KR102009204B1 - Cosmetic Composition Comprising Extracts of Pinecone of Pinus sylvestris - Google Patents

Abstract

The present invention relates to a cosmetic composition containing the old pine cone pine extract as an active ingredient, the old pine cone pine extract is characterized in that it contains 0.0001 to 30.0% by weight relative to the total weight of the cosmetic composition. According to the present invention, the old tree pine cone extract has a skin wrinkle improvement effect and skin elasticity improvement effect can provide various functional cosmetics.

Description

Cosmetic composition containing old pine cone pine extract as an active ingredient {Cosmetic Composition Comprising Extracts of Pinecone of Pinus sylvestris}

The present invention is Pinus ( Pinus) sylvestris ) relates to a cosmetic composition containing the pine cone extract as an active ingredient.

Human skin undergoes various physical and chemical changes during the aging process. The causes are largely divided into intrinsic aging and photo-aging, and research on this has been actively conducted. UV rays, stress, disease conditions, environmental factors, wounds, and free radicals may be caused by aging, which intensifies the body's ability to destroy antioxidant defenses and damage cells and tissues. And aging.

More specifically, lipids, proteins, polysaccharides, nucleic acids, and the like, which are major constituents of skin, are oxidized to destroy skin cells and tissues, resulting in skin aging. In particular, the oxidation of proteins is caused by severe cuts in the skin's connective tissues such as collagen, hyaluronic acid, elastin, proteoglycan, and fibronectin, resulting in severe over-inflammatory reactions and skin elasticity. If this becomes worse, mutations in the DNA can lead to mutations, cancer, and reduced immune function.

Therefore, cell membranes are protected by eliminating free radicals caused by metabolic processes in the body, ultraviolet rays, or inflammatory reactions.Also, damaged cells must be regenerated by active metabolism to proliferate the skin. Can recover quickly and maintain healthy skin.

Aging involves not only these free radicals but also an enzyme called matrix metalloproteinase (MMP), a collagen degrading enzyme. In other words, the synthesis and degradation of extracellular matrix such as collagen in vivo is properly controlled, but collagen synthesis decreases as aging progresses, and the expression of matrix metalloproteinase (MMP), an enzyme that degrades collagen, promotes the skin. Elasticity is reduced and wrinkles are formed. In addition, such degradation enzymes may be activated by ultraviolet irradiation.

Therefore, there is a need for development of a substance capable of regulating or inhibiting MMP expression in which activation is induced in cells. Until now, most of the raw materials used as materials for cosmetics simply inhibited enzyme activity. Therefore, we tried to regulate the expression and activity of MMP expression induced by intracellular natural aging and photoaging.

On the other hand, skin changes occur due to pigmentation. Factors that determine skin color include basic or partial pigmentation, such as tanning due to blemishes, freckles, and sun exposure, other than acne, scars, and the distribution of acne and skin, except for differences in race, region, sex, and age. Blood circulation, stress, and health. Among the above factors, the most important factor is pigmentation.

Pigments that affect skin color include melanin, melanoids, carotene, and hemoglobin, the most important of which is melanin, the most important factor affecting the biosynthesis of melanin is the amount of ultraviolet rays and hormones in the body.

Melanin plays a major role in preventing or damaging the skin from ultraviolet rays by absorbing or scattering ultraviolet rays. There is no special maximum absorption wavelength and it absorbs light in all areas. In addition, it is excellent in removing active oxygen species, but sometimes melanin itself generates free radicals, catechol or quinone in melanin structure reduces or oxidizes other substances, and melanin itself shows the properties of free radicals. Pray.

The biosynthesis of melanin is a series of oxidation processes that begin with the conversion of tyrosine, an amino acid, from the melanocytes of melanocytes to tyrosinase and into dihydroxy phenylalanine. eumelanin).

This biosynthesis process is carried out in a special type of brown cells in organelles called melanosomes. Melanosomes, including melanin granules, move from the periphery of the nucleus to the dendritic end of the dendritic projections and into the cytoplasm by the action of keratinocytes. Accumulate around the nucleus.

The synthesis of melanin, the number of melanosomes, and the migration to the surrounding keratinocytes are partially affected by hormones and ultraviolet rays, and primarily by genetic influences. In addition, it is known that interferon, prostaglandin, histamine, and other metal ions such as cytokines, copper, zinc and iron, which are intracellular regulators involved in the expression of tyrosinase and the synthesis and transfer of melanin.

Ascorbic acid, hydroquinone, kojic acid, arbutin and some extracts are already used as whitening cosmetics because they have tyrosinase inhibitory activity. Its use is limited due to efficacy, unclear effects and safety issues. The above substances have been shown to have a tyrosinase inhibitory effect, but the effect is low in experiments similar to the actual biological level. Therefore, the evaluation of the inhibitory effect by melanoma cell culture similar to the biological level is required. In addition, hydroquinone is defined as a carcinogenic substance and its use is prohibited or restricted in cosmetics. Kojic acid and ascorbic acid are very unstable substances. Cosmetics containing a small amount of the above components may cause browning when stored for several weeks at room temperature.

In order to solve these various problems, cosmetics using natural products have recently been developed to reduce skin irritation caused by various chemicals. Natural ingredients have less side effects on the skin, and as the consumer's response to cosmetics using natural ingredients has recently increased, the development value of cosmetics as a cosmetic ingredient is increasing.

On the other hand, Korean Patent Laid-Open No. 2002-0089216 discloses a whitening effect of a cosmetic composition including citron, madduyeong, betel nut, constriction, pine cone and the like.

In addition, Korean Patent Publication No. 2007-0108966 discloses a cosmetic composition having an effect of improving skin wrinkles, improving skin elasticity and skin moisturizing, including red ginseng extract, green tea extract, Pinus Sylvestris Bud Extract, ginkgo biloba extract, and machi. Is starting.

However, the effects of the old pine cone extract are not yet known.

Thus, the present inventors have studied the possibility of applying to cosmetics for a variety of natural products that have not been known so far, by selecting the pine cone pine cones and extracts prepared from the MMP-1 production inhibitory effect, collagen biosynthesis effect, melanin production inhibitory effect As a result of measuring the antioxidant effect, inflammatory cytokine expression suppression effect, moisturizing effect, skin wrinkle improvement effect, and skin elasticity improvement effect, it was found that the efficacy as a cosmetic can be expected because it is very excellent.

It is an object of the present invention to provide a cosmetic composition containing an extract of pine cone pine tree, MMP-1 production inhibitory effect, collagen biosynthesis effect, skin wrinkle improvement effect, skin elasticity improving effect.

In addition, an object of the present invention is to provide a cosmetic composition that exhibits the efficacy effect through the preparation of supercritical, ultrasonic and purifying process in order to maximize the efficacy of old pine cone pine extract.

Another object of the present invention to provide a cosmetic method using the cosmetic composition containing the old pine cone pine extract.

Therefore, the object of the present invention as described above is achieved by a cosmetic composition containing the old pine cone extract as an active ingredient.

Preferably, the cosmetic composition is characterized in that for improving the skin wrinkles.

In addition, the cosmetic composition is characterized in that the skin for whitening.

In addition, preferably, the cosmetic composition is characterized in that the antioxidant.

In addition, preferably, the cosmetic composition is characterized in that the anti-inflammatory.

In addition, preferably, the cosmetic composition is characterized in that for moisturizing.

In addition, the cosmetic composition is characterized in that for improving skin elasticity.

In addition, preferably, the old pine cone pine extract is characterized in that it contains 0.0001 to 30.0% by weight relative to the total weight of the cosmetic composition.

In addition, preferably, the old pine cone extract (a) water, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms, propylene glycol, butylene glycol, glycerin, acetone, ethyl acetate, chloroform, butyl acetate, diethyl ether , Dichloromethane, hexane, and a solvent extraction method using an extraction solvent selected from the group consisting of these, characterized in that extracted by (b) supercritical extraction or (c) ultrasonic extraction.

Old pine needles extract according to the present invention MMP-1 production inhibitory effect (Experimental Example 1), collagen synthesis enhancement effect (Experimental Example 2), melanin production inhibitory effect (Experimental Example 3), antioxidant effect (Experimental Example 4), inflammatory It has cytokine expression suppression effect (Experimental Example 5), moisturizing effect (Experimental Example 6), and skin elasticity improving effect (Experimental Example 7).

In addition, according to the present invention, in order to maximize the efficacy of the old pine cone extract, the MMP-1 production inhibitory effect, collagen synthesis inhibitory effect, melanin production inhibitory effect, skin elasticity improvement effect, etc. through ultrasonic extraction or supercritical extraction further enhanced Let it be.

The cosmetic composition of the present invention is an old tree ( Pinus) sylvestris ) contains pine cone extract as an active ingredient. The leaves of the old address tree are 2 each, 3 ~ 7cm long, twisted, grey-blue, and the resin sphere of the cross section is outer. The congregation gathers one to three lively and downward. It is short, 3 ~ 7cm long, black yellow, circumferentially long oval, flattened but flattened sometimes pyramidal, with small spines or black spines. Seeds have wings three to four times the length of the seeds.

The flowers of the old tree bloom in April-May and ripen until 3-5 years after pollination. Its height is 25 ~ 40m, its branches spread all over it, and the bark is red or light brown, thin and flat, but the bottom is black. The body is greyish yellow, and the pupil is long oval ovate brown and resinous. It is an important planting species in Europe and wood is used for building materials and pulp materials. In Korea, it was introduced for afforestation adaptation test and planted in forestry test center.

In the present invention, the old pine cone pine extract means an extract obtained from the pine cone of the old address tree.

In addition, the old tree pine cone extract of the present invention can be prepared according to a conventional method known in the art, that is, using a conventional solvent, under the conditions of conventional temperature and pressure, preferably (a) water Anhydrous or hydrous lower alcohols having 1 to 4 carbon atoms (e.g., methanol, ethanol, propanol and butanol), propylene glycol, 1,3-butylene glycol, glycerin, acetone, diethyl ether, ethyl acetate, butyl acetate, Solvent extraction using a solvent selected from the group consisting of dichloromethane, chloroform, hexane and mixtures thereof (b) extraction using supercritical extraction by reduced pressure and high temperature with carbon dioxide or (c) ultrasonic extraction.

In the present invention, a solvent extraction method using an extraction solvent is preferably used.

The old tree pine cone extract in the present invention is a variety of extraction solvents, for example, water, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (for example, methanol, ethanol, propanol and butanol), propylene glycol, 1,3-butyl Ethylene glycol, glycerin, acetone, diethyl ether, ethyl acetate, butyl acetate, dichloromethane, chloroform, hexane, and mixtures thereof, which can be obtained using at least one extraction solvent, preferably ethanol, Obtained using 70% (v / v) ethanol or water, most preferably obtained using 70% (v / v) ethanol.

On the other hand, it is apparent to those skilled in the art that the old tree pine cone extract of the present invention can obtain an extract having substantially the same effect using not only the above-described extraction solvent but also other extraction solvents.

In addition, the old tree pine cone extract of the present invention includes not only the extract by the above-described extraction solvent, but also the extract that has undergone the conventional purification and fermentation process. For example, decompression by carbon dioxide, extraction by supercritical extraction by high temperature, extraction by ultrasonic extraction, separation using ultrafiltration membranes with constant molecular weight cut-off values, various chromatography (size, charge, hydrophobicity or affinity) The active fraction obtained through various purification and extraction methods additionally carried out, such as the separation by the sex) and the extract by fermentation products using natural or various microorganisms, are also included in the extract of the present invention.

In addition, the present invention provides a cosmetic composition, characterized in that the extract is obtained by cold condensation at room temperature, heating and filtration, further obtained by concentrating the solvent under reduced pressure or lyophilization.

The decompression by carbon dioxide and the supercritical extraction by high temperature mean supercritical fluid extraction. Generally, the supercritical fluid is a liquid having a gas when the gas reaches a critical point under high temperature and high pressure. It is gaseous, chemically similar in polarity to nonpolar solvents, and because of its properties, supercritical fluids are used for extraction of fat-soluble substances (J. Chromatogr. A. 1998; 479: 200-205).

Carbon dioxide becomes a supercritical fluid with both liquid and gaseous properties as the pressure and temperature reach a critical point through the operation of a supercritical fluid device, resulting in increased solubility in fat-soluble solutes. When supercritical carbon dioxide passes through an extraction vessel containing a certain amount of sample, the fat-soluble substance contained in the sample is extracted from the supercritical carbon dioxide.

After extracting the fat-soluble substance, the supercritical carbon dioxide containing a small amount of cosolvent is passed through the sample remaining in the extraction container to extract components that were not extracted with pure supercritical carbon dioxide alone.

The supercritical fluid used in the supercritical extraction method of the present invention can effectively extract the active ingredient by using a supercritical carbon dioxide or a mixed fluid in which a co-solvent is added to carbon dioxide.

The cosolvent may be used one or a mixture of two or more selected from the group consisting of chloroform, ethanol, methanol, water, ethyl acetate, hexane and diethyl ether.

Most of the extracted samples contain carbon dioxide. Since carbon dioxide is volatilized into air at room temperature, the extract obtained by the above method may be used as a cosmetic composition, and the cosolvent may be removed by a reduced pressure evaporator.

In addition, the ultrasonic extraction method is an extraction method using the energy generated by the ultrasonic vibration, the ultrasonic wave can destroy the insoluble solvent contained in the sample in the water-soluble solvent, the reactants located around due to the high local temperature generated Since the kinetic energy of the particles is increased, sufficient energy required for the reaction is obtained, and the effect of ultrasonic energy is induced to increase the pressure by increasing the mixing effect of the substance and the solvent contained in the sample, thereby increasing the extraction efficiency.

As the extraction solvent that can be used in the ultrasonic extraction method, one or a mixture of two or more selected from the group consisting of chloroform, ethanol, methanol, water, ethyl acetate, hexane and diethyl ether can be used. The extracted sample is vacuum filtered to recover the filtrate, and then removed by a vacuum evaporator, the extract can be obtained through a conventional extract preparation method of freeze drying.

According to the invention, the old tree pine cone extract is contained 0.0001 to 30.0% by weight relative to the total weight of the cosmetic composition, more preferably, the old tree pine cone extract is contained 0.01 to 10% by weight relative to the total weight of the cosmetic composition It is characterized by. When the content of the extract is less than 0.0001% by weight, no skin improvement effect is observed, and when the content of the extract is greater than 30.0% by weight, the degree of improvement of the skin improvement effect against the increase of the content is insignificant, and there is a problem in the safety and stability of the formulation and economy. It is not an enemy.

Therefore, the cosmetic composition of the present invention containing the old pine cone pine extract having such various functions is excellent collagen synthesis promoting effect, MMP-1 production inhibitory effect, inflammatory cytokine expression inhibitory effect, antioxidant effect, skin wrinkle improvement effect, skin elasticity It is characterized by having an improvement effect, skin whitening effect, melanin production inhibitory effect, moisturizing effect, skin aging prevention effect.

Ingredients included in the cosmetic composition of the present invention as an active ingredient may include ingredients commonly used in cosmetic compositions in addition to the active ingredient, for example, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavorings Adjuvants, and carriers.

Cosmetic compositions of the present invention may be prepared in any formulation conventionally prepared in the art and include, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing , Oils, powder foundations, emulsion foundations, wax foundations, packs, massage creams and sprays and the like, but is not limited thereto. More specifically, it may be prepared in the form of a flexible lotion, nutrition lotion, nutrition cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.

When the formulation of the present invention is a paste, cream or gel, animal oils, vegetable oils, waxes, paraffins, starches, trachants, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide may be used as carrier components. Can be.

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, polyethyleneglycol 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 esters and polyoxyethylene sorbitan esters, and microcrystals are used as carrier components. Soluble cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used, 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 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.

If the cosmetic composition of the present invention is a soap, a surfactant-containing cleansing formulation or a surfactant-free cleansing formulation, it may be wiped off, peeled off or washed with water after application to the skin. As a specific example, the soap is liquid soap, powdered soap, solid soap and oil soap, the surfactant-containing cleansing formulation is a cleansing foam, cleansing water, cleansing towel and cleansing pack, the surfactant-free cleansing formulation is a cleansing cream , Cleansing lotion, cleansing water and cleansing gel, but are not limited thereto.

On the other hand, the makeup method of the present invention refers to all makeup methods using the cosmetic composition of the present invention. That is, all the methods known in the art using the cosmetic composition belong to the makeup method of the present invention.

The cosmetic composition of the present invention may be used alone or in duplicate, or may be used in combination with other cosmetic compositions other than the present invention. In addition, the cosmetic composition according to the present invention may be used according to a conventional method of use, and the number of times of use may vary depending on the skin condition or taste of the user.

When performing a cosmetic method using a cosmetic composition comprising the old pine cone pine extract of the present invention, MMP-1 production inhibitory effect, collagen synthesis enhancement effect, melanin production inhibitory effect, inflammatory cytokine expression inhibitory effect, antioxidant effect, moisturizing effect and Elasticity can be improved.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Production Example  1: Manufacture of Old Address Pine Cone Extract

After dipping, the dried old pine needles were refluxed three times for 5 hours with 70% (V / V) ethanol aqueous solution three times, cooled, and filtered through Whatman # 4 filter paper. The filtered extract was concentrated under reduced pressure and freeze dried at 50 ° C. or lower to obtain 45 g.

Production Example  2 and 3: Supercritical  Manufacture of Fluid Extracts and Ultrasonic Extracts

A supercritical extract was obtained by extraction with a conventional supercritical extraction method (extracted using ethanol as a cosolvent in a supercritical state under a pressure of about 300 atm at a temperature of about 60 ° C.) (Preparation Example 2). The extract was obtained through the extraction using ultrasonic waves (extracted for about 2 hours at 30 ℃ with a strength of 25KHz using a supersonic ultrasonic device). However, in the case of Preparation Example 3, ethanol was used as the cosolvent.

Experimental Example  1. Old tree pine cone extract MMP -1 suppression effect

Experimental Example 1 measured the effect of inhibiting the production of MMP-1, a collagen degrading enzyme, the old pine cone extract obtained in Preparation Examples 1, 2 and 3.

Fibroblasts (Korean Cell Line Bank, South Korea), which are human normal skin cells, were seeded in 48-well microplates (Nunc, Denmark) at 1 × 10 ⁶ cells per well, and treated with DMEM medium (Sigma, United States) and 37 ° C. After incubation for 24 hours in the serum-free DMEM medium to which the old pine cone pine extract of Preparation Examples 1,2,3 and the control group was further incubated for 48 hours in a serum-free DMEM medium not containing the old pine cone extract. .

After incubation, the supernatants of each well were collected and the amount of ng / ml newly synthesized MMP-1 was measured using an MMP-1 assay kit (Amersham, USA), and MMP-1 production was performed according to Equation 1 below. The percent inhibition was calculated and the results are shown in Table 1.

Sample Name Treatment concentration % Inhibition of MMP-1 production Preparation Example 1 Preparation Example 2 Preparation Example 3 Old Tree Pinecone Extract 10 ppm 21.79 22.17 24.82 50 ppm 33.93 36.55 37.95 100 ppm 49.74 52.81 54.14 500 ppm 75.57 78.23 79.55 1000 ppm 86.12 88.19 89.51 TGF-β 200 nM 76.8

As shown in the results of Table 1, it was confirmed that the old pine cone extract of the present invention inhibits the production of MMP-1 (inhibition of MMP-1 activity) in a concentration-dependent manner. Considering the difference between the concentration of TGF-β (200 nM) and the concentration of the pine cone extract (500 ppm) of the present invention, which is known to have an inhibitory effect on MMP-1 production, The same degree of inhibition of MMP-1 shows that the old pine cone extract of the present invention has an inhibitory effect on MMP-1 production.

Experimental Example  2. Collagen Synthesis Enhancement Effect of Old Pine Cone Extract

Fibroblasts, human normal epithelial cells, were seeded in 48-well microplates at 1 × 10 ⁶ cells per well and incubated in DMEM medium for 24 hours. Serum-free DMEM medium prepared with the old pine cone pine extract prepared by Preparation Examples 1, 2, and 3 and further incubated for 48 hours in a serum-free DMEM medium containing no old pine cone extract as a control. After incubation, the supernatant of each well was collected and measured in ng / ml by measuring the amount of procollagen (procollagen) type I C-peptide (PICP) using a collagen kit (Takara, Japan), whereby the amount of collagen synthesized was Measured. Collagen biosynthesis increase rate (%) was calculated according to the following equation (2), the results are shown in Table 2.

Sample Name Treatment concentration Collagen biosynthesis rate (%) Preparation Example 1 Preparation Example 2 Preparation Example 3 Old Tree Pinecone Extract 2.5 ppm 11.2 12.7 14.7 5 ppm 21.0 24.9 23.8 10 ppm 43.4 44.5 42.5 25 ppm 95.1 102.9 95.8 50 ppm 154.4 163.5 165.1 TGF-β 200 nM 163.5

According to Table 2, it was confirmed that the collagen biosynthesis rate of the old addressed pine cone extract prepared by Preparation Examples 1, 2, 3 of the present invention increases in a concentration-dependent manner. In particular, the effect of the extract prepared in Preparation Example 2 was confirmed that the effect is better than the extract of Preparation Examples 1, 3.

Experimental Example  3: inhibitory effect of melanin production using B16F1 melanogenesis cells

Experimental Example 3 is to determine the whitening effect by looking at the degree of inhibition of melanin production on B16F1 melanin forming cells in order to confirm the whitening effect of the old pine cone pine extract obtained in Preparation Examples 1, 2, 3. B16F1 melanin forming cells used in Experimental Example 3 are cell strains derived from mice, and cells that secrete a black pigment called melanin.

Samples were treated during the artificial culture of these cells to evaluate the degree of reduction of melanin black pigment. B16F1 melanin forming cells used in the present experimental example was used by being sold from the American Type Culture Collection (ATCC). The melanin biosynthesis inhibitory effect of B16F1 melanin forming cells was measured as follows.

B16F1 melanin-forming cells were dispensed in 6-well plates at a concentration of 2 X 10 ⁵ per well and attached to the cells, followed by incubation for 72 hours by treating the old pine cone extract according to Preparation Examples 1-3 at a concentration that does not cause toxicity. . After 72 hours of incubation, the cells were detached with trypsin-EDTA, and the cells were counted and centrifuged to recover the cells. Quantification of intracellular melanin was performed with a slight modification of the method of Rotan: Cancer Res., 40: 3345-3350, 1980. The cell pellet was washed once with PBS, and then 1 ml of homogenization buffer solution (50 mM sodium phosphate, pH 6.8, 1% Triton X-100, 2 mM PMSF) was added and vortexed for 5 minutes to disrupt cells. Melt the extracted melanin by adding 1N NaOH (10% DMSO) to the cell filtrate obtained by centrifugation (3,000 rpm, 10 minutes), measure the absorbance of melanin at 405 nm with a microplate reader, and quantify the melanin. Melanin production inhibition rate (%) was measured. Melanin production inhibition rate (%) of B16F1 melanin forming cells was calculated by the following equation (3), the results are shown in Table 3.

Sample Name Treatment concentration (%) Melanin production inhibitory effect (%) Preparation Example 1 Preparation Example 2 Preparation Example 3 Old Tree Pinecone Extract 0.001 22.17 23.15 21.13 0.005 35.45 40.12 38.11 0.01 52.43 56.52 54.42 0.05 73.86 76.91 74.82 Arbutin 200 ppm 68.25

As can be seen from the results of Table 3, it was found that the old tree pine cone extract prepared by Preparation Examples 1 to 3 of the present invention significantly inhibited melanin production in B16F1 melanin forming cells.

Experimental Example  4: Old Tree Pinecone Extract DPPH Radical Scavenging power  analysis

In Experimental Example 4, the antioxidant effect (free radical scavenging rate) was measured by using the DPPH method in order to measure the antioxidant effect of the old pine cone extract obtained in Preparation Examples 1-3. The DPPH method measures the antioxidant effect by reducing power using a free group called DPPH (2,2-Di (4-tert-octylphenyl) -1-picrylhydrazyl) free radical. The free radical scavenging rate (%) is measured at a wavelength of 560 nm by comparing the degree of decrease in absorbance of DPPH by the test substance (old pine cone pine extract) and the absorbance of the blank test solution. The reagent used was 0.1 mM solution of 2,2-Di (4-tert-octylphenyl) -1-picrylhydrazyl free radical (Aldrich Chem. Co., MW = 618.76) as 61.88 mg in methanol to make 100 ml.

The measurement method is as follows:

① Add 0.15 ml of 0.1 mM DPPH solution to 0.15 ml of 96-well plate, stir rapidly, and incubate for 10 minutes at 25 ° C.

(2) Then measure the absorbance St at 560nm.

③ The blank of the sample solution is measured in the same manner as when the absorbance St is measured except that methanol is used instead of the 0.1 mM DPPH solution to measure the absorbance So.

④ The blank test is performed in the same manner as in the case of measuring the absorbance St, except that distilled water is used instead of the sample solution to measure the absorbance Bt.

⑤ Blank in the blank test was measured in the same manner as when the absorbance St was measured except that methanol was used instead of the 0.1 mM DPPH solution and distilled water was used instead of the sample solution.

The result of free radical scavenging rate (%) was calculated by Equation 4, and the results are shown in Table 4 below.

St: Absorbance at 514 nm after free radical scavenging of sample solution

Bt: absorbance at 514 nm after free radical scavenging of blank test solution

So: absorbance at 514 nm before reaction when no free radical of sample solution is added

Bo: absorbance at 514 nm before reaction without free radicals in blank test solution

Sample Name SC ₅₀ (%) Preparation Example 1 0.020 Preparation Example 2 0.014 Preparation Example 3 0.022

As a result of confirming SC ₅₀ , which is a concentration of a sample required to remove 50% of DPPH free radicals, Preparation Examples 1 to 3 showed an antioxidant effect.

Experimental Example  5: Inhibitory Effect of Old Pine Cone Extract on Inflammatory Cytokine Expression by UV Irradiation

Experimental Example 5 is to evaluate the inhibitory effect of inflammatory cytokine expression expressed by UV irradiation of the pine cone pine extract obtained in Preparation Examples 1,2,3, Fibroblast isolated from human epidermal tissue 5 x 10 ⁴ pieces in a 24-well test plate and attached for 24 hours.

Each well was washed once with PBS and 500 μl of PBS was added to each well. The fibroblasts were irradiated with 10 mJ / cm 2 of UV light using an ultraviolet B (UVB) lamp (Model: F15T8, UV B 15W, Sankyo Dennki, Japan), after which PBS was removed and FBS was not added to the cell culture medium (DMEM). Medium) 350 μl was added.

After treating the old pine cone pine extract to be evaluated here was incubated for 5 hours. 150 µl of the culture supernatant was taken to quantify IL-1α to determine the inhibitory effect of inflammatory cytokine expression of pine needles extract. The amount of IL-1α was quantified using an enzyme immunoassay (Enzyme-linked Immunosorbent Assay), and the percent inhibition of expression of inflammatory cytokines (IL-1α) was calculated by Equation 5 below.

Bo: IL-1α production in wells without UV irradiation

Bt: IL-1α production in wells that were not irradiated with UV light

St: IL-1α production amount of wells that were irradiated with UV light

Sample Name Treatment concentration Inflammatory cytokines
% Inhibition of expression Preparation Example 1 Preparation Example 2 Preparation Example 3 Old Tree Pinecone Extract 10 ppm 20.11 25.79 21.53 50 ppm 33.52 37.48 35.12 100 ppm 47.15 62.25 57.75 500 ppm 53.12 66.92 65.77 1000 ppm 74.28 85.06 78.10 Indomethacin 100 ppm 61.23

According to Table 5, it was found that the extract prepared by Preparation Example 2 of the old pine cone extract inhibits the production of inflammatory cytokine IL-1α by UV at 85.06% at a concentration of 1000 ppm. Old pine needles extract according to the present invention was found to show an excellent inflammatory cytokine expression rate than indomethacin, an inhibitor of inflammatory cytokine expression at the same concentration. Therefore, it can be seen that the old tree pine cone extract of the present invention effectively protects the inflammation caused by ultraviolet irradiation even at low concentrations.

Formulation example  1: Containing Old Address Pine Cone Extract Cosmetics  Preparation of the composition

Cosmetics containing the old pine cone pine extract extracts prepared cosmetics (Formula Examples 1, 2, 3) containing the old pine cone pine extract of Preparation Examples 1 to 3, respectively, as a moisturizer instead of the old pine cone pine extract for efficacy comparison Cosmetic composition (Comparative Formulation Example 1) comprising glycerin and 1,3 butylene glycol, Cosmetic composition (Comparative Formulation Example 2) containing old pine needles extract, Cosmetic composition (Comparative Formulation Example) containing no extract or moisturizer 3) is shown in Table 6 below.

division ingredient Formulation Example 1 Formulation Example 2 Formulation Example 3 compare
Formulation Example 1 compare
Formulation Example 2 compare
Formulation Example 3 A






Old Address Pine Cone Extract (Manufacturing Example 1) 5.0 - - - - Old Tree Pinecone Extract (Manufacturing Example 2) - 5.0 - - - Old Tree Pinecone Extract (Manufacturing Example 3) - - 5.0 -  -  - Old Address Pine Needle Extract - - - - 5.0 - glycerin - - - 5.0 - - 1,3-butylene glycol - - - 5.0 - - EDTA-2Na 0.02 0.02 0.02 0.02 0.02 0.02 Purified water to 100 to 100 to 100 to 100 to 100 to 100 B









Cetostearyl alcohol 2.0 2.0 2.0 2.0 2.0 2.0 Glyceryl Stearate 1.5 1.5 1.5 1.5 1.5 1.5 Microcrystallin 0.7 0.7 0.7 0.7 0.7 0.7 Squalane 5.0 5.0 5.0 5.0 5.0 5.0 Liquid paraffin 3.0 3.0 3.0 3.0 3.0 3.0 Trioctanoine 5.0 5.0 5.0 5.0 5.0 5.0 Polysorbate 1.2 1.2 1.2 1.2 1.2 1.2 Sorbitan stearate 0.5 0.5 0.5 0.5 0.5 0.5 Tocopheryl Acetate 0.2 0.2 0.2 0.2 0.2 0.2 Cyclomethicone 3.0 3.0 3.0 3.0 3.0 3.0 BHT 0.05 0.05 0.05 0.05 0.05 0.05 C Incense, preservative Quantity Quantity Quantity Quantity Quantity Quantity

Experimental Example  6: Moisturizing Effect of Old Pine Cone Extract

The clinical moisturizing effect of the cosmetic compositions of Formulation Examples 1, 2 and 3 was measured as follows. Through a survey, 125 healthy adult men and women who feel dry skin were randomly divided into 5 groups (A, B, C, D, E, and F) of 25 people each. 2,3 cosmetic composition, group D, a cosmetic composition containing glycerin and 1,3 butylene glycol as a moisturizing agent instead of the old pine cone extract (Comparative Example 1), a cosmetic composition comprising an old pine needle extract Composition (Comparative Formulation Example 2), F group was allowed to apply the cosmetic composition (Comparative Formulation Example 3) containing no old pine cone extract and moisturizing agent to the face twice a day for 2 weeks. Moisturizing effect was evaluated using Corneometer CM 820 (Corage + Kazaka, Germany) every 1 week from the start of the practical test and after the end. The experimental results are shown in Table 7 below.

division Before use 2 weeks after use 4 weeks after use Formulation Example 1 21.4 35.6 45.7 Formulation Example 2 23.1 37.7 48.9 Formulation Example 3 24.1 38.5 47.7 Comparative Formulation Example 1 23.7 27.7 30.5 Comparative Formulation Example 2 23.8 29.4 35.9 Comparative Formulation Example 3 23.5 22.4 24.9

As shown in Table 7, the cosmetic composition containing the old pine cone pine extract of the present invention as compared to the cosmetic containing the other moisturizing agent (Comparative Formulation Example 1) and cosmetics containing the old pine needles extract (Comparative Formulation Example 2) It was confirmed that the moisturizing effect of (Formulation Examples 1, 2, 3) was excellent.

Experimental Example  7: Measure skin elasticity improvement

Experimental Example 7 is to determine the effect of improving the skin elasticity of the cosmetic composition according to the formulation examples and comparative formulations containing the old pine cone extract obtained in Preparation Examples 1, 2, 3, 30 to 40 women of reduced elasticity Forty subjects were measured. After dividing into four groups of 10 people, Formulation Example 1 in Group 1, Formulation Example 2 in Group 2, Formulation Example 3 in Group 3, and Comparative Formulation Example 1 in Group 4 each morning and evening on the face 2 After 12 weeks of application, skin elasticity was measured after 12 weeks. The elasticity of the facial area was measured using a cutometer SEM575 (MPA 580, Courage + Khazaka GmbH, Germany), and R2 (biological elasticity), which indicates skin elasticity, was measured before and after application and evaluated as improvement (%). . The results are shown in Table 8 below, and the results are average values of improvement for each group.

Experimental product Skin Elasticity Improvement (%) Formulation Example 1 23.5 Formulation Example 2 28.1 Formulation Example 3 26.8 Comparative Formulation Example 1 4.5 Comparative Formulation Example 2 12.2

As shown in Table 8, the formulation examples 1, 2 and 3 containing the old pine cone pine extract was found that the skin elasticity is significantly improved compared to the comparative formulations 1,2.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (4)

It contains pinus sylvestris pine cone extract extracted by supercritical fluid extraction method as an active ingredient in an amount of 0.0001 to 30.0% by weight based on the total weight of the cosmetic composition, the pine cone pine extract inhibits MMP-1 production or enhances collagen biosynthesis Cosmetic composition for improving skin wrinkles having an effect.

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