KR101900762B1 - A cosmetic composition comprising extract of nannochloropsis oceanica - Google Patents

Abstract

The present invention relates to the use of Nannochloropsis < RTI ID = 0.0 > The present invention also provides a cosmetic composition comprising, as an active ingredient,

Description

TECHNICAL FIELD [0001] The present invention relates to a cosmetic composition containing an extract of Nanochloprobus cisse aniina as an active ingredient,

The present invention relates to a cosmetic composition comprising a microalgae extract as an active ingredient.

There are two types of skin aging. Intrinsic aging means aging that can not be avoided as time goes by. On the other hand, photoaging refers to aging phenomenon observed in skin such as a face, a back of a hand, and a back of a neck exposed to sunlight for a long time, and is caused by interaction between the inner aging and the aging due to ultraviolet rays.

Photoaging has clinical characteristics that are deeper and wrinkle-inducing than intrinsic aging. Skin exposed to sunlight can increase pigmentation disorders such as irregular pigmentation and solar lentigo, which may result in decreased elasticity and skin sagging due to reduced moisture.

The causes of skin wrinkles have not yet been clarified, but damage to matrix proteins such as collagen and elastic fibers in the skin has been reported as a major cause. The distribution and movement of muscles in the face, genetic susceptibility, ultraviolet rays, Menopause, oxidative damage, and heat.

Ultraviolet rays reduce the number of immune cells, disrupt the skin immune system, change the genetic material DNA to cause skin cancer, and skin structure protein break down collagen, elastin, etc., Increasing ultraviolet radiation exposure due to destruction of the ozone layer as the greatest risk of skin health promotes the development of various types of functional cosmetics related to ultraviolet blocking and absorption.

Ultraviolet rays are classified into ultraviolet C (200-290 nm), B (290-320 nm) and A (320-400 nm) depending on the wavelength. It is fatal but does not reach the surface because it is 100% absorbed by the ozone layer, so there is no serious concern about the hazard. On the other hand, ultraviolet ray B is known to be a direct cause of sunburn which is frequently experienced when exposed to sunlight for a long time, and ultraviolet ray A penetrates into the dermal layer of skin, thereby causing skin cancer and aging.

UV blocking materials can be divided into organic and inorganic based on their mechanism of action. Organic blocking agents are based on chemicals (eg Avobenzone, Octylmethoxycinnamate, Benzophenone) that effectively absorb ultraviolet rays, and inorganic blockers scatter or absorb ultraviolet rays, (Eg, TiO ₂ , ZnO, and Fe ₂ O ₃ ) that can function as a metal oxide.

However, conventional organic or inorganic UV blocking materials have various problems such as lowering of stability in cosmetic formulations or causing skin troubles, and safety, efficacy and stability of sunscreen agents are important topics of cosmetics.

The cosmetics industry is developing a number of products using natural products to reduce skin irritation caused by various chemicals. In addition to low adverse effects on skin, natural materials have recently been increasingly developed as cosmetic raw materials, as consumers have become more receptive to cosmetics using natural materials. However, a cosmetic product containing a natural-derived extract obtained by a conventional method has not been sufficiently functionalized, such as photoaging effect, and the skin improving activity is not maintained constantly.

Accordingly, there is a continuing need to develop cosmetic products derived from natural products, which have excellent functionalities and excellent biostability and skin improving effect.

Disclosure of Invention Technical Problem [8] The present invention has been made to solve the above-mentioned problems of the prior art, and an object of the present invention is to provide a cosmetic composition having not only functionality but also biostability.

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

According to another aspect of the present invention, there is provided a cosmetic composition comprising, as an active ingredient, Violaxanthin derived from Nanochloprobus cisse aniina extract.

In one embodiment, the extract is selected from the group consisting of water, anhydrous or lower alcohols having 1 to 4 carbon atoms, acetone, petroleum ether, ethyl acetate, butyl acetate, trichloromethane, dichloromethane, chloroform, Glycol, < / RTI >

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

In one embodiment, the cosmetic composition may be for preventing skin aging induced by ultraviolet light blocking or UVB.

In one embodiment, the cosmetic composition may be for skin soothing or skin irritation relief.

In one embodiment, the cosmetic composition is comprised of a softening agent, a nutritional lotion, a water cream, a nutritional cream, a massage cream, an essence, an ampoule, a gel, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, Lt; / RTI > may be formulated into one or more selected from the group.

According to the present invention, since the cosmetic composition contains an effective ingredient derived from microalgae, it has not only an ultraviolet shielding effect and a wrinkle-reducing effect but also an excellent human safety.

It should be understood that the effects of the present invention are not limited to the effects described above, but include all effects that can be deduced from the description of the invention or the composition of the invention set forth in the claims.

As used herein, the terminology used herein is intended to encompass all commonly used generic terms that may be considered while considering the functionality of the present invention, but this may vary depending upon the intent or circumstance of the skilled artisan, the emergence of new technology, and the like. Also, in certain cases, there may be a term selected arbitrarily by the applicant, in which case the meaning thereof 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, not on the name of a simple term, but on the entire contents of the present invention.

Unless defined otherwise, all terms used herein, including technical or 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 commonly used dictionaries are to be interpreted as having a meaning consistent with the contextual meaning of the related art and are to be interpreted as either ideal or overly formal in the sense of the present application Do not.

The numerical range includes numerical values defined in the above range. All numerical limitations of all the maximum numerical values given throughout this specification include all lower numerical limitations as the lower numerical limitations are explicitly stated. All the minimum numerical limitations given throughout this specification include all higher numerical limitations as the higher numerical limitations are explicitly stated. All numerical limitations given throughout this specification will include any better numerical range within a broader numerical range, as narrower numerical limitations are explicitly stated.

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

According to one aspect of the present invention, there is provided a cosmetic composition comprising an extract of Nannochloropsis oceanica as an active ingredient. In addition, the cosmetic composition may contain, as an active ingredient, Violaxanthin derived from Nanochloprobus cisse aniina extract.

The cosmetic composition contains an extract derived from a natural microalgae as an active ingredient, so that it is excellent in skin improving effect and biosafety, and skin irritation can be minimized.

The cosmetic composition of the present invention is excellent in skin wrinkles and skin moisturizing effects as well as skin aging and skin irritation mitigating effects due to ultraviolet rays due to various active ingredients contained in nanochloprobasis oseanica. In particular, Violaxanthin, which is abundantly contained in nanocloprobasis oseanica, can alleviate the skin irritation induced by ultraviolet rays and effectively improve the damaged skin condition.

The " Nannochloropsis " oceanica "is a microalgae of the sea-habitat system of Nanochloropsis sp., which contains a relatively high lipid content. The nanochlroboscis oseanica extract contains a large amount of essential amino acids and polyunsaturated fatty acids and, in addition, it contains various active ingredients and can exhibit various skin improving effects such as wrinkle improvement and protection of skin cells.

The above-mentioned "Violaxanthin" is a carotenoid-based xanthophyll-based coloring material having molecular formula C ₄ OH ₅₆ O ₄ and an orange prism crystal having a melting point of 200 ° C., and is mainly distributed in higher plants and animals, green algae and diatoms. The present inventors have confirmed the photoaging prevention effect and various skin improving effects of violasanthin derived from the above-mentioned nanocholorobiossienica.

The above-mentioned " comprising as an active ingredient " means a degree capable of exhibiting a skin improving effect, for example, collagen synthesis associated with a wrinkle-reducing effect, prevention of aging of skin caused by improvement in elasticity or ultraviolet rays, alleviation of skin irritation, ≪ / RTI >

The above-mentioned "extract" refers to a solvent in which an effective ingredient contained in an extraction raw material has been transferred by contacting a solvent and an extraction raw material under specific conditions. If a substance is separated from a natural product, the extraction method, You can include them all regardless. For example, it may include extracts of components dissolved in a solvent from natural products by using water or an organic solvent, and specific components of natural products, such as oils obtained by extracting only specific components such as oil. The extract can be obtained by drying and then pulverizing the nanocloprosis oseanica, or various extraction methods known in the art such as a hot water extraction method and an ethanol extraction method.

Wherein the extract is selected from the group consisting of water, an anhydrous or lower alcohol having 1 to 4 carbon atoms, acetone, petroleum ether, ethyl acetate, butyl acetate, trichloromethane, dichloromethane, chloroform, hexane and 1,3-butylene glycol (V / v) of ethanol, and more particularly, it can be extracted with a concentration of 60 to 80% (v / v) ethanol.

The extraction ratio of the active ingredient contained in the raw material may be different depending on the polarity of the solvent. Since the ethanol is excellent in selectivity for extracting a physiologically active substance from a natural raw material, the ethanol extract can realize an optimal skin improving effect .

Since the water and ethanol have different polarities and the effective components to be extracted depend on the polarity, the concentration of ethanol can be appropriately controlled so that an optimal skin improving effect can be realized. At this time, if the concentration of ethanol is less than 60%, the effective ingredient showing skin improving effect may not be extracted sufficiently, and if it is more than 80%, an adequate yield may not be realized.

The extract is washed with water, dried and pulverized, and then extracted with a solvent having a volume of 8 to 12 times the weight of the raw material for about 1 to 24 hours by a conventional method such as circulation extraction, pressure extraction or ultrasonic extraction And filtering. In addition, the extract can be obtained in powder form by an additional process such as vacuum distillation or freeze-drying.

The extract may also contain an extract which has been subjected to a conventional purification process. For example, the extract may be subjected to various purification methods such as separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity) Lt; RTI ID = 0.0 > fractions. ≪ / RTI >

The cosmetic composition may be at least one selected from the group consisting of softening longevity, nutritional lotion, moisture cream, nutritional cream, massage cream, essence, ampoule, gel, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray and powder Can be formulated.

The cosmetic composition may be for wrinkle improvement or skin moisturizing.

The "wrinkles " refers to the scarring caused by degeneration of the skin, and may be caused by a cause of genes, a reduction of collagen and elastin present in the skin dermis, and an external environment. The "wrinkle improvement" refers to a phenomenon that inhibits or inhibits the generation of wrinkles on the skin, or alleviates the already generated wrinkles.

The term " skin moisturizing "means any action that maintains the homeostasis of the skin tissue by appropriately controlling moisture loss (moisture evaporation) of the skin and the like. The skin moisturizing effect can be achieved by the nanolocloprosis oseanica extract, in particular, violasanthin derived from the nanochloorobisis aniina extract. In addition, the skin moisturizing effect can be achieved by various aspects such as the exfoliation effect and the skin irritation reduction effect It may be accompanied by an additional skin improvement effect.

In addition, the cosmetic composition may be for preventing skin aging induced by ultraviolet blocking or UVB.

The above-mentioned " skin aging ", specifically, " photoaging " is an aging phenomenon caused by ultraviolet rays, which is an external environmental factor. The ultraviolet light activates the proteolytic enzyme, causing chain breakage of the protein and abnormal cross-linking, resulting in damage of the biocomponents, and repetition of the mechanism can cause apparent skin aging. The cosmetic composition can effectively improve skin aging induced by ultraviolet light, unlike endogenous aging caused by genetic factors regardless of the external environment.

The cosmetic composition may be at least one selected from the group consisting of softening lotion, astringent lotion, nutritional lotion, lotion, nutritional cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray, .

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

Specifically, the cosmetic composition may be prepared into a general emulsified formulation and a solubilized formulation. For example, lotion such as soft lotion or nutrition lotion; Lotion such as facial lotion, body lotion; Nourishing cream, moisture cream, cream such as eye cream; essence; Makeup ointment; spray; Gel; pack; Sunscreen; Makeup base; A foundation such as a liquid type, a solid type or a spray type; powder; Make-up removers such as cleansing creams, cleansing lotions and cleansing oils; Or a cleansing agent such as a cleansing foam, a soap, a body wash, and the like, but is not limited thereto. The external preparation for skin may be formulated into ointments, patches, gels, creams or sprays, but is not limited thereto.

The cosmetic composition of the present invention may be appropriately blended with other ingredients within the range of not compromising the object of the present invention depending on the kind of the formulation or the purpose of use, in addition to the essential ingredients in each formulation.

The cosmetic composition may contain a conventional acceptable carrier and may appropriately contain, for example, oil, water, a surfactant, a moisturizer, a lower alcohol, a thickener, a chelating agent, a colorant, no.

The acceptable carrier may vary according to the formulation. For example, when formulated into ointments, pastes, creams or gels, there may be used an animal oil, a vegetable oil, a wax, a paraffin, a starch, a tracer, a cellulose derivative, polyethylene glycol, silicon, bentonite, silica, talc, zinc oxide, Mixture can be used

When the cosmetic composition is formulated into a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder or a mixture thereof may be used as a carrier component. In the case of a spray, Propellants such as carbon, propane, butane or dimethyl ether.

When the cosmetic composition is formulated into a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent may be used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl benzoate, 1,3-butyl glycol oil may be used, and in particular, fatty acid esters of cottonseed oil, peanut oil, corn oil, olive oil, castor oil and sesame oil, glycerol aliphatic ester, polyethylene glycol or sorbitan may be used.

When formulated into a suspension, the cosmetic composition may contain, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the cosmetic composition is formulated into soap, it is preferable to use, as a carrier component, an alkali metal salt of a fatty acid, a fatty acid hemiester salt, a fatty acid protein hydrolizate, isethionate, a lanolin derivative, an aliphatic alcohol, a vegetable oil, Can be used.

The cosmetic composition may be a cosmetic or dermatological composition which is generally used in the art depending on the quality or function of the final product, such as a lipid, an organic solvent, a solubilizing agent, a thickening agent, a gelling agent, a softening agent, an antioxidant, a suspending agent, a stabilizer, Surfactants, water, ionic or nonionic emulsifiers, fillers, metal ion sequestrants, chelating agents, preservatives, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, And may further contain adjuvants conventionally used in the field of cosmetics or dermatology, such as any other ingredient.

However, the adjuvant and the mixing ratio thereof can be appropriately selected so as not to affect the preferable properties of the cosmetic composition according to the present invention.

The present invention will be further described with reference to the following examples, but it should be apparent that the present invention is not limited by the following examples.

Example  One : Nanocloprosis Oceania  Preparation of extract

Nanochloprobus cysteinica strain was purchased from the Korea Marine Microalgae Bank of Pukyong National University. F / 2 medium (Guillard and Ryther 1962, Guillard 1975) was prepared using sterile seawater. The aseptically isolated strains were subcultured in f / 2 medium for a certain period (2 to 3 weeks).

Subcultured nanocloprosis oseanica was centrifuged at 7,000 rpm for 15 minutes to separate the supernatant and solid. After removing the supernatant, the solid was freeze-dried (-80 ° C, 72h) and stored at low temperature (-40 ° C).

100 g of the stored nanocloprosis oceanica was immersed in a 10-fold volume of 70% (v / v) ethanol and reflux-extracted at a temperature of 80 ° C for 12 hours.

The extract was filtered with 250 mesh and 0.5 mu m filter paper. The extract was repeatedly extracted three times in the same manner for the remaining raw materials and then cooled at room temperature. The extract was concentrated under reduced pressure at 20 ° C and lyophilized to give a solid.

Example  2 : Nanocloprosis Oceania  origin Violaxanthin  extraction

Violaxanthin was extracted by subjecting the obtained nanocloprolysis aneica powder to an organic solvent treatment.

The solid content obtained in Preparation Example 1 was dissolved in 400 mL of distilled water, followed by addition of ethyl acetate and stirring. After 30 minutes, the ethyl acetate layer was separated. The ethyl acetate layer was repeatedly extracted until no color appeared, and then concentrated to a volume of 300 mL. Distilled water was added repeatedly to adjust the pH of the ethyl acetate layer to neutrality, followed by vacuum drying.

4 mL of acetone was added to the dried product and dissolved, followed by purification using silica gel. The viologaxin extract from the purified microalgae was vacuum dried and stored at -20 ° C.

To confirm the safety of violaxanthin, it was purified and analyzed by nuclear magnetic resonance (NMR). Violaxanthin complex extract was dissolved in CDCl ₃ and analyzed by NMR. Structural identity with plant - derived violaxanthin was confirmed.

Experimental Example  1: Skin safety test

MTT assay experiments were performed on fibroblasts (HDF), melanocytes (B16F10), and keratinocytes (HaCaT) to verify the skin safety of the samples obtained in Examples 1 and 2.

The samples of Examples 1 and 2 were suspended in purified water at concentrations of 0, 1, 5, 10, 20, 50 and 100 μg / mL, and cell viability was measured.

Cytotoxicity was performed by modifying the method of Mosmann to measure cell viability using MTT {3- (4,5-dimethylthiazol-2-yl) -2-5-diphenyltetrazolium bromide} reagent.

(HDF), melanocyte-forming cells (B16F10), and keratinocytes (HaCaT) were dispensed in 96-well plates at a concentration of 1 × 10 ⁴ cells / well and cultured at 37 ° C. and 5% CO ₂ for 48 hours Respectively.

After the culture medium was removed and the sample was cultured for 48 hours in a concentration-treated medium, the medium was removed and repeatedly washed with PBS (phosphate buffered saline). MTT was dissolved in PBS at 5 mg / mL and 50 L was added. The cells were cultured at 37 ° C and 5% CO ₂ for 48 hours. 100 L of DMSO (dimethyl sulfoxide) was added per well, and the mixture was stirred for 10 minutes and absorbance was measured at 540 nm.

No cytotoxicity was observed at all concentrations of each sample. The results suggest that the nanocloprolysis aniina extract and violaxanthin are not only harmless to human body but also have excellent human safety.

Experimental Example  2 : COL1A1  And Of MMP1  Expression test

The sample powders obtained in Examples 1 and 2 were suspended in purified water, diluted by concentration, and the effect of improving wrinkles was evaluated.

The expression of COL1A1, one of typical collagen expressed by human dermal fibroblasts, ELN, one of elastic proteins, and MMP1, a collagenase, were measured.

Human fibroblasts (Human Skin Fibroblast) for 37 ℃, 5% in the cell culture medium to maintain a constant humidity in the CO ₂ incubator with 10% FBS, Penicillin (50U / mL), DMEM (Dulbecco's containing Streptomycin (50 U / mL) The cells were cultured for 24 hours at a density of 1 × 10 ⁵ cells / ml in a 24-well plate, and cultured for 24 hours. UVB (30 mJ / cm ² ) was irradiated.

In order to confirm the extent of expression of collagen, control samples (DMEM medium) and the samples of Examples 1 and 2 diluted by concentration were treated with human dermal fibroblasts and cultured for 24 hours.

Changes in gene expression were measured by qRT-PCR (quantitative real-time PCR), which binds the fluorescent substance to the DNA product amplified by polymerase chain reaction (PCR) and continuously detects the fluorescent substance.

To quantify the changes of COL1A1 and MMP1 gene expression, fluorescence values of PCR products were measured by SYBR green I (Invitrogen). A reaction solution was prepared by mixing 0.2 μM primer, 50 mM KCl, 20 mM Tris / HCl pH 8.4, 0.8 mM dNTP, 0.5 U Extaq DNA polymerase, 3 mM MgCl ₂ and 1 × SYBR green in a PCR tube.

Denaturation, annealing, and polymerization were performed at 94 ° C for 30 seconds, at 58 ° C for 30 minutes, and then for 3 minutes at 94 ° C using Linegene K (BioER, China) Sec., 72 ° C for 30 sec., And fluorescence intensity was measured after completion of each cycle.

PCR results were verified by melting curve for each result. The threshold cycle (Ct) value of each gene was normalized to the Ct value of β-actin, and the amount of change in the Ct value was compared and analyzed.

The Ct value is the number of cycles when the amount of fluorescence generated by the PCR product (the number of amplified PCR products) reaches a certain reference value above the basal value, and the amount of gene expression can be confirmed through the Ct value. The primer sequences used in the experiments are shown in Table 1 below.

Gene Forward primer Reverse primer β-actin 5-GGATTCCTATGTGGGCGACGA-3 5-CGCTCGGTGAGGATCTTCATG-3 COL1A1 5-AGGGCCAAGACGAAGACATC-3 5-AGATCACGTCATCGCACAACA-3 MMP1 5-TCTGACGTTGATCCCAGAGAGCAG-3 5-CAGGGTGACACCAGTGACTGCAC-3

The results of measurement of the expression level by the sample treatment are shown in Table 2 below. The expression level of COL1A1 mRNA was increased in a dose dependent manner, whereas the amount of MMP1 mRNA expression was decreased in a concentration dependent manner.

division COL1A1 expression level MMP1 expression level Example 1 0 μg / mL 1.00 1.00 5 μg / mL 1.47 0.94 10 μg / mL 1.56 0.89 20 μg / mL 1.97 0.85 50 μg / mL 2.45 0.79 Example 2 0 μg / mL 1.00 1.00 5 μg / mL 1.58 0.91 10 μg / mL 1.79 0.85 20 μg / mL 2.23 0.80 50 μg / mL 2.64 0.74

 [Fold, β-actin normalized]

Skin wrinkles are caused by unbalance of synthesis and degradation of collagen, and the synthesis of type I collagen in skin and MMP-1, a degradation enzyme thereof, are normally balanced. However, in photoaged skin, the synthesis of type I and III collagen is reduced and the activity of MMP-1 is increased. These MMPs (matrix metalloproteinases) are proteolytic enzymes that degrade the extracellular matrix and are known to be involved in wound healing and tissue regeneration under normal conditions.

Therefore, the higher the inhibitory effect on MMP-1 expression, the better the skin wrinkle and anti-aging effect, and the above results show that the nanocloprolysis aniina extract and violaxanthin promote the production of collagen, And wrinkles can be improved, suggesting that the present invention can be utilized as a beauty cosmetic application.

Experimental Example  3: Antioxidant activity test

Active oxygen decomposition effect

Free radical scavenging activity using DPPH (1, 1-diphenyl-2-picryl hydrazyl).

4 mL of methanol was added to the test tube, and the concentrations of Examples 1 and 2 were varied. 1 mL of 0.15 mM DPPH solution was added, and the reaction was allowed to proceed at room temperature for 30 minutes. Then, the absorbance at 520 nm was measured.

Initial (Ai) and blank (Ab) without substrate and DPPH were measured, and α-tocopherol and BHT were used as positive control. The results are shown in Table 3 below.

division DPPH erase rate% α-tocopherol (50 μg / mL) 32.5 BHT (50 [mu] g / mL) 34.2 Example 1 0 μg / mL 0.0 5 μg / mL 3.5 10 μg / mL 10.5 20 μg / mL 21.3 50 μg / mL 42.1 Example 2 0 μg / mL 0.0 5 μg / mL 5.8 10 μg / mL 13.7 20 μg / mL 25.2 50 μg / mL 46.4

[DPPH erase rate%]

The samples of Examples 1 and 2 were evaluated to have higher free radical scavenging activity than α-tocopherol and BHT, which are well known in the art. Therefore, the decomposition of active oxygen can be promoted by the nanolocloprosis oseanica extract and violaxanthin derived therefrom, which suggests that the antioxidative activity is superior in preventing skin aging and improving skin condition .

Intracellular Active oxygen species  Measurement test ( DCF -DA assay)

After the samples of Examples 1 and 2 were treated, the reactive oxygen species (ROS) removal ability was evaluated by comparing the amount of reactive oxygen species generated in the UVB-irradiated cells with the negative control (Cho et al. , 1999; Kim et al., 2002).

Raw 264.7 cells were inoculated into 96 well plates at a concentration of 1 × 10 ⁴ cells / well. The medium was replaced with fresh medium, the samples of Examples 1 and 2 were treated, and the cells were cultured for 24 hours.

10.0 μL of WST-1 solution (EZ-CYTOX, DOGEN, Korea) was added to each well, and the cells were incubated for 1 hour. The absorbance was measured at 450 nm and 655 nm using an ELISA reader Respectively.

Samples were diluted with dimethylsulfoxide (DMSO, Sigma-Aldrich, USA) to a final concentration of 20 μM and DMSO was used as a blank test (negative control). The experiment was repeated 3 times to ensure reliability.

The experiment was carried out under the same cell line and the same cell culture conditions as the WST-1 assay, a cell activity assay. The cultured Raw 264.7 cells were washed once with phosphate buffered saline (PBS; sodium chloride 137 mM, phosphate buffer 10 mM, potassium chloride 2.7 mM, all from Biopure, Canada), and Raw 264.7 cells were harvested. The cells were suspended in 15 mL of PBS, and 15 μL of 20 mM 2 ', 7'-Dichlorofluorescein diacetate was added and incubated for 1 hour.

Centrifuged at 1200 rpm for 2 minutes, washed once with PBS solution to dilute to 1x10 ⁵ cells / mL, and the control material and the samples of Examples 1 and 2 were treated and incubated for 10 minutes. 20 mg / mL of silica was treated with 50 μL and incubated for 1 hour. The cell pellet obtained by centrifuging at 6000 rpm for 5 minutes was dispersed in 200 μL PBS solution and inoculated into a 96-well plate.

Fluorescence was measured at 485 nm excitation / 535 nm emission using a fluorescence microplate reader to evaluate the amount of reactive oxygen species.

The extracts treated for the measurement of reactive oxygen species to the concentration of the test solution were used as the concentration of the test solution set up through the cell activity measurement.

As a negative control, DMSO was used instead of the sample. As a positive control, ascorbic acid (Sigma-Aldrich) was diluted in DMSO to a concentration of 50 μg / mL.

Based on the above results, the measurement of reactive oxygen species at the same concentration was carried out, and the treated extract was used at a concentration of 50 mg / L. The experiment was repeated 4 times to ensure reliability. The measurement results are shown in Table 4 below.

division ROS generation inhibition rate (% of control) Ascorbic acid (50 μg / mL) 71.5 Example 1 0 μg / mL 0.0 5 μg / mL 9.8 10 μg / mL 22.7 20 μg / mL 47.2 50 μg / mL 62.8 Example 2 0 μg / mL 0.0 5 μg / mL 11.5 10 μg / mL 25.8 20 μg / mL 50.2 50 μg / mL 68.7

The samples of Examples 1 and 2 were evaluated to have equivalent levels of ascorbic acid and free radical scavenging activity, which are well known in the art. That is, the above results suggest that the nanolocloprosis oseanica extract and the violaxanthin derived therefrom are excellent in the ability to remove active oxygen species and have excellent antioxidative effects due to the free radical scavenging activity.

Experimental Example  4: Collagen biosynthesis induction test

The synthesis of collagen following fibroblast growth was observed.

Human dermal fibroblasts at a concentration of 1.0 × 10 ⁴ cells / mL were dispensed in a 96-well plate in an amount of 100 μL each, and cultured for 3 days under the conditions of 37 ° C., 5% CO ₂ and humidification, and then irradiated with UVB (30 mJ / cm ² ) . Medium 100 S (manufactured by Kurashiki Boshiki) was used a medium containing 10% by weight of LSGS (Low Serum Growth Supplement, Kurashiki Boshiki Co., Ltd.) per well.

The medium was exchanged with DMEM (Dulbecco's Modified Eagle's Medium, Sigma), and the samples of Examples 1 and 2 were treated at a concentration of 50 μg / mL and then cultured. Purified water was used as a negative control, and ascorbic acid (50 μg / mL) was used as a positive control.

After the cells were cultured for 7 days, the culture solution was collected and the concentration of secreted type I collagen in the culture solution was quantified by enzyme-linked immunosorbent assay (Procollagen type I c-peptide EIA Kit, Takara Bio).

The amount of collagen in each test sample culture fluid was calculated based on the amount of type I collagen in the control group (100%). The measurement results are shown in Table 5 below.

Presence or absence of UVB treatment division Collagen production (%) - Negative control group 101.2 + Negative control group 7.3 Example 1 98.5 Example 2 109.8 Ascorbic acid 85.2

Experimental Example  5: skin moisturizing effect test

The sample powders obtained in Examples 1 and 2 were suspended in purified water, diluted by concentration, and skin moisturizing effect was evaluated.

The degree of expression of HAS2 gene and AQP3 gene involved in skin moisturization in human keratinocytes was measured at the mRNA level.

Cultured human keratinocyte (HaCaT) under the conditions of Dulbecco's Modified Eagle's Medium (DMEM ), 10% Fatal bovine serum (FBS), 1% 37 ℃ at 100 mm / 60.1 cm culture dish along with Antibiotic-Antimycotic, 5% CO ₂ Respectively.

The keratinocytes were plated at a density of 1.0 x ¹⁰ < ⁶ > cells / mL into 96-well plates and cultured for 24 hours. After replacing with DMEM free medium, the samples of Examples 1 and 2 diluted by concentration were treated and cultured for 24 hours.

Cells were harvested, washed with cold phosphate buffered saline (PBS), and 1 ml of TRIZol reagent (Life Technologies, Inc.) was added and the total RNA extracted.

Changes in gene expression were measured by qRT-PCR (quantitative real-time PCR), which binds the fluorescent substance to the DNA product amplified by polymerase chain reaction (PCR) and continuously detects the fluorescent substance.

To quantify the changes of HAS2 and AQP3 gene expression, fluorescence values of the PCR products were measured by SYBR green I (Invitrogen). A reaction solution was prepared by mixing 0.2 μM primer, 50 mM KCl, 20 mM Tris / HCl pH 8.4, 0.8 mM dNTP, 0.5 U Extaq DNA polymerase, 3 mM MgCl ₂ and 1 × SYBR green in a PCR tube.

Denaturation, annealing, and polymerization were performed at 94 ° C for 30 seconds, at 58 ° C for 30 minutes, and then for 3 minutes at 94 ° C using Linegene K (BioER, China) Sec., 72 ° C for 30 sec., And fluorescence intensity was measured after completion of each cycle.

PCR results were verified by melting curve for each result. The threshold cycle (Ct) value of each gene was normalized to the Ct value of β-actin, and the amount of change in the Ct value was compared and analyzed.

The Ct value is the number of cycles when the amount of fluorescence generated by the PCR product (the number of amplified PCR products) reaches a certain reference value above the basal value, and the amount of gene expression can be confirmed through the Ct value. The primer sequences used in the experiments are shown in Table 6 below.

Gene Forward primer Reverse primer HAS-2 5'-TTTCTTTATGTGACTCATCTGTCTCACCGG-3 ' 5'-ATTGTTGGCTACCAGTTTATCCAAAGGG-3 ' AQP3 5'-ACCCTCATCCTGGTGATGTTTG-3 ' 5'-TCTGCTCCTTGTGCTTCACAT-3 '

The results of measurement of HAS2 and AQP3 mRNA expression amounts are shown in Table 7 below, and the amount of mRNA expression was increased in a concentration-dependent manner according to the treatment of the extract. The higher the ability to promote HAS2 and AQP3 expression, the better the skin moisturizing effect.

The results suggest that the extract may enhance skin moisturization by promoting the expression of aquaporin protein and the synthesis of hyaluronic acid.

division HAS2 expression level AQP3 expression level Example 1 0 μg / mL 1.00 1.00 5 μg / mL 1.07 1.04 10 μg / mL 1.12 1.09 20 μg / mL 1.23 1.16 50 μg / mL 1.37 1.25 Example 2 0 μg / mL 1.00 1.00 5 μg / mL 1.09 1.07 10 μg / mL 1.17 1.12 20 μg / mL 1.29 1.20 50 μg / mL 1.41 1.35

 [Fold of control]

Experimental Example  6: Test of cytoprotective effect by ultraviolet irradiation

The cytoprotective effect of the samples of Examples 1 and 2 against UVB was measured.

Human dermal fibroblasts at a concentration of 1.0 × 10 ⁴ cells / mL were seeded in a 96-well plate at a concentration of 1 × 10 ⁴ cells / well and cultured at 37 ° C. and 5% CO ₂ for 48 hours. The culture medium was removed and the remaining medium was removed by washing twice with PBS. The samples of Examples 1 and 2 were replaced with PBS adjusted to concentration, and irradiated with UVB (30 mJ / cm ² ) for 5 minutes.

PBS was removed and remaining samples were removed and further incubated for 24 hours. MTT was dissolved in PBS at 5 mg / mL, and 50 L was added and cultured at 37 ° C and 5% CO ₂ for 4 hours. 100 μL of dimethyl sulfoxide solution was added to each well to dissolve the stained cells, and the absorbance at 570 nm was measured with a microplate reader.

The absorbance of the UV-irradiated control group and PBS containing the samples of Examples 1 and 2 were compared and the UV-irradiated experimental groups were compared and shown in Table 8.

division Cell survival rate (%) Negative control group 100.2 Example 1 0 μg / mL 58.2 5 μg / mL 63.5 10 μg / mL 75.2 20 μg / mL 85.4 50 μg / mL 92.5 Example 2 0 μg / mL 57.6 5 μg / mL 65.3 10 μg / mL 78.9 20 μg / mL 89.1 50 μg / mL 97.3

Experimental Example  7: Ultraviolet ray Absorption capacity  Measure

Absorbance was measured in the ultraviolet region (200 to 400 nm) using a UV spectrophotometer (Hewlett Packard, 8453) in order to evaluate the ultraviolet blocking and absorption effect of the nanochlroboscis oceanica extract. Specifically, the samples of Examples 1 and 2 were diluted to a concentration of 50 μg / mL, subjected to UV scanning at 200 to 400 nm, and distilled water was used as a control.

UV spectrum analysis showed that nanolocloprosis oceanica extract and violaxanthin showed a significant absorption peak in UV-C (200-290 nm) and UV-B (290-320 nm) compared to the control, and UV-A (320- 400 nm) region. The samples of Examples 1 and 2 exhibited maximum UV absorption capacity at a wavelength of about 320 nm.

These results suggest that nanolocloprosis oseanica extract and violaxanthin can be used as raw materials for ultraviolet screening agents that inhibit skin penetration of UV-B and UV-C.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

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

Claims (7)

A cosmetic composition for ultraviolet screening comprising an extract of Nannochloropsis oceanica as an active ingredient. A cosmetic composition for protecting against ultraviolet rays comprising, as an active ingredient, Violaxanthin derived from an extract of Nanochloprobus cisse aniina. 3. The method according to claim 1 or 2,
Wherein the extract is selected from the group consisting of water, an anhydrous or a lower alcohol having 1 to 4 carbon atoms, acetone, petroleum ether, ethyl acetate, butyl acetate, trichloromethane, dichloromethane, chloroform, hexane and 1,3-butylene glycol Gt; cosmetic < / RTI > composition. 3. The method according to claim 1 or 2,
Wrinkle improving or skin moisturizing. delete 3. The method according to claim 1 or 2,
A cosmetic composition further comprising a skin soothing or skin irritation relieving application. 3. The method according to claim 1 or 2,
Cosmetics formulated with at least one selected from the group consisting of softening longevity, nutritional lotion, moisture cream, nutritional cream, massage cream, essence, ampule, gel, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray and powder Composition.