after the anticancer polysaccharide extraction process and the lower alcohol-soluble part of shiitake mushrooms discarded in the pretreatment process for the extraction of anticancer polysaccharides. Shiitake Mushroom, which can produce a large amount of ergosterol epoxide with high yield by discovering that there is a large amount of ergosterol epoxide, which is widely used as an organic solvent, and goes through the processes of extraction, concentration treatment, fractionation and purification with an organic solvent. From ergosterol epoxide and a physiologically acceptable salt thereof.

The ergosterol epoxide and physiologically acceptable salts thereof of the present invention can be obtained from shiitake mushrooms, and in particular, alcohol-soluble sites discarded in the process of isolation of immune-enhancing polysaccharides from mushrooms are preferred.

The ergosterol epoxide targeted in the present invention is a substance represented by the following formula (1). In addition, physiologically acceptable salts thereof are derived from the hydroxyl group (-OH) in the formula (1), and the ergosterol epoxide obtained by the method of the present invention and contained in shiitake mushrooms is used in a basic medium and/or basic. Includes all that are formed by treatment with a substance. The type of salt is not limited to a special one as long as it can be used in a pharmaceutical composition, cosmetic, food or the like.

[Formula 1]

The method for preparing nuts and physiologically acceptable salts made by extracting the ergosterol epoxide component from shiitake mushrooms of the present invention is to dry shiitake mushrooms, pulverize them, extract them with an organic solvent, and concentrate the extract, followed by chromatography. It consists of a process of purifying ergosterol epoxide by separating it with other separation devices. Preferably, it comprises the step of concentrating the alcohol-soluble portion obtained in the process of separating the immune enhancing polysaccharide of shiitake mushrooms, and treating the obtained extract and/or extract with an acidic medium and/or an acidic substance, or a basic medium and/or a basic substance. It consists of including the step of processing.

Each step of the method of separating ergosterol epoxide and physiologically acceptable salts thereof from shiitake mushrooms will be described separately.

First, the shiitake mushrooms are collected, pulverized, and then extracted with an extraction solvent.

Extraction can be carried out using water and/or an organic solvent. The extraction treatment may be repeatedly performed by the same method, may be performed in combination with other extraction methods, or extraction treatments in different solvents or the like may be combined. The method and conditions of the extraction treatment are not particularly limited, but in the case of an organic solvent, either a hydrophilic organic solvent or a hydrophobic organic solvent may be used.

Hydrophilic organic solvents have the advantage of excellent permeability to tissues and excellent extraction efficiency, but it is more preferable to use a hydrophilic solvent. In addition, a mixture of chloroform and methanol and/or ethanol, which is excellent in the solubility of oleanolic acid and/or ursolic acid, is preferably one or two or more selected from the group consisting of ethanol and ethyl acetate. As the hydrophilic organic solvent, one or two or more types selected from the group consisting of alcohol, a mixture of chloroform and alcohol, and acetone can be preferably used. Hydrophobic organic solvents can also be used as extraction solvents. The hydrophobic organic solvent is preferably one or two or more selected from the group consisting of ethyl acetate, hexane, diethyl ether, and chloroform.

In addition, when the water and/or organic solvent is 50° C. or higher, preferably 60° C. or higher, the solubility is improved, and since the cells expand, it is preferably extracted, and can be extracted more preferably by extraction treatment under pressure. , The extraction efficiency is improved.

In the present invention, high purity ergosterol epoxide and physiologically acceptable salts thereof can be obtained in large amounts by concentrating and/or fractionating/purifying the extract and/or extract obtained by the extraction treatment. The concentration treatment may be repeated, or other concentration treatments may be combined. Similarly, the fractionation/purification treatment may be repeated, or other fractionation/purification treatments may be combined. Further, fractionation/purification treatment may be performed after the concentration treatment is performed, or concentration treatment may be performed after performing the fractionation/purification treatment, or fractionation/purification treatment may be performed after the concentration treatment and then concentration treatment may be performed again. Of course, combinations other than those described above may be used.

Concentration treatment includes soluble matter recovery treatment and/or insoluble matter recovery treatment using solubility in water and/or organic solvent, liquid-liquid distribution treatment using water-hydrophobic organic solvent, recrystallization treatment, reprecipitation treatment, and cooling. Treatment of recovering the generated precipitate may be mentioned, and examples of the fractionation/purification treatment include recrystallization, reprecipitation, purification by normal and/or reverse phase chromatography, decolorization treatment, and deodorization treatment.

The combination of extraction treatment and concentration treatment and/or fractionation/purification treatment is not particularly limited, for example, after extracting shiitake mushrooms with water and/or a hydrophilic organic solvent, and then part or all of the hydrophilic organic solvent with respect to the obtained extract. Is removed, water is added and stirred as necessary, and the water-insoluble component precipitated in the water layer is recovered and concentrated. The precipitated water-insoluble matter can be recovered by filtration or centrifugation. However, in order to improve the recovery efficiency, water may be added to the aqueous solution, or agitation may be performed as needed. In addition, for the dry extract from which the water and/or hydrophilic organic solvent of the extract obtained from shiitake mushrooms has been removed, water is added and stirred in the same manner as above, and the water-insoluble content is recovered by filtration. can do. By fractionating and purifying these concentrates by normal and/or reverse phase chromatography and/or recrystallization, highly purified ergosterol epoxide and physiologically acceptable salts thereof can be obtained.

According to the production method of the present invention, ergosterol epoxide and physiologically acceptable salts thereof can be obtained in an amount of 85 to 100% in total. In addition, by repeating each step, the combined purity of ergosterol epoxide and physiologically acceptable salts thereof can be obtained with high purity of 90 to 100% or 95 to 100%.

Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to the examples.

Example: Method for separating ergosterol epoxide from shiitake mushrooms

Extraction was repeated twice by immersing 1 kg of shiitake powder in 10 L of 80% ethanol for 3 days, followed by concentration using a vacuum rotary concentrator. The shiitake mushroom extract was fractionated using hexane and water, and the hexane layer extract of which the volume ratio of hexane and ethyl acetate was 99: 1, 86: 14, 74: 26, 62: 38, 50: 50, 38: 62, By gradually increasing the concentration of ethyl acetate to 26:74, 14:86 and 1:99, silica gel column chromatography was performed to obtain 9 fractions. The 6th fraction was again subjected to silica gel column chromatography while gradually increasing the concentration of the ether to the volume ratio of hexane and ether to 99:1, 80:20, 60:40, 40:60, 20:80 and 1:99. It was divided into 6 small fractions, and the fourth small fraction was recrystallized using petroleum ether, and white crystals were separated as a result of purification using high performance liquid chromatography (HPLC). As a result of confirming through the proton NMR spectrum of FIG. 1a and the carbon NMR spectrum of FIG. 1b, it was confirmed that the white crystal was ergosterol epoxide. Through this separation process, 1.1 g of ergosterol epoxide having a purity of 99% or more was extracted from 1 kg of shiitake mushroom powder, and the above process is illustrated in FIG. 2.

Claims (2)

A first step of extracting the anticancer polysaccharide extraction residue of shiitake mushrooms or shiitake mushrooms with an organic solvent to obtain an organic solvent extract;
A second step of concentrating the organic solvent extract and fractionating with hexane and water to obtain a hexane layer extract;
A third step of dissolving the hexane layer extract in a mixed solvent of hexane and ethyl acetate and then performing silica gel column chromatography to collect active fractions;
A fourth step of dissolving the active fraction in a mixed solvent of hexane and ether and then performing silica gel column chromatography to collect the active fraction; And
A fifth step of recrystallizing the active fraction of the fourth step using petroleum ether and purifying it using high performance liquid chromatography (HPLC) to obtain a compound represented by the following formula (1) or a physiologically acceptable salt thereof;
A method for preparing an ergosterol epoxide or a physiologically acceptable salt thereof, characterized in that it comprises a.
[Formula 1]

The method of claim 1, wherein the organic solvent in the first step is 80% ethanol.

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