KR20180036036A - Cosmetic composition comprising the extract of cacao nibs irradiated with far-infrared ray and steamed - Google Patents

Abstract

The present invention relates to a cosmetic composition comprising an extract of cacao nibs irradiated with far-infrared ray and steamed as an active ingredient, wherein the extract of cacao nibs irradiated with far-infrared ray and steamed has excellent skin antioxidant effect, inhibitory effect on MMP-1 production, inhibitory effect on UV-induced MMP-1 production, effect on collagen synthesis, inhibitory effect on melanin production, effect of mitigating cytotoxicity by ultraviolet irradiation, inhibitory effect on the expression of inflammatory cytokines by ultraviolet irradiation, an effect of reducing skin wrinkles, an effect of improving skin elasticity, and a skin moisturizing effect.

Description

[0001] The present invention relates to a cosmetic composition comprising, as an active ingredient, an extract of steamed cacao nips after irradiation with far-infrared rays, and a cosmetic composition comprising an extract of cacao nibs irradiated with far-

The present invention relates to a cosmetic composition containing steamed cacao nips extract as an effective ingredient after far-infrared ray irradiation treatment, and more particularly to a cosmetic composition containing steamed cacao nips extract after far-infrared ray irradiation treatment to provide excellent antioxidation, Improving skin elasticity, whitening skin, suppressing skin damage due to ultraviolet rays, alleviating skin irritation, and moisturizing the skin.

The function of cosmetics has been extended through cleanliness and simple makeup to eliminate or inhibit the causes of skin aging such as pollutants, stress, ultraviolet rays, active oxygen, peroxides, and the like. Due to recent industrialization and social activities, the skin is exposed to various environmental pollution and stress, and becomes rough and dry. The aging of the skin such as dull appearance is accelerated, and this phenomenon is closely related to the moisture supply and maintenance of the skin.

In recent years, there has been actively developed cosmetics that prevent skin aging by improving skin wrinkles and relieving skin irritation and inflammation using safe natural materials. Cacao nibs will ferment, dry and roast cacao seeds, the seeds of cacao fruit, then remove the shells and crush them. Cacao is known as the world's three antioxidant foods with Aronia and turmeric. It contains a large amount of polyphenols and catechins, antioxidants that inhibit free radicals that promote cell senescence. These cacao necrosis cacao nibs have polyphenol content of 3.6 times higher than green tea and catechin content of 60 times higher than green tea, which maintains and stabilizes the active oxygen concentration by antioxidant effect, thus anticancer effect is expected.

The present inventors have made efforts to develop cacao jinx, which is a natural material, as a cosmetic, and have found that when cacao nips is steamed and then steamed and subjected to radical abrasion treatment under specific conditions, it has excellent antioxidative activity, improved skin wrinkles and elasticity, , Suppression of skin damage by ultraviolet rays, alleviation of skin irritation and skin moisturizing activity, thus completing the present invention.

(0001) Korean Patent Publication 10-2010-0010433 (2010.02.01.) (0002) Korean Patent No. 10-1009957 (Jan. 20, 2011)

The present invention relates to an antioxidant, an anti-oxidant, a skin anti-wrinkle, a skin wrinkle, a skin elasticity improvement, a skin whitening, a skin damaging by ultraviolet ray, a skin irritation easing and a skin moisturizing effect by containing a cacao nips extract steam- And a cosmetic composition containing the same.

It is another object of the present invention to provide an antioxidant composition which is excellent in skin antioxidant activity, inhibitory effect on MMP-1 production, inhibitory effect on MMP-1 production by ultraviolet ray induction, collagen synthesis promotion effect, melanin production inhibitory effect, cytotoxic relaxation effect by ultraviolet irradiation, Another object of the present invention is to provide a method for producing cacao nips extract, which exhibits an effect of inhibiting the expression of inflammatory cytokines, the effect of improving skin wrinkles and the effect of improving skin elasticity.

According to the present invention, there is provided a cosmetic composition comprising, as an active ingredient, an extract of Cacao nibs steamed after far-infrared ray irradiation treatment. The far-infrared ray irradiation treatment is a treatment of far-infrared rays having a wavelength of 5 to 20 μm at 90 to 130 ° C. for 30 to 120 minutes, more preferably far-infrared rays having a wavelength of 5 to 20 μm at a temperature of 110 to 130 ° C. for 60 to 120 minutes . Steaming with steam is carried out for 3 to 12 hours with steam at 110 to 130 ° C.

The steamed cacao nibs extract after the far-infrared ray irradiation treatment is contained in an amount of 0.001 to 98.0% by weight based on the weight of the entire cosmetic composition.

At this time, the Cacao nibs extract steam-roasted after the far-infrared ray irradiation treatment is water, anhydrous or hydrous lower alcohol (for example, methanol, ethanol, propanol and butanol) having 1-4 carbon atoms, propylene glycol, Is extracted with at least one solvent selected from the group consisting of glycerin, acetone, ethyl acetate, chloroform, butyl acetate, diethyl ether, dichloromethane and hexane. Alternatively, the extract is an extract obtained by supercritical extraction or ultrasonic extraction. Or the steamed cacao nymph extract after the far-infrared ray irradiation treatment is a fermented extract fermented by at least one fermenting bacteria selected from the group consisting of yeast, lactic acid bacteria, and Bacillus subtilis.

The cosmetic composition can be used for antioxidation, for preventing skin aging, for improving skin wrinkles and improving skin elasticity, for whitening skin, for suppressing skin damage due to ultraviolet rays, for alleviating skin irritation or for skin moisturizing.

The cosmetic composition may be at least one selected from the group consisting of a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nutrition lotion, a massage cream, a nutritive cream, a moisturizing cream, a hand cream, A lotion, a cleansing cream, a body lotion, a body cleanser, an emulsion, a press powder, a loose powder, and an eye shadow.

According to another aspect of the present invention, there is provided a method for manufacturing a cacao nib, comprising the steps of: A) treating Cacao nibs with far infrared rays having a wavelength of 5 to 20 μm at 90 to 130 ° C for 30 to 120 minutes; B) treating the far-infrared-treated cacao nips with steam at 110 to 130 ° C for 3 to 12 hours; And C) extracting steamed cacao nips. The present invention also provides a method for producing cacao nips extract.

The step c) comprises: c1) drying and pulverizing the steamed cacao nips; And c2) leaching at a temperature of 1 to 24 hours at room temperature by adding 5 to 30 times the weight of 70% ethanol.

In addition, the step c) comprises: c1) drying the steamed cacao nips by pulverizing the powder into a size of 200 to 300 mesh; c2) fermenting the cells in a culture medium of Saccharomyces cerevisiae, Lactobacillus plantarum or Bacillus subtilis culture under the conditions of 20 to 50 and pH 3 to 9; And c3) filtration after removing the culture bacteria.

In addition, the step c) comprises: c1) drying and pulverizing the steamed cacao nips; c2) shaking and shading at a temperature of 60 to 150 for 10 minutes to 2 hours; And c3) leaching at a room temperature for 1 to 24 hours in a weight of 5 to 30 times by weight of 70% ethanol.

The steamed cacao nips extract after the far-infrared ray irradiation treatment of the present invention has excellent skin antioxidative effect, inhibitory effect on MMP-1 production, inhibitory effect on UV-induced MMP-1 production, effect on collagen synthesis, An effect of suppressing cytotoxicity due to irradiation with ultraviolet rays, an effect of suppressing the expression of inflammatory cytokines by irradiation of ultraviolet rays, an effect of improving skin wrinkles and an effect of improving skin elasticity.

The present invention relates to a cosmetic composition containing, as an active ingredient, a cacao nips extract steam-steamed after far-infrared ray irradiation treatment.

Far infrared ray is a type of electromagnetic wave and refers to a long wavelength of infrared rays (0.76 to 1,000 μm). The wavelength range is usually in the range of 5 to 20 μm. It has stronger heat action than visible ray and radiative energy is directly and instantaneously transferred It is a useful microwave which is widely used because it quickly heats a black body and has a large energy saving effect. Far infrared rays have the characteristic of transferring heat evenly to the center of the material. Due to these characteristics, it is possible to reduce energy consumption by shortening the heating time and to optimize the loss of nutrients or physiologically active substances by pyrolysis have. Natural antioxidants such as polyphenols, tocopherols and flavonoids, which are polymers, have polymers. However, it has been reported that far-infrared treatment increases the antioxidant ability by liberating them with low molecular weight.

In the present invention, cacao nymphs were treated with far infrared ray irradiation under specific conditions, treated with cacao nips using steam, and extracted by various methods to evaluate the skin improving activity of the extracts.

According to the present invention, there is provided a cosmetic composition comprising, as an active ingredient, an extract of cacao nips steamed with steam after irradiation with far-infrared rays. At this time, the far-infrared ray irradiation treatment is to treat the far-infrared rays having a wavelength of 5 to 20 μm at 90 to 130 ° C. for 30 to 120 minutes, more preferably the far infrared rays having a wavelength of 5 to 20 μm at a temperature of 110 to 130 ° C. for 60 to 120 minutes The antioxidant activity was greatly increased.

The steam treatment after the far-infrared ray irradiation treatment is performed for 3 to 12 hours at a steam of 110 to 130 ° C. In the case of steam-brewing the far-infrared irradiated cacao nymphs with steam, not only the antioxidative activity, but also the effect of enhancing collagen synthesis, inhibiting melanin formation, mitigating cytotoxicity by ultraviolet irradiation, and inhibiting the expression of inflammatory cytokine by ultraviolet irradiation Respectively.

The cacao nymph extract of the present invention comprises the steps of: A) treating far-infrared rays having a wavelength of 5 to 20 탆 at 90 to 130 캜 for 30 to 120 minutes in cacao nips; B) treating the far-infrared-treated cacao nips with steam at 110 to 130 ° C for 3 to 12 hours; And C) extracting the steamed cacao nips.

According to one embodiment of the present invention, the cacao nips extract is selected from the group consisting of water, anhydrous or hydro-lower alcohols having 1-4 carbon atoms (e.g., methanol, ethanol, propanol and butanol), propylene glycol, Preferably 70% (v / v), of a solvent selected from the group consisting of acetone, acetone, diethyl ether, ethyl acetate, butyl acetate, dichloromethane, chloroform, hexane and mixtures thereof. ) Ethanol aqueous solution or water, and most preferably is a solvent extract obtained by using 70% (v / v) ethanol.

According to an embodiment of the present invention, the step c) comprises: c1) drying and pulverizing the steamed cacao nips; c2) adding 5 to 30 times the weight of 70% (v / v) ethanol and leaching at room temperature (15 to 20 ° C) for 1 to 24 hours.

In addition, the steamed cacao nips extract after the far-infrared ray irradiation treatment of the present invention can be used not only as an extract obtained by the above-mentioned extraction solvent, but also as an extract obtained by decompression by carbon dioxide, extraction by supercritical extraction at high temperature, Lt; / RTI > Further, it is also possible to carry out the separation using an ultrafiltration membrane having a constant molecular weight cut-off value, various purification methods such as separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity) The obtained active fraction is also included in the extract of the present invention.

The supercritical fluid extraction refers to supercritical fluid extraction. Generally, the supercritical fluid is extracted from the liquid and the liquid when the gas reaches the critical point at high temperature and high pressure. Gas properties, and chemically similar polarity to non-polar solvents. Due to these properties, supercritical fluids are being used to extract lipophilic materials. Carbon dioxide becomes a supercritical fluid with both liquid and gaseous properties as the pressure and temperature reach the critical point through the operation of the supercritical fluid device, resulting in increased solubility in the fat soluble solute. When supercritical carbon dioxide passes through an extraction vessel containing a certain amount of sample, the lipophilic substance contained in the sample is extracted into supercritical carbon dioxide. When the supernatant carbon dioxide containing a small amount of cosolvent is passed through the sample remaining in the extraction vessel after extracting the lipid-soluble substance, components that were not extracted by pure supercritical carbon dioxide can be extracted. The supercritical fluid used in the supercritical extraction method of the present invention can effectively extract an active ingredient by using a mixed fluid in which a cosolvent is further mixed with supercritical carbon dioxide or carbon dioxide. Such a co-solvent may be a mixture of one or more selected from the group consisting of chloroform, ethanol, methanol, water, ethyl acetate, hexane and diethyl ether. Most of the extracted samples contain carbon dioxide. Since the carbon dioxide is volatilized into air at room temperature, the extract obtained by the above method can be used as a cosmetic composition, and the cosolvent can be removed by a reduced pressure evaporator.

In addition, the ultrasonic extraction method is an extraction method using energy generated by ultrasonic vibration. Ultrasonic waves can destroy an insoluble solvent contained in a sample in a water-soluble solvent. Due to the high local temperature, Since the kinetic energy of the reactant particles is increased, sufficient energy required for the reaction is obtained. By inducing the high pressure by the shock effect of the ultrasonic energy, the mixing effect of the substance contained in the sample and the solvent is enhanced, thereby increasing the extraction efficiency. As the extraction solvent which can be used for the ultrasonic extraction method, one or a mixture of two or more selected from the group consisting of chloroform, ethanol, methanol, water, ethyl acetate, hexane and diethyl ether can be used. The extracted sample is recovered by vacuum filtration, and the filtrate is recovered, and the extract is removed by a vacuum evaporator and freeze-dried to obtain an extract.

According to another embodiment of the present invention, the steamed cacao nips extract after the far-infrared ray irradiation treatment is a fermented extract using a microorganism. The microorganism which can be used at this time is a bacterium selected from yeast, lactic acid bacteria, bacteria of the genus Bacillus, and a mixture thereof and is generally fermented for 1 to 7 days while maintaining a pH of 3 to 9 under a temperature condition of 20 to 50 캜, And may be fermented for several months or years.

According to an embodiment of the present invention, the step c) comprises: c1) drying the steamed cacao nibs to a size of 200 to 300 mesh and pulverizing the dried cacao nips; c2) adding to a culture medium of Saccharomyces cerevisiae, a culture of Lactobacillus plantarum or a culture of Bacillus subtilis, fermenting at a temperature of 20 to 50 ° C and a pH of 3 to 9; And c3) filtration after removing the culture bacteria.

The yeast used in the production of the fermentation extract of the far-infrared ray irradiation treated cacao nymph of the present invention belongs to a eukaryotic microorganism (a cell and a cell organism divided by a nuclear membrane) and has a cell organelle such as a cell wall, a cell membrane, a nucleus, . Those with no distinction of sex mainly reproduce by budding. Among many isolated yeasts, there is no efficacy, proliferation by fission instead of budding, bud-fission method, injection by non-ascospore There are also diverse groups of yeasts, including those that form spores (ballistospores) or that do not exhibit spore formation. The size is usually 5 ~ 10 ㎛, 5 ~ 10 times larger than bacteria, and the optimum pH is 4.5 ~ 5.5. Yeast surviving at pH 1 for 12 hours is stronger in acid than bacteria, so if the medium conditions are adjusted to about pH 3.5 to pH 3.8, bacteria contamination can be suppressed to some extent. Yeast is slightly different depending on the species, but most of them grow well at 20 ~ 25 ℃. Usually, it is said to die at 50 ° C or higher, but it can be overcome to some extent by adding a growth factor which is not synthesized above the temperature, directly to the medium. In the case of Saccharomyces cerevisiae, it can be prevented by adding oleic acid and ergosterol to the medium at 40 ° C or higher. Yeast cells themselves are unicellular plants and contain large amounts of proteins and trace minerals. A preferred yeast for use in fermenting the far-infrared irradiation cacao nymph of the present invention is Saccharomyces cerevisiae.

In the present invention, lactic acid bacteria may be used for producing the fermentation extract of far-infrared ray treated cacao nips. Lactic acid bacteria is a type of microorganism that is very beneficial to the human body that decomposes carbohydrates such as glucose or lactose to produce organic acids such as lactic acid (lactic acid) and acetic acid. In general, lactic acid bacteria make lactic acid from sugar is called fermentation, and through this fermentation process fermented food is produced. The lactic acid bacteria which can be used in the present invention preferably include Lactobacillus, Streptococcus, Bifidobacterium, Leuconostok, Pediococcus, Lactococcus and mixtures thereof, most preferably Lactobacillus . Among Lactobacillus, Lactobacillus plantarum is the most preferable lactic acid bacterium for use in fermenting far-infrared ray irradiation cacao nives of the present invention.

Further, in the present invention, for the production of the cacao nips fermented extract, Bacillus bacteria can be used as the fermenting bacteria. Bacillus is a spore-forming organism that lives in soil or in water and is mostly associated with decomposition of organic matter. Bacillus that can be used in the present invention preferably includes Bacillus subtilis, Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, Bacillus polyfermenticus, Bacillus meencentericus, and mixtures thereof Most preferably Bacillus subtilis. This bacterium is also known as Bacillus subtilis. It is metabolized to various saccharides such as glucose and starch, and is used as an organic decomposing microorganism to be. In particular, the Bacillus fermentation extract had excellent cell regeneration effects (Test Example 5).

Further, the steamed cacao nips extract after the far-infrared ray irradiation treatment of the present invention includes the extract obtained by the pulverization method. Fojera is a pharmaceutical technology that changes the original properties of medicines by processing the medicines based on the oriental medicine theory. Mainly refers to the process of steaming, boiling, roasting, roasting, burning, or mixing processes.

According to an embodiment of the present invention, the step c) comprises: c1) drying and pulverizing the steamed cacao nips; c2) shaking and shading at a temperature of 60 to 150 for 10 minutes to 2 hours; And c3) adding 5 to 30 times the weight of 70% ethanol and leaching at room temperature for 1 to 24 hours. The extract by the inhalation method was superior to the UV-induced inhibitory effect of inflammatory cytokine IL-1 alpha (Test Example 11) and MMP-1 production inhibitory effect (Test Example 6).

The steamed cacao nips extract in the steam after the far-infrared ray irradiation treatment exhibited antioxidative effects (Test Examples 2 to 4), skin cell regeneration effect (Test Example 5), MMP-1 generation inhibitory effect (Test Example 6) 1 production inhibitory effect (Test Example 7), collagen synthesis enhancing effect (Test Example 8), melanin formation inhibiting effect (Test Example 9), cytotoxic relaxation effect by ultraviolet irradiation (Test Example 10), inflammatory cytokine expression inhibitory effect (Test Example 11), skin wrinkle improving effect (Test Example 12), skin elasticity improving effect (Test Example 13), and moisturizing effect (Test Example 14).

The cacao nymph extract is contained in an amount of 0.001 to 98.0% by weight based on the total weight of the cosmetic composition, more preferably 0.01 to 90.0% by weight based on the total weight of the cosmetic composition. When the content of the extract is less than 0.001% by weight, no skin improving effect is exhibited.

The cosmetic composition of the present invention comprising the steamed cacao nips extract after the far-infrared ray irradiation treatment with various functions has excellent antioxidative effect, skin aging prevention effect, human fibroblast activity effect, skin whitening effect, skin damaging effect And can be used for irritation alleviation effect, skin wrinkle improvement and skin elasticity improvement.

The cosmetic composition of the present invention may contain, as an active ingredient, the ingredients commonly used in cosmetic compositions in addition to the above-mentioned effective ingredients, and may contain conventional additives such as antioxidants, stabilizers, solubilizers, vitamins, pigments and perfumes, . The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used in the form of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component . When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters. In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used. When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, / Propane or dimethyl ether. 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 Alkylamido betaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester.

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

[Example]

Hereinafter, the present invention will be described in more detail with reference to the following examples. It should be understood, however, that these embodiments are merely examples for the purpose of easily describing the scope and scope of the technical idea of the present invention, and thus the technical scope of the present invention is not limited or changed. It will be apparent to those skilled in the art that various changes and modifications can be made within the scope of the technical idea of the present invention based on these examples.

Manufacturing example  1 to 9: Cacao treated with far-infrared rays Nips  Preparation of extract

Viva Labs Raw Cacao Nibs were cleaned with purified water and then dried. The prepared cacao nives were placed in a far-infrared oven at 90, 110, and 130 ° C for 30, 60, and 120 minutes, respectively. 1000 g of 70% ethanol adjusted to a ratio of water to ethanol of 7: 3 was added to 200 g of cacao nips and the mixture was leached at room temperature for 1 day. This was filtered and the filtrate was concentrated in vacuo to give a dry extract. The dried extract was dissolved in 50% 1,3-butylene glycol solution to a concentration of 1% (v / v), filtered, and applied to the experiment of the present invention.

Far-infrared irradiation (FIR) conditions Temperature (℃) Time (min) Production Example 1 90 30 Production Example 2 90 60 Production Example 3 90 120 Production Example 4 110 30 Production Example 5 110 60 Production Example 6 110 120 Production Example 7 130 30 Production Example 8 130 60 Production Example 9 130 120

Test Example  1: Cacao treated with far infrared rays Nips  Extract NBT method  Antioxidative effect measurement using

To determine the antioxidant effect of cacao nips extract treated with far infrared rays, antioxidative effect (active oxygen scavenging ratio) was measured by NBT method using vitamin C and BHT (Butylated Hydroxytoluene), which are known to have antioxidative effects.

In order to measure the antioxidative effect, active oxygen produced by xanthine and xanthine oxidase was measured by NBT method, and the test substance (cacao nips extract, vitamin C, BHT, steamed after the far-infrared irradiation treatment) , That is, an active oxygen scavenging ratio (%). The active oxygen scavenging ratio (%) is measured by reacting the active oxygen with Nitro Blue Tetrazolium (NBT) and measuring the blue color produced by the reaction at a wavelength of 560 nm.

As a measuring method

1. 0.05 M Na ₂ CO _{3 -} 2.4 ml

2. 3 mM xanthine solution - 0.1 ml

3. 3 mM EDTA solution ------------------------------------ 0.1 ml

4. BSA solution ----------------------------------------- 0.1ml

5. 0.72 mM NBT solution - 0.1 ml

6. Xanthine oxidase solution - 0.1 ml

7. 6 mM CuCl ₂ solution - 0.1 ml

① Add 1, 2, 3, 4, and 5 to a vial bottle, add 0.1 ml of the sample solution, and allow to stand at 25 ℃ for 10 minutes.

② Add 6, stir rapidly, and start culturing at 25 ℃ for 20 minutes.

③ Then, 7 is added to stop the reaction, and the absorbance St at 560 nm is measured.

(4) The absorbance So of the blank of the sample solution is measured by operating in the same manner as that for measuring the absorbance St except that distilled water is used instead of the above-mentioned 6.

(5) The absorbance Bt is measured by operating the blank test in the same manner as in the measurement of the absorbance St except that distilled water is used instead of the sample solution.

(6) Blank of the blank test is carried out in the same manner as in the measurement of the absorbance St except that distilled water is used instead of the above sample solution and 6, and the absorbance Bo is measured.

The% of active oxygen scavenging was calculated by the following formula, and the results are shown in Table 2.

Active oxygen scavenging rate ( % ) = [1- (St-So) / (Bt-Bo)] X  100

St: Absorbance at 560 nm after enzyme reaction of sample solution

Bt: Absorbance at 560 nm after enzyme reaction of blank test solution

So: the absorbance at 560 nm before the reaction in the absence of enzyme in the sample solution

Bo: absorbance at 560 nm before the reaction in the absence of enzyme in the blank test solution

FIR condition Treatment concentration (%, v / v) Antioxidative effect(%) Temperature (℃) Time (min) Control
(Cacao nives) - - 0.01 43.1 Production Example 1 90 30 0.01 49.7 Production Example 2 90 60 0.01 55.3 Production Example 3 90 120 0.01 59.2 Production Example 4 110 30 0.01 52.6 Production Example 5 110 60 0.01 59.0 Production Example 6 110 120 0.01 66.2 Production Example 7 130 30 0.01 55.4 Production Example 8 130 60 0.01 67.9 Production Example 9 130 120 0.01 75.8 Vit. C - - 0.01 83.1 BHT - - 0.01 90.6

As shown in Table 2 above, all of the samples tested at the concentration of 0.01% showed higher antioxidative effect than Control. Production Examples 1 to 9 were prepared as described in Vit. C, but the antioxidative effect of cacao nives was increased by far-infrared treatment.

Example  1 to 4: Steamed cacao after far infrared ray irradiation treatment Nips  Preparation of extract

The far-infrared ray-treated cacao nymph of Preparation Example 9 was treated at 120 ° C for 3, 6, 9 and 12 hours. The extract was prepared by extracting in the same manner as in Preparation Example 1 above.

Test Example  2: Steamed cacao after far infrared ray irradiation treatment Nips  Extract NBT method  Antioxidative effect measurement using

The antioxidative effect was measured by the same method as in Test Example 1 to confirm the antioxidant effect of the steamed cacao nips extract after the far-infrared ray irradiation treatment. The antioxidative effect (active oxygen scavenging ratio) was measured by the NBT method using vitamin C and BHT (Butylated Hydroxytoluene), which are known to have antioxidant effects. The results are shown in Table 3 below.

Steaming time (hr) Treatment concentration
(%, v / v) Antioxidative effect(%) Control
(Cacao nives) - 0.01 43.1 Production Example 9 0 0.01 75.8 Example 1 3 0.01 82.6 Example 2 6 0.01 91.4 Example 3 9 0.01 91.8 Example 4 12 0.01 92.3 Vit. C - 0.01 83.2 BHT - 0.01 90.5

As can be seen in Table 3, the antioxidative effect of the steamed steamed steam steamed at the 0.01% concentration was higher than that of the control steamed steamed steamed steamed steam steamed steam steamed steam steamed steam steamed steam steamed steam steamed steam steamed steam steamed steam steamed steam steamed steam steamed steam steamed steam steamed steam steamed steam steamed steam steamed steam steamed steam steamed steam steamed In Examples 2 to 4, Vit. C and BHT, respectively. In the case of Example 2, Example 3, and Example 4, which showed excellent antioxidative effects, the steam-boiling time increased by 3 hours, 6 hours, 9 hours, and 12 hours, respectively. However, the effect of antioxidation was slightly increased, It was confirmed that the condition of Example 2 was steamed for 6 hours. Hereinafter, the cacao nips extract of Example 2 was used for the test.

Example  5: Steamed cacao in steam after far infrared ray irradiation treatment Nips  Preparation of extract

Extraction was carried out in the same manner as in the above Example except that the extraction solvent shown in Table 4 was used, and steamed cacao nips extract was obtained after far-infrared ray irradiation treatment. In Examples 5-14 and 5-15 described below, steamed cacao nips extract was produced by far ultraviolet ray irradiation treatment through extraction using conventional supercritical extraction method and ultrasonic extraction method. In the case of Examples 5-14, olive oil was used as a co-solvent and it was liquid.

Extraction solvent From 1 kg of cacao nips
Weight of extract (unit: g) Example 5-1 Purified water 28.5 Example 5-2 25% ethanol 31.8 Example 5-3 50% ethanol 33.2 Examples 5-4 100% ethanol 34.9 Example 5-5 80% methanol 35.9 Examples 5-6 100% methanol 33.2 Examples 5-7 Acetone 37.5 Examples 5-8 Diethyl ether 34.7 Examples 5-9 Hexane 30.1 Examples 5-10 Normal-propanol 37.2 Examples 5-11 Isopropanol 33.3 Examples 5-12 Normal-butanol 36.4 Examples 5-13 Ethyl acetate 32.6 Examples 5-14 Supercritical extraction 101.5 Examples 5-15 Ultrasonic extraction 43.7

Example  6: Steamed cacao in steam after far-infrared irradiation treatment Nips  Yeast fermentation extract preparation

Washed with purified water and dried, and steamed cacao nips (treated under the condition of Example 2) in the steam was pulverized and finely made using 300 mesh. This was added to the yeast culture (Potato Dextrose Broth, Accumedia, USA) to 10 g / L. Saccharomyces cerevisiae was used as a yeast strain used for fermentation and added at 50,000 CFU / L per culture. The cultures were incubated for 7 days at 30 ° C and pH 5 to 7 using a 5 L fermenter. After cultivation, the cocoa nymphs fermented in steam were centrifuged to remove primary cultures, followed by final filtration using 0.25 μm filter paper. The filtrate thus obtained was aged at a low temperature warehouse at 4 ° C for about 10 days. The filtrate was filtered again using 0.25 μm filter paper to be used in the present invention. For convenience, the fermentation filtrate was referred to as yeast fermentation extract . The extraction yield by this method was 291.5 g / lkg. The fermented yeast extracts used in the experiments were kept at a final concentration of 1 g / 100 mL.

Example  7: Steamed cacao in steam after far infrared ray irradiation treatment Nips  Preparation of Fermented Extract of Lactic Acid Bacteria

Washed with purified water and dried, and steamed cacao nips (treated under the condition of Example 2) in the steam was pulverized and finely made using 300 mesh. This was added to Lactobacillus MRS Broth (Difco, USA) to a concentration of 10 g / L, and the mixture was sterilized at 121 ° C and inoculated with lactic acid bacteria.

Lactobacillus plantarum was used for the lactic acid bacteria strain used for fermentation and added at 50,000 CFU / L per culture. Cultures were cultured for 3 days at 30 ° C and pH 5 to 7 using a 5 L fermenter. After cultivation, the cocoa nymphs fermented in steam were centrifuged to remove primary cultures, followed by final filtration using 0.25 μm filter paper. The filtrate thus obtained was aged for about 10 days at a low temperature warehouse at 4 DEG C, and the filtrate was filtered through a filter paper of 0.25 mu m to be used in the present invention. For convenience, the fermentation filtrate was referred to as lactic acid fermentation extract Respectively. The extraction yield by this method was 297.1 g / lkg. The fermented extracts of lactic acid bacteria used in the experiments were kept at a final concentration of 1 g / 100 mL.

Example  8: Steamed cacao in steam after far-infrared irradiation treatment Nips Bacillus  Preparation of fermented extract

Washed with purified water and dried, and steamed cacao nips (treated under the condition of Example 2) in the steam was pulverized and finely made using 300 mesh. This was added to the Bacillus culture medium to 10 g / L. Bacillus subtilis (Bacillus subtilis) was used for fermentation and added at 50,000 CFU / L per culture. The cultures were incubated for 3 days at 30 ° C with a pH of 5 to 7 using a 5 L fermenter. After cultivation, the fermentation broth of far-infrared irradiation cacao nymphs was centrifuged to remove the cultures first, followed by final filtration using 0.25 μm filter paper. The filtrate thus obtained was aged at a low temperature warehouse at 4 ° C for about 10 days. The filtrate was filtered again with 0.25 μm filter paper to be used in the present invention. For convenience, this fermentation filtrate was called a Bacillus fermentation extract Respectively. The extraction yield by this method was 229.7 g / lkg. The Bacillus fermentation extract used in the experiments was maintained at a final concentration of 1 g / 100 mL.

Example  9: Steamed cacao in steam after far-infrared irradiation treatment Nive's Clotting method  Preparation of extract

In order to obtain a crude extract of steamed cacao nips after the far-infrared ray irradiation treatment, this example was subjected to a step of steam-drying. That is, after steamed cacao nips (treated under the conditions of Example 2) were pulverized after finishing the far-infrared ray irradiation treatment which was washed with purified water and dried, it was made fine using 300 mesh, And steamed it. Extraction was carried out with 70% ethanol. As a result, 48.7g / 1kg of extract was obtained.

Test Example  3: Steamed cacao in steam after far infrared ray irradiation treatment Nive's  Extracts from various extraction methods NBT method  Antioxidative effect measurement using

In order to confirm the antioxidative effect of the extract by various extraction methods such as supercritical extraction, ultrasonic extraction, yeast fermentation extraction, lactic acid fermentation extraction, Bacillus fermentation extraction, and pulmonary extraction, steamed cacao nips after the irradiation with far infrared rays . The antioxidative effect (active oxygen scavenging ratio) was measured by NBT method using vitamin C and BHT (Butylated Hydroxytoluene), which are known to have antioxidant effect, as a comparative group. The results are shown in Table 5.

Name of sample Treatment concentration
(%, w / v) Antioxidative effect(%) Example 2
Cacao nips extract 0.01 91.4 Examples 5-14
Supercritical extract 0.01 84.6 Examples 5-15
Ultrasonic Extract 0.01 81.9 Example 6
Yeast fermented extract 0.01 83.3 Example 7
Fermented extract of lactic acid bacteria 0.01 82.1 Example 8
Bacillus fermented extract 0.01 83.8 Example 9
Herb extract 0.01 85.8 Vit. C 0.01 83.6 BHT 0.01 90.5

As shown in Table 5, excellent antioxidative effects were confirmed in all of the tested samples at the concentration of 0.01%. All of the examples of the present invention had an antioxidative effect superior to that of Vit C, and in particular, Example 2 showed a superior antioxidative effect in comparison with BHT showing the highest antioxidative effect at a concentration of 0.01%.

Test Example  4: Steamed cacao in steam after far-infrared irradiation treatment Nips  Extract DPPH method  Antioxidative effect measurement using

The antioxidative effect (free radical scavenging ratio) of vitamin C, which is known to have antioxidative effect, was measured by DPPH method to measure the antioxidative effect of steamed cacao nips extract in steam after the far - infrared irradiation treatment.

The DPPH method measures the antioxidant effect by reducing power using a free radical called DPPH (2,2-Di (4-tert-octylphenyl) -1-picrylhydrazyl) and free radical. The degree of free radical scavenging (%) is measured at a wavelength of 560 nm by comparing the degree of decrease in absorbance of DPPH by the test substance (far infrared irradiation irradiated steam cacao nips extract, vitamin C) with the absorbance of the blank test solution. As a reagent used, 61.88 mg of a 0.1 mM solution of 2,2-Di (4-tert-octylphenyl) -1-picrylhydrazyl) or free radical (Sigma Aldrich Chem. Co., MW = 618.76) is dissolved in methanol to make 100 ml.

As a measuring method

(1) Add 0.15 ml of the sample solution to 0.15 ml of 0.1 mM DPPH solution in a 96-well plate, stir rapidly, and start culturing at 25 for 10 minutes.

② Measure the absorbance St at 560 nm thereafter.

(3) The blank of the sample solution is measured by the same procedure as that for measuring the absorbance St except that methanol is used instead of the 0.1 mM DPPH solution.

(4) The absorbance Bt is measured by operating in the same manner as in the measurement of the absorbance St except that distilled water is used instead of the sample solution.

(5) Blank of the blank test is carried out in the same manner as in the measurement of the absorbance St, except that methanol is used instead of the 0.1 mM DPPH solution and distilled water is used instead of the sample solution, and the absorbance Bo is measured.

The results of the free radical scavenging rate (%) were calculated by the following equation, and the results are shown in Table 6 below.

Free radical scavenging rate % ) = [1- (St-So) / (Bt-Bo)] X  100

St: absorbance at 560 nm after free radical scavenging of the sample solution

Bt: absorbance at 560 nm after free radical scavenging of the blank test solution

So: the absorbance at 560 nm before the reaction in the absence of free radicals in the sample solution

Bo: absorbance at 560 nm before the reaction in the absence of the free radical of the blank test solution

Name of sample Treatment concentration (%, w / v) Antioxidative effect(%) Example 2
Cacao nips extract 0.01 94.8 Examples 5-14
Supercritical extract 0.01 93.4 Examples 5-15
Ultrasonic Extract 0.01 93.9 Example 6
Yeast fermented extract 0.01 93.2 Example 7
Fermented extract of lactic acid bacteria 0.01 91.1 Example 8
Bacillus fermented extract 0.01 90.0 Example 9
Herb extract 0.01 90.4 Vit C 0.01 90.2

As shown in Table 6, the examples of the present invention exhibited antioxidative effects higher or equivalent to those of vitamin C, which is known to have an antioxidative effect at a concentration of 0.01%.

Test Example  5: Steamed cacao in steam after far infrared ray irradiation treatment Nips  Cell Proliferation Effect of Extracts

(Dulbecco's Modified Eagle's Medium, Gibco) supplemented with 10% FBS at a density of 2 × 10 ⁵ cells / well was added to a humidified 37 ° C, 5 ° C % CO ₂ incubator for 1 day. After exchanging with serum-free DMEM, the samples were treated with 1.0% (v / v) and cultured for 3 days. To determine the cell-forming effect after culturing, the degree of cell formation was evaluated by MTT assay, and TGF-β (10 ng / mL) was used as a positive control. The results are shown in Table 7 below.

Name of sample Abs. At 570 nm Example 2
Cacao nips extract 1.114 Examples 5-14
Supercritical extract 1.103 Examples 5-15
Ultrasonic Extract 1.112 Example 6
Yeast fermented extract 1.107 Example 7
Fermented extract of lactic acid bacteria 1.112 Example 8
Bacillus fermented extract 1.992 Example 9
Herb extract 1.121 Positive control group
TGF-beta (10 ng / ml) 1.482 Control group 0.912

In general, skin cell regeneration was measured by the cell activity ratio, and it was confirmed that the cells regenerated in the examples of the present invention. Also, it was confirmed that the extract of the Example of the present invention has excellent cell regeneration effect by the present test results in which the cell regeneration effect is similar even considering the difference in concentration with TGF-beta (10 ng / ml) In particular, it was confirmed that the Bacillus fermentation extract of Example 8 was the most excellent in cell regeneration effect.

Test Example  6: Steamed cacao in steam after far-infrared irradiation treatment Nips  Extract MMP -1 production inhibitory effect

In order to determine whether the effect of suppressing the production of MMP-1, a collagen degrading enzyme of steamed cacao nips extract in steam after irradiation with far-infrared rays, TGF-β (a substance known to inhibit the production of MMP- 10 ng / ml) was used as a comparative group to measure the effect of inhibiting the production of MMP-1.

Human normal skin cells, fibroblasts (Korean Cell Line Bank, Korea) were inoculated into 48-well microplates (Nunc, Denmark) at a density of 1 × 10 ⁶ cells per well and cultured in DMEM medium and 37 ° C. for 24 hours Then, the above examples were further cultured in serum-free DMEM medium supplemented with 100 μg / ml of final concentration and in serum-free DMEM medium containing no sample for 48 hours as a control group. After the incubation, the supernatant of each well was collected and the amount (ng / ml) of the newly synthesized MMP-1 was measured using an MMP-1 assay kit (Amersham, USA) %) Was calculated, and the results are shown in Table 8.

Name of sample Inhibition rate of MMP-1 production (%) Example 2
Cacao nips extract 71.0 Examples 5-14
Supercritical extract 70.7 Examples 5-15
Ultrasonic Extract 71.8 Example 6
Yeast fermented extract 71.1 Example 7
Fermented extract of lactic acid bacteria 71.1 Example 8
Bacillus fermented extract 69.8 Example 9
Herb extract 74.7 Positive control group
TGF-beta (10 ng / ml) 69.6

As shown in the results of Table 8, when the inhibitory rate of MMP-1 production was measured at a concentration of 100 μg / ml, the samples of the Examples showed 69.6% of TGF-β at a suppression rate of 70.7 to 74.7% Respectively. Even considering the difference between the concentration of TGF-beta (10 ng / ml), which is a substance having an inhibitory effect on MMP-1 production, and the experimental concentration (100 μg / ml) of the samples of the present invention, -1 inhibition rate is shown by the fact that the cacao nips extract in steam after the far-infrared ray irradiation treatment of the present invention has an inhibitory effect on MMP-1 production.

Test Example  7: Steamed cacao in steam after far infrared ray irradiation treatment Nips  UV-induced induction of the extract MMP -1 production inhibitory effect

In general, it is known that the amount of MMP-1 is increased by ultraviolet light. Therefore, in this test example, experiments were conducted to examine whether the steamed cacao nips extract in the steam after the far-infrared ray irradiation treatment suppresses the amount of MMP-1 which is increased by ultraviolet rays. Measurement of inhibitory effect of UV-induced MMP-1 production was carried out in the same manner as in Test Example 6, except that a certain amount of UVB was treated prior to the sample treatment. The% inhibition of MMP-1 production of the samples was calculated according to the above formula, and the results are shown in Table 9 below. At this time, TGF-β (10 ng / ml), a substance known to have an inhibitory effect on MMP-1 production, was used as a comparative group, and samples were subjected to 0.01% (w / v) concentration.

Name of sample Inhibition rate of MMP-1 production (%) Example 2
Cacao nips extract 69.6 Examples 5-14
Supercritical extract 69.0 Examples 5-15
Ultrasonic Extract 69.1 Example 6
Yeast fermented extract 67.8 Example 7
Fermented extract of lactic acid bacteria 68.6 Example 8
Bacillus fermented extract 68.5 Example 9
Herb extract 65.1 Positive control group
TGF-beta (10 ng / ml) 63.9

The amount of collagenase MMP-1 secreted in the cultured medium supplemented with UVB-irradiated samples and TGF-β was measured in the cultured fibroblasts, and the results are shown in Table 9 above. When the inhibitory rate of MMP-1 production was measured at a concentration of 100 μg / ml, the extract of steamed cacao nymphs after the far-infrared ray irradiation treatment of the present invention showed the highest inhibition rate with an inhibition rate of 69.6%. (10 ng / ml), which is a substance known to have an inhibitory effect on MMP-1 production, and the concentration of the samples of the present invention (0.01% (w / v)), 1 shows that the cacao nips extract in steam after the far-infrared ray irradiation treatment of the present invention has an inhibitory effect on MMP-1 production due to ultraviolet induction.

Test Example  8: Steamed cacao in steam after far-infrared irradiation treatment Nips  Enhancement of collagen synthesis of extract

Human normal epithelial cells, fibroblasts, were inoculated into a 48-well microplate at a density of 1 x 10 ⁶ cells per well and cultured in DMEM medium for 24 hours. The above samples were further cultured for 48 hours in serum-free DMEM medium supplemented with 100 μg / ml final concentration and in serum-free DMEM medium containing no sample. After the incubation, the supernatant of each well was collected, and the amount of procollagen type I C-peptide (PICP) was measured using a collagen kit (Takara, Japan) and converted into ng / Respectively. As a comparative group, TGF-β (10 ng / ml), a substance known to have an effect of enhancing collagen synthesis, was used as a comparative group, and samples were subjected to a concentration of 0.01% (w / v). Collagen biosynthesis increase rate (%) was calculated according to the following formula, and the results are shown in Table 10.

Name of sample Increase in collagen biosynthesis (%) Example 2
Cacao nips extract 88.7 Examples 5-14
Supercritical extract 76.9 Examples 5-15
Ultrasonic Extract 81.1 Example 6
Yeast fermented extract 81.2 Example 7
Fermented extract of lactic acid bacteria 83.0 Example 8
Bacillus fermented extract 83.3 Example 9
Herb extract 83.1 Positive control group
TGF-beta (10 ng / ml) 89.2

Table 10 shows the results of measuring the effect of enhancing collagen synthesis in the culture medium supplemented with the samples and TGF-beta using fibroblasts. According to Table 10, Example 2 of the present invention showed a biosynthesis rate of 88.7% when the increase rate of collagen biosynthesis was measured at a concentration of 0.01%. (0.01% (w / v)) difference between the concentration of TGF-β (10 ng / ml), which is a substance known to have an effect of promoting collagen synthesis, and the steamed cacao nips extract in the steam after the far-infrared ray irradiation treatment of the present invention, The increase rate of collagen biosynthesis at the level of the extract at the extract level shows that the cacao nips extract in steam after the far-infrared ray irradiation treatment of the present invention has an effect of promoting collagen synthesis.

Test Example  9: Steamed cacao in steam after far-infrared irradiation treatment Nips  Extract B16F10  Melanin production inhibitory effect on melanin-forming cells

In order to confirm the whitening effect of steamed cacao nips extract in steam after the far-infrared ray irradiation treatment, the inhibitory effect of melanin formation on B16F10 melanin-forming cells was examined to determine the whitening effect. The B16F10 melanin-forming cell used in Test Example 9 is a mouse-derived cell line and is a cell that secretes a melanin pigment called melanin.

During the artificial culture of these cells, samples were treated to compare the degree of reduction of melanin pigment. The B16F10 melanin-forming cells used in this test were used from ATCC (American Type Culture Collection). Melanin biosynthesis inhibitory effect of B16F10 melanin-forming cells was measured as follows. B16F10 melanogenesis cells were plated at a concentration of 2 x 10 ⁶ per well in a 6-well plate, and the cells were treated at a concentration that did not induce toxicity after the cells were adhered and cultured for 72 hours. After incubation for 72 hours, the cells were detached with trypsin-EDTA, the number of cells was measured, and the cells were recovered by centrifugation. Quantification of intracellular melanin was carried out with a slight modification of the method of Lotan (Cancer Res., 40: 3345-3350, 1980). Cell pellet was washed once with PBS, and 1 ml of homogenization buffer solution (50 mM sodium phosphate, pH 6.8, 1% Triton X-100, 2 mM PMSF) was added and vortexed for 5 minutes to disrupt the cells. After centrifugation (3,000 rpm, 10 min), 1N NaOH (10% DMSO) was added to the cell filtrate to dissolve the extracted melanin, and the absorbance of melanin was measured at 405 nm with a microplate reader. The inhibition rate (%) of melanin formation was measured. B16F10 Melanin formation inhibition rate (%) of melanin-forming cells was calculated by the following formula, and the results are shown in Table 11. [ At this time, hydroquinone and arbutin, which are substances known to have an inhibitory effect on melanin production, were used as a comparative group.

] X 100Melanin inhibition rate (%) = [

] X 100

A: Amount of melanin in wells to which no sample was added

B: Amount of melanin in the well to which the sample was added

Name of sample Treatment concentration (%, w / v) Melanin inhibition rate (%) Example 2
Cacao nips extract 0.1 4.94 Examples 5-14
Supercritical extract 0.1 4.53 Examples 5-15
Ultrasonic Extract 0.1 3.69 Example 6
Yeast fermented extract 0.1 4.68 Example 7
Fermented extract of lactic acid bacteria 0.1 4.10 Example 8
Bacillus fermented extract 0.1 3.62 Example 9
Herb extract 0.1 3.86 Hydroquinone 0.1 7.57 Arbutin 0.1 3.61

As can be seen from the results of Table 11, it can be seen that the embodiments of the present invention significantly inhibit melanin production in B16F10 melanin-forming cells. The cacao nymph extract of the present invention, which was steamed after the far-infrared ray irradiation treatment, exhibited a melanin formation inhibition value of 3.62 to 4.94% at a concentration of 0.1%. Arbutin and hydroquinone, which are known to have a whitening effect at the same concentration, Compared with rice, it showed relatively low melanin formation inhibitory effect than hydroquinone but showed better melanin formation inhibitory effect than arbutin. Therefore, it can be seen that the steamed cacao nips extract after the far-infrared ray irradiation treatment of the present invention has an excellent effect on suppression of melanin production.

Test Example  10: Steamed cacao in steam after far-infrared irradiation treatment Nips  Effect of ultraviolet irradiation on the cytotoxicity of extracts

This study was carried out to evaluate the mitigation effects of cacao nips extract in steam after irradiation with ultraviolet rays.

Fibroblasts were placed in 24-well test plates at 5 × 10 ⁴ and adhered for 24 hours. Each well was washed once with PBS, and 1000 占 퐇 of PBS was added to each well. After irradiating the fibroblasts with 10 mJ / cm 2 of ultraviolet light using an ultraviolet B (UVB) lamp (Model: F15T8, UVB15W, Sankyo Dennki, Japan), PBS was removed and cultured in a cell culture medium (DMEM supplemented with 10% FBS 1 ml) was added. Here, the samples were treated and cultured for 24 hours. After 24 hours, the culture medium was removed, and 500 μl of the cell culture medium and 60 μl of MTT solution (2.5 mg / ml) were added to each well, followed by culturing in a 37 ° C. CO ₂ incubator for 2 hours. The medium was removed and 500 μl of isopropanol-HCl (0.04 N) was added. The cells were shaken for 5 minutes to dissolve the cells. 100 μl of the supernatant was transferred to a 96-well plate, and the absorbance at 565 nm was measured in a microplate reader. At this time, TGF-β was used as a positive control. Cell viability (%) and cytotoxic relaxation rate (%) by ultraviolet irradiation were calculated by the following formula, and the results are shown in Table 12 below.

〕X 100Cell survival rate (%) = [

] X 100

Bo: Absorbance at 565 nm of the well of the cell culture medium alone

Bt: Absorbance at 565 nm of a well that underwent chromogenic reaction in a sample not treated with the sample

St: absorbance at 565 nm of wells treated with the sample

〕X 100Cytotoxicity mitigation rate (%) by ultraviolet irradiation = [1-

] X 100

Bo: Cell survival rate of wells not irradiated with ultraviolet light and not treated with sample

Bt: Cell viability of wells irradiated with ultraviolet light and not treated with the sample

St: Cell viability of well treated with ultraviolet light and treated with sample

Name of sample Treatment concentration (%, w / v) Cytotoxic Relaxation Rate (%) Example 2
Cacao nips extract 0.0125 64 0.025 74 0.050 82 Examples 5-14
Supercritical extract 0.0125 60 0.025 67 0.050 76 Examples 5-15
Ultrasonic Extract 0.0125 55 0.025 64 0.050 82 Example 6
Yeast fermented extract 0.0125 54 0.025 68 0.050 71 Example 7
Fermented extract of lactic acid bacteria 0.0125 56 0.025 63 0.050 75 Example 8
Bacillus fermented extract 0.0125 54 0.025 61 0.050 70 Example 9
Herb extract 0.0125 58 0.025 64 0.050 72 Positive control group
TGF-beta 10 ng / ml 85

According to Table 12, the cytotoxic relaxation rate was increased in a concentration-dependent manner as the concentration of treatment in the Examples was increased, and it was found that the cytotoxicity by ultraviolet rays was effectively prevented. From the above experimental results, it can be seen that the steamed cacao nips extract in the steam after the far infrared ray irradiation treatment of the present invention effectively protects the cell damage by ultraviolet irradiation even at a low concentration.

Test Example  11: Steamed cacao in steam after far-infrared irradiation treatment Nips  Inhibitory Effect of Extracts on Inflammatory Cytokine Expression by UV Irradiation

To evaluate the inhibitory effect of inflammatory cytokine expression expressed by ultraviolet irradiation of steamed cacao nips extract in steam after the far-infrared ray irradiation treatment, fibroblasts isolated from human epidermal tissue were plated on a 24-well plate at 5 X 10 ⁴ Were added and allowed to attach for 24 hours. Each well was washed once with PBS and 500 占 퐇 of PBS was added to each well. After irradiating the fibroblasts with 10 mJ / cm 2 of ultraviolet light using an ultraviolet B (UVB) lamp (Model: F15T8, UVB 15W, Sankyo Dennki, Japan), PBS was removed and the cell culture medium ≪ / RTI > medium). Here, the samples to be evaluated were treated and cultured for 5 hours. 150 [mu] l of culture supernatant was taken to quantify IL-1 [gamma], and the inhibitory effect of inflammatory cytokines on the samples was evaluated. The amount of IL-1α was quantitated using Enzyme-linked Immunosorbent Assay and Ketoprofen, a known IL-1α inhibitor, was used as a positive control. The expression inhibition rate (%) of inflammatory cytokine (IL-1?) Was calculated by the following equation.

〕X 100Inhibitory rate of inflammatory cytokine expression (%) = [1-

] X 100

Bo: IL-1? Production in wells not subjected to ultraviolet irradiation and not treated

Bt: IL-1? Production in wells irradiated with ultraviolet light and not treated with the sample

St: IL-1α production in wells irradiated with ultraviolet light and treated with the sample

Name of sample Treatment concentration (%, w / v) Inhibitory rate of inflammatory cytokine expression (%) Example 2
Cacao nips extract 0.0125 40 0.025 53 0.050 62 Examples 5-14
Supercritical extract 0.0125 41 0.025 50 0.050 65 Examples 5-15
Ultrasonic Extract 0.0125 38 0.025 42 0.050 47 Example 6
Yeast fermented extract 0.0125 41 0.025 52 0.050 70 Example 7
Fermented extract of lactic acid bacteria 0.0125 44 0.025 50 0.050 58 Example 8
Bacillus fermented extract 0.0125 20 0.025 27 0.050 35 Example 9
Herb extract 0.0125 48 0.025 56 0.050 72 Ketoprofen 100 μg / ml 43

According to Table 13, the cacao nips extracts of Examples 2, 5-14, 6, 7, and 9 suppressed the production of IL-1α, an inflammatory cytokine caused by ultraviolet light, better than Ketoprofen at 0.025% concentration. In addition, the suppression rate of inflammatory cytokine expression was increased in a concentration-dependent manner as the concentration of treatment in the above Examples was increased. The steamed cacao nips extract after the far-infrared ray irradiation treatment of the present invention effectively inhibited the generation of inflammation caused by ultraviolet rays You can see the defense.

Formulation Example  1: softening longevity

The softening longevity was prepared according to a conventional production method with the composition shown in Table 14 below.

ingredient Content (% by weight) Cacao nips extract (Example 2) 30.0 glycerin 5.1 Propylene glycol 4.2 Tocopheryl acetate 3.0 Liquid paraffin 4.6 Triethanolamine 1.0 Squalane 3.1 Macadamia nut oil 2.5 Polysorbate 60 1.6 Sorbitan sesquiterate 1.6 Propylparaben 0.6 Carboxyl vinyl polymer 1.5 incense a very small amount antiseptic a very small amount Purified water Balance system 100.0

Formulation Example  2: Cream

Creams were prepared according to the usual production methods with the compositions shown in Table 15 below.

ingredient Content (% by weight) Cacao nips extract (Example 9) 30.0 Chin type clinoid monostearate  2.0 Cetearyl alcohol  2.2 Stearic acid  1.5 Wax  1.0 Polysorbate 60  1.5 Sorbitan stearate  0.6 Hardened vegetable oil  1.0 Squalane  3.0 Mineral oil  5.0 Trioctanoin  5.0 Dimethicone  1.0 Sodium magnesium silicate  0.1 glycerin  5.0 Betaine  3.0 Triethanolamine  1.0 Sodium hyaruronate  4.0 antiseptic  a very small amount incense  a very small amount Purified water Balance system 100.0

Formulation Example 3 : Efficacy evaluation cream

Creams for evaluation of efficacy were prepared according to the usual production methods with the compositions shown in Table 16 below.

Component (% by weight) Formulation Example 3 compare
Formulation Example 1 compare
Formulation Example 2 compare
Formulation Example 3 Cacao nips extract (Example 2)  3.0 - - - Adenosine - - 3.0 - White oyster extract - - -  3.0 Chin type clinoid monostearate 2.0 2.0 2.0 2.0 Cetearyl alcohol  2.2  2.2  2.2  2.2 Stearic acid  1.5  1.5  1.5  1.5 Wax  1.0  1.0  1.0  1.0 Polysorbate 60  1.5  1.5  1.5  1.5 Sorbitan stearate  0.6  0.6  0.6  0.6 Hardened vegetable oil  1.0  1.0  1.0  1.0 Squalane  3.0  3.0  3.0  3.0 Mineral oil  5.0  5.0  5.0  5.0 Trioctanoin  5.0  5.0  5.0  5.0 Dimethicone  1.0  1.0  1.0  1.0 Sodium magnesium silicate  0.1  0.1  0.1  0.1 glycerin  5.0  5.0  5.0  5.0 Betaine  3.0  3.0  3.0  3.0 Triethanolamine  1.0  1.0  1.0  1.0 Sodium hyaruronate  4.0  4.0  4.0  4.0 antiseptic  a very small amount  a very small amount  a very small amount  a very small amount incense  a very small amount  a very small amount  a very small amount  a very small amount Purified water Balance Balance Balance Balance system 100.0 100.0 100.0 100.0

Test Example  12: Steamed cacao in steam after far-infrared irradiation treatment Nips  Effect of extracts on skin wrinkles

The wrinkle-reducing effect of the cosmetic composition containing steamed cacao nips extract in the steam after the far-infrared ray irradiation treatment of the present invention was evaluated through practical use tests. (Comparative Formulation Example 1), a cream containing steamed cacao nips extract in steam after the far-infrared ray irradiation treatment shown in Table 16 (Formulation Example 3), a cream obtained by replacing steamed cacao nips extract in steam after the far-infrared ray irradiation treatment with purified water A cream containing adenosine (Comparative Formulation Example 2), which is known to have a wrinkle-reducing effect, was used in place of the steamed cacao nips extract in the steam after the far-infrared ray irradiation treatment. Sixty women were tested for wrinkle improvement after 6 weeks using both sides of the face. The results of the wrinkle-improving effect based on this evaluation are shown in Table 17 below.

Wrinkle improvement effect Effective rate (%) Great slightly none Formulation Example 3
(Cacao nips extract) 15 4 One 85.0 Comparative Formulation Example 1
(Purified water) 0 One 19 2.5 Comparative Formulation Example 2
(Adenosine) 12 6 2 75.0

As can be seen from the results of Table 17, the cream of Formulation Example 3 containing the steamed cacao nips extract in the steam after the far-infrared ray irradiation treatment of the present invention was compared with the comparative dosage form containing no steamed cacao nips extract in the steam after the far- 1 showed a wrinkle-reducing effect comparable to that of the cream of Comparative Example 2 containing adenosine instead of steamed cacao nips extract in the steam after the far-infrared irradiation treatment. In addition, skin irritation could not be observed in most of the subjects who applied the cosmetic composition containing steamed cacao nips extract to steam after the far-infrared ray irradiation treatment of the present invention.

Test Example  13: Steamed cacao in steam after far infrared ray irradiation treatment Nips  Skin elasticity improvement effect of extract

Clinical trials on the skin elasticity improvement effect of the cosmetic containing steamed cacao nips extract in the steam after the far-infrared ray irradiation treatment of the present invention were carried out. Clinical trials were conducted through actual use tests of each cosmetic product.

The experiment was carried out in the same manner as in Example 1, except that the nutrient cream (Formulation Example 3) containing the steamed cacao nips extract after the far-infrared ray irradiation treatment shown in Table 16, the cream obtained by replacing the steamed cacao nips extract with steam after the far- 1), improvement of skin elasticity due to use of cosmetics for humans, using adenosine-containing cream (Comparative Formulation Example 2), which is a substance known to have a wrinkle-reducing effect instead of steamed cacao nips extract in steam after far- The effects were confirmed and compared.

 The experiment group was divided into two groups: 20 patients (30 to 45 year old female), one group had Formulation Example 3 on the right side of the face, Comparative Formulation Example 1 on the left side of the face, Formulation Example 2 was applied twice a day for 8 consecutive weeks.

The skin elasticity after the completion of the experiment was measured by using a skin elasticity meter (cutter SEM 575, C + K Electronic Co., Germany) before and after the use of the product, SEM 575, respectively. This R8 value indicates the nature of the viscoelasticity of the skin.

Skin elasticity effect (△ R8) Formulation Example 3
(Cacao nips extract) 0.20 Comparative Formulation Example 1
(Purified water) 0.06 Comparative Formulation Example 2
(Adenosine) 0.17

As shown in Table 18, the cream of Formulation Example 3 containing the steamed cacao nips extract in the steam after the far-infrared ray irradiation treatment as the active ingredient contained the cream of Comparative Example 1 Cream showed a skin elasticity improvement effect that was about 2.6 times higher than that of cream and showed a skin elasticity improvement effect of about 1.15 times as compared with the cosmetic composition of Comparative Formulation Example 2 containing adenosine. It can be seen that the cosmetic composition containing cacao nips extract has an excellent skin elasticity improving effect.

Test Example  14: steamed cacao after far-infrared irradiation treatment Nips  Extract-containing Cosmetic  skin Moisture power  Improvement effect

14-1. Device Rating

In order to examine the skin moisturizing effect of the cosmetic composition containing the steamed cacao nips extract in the steam after the far-infrared ray irradiation treatment of the present invention, the following experiment was conducted. The nutritional cream (Formulation Example 3) containing steamed cacao nips extract in the steam after the far-infrared ray irradiation treatment shown in Table 16, divided into three groups of 20 persons per 1 person, (Comparative formulation example 1), cream containing steamed cacao nips extract in steam after the far-infrared ray irradiation treatment (comparative formulation example 1), cosmetics containing white oak extract known to have moisturizing effect instead of steamed cacao nips extract in steam after far-infrared ray irradiation treatment Formulation Example 3) were prepared together and applied to the face and forehead twice a day for one month.

The skin conductivity was measured by using a corneometer (CM820 courage Khazaka electronic GmbH, Germany) under the constant temperature and humidity conditions (24 ° C, 40% humidity) before the start of application, and after 1 week, 2 weeks and 4 weeks The skin conductivity was measured and the rate of increase was evaluated. The results are shown in Table 19 below.

After 1 week after 2 weeks After 4 weeks Formulation Example 3
(Cacao nips extract) 47 49 54 Comparative Formulation Example 1
(Purified water) 11 12 12 Comparative Formulation Example 3
(White oblong extract) 47 50 51

As shown in the results of Table 19, the cream containing the steamed cacao nips extract after the far-infrared ray irradiation treatment of the present invention (Formulation Example 3) showed a much higher rate of skin conductivity increase than Comparative Formulation Example 1. In addition, it exhibited a high skin conductivity increase similar to Comparative Formulation Example 3, which is a cosmetic containing a white elm extract known to have a moisturizing effect.

Since the skin conductivity is generally proportional to the skin moisture content, it can be seen from the above results that the cosmetic composition containing steamed cacao nips extract in the steam after the far-infrared ray irradiation treatment according to the present invention maintains the skin moisture content higher than that of the cosmetic composition containing purified water have. Therefore, the cosmetic composition containing steamed cacao nips extract in the steam after the far-infrared ray irradiation treatment of the present invention is excellent in the skin moisturizing power improving effect.

14-2. Clinical evaluation

The clinical moisturizing effect of the cosmetic composition prepared according to the above-mentioned prescription examples was measured as follows. From the questionnaire, we selected 20 healthy male and female adults who felt dry skin, and then divided them into 10 sites. Each sample was applied twice a day for 4 weeks. The moisturizing effect was evaluated by Corneometer CM 820 (Corage + Khazaka, Germany) for every 2 weeks and after the start of the test. The experimental results are shown in Table 20 below.

TEWL (g / h / m ² ) After 1 week after 2 weeks After 4 weeks Formulation Example 3
(Cacao nips extract) 32.2 42.7 49.3 Comparative Formulation Example 1
(Purified water) 32.4 33.0 33.2 Comparative Formulation Example 3
(White oblong extract) 32.1 43.0 48.6

As shown in Table 20, as shown in the evaluation results of the skin moisturizing effect improvement effect by instrument measurement in the above table, the cream containing the steamed cacao nips extract in the steam after the far-infrared ray irradiation treatment of the present invention (Formulation Example 3) The effect of improving the skin moisturizing ability was more excellent than the cosmetic composition (Comparative Formulation Example 1) which did not contain steamed cacao nips extract after irradiation treatment. In addition, when compared with the cosmetic containing the white clover extract known to have a moisturizing effect (Comparative Formulation Example 3), it was measured that the moisturizing power improving effect was similarly excellent. Also, as a result of measuring the moisturizing power improving effect after 2 weeks of use and after 4 weeks of use, it was confirmed that the skin moisturizing power improving effect was improved by using the cosmetic composition containing the steamed cacao nips extract in the steam after the far-infrared ray irradiation treatment of the present invention there was.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (15)

A cosmetic composition comprising a Cacao nibs extract steam-steamed after irradiation with far-infrared rays in an amount of 0.001 to 98.0% by weight based on the total weight of the cosmetic composition. The cosmetic composition according to claim 1, wherein the far-infrared ray irradiation treatment is a treatment of far-infrared rays having a wavelength of 5 to 20 mu m at 90 to 130 DEG C for 30 to 120 minutes. The cosmetic composition according to claim 1, wherein the steam is steamed for 3 to 12 hours at 110 to 130 ° C. The method of claim 1, wherein the steamed cacao nibs extract after the far-infrared ray irradiation treatment is water, an anhydrous or a lower alcohol having 1 to 4 carbon atoms, propylene glycol, butylene glycol, glycerin, acetone, ethyl acetate, chloroform , And a solvent extract extracted with at least one solvent selected from the group consisting of butyl acetate, diethyl ether, dichloromethane and hexane. The cosmetic composition according to claim 1, wherein the steamed cacao nibs extract after the far infrared ray irradiation treatment is an extract by supercritical extraction or ultrasonic extraction. [Claim 2] The method according to claim 1, wherein the steamed cacao nibs extract after the far-infrared ray irradiation treatment is a fermentation extract fermented by at least one fermenting bacteria selected from the group consisting of yeast, lactic acid bacteria, and Bacillus subtilis Cosmetic composition. The cosmetic composition according to claim 1, wherein the cosmetic composition is for antioxidation. The cosmetic composition according to claim 1, wherein the cosmetic composition is for improving skin wrinkles and elasticity. The cosmetic composition according to claim 1, wherein the cosmetic composition is for skin whitening. The cosmetic composition according to claim 1, wherein the cosmetic composition is for alleviating skin irritation. The cosmetic composition according to claim 1, wherein the cosmetic composition is for moisturizing the skin. A) treating cacao nibs with far infrared rays having a wavelength of 5 to 20 mu m at 90 to 130 DEG C for 30 to 120 minutes;
B) treating the far-infrared-treated cacao nips with steam at 110 to 130 ° C for 3 to 12 hours; And
C) Extracting steamed cacao nips. [12] The method of claim 12, wherein the step c) comprises: c1) drying and pulverizing the steamed cacao nips; c2) adding 5 to 30 times the weight of 70% ethanol and leaching at room temperature for 1 to 24 hours. [14] The method of claim 12, wherein the step c) comprises: c1) drying the steamed cacao nips by pulverizing the powder into a size of 200 to 300 mesh; c2) adding to a culture medium of Saccharomyces cerevisiae, a culture of Lactobacillus plantarum or a culture of Bacillus subtilis, fermenting at a temperature of 20 to 50 ° C and a pH of 3 to 9; And c3) removing the culture medium and then filtering the cacao nymph extract. [12] The method of claim 12, wherein the step c) comprises: c1) drying and pulverizing the steamed cacao nips; c2) shaking the mixture at a temperature of 60 to 150 DEG C for 10 minutes to 2 hours; And c3) adding 5 to 30 times the weight of 70% ethanol to the mixture at room temperature for 1 to 24 hours.