KR20050100879A - Process for preparing 4-(3,4-dihydroxy-phenyl)-but-3-en-2-one from inonotus obliquus - Google Patents

Abstract

The present invention relates to a method for separating 4- (3,4-dihydroxy-phenyl) -but-3-en-2-one having antioxidant activity isolated from Chaga ( Inonotus Obliquus ), and more particularly. (A) dipping chaga in alcohol to separate the extract; (b) adding alkanes and water having 5 to 8 carbon atoms to the extract to separate the alkanes and the water; (c) extracting the water layer with ethyl acetate and fractionating to separate the ethyl acetate layer; (d) 4- (3,4-dihydroxy-phenyl) -but-3-ene-2, comprising the steps of sequentially separating the ethyl acetate layer by gel filtration chromatography and hydrophilic chromatography. It relates to a separation method of -on.

Separation method of 4- (3,4-dihydroxy-phenyl) -but-3-en-2-one in Chaga ( Inonotus Obliquus ) according to the present invention is characterized by 4- (3) having antioxidant activity from Chaga. , 4-Dihydroxy-phenyl) -but-3-en-2-one can be produced in high yield and low cost, and can be used as a raw material for functional foods and pharmaceutical materials, bioactive materials and cosmetics. It can be utilized. In addition, it is possible to obtain polysaccharides exhibiting immuno-enhancing activity contained in chaga and to separate 4- (3,4-dihydroxy-phenyl) -but-3-en-2-one from fractions containing the remaining low molecular weight substances. High industrial applicability.

Description

Separation method of 4- (3,4-dihydroxy-phenyl) -but-3-en-2-one from chaga {Process for preparing 4- (3,4-dihydroxy-phenyl) -but-3-en -2-one from Inonotus obliquus}

The present invention relates to a method for separating 4- (3,4-dihydroxy-phenyl) -but-3-en-2-one having antioxidant activity isolated from Chaga ( Inonotus Obliquus ), and specifically, antioxidant. 4- (3,4-dihydroxy-phenyl) -but-3-en-2-one having an active activity can be prepared from chaga mushroom in high yield and low cost, and it is also included in chaga mushroom. 4- (3,4-dihydroxy-phenyl) -but-3-en-2-one can be separated from the fraction containing the remaining low molecular weight material to obtain an active polysaccharide, thereby increasing the industrial applicability of 4- (3 And a method for separating, 4-dihydroxy-phenyl) -but-3-en-2-one.

Modern man is exposed to harmful environment such as air pollution, irregular diet, UV exposure, stress and disease, and it is difficult to eliminate oxidative stress caused by free radicals. Continuous exposure to these oxidative stresses leads to skin aging, sunburn and inflammation caused by sunlight, contact dermatitis, and wrinkles.

Free radicals are atoms or molecules that have one or more unpaired electrons and have the property of stabilizing electrons from other molecules. The lifetime of free radicals is very short, about a few thousandths of a second. For this reason, free radicals are very unstable and aggressive. Radicals that are involved in skin aging include hydroxyl radicals, superoxide radicals, and singlet oxygen. Hydrogen peroxide, which is easily broken down into hydroxyl radicals, is also involved in skin aging.

The hydroxyl radical has a strong oxidizing power and the reaction time is very short, 10-11 seconds. Hydroxyl radicals can react with any substance in the body, attack lipids to produce lipid peroxides, create abnormal crosslinks between proteins, and attack genes (DNA). Hydroxyl radicals are one of the main factors of aging.

Superoxide radicals attack urea and cell membranes, and in particular, attack and decompose unsaturated fatty acids, one of the major components of cell membranes, to alter cell membrane properties. Changes in the properties of the cell membrane lead to changes in the delivery system of substances through the cell membrane, thereby promoting aging. The breakdown of unsaturated fatty acids also results in a lipofucin, a yellowish substance, which makes the skin look dull yellowish and look older. Fatty acid attack of superoxide radicals proceeds continuously.

Free radicals, like hydroxyl radicals, are very aggressive radicals that change all the components of skin cells.

Hydrogen peroxide is less oxidizing than radicals but has a very strong reactivity. Hydrogen peroxide is highly diffused and easily penetrates the cell membrane, and when it comes into contact with the iron (Fe) component, it easily becomes a hydroxyl radical. Hydrogen peroxide attacks the DNA of the cell nucleus and reacts with proteins to denature or prevent them from functioning normally.

The human body is equipped with a defense system to prevent damage caused by free radicals. Vitamin E, vitamin C, superoxide dismutase (SOD), catalase and various kinds of oxidases (oxydase) and the like to eliminate free radicals.

However, the substance that can eliminate free radicals can be prevented or delayed by aging the skin by eliminating oxidative stress on the skin to modern people who cannot easily remove free radicals due to harmful environment. Conventionally, substances known as free radical scavengers include ascorbic acid, dibutyl hydroxyl toluene (BHT), DL-alpha-tocopherol, beta-carotene, β-carotene, Morine and gallic acid. However, such a conventional free radical scavenger is somewhat problematic in safety for use in food or cosmetics.

For this reason, the development of more safe and high free radical scavenging effect has emerged as an important task in the field of food and cosmetics, and much research is being conducted.

Among the natural antioxidants, the extracts of various spices, such as spearamint, marjoram, sweet basil, and gagyo, reported similar antioxidant effects to tocopherol and BHA (Butylated Hydroxyanisole). Nose (sesaminol), γ-tocopherol, trans-ferulic acid, bisepoxyligan homologues, and the like have been reported. Studies on the antioxidant effects from edible plants have shown that there is an antioxidant effect from the ethanol extract of Deodeok. Flavonoid puerarin was found to be an antioxidant from the roots, benzene-based antioxidants were isolated from edible seaweeds, salicylic acid, ρ-cumaric acid, Phenolic antioxidants such as gentisic acid and caffeic acid were isolated. In the methanol extract of rice hulls, flavonoid-based substances are present and they exhibit an antioxidant effect. The active substance was found to be C-glycosyl flavonoid. The ethanol extract of Rhus japonica, which is used as an herbal medicine, also showed a strong antioxidant effect. When used with tocopherol, synergistic effect was observed. In addition, various edible plants have been reported to have antioxidant activity in Schizandra chinensis, Kernel and mustard, Sansa.

Chaga is a mushroom that has been handed down to Siberia and Eastern Europe for a very long time, and has been scientifically analyzed and clinically tested for decades in Russia and Eastern Europe, where alternative medicine has developed. In particular, the efficacy of anticancer, antitumor and hypoglycemic effect have been widely known. Joan H. Winters et al. "Comparative Plant Chemistry Studies of Polish and American Variations of Chaga" (University of Florida, Department of Pharmacy, 1960) describes the anticancer effects of chaga, and Takashi Mizuno et al. And the antitumor and hypoglycemic activity of mycelia derived polysaccharides (International Jounal of Medicinal Mushrooms, Vol. 1, pp. 301-306, 1999) describe the efficacy of chaga against cancer and diabetes. In addition, it is known to be effective in AIDS and diseases of the gastrointestinal tract.

In general, in the case of edible mushrooms, you can grind the mushroom fruit body directly, but unlike ordinary edible mushrooms, edible mushrooms that have a unique inherent scent may require a deodorizing process, even though they are edible mushrooms. It is true that the fruit recovery is a fungi belonging to the woody fungi, and the recovery of the extract is low due to the limited extraction of substances in the cells. In order to overcome these problems, researches on hot water extraction method or organic solvent method have been conducted, and only hot water or solvent extracted ingredients have been reported to have anti-cancer, immune enhancement, hypertension and diabetes treatment effects.

In the case of chaga, hot water extraction, an organic solvent method and the like are used as a method of extracting pharmacological components from chaga. For example, Korean Patent Registration No. 385739 describes the preparation of mycelium or mycelium of mushrooms in powder and extracted from hot water to prepare health supplements containing mushrooms, or pharmacological components using organic solvents such as ethanol, methanol or butanol. The method of eluting and drying the organic solvent by evaporation under reduced pressure is described. In Korean Patent Publication No. 2003-65964, the hyphae of chaga mushrooms are treated with PBS solution, butanol, ethanol, ethyl acetate or acetone in order to extract ingredients effective for prostatic hyperplasia and the like. Extraction methods are described. Korean Patent Registration No. 346063 describes a hydrothermal extraction method for extracting anticancer immune polysaccharides from chaga.

However, the above known methods are intended to extract pharmacological components and anti-cancer polysaccharides related to prostatic hypertrophy from chaga, and there is no known method of extracting antioxidants such as the present invention. There is no disclosure regarding the separation and purification of only a single compound from the antioxidant active ingredients.

The present inventors have found a method to efficiently extract and separate 4- (3,4-dihydroxy-phenyl) -but-3-en-2-one, a natural substance with antioxidant activity, from chaga The present invention has been completed.

An object of the present invention is to provide a method for separating 4- (3,4-dihydroxy-phenyl) -but-3-en-2-one having antioxidant activity isolated from Chaga ( Inonotus Obliquus ) do.

Another object of the present invention is to provide 4- (3,4-dihydroxy-phenyl) -but-3-en-2-one isolated from chaga.

In order to achieve the above object, the present invention comprises the steps of immersing chaga mushroom in (a) alcohol to separate the extract; (b) adding alkanes and water having 5 to 8 carbon atoms to the extract to separate the alkanes and the water; (c) extracting the water layer with ethyl acetate and fractionating to separate the ethyl acetate layer; (d) sequentially separating the ethyl acetate layer by gel filtration chromatography and hydrophilic chromatography, thereby obtaining 4- (3,4-dihydroxy-phenyl) -but-3-en-2-one. It is characterized by providing a separation method and 4- (3,4-dihydroxy-phenyl) -but-3-en-2-one separated by the separation method.

Hereinafter, the present invention will be described in more detail.

The scientific name of chaga is Inonotus obliqua or Fuscoporia obliqua. In Russia it is called Chaga and in Japan it is called Kabanoanatake. Mycelium forms black on the surface, cracks vertically and horizontally, yellowish brown inside. Mycelium, a hard mass made by densely packed mycelia, is mainly used for medicinal purposes. The diameter of the bacterium is large, about 10-20 cm. The fruiting body has a thickness of about 2 to 8mm, a hole on the front, and several bristle bodies. Chaga occurs in birch (Betula sp.), In particular in birch (Betula platyphylla var. Japonica). It is a resilient and reflective parasitic mushroom that is attached to the birch tree like a black rock mass, and the birch tree gradually dies as the mass grows larger.

Chaga mushroom used in the present invention is by natural or artificial culture. Artificial culture can use both liquid culture and solid culture. In liquid culture, the mycelia are obtained by culturing chaga mushroom strain in a liquid medium containing glucose, peptone, and yeast extract as essential components for 3 to 30 days. In addition to the essential components, the medium may be added with inorganic substances such as dipotassium phosphate, magnesium sulfate, and calcium chloride, and trace elements such as iron sulfate, zinc sulfate, and copper sulfate to help the growth of the mycelium. The pH of the medium is adjusted to 5-7, the temperature of about 20 ℃ to 30 ℃. As a solid culture method, a wooden culture method is used, and chaga mushroom strains are cultured in wood to obtain fruiting bodies. Chaga mushroom mycelium cultured in malt extract medium is added to a medium in which rice bran is added to wood sawdust for seed production for wood culture. It is inoculated off and incubated for 20-30 days in 25 degreeC-30 degreeC dark conditions.

4- (3,4-dihydroxy-phenyl) -but-3-en-2-one targeted in the present invention is a substance represented by the following formula (1).

A method of separating 4- (3,4-dihydroxy-phenyl) -but-3-en-2-one in the present invention will be described in detail with reference to FIG. 1.

Chaga is immersed in alcohol, extracted twice in succession, filtered and concentrated. Preferably, the present invention may be a chaga mushroom powder or chaga mushroom powder or a fraction containing the remaining low-molecular substances obtained from the polysaccharide exhibiting the immuno-enhancing activity contained in chaga mushroom. In the present invention, the alcohol used as the solvent is preferably methanol, ethanol, propanol, butanol and the like, and more preferably methanol.

To the concentrated chaga mushroom extract, an equivalent amount of alkanes having 5 to 8 carbon atoms, preferably hexane and water, were separated by phase separation with a separatory filter to extract an ethyl acetate to obtain an ethyl acetate extract. In order to remove the residues and solvents contained in the extracted material, for example, separation, concentrators such as centrifuges and rotary evaporators can be used.

The extracted extract contains an antioxidant including 4- (3,4-dihydroxy-phenyl) -but-3-en-2-one and can be directly used for various applications as an antioxidant active composition. If high purity is required, further separation and purification is desirable.

Specifically, ethyl acetate extract is passed through gel filtration chromatography to obtain a fraction showing free radical scavenging activity by DPPH. As the filler of the gel filtration chromatography, dextran gel, polyacrylamide gel, agarose gel and the like can be used. The gel filtration chromatography may preferably be Sephadex H-20 column chromatography.

Subsequently, the material of this section was analyzed by hydrophilic high performance liquid chromatography using a reversed phase column having a stationary phase of C18. 4- (3,4-dihydroxy-phenyl) -but-3-ene-2 having excellent DPPH scavenging action Separate and purify the warm. The column was performed eluting at 1.5 ml / min with 30% acetonitrile solution.

The present inventors performed nuclear magnetic resonance hydrogen spectrum, nuclear magnetic resonance carbon spectrum, mass spectrometry for identification of the 4- (3,4-dihydroxy-phenyl) -but-3-en-2-one, Diffie H (1,1-diphenyl-2-picrylhydrazyl) was used to measure the antioxidant activity.

The present inventors have improved the yield of 4- (3,4-dihydroxy-phenyl) -but-3-en-2-one separated from chaga by the present invention by more than four times compared to the case of separating from mushrooms. .

4- (3,4-dihydroxy-phenyl) -but-3-en-2-one isolated by the composition of the present invention is excellent in antioxidant activity and useful for health promotion of the human body. As a food, it is preferable to use it as a health food supplement, a beverage additive, or a food additive, for the purpose of disease prevention and health promotion. In addition, it can be used as a prophylactic or therapeutic agent for diseases caused by the oxidative power of free radicals, especially adult diseases, aging.

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

Grind or crush 1 kg of chaga mushroom in a solid mass and immerse it in 10 liters of methanol for 10 days and extract it first, then immerse in 5 liters of methanol for 3 days and collect the second extract, concentrate by filtration and dry. A weight of 45 g was obtained. Equal amount of hexane and water were added to the concentrated chaga mushroom methanol extract, and the water layer obtained by phase separation with an aliquot was extracted with ethyl acetate to obtain 5.6 g of ethyl acetate extract.

Example 2

Sepadex L-20 Column Chromatography (Column Size: 2.8cm in Diameter, Height: 75cm, Solvent: Methanol, 14g Received) of 5.6g Chaga Ethyl Acetate Extract Shows Free Radical Scavenging Activity by DPI 0.4 g of fractions were obtained.

The material in this section was analyzed by high-performance liquid chromatography (YMC-Pack-ODS-AQ column 300 ㅧ 10mm ID, acetonitrile 30%, 1.5ml / min). Excellent separation was possible, and 42 mg of compound could be separated and purified. The present substance was identified to be 4- (3,4-dihydroxy-phenyl) -but-3-en-2-one by confirming the nuclear magnetic resonance hydrogen spectrum, the nuclear magnetic resonance carbon spectrum, and the mass spectrometry.

Example 3

DPPH (1,1-diphenyl-2-picrylhydrazyl), which contains free radicals in the molecule, can easily accept electrons or hydrogen radicals and become stable molecules, thereby measuring the efficacy of free radical scavenger. Therefore, an amount of 5 × 10 ⁻⁴ mol of DPPH solution was prepared in the separated sample, and after 15 to 20 minutes, the absorbance was measured at 517 nm to confirm the activity of the antioxidant reaction. Reaction of 4- (3,4-dihydroxy-phenyl) -but-3-en-2-one with DPPH solution showed better antioxidant activity than alpha-tocopherol at 517 nm (FIG. 2).

As described and demonstrated in detail above, the present invention provides 4- (3,4-dihydroxy-phenyl) -but-3-en-2-one having antioxidant activity in high yield and low cost from chaga. From this, it can be utilized for various purposes as a raw material of functional food and pharmaceutical materials, bioactive materials, cosmetics. In addition, it is possible to obtain polysaccharides exhibiting immuno-enhancing activity contained in chaga and to separate 4- (3,4-dihydroxy-phenyl) -but-3-en-2-one from fractions containing the remaining low molecular weight substances. High industrial applicability.

1 is a schematic diagram showing a process of separating 4- (3,4-dihydroxy-phenyl) -but-3-en-2-one from chaga.

 2 is a graph comparing the antioxidant activity of 4- (3,4-dihydroxy-phenyl) -but-3-en-2-one and alpha-tocopherol.

Claims (9)

(A) dipping chaga in alcohol to separate the extract; (b) adding alkanes and water having 5 to 8 carbon atoms to the extract to separate the alkanes and the water; (c) extracting the water layer with ethyl acetate and fractionating to separate the ethyl acetate layer; (d) 4- (3,4-dihydroxy-phenyl) -but-3-ene-2, comprising the steps of sequentially separating the ethyl acetate layer by gel filtration chromatography and hydrophilic chromatography. Method of separation of on-ion. 4. The 4- (3,4-dihydroxy-phenyl) -but- characterized in that chaga is a chaga mushroom powder or a fraction containing a low molecular weight substance obtained by obtaining a polysaccharide contained in chaga mushroom. Separation method of 3-en-2-one. The method of claim 1, wherein the alcohol is selected from the group consisting of methanol, ethanol, propanol and butanol. 4-(3,4-Dihydroxy-phenyl) -but-3-en-2-one Separation method. The method for separating 4- (3,4-dihydroxy-phenyl) -but-3-en-2-one according to claim 1, wherein the filler of gel filtration chromatography is dextrangel. The method for separating 4- (3,4-dihydroxy-phenyl) -but-3-en-2-one according to claim 1, wherein the alkane having 5 to 8 carbon atoms is hexane. 2. The method of claim 1, wherein the hydrophilic chromatography is hydrophilic liquid chromatography. The method for separating 4- (3,4-dihydroxy-phenyl) -but-3-en-2-one according to claim 6, wherein the column of hydrophilic chromatography is a reversed phase column in which the stationary phase is C18. Separation of 4- (3,4-dihydroxy-phenyl) -but-3-en-2-one according to claim 6, which is eluted at 1.5 ml / min with 30% acetonitrile. Way. 4- (3,4-Dihydroxy-phenyl) -but-3-en-2-one isolated from chaga by the method of claim 1.