KR100864915B1 - Composition comprising mixed extract of japanese apricot and pear blossom having anti-oxidative activity - Google Patents

Abstract

An anti-oxidative composition comprising the mixed extract of Japanese apricot(Prunus mume SIEB. et ZUCC flower) and pear blossom(Pyrus communis Linn. var. sativa DC flower) is provided to inhibit mushroom tyrosinase activity, melanin production of melan A, lipid peroxidation, elastase activity and hyalurodinase activity, and remove free radicals. A pharmaceutical or cosmetic composition for whitening the skin, inhibiting skin aging and improving wrinkles comprises 0.1-20 wt.%, preferably 0.1-10 wt.% of the mixed extract of Japanese apricot and pear blossom in a weight ratio of 1-10:1-5 which is prepared by extracting them with water, C1-C4 lower alcohol or a mixed solvent thereof, hexane, butylenes glycol, propylene glycol or glycerin, preferably water or water and ethanol mixed solvent, most preferably 50-90% ethanol mixed solvent, wherein the daily dosage of the composition is 0.0001-100 mg/kg, preferably 0.001-10 mg/kg.

Description

 Composition comprising mixed extract of Japanese apricot and pear blossom having anti-oxidative activity}

       The present invention relates to a composition containing a mixed plant extract of plum and catabolic having antioxidant activity as an active ingredient.

       Our bodies are exposed to ultraviolet rays and harmful foreign substances and have a natural healing ability to fight them. One such feature is the endocytosis process, which is an important cell's healing system, the white blood cells that play a key role in the immune system that protects the human body, the production of melanin that protects the skin from harmful radicals, and the superoxide dismutase that removes superoxide. There is this. However, the overreaction of these natural healing abilities can cause harm to the body. One of them is the overreaction of immune devices, which can lead to hypersensitivity diseases (allergies) and excessive inflammatory reactions when judged to have an excessive immune response against non-breeding objects such as hair, dust and pollen. Cytokines, hormones, and reactive oxygen species may cause excessive pigmentation of the skin. Among these causes, especially active oxygen species, leading to skin cell and tissue damage, act as a causative agent of aging, particularly skin aging. They break the antioxidant defenses made up of antioxidant enzymes and non-enzymatic antioxidants, shifting the oxidant / antioxidant balance towards the oxidative state. The ongoing oxidative stress leads to skin hyperpigmentation, lipid peroxidation, protein oxidation, activation of proteolytic enzymes that destroy epileptic components, chain breakage and abnormal crosslinking of collagen and elastin, elastic fibers, cleavage of hyaluronic acid chains, and melanin production. It causes damage to biological components such as reaction promotion and DNA oxidation. Eventually, skin aging, characterized by decreased elasticity, wrinkles and spots, and freckles, is accelerated.

        Therefore, in order to prevent hyperpigmentation and aging, a system capable of suppressing excessive reactive oxygen species generation and efficiently removing the generated reactive oxygen species must be included in the cosmetic formulation. In other words, it is necessary to build an antioxidant defense system for the skin that maintains an oxidant / antioxidant balance.

        In vivo, oxidant attack and antioxidant defense are in close harmony. But defense is not necessarily perfect. Therefore, biomolecules such as lipids, proteins, nucleic acids, and the like are continuously oxidatively damaged by free radicals, which are oxidizing agents, and these oxidation products accumulate with age. The theory that this imbalance, which favors oxidants, causes aging itself is a free radical theory (Harman, Brunk et al. Pp1596-1992, 1992).

Free radicals, strong oxidants, are unpaired electrons. Free radicals are produced by the redox reactions of various living organisms, which can lead to the deterioration of edible fats or oxidative damage to various biomaterials (lipids, proteins, nucleic acids, carbohydrates), and mutations as a result of several steps. (Yen GC et al., J. Agric. Food Chem . , 43 , pp 27-32, 1995). Unsaturated fatty acids of phospholipids, which are constituents of biological membranes, initiate peroxidation reactions by free radicals such as reactive oxygen species and proceed in a chain. Therefore, free radical peroxidation not only promotes cell membrane permeability but also results in overall cytotoxicity, which induces aging or pathology of various diseases and is involved in carcinogenesis.

Radical action has a significant effect on the progression of various chronic diseases such as blemishes, freckles, atopic diseases related to oxidative stress, cancer, hypertension, myocardial infarction, arteriosclerosis, rheumatism, cataracts and Parkinson's disease (De Souza) LC et al ., Bioorg . Med . Cehm . Lett . , 14 , pp5859-5861, 2004), may act as a factor in weakening immune system function (Pike J et. al ., Int . J. Vitam . Nutr . Res . , 65 , pp 117-120, 1995).

DPPH is a stable free radical that is widely used to measure the antioxidant activity of various natural compounds (Yokozawa T et. al ., Biochem . Pharmacol . , 56 , pp 213-222, 1998). Antioxidants, or DPPH, which delivers electrons or hydrogen ions to DPPH, neutralize the properties of free radicals. Xanthine oxidase (XO) is an enzyme that oxidizes the purines hypoxanthine and xanthine to form uric acid, a cause known as gout. Gout is an inflammation accompanied by pain due to the deposition of uric acid on the joints. Therefore, inhibition of xanthine oxidase calms gout (Chiang et. al ., Journal of Enzyme Inhibition , 8 , pp 61-71, 1994). Xanthine oxidase is a biological cause of oxygen-derived free radicals that act on oxidative damage in living cells. These various oxidative damages cause many pathological diseases, including inflammation, arteriosclerosis, cancer and aging (Cos P et. al ., Journal of Natural Products , 61 , pp 71-76, 1998). Inhibition of xanthine oxidase has been demonstrated to be useful for the treatment of gout and other xanthine oxidase-induced diseases (Goodman Gilman A et al., Pharmacological Basis of Therapeutics (eighth edition), pp 674-681, 1990).

Cooperative defense system that protects the body from the free radical has the nutrients and enzymes that inhibit oxidation (Halliwell B, Annu. Rev. Nutr., 16, pp33-50, 1996). One effective way to protect the body from oxidative stress is to increase antioxidant levels (De Souza LC et al., Bioorg . Med . Cehm . Lett. , 14 , pp 5859-5861, 2004). Therefore, the antioxidant evaluation of alternative substances to prevent from oxidative damage is very active.

        Although there are many known anti-pigmentation agents and antioxidants that eliminate free radicals, which are used as raw materials for general cosmetics, medicines, and foods, their use is limited because they are problematic when applied to human skin for a long time as a synthetic product. Natural products are mostly weak in pigmentation inhibition and free radical scavenging effect, but the safety of human skin is guaranteed (Korean Society for Cosmetics Vol. 23, No. 1, Skin Aging and Antioxidants, pp.75, 1997).

Plum Tree ( Prunus) mume SIEB. et ZUCC) belongs to the bark family and leaves are small tall trees. Egg-shaped leaves stick out alternately and white or red flowers bloom before the early spring leaves. Fruits are round and yellow in color. In motion therapy, it was used as a cold astringent medicine, diarrhea medicine, fever drop medicine, cough medicine and sputum medicine. It is mainly used for embryo pain, vomiting and diarrhea. Its main ingredients include flavonoids, tannins and glucose. (Ingredients and Uses of Medicinal Herbs, pp358, 1984, Sun Moon Seok)

Ewha is the flower of the rose family Pyrus bretschneideri Rehd. And Pyrus pyrifolia (Burm.f. Nakai.) Pyrus ussuriensis Max. Stone pears are distributed in various parts of Korea, and in the Yangtze and Pearl River basins in China, and mountain pears are distributed in the mountains and villages of Korea, and in Northeast, Hebei, Shandong, Shanxi, Shaxi and Gansu of China.

It is a deciduous tree that blooms in April and fruiting in September. The roots, branches, leaves, and rinds of the original plant are also medicinal. The nature of the drug is sweet, slightly sour, slightly cold. Menopause, gastrointestinal, the effect of fever is damaged by the essence of the fluid is filled with chest and thirst to cause thirst, stop thirst in thirst, fever and seawater fever, antitussive action. Clinical applications are known to treat fever upset, small, pulmonary fever, keratosis, hemostasis, fever, rebellion, and constipation. (Oriental Medical Metabolism, 8 , p166, 1999)

        However, none of the above documents teaches or discloses that a mixture of plum and catabolic shows skin pigmentation inhibition, aging, and wrinkle relief.

        In the present invention, the effect of the above-mentioned mixture is to be mixed by the plum and catabolic.

        Among the raw materials having a conventional antioxidant effect, BHT or BHA is made through chemical synthesis, causing skin irritation and limiting the amount used. Ascorbic acid, a natural antioxidant, is unstable in water, heat, and air, and has good oxidation properties. Tocopherol, which is one of the fat-soluble vitamins, is also fat-soluble, so its solubility in water-soluble media is low, and its use is limited.

        Due to these shortcomings, studies to find new functional materials have been continuously attempted. Various natural natural products, which have been used as herbal medicines since ancient times, have not only been excellent in stability but also contain various beneficial drug substances, and have been a good search target.

   The present inventors use a composition containing a mixed extract including plum and catabolic as an active ingredient, inhibiting mushroom tyrosinase activity, melanin biosynthesis using melan-a cells, free radical scavenging activity, lipid peroxidation inhibitory effect, elastase Through the activity inhibitory effect and hyaluronidase activity inhibitory effect experiments to confirm the skin whitening, anti-aging and anti-wrinkle effect has been completed the present invention.

  An object of the present invention is to provide an external skin pharmaceutical composition and cosmetic composition containing a mixed plant extracts of plum and pear as an active ingredient exhibiting skin pigmentation suppression, anti-aging and anti-wrinkle effect.

        In order to achieve the above object, the present invention provides a skin external pharmaceutical composition exhibiting a skin whitening, anti-aging and anti-wrinkle effect containing a mixed plant extract of plum and catabolic as an active ingredient.

         In another aspect, the present invention provides a cosmetic composition showing the skin whitening, anti-aging and anti-wrinkle effect containing the mixed plant extracts of plum and Ewha as an active ingredient.

         Extracts as defined herein include water containing purified water, lower alcohols having 1 to 4 carbon atoms or mixed solvents thereof, hexane, butylene glycol, propylene glycol, glycerin, preferably water or purified water and ethanol mixed solvents, most preferably Preferably extracts soluble from 50-90% ethanol mixed solvent.

In addition, the plum defined above is Prunus ( Prunus) mume SIEB. et ZUCC), Ewha is a rose family Pyrus bretschneideri Rehd .), stone pear (나무) Pyrus pyrifolia (Burm.f. Nakai.) Pyrus ussuriensis Max. or Pyrus communis Linn. var. sativa. DC.), Preferably Pyrus communis Linn. var. sativa. Includes flowers of DC.)

       As a blending weight ratio of the above-mentioned mixed plant extract, the weight ratio of plum: pear flower is preferably 1 to 10: 1 to 5, and most preferably 1 to 5: 1 to 4 weight ratio.

        Hereinafter, the present invention will be described in detail.

        Mixed plant extract of the present invention can be prepared as follows.

       1 to 30 times by volume of one or more solvents selected from the group consisting of water, ethanol, methanol, hexane, butylene glycol, propylene glycol, and glycerin, including purified water, as an extraction solvent for the dry weight ratio of plum and catabol Is 10 to 20 times by volume of the cooling condenser is installed to prevent the solvent from evaporating 30 to 100 ℃, preferably 1 to 30 hours, preferably 2 to 20 hours at an extraction temperature of 50 to 95 ℃ Extraction method of cold sediment extraction, hot water extraction, ultrasonic extraction, reflux cooling extraction, heating extraction, etc., preferably 1 to 7 times, preferably 1 to 3 times extraction by hot water extraction method, filtered and concentrated under reduced pressure Mixed extracts can be obtained.

        The composition of the present invention comprises the mixed extract as a weight of the liquid, preferably 0.1 to 20% by weight, most preferably 0.1 to 10% by weight, based on the total weight.

        The concentrated weight ratio also preferably comprises a weight ratio of 0.01 to 10%, more preferably 0.01 to 5% by weight, most preferably 0.01 to 3% by weight.

        The present invention provides a skin external pharmaceutical composition for inhibiting skin pigmentation, preventing aging, and alleviating wrinkles, which contains a mixed extract of plum and catabolic obtained by the above production method as an active ingredient.

The external skin pharmaceutical composition containing the extract of the present invention as an active ingredient may further include appropriate carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.

         The external skin pharmaceutical composition containing the extract according to the present invention as an active ingredient is formulated in the form of external preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and sterile injectable solutions, respectively, according to a conventional method. It may be used in the form, preferably, it may be prepared and used as an external skin pharmaceutical composition of creams, gels, patches, sprays, ointments, warnings, lotions, linings, pasta or cataplasma, but is not limited thereto. It is not.

   The preferred dosage of the composition containing the extract of the present invention as an active ingredient depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the preferred effect, the composition of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg per day. Topical administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

        The present invention also provides a cosmetic composition having a skin pigmentation suppression, anti-aging and anti-wrinkle effect containing a mixed plant extract of plum and catabolic as an active ingredient.

       Extract of the present invention can be used in a variety of cosmetics and cleansing agents having a skin pigmentation inhibition, anti-aging and anti-wrinkle effect. Examples of products to which the present composition can be added include cosmetics such as various creams, lotions, skins, and the like, cleansing agents, face washes, soaps, treatments, and essences.

       Cosmetics of the present invention comprises a composition selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymer polysaccharides, sphingolipids and seaweed extract.

In addition, the compounding component which may be added other than this is not limited to this, Although any said component can be mix | blended within the range which does not impair the objective and effect of this invention, Preferably it is 0.01-5% weight with respect to a total weight, More preferably, Is formulated at 0.01-3% by weight.

        The cosmetic of the present invention may take the form of a solution, an emulsion, a viscous mixture, or the like.

       Ingredients included in the cosmetic composition of the present invention may include components commonly used in cosmetic compositions in addition to the compound as an active ingredient, for example, conventional auxiliary agents such as stabilizers, solubilizers, vitamins, pigments and flavorings. And carriers.

        The cosmetic composition of the present invention may be prepared in any formulation commonly prepared in the art, and includes, for example, milky lotion, cream, lotion, pack, foundation, lotion, essence, hair cosmetic, and the like.

       Specifically, the cosmetic composition of the present invention skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutrition cream, moisturizing cream, hand cream, foundation, essence, nutrition essence, Formulations of packs, soaps, cleansing foams, cleansing lotions, cleansing creams, body lotions and body cleansers.

Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

Example  1. Plum single extract ( BL -1) Preparation

      To 1 kg of dried plum (Gwangyang Plum Farm Combination) 5 kg of 70% ethanol was added. After 5 days of infiltration at room temperature and filtered with a 250 mesh filter cloth, it was left to mature for 7 days at 4 ℃, and then filtered with Whatman filter paper. The extract was concentrated on a rotary evaporator under a water bath at 50 ° C. to obtain 39.5 g of plum single extract (BL-1) in powder form, which was used as a sample for the experimental example.

Example  2. Yihua Single Extract ( BL -2) Preparation

Except for changing the dried plum to Ewha (cultivated farmhouse in Byeongcheon, Cheonan-si, Chungnam), the same reaction process as in Example 1 was carried out to obtain 39.8 g of Ewha single extract (BL-2) in powder form to be used as a sample of the experimental example. .

Example  3. Plum and catabolic extract ( BL -3) Preparation

Mixed extract in powder form by following the same reaction procedure as Example 1 except for changing 1 kg of dried plum to 0.5 kg of dried plum and 0.5 kg of catabolic. 39.9 g was obtained and used as a sample of an experiment example.

Example  4. Plum and catabolic extract ( BL -4) Preparation

The same reaction process as in Example 1 was carried out except for changing 1 kg of dried plums to 0.67 kg of dried plums and 0.33 kg of catabolic, to obtain 40.3 g of a mixed extract (BL-4) in powder form. Used as a sample.

Example  5. Plum and catabolic extract ( BL -5) Preparation

The same reaction process as in Example 1 was carried out except for changing 1 kg of dried plums to 0.75 kg of dried plums and 0.25 kg of pear blossoms, to obtain 40.0 g of a mixed extract (BL-5) in powder form. Used as a sample.

Example  6. Plum and catabolic extract ( BL -6) Preparation

The same reaction process as in Example 1 was carried out except for changing 1 kg of dried plums to 0.8 kg of dried plums and 0.2 kg of catabolic, to obtain 40.2 g of a mixed extract (BL-6) in powder form. Used as a sample.

Example  7. Plum and catabolic extract ( BL -7) Preparation

The same reaction process as in Example 1 was carried out except for changing 1 kg of dried plums into 0.33 kg of dried plums and 0.67 kg of catabolic, to obtain 39.9 g of a mixed extract (BL-7) in powder form. Used as a sample.

Example  8. Plum and catabolic extract ( BL -8) Preparation

The same reaction process as in Example 1 was carried out except for changing 1 kg of dried plums to 0.25 kg of dried plums and 0.75 kg of catabolic, to obtain 40.0 g of a mixed extract (BL-8) in powder form. Used as a sample.

Example  9. Plum and catabolic extract ( BL -9) Preparation

The same reaction process as in Example 1 was carried out except for changing 1 kg of dried plums into 0.2 kg of dried plums and 0.8 kg of catabolic, to obtain 40.1 g of a mixed extract (BL-9) in powder form. Used as a sample.

Example  10. Plum and catabolic extract ( BL -10) Preparation

0.8 kg of dried plum and 0.2 kg of pear were mixed and 5 kg of purified water was added. After immersion extraction at room temperature for 5 days, and filtered through a 250 mesh filter cloth, it is aged for 4 days at 4 ℃ and then filtered with Whatman filter paper. The extract was concentrated on a rotary evaporator under a water bath at 50 ° C. to obtain 51.3 g of a mixed extract (BL-10) in powder form, which was used as a sample for the experimental example.

Example  11. Plum and catabolic extract ( BL -11) Preparation

        0.8 kg of dried plum and 0.2 kg of catabolic were mixed and 5 kg of 30% hydrous butylene glycol was added. After immersion extraction at room temperature for 5 days, and filtered through a 250 mesh filter cloth, and then aged for 7 days at 4 ℃ filtered by Whatman filter paper to obtain 4.02 kg of extract (BL-11) was used as a sample of the experimental example.

Example  12. Plum and catabolic extract ( BL -12) Preparation

0.8 kg of dried plum and 0.2 kg of catabolic were mixed and 5 kg of 30% ethanol was added. After immersion extraction at room temperature for 5 days, and filtered through a 250 mesh filter cloth, it was aged for 7 days at 4 ℃ and then filtered with Whatman filter paper. The extract was concentrated on a rotary evaporator under a 50 ° C. water bath to obtain 50.2 g of a mixed extract (BL-12) in powder form, which was used as a sample for the experimental example.

Example  13. Plum and catabolic extract ( BL -13)

0.8 kg of dried plum and 0.2 kg of catabolic were mixed and 5 kg of 80% hydrous glycerin was added. After immersion extraction at room temperature for 5 days, filtered with a 250 mesh filter cloth, and then aged for 7 days at 4 ℃ and filtered with Whatman filter paper to obtain 3.97 kg of the mixed extract (BL-13) was used as a sample of the experimental example.

Example  14. Plum and catabolic extract ( BL -14)

0.8 kg of dried plum and 0.2 kg of catabolic were mixed and 5 kg of butylene glycol were added. After immersion extraction at room temperature for 5 days, filtered with a 250 mesh filter cloth, and then aged for 7 days at 4 ℃ and filtered with Whatman filter paper to obtain 3.86 kg of the mixed extract (BL-14), which was used as a sample of the experiment.

Example  15. Plum and catabolic extract ( BL -15)

0.8 kg of dried plum and 0.2 kg of catabolic were mixed and 5 kg of 95% ethanol was added. After immersion extraction at room temperature for 5 days, and filtered through a 250 mesh filter cloth, it was aged for 7 days at 4 ℃ and then filtered with Whatman filter paper. The extract was concentrated on a rotary evaporator under a 50 ° C. water bath to obtain 28.9 g of a mixed extract (BL-15) in powder form, which was used as a sample for the experimental example.

Example  16. Plum and catabolic extract ( BL -16)

0.8 kg of dried plum and 0.2 kg of catabolic were mixed and 5 kg of 30% hydrous propylene glycol was added. After immersion extraction at room temperature for 5 days, filtered with 250 mesh filter cloth, and then aged for 7 days at 4 ℃, filtered with Whatman filter paper to obtain 4.01 kg of mixed extract (BL-16) was used as a sample of the experimental example.

Experimental Example 1. Measurement of free radical scavenging efficacy

In order to determine the free radical scavenging effect of the plum and catabolic extracts of Examples 1 to 16, the following experiment was conducted using the method described in the literature (Y. Fugita et al., Yakugak Zasshi , 108 , p129, 1988). .

         To 0.5 mL of 0.2 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) solution, 1 mL of each of the plum and catabolic extracts of Examples 1 to 16 was added at each concentration, and solution A, which was reacted at room temperature for 10 minutes, at 520 nm. Maximum absorbance was measured at. The experimental group color correction A 'used 95% ethanol instead of DPPH, and the control group B used ethanol instead of DPPH, and the color correction control group B' added 95% EtOH instead of the sample. Free radical scavenging effect of each extract was calculated by the following equation 1, the results are shown in Table 1.

% Free radical scavenging effect = [1- (A-A ') / (B-B')] x 100

extract SC ₅₀ (ug / mL) BL-1 39.78 BL-2 96.41 BL-3 37.86 BL-4 35.20 BL-5 10.12 BL-6 41.20 BL-7 65.86 BL-8 75.27 BL-9 92.04 BL-10 1001.75 BL-11 100.87 BL-12 28.01 BL-13 530.92 BL-14 819.38 BL-15 20.69 BL-16 151.28

As a result, it was confirmed that the free radical scavenging efficacy of the BL-5 extract as shown in Table 1 was excellent.

Experimental Example  2. Mushroom Tyrosinase  Determination of Activity Inhibition

In order to measure the activity of inhibiting mushroom tyrosinase activity of the plum and catabolic extracts of Examples 1 to 16, the following experiment was conducted using the method described in the literature (A. Vanni, Annali di Chimica , 80, p35, 1990).

  1.0 ml of 0.1 M potassium phosphate buffer (pH 6.8), 0.3 mg / ml 1.0 ml of L-tyrosine aqueous solution, 1 ml of 1,250 U / ml mushroom tyrosinase (SIGMA, T-7755), followed by sample solution Each of 0.2 ml was added to proceed the enzyme reaction at 37 ° C. for 10 minutes, and arbutin (Bioland) was used as a positive control. The absorbance of the reaction solution was measured at 480 nm. Experimental group A's color correction used the same amount of buffer instead of tyrosinase, control group B used the solvent instead of the sample, and color control group 'B' added the same amount of buffer instead of solvent and tyrosinase. Mushroom tyrosinase activity inhibitory effect of each extract was calculated by the following equation (2), the results are shown in Table 2.

Inhibition of mushroom tyrosinase activity (%) = = [1- (A-A ') / (B-B')] x 100

extract IC ₅₀ (μg / mL) BL-1 206.59 BL-2 299.01 BL-3 197.46 BL-4 195.02 BL-5 84.57 BL-6 210.21 BL-7 228.05 BL-8 289.42 BL-9 291.68 BL-10 > 600.00 BL-11 307.00 BL-12 133.52 BL-13 464.52 BL-14 550.85 BL-15 85.29 BL-16 339.41

As a result, as shown in Table 2, it was confirmed that the mushroom tyrosinase activity inhibitory effect of the BL-5 and BL-15 extract.

Experimental Example  3. Melan -a( melan -a) Cell viability measurement using cells

In order to measure the cell viability of melanin-A (melan-a, sold from St. George's University, UK) of the plum and catabolic extracts of Examples 1 to 16, the following experiment was conducted using the method described in the literature ( Maeda and Fukuda, J. Soc. Cosmetic Chem ., 42, 361, 1991).

RPMI 1640 medium (GIBCO, USA) supplemented with melan-a cells with 10% bovine serum (GIBCO, USA), 1% antibiotic (GIBCO, USA), 200 nM Tetradecanoyl phorbol acetate (Sigma, USA) USA) was inoculated in a 24 well culture dish at a density of 1 × 10 ⁵ cells and incubated at 10% CO ₂ , 37 ° C. for 1 day. Subsequently, it was replaced with fresh medium, and the samples were treated by concentration and incubated for 3 days, and the number of wells according to all concentrations was prepared in triplicate. After 3 days, the medium was removed, and 1.0 ml of 0.25 mg / ml MTT (3- [4,5-dimethylthiazole-2-yl] -2,5-diphenyltetrazolium bromide (Sigma, USA) was treated with 4 ml at 37 ° C. After the reaction, the MTT was removed, and then 1.0 ml of DMSO (dimethylsulfoxide) was added to measure absorbance at 570 nm. For the control group B, a solvent was used instead of the sample, and the results are shown in Table 3.

 Cell viability (%) = (A / B) × 100

extract Experimental concentration (μg / mL) Survival rate (%) BL-1 10 101.15 100 100.80 BL-2 10 100.87 100 101.64 BL-3 10 100.02 100 103.56 BL-4 10 105.68 100 105.66 BL-5 10 100.41 100 96.95 BL-6 10 109.42 100 102.41 BL-7 10 100.86 100 100.53 BL-8 10 106.81 100 110.51 BL-9 10 101.33 100 106.41 BL-10 10 103.58 100 102.15 BL-11 10 103.11 100 109.62 BL-12 10 99.64 100 103.18 BL-13 10 98.18 100 102.61 BL-14 10 103.28 100 106.24 BL-15 10 105.62 100 101.51 BL-16 10 106.21 100 101.08

As a result, it was confirmed that all extracts are not cytotoxic as shown in Table 4 above.

Experimental Example  4. Melan -a( melan -a) Determination of melanin biosynthesis effect using cells

In order to determine the effect of inhibiting melanogenesis in melan-a cells of the plum and catabolic extracts of Examples 1 to 16, experiments were performed as follows using the method described in the literature (Maeda and Fukuda, J. Soc. Cosmetic Chem ., 42, 361, 1991).

Melan-a cells were inoculated in a 6-well culture plate at a density of 1 × 10 ⁵ cells in RPMI 1640 medium supplemented with 10% bovine serum, 1% antibiotic and 200 nM TPA, followed by 10% CO ₂ for one day. , And incubated at 37 ℃. Two sets of well culture plates inoculated with cells were used to measure melanin biosynthesis and cell viability. After culturing the cells of the cultured cells to a new medium for 1 day, the samples were incubated for 3 days by treating the wells by concentrations, and the number of wells according to all concentrations was prepared by three-fold. After 3 days, the set of cells to measure the amount of melanin was washed with PBS, treated with 0.5 ml of 1N sodium hydroxide, collected into a 1.5 ml tube, and sonicated to completely dissolve melanin, and then absorbance was measured at 405 nm. One set was treated with MTT and measured the cell viability to divide the melanin produced by the number of cells to determine the percentage of melanin biosynthesis per unit cell (%) is shown in Table 4.

Melanin biosynthesis inhibition rate (%) = [(A-B) / A] × 100

A: cell survival rate B: count the amount of melanin produced

extract Experimental concentration (μg / mL) Inhibition Rate (%) BL-1 10 7.05 100 30.64 BL-2 10 5.23 100 42.08 BL-3 10 8.69 100 58.64 BL-4 10 9.54 100 60.49 BL-5 10 15.67 100 68.46 BL-6 10 6.59 100 50.46 BL-7 10 5.94 100 48.21 BL-8 10 5.36 100 47.99 BL-9 10 5.33 100 42.61 BL-10 10 0.29 100 30.85 BL-11 10 5.12 100 40.69 BL-12 10 10.59 100 61.74 BL-13 10 5.00 100 38.94 BL-14 10 4.01 100 30.59 BL-15 10 12.18 100 65.99 BL-16 10 5.06 100 39.86

As a result, as shown in Table 4, the BL-5 extract was confirmed that the melanin biosynthesis effect is excellent.

Experimental Example  5. Inhibition of Lipid Peroxidation Effect

In order to determine the lipid peroxidation inhibitory effect of the plum and catabolic extracts of Examples 1 to 16, the following experiment was conducted using the method described in the literature (Ohkawa et. al ., Anal . Biochem . 95 , pp. 351 , 1979).

20 uL of 2% linolenic acid, 160 uL of 0.5 M glycine-HCl buffer (pH 3.6), 640 uL of ethanol, 80 uL of plum and catabolic extracts of Examples 1-16, 588 uL of purified water, 32 uL of 30 mM ferric chloride (FeCl _{3 ),} and 80 uL of 1.5% TBA was mixed well, and then heated in boiling water for 1 hour.

       After cooling in iced water, distilled liquid was supplemented with purified water and mixed. 1,600 uL of n-butanol was mixed well and centrifuged at 3,500 rpm for 5 minutes, and the absorbance was measured at 534 nm for A having the n-butanol layer as an upper layer.

       In the same way as above, a color correction liquid containing 20 uL of purified water instead of linolenic acid is called A '. And the control group B was tested in the same manner as above by adding 80uL of solvent instead of the plum and catabolic extract. B ', a color correction solution of the control group, was tested in the same manner by adding the same amount of solvent and purified water instead of the sample and linolenic acid.

  Lipid peroxidation inhibitory effect of each extract was calculated by the following equation (5), the results are shown in Table 5.

% Inhibition of lipid peroxidation = [1- (A-A ') / (B-B')] x 100

extract IC ₅₀ (ug / mL) BL-1 321.68 BL-2 398.99 BL-3 245.57 BL-4 191.11 BL-5 102.72 BL-6 391.64 BL-7 348.29 BL-8 361.15 BL-9 375.06 BL-10 2051.47 BL-11 507.19 BL-12 188.82 BL-13 909.00 BL-14 1001.21 BL-15 150.97 BL-16 301.28

As a result, as shown in Table 5, it was confirmed that the lipid peroxidation inhibitory effect of the BL-5 extract is excellent.

Experimental Example  6. Elastase  Activity inhibition effect measurement

In order to determine the effect of inhibiting the elastase activity of the plum and catabolic extracts of Examples 1 to 16, the following experiment was conducted using the method described in the literature (James et. al ., Biochemistry , 35 , pp.9090-9096, 1996).

       Substrate solution containing 8.8 mM Elastase substrate Succ-Ala-Ala-A-p-nitroanilide standard in 60 µl of a buffer adjusted with 0.267 M Tris solution with 0.267 M hydrochloric acid (HCl) to pH 8.0. 100 μl of each of the plum and catabolic extracts of Examples 1 to 16 were mixed, and 20 μl of enzyme solution containing 10 μg / ml of Porcine Pancreatic Elastase was added and mixed. After reacting at 25 ° C. for 15 minutes, absorbance A was measured at a wavelength of 410 nm for the amount of para-nitroaniline produced.

      Color correction 'A' was measured for absorbance using a buffer solution instead of the enzyme solution in the same manner as described above.

      In addition, the absorbance B was measured using 100 μl of solvent instead of the sample in the same manner as the test solution, and the absorbance B was measured. 20 uL of buffer solution was used instead of the enzyme solution. The absorbance B 'was measured using the liquid obtained by the method as the color correction liquid of a control group.

The inhibitory effect of elastase activity of each extract was calculated by the following Equation 6, and the results are shown in Table 6.

% Elastase inhibition = [1- (A-A ') / (B-B')] x 100

extract IC ₅₀ (ug / mL) BL-1 193.81 BL-2 262.69 BL-3 132.12 BL-4 91.94 BL-5 55.26 BL-6 423.62 BL-7 531.68 BL-8 555.71 BL-9 605.99 BL-10 1222.35 BL-11 553.92 BL-12 83.14 BL-13 903.48 BL-14 971.74 BL-15 62.55 BL-16 353.68

As a result, it was confirmed that the elastase activity inhibitory effect of the BL-5 extract as shown in Table 6.

Experimental Example  7. Hyaluronidase  Activity inhibition effect measurement

In order to determine the effect of inhibiting the hyaluronidase activity of the plum and catabolic extracts of Examples 1 to 16, the following experiment was conducted using the method described in the literature (Reissig et. al ., J. Biol . Chem . 217 , pp. 959-963, 1955).

50 uL of 7,900units of hyaluronidase and 100 uL of the plum and catabolic mixture extracts of Examples 1 to 16 were added and left in a water bath at 37 ° C. for 20 minutes, and then 100 uL of 12.5 mM calcium chloride (CaCl _{2 )} was added thereto, followed by a water bath at 37 ° C. It was left for 20 minutes. 250 uL of sodium hyaluronic acid which is a substrate was put, and it reacted for 20 minutes on 37 degreeC water bath. Then, 100NL of 0.4N NaOH and 100uL of 0.4M potassium borate were added and reacted in boiling water for 3 minutes.

      After the reaction solution was cooled to room temperature, 3 mL of DMAB solution was added thereto, and the reaction solution was allowed to react for 20 minutes on a 37 ° C water bath. Absorbance of the reaction solution A was measured at 585 nm. Color calibration A 'was the same experiment using 0.1M acetic acid buffer instead of hyaluronidase. The control group B used the solvent of the sample instead of the sample, and the color correction of the control group B 'was tested in the same manner as above using the solvent of the sample instead of the sample, 0.1M acetic acid buffer instead of hyaluronidase.

 The inhibitory effect of hyaluronidase activity of each extract was calculated by the following equation, and the results are shown in Table 7.

% Hyaluronidase inhibition = [1- (A-A ') / (B-B')] x 100

extract IC ₅₀ (ug / mL) BL-1 363.81 BL-2 414.29 BL-3 293.75 BL-4 201.68 BL-5 122.84 BL-6 703.42 BL-7 447.37 BL-8 553.31 BL-9 671.97 BL-10 2100.74 BL-11 533.08 BL-12 183.27 BL-13 903.44 BL-14 1034.81 BL-15 132.98 BL-16 563.65

 As a result, it was confirmed that the hyaluronidase activity inhibitory effect of the BL-5 extract as shown in Table 7.

Experimental Example  8. Moisturizing effect measurement (1)

The moisturizing effect of the BL-10 extract was measured as follows using Corneometer CM 825 by Courage + Khazaka.

    The experiment was conducted on 20 men and women (5 males and 15 females) aged 23 to 32 who had no skin disease that could affect the test results. Each subject was washed with water for 30 minutes before the test. While adapting to the external environment. The experimental environmental conditions were kept the same at room temperature 20 ~ 22 ℃ and room humidity 20 ~ 25%.

Samples were each applied with 10 uL of 2 × 2 cm ² of the inner arm and dried for 1 minute. Afterwards, apply 20uL of purified water to each coating site, and after every minute of cleaning with Kleenex, every 1 minute (0 minutes, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes) The capacitance of the skin surface of each application site was measured by Corneometer (CM 825), and the conductivity (%) was calculated by the following Equation 8, and the results are shown in Table 8.

Conductivity (%) = {(Measured average Cv value 5 times per minute-Cv value before application)

          / (0 min) immediately after sample application-Cv before sample application) × 100

 As a result, it was confirmed that the moisturizing effect of the BL-10 extract is superior to the purified water as shown in Table 8.

Experimental Example  9. Moisturizing effect measurement (2)

The moisturizing effect of the BL-10 extract was measured using Delfin's Vapometer SWL as follows.

Twenty females, 20-30 years old, and 20 subjects were tested for TEWL. 20 μl each of BL-10 extract and purified water were applied to the upper arm 2cm × 2cm (4cm ² ) of the upper arm and then dried.

After that, TEWL (Transepidermal water loss) of each coating site was measured for 6 hours in units of 1 hour using a Vapometer (Delfin technologies Ltd.), and the results are shown in Table 9. (Unit: g / m ² hr)

      As a result, it was confirmed that the moisturizing effect of the BL-10 extract is superior to the purified water as shown in Table 9.

Examples of the formulation of the composition comprising the extract of the present invention will be described, but the present invention is not intended to limit it, but is intended to explain in detail only.

Formulation example  1. Flexible Lotion

Number Raw material Unit (wt%) One BL-5 4.0 2 glycerin 3.0 3 Butylene glycol 2.0 4 Polyoxyethylene (60) Cured Castor Oil 0.5 5 ethanol 6.0 6 Flavoring, preservative, coloring Quantity 7 Purified water Remaining amount Sum 100.0

Formulation example  2. Nutrition Lotion

number Raw material Unit (wt%) One BL-5 10.0 2 Squalane 4.0 3 Liquid paraffin 3.0 4 Caprylic / Capric Glyceride 4.0 5 Stearic acid 1.0 6 Cetyl alcohol 1.0 7 Glyceryl Monostearate 0.5 8 Polysorbate 60 1.5 9 Sorbitan sesquioleate 0.5 10 Glyceryl Stearate / Pig-100 Stearate 1.0 11 glycerin 3.0 12 Butylene glycol 2.0 13 Carbomer 0.1 14 Triethanolamine 0.1 15 Preservative, coloring, flavoring Quantity 16 Purified water Remaining amount Sum 100.0

Formulation Example 3 Nutrition Cream

number Raw material Unit (wt%) One BL-5 10.0 2 Squalane 5.0 3 Liquid paraffin 3.5 4 Caprylic / Capric Glyceride 4.5 5 Stearic acid 1.5 6 Cetyl alcohol 2.0 7 Beeswax 1.0 8 vaseline 2.0 9 Glyceryl Monostearate 1.0 10 Polysorbate 60 1.5 11 Sorbitan sesquioleate 0.5 12 Glyceryl Stearate / Pig-100 Stearate 1.5 13 glycerin 5.0 14 Butylene glycol 5.0 15 Carbomer 0.2 16 Triethanolamine 0.2 17 Preservative, coloring, flavoring Quantity 18 Purified water Remaining amount Sum 100.0

Formulation example  4. Essence

number Raw material Content (% by weight) One BL-5 0.5 2 Cytostolol 1.70 3 Polyglyceryl 2-oleate 1.50 4 Ceramide 0.7 5 Ceteares-4 1.2 6 cholesterol 1.5 7 Dicetylphosphate 0.4 8 Concentrated glycerin 5.0 9 Sunflower oil 15.0 10 Carboxy Vinyl Polymer 0.2 11 Xanthan Gum 0.2 12 antiseptic a very small amount 13 Spices a very small amount 14 Purified water Remaining amount system 100.0

Formulation example  5. Hydrophilic Ointment

number Raw material Content (% by weight) Content (% by weight) One   BL-5 0.5 - 2   BL-5 - 0.5 3   White vaseline 250 250 4   Stearyl alcohol 220 220 5   Ethyl (or methyl) p-oxybenzoate 0.25 0.25 6   Propylene glycol 120 120 7   Sodium lauryl sulfate 15 15 8   Propyl p-oxybenzoate 0.15 0.15

As described above, the mixed plant extract of plum and catabolic of the present invention is free radical scavenging activity, mushroom tyrosinase activity inhibitory activity, melanin biosynthesis inhibitory activity using melan-a cells, lipid peroxidation inhibitory effect, elastase inhibitory activity It can be usefully used as an external skin pharmaceutical composition and cosmetic composition for inhibiting skin pigmentation, preventing skin aging, and alleviating wrinkles by showing an effect and inhibiting hyaluronidase activity.

Claims (8)

Prunus mume SIEB. Et ZUCC Flowers: Pyrus communis Linn. Var. Sativa. DC. Flowers are mixed plants of 50 to 90% ethanol with a weight ratio of 1 to 10: 1 to 5 Wrinkle improvement skin external pharmaceutical composition containing the extract as an active ingredient. delete delete delete delete delete Prunus mume SIEB. Et ZUCC Flowers: Pyrus communis Linn. Var. Sativa. DC. Flowers are mixed plants of 50 to 90% ethanol with a weight ratio of 1 to 10: 1 to 5 Wrinkle improvement cosmetic composition containing an extract as an active ingredient. delete