KR102390656B1 - A cosmetic composition comprising chlorella extract for skin-whitening - Google Patents

Abstract

The present invention provides a cosmetic composition for skin whitening comprising a chlorella extract as an active ingredient.

Description

A cosmetic composition for skin whitening containing chlorella extract as an active ingredient {A COSMETIC COMPOSITION COMPRISING CHLORELLA EXTRACT FOR SKIN-WHITENING}

The present invention relates to a cosmetic composition containing a chlorella extract as an active ingredient.

The skin is the most basic defense organ that protects the body from stimuli in contact with the external environment, and is an important organ that expresses external beauty.

Since external beauty is not only a standard of judgment symbolizing youth and health, but also a competitive advantage that affects work ability by expressing confidence, the tendency to take care of skin is increasing in modern society.

As the industry develops, environmental pollution becomes serious, and interest in skin aging prevention, whitening, and the like is rising.

In particular, human skin constantly undergoes changes, and the most representative changes are deterioration of skin function and loss of visual beauty due to aging. The final skin appearance phenomenon that appears as a result of skin aging includes the formation of wrinkles, deterioration of skin elasticity, and the formation of senile spots, and the formation of wrinkles is one of the most representative phenomena.

Lipids, proteins, polysaccharides, nucleic acids, etc., which are major constituents of the skin, are oxidized, and skin cells and tissues may be destroyed, and eventually the skin may age.

In particular, due to the oxidation of proteins, collagen, hyaluronic acid, elastin, proteoglycan, fibronectin, etc., which are connective tissues of the skin, are cut and an excessive inflammatory reaction may appear. Induction of cancer may be promoted.

Therefore, it is important to protect cell membranes by removing reactive oxygen species generated during the body's metabolic process, UV irradiation, and inflammatory reactions, and to restore skin health by regenerating damaged cells through active metabolism. desirable.

On the other hand, melanin (Melanin) refers to the black or brown pigment present in the skin, hair, and the like. Melanin exists in the skin, protects the skin by blocking UV rays, and maintains the body temperature of the skin.

Melanin is converted from tyrosine to dopa and dopaquinone by the action of tyrosinase present in pigment cells, and is produced through dopachrome and the like. Melanin plays a role in protecting the body from harmful factors such as UV rays and regulating the secretion of hormones in the body.

However, melanin is overproduced and can promote the formation of spots, freckles, and skin aging, and can be involved in the induction of skin cancer.

Recently, the cosmetic industry is developing a number of products using natural products to reduce skin irritation caused by various chemicals.

Natural ingredients have fewer side effects on the skin, and their development value as a cosmetic raw material is gradually increasing as consumers' response to cosmetics using natural ingredients increases.

As described above, the present inventors tried to develop a cosmetic composition with excellent functionality using a natural extract in a situation in which the need for the development of cosmetic products derived from natural products with excellent biosafety and skin improvement effects is constantly emerging.

The present invention is to solve the problems of the prior art described above, an object of the present invention is to provide a functional cosmetic composition comprising a chlorella extract.

According to one aspect of the present invention, there is provided a cosmetic composition comprising a chlorella extract as an active ingredient.

In one embodiment, the chlorella is Chlorella sp . It may be HS-V (Accession No.: KCTC 13850BP).

In one embodiment, the cosmetic composition may further include a barley sprout extract.

In one embodiment, the chlorella extract may be stabilized by a water-in-oil (O / W) emulsion.

In one embodiment, the diameter of the emulsion may be 1.0 μm or less.

In one embodiment, the cosmetic composition may be for skin whitening or skin wrinkle improvement.

In one embodiment, the cosmetic composition may be for antioxidant or anti-inflammatory.

In one embodiment, the cosmetic composition is skin lotion, skin softener, skin toner, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, foundation, essence, nourishing essence, pack , it may be formulated with one or more selected from the group consisting of soap, cleansing foam, cleansing lotion, and cleansing cream.

The cosmetic composition according to one aspect of the present invention contains abundant minerals and calcium components and has excellent skin improvement effect.

It should be understood that the effects of the present invention are not limited to the above-described effects, and include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

1 is a Chlorella sp. using a plate-type photobioreactor. Results of outdoor culture of HS3.
2 is a result of evaluating the cytotoxicity in melanocytes of the chlorella extract according to an embodiment of the present invention.
3 is a result of evaluating the melanogenesis inhibitory efficacy of the chlorella extract according to an embodiment of the present invention.
4 is a result of evaluating the MITF mRNA expression inhibitory efficacy of the chlorella extract according to an embodiment of the present invention.
5 is a result of evaluating the effect of Tyr mRNA expression inhibition of the chlorella extract according to an embodiment of the present invention.
6 is a result of evaluating the effect of Tyrp-1 mRNA expression inhibition of the chlorella extract according to an embodiment of the present invention.
7 is a result of evaluating the effect of Tyrp-2 mRNA expression inhibition of the chlorella extract according to an embodiment of the present invention.
8 is a result of evaluating the CREB phosphorylation inhibitory effect of the chlorella extract according to an embodiment of the present invention.
9 is a result of evaluating the NF-kb activity inhibitory efficacy of the chlorella extract according to an embodiment of the present invention.

The terms used in this specification have been selected as currently widely used general terms as possible while considering the functions in the present invention, but these may vary depending on the intention or precedent of a person skilled in the art, the emergence of new technology, and the like.

In addition, in a specific case, there is a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the corresponding invention. Therefore, the term used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, rather than the name of a simple term.

Unless defined otherwise, all terms used herein, including technical and scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not

Numerical ranges are inclusive of the values defined in that range. Every maximum numerical limitation given throughout this specification includes all lower numerical limitations as if the lower numerical limitation were expressly written. Every minimum numerical limitation given throughout this specification includes all higher numerical limitations as if the higher numerical limitation were expressly written. Any numerical limitation given throughout this specification shall include all numerical ranges within the broader numerical range, as if the narrower numerical limitation were expressly written down.

Hereinafter, examples of the present invention will be described in detail, but it is obvious that the present invention is not limited by the following examples.

One aspect of the present invention provides a cosmetic composition comprising a chlorella extract as an active ingredient.

The Chlorella is a kind of freshwater algae belonging to the Chlorella family of green algae plants, and is commonly found in green freshwater and wetlands, and has two growth properties: autotrophic growth and proliferation by photosynthesis, and heterotrophic growth using organic carbon sources. As a single-celled plant with

The protein, which accounts for about 55% by weight of chlorella, contains not only essential amino acids evenly, but also contains a large amount of vitamins A and B groups and minerals, and is known as a health food. In addition, the chlorella has been studied to solve the human food problem in the early days because it has an excellent photosynthetic ability and is very fast and easy to cultivate compared to other plants.

Physiological actions of chlorella include detoxification of dioxins (Morita K., et al., J. Nutr., 129, pp1731-1736, 1999), cholesterol lowering action (Shibata S. et al., J. Nutr. Sci. Vitaminol. (Tokyo), 47(6), pp373-377, 2001; Sano T. et al., Artery, 14(2), pp76-84, 1987) were reported to have physiologically active effects, and chlorella extracts from the growth promoting effect on microorganisms (Kim Dong-ho, Food Journal, 1, pp64-69, 1998) and antibacterial activity enhancing effect (Takashi H., et al., International J. of Immunopharmacology, 22, pp877-885, 2000) ) has been reported.

The chlorella may be a chlorella sp. green algae, preferably chlorella minutissima , chlorella vulgaris , chlorella emersonii .

The "comprising as an active ingredient" may mean containing an effective amount that can exhibit a skin improvement effect, for example, skin whitening, anti-inflammatory, antioxidant effect, and the like.

The chlorella is Chlorella sp . HS-V (KCTC 13850BP).

The Chlorella sp. For HS-V, the present inventors selected a strain with the best activity among chlorella strains isolated from domestic rivers and obtained a large amount through an optimized culture method.

The chlorella is Chlorella sp . It was named HS-V, and it was deposited at the Korea Microorganism Conservation Center on May 21, 2019, and was given a deposit number KCTC 13850BP.

The extract is selected from the group consisting of water, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms, acetone, petroleum ether, ethyl acetate, butyl acetate, trichloromethane, dichloromethane, chloroform, hexane and 1,3-butylene glycol It can be extracted with one or more solvents.

The "extract" is a natural extract using various natural substances as extraction raw materials, and is an active ingredient contained in the extraction raw material by contacting the solvent and the extraction raw material under specific conditions. However, it can be included regardless of the type of ingredient.

For example, extracts of components soluble in solvents from natural products using water or organic solvents, specific components of natural products, for example, products obtained by extracting only specific components such as oil may be included. The extract may be powdered after drying, or obtained by various conventionally known extraction methods such as hot water extraction and ethanol extraction.

The natural extract is extracted by conventional methods such as reflux circulation extraction, pressure extraction, ultrasonic extraction, etc. for about 1 to 24 hours with a solvent in a volume equivalent to 10 to 20 times the weight of the raw material by washing the raw material for extraction with water, drying and pulverizing it And it can be prepared by filtration. In addition, the extract may be obtained in a powder state by an additional process such as distillation under reduced pressure or freeze-drying.

The extract may also include an extract that has undergone a conventional purification process. For example, the extract is separated using an ultrafiltration membrane having a constant molecular weight cut-off value, and various purification methods are additionally performed, such as separation by various chromatography (prepared for separation according to size, charge, hydrophobicity or affinity). It may include a fraction obtained through

In one embodiment, the chlorophyll present in the cell can be effectively eluted in the solvent by completely drying and pulverizing the chlorella.

Various useful components including chlorophyll can be extracted by mixing the chlorella and the solvent and heating at a temperature of 60 to 100° C. for 2 to 15 hours.

If the extraction temperature is less than 60 ℃, chlorophyll may not be effectively extracted into the solvent, and if it is more than 100 ℃, various useful components contained in chlorella may be denatured.

In addition, if the extraction time is less than 2 hours, a sufficient yield may not be realized for extraction, and if it is more than 15 hours, the process efficiency may be reduced.

The content of the chlorella extract may be 0.1 wt% to 10.0 wt% based on the total weight of the cosmetic composition.

If the content of the chlorella extract is less than 0.1% by weight, the skin improvement effect may not be sufficiently realized, and if it exceeds 10.0% by weight, the specific gravity of other components required for commercialization is reduced, thereby lowering the product's original marketability or cost-effectiveness can be lowered

The cosmetic composition may further include a barley sprout extract.

The “barley sprout” refers to a young shoot of barley ( Hordeumvulgare L. ), and refers to a young plant grown by about 10 to 20 cm after sprouting on barley. Typically, the barley sprout can be obtained by immersing barley in water for about 1 day, removing moisture, sown under conditions that prevent moisture loss, germinate, and then grow for 7 to 10 days.

The barley sprout can be used as it is, or after performing a pretreatment process in consideration of the process speed and process (manufacturing) efficiency intended by those skilled in the art, the pretreatment process includes, for example, conventional selection, washing with water, and cutting , powdering, drying, etc. may be performed. The barley sprout is known to promote growth hormone secretion to help growth, strengthen immune function, and have a strong antioxidant action.

Meanwhile, the chlorella extract may be stabilized by a water-in-oil (O/W) emulsion.

The water-in-oil (O/W) emulsion may contain 60 wt% or less of polyol, 1 to 10 wt% of an emulsifier, 20 wt% or less of oils, and 5 wt% or less of additives.

The polyol may be one or more selected from the group consisting of Glycerin, 1,3-Butyleneglycol, 1,2-Hexandiol, Methylenepropandiol, Propanediol, Propyleneglycol, and Pentyleneglycol, but is not limited thereto.

The emulsifiers include Lecithin, Hydrogenated Lecithin, Polyglyceryl-3 Methylglucose Distearate, Glyceryl Stearate/PEG-100 Stearate, cetyl stearyl alcohol, Stearic acid, Palmitic acid, Lauric acid, Cetearyl Olivate/sorbitan olivate, Ceteareth-6 Olivate, Olive Oil Peg- 7 Esters, PEG-60 Hydrogenated castor oil, PEG-40 Hydrogenated castor oil, Polysorbate 80, Polysorbate 60, C14-22 Alcohols/C12-20 Alkyl Glucoside, Cetearyl Alcool/Cetearyl Glucoside, and at least one selected from the group consisting of Bees wax may be, but is not limited thereto.

The oil may be selected from the group consisting of Olive oil, Macadamianut oil, Caprylic/capric triglyceride, Dimethicone, Sunflower oil, Grape seed oil, and Sweet almond oil, but is not limited thereto.

The additive may be one or more selected from the group consisting of Ethylhexyleglycerin, Phenoxyethanol, Xanthan gum, Guar gum, Cellulose, Carbomer, Carrageenan, Hyaluronic acid, Topherol E acetate, and Ascorbic acid, but is not limited thereto.

The diameter of the emulsion may be 1.0 μm or less so that the penetration power of the contained active ingredient can be maximized.

The emulsion is a step of heating the polyol to 60 ~ 70 ℃ (加溫), the step of completely dissolving by adding an emulsifier, oils and Chlorella sp. To be prepared through; adding HS-V and stirring, adding water to form an O/W emulsion, adding an additive, and adjusting the size of the O/W emulsion through HOMO MIXER A micro-emulsion with a diameter of 1.0 μm or less can be prepared by emulsifying for 3 minutes at 1,500 ~ 2,000 rpm with a HOMO MIXER based on 200 g of emulsion, and performing a maximum of 3 passes at 1,000 ~ 1,500 bar through a high pressure homogenizer.

The cosmetic composition may implement a skin whitening effect by suppressing melanin production.

The "skin whitening" suppresses the production of various biomolecules such as melanin, oxidation-reduction hemoglobin, carotene, and melanoids that affect skin color, thereby alleviating skin pigmentation phenomena such as blemishes, freckles, and spots, and It refers to the effect of improving skin brightness and uniformity by alleviating redness.

Since the cosmetic composition effectively inhibits melanin production, it has excellent whitening effect on the skin as well as removing blemishes in the skin.

The cosmetic composition may be used for improving wrinkles.

The “wrinkle improvement” refers to a phenomenon in which elastic fibers composed of elastin exist together with collagen, and skin elasticity is maintained or improved in a state in which the elastin and collagen are sufficiently present. In addition, the "wrinkle improvement" refers to a phenomenon in which wrinkles are inhibited or inhibited from being generated on the skin, or wrinkles that have already been created are alleviated.

The cosmetic composition may be for antioxidant or anti-inflammatory.

The “antioxidation” refers to the action of inhibiting oxidation, and the human body has a balance between an oxidation promoting substance and an oxidation inhibitory substance. , oxidative stress is induced in the living body, which may cause cell damage and pathological diseases.

Reactive oxygen species (ROS), the direct cause of oxidative stress, are chemically unstable and highly reactive, so they can easily react with various biological materials such as DNA, proteins, lipids and carbohydrates, and attack macromolecules in the living body. It causes irreversible damage to cells and tissues, mutation, cytotoxicity, cancer, etc., and is also a direct cause of aging. By removing or reducing the reactive oxygen species, it is possible to obtain an antioxidant effect to prevent aging and maintain health.

The “anti-inflammatory” refers to the action of inhibiting inflammation, and the regulation of the inflammatory response is known to be very complex, which reduces the enhancement and damage of the repair system in vivo.

If the inflammatory response continues due to repeated tissue damage or regeneration, ROS and RNS are overproduced in inflammation-related cells, and permanent gene modification can be caused. That is, ROS and RNS are deeply related to the inflammatory response that regulates the actions of various cells in vivo.

The cosmetic composition may exhibit excellent anti-inflammatory activity by regulating the expression of inflammatory mediators generated due to the activation of NF-kB.

The cosmetic composition includes skin lotion, skin softener, skin toner, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, foundation, essence, nourishing essence, pack, soap, cleansing foam, It may be formulated with one or more selected from the group consisting of a cleansing lotion, and a cleansing cream.

The cosmetic composition may be formulated by a conventional method. In the formulation of external skin preparations, reference may be made to the contents disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA, and in the formulation of cosmetic compositions, International cosmetic ingredient dictionary, 6th ed (The cosmetic, Toiletry and Fragrance Association) , Inc., Washington, 1995) may be referred to.

Specifically, the cosmetic composition can be prepared in general emulsified formulations and solubilized formulations. For example, lotion, such as a softening lotion or a nourishing lotion; emulsions such as facial lotions and body lotions; creams such as nourishing creams, moisturizing creams, and eye creams; essence; makeup ointment; spray; gel; pack; sunscreen; makeup base; foundations such as liquid type, solid type or spray type; powder; makeup removers such as cleansing creams, cleansing lotions, and cleansing oils; Alternatively, it may be formulated as a cleaning agent such as a cleansing foam, soap, body wash, but is not limited thereto. In addition, the external preparation for skin may be formulated as an ointment, patch, gel, cream or spray, but is not limited thereto.

In the cosmetic composition, in addition to the essential components in each formulation, other components may be appropriately blended within the range that does not impair the purpose according to the present invention according to the type or purpose of use, etc. of the formulation.

The cosmetic composition may include a conventionally acceptable carrier, for example, oil, water, surfactant, humectant, lower alcohol, thickener, chelating agent, colorant, preservative, fragrance, etc. may be appropriately blended, but is limited thereto not.

The acceptable carrier may vary depending on the formulation. For example, when formulated into an ointment, paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide or their Extracts may be used.

When the cosmetic composition is formulated as a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder or extracts thereof may be used as a carrier component, and in the case of a spray, chlorofluorohydro It may further contain a propellant such as carbon, propane, butane or dimethyl ether.

When the cosmetic composition is formulated as a solution or emulsion, a solvent, solubilizer, or emulsifier may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl benzoate, propylene glycol, 1,3-Butylglycol oil may be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol fatty esters, fatty acid esters of polyethylene glycol or sorbitan may be used.

When the cosmetic composition is formulated as a suspension, a liquid diluent such as water, ethanol or propylene glycol as a carrier component, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester; Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth and the like can be used.

When the cosmetic composition is formulated as a soap, alkali metal salt of fatty acid, fatty acid hemiester salt, fatty acid protein hydrolyzate, isethionate, lanolin derivative, aliphatic alcohol, vegetable oil, glycerol, sugar, etc. can be used

The cosmetic composition is a fatty substance, organic solvent, solubilizer, thickening agent, gelling agent, softening agent, antioxidant, suspending agent, stabilizing agent, foaming agent, and fragrance commonly used in the industry depending on the quality or function of the final product. , surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, commonly used in cosmetics It may additionally contain adjuvants commonly used in the field of cosmetics or dermatology, such as any other ingredients.

However, the adjuvant and its mixing ratio may be appropriately selected so as not to affect the desirable properties of the cosmetic composition according to the present invention.

Through the following examples, the present invention will be described in more detail, but it is obvious that the present invention is not limited by the following examples.

Preparation Example 1: Preparation of Chlorella Extract

Chlorella sp. HS-V strain (KCTC 13850BP) was isolated as an isolated strain with remarkable activity in domestic rivers.

The dilution ratio was 0.33/day with a working volume of 3L in a 5L incubator.

Complex fertilizer (High N S-feed, Farm Hannong) 1 g/L, yeast extract (BD BactoTM Yeast extract, Cat. No. 212750) 1 g/L, glucose (Sigma-Aldrich, Cat. No. G7021) ) was added to distilled water at a concentration of 20 g/L, and stirred at a temperature of 30 ° C., a light quantity of 120 μmol photons/m ² /s, and 120 rpm per minute to obtain Chlorella sp. HS-V strains were cultured.

Cultured Chlorella sp. HS-V was harvested at 10,000 rpm using a continuous centrifuge, and the harvested microalgal solids were freeze-dried (-80° C., 72 h) to obtain dry biomass.

Chlorella sp. 100 g of HS-V dry biomass was placed in 1 kg of 70% (w/w) ethanol and extracted in an ultrasonic extractor at 60° C. for 2 hours. After low-temperature immersion at 4° C. for 72 hours, it was filtered through a 250 mesh filter cloth.

After the solvent was completely removed from the filtrate in a vacuum rotary evaporator, an extract was obtained.

Preparation Example 2: Chlorella Extract Encapsulation

A water-in-oil emulsion was prepared by using the solubility in MCT of the crude extract from which ethanol was completely removed.

Polyol, Glycerin 50% parts by weight, 1,3-Butyleneglycol 10 parts by weight, Propylene glycol 10 parts by weight was heated to 60°C, and then, as an emulsifier, Lipoid S 75 1.5 parts by weight, Tego care 450 3 parts by weight, Palmitic acid 0.3 parts by weight , Stearic acid 0.2 parts by weight, Chlorella sp. 1.0 parts by weight of HS-V extract, 9.0 parts by weight of MCT, 14.9 parts by weight of purified water, and 0.1% by weight of antioxidant (Tocopherol E Acetae) were mixed.

After emulsifying at 1,500~2,000 rpm with HOMO MIXER to control the dispersion power and size of the O/W emulsion, 3 passes are performed under 1,000 bar conditions through a high pressure homogenizer to determine the size of the O/W emulsion. It was adjusted to 1 μm or less.

Preparation Example 3: Preparation of barley sprout extract

100 g of dried and crushed barley sprouts were mixed in 1 L of 70% ethanol at a ratio of 10% by weight.

The mixed raw materials were extracted at 60° C. for 2 hours using an ultrasonic extractor, and then filtered with 3.0 μm filter paper to obtain a liquid extract.

A barley sprout extract was obtained from the obtained liquid extract using a rotary vacuum concentrator and a freeze dryer.

Comparative Preparation Example: Preparation of Chlorella Extract

Chlorella ( Chlorella sp. ) Raw powder was provided from Daesang Co., Ltd. Gunsan plant.

Chlorella Powder 100 g of 70% (w/w) ethanol was put into 1 kg and extracted in an ultrasonic extractor at 60° C. for 2 hours. After immersion at 4° C. for 72 hours, it was filtered through a 250 mesh filter cloth.

After the solvent was completely removed from the filtrate in a vacuum rotary evaporator, an extract was obtained.

Examples and Comparative Examples

In order to verify the skin improvement effect of the obtained sample, the sample was mixed as follows to set Examples and Comparative Examples.

[Content (parts by weight)] division Example comparative example One 2 3 4 One 2 Chlorella Extract
(Production Example 1) 100 50 - - - - Chlorella extract (Preparation Example 2) - - 100 50 - - Chlorella Extract
(Comparative manufacturing example) - - - - 100 - Barley sprout extract (Preparation Example 3) - 50 - 50 - 100

Experimental Example 1: Skin safety test

B16F10 melanoma cells were seeded in a 96-well plate at 2×10³ cells/well and cultured for 24 hours. Then, the test substance was treated with an appropriate concentration and cultured for 48 hours.

WST-1 assay solution (EZ-CYTOX, DOGEN, Korea) was added at a rate of 10% of the medium and reacted at 37°C for additional 0.5 to 1 hour, followed by Microplate reader system (SpectraMax® i3x Multi-Mode Detection Platform, Molecular Devices, Sunnyvale, CA, USA) was used to measure absorbance at 450 nm.

Reference absorbance was measured at 650 nm and the result was corrected. The results are expressed as the mean ± standard deviation from three independent experiments.

P<0.05 was verified through Student's t-test, and the significance of the results was indicated. From the result value, the concentration section in which the cell viability according to the test substance treatment did not decrease compared to the negative control group was set as the concentration of the subsequent experiment.

Referring to Figure 2, Chlorella sp. HS-V extract showed toxicity to B16F10 melanocytes at 75 μg/mL or more, and 50 μg/mL was set as the optimal concentration.

Experimental Example 2: Measurement of melanin biosynthesis inhibitory activity

Measurement of the amount of melanin pigment production confirmed the degree of melanin production in melanocytes through the melanin contents analysis method.

Melanin-forming cells were aliquoted at 2× ^{10 5} cells/dish in a 60 mm culture dish and cultured for 24 hours.

After melanin production was induced with α-MSH (alpha-melanocyte-stimulating hormone; Sigma-Aldrich), which is a melanin production inducer, it was treated with a certain concentration of the test substance and further cultured for 48 hours.

After harvesting the cultured cells, the color change of the pellet of melanocytes was observed, and cell lysis and centrifugation were performed by adding 150 μL 1N NaOH solution.

Only the supernatant was taken and dispensed in a 96-well plate, and absorbance was measured at 490 nm using a Microplate reader system (SpectraMax ^® i3x Multi-Mode Detection Platform, Molecular Devices, Sunnyvale, CA, USA).

Arbutin was used as a positive control in the melanin production confirmation test. The measured intracellular melanin production was compared and analyzed using the calibration curve of the standard solution prepared above, and the total protein amount was calculated using the BCA Protein assay Kit (Thermo Fisher Scientific, MA, USA).

The amount of melanin production was converted to the amount of melanin per certain protein, and as the measured value decreased, it means that there was an inhibitory effect on melanin production in melanocytes. The results are expressed as the mean ± standard deviation from three independent experiments.

P<0.5 was verified through Student's t-test, and the significance of the results was indicated.

Referring to Figure 3, Chlorella sp. When HS-V extracts 5, 10, 25, and 50 μg/mL were treated, melanin biosynthesis in B16F10 melanogenic cells was decreased in a concentration-dependent manner of the extract.

Experimental Example 3: Measurement of gene expression inhibition activity of enzymes related to melanin biosynthesis

Quantitative real-time PCR (quantitative real-time PCR) (quantitative real-time PCR ( qRT-PCR) was confirmed through analysis.

B16F10 melanocytes were aliquoted in a 6-well culture plate at 2×105 cells/well and cultured for 24 hours.

The test substance was treated with a certain concentration and further cultured for 24 hours. After harvesting the cultured cells, cell lysis and total mRNA extraction were performed by adding 1 mL TRIzol reagent (Life Technologies).

The concentration and purity of total mRNA (A260/A280 and A260/A230 ratio) were verified using a MaestroNano ^*?* microvolume spectrophotometer (Maestrogen, Las Vegas, NV, USA), and only total mRNA with a purity of 2.0 or higher was selected.

Total mRNA was substituted with cDNA using M-MLV reverse transcriptase (Life Technologies) and used for qRT-PCR.

qRT-PCR was performed using HOT FIREPol EvaGreen PCR Mix Plus (Solis BioDyne, Estonia), and the expression of the corresponding gene was analyzed using Line-Gene K software (Bioer Technology Co. Ltd., Hangzhou, China).

The PCR reaction conditions were denaturation at 94°C for 5 minutes, followed by denaturation (94°C, 30 seconds), annealing (60°C, 30 seconds), and polymerization (72°C, 30 seconds) processes for 40 cycles. Changes in gene expression were measured in comparison with the expression level of the β-ACTIN control gene.

The Cт values of tyrosinase, Tyrp-1, and Tyrp-2 were normalized to the Cт values of β-ACTIN, and the relative expression levels of COL1A1 and MMP1 were calculated through the 2-ΔΔCt method.

The results are expressed as the mean ± standard deviation from three independent experiments. P<0.5 was verified through Student's t-test, indicating the significance of the results.

Referring to Figure 3, Chlorella sp. When HS-V extracts 5, 10, 25, and 50 μg/mL were treated, melanin biosynthesis in B16F10 melanogenic cells was decreased in a concentration-dependent manner of the extract.

4 to 7, Chlorella sp. When the HS-V extract was treated with 25 and 50 μg/mL, MITF, tyrosinase, TyRP-1, and Tyrp-2 mRNA expression in B16F10 melanogenic cells was reduced in a concentration-dependent manner.

Experimental Example 4: Measurement of PKA, CREB phosphorylation inhibitory activity

B16F10 melanogenic cells were seeded in a 60-mm plate at 3 × 105 cells/well and then stabilized in an incubator for 24 hours. α-MSH (100 nM) and samples were treated at a concentration of 5 μg/mL and further cultured in an incubator.

Cells were recovered, washed with PBS, centrifuged at 4°C and 12,000 rpm, and the cell pellet obtained was lysed by heating twice at 95°C for 10 minutes with 1% SDS lysis buffer.

5X SDS sample buffer containing 1 M Tris-HCl (pH 6.8), 50% glycerol, 25% β-mercaptoethanol, 10% SDS, and 1% bromophenol blue was added and heated at 95℃ for 10 minutes, followed by 12% SDS After electrophoresis on polyacrylamide gel, it was transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA).

As the primary antibody, Mitf, Tyrosinase, β-Actin (Santa Cruz, CA, USA) and p-CREB, CREB, p-PKA, PKA (Cell Signaling, Beverly, MA, USA) were diluted and used.

The secondary antibody was used by diluting anti-mouse IgG-HRP, anti-rabbit IgG-HRP (Cell Signaling) and bovine anti-goat-HRP (Santa Cruz).

To check the protein expression level, the membranes were treated with Clarity Western ECL Substrate (BioRad, Hercules, CA, USA) solution, and the results were analyzed through ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA).

Referring to Figure 8, Chlorella sp. When the HS-V extract was treated with 25 and 50 μg/mL, the phosphorylation of CREB and PKA in B16F10 melanocytes decreased in a concentration-dependent manner of the extract.

Experimental Example 5: Measurement of NF-κB activity inhibition

In this test, the NF-кB promoter luciferase assay was used to evaluate the inhibitory ability of the NF-кB activity for the test substance.

NF-кB Luciferase Reporter NIH3T3 stable cells were aliquoted in a 6-well culture plate at 1.5×105 cells/well and cultured for 24 hours. After pre-treatment with a certain concentration of the test substance for 24 hours, it was treated with TNF-α (50 ng/mL), an inflammation inducer, and further cultured for 8 hours.

After harvesting the cultured cells, cell lysis and centrifugation were performed by adding a passive lysis buffer (Promega, USA) to extract intracellular luciferase.

After taking only the supernatant, dispensing it into a black 96-well plate, adding luciferin (Promega, USA), and measuring the luminescence level of luciferin using a Microplate reader system (SpectraMax® i3x Multi-Mode Detection Platform; Molecular Devices, USA) did

The measured luciferin luminescence level was calculated using the BCA Protein Assay Kit (Thermo) to calculate the total amount of protein. NF-кB luciferase activity was converted to the degree of luciferin luminescence per certain protein, and as the measured value decreased, it means that there was an inhibitory effect on NF-кB activity in NF-кB Luciferase Reporter NIH3T3 Stable Cells.

The results are expressed as the mean ± standard deviation from three independent experiments. P<.05 was verified through Student's t-test, indicating the significance of the results.

Referring to Figure 9, Chlorella sp. When the HS-V extract was treated with 50 μg/mL, the NF-κB activity was decreased.

Experimental Example 6: Comparative measurement of skin improvement activity

The skin improvement activity of the samples of Examples and Comparative Examples was comparatively evaluated at the same concentration (50 μg/mL). The skin improvement activity was evaluated in the same manner as in Experimental Examples 2 to 6 (Table 2).

division Example comparative example One 2 One 2 Melanin biosynthesis inhibitory activity (Experimental Example 2) 162.2 155.7 220.2 230.5 Efficacy of inhibiting MITF mRNA expression (Experimental Example 3) 208 185 335 376 Tyr mRNA expression inhibition efficacy (Experimental Example 3) 110 98 170 177 Tyrp-1 mRNA expression inhibition efficacy (Experimental Example 3) 72 58 135 152 Tyrp-2 mRNA expression inhibition efficacy (Experimental Example 3) 70 61 124 155 Efficacy of inhibiting NF-kb activity (Experimental Example 5) 435 357 590 683

The above results were obtained from Chlorella sp. HS-V has significantly superior skin improvement activity compared to conventional chlorella, suggesting that whitening activity and anti-inflammatory activity can be further improved when simultaneously applied with barley sprout.

Experimental Example 7: Measurement of skin wrinkle improvement activity

In order to evaluate the skin wrinkle improvement effect, an experiment was conducted as follows.

60 adult women over 30 years of age were divided into 6 groups of 10 people each, and the test subject was thoroughly absorbed by applying the formulated cosmetic to the face after cleansing every morning and evening for 4 weeks.

At this time, in Examples 3 and 4, the chlorella extract was formulated as a water-in-oil emulsion according to Preparation Example 2, and the remaining samples were formulated as a conventional water type.

ANTERA 3D (Miravex, Ireland) was applied to evaluate wrinkle improvement, and device measurements were made before, 2 weeks, and 4 weeks after use of the test substance.

The same test person measured the left corner of the eye of all subjects, and for reproducibility of the measurement, the same part was measured by overlapping the image measured before using the test substance.

The captured images were matched using ANTERA pro software, which is a dedicated software for ANTERA 3D, and then the matched measurement area was used for analysis.

The measured value was analyzed using the indentation index, which is a measurement variable, to analyze the Wrinkles small value, which indicates the wrinkles of the skin.

The amount of change in Wrinkles small value after 4 weeks of the test subject according to the use of cosmetics formulated with the samples of Examples and Comparative Examples is shown (Table 3). The greater the wrinkle improvement effect, the greater the change in the Wrinkles small value.

division Wrinkles small average Example 1 1.35 Example 2 1.38 Example 3 1.43 Example 4 1.47 Comparative Example 1 1.19 Comparative Example 2 1.07

Referring to Table 3, the skin wrinkle improvement effect of the cosmetic composition formulated with the samples of Examples 1 to 4 was remarkably excellent, and in particular, the skin wrinkle improvement effect of Examples 3 and 4 formulated with a water-in-oil emulsion was even better did

Experimental Example 8: Measurement of skin brightness improvement activity

Cosmetics containing the samples of Examples and Comparative Examples were prepared, and 60 subjects were divided into 10 groups and applied to each, then the change in skin brightness was confirmed.

At this time, in Examples 3 and 4, the chlorella extract was formulated as a water-in-oil emulsion according to Preparation Example 2, and the remaining samples were formulated as a conventional water type.

After washing the face before taking it, a formulated cosmetic was applied, and it was used every day for 3 weeks. Skin brightness was checked with a LAB measuring device (Konica Minolta CR-300 Chroma Meter).

The difference (L*) in skin color between the measurement time and the application start time after application of each test substance was calculated according to Equation 1 below, and the results are shown in Table 4 below.

The control group was formulated except for the samples of Examples and Comparative Examples, and arbutin was added to the positive control group instead of the samples.

The whitening effect is determined by comparing the L* between the sample application site and the control site. If the L* value is about 2, whitening of the deposited pigment is clear, and if it is about 1.5 or more, it can be determined that there is a whitening effect.

[Equation 1]

ΔL = L value at the time of measurement after application - L value at the beginning of application

division Brightness change after 3 weeks (ΔL) control - positive control 2.8 Example 1 2.3 Example 2 2.6 Example 3 2.8 Example 4 2.9 Comparative Example 1 1.8 Comparative Example 2 1.6

Referring to Table 4, the cosmetic compositions formulated with the samples of Examples 1 to 4 effectively improved skin brightness, and the skin improvement activities of Examples 3 and 4 formulated with a water-in-oil emulsion were even better.

Experimental Example 9: Formulation stability evaluation

The emulsion stability of the emulsion composition encapsulated according to Preparation Example 2 was evaluated. As a result of the evaluation, layer separation did not occur for 4 weeks.

The discoloration stability of the emulsion composition of Preparation Example 2 was evaluated. As a result of the evaluation, no discoloration occurred for 4 weeks.

Lutein stability of the emulsion composition of Preparation Example 2 was evaluated. Analysis of the component content of lutein was performed using HPLC equipment, and was not performed when layer separation and discoloration occurred.

In the preparation of the emulsion composition according to Preparation Example 2, as encapsulation is performed, colloids are formed, and for stabilization of encapsulation, nano-sized micelles are stabilized through a high-pressure homogenizer. made it

The encapsulated emulsion composition was diluted in 0.1% (w/w) purified water and the particle size was measured with DLS (Dynamic Light Scattering) equipment, and as a result, it was measured to be about 120 nm to 1 μm.

In addition, the narrower the particle size distribution, the more proportional to the stability of the emulsion.

The description of the present invention described above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. For example, each component described as a single type may be implemented in a dispersed form, and likewise components described as distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present invention.

Name of deposit institution: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC13850BP

Deposit date: 2019521

Claims (8)

Contains chlorella extract as an active ingredient,
The chlorella is Chlorella sp. HS-V (Accession No.: KCTC 13850BP),
The chlorella extract is stabilized by a water-in-oil (O/W) emulsion having a diameter of 1.0 μm or less, a cosmetic composition for skin whitening and wrinkle improvement. delete The method of claim 1,
A cosmetic composition further comprising a barley sprout extract. delete delete delete The method of claim 1,
A cosmetic composition further comprising an antioxidant or anti-inflammatory use. The method of claim 1,
Skin lotion, skin softener, skin toner, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, foundation, essence, nourishing essence, pack, soap, cleansing foam, cleansing lotion, and A cosmetic composition formulated with one selected from the group consisting of cleansing cream.

Images