KR102000687B1 - 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 unripe apple extract and Baikal, the composition comprising a mixture of immature apple extract and Baikal of the present invention as an active ingredient inhibits tyrosinase, elastase and collagenase It has excellent activity and is effective in antioxidation, and has no cytotoxic and skin side effects, so it can be safely used in cosmetic compositions, pharmaceutical compositions and food compositions.

Description

Antioxidant composition comprising immature apple extract and Baikalin as active ingredients {A COMPOSITION FOR ANTI-OXIDATING COMPRISING UNRIPE APPLE EXTRACTS AND BAICALIN}

The present invention relates to a composition containing immature apple extract (unripe apple) and Baikal (baicalin), and more particularly to an antioxidant composition comprising immature apple extract and Baikalin as an active ingredient.

Recently, in developed countries such as Europe, the United States, and Japan, the development of mad cow disease has led to the rapid replacement of animal-derived cosmetics with plant-derived materials. Plant-derived materials such as olive oil, palm oil and saponins, wheat germ oil, rice bran oil or phospholipids; Water-soluble components containing amino acids, proteins or sugars; Various natural products, such as rosemary leaves, willow bark extracts, rapeseed roots, honey or carrots, are used as moisturizers, film formers, skin protectants, lipsticks, and perfumes.

The domestic cosmetics market is worth 3 trillion won, of which the market for skin whitening cosmetics is about 10 billion. Development of functional raw materials using natural resources is required.

As a skin whitening factor, melanin is a skin pigment factor that is produced against skin damage caused by UV rays and protects the skin.Melanin production begins with oxidation of tyrosine by tyrosinase, which leads to DOPA and DOPA chromium (chrome). ) Is produced through DHICA and DHI, and until now, three species such as DHI-eu melanin, DHICA-eu melanin, and pheomelanin are known to be produced.

Melanin pigment overdeposition in the skin caused by UV exposure causes blemishes and freckles, black spots. Although it has been developed as a skin whitening agent, it has recently been reported that Nurukic acid has less inhibitory effect on melanin production in cells or in vivo than on tyrosinase inhibitor in vitro and can cause liver cancer in long-term use. . In addition, arbutin has a problem of cytotoxicity of the hydroquinone series, and vitamin C also has a problem of chemical stability, making it difficult to store for a long time.

As the side effects of such skin whitening active substances are known, researches on functional skin whitening cosmetics or drugs using various natural extracts have been actively conducted as alternative raw materials, but there is not sufficient verification of their efficacy or toxicity. Since the clinical effect is not satisfactory, the development of new alternative raw materials and the scientific and systematic efficacy verification are urgently needed.

The processed by-products of agricultural products in natural resources are currently classified as industrial wastes and are not processed and used. Among them, apples are a special product of Gyeongbuk province, with an annual output of 610,000 tons, accounting for 30% of the whole fruit. Apple apples are dumped at about four times their volume and about 2.4 million tonnes a year.

On the other hand, unripe apple (unripe apple) is known to contain a large amount of a compound of flavan-3-ol (flavan-3-ol) that shows a variety of physiological activity compared to mature apples, Korean Patent No. 10-1065558 No. discloses that the extract of the unripe fruit family can be used as a pharmaceutical composition for improving the skin condition because the content of the active ingredient is higher than that of the ripe fruit fruit and the skin whitening effect is excellent. Therefore, the skin whitening, antioxidant, and wrinkle improvement are made by using immature apples (red apples) containing 10 times more polyphenols than mature apples for efficient utilization of agricultural processed by-products that are disposed of in large quantities and revitalization of local economy. When developing functional biomaterials by verifying functionalities such as efficacy, high value-added cosmetics or drugs can be produced.

In addition, baicalin is a type of flavonoid (flavonoid) that is a plant of the plant Lamiaceae Scutellaria ( Scutellaria) baicalensis It is mainly contained in the root of Georgi ), and is known to have pharmacological effects such as anti-infection, anti-allergic, antihypertensive and the like.

Accordingly, the present inventors have continued to develop natural substances having an antioxidant effect and less human side effects, and compared with using only immature apple extracts without the cytotoxicity when using bikal in combination with immature apple extracts. The present invention has been completed by the discovery of an excellent effect on antioxidants.

The technical problem to be solved in the present invention is to provide an excellent human stability material having an antioxidant effect and no cytotoxicity derived from natural materials.

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

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

The term “mature apple” of the present invention means apples that have been loaded 1 to 3 weeks after flowering fertilization.

The term "extract" of the present invention refers to a formulation prepared by squeezing the herbal medicine with a suitable leach solution and evaporating the leach solution, but not limited thereto, but the extract obtained by the extraction treatment, the dilution or concentrate of the extract, the extract It may be a dried product obtained by drying, these adjustment products, or a refined product.

The immature apple extract of the present invention can be extracted using a conventional extraction solvent known in the art, the extracted liquid can be used in liquid form or concentrated and / or dried. At this time, the extraction solvent is, for example, (a) water, (b) anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), (c) mixing the lower alcohol with water A solvent, (d) acetone, (e) ethyl acetate, (f) chloroform or (g) 1,3-butylene glycol can be obtained as an extraction solvent. Preferably, the extraction is performed using methanol, ethanol or butane. Depending on the extraction solvent, the extraction degree and loss degree of the active ingredient of the extract may vary, so select an appropriate extraction solvent. The extraction method is not particularly limited, and examples thereof include cold needle extraction, ultrasonic extraction, reflux cooling extraction, and the like.

In addition, the immature apple extract can be obtained through a conventional purification process in addition to the extraction method by the extraction solvent. Obtained by various additional purification methods, such as separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity). Immature apple extract or golden extract can also be obtained through the fraction.

In one specific embodiment of the present invention, the immature apple extract is pulverized immature apple and then extracted with a solvent selected from the group consisting of an extraction solvent, such as water, anhydrous or hydrous lower alcohols having 1 to 3 carbon atoms and mixed solvents thereof. It can be extracted using, the amount of the extraction solvent may be in a volume ratio of 2 to 4 relative to the immature apple.

In one specific embodiment of the present invention, the immature apple extract is ground to 20 to 30 hours, preferably 22 to 20 hours at room temperature by adding 60 to 80% of ethanol (EtOH) in a volume ratio of 2 to 4 times after grinding the immature apple After dipping for 26 hours, the extract is recovered, and concentrated with a filter paper to obtain an immature apple extract.

"Baicalin" of the present invention is a kind of flavonoids, which may be isolated and purified from plants, commercially produced, or produced by microorganisms.

The plant for isolating and purifying the Baikallin is Lamiaceae plant Koganevana ( Scutellaria baicalensis) Georgi ), preferably Scutellaria baicalensis .

In one specific embodiment of the present invention, the Baikal is extracted from the root of Lamiaceae plant ( Scutellaria baicalensis ).

In one specific embodiment, Baikal is 68 to 76 hours, preferably 70 to 74 hours, even more preferably at room temperature by crushing the gold evenly and adding 60 to 80% ethanol at a volume ratio of 2 to 4 times. After extracting for 72 hours, the final extract was solubilized with water (H ₂ O), passed through a refiner, and concentrated under reduced pressure using a vacuum evaporator at a temperature of 35 to 45 ° C., preferably 40 ° C., followed by temperature Crystalline can be induced from the powder obtained by utilizing the change to obtain high purity crystalline Baikallin.

The composition of the present invention preferably comprises 3 to 10 parts by weight of bicalin per 100 parts by weight of immature apple extract, more preferably 5 to 7 parts by weight of bicalin per 100 parts by weight of immature apple extract. At this time, if the amount of bicalin per 100 parts by weight of immature apple extract is less than 3 parts by weight, the antioxidant effect of the present invention could not be remarkably obtained. If the amount of bicalin per 100 parts by weight of immature apple extract exceeds 10 parts by weight, the effect of increasing the content The difference is small.

The composition comprising immature apple extract and Baikal of the present invention exhibits an antioxidant effect and is virtually free from cytotoxicity.

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

In one specific embodiment of the present invention, cosmetic compositions include ingredients typically added to cosmetic compositions in addition to immature apple extracts and Baikal as active ingredients, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavorings. Adjuvants and carriers may be further added.

The cosmetic composition of the present invention may be prepared in any formulation commonly prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing , Oils, powder foundations, emulsion foundations, wax foundations, sprays, emulsions, multiple emulsions and liquid crystals, and the like, but are not limited thereto. More specifically, it may be prepared in the form of a nourishing cream, astringent lotion, a flexible lotion, a lotion, an essence, a nourishing gel or a massage cream.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant gum, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide are used as carrier components. Can be.

Toss, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as the carrier component when the formulation of the present invention is a powder or a spray, and in particular in the case of a spray, additionally chlorofluorohydrocarbon, propane Propellant such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 Fatty acid esters of, 3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Castle cellulose, aluminum metahydroxy, bentonite, agar or tracant gum can be used.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component is an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, an isethionate, an imidazolinium derivative, a methyltaurate, a sarcosinate, a fatty acid amide. Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

In one specific embodiment of the invention, the pharmaceutical composition comprises a pharmaceutically acceptable carrier in addition to the immature apple extract and Baikal. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. 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 may be administered orally or parenterally, and is preferably applied by topical application by parenteral administration, more preferably by application.

Suitable dosages of the pharmaceutical compositions of the present invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. The dosage of the pharmaceutical composition of the present invention is in the range of 0.001-100 mg / kg on an adult basis. In addition, in the case of external preparations, it is recommended to continue the application for 1 month to 5 times a day in an amount of 1.0 to 3.0 ml on an adult basis. However, the dosage does not limit the scope of the present invention.

The pharmaceutical compositions of the present invention may be prepared in unit dosage form or in multidose form by formulating with a pharmaceutically acceptable carrier and / or excipient, according to methods which can be easily carried out by those skilled in the art. It may be prepared by incorporation into a container. In this case, the formulation may be in the form of a solution, suspension, syrup or emulsion in an oil or an aqueous medium, or may be in the form of an exir, powder, powder, granule, tablet or capsule, and may further include a dispersing or stabilizing agent.

In one specific embodiment of the present invention, the food composition comprises immature apple extracts and bicalin as active ingredients, as well as ingredients commonly added in food preparation, such as proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents. It may further include.

Examples of such carbohydrates are monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And sugars such as conventional sugars such as polysaccharides such as dextrin, cyclodextrin and the like and xylitol, sorbitol, erythritol. As the flavoring agent, natural flavoring agents (tauumatin, stevia extract (for example rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used.

For example, when the food composition of the present invention is prepared with a drink, in addition to citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, tofu extract, jujube extract, licorice extract, etc., in addition to the mature apple extract and baikalline of the present invention It can be included as.

On the other hand, the immature apple extract and Baikal of the present invention is a natural substance harmless to the human body, there is little toxicity and side effects, so can be used safely even in the long-term use, especially in the cosmetic composition, pharmaceutical composition and food composition as described above It can be safely applied.

As such, the composition comprising the immature apple extract and Baikal of the present invention as an active ingredient has an antioxidant effect, there is no cytotoxicity and skin side effects can be useful as a cosmetic composition, pharmaceutical composition and food composition. In addition, by utilizing agricultural processing by-products that are disposed of in large quantities, it is useful not only for environmental conservation but also for economic reasons.

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

Hereinafter, examples and the like will be described in detail to help understand the present invention. However, embodiments according to the present invention can be modified in many different forms, the scope of the invention should not be construed as limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

Production Example  1: Preparation of Immature Apple Extract

The immature apples used in this experiment were obtained from Osan-ri, Hwabuk-myeon, Yeongcheon-si, Gyeongsangbuk-do, Korea.

After crushing the immature apple, 70% ethanol (EtOH) was added in three times the volume ratio of the immature apple crushed and immersion extraction was repeated three times for 24 hours at room temperature. One filter paper was filtered, and each extract was concentrated under a temperature of 40 ℃ with a rotary vacuum evaporator, and then used as a sample of this experiment while being stored in the refrigerator after freeze-drying. Figure 1 shows the manufacturing process and yield of the mature apple extract. Figure 2 shows the production process and yield of immature apple extract.

Production Example  2: Baikalin  Produce

Scutellaria produced in July 2010 at Imgo-myeon, Yeongcheon-si ethanol (EtOH) was added to the powder obtained by crushing the baicalensis evenly, and the resultant was repeatedly extracted at room temperature for 3 days, and the final extract was solubilized with water (H ₂ O) and passed through a refiner to use a vacuum evaporator at 40 ° C. After concentrating under reduced pressure, crystallization was induced from the powder obtained by utilizing the temperature change to prepare crystalline bicalin of high purity.

Example  1: immature apple extract and Baikalin  Mixture manufacturing

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

Hereinafter, Sample 1 is a mixture of 100 parts by weight of immature apple extract and 1 part by weight of Baikal, Sample 2 is a mixture of 100 parts by weight of immature apple extract and 3 parts by weight of Baikal, Sample 3 is 100 parts by weight of immature apple extract and 5 wt. A mixture of parts, Sample 4 is a mixture of 100 parts by weight of immature apple extract and 7 parts by weight of Baikal, Sample 5 is a mixture of 100 parts by weight of immature apple extract and 10 parts by weight of Baikal, Sample 6 is 100 parts by weight of immature apple extract and Baikal 12 Each weight part means a mixture.

Test Example  One: Skin whitening  To verify effectiveness Tyrosinase  Inhibition activity measurement

Tyrosinase is an enzyme that contains copper and L-3,4-dihydroxyphenylalanine (DOPA) in L-tyrosine, an early stage of melanin synthesis. , Acts on the transition from DOPA to L-dopaquinone. Ultraviolet mitosis of melanocytes occurs followed by activation of melanocytes. In activated melanocytes, tyrosinase synthesis is promoted and melanin is produced and transported out of the epidermis, causing pigmentation such as blemishes and freckles. Therefore, tyrosine activity inhibitors can effectively inhibit the synthesis of melanin polymers in the skin. Tyrosinase activity inhibition experiments have been recognized as useful evaluation methods in the development of skin whitening agents. Therefore, tyrosinase plays an important role in melanin biosynthesis, so tyrosinase inhibitors can be used as substances that can control the production of melanin in the skin.

Tyrosinase inhibitory activity was measured according to the Yagi method. The reaction zone was 0.2 mL of mushroom tyrosinase (110 U / mL) in a mixture of 0.2 mL of substrate solution dissolved in 10 mM DOPA in 0.5 mL of 1/15 M sodium phosphate buffer (pH 6.8) and 0.1 mL of sample solution. The reaction was added and reacted at 25 ° C. for 2 minutes, and the DOPA chromium produced in the reaction solution was measured at 475 nm wavelength light. The tyrosinase inhibitory activity was expressed by the decrease in absorbance of the added and non-added samples. Comparative analysis was performed using Nurukic acid as a positive control.

  Table 1 shows the tyrosinase inhibitory activity of the mixture (samples 1 to 6) in which Baikal was mixed at a concentration of 1, 3, 5, 7, 10 and 12 parts by weight based on 100 parts by weight of immature apple 70% ethanol extract. . As the content of the bicalin in the sample increased, it was confirmed that the tyrosinase inhibitory activity of the mixture was strongly expressed.

However, when the amount of Baikal is less than 3 parts by weight (sample 1), the tyrosinase inhibitory activity was weaker than that of using immature apple extract alone, and when it was more than 10 parts by weight (sample 6), the effect of tyrosinase inhibitory activity was slowed. It could be confirmed. In the following Table 1 Nurukic 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 Apples 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 Kojiic acid 94.4 84.8 70.5 50.7 31.7 12.7

Test Example  2: Antioxidant Effect Verification

(1) DPPH radical scavenging activity measurement

DPPH, which is used for measuring electron donating ability, is a substance with chemically stabilized free radicals, which is very stable in organic solvents, and has a maximum absorbance around 515 ~ 520nm. When it encounters a substance with antioxidant activity, the radicals are dissipated and discolored while giving out electrons, so the antioxidant activity can be easily observed with the naked eye. It is widely used to search for antioxidants from various materials. After initiation of oxidation, the scavenging activity of free radicals is measured by absorbance by each sample. If the ability to reduce or offset free radicals is high, scavenging activity for high radicals can be expected, which can also be used as a measure of inhibiting aging by free radicals in the human body.

Electron donating ability (EDA) was measured by modifying the method of Blois. 1 mL of 0.2 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) was added to 2 mL of each sample solution, stirred for 30 minutes, and then absorbed at 517 nm. It was. The electron donating ability was expressed as the absorbance reduction rate of the sample solution addition and no addition solution.

DPPH scavenging activity was measured to determine the antioxidant effects of the extracts and samples 1 to 6 of immature apples according to the above method, and the results are shown in Table 2 below.

The IC ₅₀ value of Table 2 means a concentration value for reducing the DPPH by 50% at the time 10 minutes after the start of the reaction, measured as the highest IC ₅₀ value of Sample 1 as shown in Table 2 below And the IC ₅₀ value of Sample 6 was the lowest. Therefore, it was confirmed that the antioxidant effect was excellent as the content of Baikal in the immature apple extract increased.

However, when the content of Baikal is less than 3 parts by weight (sample 1), the IC ₅₀ value is higher than that of the immature apple extract alone, indicating that the sulfated effect is lower than that of the immature apple extract alone. In case of exceeding 10 parts by weight (sample 6), the difference from the IC ₅₀ value of Sample 5 was insignificant, and it was confirmed that the sulfated effect was slowed down with the increase in the content of bicalin. In Table 2 (+)-Catechin was used as a positive control.

Sample IC ₅₀ (ug / mL) Immature Apples 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) Determination of ABTS radical scavenging activity

A commonly used ABTS radical cation scavenging activity, such as the scavenging activity of DPPH radicals, is the reaction of 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) with potassium persulfate When ABTS + radicals are produced, they have a distinctive index of turquoise, which is a method of measuring the discoloration of light green with the addition of a sample. ) Can be measured.

Antioxidant activity using ABTS radicals was determined by 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 for 24 hours in the dark at room temperature to form ABTS + and diluted with ethanol to ABTS + 100 ul of sample was added to 100 ul and left for 1 minute, and then absorbance was measured at 732 nm.

According to the method, ABTS radical scavenging activity was measured to determine the antioxidant effects of the extracts of immature apples and samples 1 to 6, and the results are shown in Table 3 below.

The IC ₅₀ value of Table 3 means a concentration value for reducing the ABTS by 50% at 6 minutes after the start of the reaction. As shown in Table 2, the IC ₅₀ value of Sample 1 was determined to be the highest. And the IC ₅₀ value of Sample 6 was the lowest. Therefore, it was confirmed that the antioxidant effect was excellent as the content of Baikal in the immature apple extract increased.

However, when the content of Baikal is less than 3 parts by weight (sample 1), the IC ₅₀ value is higher than that of the immature apple extract alone, indicating that the sulfated effect is lower than that of the immature apple extract alone. In case of exceeding 10 parts by weight (sample 6), the difference from the IC ₅₀ value of Sample 5 was insignificant, and it was confirmed that the sulfated effect was slowed down with the increase in the content of bicalin. In Table 2 (+)-Catechin was used as a positive control.

Sample IC ₅₀ (ug / mL) Immature Apples 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) Determination of Elastase Inhibitory Activity

Elastase present in human neutrophil granulocytes is an enzyme that breaks down elastin, an important substrate protein that maintains skin elasticity in the dermis, and a nonspecific hydrolase that can break down collagen, another important substrate protein. Therefore, elastase inhibitors have an effect of improving skin wrinkles, and ursolic acid is used as an elastase inhibitor. In addition, it is one of the leukocyte granule enzymes that break down the elastin in the body, the abnormal tissue is extremely high activity of the enzyme directly causes tissue destruction, causing 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 nitroalides was measured in light at 405 nm wavelength. In other words, each test solution was prepared to a certain concentration, 0.5 mL each was taken in a test tube, 0.5 mL of elastase (2.5 U / mL) solution dissolved in 50 mM tris-HCl buffer solution (pH 8.6) was added, and then 50 mM tris-HCl as a substrate. N-succinyl- (L-alanine) 3-p-nitroanilide (0.5 mg / mL) dissolved in buffer (pH 8.6) was added and reacted for 20 minutes.

The experimental results of the immature apple 70% ethanol extract and the 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 mixture exhibits a tendency to exhibit an elastase inhibitory activity of the mixture as the content of the Baikallin increases. However, when the amount of bacalin is less than 3 parts by weight, the elastase inhibitory activity was weaker than that of using immature apple extract alone. The effect was 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 Apples 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 collagenase inhibitory activity

Collagen, a major component of the extracellular matrix, is the major substrate protein produced in fibroblasts of the skin. It is also an important protein, accounting for about 30% of the total weight of biological proteins, and has a strong triple helix structure. Collagen forms most of the organic matter in the skin, tendons, bones and teeth, especially in the dermis of bone and skin. The main functions of collagen are known as mechanical firmness of skin, resistance of connective tissue and binding of tissue, support of cell adhesion, induction of cell division and differentiation. This collagen is reduced by photoaging by age and UV irradiation, which is known to be closely related to the wrinkle formation of the skin. In addition, collagen is reported to be degraded by collagen, although it is not affected by protease such as trypsin.

Collagen inhibitory activity was measured according to the method of Wnsch et al. In other words, the reaction zone was prepared by adding 4 mM CaCl ₂ to 0.1 M tris-HCl (pH 7.5) to dissolve 4-phenylazobenzyloxycarbonyl l-proline-leucine-glycine-proline-D-arginine (0.3 mg / mL). 0.15 mL of collagen (0.2 mg / mL) was added to a mixed solution of 0.25 mL of solution and 0.1 mL of sample solution. The mixture was left at room temperature for 20 minutes, and then 0.5 mL of 6% citric acid was added to stop the reaction. 1.5 mL of acetate was added to measure absorbance at 320 nm. Collagen inhibitory activity was expressed by the decrease in absorbance of the added and non-added samples.

The experimental results for 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 mixture showed a tendency to strongly exhibit the collagenase inhibitory activity of the mixture as the content of the Baikallin increased. However, when the content of the baccalin is less than 3 parts by weight, the collagenase inhibitory activity was weaker than that of the immature apple extract alone. It was confirmed that the slowdown.

Sample No. Inhibitory activity (%) 1000 ug / mL 500 ug / mL 250 ug / mL 1255 ug / mL 62.5 ug / mL Immature Apples 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)

Antioxidant cosmetic composition comprising 3 to 10 parts by weight of Baikal based on 100 parts by weight of immature apple extract. delete Antioxidant food composition comprising 3 to 10 parts by weight of bicalin based on 100 parts by weight of immature apple extract. delete

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