JP7597353B2 - Melanin production inhibitors, collagen production promoters, hyaluronic acid production promoters, MMP-2 inhibitors and internal medicines - Google Patents

Description

The present invention relates to a melanin production inhibitor, a collagen production promoter, a hyaluronic acid production promoter, an MMP-2 inhibitor, and an internal preparation.

Generally, skin pigmentation such as age spots, freckles, and sunburn is thought to be caused by hormonal abnormalities and UV stimulation causing melanin pigment to be produced excessively by melanin-producing cells in the skin, which then deposits in the skin. One method for preventing such pigmentation is known to be a method of suppressing the excessive production of melanin. Traditionally, ascorbic acid (vitamin C) and the like have been used internally and externally as whitening agents to treat pigmentation (Patent Document 1).

In addition to ultraviolet rays, the skin is exposed to various physical and chemical stresses such as dryness, cold, heat, and drugs every day. As a result, the skin's functions are impaired, and various skin aging phenomena become apparent. One of the skin aging phenomena is wrinkles. It is known that there are two types of wrinkles: epidermal wrinkles and dermal wrinkles. Epidermal wrinkles are called fine wrinkles, and are temporary wrinkles that occur when the moisture content in the epidermal stratum corneum decreases due to dry skin. On the other hand, dermal wrinkles are wrinkles that are formed by ultraviolet rays contained in sunlight and aging. The mechanism of their formation includes a decrease in the collagen synthesis ability of dermal fibroblasts due to ultraviolet rays and aging, and promotion of collagen decomposition due to an increase in matrix metalloproteinase (MMP).

Epidermal wrinkles caused by dryness and dermal wrinkles have different histological morphology, onset mechanisms, and treatment methods, and dermal wrinkles caused by ultraviolet rays and aging are difficult to improve with the use of cosmetics with moisturizing effects.

To date, there have been reports of an agent for preventing and improving skin wrinkle formation, which contains hydrolyzed almonds as an active ingredient, for the purpose of improving dermal wrinkles caused by ultraviolet light (Patent Document 2), and an agent for improving wrinkles caused by ultraviolet light exposure, which contains extracts of Jochou-kei, Tensho, and Xylocentrotus nigricans as active ingredients (Patent Document 3).

The dermis also contains fibroblasts and collagen, with type I collagen accounting for 80% of the total. In addition to type I collagen, types III, V, XII, and XIV are known to exist. One of the causes of wrinkles and sagging is a decrease in type I collagen. Therefore, promoting the production of type I collagen is thought to be effective in preventing and improving wrinkles and sagging. Promoting the production of type I collagen is also effective in improving wound healing in the skin.

Fibroblasts also produce proteins such as collagen and glycosaminoglycans such as hyaluronic acid to form dermal connective tissue, which maintains skin firmness. It is believed that when this connective tissue loses its contractile force and further its elasticity, wrinkles and sagging of the skin occur.

In particular, hyaluronic acid is known as a polymeric polysaccharide widely distributed in connective tissues, and takes on a gel-like form in the dermis, maintaining the elasticity of the skin. Therefore, the deterioration and reduction of hyaluronic acid are considered to be important in skin aging. In addition, since hyaluronic acid is a polymer, there is a problem that cosmetics containing it are not easily absorbed even when applied directly to the skin. Therefore, up until now, skin external preparations that can promote the production of collagen and hyaluronic acid by activating fibroblasts have been sought. A hyaluronic acid production promoter containing an extract of Acanthus arbutus as an active ingredient (Patent Document 4), a hyaluronic acid production promoter containing an extract of sprouted and roasted Job's tears as an active ingredient (Patent Document 5), and a hyaluronic acid production promoter containing an extract of the fruiting body of Tremella fuciformis as an active ingredient (Patent Document 6) have been reported.

Hyaluronic acid is also present in joints, and is known to function to cushion the impact of loads on joints and to smooth joint movements. The concentration of hyaluronic acid in normal human synovial fluid is about 2.3 mg/mL, but in the case of chronic rheumatoid arthritis, the concentration of hyaluronic acid in synovial fluid is reduced to about 1.2 mg/mL, and the viscosity of the synovial fluid is also significantly reduced (Non-Patent Document 1). It is also known that the content of hyaluronic acid decreases in purulent arthritis, gouty arthritis, and the like, as in the case of chronic rheumatoid arthritis (Non-Patent Document 2). In the above diseases, it is considered to increase the amount of hyaluronic acid in synovial fluid in order to improve lubrication function, cover and protect articular cartilage, suppress pain, and improve pathological synovial fluid. For example, it is known that when sodium hyaluronate is injected into the joint of a patient with chronic rheumatoid arthritis, the above symptoms are improved (Non-Patent Document 3). However, the treatment of the above diseases takes a long time. Therefore, there is a demand for topical skin preparations, foods, and medicines that contain hyaluronic acid production promoters so that prevention and treatment can be easily carried out in daily life.

Floaters are a symptom where a thin shadow resembling a lint or a mosquito appears in the field of vision. They occur when opacity in the vitreous body that fills the inside of the eye casts a shadow on the retina. Floaters can be broadly divided into two types: physiological floaters, which develop due to aging, ultraviolet rays, active oxygen, etc., and pathological floaters, which appear as a symptom of diseases such as retinal detachment, retinal tear, vitreous hemorrhage, and uveitis. Physiological floaters are caused by liquefaction due to a decrease in hyaluronic acid, the main component of the vitreous body, and the resulting collapse of collagen fibers, causing the vitreous body to become opaque. Treatments include vitrectomy surgery and laser treatment, but these procedures are not often performed in Japan due to safety concerns, and treatment abroad requires a large amount of money. Therefore, in order to prevent and improve physiological floaters, foods and medicines containing hyaluronic acid production promoters that can be used on a daily basis are desired.

Gelatinase (MMP-2), which belongs to MMPs, is an enzyme produced by fibroblasts, endothelial cells, cancer cells, etc., and breaks down substrates such as collagen, gelatin, and elastin (structural proteins that are special components of elastic tissues such as arteries, tendons, and skin). Therefore, substances that have inhibitory activity against gelatinase are expected to have the effect of suppressing angiogenesis and cancer metastasis in cancer tissue, and are considered to be useful in the prevention and treatment of cancer diseases. Furthermore, inhibition of MMPs is useful not only for cancer diseases, but also for the prevention, treatment, and improvement of diseases caused by increased MMPs, such as ulcer formation, chronic rheumatoid arthritis, osteoporosis, and periodontitis.

Korean pine (scientific name: Pinus koraiensis) is an evergreen coniferous tree belonging to the genus Pinus, family Pinaceae, order Pinales. In Japan, it grows naturally in the mountainous areas of central Honshu and Shikoku, as well as on the Korean Peninsula, northeastern China, Amur, and Ussuri. Mature trees reach a height of over 30m and a diameter of 1.5m. The needles are five-lobed, with five needles growing in clusters on short branches. The cones are large, measuring 9-15cm in length and 5-7cm in diameter, and each cone scale contains two seeds measuring about 1.2cm in size. The herbal medicine kaishoushi is made from the dried seeds, which contain 60-70% fatty oil. The seeds are also used for food. It has been reported that Korean pine seed oil has a cholesterol-lowering effect (Patent Document 7), that extracts of the leaves, bark and cones have a hair-growth effect (Patent Document 8), and that extracts of the cones, bark and leaves have a blood fluidity improving effect (Patent Document 9). However, it was not known that Korean pine seed extracts have melanin production suppressing effects, collagen production promoting effects, hyaluronic acid production promoting effects, and MMP-2 inhibitory effects.

Japanese Patent Application Publication No. 5-229931 JP 2000-119125 A JP 2006-199611 A JP 2007-1924 A JP 2012-153669 A JP 2019-99530 A Japanese Unexamined Patent Publication No. 61-238729 JP 2004-224783 A JP 2008-161077 A

Arthritis Rheumatism, vol. 10, pp 357, 1967 "Bonding Composition", Kanehara Publishing, 481 pages, 1984 "Inflammation", Japanese Society of Inflammation, Vol. 11, No. 16, 1991

There is a demand for a material that is safe, stable, and has excellent melanin production suppression, collagen production promotion, hyaluronic acid production promotion, and MMP-2 inhibition effects, but currently no fully satisfactory material has been provided.

In light of these circumstances, the inventors conducted extensive research and found that an extract from Korean pine seeds has excellent melanin production inhibitory effects, collagen production promoting effects, hyaluronic acid production promoting effects, and MMP-2 inhibitory effects, and is also highly stable. Furthermore, they found that topical or internal preparations containing the extract are safe and stable, and have excellent melanin production inhibitory effects, collagen production promoting effects, hyaluronic acid production promoting effects, and MMP-2 inhibitory effects, and can be used as multifunctional beauty and health materials and pharmaceuticals, leading to the completion of the present invention.

That is, the present invention includes the following inventions.
(1) A melanin production inhibitor characterized by containing an extract of Korean pine seeds.
(2) A collagen production promoter characterized by containing an extract of Korean pine seeds.
(3) A hyaluronic acid production promoter characterized by containing an extract of Korean pine seeds.
(4) An MMP-2 inhibitor comprising an extract of Korean pine seeds.
(5) A skin whitening agent comprising an extract of Korean pine seeds.
(6) A food composition for preventing and improving various diseases caused by increased MMP activity, characterized by containing an extract of Korean pine seeds.

According to the present invention, there are provided a melanin production inhibitor (skin whitening agent), a collagen production promoter, a hyaluronic acid production promoter, and an MMP-2 inhibitor, each of which contains an extract of Korean pine seeds as an active ingredient. In addition, Korean pine seeds are consumed as food, so they have no side effects and are highly safe. Therefore, external or internal preparations containing Korean pine seed extract can be used safely.

The Korean pine (scientific name: Pinus koraiensis, herbal medicine name: Kaishoushi) used in the present invention is an evergreen coniferous tree belonging to the genus Pinus, family Pinaceae, order Pinales, and grows naturally in the mountainous areas of central Honshu and Shikoku in Japan, as well as the Korean peninsula, northeastern China, Amur, and Ussuri, but the place of origin is not particularly limited. In the present invention, the seeds of Korean pine can be extracted by using the seeds as they are, or by drying, crushing, or other processing.

The extraction method is not particularly limited, but may be carried out by stirring or column extraction using water or hot water, or a mixed solvent of water and an organic solvent. Examples of the extraction solvent include water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (1,3-butylene glycol, propylene glycol, glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, liquid paraffin, etc.), and ethers (ethyl ether, tetrahydrofuran, propyl ether, etc.). Preferably, polar solvents such as water, lower alcohols, and liquid polyhydric alcohols are good, and more preferably, water, ethanol, 1,3-butylene glycol, and propylene glycol are good. These solvents may be used alone or in combination of two or more. Particularly preferred extraction solvents include water, a mixed polar solvent of water-ethanol, or a mixed polar solvent of water-1,3-butylene glycol. There is no particular limit to the amount of solvent used; for example, it is sufficient if the amount is 10 times or more, and preferably 20 times or more, relative to the Korean pine seeds (dry weight), but it is preferable that the amount is 100 times or less for convenience of operations when concentrating or isolating after extraction. In addition, the extraction temperature and time can be appropriately selected depending on the type of solvent used, the pressure during extraction, etc.

The above extract may be used as it is in the form of an extracted solution, or may be used after subjecting it to processes such as concentration (vacuum concentration, membrane concentration, etc.), dilution, filtration, decolorization with activated carbon, deodorization, ethanol precipitation, etc., as long as the effects of the present invention are achieved. Furthermore, the extracted solution may be subjected to processes such as concentration to dryness, spray drying, freeze drying, etc., and used as a dried product.

In the present invention, the above extract may be used as is, or may contain ingredients such as fats and oils, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, metal soaps, pH adjusters, preservatives, fragrances, moisturizers, powders, UV absorbers, thickeners, pigments, antioxidants, whitening agents, chelating agents, excipients, coating agents, sweeteners, acidulants, etc., which are used in cosmetics, quasi-drugs, medicines, or foods, to the extent that the effects of the extract are not impaired.

The present invention can be used in any of cosmetics, quasi-drugs, medicines, and foods, and examples of the dosage forms include lotions, creams, milky lotions, gels, aerosols, essences, packs, cleansers, bath products, foundations, dusting powders, lipsticks, ointments, poultices, candy tablets, chocolates, gums, candies, beverages, powders, granules, tablets, sugar-coated tablets, capsules, syrups, pills, suspensions, liquids, emulsions, suppositories, and injectable solutions.

When used externally, the content of the above extract used in the present invention is preferably 0.0001% by weight or more, and more preferably 0.001 to 10% by weight, calculated as solid matter. Furthermore, 0.01 to 5% by weight is most preferable. If it is less than 0.0001% by weight, it is difficult to expect a sufficient effect. If it exceeds 10% by weight, it is difficult to see an increase in effect, and it is uneconomical.

When taken internally, the dosage varies depending on age, body weight, symptoms, therapeutic effect, administration method, treatment time, etc. In general, the daily dosage per adult is preferably 5 mg or more, more preferably 10 mg to 5 g. Furthermore, 20 mg to 2 g is most preferable.

In the following, in order to explain the present invention in detail, manufacturing examples, experimental examples and formulation examples of the extract used in the present invention are given as examples, but the present invention is not limited to these. The percentages shown in the manufacturing examples are % by weight, and the parts of the content shown in the formulation examples are parts by weight.

Example of Preparation of Korean Pine Seed Extract Korean Pine seed extract was prepared as follows.

(Production Example 1) Preparation of hot water extract of Korean pine seeds 200 mL of water was added to 10 g of dried Korean pine seeds, and extraction was carried out for 2 hours at 95 to 100° C. The obtained extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 1.1 g of hot water extract of Korean pine seeds.

(Preparation Example 2) Preparation of 50% ethanol extract of Korean pine seeds 200mL of 50% ethanol aqueous solution was added to 10g of dried Korean pine seeds, and extraction was carried out by stirring at 180 rpm at room temperature for 7 days. The obtained extract was filtered, and then concentrated and dried using an evaporator to obtain 0.6g of 50% ethanol extract of Korean pine seeds.

(Preparation Example 3) Preparation of Korean Pine Seed Ethanol Extract 200 mL of ethanol was added to 10 g of dried Korean Pine seeds, and the mixture was stirred at 180 rpm for 7 days at room temperature for extraction. The resulting extract was filtered, and then concentrated to dryness using an evaporator to obtain 0.2 g of Korean Pine seed ethanol extract.

(Production Example 4) Preparation of 1,3-butylene glycol extract of Korean pine seeds 200 mL of 1,3-butylene glycol was added to 10 g of dried Korean pine seeds, and extraction was carried out at room temperature for 7 days with stirring at 180 rpm. The obtained extract was filtered to obtain 194 g of 1,3-butylene glycol extract of Korean pine seeds.

(Production Example 5) Preparation of Korean Pine Seed Hexane Extract 200 mL of hexane was added to 10 g of dried Korean Pine seeds, and the mixture was stirred and extracted at 180 rpm at room temperature for 7 days. The resulting extract was filtered, and then concentrated and dried using an evaporator to obtain 0.1 g of Korean Pine seed hexane extract.

(Formulation Example 1) Lotion Formulation Content (parts)
1. Hot water extract of Korean pine seeds (Production Example 1) 2.0
2. 1,3-butylene glycol 8.0
3. Glycerin 2.0
4. Xanthan gum 0.02
5. Citric acid 0.01
6. Sodium citrate 0.1
7. Ethanol 5.0
8. Methyl parahydroxybenzoate 0.1
9. Polyoxyethylene hydrogenated castor oil (40 E.O.) 0.1
10. Fragrance (as needed) 11. Add purified water to make the total volume 100 [Manufacturing Method] Dissolve ingredients 1-6 and 11 and ingredients 7-10 uniformly, mix the two, and filter to obtain the product.

(Comparative Formulation Example 1) Conventional lotion A conventional lotion was prepared by replacing the hot water extract of Korean pine seeds in Formulation Example 1 with purified water.

(Formulation Example 2) Cream Formulation Content (parts)
1. 50% ethanol extract of Korean pine seeds (Preparation Example 2) 1.0
2. Squalane 5.5
3. Olive oil 3.0
4. Stearic acid 2.0
5. Beeswax 2.0
6. Octyldodecyl myristate 3.5
7. Polyoxyethylene cetyl ether (20 E.O.) 3.0
8. Behenyl alcohol 1.5
9. Glyceryl monostearate 2.5
10. Fragrance 0.1
11. Methyl parahydroxybenzoate 0.2
12. 1,3-butylene glycol 8.5
13. Make the total volume 100 with purified water [Manufacturing method] Heat and dissolve ingredients 2-9, mix, and maintain at 70°C to make the oil phase. Heat and dissolve ingredients 1 and 11-13, mix, and maintain at 75°C to make the water phase. Add the water phase to the oil phase to emulsify, cool with stirring, add ingredient 10 at 45°C, and further cool to 30°C to make the product.

(Comparative Formulation Example 2) Conventional Cream A conventional cream was prepared by replacing the 50% ethanol extract of Korean pine seeds in Formulation Example 2 with purified water.

(Formulation Example 3) Emulsion Formulation Content (parts)
1. Korean pine seed hexane extract (Production Example 5) 0.01
2. Squalane 5.0
3. Olive oil: 5.0
4. Jojoba oil 5.0
5. Cetanol 1.5
6. Glyceryl monostearate 2.0
7. Polyoxyethylene cetyl ether (20 E.O.) 3.0
8. Polyoxyethylene sorbitan monooleate (20 E.O.) 2.0
9. Fragrance 0.1
10. Propylene glycol 1.0
11. Glycerin 2.0
12. Methyl parahydroxybenzoate 0.2
13. Make the total volume 100 with purified water. [Manufacturing method] Heat and dissolve ingredients 1-8, mix, and keep at 70°C to make the oil phase. Heat and dissolve ingredients 10-13, mix, and keep at 75°C to make the water phase. Add the water phase to the oil phase to emulsify, cool with stirring, add ingredient 9 at 45°C, and cool further to 30°C to make the product.

(Formulation Example 4) Gel Formulation Content (parts)
1. Korean pine seeds
1,3-Butylene glycol extract (Production Example 4) 1.0
2. Ethanol 5.0
3. Methyl parahydroxybenzoate 0.1
4. Polyoxyethylene hydrogenated castor oil (60 E.O.) 0.1
5. Fragrance (appropriate amount) 6. 1,3-butylene glycol 5.0
7. Glycerin 5.0
8. Xanthan gum 0.1
9. Carboxyvinyl polymer 0.2
10. Potassium hydroxide 0.2
11. Add purified water to make the total volume 100. [Manufacturing method] Dissolve ingredients 2 to 5, 1, and 6 to 11 uniformly, and mix the two to make the product.

(Formulation Example 5) Pack Formulation Content (parts)
1. Hot water extract of Korean pine seeds (Production Example 1) 1.0
2. Korean pine seeds
1,3-Butylene glycol extract (Production Example 4) 5.0
3. Polyvinyl alcohol 12.0
4. Ethanol 5.0
5. 1,3-butylene glycol 8.0
6. Methyl parahydroxybenzoate 0.2
7. Polyoxyethylene hydrogenated castor oil (20 E.O.) 0.5
8. Citric acid 0.1
9. Sodium citrate 0.3
10. Fragrance (as needed) 11. Add purified water to make a total of 100 parts. [Manufacturing Method] Dissolve ingredients 1 to 11 uniformly to make the product.

(Formulation Example 6) Foundation Formulation Content (parts)
1. 50% ethanol extract of Korean pine seeds (Preparation Example 2) 1.0
2. Stearic acid 2.4
3. Polyoxyethylene sorbitan monostearate (20 E.O.) 1.0
4. Polyoxyethylene cetyl ether (20 E.O.) 2.0
5. Cetanol 1.0
6. Liquid Lanolin 2.0
7. Liquid paraffin 3.0
8. Isopropyl myristate 6.5
9. Sodium carboxymethylcellulose 0.1
10. Bentonite 0.5
11. Propylene glycol 4.0
12. Triethanolamine 1.1
13. Methyl parahydroxybenzoate 0.2
14. Titanium dioxide 8.0
15. Talc 4.0
16. Bengala 1.0
17. Yellow iron oxide 2.0
18. Flavoring, appropriate amount 19. Make the total volume 100 with purified water [Manufacturing method] Heat and dissolve ingredients 2 to 8, and keep at 80°C to make the oil phase. Allow ingredient 9 to swell well in ingredient 19, then add ingredients 1 and 10 to 13 and mix uniformly. Add ingredients 14 to 17, which have been ground and mixed in a grinder, and stir with a homomixer to make the water phase, keeping the temperature at 75°C. Add the water phase to the oil phase while stirring, and emulsify. Then cool, add ingredient 18 at 45°C, and cool to 30°C while stirring to make the product.

(Formulation Example 7) Bath additive Formulation Content (parts)
1. Ethanol extract of Korean pine seeds (Preparation Example 3) 1.0
2. Sodium bicarbonate 50.0
3. Yellow No. 202 (1) appropriate amount 4. Fragrance appropriate amount 5. Sodium sulfate to make the total amount 100 [Manufacturing method] Mix ingredients 1 to 5 uniformly to make the product.

(Formulation Example 8) Ointment Formulation Content (parts)
1. Hot water extract of Korean pine seeds (Production Example 1) 5.0
2. Korean pine seeds
1,3-Butylene glycol extract (Production Example 4) 1.0
3. Polyoxyethylene cetyl ether (30 E.O.) 2.0
4. Glyceryl monostearate 10.0
5. Liquid paraffin 5.0
6. Cetanol 6.0
7. Methyl parahydroxybenzoate 0.1
8. Propylene glycol 10.0
9. Make the total volume 100 with purified water. [Manufacturing method] Heat and dissolve ingredients 3-6, mix, and keep at 70°C to make the oil phase. Heat and dissolve ingredients 1, 2, and 7-9, mix, and keep at 75°C to make the water phase. Add the water phase to the oil phase to emulsify, and cool to 30°C while stirring to make the product.

(Formulation Example 9) Powder Formulation Content (parts)
1. Hot water extract of Korean pine seeds (Production Example 1) 1.0
2. Dried cornstarch 39.0
3. Microcrystalline cellulose 60.0
[Manufacturing method] Mix ingredients 1 to 3 to make a powder.

(Formulation Example 10) Tablets Formulation Content (parts)
1. Ethanol extract of Korean pine seeds (Production Example 3) 5.0
2. Dried cornstarch 25.0
3. Calcium carboxymethylcellulose 20.0
4. Microcrystalline cellulose 40.0
5. Polyvinylpyrrolidone 7.0
6. Talc 3.0
[Manufacturing method] Components 1 to 4 are mixed, and then an aqueous solution of component 5 is added as a binder to form granules. Component 6 is added to the formed granules and compressed into tablets. Each tablet weighs 0.52 g.

(Formulation Example 11) Tablets Formulation Content (parts)
1. Ethanol extract of Korean pine seeds (Preparation Example 3) 2.0
2. Dry cornstarch 49.8
3. Erythritol 40.0
4. Citric acid 5.0
5. Sucrose fatty acid ester 3.0
6. Fragrance 0.1
7. Purified water 0.1
[Manufacturing method] Mix ingredients 1 to 4 and 7 and mold into granules. Add ingredients 5 and 6 to the molded granules and compress into tablets. Each tablet weighs 1.0 g.

(Formulation Example 12) Beverage Formulation Content (parts)
1. Hot water extract of Korean pine seeds (Production Example 1) 0.05
2. Stevia 0.05
3. Malic acid 5.0
4. Fragrance 0.1
5. Add purified water to make the total volume 100. [Production method] Dissolve components 1 to 3 in a small amount of water. Next, add components 4 and 5 and mix.

Next, experimental examples will be presented to explain the effects of the present invention in detail.

Experimental Example 1: Melanin production inhibition test using B16 mouse melanoma B16 mouse melanoma cells were seeded in a 60 mm dish at 3 x 10 ⁴ cells, and cultured for 5 days in MEM culture medium containing 10% FBS to which each sample was added so as to have a final concentration of 1, 10 or 100 μg/mL, under conditions of 37°C and 5% CO _2. After culture, the cells were detached, centrifuged, and the pellet obtained was dissolved in PBS (-) by ultrasonication. Protein quantification was performed using the Lowry method (J. Biol. Chem., 193, 265-275, 1951). In addition, when measuring the amount of melanin, 4N NaOH was added to the remaining cell disruption solution taken for protein quantification, and the solution was heated at 60°C for 2 hours, and then the absorbance at 475 nm was measured using a spectrophotometer (Shimadzu Corporation), the amount of melanin was obtained from the calibration curve, and the amount of melanin per mg of protein was calculated. The melanin production inhibition rate was calculated from the ratio of the reduction in the amount of melanin in the sample-added group to the control (no sample-added) group.

These experimental results are shown in Table 1. The Korean pine seed extract of the present invention was found to have an excellent melanin production inhibitory effect (skin whitening effect). In particular, the ethanol extract of Korean pine seed (Production Example 3) was remarkably effective.

Experimental Example 2 Measurement of mRNA expression levels of type I collagen (COL1A1), hyaluronic acid synthase 2 (HAS2), and MMP-2 The mRNA expression levels of COL1A1, HAS2, and MMP-2 were measured. Human fibroblasts NB1RGB were seeded at 1×10 ⁵ cells on a 60 mm dish and cultured in DMEM culture medium containing 10% FBS under conditions of 37° C. and 5% CO _2. When the cells were confluent, they were cultured for 24 hours in DMEM(−) culture medium to which each sample was added so as to have a final concentration of 10 or 100 μg/mL, and then total RNA was extracted. Total RNA was extracted from the cells using RNAiso Plus (Takara Bio), and the amount of total RNA was determined by absorbance at 260 nm using a spectrophotometer (Nanodrop). The amount of mRNA expression was measured by real-time RT-PCR based on the total RNA extracted from the cells. For the real-time RT-PCR method, High Capacity RNA-to-cDNA Kit (Applied Biosystems) and SYBR Select Master Mix (Life Technologies) were used. That is, 500 ng of total RNA was subjected to reverse transcription reaction, followed by PCR reaction (95°C: 15 seconds, 60°C: 60 seconds, 40 cycles). Other operations were performed according to the prescribed method, and the expression levels of COL1A1, HAS2 and MMP-2 mRNA were calculated as a ratio to the expression level of GAPDH mRNA, which is an internal standard. The COL1A1 expression promotion rate was calculated as the ratio of the expression level of COL1A1 mRNA in the sample addition group to the expression level of COL1A1 mRNA in the control (no sample addition) group. The HAS2 expression promotion rate and the MMP-2 expression inhibition rate were also calculated in the same manner. The primers used to measure the expression level of each gene are as follows:

Primer set for COL1A1 AGGACAAGAGGCATGTCTGGTT (SEQ ID NO: 1)
TTGCAGTGGTAGGTGATGTTCTG (SEQ ID NO: 2)
Primer set for HAS2 TGGATGACCTACGAAGCGATT (SEQ ID NO: 3)
GCTGGATTACTGTGGCAATGAG (SEQ ID NO: 4)
Primer set for MMP-2 CCGTCGCCCATCATCAA (SEQ ID NO:5)
CTTCTGCATCTTCTTTAGTGTGTCCTT (SEQ ID NO: 6)
Primer set for GAPDH: TGCACCACCAACTGCTTAGC (SEQ ID NO: 7)
TCTTCTGGGTGGCAGTGATG (SEQ ID NO: 8)

These experimental results are shown in Tables 2 to 4. As a result, the Korean pine seed extract of the present invention was found to have excellent COL1A1 expression promoting effects (collagen production promoting effects), HAS2 expression promoting effects (hyaluronic acid production promoting effects), and MMP-2 expression suppressing effects (MMP-2 inhibitory effects). In particular, the hexane extract of Korean pine seed (Production Example 5) was significantly more effective in promoting COL1A1 expression, and the 50% ethanol extract of Korean pine seed (Production Example 2) was significantly more effective in suppressing MMP-2 expression and promoting HAS2 expression.

Experimental Example 3: Use test A one-month use test was conducted on five subjects (aged 26 to 66) with wrinkles and sagging skin using the cream of Formulation Example 2 and the conventional cream of Comparative Formulation Example 2. After use, the degree of wrinkles and sagging skin was assessed by questionnaire.

As a result, wrinkles and sagging skin were reduced by the cream containing the extract of the present invention. Furthermore, during the test period, not a single subject experienced skin troubles, and there were no safety issues. There were also no problems with deterioration of the prescribed ingredients.

A similar usage test was also conducted using the lotion of Formulation Example 1 and the conventional lotion of Comparative Formulation Example 1. As a result, it was found that the lotion containing the extract of the present invention reduced wrinkles and sagging.

From the above, the Korean pine seed extract of the present invention has excellent melanin production inhibitory effects, collagen production promoting effects, hyaluronic acid production promoting effects, and MMP-2 inhibitory effects, and is also highly stable. Therefore, the Korean pine seed extract of the present invention can be used not only in the cosmetic field, such as skin aging, but also in the medical field, such as inhibiting functional decline due to aging, and preventing and treating cancer, and is expected to be applied to cosmetics, foods, quasi-drugs, and pharmaceuticals.

Claims (2)

A melanin production inhibitor characterized by containing an ethanol extract of Korean pine seeds (excluding the outer seed coat) . A skin whitening agent comprising an ethanol extract of Korean pine seeds (excluding the outer seed coat) .