KR101570123B1 - Cosmetic Composition Comprising Extract of testa of Pinus koraiensis Siebold et Zucc - Google Patents

Abstract

The present invention relates to a cosmetic composition comprising an external seed coat extract of Pinus koraiensis Siebold et Zucc as an active ingredient and an external dermal agent comprising the same. In addition, the present invention relates to a food composition and a pharmaceutical composition comprising an external seed coat extract of Pinus koraiensis Siebold et Zucc as an active ingredient. The external seed coat extract of Pinus koraiensis Siebold et Zucc of the present invention shows high effects on anti-oxidation, whitening, or wrinkle improvement. The external seed coat extract of Pinus koraiensis Siebold et Zucc according to the present invention can be used as an functional material having useful anti-oxidant, whitening, and wrinkle improving effects.

Description

(Cosmetic Composition Comprising Extract of Testis of Pinus koraiensis Siebold et Zucc) as an Active Ingredient < RTI ID = 0.0 >

The present invention relates to a cosmetic composition comprising an extract of Pine nut Extract as an active ingredient and a skin external preparation containing the same.

The present invention also relates to a food composition and a pharmaceutical composition comprising an extract of Pine nut Extract as an active ingredient.

As well-being culture has become a trend in recent years, research on various fields such as cosmetics, foods, and pharmaceuticals has been actively carried out with the aim of improving the skin problems caused by ultraviolet rays, food, environment, etc., .

As a protective film of the outermost layers of the body, the skin protects the internal organs from the external environment such as ultraviolet rays and harmful substances, and functions to prevent the loss of water and electrolytes. The skin is always exposed to the external environment. Excessive ultraviolet exposure causes skin burns, melanin deposition, wrinkling and promoting photoaging, skin inflammation, and, in severe cases, skin cancer.

In general, melanin is made to protect the skin from external stimuli such as ultraviolet light. Over-deposition of melanin pigment on skin may result in hyperpigmentation, phototoxic response or other similar small-sized post-inflammatory reactions due to scarring or dermatitis, including unwanted freckles, senile spots, liver, spots, brown or black spots, It can cause fixed pigmented lesions.

Melanogenesis is regulated by three enzymes, tyrosinase, TRP-1 tyrosinase related protein-1 and TRP-2. The mechanism of biosynthesis of melanin is transformed into DOPA (3,4-dihydroxyphenylalanine) and DOPA quinone by the tyrosinase tyrosinase, which is the starting material, tyrosine, a rate-limiting enzyme, and DOPA chromium is used as the starting material for the biosynthesis of melanin. (5,6-dihydroxyindole-2-carboxylic acid) to produce eumelanin. When keratinocytes in the skin are stimulated by ultraviolet rays, signaling substances such as α-MSH (α-melanocyte-sitmulating hormone) and ACTH (adrenocorricotropic hormone) are secreted into melanocytes. Melanocortin type 1 receptor (MC1R) receptors increase the level of cyclic adenosine monophosphate (cAMP) by inducing these stimuli. The increase of cAMP causes the activation of PKA (protein kinase A), followed by CREB element-binding protein). The phosphorylated CREB causes the expression of MITF in the microphthalmia associated transcription factor (MITF), a specific transcription factor of melanocytes. The expression of MITF eventually promotes the binding of tyrosinase, TRP-1, and TRP-2, the major enzymes involved in melanin production, to melanin biosynthesis.

MMPs (matrix metalloproteinase), which degrades substrates among various proteolytic enzymes, play an important role in the development of skin photo-aging as the ultraviolet rays exposed to the skin increase. Extracellular matrix proteins in the dermis play an important role in maintaining the structure and resilience of the skin, 80% to 85% of which is composed of type Ⅰ procollagen. Type I procollagen is synthesized in the form of procollagen, which is a precursor, and then released out of the cell, resulting in a complete collagen after the amino and carboxyl ends are cleaved. In particular, MMPs play an important role in the degradation of matrix proteins, including intestitial collagenase (MMP-1), 72 kD gelatinase (MMP-2), 92 kD gelatinase (MMP-9), neutrophil collagenase (MMP-8), and collagenase (MMP-13) In skin exposed to ultraviolet light, the expression of MMPs is increased, and the increased MMPs break down the collagen that constitutes the skin, resulting in the deficiency of the substrate protein, resulting in skin aging and wrinkles.

Pinus pinus koraiensis Siebold et Zucc) is an evergreen tree belonging to the pine tree family. It grows at an elevation of 1,000 m above sea level and grows up to 30 m in height and 1 m in diameter. The bark is gray-brown and the sculpture falls. The leaves are 3 ~ 5 at the end of the short branch, and there is a white pore line on the back, light green color, with serrate on the edge.

Traditionally, pine wood has been classified as a waste resource except for pine nuts. In particular, in the case of the cones which are the pine nut of the pine by-product, the husks (seeds) enclosed in the outer husks can be harvested only by removing the shells (outer shells), and the white nuts (endosperm) (Shells and outermost shells) are left untouched or discarded. Therefore, it is necessary to study the effective utilization methods.

Accordingly, the present inventors have found that there is a substance having an effect of whitening and wrinkle-removing in extracts of zygote and shell (zucchini and exocytosis), which are abandoned after being stripped for harvesting white nuts And have completed the present invention.

An aspect of the present invention is to provide a cosmetic composition comprising an extract of P. japonicus as an active ingredient and a skin external preparation containing the same.

Another aspect of the present invention is to provide a food composition and a pharmaceutical composition comprising an extract of P. japonicus as an active ingredient.

An aspect of the present invention provides a cosmetic composition comprising an extract of Pine nut Extract as an active ingredient.

Hereinafter, the present invention will be described in detail.

The pine nuts of the present invention is pine (Pinus as fruit of pine koraiensis Siebold et Zucc) is an evergreen tree belonging to the pine tree family. In the present invention, the exocrine pine nut refers to the remaining seed after removing the white pine (endosperm) from the yellow pine surrounded by the outer pine in the pine nut.

The exotoxin of the pine nut may be an exogenous patch of mature pine nut.

The pine nut extract of the present invention may be extracted with water, C1 to C4 lower alcohols or a mixed solvent thereof. In the specific example of the present invention, extraction was performed using hot water and ethanol. The above-mentioned extract is a concept that includes the extract of the pine nut, its fraction or the active fraction isolated from the fraction.

The pine nut extract of the present invention can be used for antioxidant, whitening, shrinkage of pores or wrinkle improvement. Specifically, the extracts of P. japonica inhibited DPPH, PF, ABTS and TBARS, inhibited tyrosinase, inhibited tyrosine enzyme activity in B16F10 cells, And MITF protein expression to inhibit melanin production and to exhibit a whitening effect. In addition, the extract of P. japonica showed wrinkle-reducing effects by inhibiting esterase activity, maintaining elastin molecular shape, activating ProCOL1A2, and inhibiting the expression of MMP-1 and MMP-9. Therefore, the extract of Pine nut Extract according to the present invention can be used as a functional raw material useful for antioxidative, whitening and wrinkle-reducing effects.

The cosmetic composition of the present invention may further contain conventional auxiliary agents and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments, fragrances and the like which are conventionally used in cosmetic compositions, in addition to the above-mentioned effective ingredients. For example, the cosmetic composition may further contain an auxiliary component such as glycerin, butylene glycol, polyoxyethylene hardened castor oil, tocopheryl acetate, citric acid, panthenol, squalane, sodium citrate, allantoin and the like .

The pine nut extract may be contained in an amount of 0.1 to 50% by weight, more preferably 0.3 to 10% by weight based on the total weight of the cosmetic composition, and may be freely selected depending on the formulation, use and purpose.

For example, on the basis of a single cosmetic product (30 g) containing an extract of Pine nuts,

Based on the total weight of the cosmetic composition

1) The amount of powder of extrudate extract of Pine nuts can be 0.03g at 0.1 wt%, 0.09g at 0.3 wt%, 3g at 10 wt%, and 15g at 50 wt%.

2) The amount of phenolic compounds may be 0.1767 mg at 0.1% content, 0.531 mg at 0.3% content, 17.67 mg at 10% content, and 88.35 mg at 50% content,

3) When the weight of raw material is 50%, about 390g of exocytosis is used.

The cosmetic of the present invention may be blended with other essential ingredients, if necessary, in combination with the essential ingredients.

Examples of the compounding ingredients that may be added include organic solvents such as a preservative component, a moisturizer, an emollient, a surfactant, an organic and inorganic pigment, an organic powder, an ultraviolet absorbent, a preservative, a bactericide, an antioxidant, a plant extract, a pH adjuster, A blood circulation accelerator, a cold agent, an antiperspirant agent, and purified water.

Examples of the fat-soluble ingredients include ester-based fats, hydrocarbon-based fats, silicon-based fats, fluorine-based fats, animal fats and plant fats and the like.

Examples of ester-based fats include glyceryl tri-2-ethylhexanoate, cetyl 2-ethylhexanoate, isopropyl myristate, butyl myristate, isopropyl palmitate, ethyl stearate, octyl palmitate, isocetyl isostearate, But are not limited to, ethylbutyl, butylbutyl, ethylbutylbutyl, butylbutyl, isobutylbutyl, isobutylbutyl, isobutylisobutyl, isobutylisobutyl, isobutylisobutyl, Trimethylol propane, triisostearic acid trimethylol propane, tetra-2-ethylhexanoic acid pentaerythritol tri (2-ethylhexanoic acid) Terephthalic acid, stearic acid, tallow, caprylic acid, decyl lauric acid, hexyl laurate, myristic acid myristate, myristyl myristate, myristyl acid stearyl, stearyl stearate, decyl oleate, cetyl ricinoleate, Octyldodecyl oleate, octyldodecyl oleate, octyldodecyl linoleate, isopropyl isostearate, isopropyl stearate, isostearyl stearate, isocetyl stearate, isodecyl oleate, isodecyl oleate, octyldodecyl oleate, Ethylhexanoate, stearylhexyl isostearate, stearyl 2-ethylhexanoate, hexyl isostearate, ethylene glycol dioctanoate, ethylene glycol dioleate, propylene glycol dicaprate, di (capryl, capric acid) propylene glycol, dicapryl Diethylene glycol monobutyl ether, diethylene glycol propylene glycol, dicapric acid neopentyl glycol, dioctanoic acid neopentyl glycol, tricarboxylic acid glyceryl, triunsaturated glyceryl, triisopalmitic acid glyceryl, triisostearic acid glyceryl, Octanoic acid octanoate, octanoic acid octanoate, octanoic acid octanoate, octanoic acid octanoate, octanoic acid octanoate, octanoic acid octanoate, octanoic acid octanoate, octanoic acid octanoate, octanoic acid octanoate, Octyldecyl lactate, octyldecyl stearate, polyglycerin oleic acid ester, polyglycerin isostearic acid ester, triisocetyl citrate, triisobutyl citrate, triisooctyl citrate, myristyl lactate, cetyl lactate, octyldecyl lactate, triethyl citrate , Acetyl triethyl citrate, acetyl tributyl citrate, trioctyl citrate, diisostearyl malate, 2-ethylhexyl hydroxystearate, di-2-ethylhexyl succinate, diisobutyl adipate, diisopropyl sebacate, Cholesteryl isostearate, chitostearic acid, cholestearyl isostearate, cholestearyl hydroxystearate, cholesteryl oleate, dihydrocholestearyl oleate, pitostearyl isostearate, pitostearyl oleate, 12 -Stearoylhydroxystearic acid isostearyl, 12-stearoylhydroxystearic stearyl, 12-stearoyl And esters such as hydroxystearic acid and sostearyl.

Hydrocarbon-based fats and oils such as squalene, liquid paraffin, alpha-olefin oligomer, isoparaffin, ceresin, paraffin, floating isoparaffin, polybutene, microcrystalline wax, and hydrogenated hydrocarbon such as vaseline.

Examples of silicone based oils include polymethyl silicone, methylphenyl silicone, methyl cyclopolysiloxane, octamethylpolysiloxane, decamethylpolysiloxane, dodecamethylcyclosiloxane, dimethylsiloxane-methylcetyloxysiloxane copolymer, dimethylsiloxane-methylstarchoxysiloxane copolymer, Alkyl-modified silicone oil, and amino-modified silicone oil.

Examples of the fluorine-based oil include perfluoropolyether and the like.

Examples of the animal or vegetable oil include avocado oil, almond oil, olive oil, sesame oil, rice bran oil, new flower oil, soybean oil, corn oil, rape oil, apricot kernel oil, palm kernel oil, palm oil, castor oil, sunflower oil, The present invention relates to a process for producing a starch-containing starch, which is selected from the group consisting of seed oil, cottonseed oil, palm oil, palm oil, cucumber nut oil, wheat germ oil, rice germ oil, Animal or vegetable fats such as oil, can-pick wax, carnauba wax, liquid lanolin, and hardened castor oil.

Examples of the moisturizing agent include water-soluble low-molecular moisturizing agents, oil-soluble molecular moisturizing agents, water-soluble polymers, and oil-soluble polymers.

Examples of the water-soluble low molecular weight moisturizing agent include serine, glutamine, sorbitol, mannitol, sodium pyrrolidone-carboxylate, glycerin, propylene glycol, 1,3-butylene glycol, ethylene glycol, polyethylene glycol B Propylene glycol (polymerization degree n = 2 or more), polyglycerin B (polymerization degree n = 2 or more), lactic acid, lactic acid salt and the like.

Examples of the lipid-soluble low-molecular moisturizing agent include cholesterol and cholesterol ester.

Examples of the water-soluble polymer include carboxyvinyl polymer, polyaspartic acid, tragacanth, xanthan gum, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, water-soluble chitin, chitosan, .

Examples of the oil-soluble polymer include a polyvinylpyrrolidone / eicosene copolymer, a polyvinylpyrrolidone / hexadecene copolymer, a nitrocellulose, a dextrin fatty acid ester, and a polymeric silicone.

Examples of the emollients include long chain acyl glutamic acid cholesteryl ester, hydroxystearic acid cholesteryl, 12-hydroxystearic acid, stearic acid, rosin acid and lanolin fatty acid cholesteryl ester.

Examples of the surfactant include a nonionic surfactant, an anionic surfactant, a cationic surfactant, and a positive surfactant.

Examples of nonionic surfactants include self emulsifying monostearic acid glycerin, propylene glycol fatty acid esters, glycerin fatty acid esters, polyglycerin fatty acid esters, sorbitan fatty acid esters, POE (polyoxyethylene) sorbitan fatty acid esters, POE sorbit fatty acid esters, POE-POP fatty acid ester, POE alkyl ether, POE fatty acid ester, POE hardened castor oil, POE castor oil, POE.POP (polyoxyethylene.polyoxypropylene) copolymer, POE.POP alkyl ether, polyether- Acid alkanolamides, alkylamine oxides, hydrogenated soybean phospholipids, and the like.

Examples of the anionic surfactant include fatty acid soap, alpha-acylsulfonate, alkylsulfonate, alkylallylsulfonate, alkylnaphthalenesulfonate, alkylsulfate, POE alkyl ether sulfate, alkylamide sulfate, alkyl phosphate, POE alkyl phosphate, Alkylsulfosuccinic acid salts, acylated hydrolyzed collagen peptide salts, and perfluoroalkyl phosphoric acid esters, and the like can be mentioned. have.

Examples of the cationic surfactant include alkyl trimethyl ammonium chloride, stearyl trimethyl ammonium chloride, stearyl trimethyl ammonium bromide, cetostearyl trimethyl ammonium chloride, distearyl dimethyl ammonium chloride, stearyl dimethyl benzyl ammonium chloride, behenyl trimethyl ammonium bromide, Benzalkonium chloride, diethylaminoethylamide stearate, dimethylaminopropylamide stearate, quaternary ammonium salts of lanolin derivatives, and the like.

Examples of amphoteric surfactants include carboxybetaine, amidebetaine, sulfobetaine, hydroxysulfobetaine, amidosulfobetaine, phosphobetaine, aminocarboxylate, imidazoline derivative, amideamine, etc. , And the like.

Examples of the organic and inorganic pigments include inorganic pigments such as silicic acid, anhydrous silicic acid, magnesium silicate, talc, sericite, mica, kaolin, Bengala, clay, bentonite, titanium mica, titanium oxide mica, bismuth oxychloride, zirconium oxide, Inorganic pigments such as aluminum, calcium sulfate, barium sulfate, magnesium sulfate, calcium carbonate, magnesium carbonate, iron oxide, chromium oxide, chromium oxide, chromium oxide, But are not limited to, polyamide, polyester, polypropylene, polystyrene, polyurethane, vinyl resin, urea resin, phenol resin, fluororesin, silicon resin, acrylic resin, melamine resin, epoxy resin, polycarbonate resin, Silk powder, cellulose, CI Pigment Yellow, CI Pigment Orange, and composite pigments of inorganic pigments and organic pigments thereof.

Examples of the organic powder include metal soaps such as calcium stearate; Metal salts of alkyl phosphates such as sodium zinc cetylate, zinc laurylate and calcium lauryl laurate; Acylamino acid polyvalent metal salts such as N-lauroyl-beta-alanine calcium, N-lauroyl-beta-alanine zinc and N-lauroylglycine calcium; Amidosulfonic acid multivalent metal salts such as N-lauroyl-taurine calcium and N-palmitoyl-taurine calcium; Such as N-epsilon-lauroyl-L-lysine, N-epsilon-palmitoylidene, N-alpha-paratyylnitine, N-alpha-lauroyl arginine, Acyl basic amino acids; N-acylpolypeptides such as N-lauroylglycylglycine; Alpha-amino fatty acids such as alpha-aminocaprylic acid, alpha-aminoaurauric acid, and the like; Polyethylene, polypropylene, nylon, polymethylmethacrylate, polystyrene, divinylbenzene-styrene copolymer, ethylene tetrafluoride, and the like.

Examples of the ultraviolet absorbing agent include pamoaminobenzoic acid, ethyl p-aminobenzoate, amyl p-aminobenzoate, octyl p-aminobenzoate, ethylene glycol salicylate, phenyl salicinate, benzyl salicylate, benzyl salicylate, butylphenyl salicylate, homomenthyl salicylate, Benzyl, 2-ethoxyethyl methoxycinnamate, octyl methoxycinnamate, mono-2-ethylhexane glyceryl dipyrromethoxycinnamate, isopropyl methoxycinnamate, diisopropyl-diisopropyl cinnamate ester mixture, Dihydroxymethoxybenzophenone sulfonic acid, dihydroxybenzophenone sulfonic acid, dihydroxybenzophenone sulfonic acid and its salt, dihydroxymethoxybenzophenone, sodium dihydroxymethoxybenzophenone disulfonate, dihydroxybenzo Phenanone, tetrahydroxybenzophenone, 4- tert -butyl-4'-methoxydibenzoylmethane, 2,4,6-trianylino- p- (carbo-2'-ethylhexyl- 1,3,5-triazine, 2- (2- When the like can be mentioned 5-methylphenyl) benzotriazole.

Examples of the bactericide include hinokitiol, trichloroacetic acid, trichlorohydroxydiphenyl ether, crohexidine gluconate, phenoxyethanol, resorcin, isopropylmethylphenol, azulene, salicylic acid, No. 301, mononitro and eicoll sodium, undecylenic acid, and the like.

Examples of the antioxidant include butylhydroxyanisole, gallic acid propyl, and eicosorbic acid.

Examples of the pH adjusting agent include citric acid, sodium citrate, malic acid, sodium malate, fumaric acid, sodium fumarate, succinic acid, sodium succinate, sodium hydroxide, sodium monohydrogenphosphate and the like.

Examples of the alcohol include higher alcohols such as cetyl alcohol.

Any of the above components may be blended to the extent that the object and effect of the present invention are not impaired, but is preferably 0.01-5% by weight, more preferably 0.01-5% by weight based on the total weight of the composition Lt; RTI ID = 0.0 >% < / RTI >

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

The ingredients contained in the cosmetic composition of the present invention may contain, in addition to the above extracts, the components commonly used in cosmetic compositions, for example, conventional additives such as stabilizers, solubilizers, vitamins, Carrier.

In addition, one aspect of the present invention provides an external preparation for skin comprising the pine nut extract. The external preparation may be used as a cosmetic for antioxidant, whitening, reduction of pores or wrinkle improvement, and may be used as a medicine for preventing or treating pigmentation diseases.

The external preparation of the present invention can be prepared into any formulation conventionally produced by referring to the known art in the art. For example, the compositions may be formulated as solutions, suspensions, emulsions, suppositories, suppositories, pastes, gels, creams, lotions, essences, emulsions, packs, cleansers, gels, powders, Emulsion foundation, wax foundation and spray, but are not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritive cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a mask pack, a spray or a powder.

In order to use the extract as an external preparation for skin, it may further comprise at least one selected from the group consisting of fatty substances, organic solvents, solubilizers, thickeners and gelling agents, softening agents, antioxidants, suspending agents, stabilizing agents, foaming agents, Any of those commonly used in emulsifying or emulsifying agents, fillers, sequestering and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, ≪ / RTI > and other ingredients of the skin. In addition, the components can be introduced in amounts commonly used in the dermatology field.

When the formulation of the present invention is a paste, cream or gel, the carrier component may include an animal oil, vegetable oil, wax, paraffin, starch, tracer, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, have.

When the formulation of the present invention is a powder or a spray, the carrier component may include lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder and the like. Particularly in the case of a spray, a mixture of chlorofluorohydrocarbons, propane / Propane, dimethyl ether, and the like.

When the formulation of the present invention is a solution or an emulsion, the carrier component may include a solvent, a solubilizing agent, and an emulsifying agent. Specific examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, Glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, fatty acid esters of sorbitan, and the like.

When the formulation of the present invention is a suspension, a liquid diluent such as water, ethanol, propylene glycol or the like as a carrier component; Suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester; Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, trachant, and the like.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide ether Aliphatic alcohols, fatty acid glycerides, fatty acid diethanol amides, vegetable oils, lanolin derivatives, ethoxylated glycerol fatty acid esters, and the like.

Further, one aspect of the present invention provides a food composition for antioxidant, whitening or antioxidation comprising an extract of Pine Pine Nutrient Extract as an active ingredient.

When the extract of the present invention is used as a food additive, it can be suitably used according to a conventional method such as adding the extract as it is, mixing it with another food or food ingredient, and the like.

The amount of the extract as the active ingredient may be suitably changed according to the purpose of use (prevention, health or therapeutic treatment), and the extract may be contained in an amount of 0.001 to 50% by weight based on the total weight of the food composition By weight, more preferably 0.1 to 30% by weight.

As a specific example, the extract of the present invention is added in an amount of not more than 30% by weight, preferably not more than 10% by weight based on the raw material. However, when it is intended for health and hygiene purposes or for the purpose of controlling health, it can be added in an amount below the above range, and there is no problem in terms of safety. Therefore, the active ingredient may be used in an amount exceeding the above range have.

There is no particular limitation on the kind of the food. Examples of the food to which the extract of the present invention can be added include meat products, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, Drinks, alcoholic beverages, vitamin complexes, and the like, all of which include health foods in a conventional sense.

When the food composition of the present invention is prepared as a beverage, it may contain additional ingredients such as various flavors or natural carbohydrates such as ordinary beverages. Examples of the natural carbohydrate include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharin and aspartame, and the like. The natural carbohydrate is contained in an amount of 0.01 to 10% by weight, preferably 0.01 to 0.1% by weight based on the total weight of the food composition of the present invention.

In addition to the above, the food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, , Carbonating agents used in carbonated beverages, and the like. In addition, the compositions of the present invention may include flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of the above additives is not limited to a great extent, but may be in the range of 0.01 to 0.1% by weight based on the total weight of the food composition of the present invention.

In addition, one aspect of the present invention provides a pharmaceutical composition for treating or preventing a pigmentation disorder comprising an extract of Pine Pine (Pine Nut) as an active ingredient. These pigmentation disorders include, but are not limited to, freckles, aging spots, liver, spots, dark spots, sunspots, daylight pigmentation, cyanic melasma, hyperpigmentation after drug use, gravidic chloasma, Hyperpigmentation after inflammation due to wounds or dermatitis involved, and the like.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. In addition, it can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral preparations such as syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to a conventional method. Suitable formulations known in the art are preferably those as disclosed in Remington ' s Pharmaceutical Science, recently, Mack Publishing Company, Easton PA. Examples of carriers, excipients and diluents which may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like. When the composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.

The term "administering" as used herein is meant to provide any desired composition of the invention to a subject in any suitable manner.

The pharmaceutical composition of the present invention is useful as an effective ingredient or amount of a pharmaceutical composition that induces a biological or medical response in a tissue system, an animal or a human being considered by a researcher, veterinarian, physician or other clinician, RTI ID = 0.0 > effective < / RTI > amount. It will be apparent to those skilled in the art that the therapeutically effective dose and frequency of administration for the pharmaceutical compositions of the present invention will vary with the desired effect. Thus, the optimal dosage to be administered can be readily determined by those skilled in the art and will vary with the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, The age, body weight, sex, diet, time of administration, route of administration and fraction of the composition, duration of treatment, concurrent medication, and the like. For a desired effect, the pharmaceutical composition of the present invention may be administered in an amount of 0.001 to 100 mg / kg / day, and may be administered once a day, or divided into several doses.

The pharmaceutical compositions of the present invention can be administered to a subject in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

According to the present invention, the extract of P. japonica inhibits DPPH, PF, ABTS and TBARS, inhibits tyrosinase, inhibits tyrosine enzyme protein and MITF protein expression in B16F10 cells, inhibits melanin production, . In addition, the extract of P. japonica exerts an effect of suppressing esterase activity, thereby maintaining the elastin molecular form, activating ProCOL1A2, suppressing the expression of MMP-1 and MMP-9, and improving wrinkles. Therefore, the extract of P. japonica according to the present invention can be used as a functional raw material useful for antioxidative, whitening and wrinkle-reducing effects.

Brief Description of the Drawings Fig. 1 is a graph showing the contents of phenolic compounds in the extrudate extract of pine nut.
FIG. 2 is a graph showing oxidative free radical reactivity of water and ethanol extracts of extruded pine nut using the DPPH method.
FIG. 3 is a graph showing changes in DPPH scavenging ability according to the Phenolic concentration of the extract of P. japonicus.
FIG. 4 shows the results of antioxidative activity of the extract of Porphyra zeaxus using ABTS radical cation decolorization.
FIG. 5 shows the results of ABTS radical cation decolorization measured according to the phenolic concentration of the extract of P. japonicus.
FIG. 6 shows the result of measuring the antioxidant protection factor (PF) of the extract of P. japonicus.
FIG. 7 shows the result of measuring the antioxidant protection factor (PF) according to the phenolic concentration of the extract of P. japonicus.
FIG. 8 is a graph showing the results of measurement of TBRAs of the extrudate extract of Pine nuts.
FIG. 9 is a graph showing the results of measurement of TBRAs according to Phenolic concentration in the extract of P. japonicus.
FIG. 10 is a graph showing convergence effects of the extract of Pine Pine Jellyfish.
Fig. 11 is a graph showing tyrosinase inhibitory activity of the extract of P. japonicum exoc.
12 is a graph showing the survival rate of the melanoma cell (B16F10) according to the treatment of the extragranular extrudate extract. A: water extract, B: ethanol extract.
FIG. 13 is a graph showing tyrosinase inhibitory activity in melanoma cell (B16F10) according to the treatment of extrudate extract of Pine nuts. A: water extract, B: ethanol extract.
FIG. 14 shows inhibition of melanin biosynthesis in melanoma cell (B16F10) according to the treatment of extruded pine nut extract. A: water extract, B: ethanol extract.
Fig. 15 is a graph showing the inhibitory effect of tyrosinase on the synthesis of melanin in the extragranular extrudate extract. 1, 5, and 10 μg / mL of pine nut extract were treated for 24 hours. Tyrosinase was detected by Western blot using antibody. Diagrams were derived from normalization based on β-actin. Each value was expressed as mean ± SD of three independent experiments.
FIG. 16 is a graph showing the inhibitory effect of MITF on the expression of melanin in the extract of P. japonica.
FIG. 17 is a view showing the elastase inhibitory activity of the extract of P. japonicus.
Fig. 18 is a graph showing collagenase inhibitory activity of the extract of P. japonicus exocarpae.
FIG. 19 is a graph showing the results of MTT measurement of CCD986sk fibroblast cells according to the treatment of extrudate extract of Pine nuts. A: water extract, B: ethanol extract.
FIG. 20 shows the results of confirming the pro-collagen biosynthesis rate according to the treatment of the extrudate extract of Pine nuts. A: water extract, B: ethanol extract.
FIG. 21 is a graph showing the inhibitory effect of procollagenase protein expression on the treatment of extrudate extract of Pine nuts.
22 shows the results of confirming the amount of wrinkle-related protein (MMP-1) and mRNA expression of fibroblast cells (CCD-986sk) treated with the extract of P. japonica exotrophs. . A: water extract, B: ethanol extract.
23 and 24 are graphs showing inhibitory effects of intestitial collagenase (MMP-1) and 92 kD gelatinase (MMP-9) on protein expression in fibroblast (CCD-986sk)
FIG. 25 shows the results of TIMP-1 activity according to the treatment with the extract of P. japonica. A: water extract, B: ethanol extract.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1. Preparation of materials

The materials used in this study were naturally grown in Pinus koraiensis in Gapyeong, Gyeonggi Province, and the matured pine nuts were collected from the pine nuts and separated from the shells and shells surrounding the outer surface of the pine nuts. . The seeds of the seeds were removed by removing the seed coat, which was a hard shell. The seeds were washed with water, dried and then pulverized to about 40 mesh.

Example 2. Outer Pine Patch  Extract preparation

For the hot-water extract of Pine Pine Nut, 100 g of distilled water was added to 10 g of distilled water, and the mixture was heated at 85 ° C for 3 hours under reflux and cooling for extraction. The supernatant and precipitate were separated and extracted three times. In the case of ethanol extract, After immersion for 24 hours at room temperature, the supernatant was separated from the precipitate. Each extract was centrifuged at 12,000 rpm for 10 minutes. 1 filter paper, and each extract was concentrated and stored in the freezer after freeze drying, and used as a sample of this experiment.

Example  3. Total polyphenol content measurement

Polyphenol compounds were measured by the Folin-Denis method, the sample 1 mL added to 95% ethanol 1 mL) and distilled water (5 mL to give mixed well into a 1N Folin-ciocalteu reagent 0.5 mL, and then allowed to stand for 5 minutes, Na ₂ After adding 1 mL of CO ₃ , the absorbance was measured at 725 nm within 1 hour and the amount was converted from the standard curve using gallic acid.

Example  4. Outer Pine Patch  Extract electron donating ability  Measure

The electron donating ability (EDA) is measured by modifying the Blois method. Add 1 ml of 0.2 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) to 2 ml of each sample solution, stir and incubate for 30 minutes, and measure the absorbance at 517 nm. The electron donating ability is represented by the absorbance reduction rate of the sample solution and the no-added solution.

Example  5. ABTS radical cation decolorization ( ABTS ) Measure

ABTS radical cation decolorization was measured by Pellegrin et al. That is, 7 mM ABTS and 140 mM K ₂ S ₂ O ₈ were mixed with 5 μL of 88 μL, left in the dark for 14-16 hours, and then mixed with absolute ethanol at a ratio of 1:88. The absorbance value of the control was 734 nm 0.7 ± 0.002 was used. As a positive control, 50 μL of BHT and 1 mL of ABTS solution were mixed for 30 seconds, incubated for 2.5 minutes, and the absorbance was measured at 734 nm. The inhibition rate (%) was calculated by the following equation.

Example  6. Antioxidant protection factor ( PF ) Measure

PF Andarwulan and Shetty . 10 mL of β-carotene was dissolved in 50 mL of chloroform and 1 mL of the solution was added to the evaporator. The chloroform was distilled in a water bath at 40 ° C., and 20 μL of linoleic acid, 184 μL of Tween 40 and 50 mL of H ₂ O ₂ emulsion was prepared, and 100 μL of the sample solution was mixed with 5 mL of the emulsion. After vortexing, the reaction was allowed to proceed at 50 ° C. for 30 minutes, and the absorbance was measured at 470 nm.

Example 7. Thiobarbituric acid reaction substance ( TBARs ) Measure

TBARS was measured according to the method of Blurge and Aust. The emulsion was prepared with 1% linoleic acid and 1% Tween 40, mixed with 0.8 mL of emulsion and 0.2 mL of sample, and reacted in a water bath at 50 ° C for 10 hours. After the reaction, add 2 mL of TBA regent [3 mL of 2% BHT / EtOH mixture] to 100 mL stock solution (15% trichloroacetic acid (TCA), 0.375% thiobarbituric acid, 0.25 M HCl mixture) After boiling and cooling for 10 minutes, the mixture was centrifuged for 15 minutes at 1000 rpm (vision, VS-5500N), and incubated at room temperature for 10 minutes. Absorbance was measured at 532 ㎚ for TBARS. The product was labeled with μM of 1,1,3,3-tetraethoxy propane (TEP) produced.

Example  8. Measurement of convergence effect

Astringent activity was measured by Lee et al. Using hemoglobin similar to skin protein, each sample solution and hemoglobin solution were mixed in a ratio of 1: 1 into a centrifuge tube, mixed with shaking, centrifuged at 1,500 rpm for 3 minutes, and absorbance was measured at 576 nm. The astringent activity measurement was expressed by the absorbance reduction rate of the sample solution addition group and the no addition group.

Example  9. Measurement of whitening effect

(One) Tyrosinase  Measurement of inhibitory activity

Tyrosinase inhibitory activity was measured by Yagi et al. To the reaction mixture, 0.2 mL of mushroom tyrosinase (110 U / mL) was added to a mixture of 0.2 mL of the substrate solution in which 10 mM L-DOPA was dissolved in 0.5 mL of 0.175 M sodium phosphate buffer (pH 6.8) and 0.1 mL of the sample solution and the mixture was reacted at 25 ° C. for 2 minutes And the DOPA chrome produced in the reaction solution was measured at 475 nm. The tyrosinase inhibitory activity was expressed as the absorbance reduction ratio of the sample solution and the non - added sample.

(2) Outer Pine Patch  Extract cell line  Whitening efficacy test

1) Cell viability measurement

(A) Cell culture

Cultures of each cell used for this experiment is 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (100U / mL) a dulbeco's modified eagle's medium (DMEM ) was used as the medium, 37 ℃, 5% CO ₂ was added to incubator and subcultured.

(I) MTT assay Of cell survival

Cell viability was measured by the method of Carmichael et al. Each cell line (melanoma (B16F10), fibroblast (CCD-986sk), and macrophage (Raw 264.7) cells) was dispensed in a 96-well plate at a concentration of 0.6 to 8 × 10 ³ cells / mL, and incubated at 37 ° C in a 5% CO ₂ incubator for 24 hours. In the control group, the same amount of distilled water as that of the sample was added and the cells were cultured under the same conditions. After adding 0.02 mL of MTT solution (5 mg / mL) for 4 hours, the culture solution was removed and 0.15 mL of DMSO: ethanol (1: 1) was added to each well. Absorbance at 550 nm.

2) Measurement of cellular tyrosinase inhibitory activity

Melanoma (B16F10) cells were cultured in each culture dish, and the medium was removed and washed with PBS. 100 μl of lysis buffer (67 mM sodium phosphate buffer, 1% triton X-100, 0.1 mM phenylmethyl sulfonyfluoride) was added to each dish, and the cells were disrupted by ice, and cells were collected and subjected to ultra sonication. The supernatant obtained by centrifugation at 13,200 rpm for 20 minutes was used as an enzyme solution. 120 uL of 8.0 mM L-DOPA dissolved in 67 mM phosphate buffer (pH 6.8) and 40 uL of the sample solution were added to a 96-well plate and then 40 uL of the enzyme solution dissolved in 67 mM phosphate buffer (pH 6.8) After minute incubation, the amount of DOPA chrome produced was measured at 490 nm.

3) Inhibition rate of melanin biosynthesis in melanoma cell (B16F10)

Measurement of inhibition of melanin biosynthesis from skin melanoma cells was performed according to the method of Hosoi et al. B16F10 cells cultured in DMEM medium were divided into 1 × 10 ⁶ cells / dish in a 100 mm culture dish. After culturing for 24 hours, 2 mL of each sample was prepared and the cells were washed with phosphate buffer (pH 7.4) 48 hours later. After the cells were desorbed with 0.25 M trypsin-EDTA solution, the harvested cells were treated with 1 mL of 5% TCA per 1 × 10 ⁶ cells, centrifuged twice at 2,500 rpm, and the separated melanin was washed with phosphate buffer Then, 1 mL of ether: ethanol (1: 3) was added, and the mixture was centrifuged twice. 1N NaOH was added to the dried melanin, reacted at 80 ° C for 1 hour, and the absorbance was measured at 405 nm by a spectrophotometer. The inhibition of melanin biosynthesis was expressed by the absorbance reduction ratio of the sample solution and the non - added sample.

4) Expression of intracellular pigmentation-related proteins by Western blot

The amount of MITF and tyrosinase protein expression was measured by the following method. In other words, B16F10 was cultured in a culture dish, and the sample was treated. After 48 hours, the cells were washed with PBS, centrifuged, and cell lysate was prepared using RIPA buffer. The amount of protein contained in the cell lysate was measured by the Bradford method. 15 μg of protein was separated by SDS-PAGE containing 8% polyacrylamide and electroblotted with PVDF membrane at 120 V for 1 hour. The PVDF membrane is then left in 5% skim milk for 1 hour and then overnight at 4 ° C in primary antibody. After washing three times with TBST, the secondary antibody was reacted at room temperature for 1 hour, washed three times with TBST and exposed to X-ray film by reaction with ECL kit (Amersham-Pharmacia). The developed bands on the X-ray film were quantified using a LAS 4000 image analyzer.

Example  10. Wrinkle improvement effect

(One) Elastase  Measurement of inhibitory activity

Elastase inhibitory activity was measured by Cannell et al. N-succinyl- (L-Ala) ₃ - p- nitroanilide was used as a substrate and the amount of p- nitroanilide produced from the substrate at 37 ° C for 20 minutes was measured at 405 nm. 0.5 mL of each test solution was added to 0.5 mL of each test solution. 0.5 mL of porcine pancreas elastase (2.5 U / mL) dissolved in 50 mM tris-HCl buffer (pH 8.6) was added to the test tube. 50 mM tris- N-succinyl- (L-Ala) ₃ - p- nitroanilide (0.5 mg / mL) dissolved in HCl buffer (pH 8.6) was added and the reaction was allowed to proceed for 20 minutes. Elastase inhibitory activity was expressed as the absorbance reduction ratio of the sample solution and the non - added sample.

(2) Collagenase  Measurement of inhibitory activity

Collagenase inhibitory activity was measured according to the method of Choshi et al. That is the reaction substrates dissolved phrase 0.1M tris-HCl buffer subject to 4mM CaCl ₂ in (pH 7.5) was added, 4-phenylazobenzyloxy carbonyl-Pro- Leu-Gly-Pro-D-Arg (0.3mg / mL) solution, and 0.25mL 0.1 mL of collagenase (0.2 mg / mL) was added to the mixture of 0.1 mL of the sample solution. The mixture was allowed to stand at room temperature for 20 minutes, and then the reaction was stopped by adding 0.5 mL of 6% citric acid. Absorbance was measured. Collagenase inhibitory activity was expressed as the absorbance reduction ratio of the sample solution and the non - added sample.

(3) Outer Pine Patch  Extract cell line  Wrinkle improvement efficacy test

1) Cell viability measurement

A) Cell culture

Cultures of each cell used for this experiment is 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (100U / ml) a dulbeco's modified eagle's medium (DMEM ) was used as the medium, 37 ℃, 5% CO ₂ was added to incubator and subcultured.

B) Measurement of cell viability by MTT assay

Cell viability was measured by the method of Carmichael et al. Each cell line (melanoma (B16F10), fibroblast (CCD-986sk), and macrophage (Raw 264.7) cells) was dispensed in a 96-well plate at a concentration of 0.6 to 8 × 10 ³ cells / mL, and incubated at 37 ° C in a 5% CO ₂ incubator for 24 hours. In the control group, the same amount of distilled water as that of the sample was added and the cells were cultured under the same conditions. After adding 0.02 mL of MTT solution (5 mg / mL) for 4 hours, the culture solution was removed and 0.15 mL of DMSO: ethanol (1: 1) was added to each well. Absorbance at 550 nm.

2) Skin regeneration effect of Extract of Pine Pine

A) Measurement of wrinkle improvement effect using ELISA kit

i) Procollagen type I biosynthetic kit measurement

Cells were inoculated into 12 well plates at a concentration of 5x10 ⁴ cells / well, and samples were added to each well and cultured in a CO ₂ incubator for 48 hours. The cultures of the cells thus obtained were collected and used for the experiments. The degree of collagen biosynthesis in the cell culture medium was measured by using procollagen type-Ⅰ C peptide (PIP) EIA kit.

ii) Measurement of MMP-1 & TIMP-1 active kit

Cells were inoculated into 12 well plates at a concentration of 5x10 ⁴ cells / well, and samples were added to each well and cultured in a CO ₂ incubator for 48 hours. At this time, TNF-α was added at a concentration of 10 ng / mL to increase the activity of MMP-1 and TIMP-1. Cell cultures were collected and MMP-1 was measured using Amersham matrix metallproteinase-1, Human, Biotrak ELISA system (GE Healthcare).

B) Expression of intracellular wrinkle-related protein by Western blot

The amounts of MMP-1, MMP-2, and ProCOL1A2 proteins were measured using Western blot. After culturing in a culture dish using CCD-986sk, the cells were sampled and washed for 48 hours with PBS. Cells were collected by centrifugation and cell lysate was prepared using RIPA buffer. The amount of protein contained in the cell lysate was measured by the Bradford method. 20 μg of protein was separated by SDS-PAGE containing 10% polyacrylamide and electroblotted with PVDF membrane at 120 V for 1 hour. The PVDF membrane is then left in 5% skim milk for 1 hour and then overnight at 4 ° C in primary antibody. After washing three times with TBST, secondary antibody was reacted at room temperature for 1 hour, washed three times with TBST, and exposed to X-ray film by reaction with ECL kit (Amersham-Pharmacia). The developed bands on the X-ray film were quantified using a LAS4000 image analyzer.

Example 11: Pinus koraiensis Siebold meat Zucc ) From the seed coat (testa) phenol Extraction of sex material

In order to harvest the pine seedlings from the pine nuts harvested from the main pine trees cultivated in Gapyeong, Gyeonggi province, the testa, which is abandoned and abandoned, is extracted with the extraction solvent. In order to more efficiently extract the effective components of the sample, water and ethanol were selected as a non-harmful solvent. The physiologically active components eluted by ethanol concentration were measured by using phenolic compound as a main target Respectively. The results are shown in Fig.

As shown in FIG. 1, when 50% ethanol was used as a solvent, the elution of the phenolic compound was the highest. In the concentration range of 40 to 50%, the dissolution rate was similar. As the concentration of the extraction solvent increased, It was confirmed that the elution yield was lowered.

The elution amount of the phenolic compound under the optimum conditions was confirmed, and the results are shown in Table 1 below. As shown in Table 1, the content of water extract was 4.92 ± 0.08 mg / mL and that of 50% ethanol extract was 5.89 ± 0.06 mg / mL.

Sample Phenolic content (mg / g) Water extracts 50% ethanol extracts Pinus koraiensis Siebold meat Zucc .
testa 4.92 ± 0.08 5.89 ± 0.06

Example  12. Outer Pine Patch  Antioxidant activity of extract

(One) Outer Pine Patch  Extract DPPH Scatters  Measure

The DPPH method, which is an analysis of reducing materials using the oxidative free radical reaction of exocrine papillae and ethanol extracts, was carried out and the results are shown in FIG.

As shown in FIG. 2, the highest DPPH elimination ability was obtained in the 50% ethanol extract of the phenolic compound, and the ethanol extract and water extract were excellent in the effect.

The change of DPPH scavenging ability according to the phenolic concentration was examined, and the results are shown in FIG.

As shown in FIG. 3, DPPH elimination was found to increase in a concentration dependent manner as the amount of phenolic added was increased. In the case of the concentration of about 150 μg / mL, 87.25% of water extract and 97.10% It was confirmed that the extract of P. japonica had very good antioxidant ability against water soluble substances. In addition, ethanol extract showed better antioxidant ability than BHT, which is a control antioxidant, and could be used as a material for anti - aging cosmetics.

(2) Outer Pine Patch  Extract ABTS radical cation decolorization  Measure

Relative antioxidant activities of extracts were measured by ABTS radical cation decolorization, which can measure both hydrogen-donating antioxidants and chain breacking antioxidants, and is applicable to both aqueous and organic phases. This is based on the principle that ABTS ⁺ free radicals generated by the reaction with potassium persulfate are removed by antioxidants in the extract so that relative comparison of the extracts is possible by the use of standard substances, and the radical specific cyan color is discolored.

The experimental results are shown in Fig.

As shown in FIG. 4, 29.93% inhibitory activity was shown in the case of hot water extract and ethanol extract showed a maximum inhibitory effect in 50% ethanol extract at 42.15%.

The antioxidant activity was examined according to the content of the phenolic compound. The results are shown in FIG.

As shown in FIG. 5, the higher the concentration of the phenolics added, the higher the ABTS inhibitory effect was, and the higher the antioxidative activity than the control, BHT, 50-200 μg / At low concentrations of μg / mL phenolics, water extract and ethanol extract showed 92.13% and 90.04%, respectively. Therefore, it was confirmed that the extract of P. japonicus extract showed antioxidant activity against water - soluble materials at low concentration and that the concentration of the additive increased the concentration - dependent antioxidant ability.

(3) Outer Pine Patch  Extract antioxidant protection factor  ( PF ) Measure

β-carotene was added to the linoleic acid emulsion to determine the antioxidant activity of lipase-exudate extracts of Pine nuts.

As shown in FIG. 6, the ethanol extract showed an antioxidant power of about 1.1 to 1.2 PF in the whole range according to the concentration, while the water extract showed about 1.1 PF.

The antioxidant activity was examined according to the content of the phenolic compound added. The results are shown in FIG.

As shown in FIG. 7, it was confirmed that the concentration of phenolics added increases the concentration-dependent PF. The water extracts and alcohol extracts showed a higher PF of 1.15-1.76 PF and 1.09-1.52 PF, respectively, than the control BHT (0.88-1.46 PF) at 50 ~ 200 μg / mL phenolics. The antioxidant activity Was very high.

(4) Outer Pine Patch  Extract TBARS  Measure

In order to measure the antioxidative activity against lipid-soluble substances, TBARS of the extract of P. japonicus was measured and compared. The results are shown in FIG.

As shown in FIG. 8, inhibition of water extract and 50% ethanol extract was confirmed to be 41.59% and 66.94%, respectively.

The antioxidant activity was confirmed by the content of the phenolic compound added, and the results are shown in FIG.

As shown in FIG. 9, the TBARS inhibition was increased in a concentration-dependent manner as the concentration of the added phenolics was increased, and the antioxidant activity was 90% or more even at a low concentration of 50 μg / mL of phenolics. Compared with the control, BHT, both water extract and ethanol extract showed better antioxidative activity than the control BHT in the concentration range of 50 ~ 200 μg / mL phenolics.

Example  13. Outer Pine Patch  Convergence effect of extract

The principle of convergence is the phenomenon that the skin is contracted by binding with the polymer flavonoids of the skin protein to form crosslinks. Convergence means basically wrinkled or funny. Therefore, astringent agent has a function of contraction of skin and mucosal blood vessels, and has an effect of inhibiting secretion of mucus by blocking cell gap and lymphatic gap. In addition, since astringent agent has a property of binding to protein, it can generally judge the degree of astringent effect according to the degree of binding of hemoglobin protein to the extract. Such convergent action has the effect of forming an insoluble film on the surface of skin and mucous membranes by external use or contents and consequently protecting the structure or densifying the tissue to reduce the permeability of the cell membrane.

Fig. 10 shows the result of measuring the concentration of the extract of P. japonica by controlling the concentration of 50 ~ 200 μg / mL phenolics.

As shown in FIG. 10, the ethanol extract had a convergence effect of 66.3%, which was similar to 62.4% of the control tannic acid when added at a concentration of 200 μg / mL phenolics. And it can be used as a cosmetic material for reducing pores.

Example  14. Outer Pine Patch  The result of whitening effect measurement of extract

(One) Mushroom  Derived tyrosinase  Confirm inhibitory activity

Melanin, a key determinant of skin tone, is biosynthesized in melanosomes in pigmented cells called melanocytes in the epidermal basal layer. The starting material for the synthesis of melanin is tyrosine, an amino acid. Tyrosine is oxidized by tyrosinase in melanocytes to L-3,4-dihydroxyl phenylalanine (DOPA) and DOPA quinone. It is known that DOPAquinone then becomes DOPA chrome, 5,6-dihydroxyindole, and indole-5,6-quinone, and then melanin by polymerization to indole-5,6-quinone. Tyrosinase plays an important role in the formation of melanin in the skin, tyrosine hydroxylase which oxidizes tyrosine in the melanosome to form DOPA, which acts as a DOPA oxidase which oxidizes DOPA to form DOPA quinone and is an important enzyme for synthesizing melanin polymer .

The skin color of a person is determined by the pigment content such as melanin or carotene, the distribution of blood vessels, the thickness of the stratum corneum, etc. This is because melanin is the most important factor for determining skin color, Excessive synthesis or anomalies in melanin production may lead to melasma, freckle and hyper-pigmentation. Therefore, by inhibiting tyrosinase, which degrades tyrosine, a substrate of melanin synthesis, it inhibits the synthesis of melanin, and thus the whitening effect of the skin can be seen. Recently, natural and synthetic whitening agents such as vitamin C, kojic acid, arbutin, and hydroquinone have been developed and applied to whitening cosmetics for the tyrosinase inhibition effect, which is closely related to antioxidant activity.

In this study, tyrosinase inhibition test was carried out by adjusting the concentrations of extracts of P. japonica exotrophs to 50, 100, 150, and 200 μg / mL. The results are shown in FIG.

As shown in FIG. 11, relatively high inhibitory effect of 26.4 ~ 49.9% in the extract of P. japonicus and relatively high tyrosinase inhibitory effect of 21.5 ~ 47.5% in extract of 50% ethanol similar to that of water extract. The higher concentration of phenolic compounds added to water extracts and ethanol extracts increased the whitening effect. At low concentrations of 100 μg / mL, whitening was slightly superior to that of kojic acid, which is currently applied to commercial whitening cosmetics. The inhibitory effect of tyrosinase was similar to that of kojic acid at 150 μg / mL and above.

(2) Melanoma cell  (B16F10) survival rate

MTT detection method is widely used as a cytotoxicity and cell proliferation detection method because many samples can be easily read using an ELISA reader using a 96-well plate, and the dehydrogenase activity of mitochondria in cell metabolism To reduce the yellow water-soluble MTT tetrazolium to purple colored water-insoluble MTT formazan.

The survival rate of melanoma cells by the extract of P. japonica was treated by MTT assay at a concentration of 1, 5, 10, 25 μg / mL in B16F10 cells. The results are shown in FIG.

As shown in FIG. 12, cytotoxicities of 46% and 37% were obtained at 25 μg / mL of water and alcohol extracts of pine nut exocytosis, respectively, and cell survival rates were close to 90% at 1, 5 and 10 μg / mL Respectively.

(3) Melanoma cell (B16F10) tyrosinase  Confirm inhibitory activity

Tyrosinase is an enzyme that is most important for melanin production in tyrosine when skin is exposed to ultraviolet light. By inhibiting tyrosinase activity, it can inhibit melanin production, thereby preventing the occurrence of spots, freckles, and erythema .

Tyrosinase is an enzyme that binds to Cu ^{2 +} and is a polyphenol oxidase widely distributed in plants, microorganisms, and humans. It is an enzyme that represents a major regulatory step in melanin synthesis, such as limiting the rate of melanin synthesis.

Fig. 13 shows the results of measuring the amount of L-DOPA produced by adding the extract of P. japonica extracellularis at concentrations of 1, 5 and 10 μg / mL. As shown in FIG. 13, inhibition rates of 12% and 11% at 10 μg / mL concentration of water extract of Pine nuts and alcohol extract were respectively shown in FIG. 12.

(4) Melanoma cell  (B16F10) melanin  Confirmation of biosynthesis inhibition

Melanin, which is the most important factor in determining the skin color of a person, not only suppresses skin photoaging and sunburn keratosis but also causes partial hyperpigmentation of spots, freckles, and black spots. Melanin is transmitted to the surrounding keratinocyte through the dendrites of the melanocyte of the epidermal basal layer and migrates to the stratum corneum of the skin. Skin blackening is due to the fact that melanocytes present in the skin increase the production of melanin in response to external conditions such as UV exposure. In order to confirm the whitening effect of raw materials and substances, the inhibition of enzyme activity of tyrosinase, which is a melanin-producing enzyme, has been mainly used. However, in recent years, in addition to suppression of enzyme activity, whitening agents have been shown to increase tyrosinase and related enzyme expression in melanocyte Which is known to be involved in the development of neurodegenerative diseases.

In order to measure the content of melanin in B16F10 cells, the water extract of P. japonica and the alcohol extract were treated at the concentrations of 1, 5, and 10 μg / mL, and the results are shown in FIG.

As shown in FIG. 14, it was confirmed that the melanin biosynthesis was inhibited as compared with the control group as the treatment concentration of the extract increased. In addition, although the inhibitory effect is not high even at a low concentration of 1 and 5 μg / mL, inhibition of biosynthesis of melanin is confirmed, and if the purity of the extract is increased, it can be industrialized as a functional cosmetic material utilizing a whitening effect .

(5) Outer Pine Patch  Extract melanin  Involved in synthesis tyrosinase  Expression inhibitory effect

The extracts of P. japonica excelled in pro-collagen, confirming the expression of wrinkle-related proteins and mRNA. To investigate the effect of tyrosinase, an enzyme involved in the synthesis of melanin, tyrosinase protein expression was determined by western blotting after 48 h of treatment with B10F10 mouse melanoma cells at a concentration of 1 to 10 μg / mL. Β-actin, a house keeping gene, was used as a positive control with little difference in the expression level of the cells under various conditions.

The results are shown in Fig.

As shown in FIG. 15, the protein expression of each transcription factor was increased in the B16F10 group not treated with the extract, but the expression of tyrosinase protein was decreased in the B16F10 group treated with the extracellular extract of pine nuts, At the concentration of μg / mL, the water and alcohol extract showed 30% or more inhibitory effect. Therefore, it was concluded that the extract of P. japonica extract decreased the expression of mushroom tyrosinase in a dose - dependent manner. Thus, it was confirmed that the extract of P. japonicum excellently inhibited the production of tyrosinase by inhibiting the expression of tyrosinase.

(6) Melanin  Involved in synthesis microphthalmia - associated transcription factor  (MITF) expression inhibitory effect

A wide variety of signaling molecules are involved in the melanin biosynthetic signaling system. Melanin is synthesized through several intracellular signal transduction mechanisms. Among them, cAMP (cyclic adenosine monophosphate) / PKA (protein kinase A) pathway is the main pathway for melanin synthesis. And activates PKA, a downstream signaling substance, and increases the expression of MITF (microphthalmia -associated transcription factor) via CREB (cAMP response element binding protein). MITF promotes the transcription of tyrosinase as an important transcription factor in melanin synthesis.

To examine the effect of MITF on the expression of melanin for the whitening effect of the extract of P. japonicus, the B16F10 mouse melanoma cells were treated with a concentration of 1 ~ 10 μg / mL for each concentration, and the expression of MITF protein was western blotted Respectively. Β-actin, a house keeping gene, was used as a positive control with little difference in the expression level of the cells under various conditions. The results are shown in Fig.

As shown in Fig. 16, the protein expression of each transcription factor was increased in the B16F10 group without the extract treatment, but the expression of MITF protein was decreased in the B16F10 group treated with the extract of Porphyrae japonica extract in a concentration-dependent manner, mL, water extracts showed about 40% inhibition and alcohol extracts showed about 30% inhibition. Water extracts and alcohol extracts were effective in inhibiting the expression of MITF, a transcription factor. Thus, the extract of P. japonica extract showed a concentration - dependent decrease in MITF expression, indicating that the extract of P. japonica was effective in inhibiting melanogenesis. It was confirmed that the protein expression of tyrosinase was decreased by suppression of MITF expression in a study on the whitening effect of pine nut extract.

Example  15. Outer Pine Patch  Wrinkle improvement effect measurement result of extract

(One) Elastase  Confirm inhibitory activity

Skin is a representative form of aging, such as wrinkles increase, elasticity decrease, pigmentation, etc. The most typical phenomenon of aging is wrinkles. The elastase present in the neutrophil granules of the human body is an enzyme that decomposes elastin, which is the main substrate protein for maintaining skin elasticity in the dermis. As the elastase acts, it decomposes elastin in the skin tissue, thereby losing elasticity and freshness of the skin. It causes roughness and promotes aging of the skin. Thus, inhibition of elastase activity may delay skin aging.

Elastase is a nonspecific hydrolytic enzyme capable of degrading collagen, another important substrate protein. Elastase inhibitors improve skin wrinkles. Ursolic acid is currently used as an elastase inhibitor. In addition, Cannell et al. Is one of the leucocyte granulase enzymes that degrade elastin in the body. In the abnormal tissues, the activity of the enzymes is extremely high, which is a direct cause of tissue destruction, resulting in wrinkles and loss of elasticity. Therefore, the effect of improving wrinkles can be expected by measuring the elastase inhibitory activity. Elastase activity is measured by measuring the change in color caused by the decomposition of nitroanilide (NA) using the substrate N-succinyl- (Ala) -3-p-nitroanilide using absorbance. It has the disadvantage of measuring the activity using the most widely used method or synthesized substrate instead of bioelastic.

Elastase inhibitors include ursolic acid is used as an elastase inhibitor. ursolic acid is difficult to formulate because it has insoluble properties in a solvent such as water or oil, so that it is generally difficult to use.

The elastase inhibitory activity was measured by adjusting the concentration of the extrudate extract of Pine nuts to 50-200 μg / mL phenolics. The results are shown in FIG.

As shown in FIG. 17, it was confirmed that the inhibitory activity against elastase was increased in a concentration-dependent manner as the concentration of 50 μg / mL phenolis was increased from 200 μg / mL to 50 μg / mL of phenolics The inhibitory power of 28.47% was confirmed.

(2) Collagenase  Inhibitory activity

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

The concentration of the extract of P. japonica was adjusted to 50 ~ 200 μg / mL phenolics to measure collagenase inhibitory activity. The results are shown in FIG.

As shown in Fig. 18, the collagenase inhibitory effect was 3.04 ~ 7.39% in the extract of P. japonicus extract and 3.53 ~ 51.69% in the ethanol extract, and the inhibitory effect of the alcohol extract on collagenase Respectively. These results suggest that the extracts of P. japonica inhibit collagenase activity in the skin and inhibit degradation of collagen, so that it can be used as a functional cosmetic material to improve wrinkles.

(3) Fibroblast cell  ( CCD -986 sk ) Survival rate confirmation

1) MTT measurement results on CCD986sk fibroblst cells

MTT detection method is widely used as a cytotoxicity and cell proliferation detection method because many samples can be easily read using an ELISA reader using a 96-well plate, and the dehydrogenase activity of mitochondria in cell metabolism To reduce the yellow water-soluble MTT tetrazolium to purple colored water-insoluble MTT formazan.

In order to measure the cell survival rate of the extract of P. japonicus, the concentration of 1, 5, 10, 25 μg / mL was applied to CCD-986sk cells, and the result is shown in FIG.

As shown in FIG. 19, both the water extract and the alcohol extract showed cytotoxicity of about 25% at a concentration of 25 μg / mL, but the survival rate was high at the concentrations of 1, 5, and 10 μg / mL.

(4) ELISA kit Using fibroblast  ( CCD -986 sk ) In procollagen type  I Biosynthesis kit Measurement result

Most of the collagen is present in the dermis of the skin and accounts for about 70 to 80% of the dry weight of the skin. It plays a role of supporting the skin occupying most of the extracellular matrix. However, degradation of collagen is accelerated by extrinsic factors such as decrease of collagen biosynthesis due to internal factors such as decrease of cell activity due to natural aging, increase of stress due to various harmful environments, and increase of active oxygen species by sunlight And the skin substrate is destroyed to form wrinkles. Collagen also plays an important role in healing wounds and can quickly restore scars without scarring, promoting the synthesis of collagen in damaged epithelium. The most prominent examples of collagen synthesis promoting substances that have been found so far include asiaticoside, asiatic acid, and madecasic acid, which are the major components of centella asiatica.

The results of measurement of the pro-collagen synthesis at concentrations of 1, 5, and 10 μg / mL of extraneous pine nut and alcohol extracts in CCD-986sk cells are shown in FIG.

As shown in FIG. 20, the extracts showed pro-collagen biosynthesis in a concentration dependent manner at a concentration of 1 to 10 μg / mL. In particular, the biosynthesis rate was 50% and 62% at a concentration of 10 μg / (EGCG), but the pro-collagen biosynthesis was relatively superior to epigallocatechin-3-O-gallate (EGCG).

(5) Western blotting 분석 Using fibroblast  ( CCD -986 sk ) In procollagenase protein  Expression inhibitory effect

The wrinkles of the skin are formed by collagen biosynthesis being reduced, collagen degradation accelerated by various extrinsic factors such as harmful environment, and destruction of the skin substrate. The biosynthesis of collagen plays an important role in wound healing and promotes the synthesis of collagen in the damaged epithelium, thus promptly restoring the wound.

Therefore, we investigated the effect of the extract of P. japonica on the expression of the enzyme protein of procollagenase, an enzyme involved in biosynthesis of collagen, by Western blotting the expression of procollagenase protein in CCD-986sk cells. GAPDH, a house keeping gene, was used as a positive control with little difference in the expression level even in various cell conditions. The results are shown in Fig.

As shown in Fig. 21, in the control group irradiated with UV-B, it was confirmed that the degree of procollagenase expression was remarkably decreased compared to the normal group. In the CCD-986sk cell line treated with pine nut extract, the protein expression of each transcription factor was increased in a concentration-dependent manner and the water extract had little effect. However, at 10 μg / And the expression level of the protein was restored to the expression level. Therefore, it was confirmed that the alcohol extract of P. japonica exerts an effect of promoting the synthesis of collagen by activating the damaged procollagenase, improving the wrinkles generated, and promoting the recovery from damaged epithelium.

(6) Fiber  cell( CCD -986 sk ) Wrinkle-related protein ( MMP -1) and mRNA  Check the expression level

Collagen is a major constituent protein that accounts for 90% of skin dry weight. Therefore, degradation of collagen directly affects the elasticity of the connective tissue and wrinkle formation. In addition, the factor most affected by MAPK, which is an aging-related factor, is c-fos, which is affected by p38. Activation of these factors strongly regulates the expression of MMPs. Among the several MMPs produced in the body, MMP-1 is a protease specifically acting on collagen. It is known that inhibition of MMP-1 activity reduces collagen degradation, maintains elasticity of skin tissue and prevents wrinkle formation .

The results of measuring the activity of MMP-1 in CCD-986sk cells are shown in Fig.

As shown in FIG. 22, it was confirmed that both the extrudate and the alcohol extract showed inhibition rates in a concentration-dependent manner. In addition, it was confirmed that the alcohol extracts showed more excellent inhibitory effect on the expression of MMP-1 than water extracts.

(7) Western blotting 분석 Using fibroblast  ( CCD -986 sk ) In intestinal  collagenase ( MMP -1) and 92 kD gelatinase  ( MMP -9) of protein  Expression inhibitory effect

Reconstruction of extracellular matrix proteins in the dermis involves a variety of biological reactions, and these substrate proteins play an important role in maintaining the structure and resilience of the skin. (MMP-1), 72 kD gelatinase (MMP-2), 92 kD gelatinase (MMP-9), neutrophil collagenase (MMP-8) and collagenase (MMP-9) 13) and more than 26 MMPs are already known. In skin exposed to excessive ultraviolet rays, the expression of MMPs is increased, and the increased MMPs break down the collagen that constitutes the skin, resulting in the deficiency of the substrate protein, thereby causing wrinkles along with aging of the skin.

Inhibition of MMP-1 activity as a protease specifically acting on collagen, which accounts for 90% of skin connective tissues, can reduce collagen degradation, maintain skin elasticity and prevent wrinkle formation. In addition, MMP-9, a 92 kD gelatinase in the same order as 72 kD gelatinase (MMP-2) among enzymes, induces the activity of gelatinase, which causes decomposition of gelatin existing as an intercellular material of the dermal layer. Is an important factor. Therefore, inhibiting the activity of MMP-9 in combination with MMP-1 has been shown to reduce collagen degradation, maintain skin elasticity and prevent wrinkle formation. Therefore, the expression of MMP-1 and MMP-9 protein in CCD-986sk cells was confirmed by western blotting in order to investigate the effect of the extract of P. japonica on MMP-1 and MMP-9 protein expression. Β-actin, a house keeping gene, was used as a positive control with little difference in the expression level of the cells under various conditions. The results are shown in Fig.

As shown in FIG. 23, it was confirmed that the expression level of MMP-1 was markedly increased in the control group irradiated with UV-B compared with the normal group. In the CCD-986sk cell line treated with the extract of Porphyra japonica, the protein expression level of each transcription factor was decreased depending on the concentration of the extract added. The alcohol extract was more effective than the water extract and the concentration of alcohol extract was 10 μg / mL , It was confirmed that the inhibition rate is better than that of EGCG which is currently used in functional cosmetics in the market. Therefore, it is possible to confirm that the extract of exocytotic papillae inhibits the expression of MMP-1 protein and consequently inhibits collagenase, thereby exhibiting an effect of improving wrinkles and promoting the recovery of damaged epithelium there was.

In the case of MMP-9, it was also confirmed that the expression level of protein in the control group was significantly increased as compared with that of the normal group.

In the CCD-986sk cell group treated with the extract of P. japonica, the expression level of the protein expressed by each transcription factor was decreased in a concentration-dependent manner. As shown in Fig. 24, the expression level of 10 μg / The inhibitory effect was similar to that of EGCG used in functional cosmetics. The alcohol extract showed better inhibitory effect than EGCG, which is a control. Therefore, it was confirmed that the extract of P. japonica exerts an effect of suppressing the expression of MMP-9 protein and consequently improving wrinkles.

(8) TIMP -1 activity kit  Measurement result

MMP activation can be specifically inhibited by tissue inhibitors of metallo-proteinase as well as non-specific inhibitors such as α2-macroglobulin and RECK. TIMPs are specific inhibitors that specifically bind 1: 1 in stoichiometry. TIMP-1 is an important inhibitor of MMP-1 in tissues. In order to measure the activity of TIMP-1, CCD-986sk cells were irradiated with UV-B, and the extracts were treated with 1, 5, and 10 μg / mL concentrations and cultured for 48 hours. The cell culture was taken and the activity of TIMP-1 was measured. The results are shown in FIG.

As shown in FIG. 25, at a treatment concentration of 10 μg / mL, a relatively high expression rate of 74% in the extract of Pine nuts and 75% in the alcohol extract was confirmed. These results indicate that TIMP-1 activity is restored to normal by the treatment of exocrine pine nut extract, so that the activity of MMP-1 is inhibited to reduce collagen degradation, maintain skin elasticity and prevent wrinkle formation Respectively.

Formulation example  One. Cosmetics  Manufacture of pharmaceutical preparations

Flexible longevity manufacturing

0.1% by weight of the extrudate extract of pine nut, 5.2% by weight of 1,3-butylene glycol, 1.5% by weight of oleyl alcohol, 3.2% by weight of ethanol, 3.2% by weight of polysorbate 20, 2.0% 1.0% by weight of a carboxyl vinyl polymer, 3.5% by weight of glycerin, a small amount of fragrance, a small amount of preservative, and a remaining amount of purified water were mixed to prepare a softening water by a conventional method.

Milk lotion  Produce

0.1% by weight of the extrudate extract of pine nut, 5.1% by weight of glycerin, 4.2% by weight of propylene glycol, 3.0% by weight of tocopheryl acetate, 4.6% by weight of liquid paraffin, 1.0% by weight of triethanolamine, 3.1% by weight of squalane, , 1.6% by weight of polysorbate 60, 1.6% by weight of sorbitan sesquioleate, 0.6% by weight of propylparaben, 1.5% by weight of carboxyl vinyl polymer, a small amount of fragrance, a small amount of preservative, Milk lotion was prepared by the method.

Nutrition cream manufacturing

Example 2 was conducted in the same manner as in Example 2 except that 0.5% by weight of the extrudate extract of pine nut, 4.0% by weight of glycerin, 3.5% by weight of petroleum jelly, 2.1% by weight of triethanolamine, 5.3% by weight of liquid paraffin, 3.0% by weight squalane, 2.6% by weight of beeswax, 3.2% by weight of Polysorbate 60, 1.0% by weight of carboxyl vinyl polymer, 3.1% by weight of sorbitan sesquioleate, a small amount of fragrance, a small amount of preservative and remaining amount of purified water were mixed to prepare a nutritional cream by a conventional method.

Massage cream manufacturing

Example 2 was conducted in the same manner as in Example 2 except that the pine nut extract of Example 2 was used in an amount of 0.5 wt%, glycerin 4.0 wt%, vaseline 3.5 wt%, triethanolamine 0.5 wt%, liquid paraffin 24.0 wt%, squalane 3.0 wt%, wax 2.1 wt%, tocopheryl acetate 0.1 wt% 2.4% by weight of Polysorbate 60, 1.0% by weight of carboxyl vinyl polymer, 2.3% by weight of sorbitan sesquioleate, a small amount of fragrance, a small amount of preservative and remaining amount of purified water were mixed to prepare a massage cream by a conventional method.

Formulation example  2. Manufacture of food preparation

Health food manufacturing

100 mg of pine nutritional extract of Example 2, a proper amount of vitamin A, 70 g of vitamin A acetate, 1.0 mg of vitamin E, 0.13 mg of vitamin B1, 0.15 mg of vitamin B2, 0.5 mg of vitamin B6, 0.2 g of vitamin B12, 10 mg of niacinamide, 1.7 mg of nicotinic acid amide, 50 g of folic acid, 0.5 mg of calcium pantothenate, 1.75 mg of ferrous sulfate, 0.82 mg of zinc oxide, 25.3 mg of magnesium carbonate, 15 mg of potassium phosphate monobasic, 90 mg of potassium citrate, 100 mg of calcium carbonate and 24.8 mg of magnesium chloride were mixed and granules were prepared and a health food was prepared according to a conventional method. At this time, although the composition ratio of the vitamin and mineral mixture is relatively mixed with the ingredient suitable for health food, it may be arbitrarily modified.

Health drink  Produce

100 mg of vitamin C, 100 g of vitamin E (powder), 19.75 g of iron lactate, 3.5 g of zinc oxide, 3.5 g of nicotinic acid amide, 0.2 g of vitamin A, 0.2 g of vitamin A B1, 0.3 g of vitamin B2 and a predetermined amount of water were mixed and heated at 85 DEG C for about 1 hour with stirring. The resulting solution was filtered and sterilized in a sterilized 2 L vessel, . At this time, although the composition ratio of the ingredients suitable for the beverage is comparatively mixed, the mixture ratio may be arbitrarily varied according to the demand, the demanded country, the intended use, and the regional or national preference.

Formulation example  3. Manufacture of pharmaceutical preparations

Powder  Produce

20 mg of the crude extract of Pine nuts, 100 mg of lactose and 10 mg of talt were mixed and filled in an airtight container to prepare powders by a conventional method.

Tablet manufacture

10 mg of pine nut extract of Example 2, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate were mixed and tableted by a conventional method.

Capsule  Produce

10 mg of pine nut extract of Example 2, 3 mg of crystalline cellulose, 14.8 mg of lactose and 0.2 mg of magnesium stearate were mixed and filled in gelatin capsules by a conventional method to prepare capsules.

Injection manufacturing

(2 mL) per ampoule: 10 mg of the crude extract of Example 2, 180 mg of mannitol, 2,974 mg of sterilized distilled water for injection, and Na ₂ HPO ₄ ? 2H ₂ O (26 mg).

Liquid  Produce

To the purified water was added 20 mg of the pine nut extract of Example 2, 10 g of isomerized sugar and 5 g of mannitol and dissolved, and the lemon flavor was added in an appropriate amount, and the above components were mixed. Then, purified water was further added thereto, adjusted to a total volume of 100 mL, filled in a brown bottle, and sterilized to prepare a liquid preparation by a conventional method.

Claims (9)

A cosmetic composition for whitening and wrinkle improvement, which contains an extract of Pine Pine Zoo as an active ingredient. [Claim 2] The cosmetic composition according to claim 1, wherein the extrudate of the pine nut is an outer skin of mature pine nut. [Claim 2] The cosmetic composition according to claim 1, wherein the extract of Pine Pine Zoo is extracted with water, C1 to C4 lower alcohol or a mixed solvent thereof. delete A skin external preparation for whitening and wrinkle improvement, comprising an extract of Pine Pine Nectarifera as an active ingredient. [Claim 7] The external preparation for skin according to claim 5, wherein the external preparation for skin is selected from the group consisting of a soft lotion, a nutritional lotion, a gel, a cream, an essence, an emulsion, a pack, a cleanser and a powder for bath. A composition for preventing whitening and antiaging of aging comprising an extract of P. japonica as an active ingredient. delete delete

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