KR100836941B1 - Antioxidant Compound Isolated from Chaga and Composition Comprising the Same - Google Patents

Abstract

The present invention relates to an antioxidant compound isolated from chaga, and more particularly, to antioxidant compounds isolated from Chaga ( Inonotus obliquus ), that is, phelligridin D, phelligridin E, Phelligridin G, inonoblin A, and inonoblin B exhibited superoxide radical scavenging effect, ABTS radical scavenging effect, and DPPH radical scavenging effect, indicating antioxidant activity. It is useful as a medicine, cosmetic material, functional food, or food additives for the prevention and treatment of various diseases such as arteriosclerosis, arthritis, stroke, Parkinson's disease, Alzheimer's disease, autoimmune disease and cancer and suppressing skin aging. It relates to an antioxidant compound isolated from Chaga ( Inonotus obliquus ).

Description

Antioxidant Compound Isolated from Chaga Mushroom and Composition Comprising the Amino Acids from Mushroom Inonotus obliquus and composition containing them

1 shows the ¹ H NMR spectrum of Innoblin A of the present invention,

Figure 2 shows the ¹³ C NMR spectrum of Innoblin A of the present invention.

Figure 3 shows the ¹ H NMR spectrum of the innoblin B of the present invention,

Figure 4 shows the ¹³ C NMR spectrum of the innoblin B of the present invention.

The present invention relates to an antioxidant compound isolated from chaga, and more particularly, to antioxidant compounds isolated from Chaga ( Inonotus obliquus ), that is, phelligridin D, phelligridin E, Phelligridin G, inonoblin A, and inonoblin B exhibit antioxidant peroxidation, ABTS radical scavenging, and DPPH radical scavenging. As a medicine, cosmetic materials, functional foods, or food additives for the prevention and treatment of various diseases such as arteriosclerosis, arthritis, stroke, Parkinson's disease, Alzheimer's disease, autoimmune diseases and cancer and suppressing skin aging An antioxidant compound isolated from useful Chaga ( Inonotus obliquus ).

The human body acquires energy through breathing, which uses oxygen as the final electron acceptor. However, this way superoxide radical (O ₂ ^-) by the absolute oxygen but stable molecular state of the base triplet oxygen required to sustain life for various physical, chemical and environmental factors, the hydroxyl radical (HO and), hydrogen peroxide (H ₂ O ₂ ), which is converted into highly reactive free radicals or reactive oxygen species such as singlet oxygen ( ¹ O ₂ ), has a double sided effect that causes fatal oxygen toxicity to the living body. That is, these free radicals are non-selective and irreversible destructive action on cell components lipids, proteins, sugars, DNA, etc., such as cancer, arteriosclerosis, arthritis, stroke, Parkinson's disease, Alzheimer's disease, autoimmune diseases It is known to act as a cause of disease as well as to cause aging. Therefore, antioxidants that remove free radicals or inhibit free radical production include cancers caused by excess free radicals, including arteriosclerosis, arthritis, stroke, Parkinson's disease, Alzheimer's disease, and autoimmune diseases. It can be used for the purpose of prevention and treatment and to suppress skin aging [Hajieva, P .; Behl, C. Antioxidant as a potential therapy against age-related neurodegenerative diseases: amyloid Beta toxicity and Alzheimer's disease. Curr. Pharm. Des. 12 (6): 699-704. 2006. Cherubini, A .; Vigna, GB; Zuliani, G .; Ruggiero, C .; Senin, U .; Fellin, R. Role of antioxidants in atherosclerosis. Curr. Pharm. Des. 11 ((16): 2017-2032. 2005. Zhao, B. Natural antioxidants for neurodegenerative diseases.Mol. Neurobiol. 31: 283-293, 2005. Murakami, A .; Ohigashi, H. Cancer-preventive antioxidants that attenuate free radical generation by inflammatory cells.Biol. Chem. 387: 387-392. 2006].

Synthetic antioxidants known to date include butylated hydroxy anisole (BHA), butylated hydroxy toluene (BHT), and nordihydro-guaiaretic acid (NDGA) .Natural antioxidants include superoxidide dimustase, peroxidase, catalase, and glutathione. Antioxidant enzymes such as peroxidase and non-enzymatic antioxidants such as vitamin E, vitamin C and glutathione. However, synthetic antioxidants are toxic in vivo and can cause allergies and tumors. On the other hand, natural antioxidants have the advantages of being safe to the living body compared to synthetic antioxidants, but have the disadvantage of their weakness. Therefore, there is an urgent need for the development of new natural antioxidants that are excellent in antioxidant activity and safer in vivo. In fact, through the development of natural antioxidants that can eliminate free radicals, efforts to use them as therapeutic agents and aging inhibitors for cancer, arteriosclerosis, arthritis, stroke, Parkinson's disease, Alzheimer's disease, autoimmune diseases, etc. It has been constantly.

Recently, mushrooms, which have been spotlighted as health foods along with the well-being fever, are being used as medicines as well as food. Mushrooms appear as a form of fruiting body where metabolites accumulate as a result of nutritional metabolism of Bacillidiomycotina, Ascomycotina and Imperfect fungi among higher fungi. It is known that there are female species and 992 species of 68 and 261 genera are reported in Korea, and 328 of them are known as edible and medicinal mushrooms. The pharmacological effects of the mushrooms are known to be anti-cancer, lowering blood cholesterol, lowering blood pressure, antiviral, immune boosting, antibacterial, interferon-inducing effect, so far known medicinal mushrooms are the main anticancer activity of the polysaccharide. The pharmacological effect of the mushrooms as described above is believed to be inferred from the antioxidant activity of the mushrooms.

The main agent of chaga is black birch. Chaga mushrooms native to the Kamchatka Peninsula are reproductive parasitic mushrooms that are attached to a living birch like black lumps of black lumps, and the lumps of black lumps grow gradually. Fruiting bodies are formed on the bark of birch trees, and the epidermis is black, and the genus has a yellowish inner tissue.

In particular, chaga produces yellow pigments, and these pigments are thought to be related to various biological activities of chaga. The present study isolated the novel antioxidant compounds Innoblin A and B from the yellow pigment. In addition, the antioxidant activity of the compounds identified in the present invention has not been found in any literature.

Accordingly, the present inventors separated and purified the pigment material showing antioxidant activity from chaga mushroom, and determined the structure, the active material is two kinds of innooblin A (inonoblin A) and inonoblin B (inonoblin B) Novel compounds have been reported and isolated from Phellinus ignarius , with phelligridin D, phelligridin E, and phelligridin G, as reported. It is the first time that chaga, E and G are contained in chaga, and the antioxidant activity of the five compounds was confirmed by identifying diseases such as arteriosclerosis, cancer, arthritis, stroke, Parkinson's disease, Alzheimer's disease and autoimmune diseases. The present invention has been completed by revealing that it can be used for the purpose of preventing and treating and inhibiting skin aging.

Accordingly, an object of the present invention is to provide novel inonoblin A and inonoblin B.

In addition, the present invention is made from inonoblin A, inonoblin B, phelligridin D, phelligridin E and phelligridin G. Another object is to provide an antioxidant composition containing at least one selected as an active ingredient.

The present invention is characterized by inonoblin A represented by the following formula (1) and inonoblin B (inonoblin B) represented by the following formula (2).

In another aspect, the present invention is characterized by an antioxidant composition containing one or two or more selected from the compounds represented by the following formulas (1) to (5) as an active ingredient.

[Formula 1]

[Formula 2]

Referring to the present invention in more detail as follows.

The present invention relates to antioxidant compounds isolated from Chaga ( Inonotus obliquus ), that is, inonoblin A, inonoblin B, phelligridin D, phelligridin E E), phelligridin G has antioxidant activity by showing superoxide radical scavenging effect, ABTS radical scavenging effect and DPPH radical scavenging effect. And antioxidant compounds isolated from Chaga ( Inonotus obliquus ) useful as medicines, cosmetic materials, functional foods, or food additives for the prevention and treatment of various diseases such as Alzheimer's disease, autoimmune diseases and skin aging. .

In order to separate the active substance from chaga mushroom characterized by the present invention, a well-known method used for extracting fruiting body methanol extract with organic solvents such as hexane, chloroform, ethyl acetate, separation of components such as water distribution, column chromatography, etc. It can be obtained easily by combining single or suitably. The crude extract can be further purified according to a commercial method as needed.

Chaga mushroom fruiting body is used in the present invention. A preferred method for efficiently separating the antioxidant active substance from chaga according to the present invention is as follows:

The present invention

1) extracting the pulverized chaga mushroom with a solvent selected from aliphatic alcohols having 1 to 6 carbon atoms and aliphatic hydrocarbons having 1 to 10 carbon atoms to obtain a solvent extract;

2) fractionating the solvent extract with hexane, chloroform and ethyl acetate in order to obtain an ethyl acetate fraction; And

3) Separation of the ethyl acetate fraction with 100% methanol was performed by Sephadex LH-20 column chromatography, and octadecylylated silica gel (ODS) column chromatography was used to separate pelicidin D from the 70% methanol eluate fraction. The process consists of separating the pelicidine D from chaga.

In addition, the present invention

1) extracting the pulverized chaga mushroom with a solvent selected from aliphatic alcohols having 1 to 6 carbon atoms and aliphatic hydrocarbons having 1 to 10 carbon atoms to obtain a solvent extract;

2) fractionating the solvent extract with hexane, chloroform and ethyl acetate in order to obtain an ethyl acetate fraction; And

3) The ethyl acetate fraction of 2) was subjected to Sephadex LH-20 column chromatography using 100% methanol, followed by Sephadex LH-20 column chromatography with 70% aqueous methanol solution. The separation step consists in separating pelicidin E from chaga.

The present invention

1) extracting the pulverized chaga mushroom with a solvent selected from aliphatic alcohols having 1 to 6 carbon atoms and aliphatic hydrocarbons having 1 to 10 carbon atoms to obtain a solvent extract;

2) fractionating the solvent extract with hexane, chloroform and ethyl acetate in order to obtain an ethyl acetate fraction; And

3) The ethyl acetate fraction of 2) was subjected to Sephadex LH-20 column chromatography using 100% methanol, and then the active eluate was subjected to preparative high performance liquid chromatography (preparative HPLC) with an aqueous solution of 52% methanol. And separating the pelicidine G at the retention time of 48 minutes to separate the pelicidine G from chaga.

The present invention

1) extracting the pulverized chaga mushroom with a solvent selected from aliphatic alcohols having 1 to 6 carbon atoms and aliphatic hydrocarbons having 1 to 10 carbon atoms to obtain a solvent extract;

2) fractionating the solvent extract with hexane, chloroform and ethyl acetate in order to obtain an ethyl acetate fraction; And

3) The ethyl acetate fraction of 2) was subjected to Sephadex LH-20 column chromatography with 100% methanol, followed by Sepadex LH-20 column chromatography with 70% aqueous methanol solution. The separation step consists of separating Innobulin A from chaga.

The present invention

1) extracting the pulverized chaga mushroom with a solvent selected from aliphatic alcohols having 1 to 6 carbon atoms and aliphatic hydrocarbons having 1 to 10 carbon atoms to obtain a solvent extract;

2) fractionating the solvent extract with hexane, chloroform and ethyl acetate in order to obtain an ethyl acetate fraction; And

3) Ethyl acetate fraction of 2) was subjected to octadecylylated silica gel (ODS) column chromatography with 50-90% methanol solution to obtain 50-60% methanol eluate, and 60% methanol solution was used as a developing solvent. Octadecylsilylated silica gel (ODS) thin layer chromatography (TLC), and then eluted with a band of rf value of 0.3 with methanol, and then eluting the solution with 70% aqueous methanol solution. Separation of Innoblin B consists of separating inooblin B from chaga mushroom.

In each step 1) of the separation method, preferably, methanol, ethanol, acetone, propanol, or the like is suitable.

In addition, the compound of the present invention can be obtained by combining a known method used for separate extraction of mushroom components alone or in a suitable combination.

The physical and chemical properties of the compounds represented by Chemical Formulas 1 to 5 thus obtained are as follows.

All compounds were obtained as a yellow powder.

As a result of measuring the ESI-mass to determine the molecular formula of the compound, the Feligridine D was the molecular formula C ₂₀ H ₁₂ O ₈ , molecular weight 380, the peligridine E is the molecular formula C ₂₅ H ₁₄ O ₁₀ , molecular weight 474, Peli Gridine G is determined by the molecular formula C ₃₂ H ₁₈ O ₁₂ , molecular weight 594, Innoblin A is the molecular formula C ₃₃ H ₂₀ O ₁₃ , molecular weight 624, Innoblin B is the molecular formula C ₂₃ H ₁₄ O ₁₀ , molecular weight 450 .

As a result of measuring the UV spectrum, all of these compounds showed the maximum absorption peak in the vicinity of 250 ~ 260 nm, 380 ~ 400 nm.

Compounds according to the invention are 1) superoxide radicals using xanthine / xanthine oxidase enzyme system, 2) ABTS radicals, 3) caffeic acid known as antioxidants using DPPH radicals, BHA, trolox ( compared with trolox).

From these antioxidant activities, it is applicable to medicines, cosmetic materials or foods for the prevention and treatment of various diseases such as cancer, arteriosclerosis, arthritis, stroke, Parkinson's disease, Alzheimer's disease, autoimmune diseases and skin aging.

Therefore, the present invention includes an antioxidant composition containing one or two or more selected from the compounds of Formulas 1 to 5 obtained from chaga as an active ingredient.

In addition, as a result of the acute toxicity test for the antioxidant compounds of the formula 1 to 5 obtained from the chaga mushroom of the present invention, 50% lethal dose (LD ₅₀ ) was found to be at least 1 g / kg or more it was found to be a very safe substance.

In case of using the antioxidant compounds of the present invention as a medicament, 1 to 600 mg / kg may be administered to adults per day, and in case of using the extract, the extract is evaporated to 10 to 100 mg / kg based on the residue. It can be administered once or divided into several times. Inorganic or organic carriers may be added to the antioxidant compounds of the present invention and administered orally or parenterally in solid, semisolid or liquid form.

Examples of preparations for oral administration include tablets, pills, granules, soft and hard capsules, powders, fine granules, powders, emulsions, syrups and pellets. There is a number. The pharmaceutical composition of the present invention is a formulation for oral administration, such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin for preparation in formulations such as 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.

Preparations for parenteral administration include injections, drops, fluids, ointments, lotions, sprays, suspensions, emulsions, emulsions, suppositories, and the like. In order to formulate the active ingredient of the present invention, it can be easily formulated according to the conventional method. A surfactant, an excipient, a coloring agent, a spice, a preservative, a stabilizer, a buffer, a suspending agent, and other commonly used auxiliaries are mixed with water as appropriate. Or prepared in suspension and formulated into unit dosage forms of ampoules or vials.

Hereinafter, the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited to these Examples.

Example 1 Preparation of Chaga Extract

3 kg of dry and fine chaga fruiting bodies were immersed in 15 liters of a solvent such as methanol, ethanol or acetone for 1 day and extracted at room temperature to obtain chaga extract.

Example 2: Preparation of Ethyl Acetate Fraction of Chaga Methanol Extract

The methanol extract of Example 1 was concentrated to remove methanol, and then partitioned and extracted with hexane to separate the water layer and the hexane layer.

The water layer was extracted again with chloroform, divided into a water layer and a chloroform layer. The water layer was extracted with ethyl acetate, and the ethyl acetate fraction was prepared by dividing the water layer with an ethyl acetate layer.

Example 3: Preparation of Feligridine D

The fraction of Example 2 was subjected to Sephadex LH-20 column chromatography using 100% methanol, and then subjected to ODS column chromatography to obtain 10 mg of Feligridin D in 70% methanol aqueous solution fraction. Separated.

[Formula 3]

① Appearance: Yellow powder

② Molecular Weight: 380

③ Molecular Formula: C ₂₀ H ₁₂ O ₈

④ Mass Spectrometry Spectrum: ESIMS m / z 379 [MH] ^-

⑤ UV absorption spectrum: UV λ _max (MeOH) 250, 388 nm

⑥ Hydrogen nuclear magnetic resonance spectrum: ppm (400MHz, CD ₃ OD) 6.40 (s), 6.66 (d, J = 15.6), 7.37 (d, J = 15.6), 7.05 (d, J = 1.6), 6.77 (d , J = 8), 6.96 (dd, J = 1.6, 8), 7.57 (s), 8.38 (s)

Example 4 Preparation of Feligridine E

The fraction of Example 2 was subjected to Sephadex LH-20 column chromatography using 100% methanol, followed by Sephadex LH-20 column chromatography using a 70% aqueous methanol solution, which is called Feligridine. 7 mg of Phelligridin E was isolated.

[Formula 4]

① Appearance: Yellow powder

② Molecular Weight: 474

③ Molecular Formula: C ₂₅ H ₁₄ O ₁₀

④ Mass Spectrometry Spectrum: ESIMS m / z 473 [MH] ⁺ , m / z 497 [M + Na] ⁺

⑤ UV absorption spectrum: UV λ _max (MeOH) 257, 389 nm

⑥ Hydrogen nuclear magnetic resonance spectrum: ppm (400MHz, CD ₃ OD) 6.08 (s), 6,17 (s), 6,62 (s), 6.98 (s), 7.51 (s), 7.86 (s), 8.30 (s)

Example 5: Preparation of Feligridine G

The fraction of Example 2 was subjected to Sephadex LH-20 column chromatography using 100% methanol, and then the active eluate was subjected to preparative high performance liquid chromatography (preparative HPLC) using an aqueous solution of 52% methanol. 8 mg of Phelligridin G was isolated at 48 minutes of retention time.

[Formula 5]

① Appearance: Yellow powder

② Molecular Weight: 594

③ Molecular Formula: C ₃₂ H ₁₈ O ₁₂

④ Mass spectrometry spectrum: ESIMS m / z 593 [MH] ^- , m / z 617 [M + Na] ⁺

⑤ UV absorption spectrum: UV λ _max (MeOH) 267, 400 nm

⑥ Infrared Absorption Spectrum: IR (KBr) ν _max 3390, 3240, 1589, 1545, 1518, 1298, 1149 cm ^-1

⑦ Hydrogen nuclear magnetic resonance spectrum: ppm (500MHz, CD ₃ OD) 6.02 (s), 6.09 (s), 6.60 (s), 6.80 (d, J = 8.7), 6.91 (s), 6.94 (d, J = 16.5), 7.05 (d, J = 1.8, 8.7), 7.16 (s), 7.49 (s), 7.64 (d, J = 16.5), 7.68 (s), 8.33 (s)

Example 6: Preparation of Innoblin A

The fraction of Example 2 was subjected to Sephadex LH-20 column chromatography using 100% methanol, followed by Sephadex LH-20 column chromatography again using 70% aqueous methanol solution. 10 mg of inonoblin A) was isolated (see FIGS. 1 and 2).

[Formula 1]

① Appearance: Yellow powder

② Molecular Weight: 624

③ Molecular Formula: C ₃₃ H ₂₀ O ₁₃

④ Mass Spectrum Spectrum: ESIMS m / z 623 [M ⁻ H] ⁻

⑤ UV absorption spectrum: UV λ _max (MeOH) 253, 389 nm

⑥ Infrared Absorption Spectrum: IR (KBr) ν _max 3390, 3240, 1589, 1545, 1518, 1298, 1149 cm ^-1

⑦ Hydrogen nuclear magnetic resonance spectrum: ppm (400MHz, CD ₃ OD) 6.37 (s), 6.61 (s), 6.74 (s), 6.74 (d, J = 15.6), 7.20 (d, J = 15.6), 7.21 ( s), 7.01 (d, J = 8.0, 1.6), 7.10 (d, J = 1.6), 6.79 (d, J = 8.0), 6.80 (d, J = 16), 7.09 (d, J = 16), 7.51 (s), 8.26 (s)

⑧ Carbon nuclear magnetic resonance spectrum: ppm (100MHz, CD ₃ OD) 160.1 (C-1), 159.1 (C-3), 100.0 (C-4), 161.4 (C-4a), 159.4 (C-6), 112.1 (C-6a), 115.0 (C-7), 147.6 (C-8), 154.3 (C-9), 111.3 (C-10), 127.7 (C-10a), 99.5 (C-10b), 116.5 (C-1 '), 134.5 (C-2'), 126.5 (C-3 '), 111.9 (C-4'), 146.1 (C-5 '), 148.2 (C-6'), 111.3 (C -7 '), 126.5 (C-8'), 159.0 (C-2 "), 102.1 (C-3"), 159.1 (C-4 "), 100.9 (C-5"), 159.0 (C-6 "), 116.9 (C-7"), 135.5 (C-8 "), 127.6 (C-9"), 121.1 (C-10 "), 115.1 (C-11"), 148.1 (C-12 ") , 146.3 (C-13 "), 116.5 (C-14")

Example 6: Preparation of Innoblin B

Separation of the fraction of Example 2 was performed by Sepadex LH-20 column chromatography with 100% methanol, the active fraction was subjected to ODS column chromatography with 50 ~ 90% methanol aqueous solution to obtain a 50 ~ 60% methanol eluate, 60 After developing with ODS TLC using a% methanol aqueous solution as a developing solvent, a band corresponding to an rf value of 0.3 was eluted with methanol. The eluate was subjected to LH-20 column chromatography with an aqueous 70% methanol solution to separate 5 mg of inonoblin B (see FIGS. 3 and 4).

[Formula 2]

① Appearance: Yellow powder

② Molecular Weight: 450

③ Molecular Formula: C ₂₃ H ₁₄ O ₁₀

④ Mass Spectrometry Spectrum: ESIMS m / z 449 [MH] ^-

⑤ UV absorption spectrum: UV λ _max (MeOH) 253, 389 nm

⑥ Infrared Absorption Spectrum: IR (KBr) ν _max 3390, 3240, 1589, 1545, 1518, 1298, 1149 cm ^-1

⑦ Hydrogen nuclear magnetic resonance spectrum: ppm (400MHz, CD ₃ OD) 6.39 (s), 6.78 (s), 6.99 (s), 7.52 (s), 7.66 (s), 8.29 (s)

⑧ Carbon Magnetic Resonance Spectrum: ppm (100MHz, CD ₃ OD) 161.7 (C-1), 155.4 (C-3), 98.8 (C-4), 162.0 (C-4a), 160.9 (C-6), 113.4 (C-6a), 115.5 (C-7), 148.4 (C-8), 154.9 (C-9), 111.9 (C-10), 128.7 (C-10a), 101.2 (C-10b), 137.1 (C-1 '), 140.1 (C-2'), 134.8 (C-2a '), 111.9 (C-3'), 148.3 (C-4 '), 148.7 (C-5'), 111.3 (C -6 '), 139.8 (C-6a'), 90.4 (C-7 '), 206.5 (C-8'), 22.6 (C-9 ')

Test Example 1 Superoxide Radical Scavenging Effect

To investigate the superoxide radical scavenging ability of the compounds, xanthine (0.5 mM), NBT (nitrobluetetrazolium, 0.1 mM) 1: dissolved in potassium phosphate buffer (50 mM, pH 7.8) per well of a 96 well microplate. 1 75 μl of a mixed solution and 5 μl of a sample of each concentration of hispidine-based compound were added to DMSO (dimethylsulfoxide), and then 25 μl of 1 unit / ml xanthine oxidase was added thereto, followed by reaction at 37 ° C. for 30 minutes. The free radical scavenging activity (%) was calculated from the following equation (1). As a positive control, vitamin E, caffeic acid, and BHA were used. The superoxide radical scavenging activity is shown in Table 1 below, with the concentration of IC ₅₀ decreasing the absorbance value by 50% compared to the control without the sample.

In the following equation 1, A is the absorbance of the test group to which the compound of the present invention is not added, and B is the absorbance of the test group to which the compound of the present invention is added.

Test Example 2: ABTS radical scavenging effect

In order to investigate the ABTS radical scavenging activity of the compounds, 5 μl sample was added to 95 μl of ABTS radical cation solution, and left at room temperature for 5 minutes, and then the absorbance was measured at 734 nm using a microplate reader. Compared with. The activity was measured for each concentration, and the concentration showing 50% scavenging activity was calculated by the following equation (2) as the relative value of Trolox (Trolox equivalen antioxidant capacity), and the positive controls caffeic acid and BHA and In comparison, it is shown in Table 1 below.

Test Example 3: DPPH radical scavenging effect

In order to measure DPPH radical scavenging activity of the compounds, 5 μl of 95 μl 150 μM DPPH solution and 5 μl of sample per microplate well were added and allowed to stand at room temperature for 20 minutes, and then the absorbance was measured at 517 nm using a microplate reader. The scavenging activity was shown in comparison with the control. Activity was measured for each concentration, the TEAC was calculated by Equation 2, and compared with the positive control caffeic acid, BHA is shown in Table 1 below.

As shown in Table 1, the free radical scavenging activity of the chaga antioxidant compound was examined, and the compounds showed similar scavenging activity as the positive control caffeic acid and BHA.

Test Example 4: Parenteral Administration Acute Toxicity in Rats

In order to determine the acute toxicity of the compounds of Formulas 1 to 5 isolated from chaga mushroom, the following experiment was performed.

Acute toxicity test was performed using 6-week-old SPF SD rats. Each compound of Formulas 1 to 5 was suspended in 10 ml of saline and injected intramuscularly into the two anterior tibialis of the rats at a dose of 1 g / kg. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed, and hematological and blood biochemical tests were performed. Necropsy was performed to visually examine the abdominal and thoracic organ abnormalities. As a result, there were no clinical symptoms or deaths in all animals treated with the test substance, and no toxicity change was observed in weight change, blood test, blood biochemistry test, autopsy findings, etc. As a result, the compounds of Formulas 1 to 5 did not show toxic changes up to 1 g / kg in rats, and the parenteral administration minimum dose (LD ₅₀ ) was determined to be a safe substance of 1 g / kg or more.

Formulation Example 1 Preparation of Syrup

A syrup containing 2% (weight / volume) of an antioxidant compound of Formula 1 to 5 or a pharmaceutically acceptable salt thereof as an active ingredient was prepared by the following method.

Acid addition salts, saccharin and sugars of the antioxidant compounds of Formulas 1 to 5 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.

The components of the syrup are as follows.

Active ingredient ·············· 2 g

Saccharin 0.8 g

25.4 g of sugar

Glycerin · 8.0 g

Spices ················ 0.04 g

Ethanol 4.0 g

0.4 g of sorbic acid

Distilled water ··············

Formulation Example 2: Preparation of Tablet

Tablets containing an antioxidant compound of Formula 1 to 5 or a pharmaceutically acceptable salt thereof as an active ingredient are prepared by the following method.

250 g of active ingredient was 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 Injection

Injection solution containing an antioxidant compound of Formula 1 to 5 or a pharmaceutically acceptable salt thereof as an active ingredient was prepared by the following method.

1 g of the active ingredient, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

As described in detail above, the compounds of Chemical Formulas 1 to 5 obtained from chaga have been shown to have an antioxidant activity that strongly eliminates free radicals, including cancer, arteriosclerosis, arthritis, stroke, Parkinson's disease, Alzheimer's disease, autologous It can be widely used industrially as a material of food, cosmetics and medicines for the prevention and treatment of various diseases such as immune diseases, and for inhibiting skin aging.

Claims (5)

A compound characterized by the following formula (1). [Formula 1]

A compound characterized by the following formula (2). [Formula 2]

The antioxidant composition characterized by containing one or two or more selected from the compounds represented by the following formula (1), (2), (4) and (5) as an active ingredient. [Formula 1]

[Formula 2]

[Formula 4]

[Formula 5]

The composition of claim 3, wherein the compound is isolated from Chaga ( Inonotus obliquus ). delete

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