KR20050074070A - Novel antioxidant from phellinus baumi and its manufacture and use - Google Patents

Abstract

The present invention relates to a novel compound having an antioxidant activity isolated from a situation mushroom, a preparation method and a use thereof, and more particularly, to a novel compound having the antioxidant activity isolated from a situation mushroom ( Phellinus baumi ), which is a kind of mushroom. A compound, a method for preparing the compound, and a use of the novel compound in which the compound can be used in various therapeutic agents and health foods related to antioxidant activity.

Description

Novel antioxidant from Phellinus baumi and its manufacture and use

The present invention relates to a novel compound having an antioxidant activity isolated from a situation mushroom, a preparation method and a use thereof, and more particularly a novel compound having an antioxidant activity isolated from a situation mushroom ( Phellinus baumi ), which is a kind of a mushroom. It relates to a process for the preparation of and to the use of new compounds which can be used in various therapeutic agents and health foods related to the antioxidant activity.

Aerobic organisms gain energy by breathing oxygen, the most abundant element on Earth, as their final electron acceptor. However, the basic triplet oxygen, which is oxygen essential for this life phenomenon but stable molecular state, produces free radicals of oxygen toxicity which are fatal to the living body due to physicochemical and environmental factors. Representative free radicals, which are the biggest problem in vivo, are generated from enzymes, reduction metabolism, chemicals, pollutants and photochemical reactions from the base triplet oxygen, a stable molecular state widely available in nature as active oxygen. As a product are O ₂ ^- (superoxide), H ₂ O ₂ (dihydro genpa oxide), OH and (hydroxyl radical), ¹ O ₂ (singlet oxy gen) etc., and these producing cells in the granular and cytoplasmic do. Free radicals are indispensable substances used in the sterilization of macrophages in vivo, removal of old proteins, etc., but they can exhibit harmful effects in vivo due to their high reactivity.

However, in the case of abnormality in the active oxygen defense mechanism or when the active oxygen is excessively increased by physical or chemical factors, the active oxygen may cause fatal damage to each part of the body. Because of this, it is known to cause various diseases such as brain diseases such as stroke, Parkinson's disease, heart disease, ischemia, arteriosclerosis, skin disease, digestive diseases, inflammation, rheumatism, autoimmune diseases, and other cancers. Antioxidants that remove free radicals or inhibit free radical production may be used for the prevention and treatment of various diseases caused by excessively produced free radicals and to inhibit skin aging.

Recently, the development of antioxidants as food additives, health foods for the prevention of various diseases, and the pharmaceutical and cosmetics industry for the purpose of inhibiting aging is diversified. Currently used antioxidants include synthetic antioxidants such as t-butylhydroxytoluene (BHT) and t-butylhydroxyanisole (BHA), and natural antioxidants such as tocopherol, carotenoid, flavonoid and tannin. And antioxidant enzymes such as SOD are used. Synthetic antioxidants are limited to use due to the toxicity and the expensive price to the human body, it is necessary to develop a natural antioxidant that is harmless to the human body and shows a stronger antioxidant effect.

Among the natural antioxidants, the extracts of various spices, such as speamint, marjoram, sweet basil, and gagyo, have reported similar antioxidant effects to tocopherol and BHA, and sesameol (sesaminol) as an antioxidant component. , γ-tocopherol, trans-ferulic acid, and bisepoxyligan homologues were 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.

Accordingly, the present inventors have completed the present invention by finding a substance showing antioxidant activity in a situation mushroom while searching for an antioxidant substance from a medicinal plant material and analyzing the structure by separating the antioxidant substance.

It is an object of the present invention to provide a novel compound having an antioxidant activity isolated from a situation mushroom ( phellinus baumi ) and a preparation method thereof.

In addition, the present invention has another object to provide an antioxidant composition containing a novel compound showing an excellent antioxidant activity effect as an active ingredient, and a pharmaceutical or health food comprising the same.

The present invention is characterized by a novel compound represented by the following formula (1) having an antioxidant activity isolated from phellinus baumi , and a preparation method thereof.

[Formula 1]

In addition, it includes an antioxidant composition containing the compound as an active ingredient, and pharmaceuticals or health foods containing the composition.

Referring to the present invention in more detail as follows.

The present invention is isolated from a situation mushroom, which is a kind of medicinal mushroom, and then represented by the following formula (1), a method for preparing the compound, and the use of the novel compound which can be used in various therapeutic agents and health foods related to the antioxidant activity It is about.

The preparation method of the novel compound according to the present invention

1) collecting the situation mushroom, dried in the shade, extracted with methanol, filtered and the filtrate was concentrated under reduced pressure to obtain a methanol extract;

2) suspending the methanol extract in distilled water and shaking with ethyl acetate to obtain an ethyl acetate layer;

3) performing column chromatography on the ethyl acetate layer to obtain an active fraction;

4) drying the active fractions under reduced pressure, and performing high performance liquid chromatography with acetonitrile aqueous solution.

As a result of purifying the separated active fraction, the structure of the new compound was analyzed by NMR, and three groups had a benzene ring substituted with two groups, two of which were substituted with hydroxyl groups, and the other was composed of trans-type olefins, ketones, and methyl groups. As a compound, C ₁₀ H ₁₀ O ₃ has a molecular weight of 178 and is represented by Chemical Formula 1.

Investigation of the antioxidant activity of this compound was confirmed by performing DPPH (1,1-diphenyl-2-picrylhydrazyl) reduction method. Therefore, the compound represented by Chemical Formula 1 may be usefully used to treat various diseases caused by harmful oxygen or free radicals as an active ingredient having an antioxidant effect.

Antioxidant composition comprising the compound as an active ingredient can be administered orally or parenterally, for example, intravenous, subcutaneous, intraperitoneal or topical in clinical administration, can be used in the form of general medicines and health foods .

The antioxidant composition may be formulated for oral administration, eg, tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs. Formulated). Binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin for formulation into tablets and capsules; Excipients such as dicalcium phosphate; Disintegrants such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. Capsules contain liquid carriers, such as fatty oils, in addition to the substances mentioned above. In addition, the pharmaceutical composition of the present invention can be administered parenterally, and parenteral administration is by subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection. To formulate into a parenteral formulation, the composition is prepared in solution by mixing in water with stabilizers or buffers and formulated into unit dosage forms of ampoules or vials.

Effective dosages of the compounds may vary depending on the age, physical condition, weight, etc. of the patient, but are generally administered within the range of 1-100 mg / kg body weight per day. In addition, within a daily effective dosage range is divided into once a day or several times a day.

In addition, the present invention includes a health food containing the compound represented by the formula (1) as an active ingredient.

The health food is a food prepared by adding the compound represented by Chemical Formula 1 to food materials, such as beverages, teas, spices, gums, confectionery, or the like, encapsulated, powdered, or suspension, and when ingested, have a specific health effect. Means to bring, but unlike the general medicine has the advantage that there is no side effect that can occur during long-term use of the drug as a raw material of the food.

Antioxidant composition comprising such a compound represented by the formula (1) is useful for preventing or treating diseases related to the antioxidant action.

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

Example 1 Isolation of Antioxidant Active Substances from Situary Mushrooms

The present inventors used extraction and chromatography to isolate new antioxidants with potent antioxidant activity from mushrooms.

First, 1.5 kg of the cultivated dried mushroom mushroom was crushed, extracted with 100% methanol for 3 days, filtered under reduced pressure, and the filtrate was concentrated under reduced pressure in a 60 ° C water bath, and the concentrated methanol concentrate was suspended in 1 L of distilled water. The methanol extract suspended in distilled water was shaken and extracted with 2 L of ethyl acetate in a separatory filter to obtain an ethyl acetate layer, and then the ethyl acetate layer was concentrated under reduced pressure in a 60 ° C water bath. The concentrate of the ethyl acetate layer was dissolved in a small amount of methanol and developed on a Sephadex LH-20 gel column chromatography equilibrated in methanol to obtain an active fraction, and the fraction was subjected to YMC gel ODS column chromatography. ODS column chromatography) was carried out using 10% acetonitrile aqueous solution as a solvent to perform high performance liquid chromatography.

The separated and purified material was dissolved in methanol (CD ₃ OD) to determine the chemical shift of hydrogen and carbon in Table 1 through ¹ H-NMR, ¹³ C-NMR, two-dimensional NMR. In addition, ¹ H-NMR, ¹³ C-NMR, two-dimensional NMR spectrum results are shown in the accompanying drawings.

Carbon number Chemical shift of hydrogen NMR (ppm) Chemical shift of carbon NMR (ppm) One - 127.7 2 7.07 115.3 3 - 150.1 4 - 147.0 5 6.78 116.6 6 6.99 123.5 7 7.52 146.9 8 6.55 124.7 9 - 201.6 10 2.32 27.0

The separated and purified material is a compound in which two hydroxyl groups are substituted on a benzene ring, and a ketone group and a methyl group are attached to a trans-type olefin group. 2-one (4- (3,4-Dihydroxy-phenyl) -but-3-en-2-one, sanghone) was named, and the structure is represented by the following formula (1).

[Formula 1]

Example 3: Confirmation of Antioxidant Activity of Purified Compounds

4- (3,4-dihydroxy-phenyl) -but-3-en-2-one, which contains free radicals in the molecule, readily accepts electrons or hydrogen radicals and thus becomes a stable molecule. Removal efficiency can be measured. The hydrogen donating ability of DPPH of the sample was measured as follows. After 12 mg of DPPH was completely dissolved in 100 ml of ethanol, 100 ml of distilled water was added to make 0.5 mM DPPH solution, and 200 µl of DPPH solution was added to 2 µl of the sample in each separated tablet. After 30 minutes, the absorbance was measured at 517 nm using a spectrophotometer to indicate the antioxidant activity as a DPPH radical scavenging activity. At this time, 4- (3,4-dihydroxy-phenyl) -but-3-en-2-one was reacted at 517 nm as a result of reacting 2 µl with 0.5 mM DPPH solution to a final concentration of 50 µg / ml. The decrease in absorbance over time is shown in FIG. 4, and 4- (3,4-dihydroxy-phenyl) -but-3 is used to add methanol as a negative control and alpha tocopherol as a positive control. As a result of comparison with -en-2-one, 4- (3,4-dihydroxy-phenyl) -but-3-en-2-one showed excellent antioxidant activity compared to alpha-tocopherol.

Example 4: Toxicity Test

The compound represented by Chemical Formula 1 was dissolved in dimethylsulfoxide (DMSO), diluted with water, and then administered to the mice (10 mice per group) at 100 mg / kg each, and observed for 7 days, but no rats died.

Formulation Example 1 Preparation of Syrup

Syrup containing the new compound according to the present invention and a pharmaceutically acceptable salt thereof as an active ingredient of 2% (weight / volume) was prepared by the following method.

Acid addition salts, saccharin and sugars of the novel compounds were dissolved in 80 g of warm water. After the solution was cooled, a solution consisting of glycerin, saccharin, spices, ethanol, sorbic acid and distilled water was prepared and mixed thereto. Water was added to this mixture to 100 ml. The addition salt can be replaced with other salts according to the examples.

Formulation Example 2: Preparation of Tablet

A tablet containing 15 mg of active ingredient is prepared by the following method.

250 g of the novel compound according to the invention were mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet.

Formulation Example 3 Preparation of Capsule

500 mg of the novel compound according to the present invention was filled into hard gelatin capsules to prepare capsules.

Formulation Example 4 Preparation of Injection

200 mg of the novel compound according to the present invention was heat-dissolved in 200 mg of physiological saline containing polyoxyethylene hydrogenated Castro oil to prepare an injection containing the mixed extract at a concentration of 0.1.

Formulation Example 5: Beverage Preparation

After dissolving 500 mg of the novel compound according to the present invention in an appropriate amount of water, vitamin C as an auxiliary component, citric acid, sodium citrate, and oligosaccharide as an auxiliary component were added, and an appropriate amount of sodium benzoate was added as a preservative, followed by adding water to the whole amount. 100 ml was prepared to prepare a beverage composition. At this time, taurine, myo-inositol, folic acid, pantothenic acid, etc. may be added alone or together.

As can be seen from the above description, the novel compound according to the present invention is excellent in antioxidant activity, and thus it is useful to use as a therapeutic or health food for diseases related to antioxidant activity.

Figure 1 shows the hydrogen nuclear magnetic resonance spectrum of the compound represented by the formula (1).

Figure 2 shows the carbon nuclear magnetic resonance spectrum of the compound represented by formula (1).

Figure 3 shows the two-dimensional nuclear magnetic resonance spectrum of the compound represented by the formula (1).

Figure 4 shows the antioxidant activity of the compound represented by the formula (1).

Claims (5)

A compound characterized by the following formula (1). [Formula 1] 1) collecting the situation mushroom, dried in the shade, extracted with methanol, filtered and the filtrate was concentrated under reduced pressure to obtain a methanol extract; 2) suspending the methanol extract in distilled water and shaking with ethyl acetate to obtain an ethyl acetate layer; 3) performing column chromatography on the ethyl acetate layer to obtain an active fraction; 4) A method for preparing a compound represented by the following Chemical Formula 1, which comprises drying the active fraction under reduced pressure and performing high performance liquid chromatography with an acetonitrile aqueous solution. [Formula 1] The antioxidant composition characterized by containing the compound represented by the following formula (1) as an active ingredient. Formula 1 Antioxidant, characterized in that the composition of claim 3. Health food comprising the composition of claim 3.