KR20140106255A - Novel compound having antioxidant activity isolated from mycelial culture of Coprinus echinosporus and antioxidant composition comprising the same - Google Patents

Abstract

The present invention relates to a novel compound having an antioxidant activity and isolated from a mycelial culture of Coprinus echinosporus and to an antioxidant composition comprising the same as an active ingredient. The compound isolated and purified from the mycelia culture of Coprinus echinosporus according to the present invention is excellent in ABTS and DPPH radical scavenging activity, reducing power, and single strand breakage (SSB) suppressive activity, thereby having excellent antioxidant efficacy, and thus can be very useful as an antioxidant, and a medicine, a cosmetic material, functional food, or food additive for preventing and treating diseases caused by excessive accumulation of active oxygen and inhibiting skin damage and skin aging.

Description

TECHNICAL FIELD [0001] The present invention relates to a novel antioxidant compound having antioxidative activity and a novel antioxidant composition comprising the antioxidant composition isolated from the mushroom mycelial culture.

The present invention relates to a novel compound having antioxidative activity isolated from a mushroom mycelial culture solution, and to an antioxidative composition containing the compound as an active ingredient.

The reactive oxygen species (ROS) are produced from the aerobic metabolism in the human body, about 5% of which is transformed into free radicals (the generic name of atoms or molecules with free electrons). These radicals are generated in immune cells such as macrophages and neutrophils, and kill pathogens that enter from the outside. However, the radicals are very unstable and highly reactive, so that proteins, lipid molecules In addition, it may cause oxidative damage to DNA containing genetic information. As a result, the active oxygen generated in the body causes various diseases such as cancer, inflammation including anemia, myocardial infarction, cerebral palsy, arteriosclerosis, diabetes, rheumatoid arthritis, Parkinson's disease, skin damage, and autoimmune disease It is also known to act as an important cause of aging (Halliwell et al., 1999; Diolock et al., 1998; Wickens, et al, 2001). There are antioxidant enzymes such as peroxidase, catalase and antioxidants such as vitamin E and vitamin C in the body to protect themselves against this reactive oxygen. If the production of active oxygen exceeds the capacity of the bio-defense system due to physical and chemical factors, damage due to oxidative stress can not be avoided.

Recently, studies on various antioxidants to protect the body from oxidative stress have been actively conducted and reported (Diolock et al., 1998; Mandade et al., 2011), among which synthetic antioxidants (Butylated Hydroxy Anisole), BHT (Butylated Hydroxy Toluene) and NDGA (Nordihydro-guaiaretic acid). However, these synthetic antioxidants are toxic in vivo and can cause allergies and tumors. When they are weak at high temperature, they are easily destroyed by heat.

Therefore, the need and preference for safe and in vivo stable new natural antioxidants that can be widely used for medicines and health functional foods are gradually increasing (Liu et al., 2012; Guo et al., 2012; Li et al , 2012; Ramaratham et al., 1995). Saito et al., 2003).

On the other hand, it is well known that mushrooms contain a wide variety of pharmacological effects including antibiotics, antioxidants, anti-tumor, cholesterol lowering, hypoglycemic, antiviral, Among them, mushrooms belonging to the genus Coprinus have various useful properties such as anti-inflammation, antioxidant, antitumor, anti-bacterial and antifungal activity as well as activity for improving cerebral blood flow disorder.

Among the mushrooms belonging to Coprinus genus, Coprinus echinosporus is white and covered with roughly fibrous scales of light gray and dark yellowish brown. The spores are elliptical of 13 ~ 18 × 7 ~ 8 ㎛, the surface is smooth, and the foam is black. In the spring and autumn, grasses, fields, and gardens grow on land rich in humic substances.

Therefore, the present inventors isolated and purified novel compounds of the formula (I) or ( II ) from a mycelial culture of Coprinus echinosporus . The ABTS radical scavenging activity, DPPH radical scavenging activity, DNA single strand breakage (SSB) Inhibitory activity and reducing power, and thus it has been confirmed that the present invention has an excellent antioxidative activity.

The present invention is to provide a compound having antioxidative activity separated and purified from a mushroom mycelium culture solution.

The present invention also provides a pharmaceutical composition for antioxidation comprising the compound as an active ingredient.

The present invention also provides a pharmaceutical composition for preventing or treating diseases caused by excessive accumulation of active oxygen containing the above compound as an active ingredient.

The present invention also provides an antioxidant food composition comprising the compound as an active ingredient.

The present invention also provides an anti-aging cosmetic composition comprising the compound as an active ingredient.

The present invention provides a compound having an antioxidative activity separated and purified from a mushroom mycelium culture solution.

The present invention also provides a pharmaceutical composition for antioxidation comprising the compound as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating a disease caused by excessive accumulation of active oxygen containing the above-mentioned compound as an active ingredient.

The present invention also provides a food composition for antioxidation comprising the compound as an active ingredient.

The present invention also provides an anti-aging cosmetic composition comprising the compound as an active ingredient.

The compounds isolated and purified from the mushroom mycelial culture according to the present invention exhibit excellent antioxidative activity by exhibiting ABTS and DPPH radical scavenging activity, reducing power and DNA single strand breakage (SSB) inhibitory activity, Can be very usefully used as pharmaceuticals, cosmetic materials, functional foods, or food additives for prevention and treatment of diseases caused by excessive accumulation, skin damage, or inhibition of skin aging.

1 shows (a) ¹ H NMR spectra (600 MHz, CD ₃ OD) and (b) ¹³ C NMR spectra (150 MHz, CD ₃ OD) of Compound 1 of the present invention.
2 is a diagram showing (a) ¹ H NMR spectrum (600 MHz, CD ₃ OD) and (b) ¹³ C NMR spectrum (150 MHz, CD ₃ OD) of Compound 2 of the present invention.
3 is a diagram showing (a) ¹ H NMR spectrum (600 MHz, CD ₃ OD) and (b) ¹³ C NMR spectrum (150 MHz, CD ₃ OD) of the compound 3 of the present invention.
4 shows (a) ¹ H NMR spectra (600 MHz, CD ₃ OD) and (b) ¹³ C NMR spectra (150 MHz, CD ₃ OD) of Compound 4 of the present invention.
FIG. 5 shows the ABTS radical scavenging activity of Compounds 1 to 4 isolated and purified from Mycelia mushroom mycelium culture.
FIG. 6 shows the DPPH radical scavenging activity of Compounds 1 to 4 isolated and purified from the culture medium of the mushroom mycelium mycelium.
Fig. 7 is a diagram showing the DNA-protecting activity of the compounds 1 to 4 isolated and purified from the culture medium of the mushroom mycelium mycelium.

The present invention provides a compound having an antioxidative activity represented by the following Formula 1:

[Chemical Formula 1]

In the above formula (1), R is H, OH, or C ₁ -C ₁₀ alkoxy.

Preferably, the compound of formula 1 may be represented by the following formulas 1-1 to 1-3:

[Formula 1-1]

[Formula 1-2]

[Formula 1-3]

 The present invention also provides a compound having an antioxidative activity represented by the following general formula (2).

(2)

The compounds of the formulas 1-1 to 1-3 and the compounds of the formula 2 of the present invention are pharmaceutically acceptable such as a salt with a metal salt, an organic base, a salt with an inorganic acid, a salt with an organic acid, a salt with a basic or an acidic amino acid It can be used in the form of possible salts. Suitable metal salts include alkali metal salts such as sodium salts, potassium salts and the like; Alkaline earth metal salts such as calcium salts, magnesium salts, barium salts and the like; Aluminum salts and the like. Suitable salts with organic bases include, for example, salts with organic bases such as trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexyl Amine, dicyclohexylamine, N, N-dibenzylethylenediamine and the like. Examples of suitable salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like. Examples of suitable salts with organic acids include formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, Maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and the like. Suitable examples of salts with basic amino acids include salts with, for example, arginine, lysine, ornithine and the like. Suitable examples of salts with acidic amino acids include salts with, for example, aspartic acid, glutamic acid and the like. Particularly preferred salts include inorganic salts such as alkali metal salts (for example, sodium salts and potassium salts) and alkaline earth metal salts (for example, calcium salts, magnesium salts and barium salts) when the compound has an acidic functional group therein, And salts with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like, organic acids such as acetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, Succinic acid, methanesulfonic acid, p-toluenesulfonic acid and the like.

The present invention also provides a composition comprising the compound of Formula 1 or 2 as an active ingredient. The composition comprises a pharmaceutical composition, a food composition, and a cosmetic composition.

The compound represented by Formula 1 or Formula 2, which is an active ingredient of the composition of the present invention, can be obtained by extracting and separating from a herbal medicine, and can also be obtained by a conventional organic synthesis method.

In the present invention, the compound represented by the formula (1) or (2) can be obtained by extracting and isolating from a culture solution of mushroom mycelium, and the method for extracting and separating the compound is as follows.

First, on the PDB medium, the mushroom ( Coprinus echinosporus , KACC 52101) and incubated at 27 ° C for one month. The mycelia of the cultivated strains are finely pulverized and then extracted by immersing in acetone for 1 to 3 days. After the extraction, the extract is filtered and concentrated under reduced pressure to remove acetone. The concentrated extract was partitioned with ethyl acetate, and the organic solvent fraction was concentrated under reduced pressure to obtain an ethyl acetate fraction.

Then, the ethyl acetate fraction of the mushroom mycelial culture was subjected to Sephadex LH-20 column chromatography using methanol, and the easy-to-separate fraction in the fraction was subjected to HPLC using 50% Separate pure compounds.

The compound of formula (I) or (II) of the present invention isolated as described above decreases ABTS cation radicals produced by the reaction of ABTS with potassium persulfate in a concentration-dependent manner, exhibits a concentration-dependent inhibitory activity in the evaluation of DPPH radical scavenging activity, As a result of the test for reducing power, the reducing ability and the ability to inhibit DNA damage were comparatively superior to those of the positive control, the antioxidant can be effectively used for medicines, health functional foods, and cosmetics.

The present invention provides a pharmaceutical composition for antioxidation, which comprises a compound isolated from the mushroom mycelium culture solution as an active ingredient, and a pharmaceutical composition for preventing or treating diseases caused by excessive accumulation of active oxygen.

Diseases caused by excessive accumulation of active oxygen include, but are not limited to, cancer, inflammation, anemia, myocardial infarction, cerebral palsy, arteriosclerosis, diabetes, rheumatoid arthritis, Parkinson's disease, and autoimmune diseases.

The pharmaceutical composition of the present invention may contain one or more known active ingredients having the effect of preventing or treating disease caused by excessive accumulation of active oxygen together with the compound isolated from the mushroom mycelia culture solution.

The pharmaceutical composition of the present invention may further comprise at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components. If necessary, an antioxidant, , And other conventional additives such as a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using appropriate methods in the art or by the method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the intended method, and the dose may be appropriately determined depending on the patient's body weight, The range varies depending on the health condition, diet, administration time, method of administration, excretion rate, and severity of the disease. The daily dose of the compound isolated from the mushroom mycelium culture solution is about 1 to 1000 mg / kg, preferably about 50 to 500 mg / kg, and is preferably administered once or several times a day.

The pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, hormone therapy, drug therapy and biological response modifiers for prevention or treatment and improvement of diseases caused by excessive accumulation of active oxygen .

The present invention also provides an antioxidant food composition comprising the compound of formula (I) or (II) isolated from the culture medium of the mushroom mycelium of the present invention as an active ingredient.

In addition, the compound isolated from the mushroom mycelium culture solution of the present invention can be added to health foods and beverages for the purpose of preventing the accumulation of active oxygen or removing the generated active oxygen. When the compound isolated from the mushroom mycelium culture solution of the present invention is used as a food additive, the compound can be added as it is or can be used together with other food or food ingredients, and can be suitably used according to a conventional method.

The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, in the production of food or beverage, the compound of the present invention is added in an amount of 10% by weight or less, preferably 5% by weight or less, based on the raw material. However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range .

There is no particular limitation on the kind of the food. Examples of the food to which the above substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, A carbonating agent used in a carbonated beverage, and the like. In addition, the composition of the present invention may include natural fruit juice, fruit juice beverage, and flesh for the production of vegetable beverages. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

The present invention also provides an anti-aging cosmetic composition comprising the compound of formula (1) or (2) as an active ingredient isolated from the mushroom mycelium culture solution.

The cosmetic composition of the present invention may be prepared by mixing with a skin external agent such as water, physiological saline, glycerol, a surfactant, a moisturizer, a thickener, a chelating agent and a pigment, if necessary, in addition to the compound separated from the mushroom mycelium culture solution. And can be produced in various forms according to a conventional method for producing a skin cosmetic composition for external use. For example, the cosmetic composition may contain an ointment, a cream, a softening liquor (skin), a convergent lotion (lotion), an emulsion, an astringent, a massage cream, Powders, suspensions, sprays or essences, for example.

In addition, the cosmetic composition of the present invention may contain customary adjuvants such as stabilizers, solubilizers, vitamins, pigments, and flavorings commonly used in the field of the composition. In the composition, the content of the compound of formula (1) or (2) may be an amount effective to achieve an antioxidant or antioxidant effect, for example, 0.001 to 10% by weight based on the total weight of the composition, 0.01 to 5% by weight, but not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Examples. Preparation of ethylacetate fractions from the mushroom mycelial culture and separation and purification of the compounds 1 to 4 therefrom

1. Culture of mushroom mycelia

400 mL of PDB medium was placed in a 1 L sized flask and inoculated with Coprinus echinosporus (KACC 52101), which was distributed from RDA, and cultured at 27 ° C for 30 days. A total of 14 L mycelium mushroom cultivars were used for the experiment.

2. Preparation of ethylacetate fractions of the mycelia of the mushroom mycelium

The mycelium of the strain cultured in 1 above was finely pulverized and extracted with acetone. After the extraction of the extracted liquid was completed, the filtrate was concentrated under reduced pressure to remove acetone. An equal volume of ethyl acetate was added to the concentrated extract, followed by mixing and fractionation. This procedure was repeated three times to obtain 5 L of the ethyl acetate soluble fraction. The ethyl acetate soluble fraction was concentrated under reduced pressure to obtain 3 g of ethyl acetate soluble extract, which was used as the ethyl acetate fraction.

3. Isolation and purification of compounds 1 to 4

3 g of the ethyl acetate fraction obtained in 2 above was dissolved in methanol and subjected to Sephadex LH-20 column chromatography in order to separate it by molecular size. The fractions thus obtained were analyzed by silica gel TLC under the condition of using chloroform and methanol as a solvent. The fractions obtained by using TLC were analyzed by using concentration gradient conditions in which methanol was gradually increased from 10% to 100% HPLC was performed. The fractions, which were easy to separate out, were subjected to HPLC (Cosmosil, Rp-18, 10 x 150 mm, Japan) while flowing 50% methanol at 3 mL / min to obtain pure compound 1 (4.1 mg) mg), 3 (1.3 mg), and 4 (3.4 mg).

4. Structural analysis of compounds 1 to 4

In order to analyze the structures of the compounds 1 to 4, the molecular weights, EI-MS, UV spectrum, ¹ H / ¹³ C NMR spectrum, and HMBC spectrum of each of the compounds 1 to 4 were confirmed.

(1) Structural analysis of Compound 1

Completely separated Purified Compound 1 was a white powder which was well dissolved in methanol and DMSO. The molecular weight was [M] ⁺ m / z 246.1254 and the molecular formula was estimated as C ₁₅ H ₁₈ O ₃ in HREI-MS. The maximum absorbance at 305.5 and 204.0 nm in the UV spectrum and the small absorbance at 258.5 nm were observed in the shoulder.

^OneThe 1 H NMR spectrum showed three methyl protons (0.92, 1.08, and 2.15 ppm), two aromatic methine protons (6.51 to 6.56 ppm), two methylene protons (2.54, 2.38 ppm) and one methine proton (4.11, 4.75 ppm).

¹³C NMR spectra showed that one ketone (218.1 ppm), six quaternary carbons (41.3, 59.9, 126.6, 128.5, 150.9, 155.0 ppm), two methylene carbons (50.0, 52.3 ppm) Methylene carbon of furan (79.0 ppm) and methine carbon of benzene (109.0, 111.3 ppm) were observed.

In the HMBC spectrum, the methyl proton of 52 ppm (C-2), 41.3 (C-3), 59.7 (C-4), and 23.6 2), 59.9 (C-3), 41.3 (C-4), 22.1 (C-15) ppm carbon and 2.15 ppm methyl protons were 150.9 (C-8), 126.6 -10) ppm carbon and long-rang coupling was observed. The methylene protons of 2.38 ppm were 218.1 (C-1), 59.9 (C-3), 41.3 (C-4), 23.6 (C-14), 22.1 (C-1), 59.9 (C-3), 41.3 (C-4), 128.5 (C-6), and 79.0 Far distance bonds with carbon of 59.9 (C-3), 41.3 (C-4), 50.0 (C-5), 128.5 (C-6), and 155.0 (C-7), 150.9 (C-8) ppm carbon and 6.56 ppm methine proton were 126.6 (C-9), 155.0 (C-11), and 15.3 ppm of carbon and long-distance bonding was observed, and the structure of Compound 1 was estimated as shown in Formula 1-1 below.

[Formula 1-1]

This compound was named as 5-hydroxy-2 ', 2', 6-trimethyl-2H-spiro [benzofuran-3,1'-cyclopentan] -4'-one as a new compound of the benzofuran series.

(2) Structural analysis of Compound 2

Completely separated and purified Compound 2 was a white powder which was well dissolved in methanol and DMSO. The molecular weight of [M] ⁺ was found to be m / z 262 and the molecular formula was estimated as C ₁₅ H ₁₈ O ₄ in EI-MS . UV spectra showed maximum absorbance at 303.5 and 203.5 nm and small absorbance at 256.5 nm.

^OneAs shown in FIG. 2A, in the 1 H NMR spectrum, three methyl protons (0.92, 1.08, and 2.15 ppm), two aromatic methine protons (6.51 to 6.56 ppm), two methylene protons (2.49, 2.32, 2.41 ppm) and one methine proton (5.8 ppm).

¹³C NMR spectra showed that one ketone (218.0 ppm), six quaternary carbons (41.3, 59.7, 126.6, 129, 149.1, 150.9 ppm), two methylene carbons (42.8, 52.2 ppm) Methine carbon (102.4 ppm) of furan and methine carbon (110.7, 111.3 ppm) of benzene were observed.

In the HMBC spectrum, the methyl proton of 52 ppm (C-2), 41.3 (C-3), 59.7 (C-4), and 22.6 2), 41.3 (C-3), 59.7 (C-4), 23.6 (C-14) ppm carbon and 2.15 ppm methyl protons were 149.1 (C-8), 124.8 -10) ppm carbon and long-rang coupling was observed. The methylene protons at 2.32 and 2.41 ppm were 218.1 (C-1), 41.3 (C-3), 59.7 (C-4), 23.6 (C- (C-1), 41.3 (C-3), 59.7 (C-4), 126.6 (C-6), 102.4 (C-12) ppm carbon and 5.84 ppm methine proton (C-3), 59.7 (C-4), 150.9 (C-11) and 22.6 Respectively.

[Formula 1-2]

(3) Structural analysis of Compound 3

Fully separated and purified Compound 3 was a white powder that was well dissolved in methanol and DMSO. The molecular weight was [M] ⁺ m / z 276.1358 and the molecular formula was estimated as C ₁₆ H ₂₀ O ₄ in HREI-MS. The maximum absorbance at 301.0 and 204.5 nm in the UV spectrum and the small absorbance at 258.0 nm were observed. The maximum absorbance at 303.5 and 203.5 nm in the UV spectrum and the small absorbance at 256.5 nm were observed.

^OneIn the 1 H NMR spectrum, three methyl protons (0.89, 1.08, 2.14 ppm), two aromatic methine protons (6.48 to 6.56 ppm), two methylene protons (2.44, 2.38 ppm), one methine proton (5.46 ppm) and one methoxyproton (3.54 ppm) present in the furan ring.

¹³In the C NMR spectrum, one ketone (218.0 ppm), six quaternary carbons (46.0, 60.0, 130.0, 132.0, 154.0, 156.0 ppm), two methylene carbons (48.0, 52.0 ppm) Furan methine carbon (110.0 ppm) and benzene methine carbon (112.0, 116.0 ppm) were observed.

In the HMBC spectrum, the methyl protons of 0.89 ppm were 48.0 (C-2), 46.0 (C-3), 50.0 (C-4), and 28.0 (C-8), 130.0 (C-9), 116.0 (C-12) C-10) ppm carbon. The methylene protons of 2.36 ppm were 2.48 ppm (C-1), 46.0 (C-3), 50.0 (C-4), 28.0 (C-1), 46.0 (C-3), 50.0 (C-4), 132.0 (C-6), 110.0 (C-12) ppm of methylene proton, and 3.54 ppm of methoxyproton were 110.0 (C-12) ppm of carbon and long-distance bonds were observed. The methine protons at 5.48 ppm were 50.0 (C-40), 52.0 (C-5), 132.0 (C-6), 156.0 (C-11), 56.0 (C-6), 154.0 (C-8) ppm of carbon and 6.56 ppm of methine proton are far away from carbon at 130.0 (C-9), 156.0 (C-11), and 18.0 Was observed, and the structure of the compound 3 was estimated as shown in the following formula 1-3.

[Formula 1-3]

This compound is a new compound of benzofuran series in which hydroxyl group is substituted with methoxy group at the 2-position of compound 2, and 5-hydroxy-2-methoxy-2 ', 2', 6-trimethyl- benzofuran-3,1'-cyclopentan] -4'-one.

(4) Structural analysis of compound 4

Completely separated and purified Compound 4 was a white powder which was well dissolved in methanol and DMSO. The molecular weight was [M] ⁺ m / z 274.1201 and the molecular formula was estimated as C ₁₆ H ₁₈ O ₄ in HREI-MS. The maximum absorbance at 334.5, 263 and 209 nm in the UV spectrum and the small absorbance at 315 and 233 nm were observed.

^OneIn the 1 H NMR spectrum, three methyl protons (1.31, 1.34, and 2.17 ppm), two aromatic methine protons (6.80 to 7.51 ppm), one methylene proton (2.48 ppm) one methine carbon (5.84 ppm) and one methoxyproton (3.51 ppm) present in the pyran ring were identified.

¹³C NMR spectrum showed that one ketone (218.0 ppm), seven quaternary carbons (36.0, 116.0, 130.0, 134.0, 140.0, 146.0, 162.0 ppm), one methylene carbon (50.0 ppm) Pyran methine carbon (96.0 ppm) and benzene methine carbon (110.0, 118.0 ppm) were observed.

In the HMBC spectrum, the methyl protons of 1.31 ppm were 50.0 (C-2), 36.0 (C-3), 162.0 (C-4), 27.0 2), 36.0 (C-3), 162.0 (C-4), 26.5 (C-15) ppm carbon and 2.17 ppm methyl protons were 1146.0 (C-8), 130.0 -10) ppm carbon and long-rang coupling was observed. (C-1), 36.0 (C-3), 162.0 (C-4), 26.5 (C-15) ppm carbon, 3.51 ppm methoxyproton was 96.0 (C-4), 134.0 (C-5), 140.0 (C-11), and 54.0 (C-16) ppm of carbon and 5.84 ppm of methine proton. (C-6), 146.0 (C-8) and 140.0 (C-11) ppm of methine proton at 6.80 ppm and 134.0 (C-5) , And 130.0 (C-9) ppm of carbon were observed, and the structure of Compound 4 was estimated as shown in Chemical Formula 2 below.¹³C NMR and HMBC spectra show that 162.0 (C-4) ppm and 134.0 (C-5) ppm are double bonds.

(2)

This compound is a new compound in which a pyran group is present instead of the furan group of compound 2 and is a compound of 8-hydroxy-4-methoxy-3,3,7-trimethyl-2,3-dihydrocyclopenta [ -1 (4H) -one. ≪ / RTI >

Experimental Example . Mushroom mushroom  Antioxidant Activity Measurement of Compounds 1 to 4 Isolated and Purified from Mycelial Culture Solution

One. ABTS Radical  Scavenging activity

The antioxidative activity of ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)) radicals was measured by the antioxidant activity of ABTS free radicals produced by the reaction with potassium persulfate And the radical-specific cyan color is discolored (Nagmoti et al . 2012).

To evaluate the antioxidative activity of the compounds 1 to 4 of the present invention (Formulas 1-1 to 1-3 and Formula 2), the ABTS radical scavenging activity was measured according to the following procedure.

First, ABTS was dissolved at a concentration of 7 mM, and 2.45 mM potassium persulfate was added to make an ABTS cation radical, and the mixture was stored in a dark room at room temperature for 12 hours to produce a chromatic color blue. The resulting ABTS cation radical was diluted in distilled water and prepared to have an absorbance of about 0.7-1.1 at 734 nm. Then, 10 μL of each of compounds 1 to 4 dissolved in DMSO was added to a 96-well plate, and 190 μL of ABTS cation radical solution Was added and reacted for 7 minutes. After completion of the reaction, light absorption was measured at 734 nm through a microplate reader (BMG Labtech). The control group was confirmed by adding DMSO without adding samples. Trolox and BHA (butylated hydroxyanisole) were used as positive control.

The results are shown in Fig.

As shown in FIG. 5, the compounds 1 to 4 exhibited a concentration-dependent ATBS radical scavenging activity, and the compounds 2 and 3 showed similar activities to Trolox and BHA used as positive control.

The IC ₅₀ values of the compounds 1 to 4 of the present invention were measured as 64.1 ± 3.1 μM (1), 52.4 ± 6.0 μM (2), 58.4 ± 3.1 μM (3), and 87.9 ± 4.3 μM (4) The IC ₅₀ values of the control broths and BHA were measured at concentrations of 57.0 ± 6.2 and 45.3 ± 2.3 μM, respectively.

Therefore, it can be seen that the compounds 1 to 4 of the present invention exhibit excellent antioxidant efficacy.

2. DPPH Radical  Scavenging activity

The DPPH radical is a very stable free radical and a deep violet compound at about 520 nm. Antioxidant activity can be easily measured since it is decolorized by antioxidants (Zhang et al. 2012).

To evaluate the antioxidative activity of the compounds 1 to 4 of the present invention (Formulas 1-1 to 1-3 and Formula 2), DPPH radical scavenging activity was measured according to the following procedure.

First, add 10 μL of the sample dissolved in DMSO to a 96-well plate, add 90 μL of DPPH solution (150 μM, ethanol), react at room temperature for 10 minutes, measure the light absorbance at 517 nm through a microplate reader Respectively. At this time, only DMSO was added without adding sample, and Trolox and BHA were used as positive control.

The results are shown in Fig.

As shown in FIG. 6, the compounds 1 to 4 exhibited concentration-dependent DPPH radical scavenging activity, and in particular, Compound 2 showed equivalent activity as compared with the control.

The IC ₅₀ values of the compounds 1 to 4 of the present invention were measured as 226.9 ± 36.5 μM (1), 125.8 ± 5.9 μM (2), 335.7 ± 15.8 μM (3), and 834.3 ± 93.5 μM (4), respectively. The IC ₅₀ values were measured at concentrations of 103.1 ± 3.2 and 137.0 ± 3.8 μM in the positive control, Trolox and BHA, respectively.

Therefore, it can be seen that the compounds 1 to 4 of the present invention exhibit excellent antioxidant efficacy.

3. Reduction power test

In order to evaluate the antioxidative activity of the compounds 1 to 4 of the present invention (Formulas 1-1 to 1-3, and Formula 2), the reducing power was tested according to the following procedure.

First, 50 μL of potassium ferricyanide and 20 μL of sample were added to 50 μL of a 0.2 M phosphate buffer solution (pH 6.6), followed by reaction at 50 ° C. for 20 minutes. After the reaction was completed, 50 μl of 10% TCA solution was added and centrifuged at 3000 × g for 20 minutes. 100 μL of the supernatant, 100 μL of tertiary distilled water and 20 μL of 0.1% ferric chloride were added, and the absorbance at 700 nm was measured. Trolox and BHA were used as positive control.

The results are shown in Table 1.

The compound (formula) Reducing power A (%) ^a Reduction power B (%) ^b 1 (1-1) 88.9 ± 0.8 71.1 ± 0.6 2 (1-2) 116.4 ± 12.6 93.1 ± 10.1 3 (1-3) 81.0 ± 1.8 64.7 ± 1.5 4 (2) 53.8 ± 1.5 43.0 ± 1.2

^a value obtained when the reducing power of Trolox is taken as 100%;

^{b The} value obtained when the reducing power of BHA is 100%

As shown in Table 1, the reductive power test showed that Compound 2 showed higher activity than Trolox, and Compounds 1, 3, and 4 were lower than positive control but showed excellent reducing power.

Therefore, it can be seen that the compounds 1 to 4 of the present invention exhibit excellent antioxidant efficacy.

4. DNA SSB ( single strand breakage ) Inhibitory activity

In order to evaluate the DNA damage inhibiting ability of the compounds 1 to 4 of the present invention (Formulas 1-1 to 1-3 and Formula 2), the following experiment was conducted.

First, the supercoiled DNA of pBR322 (Roche) was dissolved in 10 mM Tris-HCl buffer (pH 7.5) and prepared. Add 20 μL of a different concentration of each sample, 40 μL of 0.25 mM ferrous sulfate, and 40 μL of 1 mM H ₂ O ₂ to 60 μL of Tris-HCl buffer, add 16 μL each to a new Eppendorf tube And 0.2 μg of DNA was added, followed by reaction for 30 minutes. After 30 minutes, the reaction was stopped by the addition of 10 μL of the reaction stop solution (4.0 M urea, 50% sucrose, 50.0 mM EDTA, 0.1% bromophenol blue). To 10 μL of the reaction sample, 2 μL of a DNA dye, loading star, was added and electrophoresed on 1% agarose gel using TAE buffer (40.0 mM Tris acetate, 2.0 mM EDTA, 20.0 mM sodium acetate). Hydroxy radicals induced from the Fenton reaction damage supercoiled DNA and convert it into loose DNA (Lee et al., 2011; Han et al., 2010). BHA, Trolox, and clinically used iron chelator, DFO (deferoxamine), were used as positive control.

The results are shown in Fig.

As shown in FIG. 7, Compounds 1 to 4 of the present invention inhibit DNA single strand breakage (DNA SSB) at a concentration of 50 μM in a manner similar to the control Trolox and DFO.

Therefore, it can be seen that the compounds 1 to 4 of the present invention can be effectively used for the prevention and treatment of various diseases caused by DNA damage.

From the above results, the compounds 1 to 4 of the present invention isolated and purified from the mushroom mycelial culture showed excellent antioxidative activity, exhibiting ABTS and DPPH radical scavenging activity, reducing power and DNA single strand breakage (SSB) inhibitory activity, A medicament for the prevention and treatment of various diseases such as inflammation, anemia, myocardial infarction, cerebral palsy, arteriosclerosis, diabetes, rheumatoid arthritis, Parkinson's disease and autoimmune diseases, skin damage, Food, or food additive.

Examples of formulations for the composition of the present invention are illustrated below.

Formulation example  1. Preparation of pharmaceutical preparations

1. Preparation of Syrup

Syrups containing 2% (weight / volume) of the compounds 1 to 4 of the present invention (Formula 1-1 to 1-3 and Formula 2) or a pharmaceutically acceptable salt thereof as an active ingredient were prepared by the following method Respectively.

The acid addition salts, saccharin and saccharides of the compounds 1 to 4 of the present invention were dissolved in 80 g of hot water. After the solution was cooled, a solution composed of glycerin, saccharin, spices, ethanol, sorbic acid and distilled water was prepared and mixed. Water was added to the mixture to make 100 ml. The addition salt may be replaced with another salt according to the embodiment.

The components of the syrup are as follows.

Active ingredient ··············· 2 g

Saccharin ················· 0.8 g

Per day ····················· 25.4 g

Glycerin · · · · · · · · · · · · · 8.0 g

Spices · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

Ethanol ····················· 4.0 g

Sorbic acid ························· 0.4 g

Distilled water ·················································

2. Preparation of tablets

Tablets containing the compounds 1 to 4 of the present invention (Formula 1-1 to 1-3 and Formula 2) or a pharmaceutically acceptable salt thereof as an active ingredient were prepared as follows.

250 g of the active ingredient was mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. To this mixture was added a 10% gelatin solution, which was pulverized and passed through a 14-mesh sieve. This was dried, and a mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into tablets.

3. Preparation of injection solutions

Injection solutions containing the compounds 1 to 4 of the present invention (Formulas 1-1 to 1-3 and Formula 2) or a pharmaceutically acceptable salt thereof as an active ingredient were prepared by the following method.

1 g of active ingredient, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. This solution was placed in a bottle and sterilized by heating at 20 DEG C for 30 minutes.

Formulation example  2. Manufacturing of food

Foods containing the compounds 1 to 4 of the present invention (Formulas 1-1 to 1-3 and Formula 2) were prepared as follows.

1. Preparation of cooking seasoning

The cooking sauce for health promotion was prepared in the ratio of 0.001 to 10% by weight of the compounds 1 to 4 of the present invention.

2. Manufacture of tomato ketchup and sauce

Compounds 1 to 4 of the present invention were added to tomato ketchup or sauce at a ratio of 0.001 to 10% by weight to prepare healthy tomato ketchup or sauce.

3. Manufacture of flour food

Compounds 1 to 4 of the present invention were added to wheat flour at a ratio of 0.001 to 5.0% by weight, and breads, cakes, cookies, crackers and noodles were prepared using the mixture to prepare foods for health promotion.

4. Manufacture of soups and gravies

Compounds 1 to 4 of the present invention were added to soups and juices at a ratio of 0.001 to 5.0% by weight to prepare meat products for health promotion, soups of noodles and juices.

5. Manufacture of ground beef

The ground beef for health promotion was prepared by adding the compounds 1 to 4 of the present invention to ground beef at a ratio of 0.001 to 5.0% by weight.

6. Manufacture of dairy products

Compounds 1 to 4 of the present invention were added to milk in a proportion of 0.001 to 10% by weight, and various dairy products such as butter and ice cream were prepared using the milk.

Formulation example  3. Manufacturing of beverage

1. Manufacture of health drinks

(Such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%) and water (75% , And Formula (2) were uniformly blended and instant sterilized, and then packaged in small containers such as glass bottles and PET bottles to produce health drinks.

2. Manufacture of vegetable juice

1 to 4 and 5 g of the compounds of the present invention were added to 1,000 ml of tomato or carrot juice to prepare vegetable juice for health promotion.

3. Manufacture of fruit juice

1 to 4 and 1 g of the compounds of the present invention were added to 1,000 ml of apple or grape juice to prepare health promotion fruit juice.

Claims (9)

A compound having an antioxidative activity represented by the following formula (1):
[Chemical Formula 1]

In the above formula (1), R is H, OH, or C ₁ -C ₁₀ alkoxy.
The compound according to claim 1, wherein the compound of formula (1) is selected from the following formulas (1-1) to (1-3).
[Formula 1-1]

[Formula 1-2]

[Formula 1-3]
A compound having an antioxidative activity represented by the following formula (2).
(2)

4. The method according to any one of claims 1 to 3, wherein the compound represented by Formula 1 or Formula 2 is selected from the group consisting of Coprinus echinosporus hyphae culture broth.
A pharmaceutical composition for antioxidation comprising the compound of any one of claims 1 to 3 as an active ingredient.
A pharmaceutical composition for preventing or treating a disease caused by excessive accumulation of active oxygen containing the compound of any one of claims 1 to 3 as an active ingredient.
The method according to claim 6, wherein the disease caused by excessive accumulation of active oxygen is selected from the group consisting of cancer, inflammation, anemia, myocardial infarction, cerebral palsy, arteriosclerosis, diabetes, rheumatoid arthritis, Parkinson's disease, Lt; RTI ID = 0.0 > 1, < / RTI >
A food composition for antioxidation comprising the compound of any one of claims 1 to 3 as an active ingredient.
A cosmetic composition for preventing aging comprising the compound of any one of claims 1 to 3 as an active ingredient.

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