KR20180092305A - A composition for anti-oxidating comprising unripe apple extracts and baicalin - Google Patents

Abstract

The present invention relates to a composition containing a mixture of an unripe apple extract and baicalin. The composition containing the mixture of the unripe apple extract and baicalin as an active ingredient according to the present invention is effective in antioxidation owing to excellent inhibitory activities on tyrosinase, elastase and collagenase, without cytotoxicity and side effects on skin. Thus, the composition can be safely used in food compositions, pharmaceutical compositions, and cosmetic compositions.

Description

TECHNICAL FIELD [0001] The present invention relates to an antioxidant composition comprising an immature apple extract and bicalin as an active ingredient. BACKGROUND ART < RTI ID = 0.0 >

The present invention relates to a composition containing an unripe apple extract and baicalin, and more particularly, to a composition for antioxidation comprising an immature apple extract and baicalin as an active ingredient.

In recent years, in advanced countries such as Europe, America, and Japan, cosmetic materials derived from animals have been rapidly replaced with materials derived from plants due to the incidence of mad cow disease. Plant derived materials include surfactants such as olive oil, palm oil and saponin, wheat germ oil, rice bran oil or phospholipids; A water-soluble component containing an amino acid, a protein, or a saccharide; Various natural products such as rosemary leaves, willow bark extract, niraji root, honey or carrot are used as moisturizers, film formers, skin protectants, lipsticks, and celery.

The domestic cosmetics market is worth KRW 3 trillion, of which the market for skin whitening cosmetics is about 10 billion KRW. In response to the increasing demand for skin whitening and wrinkle improvement in domestic cosmetics, Development of functional raw materials using natural resources is required.

Melanin, a skin pigment factor, is produced against skin damage caused by ultraviolet light and protects the skin. Melanin production starts with the oxidation of tyrosine by tyrosinase, and it causes DOPA and DOPA chrome ), And then DHICA and DHI. DHI-eu melanin, DHICA-eu melanin, and pheo melanin are known to be produced.

The skin melanin pigmentation and deposition caused by exposure to ultraviolet rays are a cause of black spots and the like of spiny freckles. In order to prevent and improve the blackening of such skin, kojic acid, arbutin, vitamin C derivatives Has been developed as a skin whitening agent. However, it has recently been reported that the effect of inhibiting melanogenesis in cell and in vivo is less than that of in vitro tyrosinase, . In addition, arbutin has a problem of cytotoxicity of hydroquinone series, and vitamin C also has a problem of chemical stability, which makes it difficult to keep for a long time.

Since it is known that the skin whitening active substances as described above are known to have adverse effects, studies on functional skin whitening cosmetics or drugs using various natural extracts as an alternative raw material have been actively conducted, but there is insufficient verification of its efficacy and toxicity Since the clinical effect is not satisfactory, the development of new alternative raw materials and the scientific and systematic validation of efficacy are urgent.

Most of the processing byproducts of agricultural products among natural resources are currently classified as industrial wastes and are not used. Among them, apples are local specialties of Gyeongbuk Province and their production amount is 610 thousand tons per year, accounting for 30% Approximately four times as many of these apples are abandoned and about 2.4 million tons per year are being discarded.

On the other hand, it is known that unripe apple contains a large amount of flavan-3-ol based compound which exhibits various physiological activities as compared with mature apple, and Korean Patent No. 10-1065558 Discloses that the extract of the immature apple has a higher active ingredient content than the ripe apple and has excellent skin whitening effect and can be used as a pharmaceutical composition for improving the skin condition. In order to effectively utilize agricultural processing by-products, which are disposed of in large quantities, and to activate the local economy, skin is whitened, antioxidant, and wrinkled using immature apples containing 10 times or more polyphenol than mature apples When a functional biomaterial is developed by verifying the functionality such as efficacy, a high value-added cosmetic or pharmaceutical product can be produced.

In addition, baicalin is a kind of flavonoid, which is a plant of the family Lamiaceae ( Scutellaria baicalensis Georgi ), and it is known to have pharmacological effects such as anti-infective, anti-allergic and antihypertensive effects.

Accordingly, the inventors of the present invention have continued to develop a natural substance having an antioxidant effect and less side effects on human body. As a result, when mixed with an immature apple extract and mixed with bicalin, compared with the case of using only an immature apple extract And exhibits an excellent effect in antioxidation.

The technical problem to be solved by the present invention is to provide a substance having an antioxidant effect and excellent human stability without cytotoxicity derived from natural materials.

In order to solve the above technical problems, the present invention provides an antioxidant composition comprising an immature apple extract and bicalin as an active ingredient.

Preferably, the composition comprising the immature apple extract and baicalin in the present invention may be a cosmetic composition, a pharmaceutical composition or a food composition.

The term " immature apple " of the present invention means an apple that has been fouled one to three weeks after flowering and fertilization.

The term "extract " of the present invention means a preparation which is obtained by squeezing a herbal medicine with an appropriate leaching solution and concentrating by evaporating the leaching solution, and is not limited thereto. The extract, the diluted solution, A dried product obtained by drying, a controlled preparation thereof, or a purified product thereof.

The immature apple extract of the present invention can be extracted using a conventional extraction solvent known in the art, and the extracted solution can be used in a liquid form or can be used by concentration and / or drying. The extraction solvent may be, for example, (a) water, (b) anhydrous or hydrated lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), (c) Solvent, (d) acetone, (e) ethyl acetate, (f) chloroform or (g) 1,3-butylene glycol as the extraction solvent. Preferably, extraction is carried out using methanol, ethanol or butane. Depending on the extraction solvent, the degree of extraction and the degree of loss of the active ingredient of the extract may differ. Therefore, an appropriate extraction solvent should be selected and used. The extraction method is not particularly limited, and examples thereof include cold extraction, ultrasonic extraction, and reflux cooling extraction.

In addition, the immature apple extract can be obtained through a conventional purification process in addition to the extraction method using the extraction solvent. For example, by separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatographies (made for separation by size, charge, hydrophobicity or affinity), and the like, Through the fractionation, an immature apple extract or a gold extract can be obtained.

In one specific embodiment of the present invention, the immature apple extract is prepared by pulverizing immature apple and then extracting a solvent selected from the group consisting of water, an anhydrous or low-boiling alcohol having 1 to 3 carbon atoms, or a mixed solvent thereof, , And the amount of the extraction solvent may be 2 to 4 parts by volume relative to immature apple.

In one specific embodiment of the present invention, the immature apple extract is prepared by pulverizing immature apples and then adding 60 to 80% ethanol (EtOH) at a volume ratio of 2 to 4 times at room temperature for 20 to 30 hours, After immersed and extracted for 26 hours, it is recovered and concentrated with a filter paper to obtain an immature apple extract.

The term " baicalin " of the present invention is a kind of flavonoid, and the baicalin can be purified from plants, produced commercially, or produced by microorganisms.

Plants for isolating and purifying the baicalin were Scutellaria baicalensis Georgi ), and may be preferably golden ( Scutellaria baicalensis ).

In one specific embodiment of the present invention, the baicalin is extracted from the roots of Scutellaria baicalensis , a plant of Lamiaceae.

In one specific embodiment, baicalin is prepared by pulverizing the gold evenly and then adding 60 to 80% ethanol at a volume ratio of 2 to 4 times, at 68 to 76 hours at room temperature, preferably 70 to 74 hours, After 72 hours of extraction, the final extract is solubilized with water (H ₂ O), passed through a purifier, and concentrated under reduced pressure using a vacuum evaporator at 35 to 45 ° C, preferably 40 ° C. Crystallization can be induced from the powder obtained by utilizing the change to obtain a high purity crystalline bacalin.

The composition of the present invention preferably contains 3 to 10 parts by weight of bicalyin per 100 parts by weight of the immature apple extract, more preferably 5 to 7 parts by weight of bicalin per 100 parts by weight of the immature apple extract. When the amount of bicalkine is less than 3 parts by weight per 100 parts by weight of the immature apple extract, the antioxidant effect of the present invention can not be obtained remarkably. When the amount of bicalin is more than 10 parts by weight per 100 parts by weight of the immature apple extract, .

The composition comprising the immature apple extract of the present invention and the baicalin exhibits an antioxidative effect, and has little cytotoxicity as a natural substance.

In one specific embodiment of the present invention, there is provided a cosmetic composition comprising an immature apple extract and baicalin as an active ingredient.

In one specific embodiment of the present invention, the cosmetic composition may contain, in addition to the immature apple extract as an active ingredient, and bicalin, ingredients commonly added to cosmetic compositions such as antioxidants, stabilizers, solubilizers, vitamins, Adjuvants, and carriers may be further added.

The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used as a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, , Oil, powder foundation, emulsion foundation, wax foundation, spray, emulsion, multiple emulsion, liquid crystal, and the like. More specifically, it can be prepared as a nutritional cream, a convergent lotion, a soft lotion, a lotion, an essence, a nutritional gel or a massage cream.

When the formulation of the present invention is a paste, cream or gel, use is made of an animal oil, vegetable oil, wax, paraffin, starch, tragacanth gum, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide .

When the formulation of the present invention is a powder or a spray, tosse, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, chlorofluorohydrocarbons, propane / Propane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Castellulose, aluminum metahydroxide, bentonite, agar or tracert, etc. may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.

In one specific embodiment of the invention, the pharmaceutical composition comprises an immature apple extract and a pharmaceutically acceptable carrier other than baicalin. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc., in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, and is preferably applied by parenteral administration, more preferably topical application by application.

A suitable dosage of the pharmaceutical composition of the present invention may vary depending on such factors as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate, . The dosage of the pharmaceutical composition of the present invention is in the range of 0.001-100 mg / kg on an adult basis. When the composition is an external preparation, it is preferably applied in an amount of 1.0 to 3.0 ml on an adult basis once to five times a day, and continued for 1 month or more. However, the dose is not intended to limit the scope of the present invention.

The pharmaceutical composition of the present invention may be prepared in unit dosage form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art, Into the container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in an oil or aqueous medium, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

In one specific embodiment of the present invention, the food composition contains not only an immature apple extract and baicalin as an active ingredient, but also ingredients normally added in food production such as protein, carbohydrate, fat, nutrients, . ≪ / RTI >

Examples of such carbohydrates are monosaccharides, such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavorings such as tau martin and stevia extract (e.g., rebaudioside A and glycyrrhizin) and synthetic flavorings (saccharine, aspartame, etc.) can be used as flavorings.

For example, when the food composition of the present invention is prepared as a drink, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, mulberry extract, jujube extract and licorice extract are added in addition to mature apple extract of the present invention and baicalin .

Meanwhile, the immature apple extract and baicalin of the present invention are harmless to the human body and have little toxicity and side effects, so that they can be safely used for a long period of use. In particular, the cosmetic composition, the pharmaceutical composition and the food composition It can be safely applied.

As described above, the composition comprising the immature apple extract of the present invention and baicalin as an active ingredient has antioxidative effect, and is free from cytotoxicity and skin side effect, and can be usefully used as a cosmetic composition, a pharmaceutical composition and a food composition. In addition, it is useful not only for environmental conservation but also for economic purposes, by utilizing agricultural processing by-products which are abolished in large quantities.

Figure 1 shows the production process and yield of mature apple extract.
Fig. 2 shows the production process and yield of immature apple extract.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

Manufacturing example  1: Preparation of immature apple extract

The immature apple used in this experiment was obtained in May 2011 in Osanri area, Yeongcheon, North Gyeongsang Province, and immature apple was used as the material after 2 weeks of flowering.

After immature apple was pulverized, 70% ethanol (EtOH) was added at a volume ratio of 3 times of the immature apple crumb, and the immersion extraction was repeated three times at room temperature for 24 hours. 1 filter paper, and the extracts were concentrated at 40 ° C or lower using a rotary vacuum evaporator, and then stored in a refrigerated room after lyophilization. Figure 1 shows the production process and yield of mature apple extract. Fig. 2 shows the production process and yield of immature apple extract.

Manufacturing example  2: Baikal's  Produce

The gold ( Scutellaria) produced in July 2010 The resulting extract was dissolved in water (H ₂ O) and passed through a purifier. A vacuum evaporator was used at a temperature of 40 ° C to utilize the extract. After concentration under reduced pressure, crystallization was induced from the powder obtained by using the temperature change to prepare high purity crystalline bacalin.

Example  1: immature apple extract and Baikal's  Preparation of mixture

1, 3, 5, 7, 10 and 12 parts by weight of Baikalin prepared in Preparation Example 2 were added to 100 parts by weight of the immature apple extract prepared in Preparation Example 1 to prepare a mixture.

Sample 1 was a mixture of 100 parts by weight of an immature apple extract and 1 part by weight of bicalyin, Sample 2 was a mixture of 100 parts by weight of an immature apple extract and 3 parts by weight of bicalin, Sample 3 contained 100 parts by weight of an immature apple extract, A mixture of 100 parts by weight of an immature apple extract and 7 parts by weight of bicalin, a mixture of 100 parts by weight of an immature apple extract and 10 parts by weight of bicalin, a sample 5, a mixture of 100 parts by weight of an immature apple extract and bicalin 12 By weight, respectively.

Test Example  One: Skin whitening  For effect verification Tyrosinase  Measurement of inhibitory activity

Tyrosinase is an enzyme containing copper, which is an L-tyrosine (L-3,4-dihydroxyphenylalanine (DOPA)) in the early stage of melanin synthesis. , DOPA to L-dopaquinone (L-dopaquinone). The mitosis of the melanocyte occurs by ultraviolet light and then the melanocyte is activated. In activated melanocytes, tyrosinase synthesis is promoted and melanin is produced and transported out of the epidermis, causing pigmentation such as spots and freckles. Therefore, tyrosine activity inhibitors can effectively inhibit the synthesis of melanin polymers in the skin, so that inhibition of tyrosinase activity in the development of skin whitening agents has been recognized as a useful evaluation method. Thus, tyrosinase plays an important role in the melanin biosynthesis process, so that tyrosinase inhibitors can be used as a substance capable of controlling the melanin pigment production of the skin.

The tyrosinase inhibitory activity was measured according to the Yagi method. To the reaction mixture was added 0.2 mL of mushroom tyrosinase (110 U / mL) to a mixed solution of 0.5 mL of a 1 / 15M sodium phosphate buffer solution (sodium phosphate buffer, pH 6.8), 0.2 mL of a substrate solution in which 10 mM of DOPA had been dissolved and 0.1 mL of a sample solution And the mixture was reacted at 25 DEG C for 2 minutes to measure DOPA chromium generated in the reaction solution in the light having a wavelength of 475 nm. The tyrosinase inhibitory activity was expressed by the absorbance reduction rate of the sample solution added and the non-added sample. Nuruk acid was used as a positive control for comparative analysis.

  Table 1 below shows the results of measuring tyrosinase inhibitory activity of a mixture (samples 1 to 6) obtained by mixing 100 parts by weight of an extract of an immature apple with 70% ethanol and a mixture of bikalin at a concentration of 1, 3, 5, 7, 10 and 12 parts by weight . As the content of baicalin in the sample increased, the tyrosinase inhibitory activity of the mixture was strongly shown.

However, when the content of BaCalin was less than 3 parts by weight (Sample 1), the tyrosinase inhibitory activity was weaker than that of the immature apple extract alone, and when the amount was more than 10 parts by weight (Sample 6), the effect of tyrosinase- . In Table 1, nuruk acid was used as a positive control.

Samples Inhibitory activity (%) 1000ug / mL 500 ug / mL 250 ug / mL 125 ug / mL 62.5 ug / mL 31.3 ug / mL Immature apple 70% EtOH ext. 100 parts by weight 15.9 13.5 8.4 6.3 4.0 1.2 Sample 1 13.7 10.7 6.1 0.7 0.6 0.0 Sample 2 17.4 14.1 9.4 6.9 4.2 1.4 Sample 3 21.7 16.8 12.1 8.7 6.5 4.7 Sample 4 25.5 18.7 15.8 12.4 8.1 6.1 Sample 5 29.3 20.5 19.3 14.5 10.0 7.8 Sample 6 30.1 21.0 19.6 14.7 10.2 7.9 Kojic acid 94.4 84.8 70.5 50.7 31.7 12.7

Test Example  2: Verification of antioxidant effect

(1) Measurement of DPPH radical scavenging ability

DPPH used in the determination of electron donating ability is a chemically stabilized free radical material which is very stable to organic solvents and has a maximum absorbance in the vicinity of 515 to 520 nm. The antioxidant activity of the antioxidant can be easily observed with the naked eye because the antioxidant activity of the antioxidant can be easily detected by the decolorization. After the initiation of oxidation, the scavenging activity of free radicals is measured by absorbance of each sample. If the ability to reduce or offset the free radicals is high, the scavenging activity against high radicals can be expected, which can also be used as a measure to inhibit free radical aging in the human body.

The electron donating ability (EDA) was measured by modifying the Blois method. 1 mL of 0.2 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) was added to 2 mL of each sample solution, and the mixture was allowed to stand for 30 minutes, and the absorbance was measured at 517 nm Respectively. The electron donating ability was expressed by the absorbance reduction ratio of the sample solution and the non-additive solution.

DPPH scavenging activity was measured in order to examine the antioxidant effect of the immature apple extract and Samples 1 to 6 according to the above method. The results are shown in Table 2 below.

The IC ₅₀ value in Table 2 below means a concentration value for reducing the DPPH by 50% at the point of 10 minutes after the start of the reaction. As shown in the following Table 2, the IC ₅₀ value of the sample 1 was the highest And the IC ₅₀ value of the sample 6 was the lowest. Therefore, it was confirmed that the antioxidative effect was superior to the immature apple extract when the content of bacalin was increased.

However, when the content of BaCalin was less than 3 parts by weight (Sample 1), the IC ₅₀ value was higher than that of the immature apple extract alone, indicating that the sulfation effect was lower than using the immature apple extract alone (Sample 6) and the IC ₅₀ value of the sample 5 was small, it was confirmed that the effect of sulfation was slowed down with an increase in the content of bicanal. In Table 2 below, (+) - Catechin was used as a positive control.

Sample IC ₅₀ (ug / mL) Immature apple 70% EtOH ext. 115.4 ± 6.4 Sample 1 117.5 ± 5.4 Sample 2  78.9 ± 3.1 Sample 3  64.8 ± 4.3 Sample 4  51.2 ± 1.9 Sample 5  49.7 ± 2.1 Sample 6  49.5 ± 2.2 (+) - Catechin   4.6 ± 0.3

(2) Measurement of ABTS radical scavenging activity

The ABTS latticidal cation scavenging activity, which is commonly used, such as the scavenging activity of DPPH radicals, is caused by the reaction of 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) with potassium persulfate ABTS + · When radicals are produced, they have a unique index of cyan color. It is a method to measure discoloration of light green by the addition of a sample. It is a method of measuring hydrogen-donating antioxidant and chain breaking antioxidant ) Can be measured.

Antioxidant activity using ABTS radicals was measured by the ABTS + cation decolorization assay (Roberta, et al., 1999). 7 mM 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) and 2.4 mM potassium persulfate were mixed and incubated at room temperature for 24 hours to form ABTS +, diluted with ethanol, 100 μl of the sample was added to the wells and left for 1 minute, and the absorbance was measured at 732 nm.

In order to examine the antioxidative effect of the immature apple extract and Samples 1 to 6 according to the above method, the ABTS radical scavenging activity was measured and the results are shown in Table 3 below.

The IC ₅₀ values shown in Table 3 below represent concentrations at which the ABTS is reduced by 50% at 6 minutes after the start of the reaction. As shown in Table 2 below, the IC ₅₀ value of the sample 1 is the highest And the IC ₅₀ value of the sample 6 was the lowest. Therefore, it was confirmed that the antioxidative effect was superior to the immature apple extract when the content of bacalin was increased.

However, when the content of BaCalin was less than 3 parts by weight (Sample 1), the IC ₅₀ value was higher than that of the immature apple extract alone, indicating that the sulfation effect was lower than using the immature apple extract alone (Sample 6) and the IC ₅₀ value of the sample 5 was small, it was confirmed that the effect of sulfation was slowed down with an increase in the content of bicanal. In Table 2 below, (+) - Catechin was used as a positive control.

Sample IC ₅₀ (ug / mL) Immature apple 70% EtOH ext. 45.4 ± 2.3 Sample 1 47.7 ± 2.8 Sample 2 41.5 ± 1.6 Sample 3 34.3 ± 1.5 Sample 4 30.8 ± 2.1 Sample 5 28.3 ± 1.5 Sample 6 28.1 ± 1.4 (+) - Catechin 1.34 ± 0.2

Test Example  3: Wrinkle improvement effect verification

(1) Measurement of Elastase Inhibitory Activity

The existing elastase in the neutrophil granules of the human body is an enzyme that degrades elastin, an important substrate protein that maintains skin elasticity in the dermis, and is a nonspecific hydrolytic enzyme capable of degrading collagen, another important substrate protein. Thus, the elastase inhibitor has a function of improving the wrinkles of the skin, and ursolic acid is used as the elastase inhibitor. It is also one of the leucocyte granulase enzymes that degrade elastin in the body. In the abnormal tissues, the activity of the enzymes is extremely high, which is a direct cause of tissue destruction, resulting in wrinkles and loss of elasticity of the skin.

Formate new plate Crescent Az elastase (Porcine pancreas elastase) inhibitory activity is measured as the substrate N - succinyl - (L- alanine) ₃ - p - a by 20 min at 37 ℃ using a fluoride generating nitro not p - The amount of nitroalide was measured in light of 405 nm wavelength. 0.5 mL of each test solution was added to 0.5 mL of each test solution. 0.5 mL of elastase (2.5 U / mL) dissolved in 50 mM tris-HCl buffer (pH 8.6) was added to the test tube. 50 mL of tris-HCl N-succinyl- (L-alanine) 3-p-nitroanilide (0.5 mg / mL) dissolved in a buffer solution (pH 8.6) was added and reacted for 20 minutes.

The results of the above test on the 70% ethanol extract of immature apple and Samples 1 to 6 are shown in Table 4 below. Ursolic acid was used as a positive control.

As a result of the experiment, it was confirmed that the above-mentioned mixture shows a stronger inhibitory activity of the mixture with elastin than with the content of baicalin. However, when the content of baicalin is less than 3 parts by weight, the inhibitory activity of the elastase is weaker than that of the immature apple extract alone. When the content of the ingredient is more than 10 parts by weight, the activity of inhibiting elastase It is confirmed that the effect is slowed down.

Sample No. Inhibitory activity (%) 1000ug / mL 500 ug / mL 250 ug / mL 125 ug / mL 62.5 ug / mL 31.3 ug / mL Immature apple 70% EtOH ext. 100 parts by weight 63.1 56.2 52.2 30.3 12.0 0.0 Sample 1 56.1 49.5 49.2 41.7 4.6 0.0 Sample 2 65.0 57.8 52.8 30.5 15.5 0.0 Sample 3 67.0 59.8 54.6 37.9 22.3 1.4 Sample 4 69.5 60.6 56.2 45.5 27.2 1.8 Sample 5 71.2 65.1 58.8 49.1 31.1 2.0 Sample 6 71.5 65.5 59.0 49.2 31.1 1.9 Ursolic acid 76.1 71.3 66.4 62.8 30.2 6.5

(2) Measurement of inhibitory activity of collagenase

Collagen, a major constituent of the extracellular matrix, is a major substrate protein produced in fibroblasts of the skin. It is also an important protein, accounting for about 30% of the total weight of bioprotein, and has a solid triple structure. Collagen forms most of the organic matter in the skin, tendons, bones and teeth, especially in the bones and dermis of the skin. The main functions of collagen are known to be mechanical durability of the skin, resistance of connective tissue and binding force of tissue, support of cell adhesion, induction of cell division and differentiation. Such collagen is reduced by aging and photoaging by ultraviolet irradiation, which is known to be closely related to wrinkling of the skin. In addition, collagen is not affected by proteases such as trypsin, but is reported to be degraded by collagenase.

The collagenase inhibitory activity was measured according to the method of Winsch et al. That is, 4 mM CaCl ₂ was added to 0.1 M tris-HCl (pH 7.5) to prepare a reaction substrate. The substrate was dissolved in 4-phenylazo benzyloxycarbonyl 1-proline-leucine-glycine-proline-D-arginine (0.3 mg / 0.15 mL of collagenase (0.2 mg / mL) was added to a mixture of 0.25 mL of the solution and 0.1 mL of the sample solution, and the mixture was allowed to stand at room temperature for 20 minutes. Then, 0.5 mL of 6% citric acid was added to stop the reaction, 1.5 mL of acetate was added and the absorbance at 320 nm was measured. The collagenase inhibitory activity was expressed by the absorbance reduction ratio of the sample solution and the non-added sample.

The results of the above test on the immature apple 70% ethanol extract and Samples 1 to 6 are shown in Table 5 below. Epigallocatechin gallate (EGCG) was used as a positive control.

As a result of the experiment, it was confirmed that the above mixture exhibited a strong collagenase inhibitory activity as the content of bicalyin increased. However, when the content of bacalin was less than 3 parts by weight, the collagenase inhibitory activity was weaker than that of the immature apple extract alone. When the content of bacalin was more than 10 parts by weight, the effect of collagenase inhibitory activity It can be confirmed that it is slowing down.

Sample No. Inhibitory activity (%) 1000 ug / mL 500 ug / mL 250 ug / mL 1255 ug / mL 62.5 ug / mL Immature apple 70% EtOH ext. 100 parts by weight 54.6 35.9 29.0 20.4 .2 Sample 1 54.5 36.0 28.4 19.9 15.0 Sample 2 57.3 53.1 48.3 32.6 17.8 Sample 3 68.2 60.7 52.8 36.5 22.3 Sample 4 82.9 78.2 72.0 61.3 28.0 Sample 5 93.3 88.0 79.7 69.8 44.0 Sample 6 93.4 88.2 79.6 69.8 44.1 EGCG 87.4 87.5 83.5 81.0 61.9

Claims (4)

An antioxidant cosmetic composition comprising an immature apple extract and baicalin as an active ingredient. The method according to claim 1,
Wherein the composition comprises 3 to 10 parts by weight of bicalkine based on 100 parts by weight of an immature apple extract. An extract for antioxidant composition containing an immature apple extract and baicalin as an active ingredient. The method of claim 3,
Wherein the composition comprises 3 to 10 parts by weight of bicalin based on 100 parts by weight of an immature apple extract.

Images