KR101612509B1 - Mutant Arthrospira platensis Having Skin Condition Improvement Effect - Google Patents

Abstract

The present invention provides a spirulina mutant strain (KCTC 18254P) having an effect of improving skin condition. The spirulina mutant strain of the present invention has remarkable elastase inhibitory activity, MMP-1 expression inhibiting ability and procollagen combining ability as compared with the wild type Spirulina strain. The present invention provides a skin condition improving composition comprising the spirulina mutant strain of the present invention, the culture thereof or an extract thereof.

Description

A mutant Arthrospira platensis Having Skin Condition Improvement Effect < RTI ID = 0.0 >

The present invention relates to a Spirulina mutant strain having a skin condition improving effect.

Spirulina is a commercially important filamentous microalgae, spiral with a size of 0.5 nm, thin in cell walls and excellent in growth in alkaline environment. Spirulina is known to have various physiological functions such as antioxidation, heavy metal poisoning, immunity enhancement, and cholesterol lowering, and carotenoids, chlorophyll, phycocyanin, etc. are known as the main active ingredients.

Recently, the food and pharmaceutical industry has actively pursued research for the development of functional materials for anti-aging, whitening, and skin treatment caused by aging society and eating habits, and its market size is rapidly increasing. Spirulina is a promising material for this It has attracted much attention.

Spirulina is two cyanobacteria used as a food or animal feed additive, including Arthrospira platensis and Arthrospira maxima . The main habitat of Spirulina is high carbonate and alkaline water quality in the tropical and subtropical regions. Recently, it has been commercialized as a human health functional food additive and artificial cultivation has been done.

Spirulina is a high-level natural chlorophyll nutrient rich in nutrients, especially protein and green-yellow vegetables, needed by living organisms. It contains the five nutrients (protein, carbohydrate, various minerals, fat and vitamins) - 30,000 kinds of evenly distributed nutrients are implied. In addition, it has a very high protein content (55-77%), and its various constituents are composed of almost the same ratio as the human body. It has a digestion rate of 95% or more in the body and can be consumed by patients suffering from weak digestive function, It is a strong alkaline whole food that does not cause imbalance in nutrition even when used for a long time. In particular, it has been officially recognized by the United Nations, the World Health Organization and the Food and Drug Administration (FDA) as a complete food product in terms of safety and nutrition as a health food. It is also a decided food.

Until recently, more than 1,000 papers were published by scholars from around the world and 730 patents were registered for research on nutritional aspects of spirulina, its relation to diseases, and its relationship to air pollution. Currently, more than 70 countries in the world is receiving good reputation as a functional food. Furthermore, studies on various effects such as anticancer effect and various immune function enhancement effects have been reported, and it is possible to be developed as an excellent functional cosmetic material because it contains a high amount of antioxidant components and vitamins A, E, and C Recently, various methods of processing such as hydrolysis and fermentation have been applied for use as functional materials for cosmetics.

Development of functional cosmetic materials is largely focused on the development of anti-aging materials that inhibit skin aging, the development of whitening agents that inhibit the melanin biosynthesis of melanin-forming cells of the epidermal layer or block the pathway of the produced melanin to epidermal cells, Antioxidant and antioxidant ingredients that inhibit skin inflammation are used as a major search tool in the development of functional cosmetic materials as an antioxidant material category.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made efforts to develop a novel strain capable of improving skin conditions and found that Spirulina mutant strains inducing genetic mutations using ultraviolet rays have a remarkable skin condition improving effect as compared with wild type strains Thereby completing the present invention.

It is therefore an object of the present invention to provide a Spirulina mutant strain (KCTC 18254P).

Another object of the present invention is to provide a composition for improving skin condition.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention and claims.

According to one aspect of the present invention, there is provided a mutant strain of Arthrospira platensis (KCTC 18254P) having an effect of improving skin condition.

The present inventors have made efforts to develop a novel strain capable of improving skin conditions and found that Spirulina mutant strains inducing genetic mutations using ultraviolet rays have a remarkable skin condition improving effect as compared with wild type strains Respectively.

The spirulina mutant strain of the present invention induces a genetic mutation through ultraviolet irradiation. Cytosine and thymine in DNA can easily change properties due to ultraviolet irradiation. Ultraviolet light induces covalent bonding of pyrimidine bases in DNA strands to pyrimidine dimers.

According to one embodiment of the present invention, a mutant strain is produced by using UV-B (254 nm) wavelength capable of introducing a stronger energy than general near-ultraviolet rays.

According to a specific embodiment of the present invention, the Spirulina mutant strains of the present invention are prepared by directly irradiating the cells with UV-B (254 nm) for 1 minute.

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The Spirulina mutant strains of the present invention have skin condition improving, antioxidant and anti-inflammatory effects.

According to one embodiment of the present invention, the mutant strain of the present invention has an elastase inhibitory activity increased by 20% to 45% as compared with the wild-type Spirulina strain.

According to another embodiment of the present invention, the mutant strain has an elastase inhibitory activity which is increased by 30% to 35% as compared with the wild-type Spirulina strain.

Elastase is an enzyme that decomposes skin elasticity fibers. It decomposes collagen such as elastin, which plays an important role in suppressing skin elasticity and wrinkle formation, and has an effect of improving wrinkles when it inhibits activity of elastase .

The Spirulina mutant strain of the present invention has a remarkable inhibitory effect on elastase as compared with the wild type Spirulina strain, and thus is more effective in improving wrinkles.

According to one embodiment of the present invention, the mutant strain of the present invention has a 1.8 to 2.5-fold increase in inhibition of MMPs (matrix metalloproteinases) -1 expression as compared with the wild-type Spirulina strain.

According to another embodiment of the present invention, the mutant strain of the present invention has a 2-fold to 2.5-fold increased inhibitory effect on MMP-1 expression as compared with the wild-type Spirulina strain.

When the skin is exposed to ultraviolet rays, the amount of MMP, an enzyme that degrades collagen, is increased. As a result, when collagen decomposition is greater than collagen synthesis, wrinkles are formed as a result. Among the MMP enzymes, the most important role in the skin aging process is the MMP-1 enzyme, an enzyme essential for collagen degradation and acts to start collagen decomposition first.

The Spirulina mutant strain of the present invention has a remarkable inhibitory effect on MMP-1 expression as compared with the wild-type Spirulina strain, and thus is more effective in improving wrinkles.

According to one embodiment of the present invention, the mutant strain of the present invention increases the synthesis of pro-collagen by 15% to 35% as compared to the wild-type Spirulina strain.

According to another embodiment of the present invention, the mutant strain of the present invention increases the synthesis of procollagen by 25% to 32% as compared to the wild-type Spirulina strain.

The procollagen is an inducer of collagen production and plays an important role in aging and prevention of skin wrinkles. The spirulina mutant strains of the present invention, which have a remarkable ability of procollagen aggregation in comparison with wild-type spirulina strains, are more effective in preventing aging and improving wrinkles.

According to another aspect of the present invention, there is provided a skin condition improving composition comprising the spirulina mutant strain of the present invention, the culture of the strain, or the extract of the strain.

Since the composition of the present invention includes the spirulina mutant strain of the present invention described above as an effective ingredient and is produced using the same, common description between them is omitted in order to avoid the excessive complexity of the present specification.

According to one embodiment of the present invention, improvement of the skin condition by the composition of the present invention includes improvement of wrinkles, skin regeneration, whitening, improvement of skin elasticity, prevention of skin aging, improvement of skin moisturizing, removal of black spot, treatment of acne, However, the present invention is not limited thereto.

According to a preferred embodiment of the present invention, the composition of the present invention is made from a cosmetic composition.

The components contained in the cosmetic composition of the present invention include components commonly used in cosmetic compositions in addition to the spirulina mutant strain as an active ingredient, the culture of the strain or the extract of the strain, and examples thereof include antioxidants, stabilizers, Vitamins, pigments and flavors, and carriers.

The composition for improving the skin condition of the present invention may be prepared in any formulations conventionally produced in the art and may be in the form of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, -Containing cleansing, oils, powder foundations, emulsion foundations, wax foundations and sprays, but are 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 .

In the case where 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. Especially, in the case of a spray, a mixture of chlorofluorohydrocarbons, propane / Propane or dimethyl ether.

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 an interfacial active agent-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 sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters.

The cosmetic composition of the present invention has a use for improving skin condition. As used herein, the term " improvement of skin condition " has a meaning inclusive of prevention of skin aging, improvement of skin wrinkles and improvement of skin moisturizing. The cosmetic composition of the present invention prevents aging of the skin through improvement of skin wrinkles, improvement of moisturizing power and improvement of skin elasticity, and improves the condition of the skin as a whole, and this fact is proved in the following examples.

The features and advantages of the present invention are summarized as follows:

(I) The present invention provides a Spirulina mutant strain (KCTC 18254P) having an improved skin condition, antioxidant or anti-inflammatory effect.

(Ii) The spirulina mutant strain of the present invention has remarkable elastase inhibitory activity, MMP-1 expression inhibiting ability and procollagen combining ability as compared with the wild-type Spirulina strain.

(Iii) The present invention provides a skin condition improving composition comprising the Spirulina mutant strain of the present invention, a culture thereof or an extract thereof.

Figure 1 shows the results of measurement of chlorophyll-a and chlorophyll-b concentrations of (a) chlorophyll a and (b) A. platensis in the extraction solvent (MeOH 40-80%) and temperature.
Figure 2 shows the effect of various A. platensis . It is the result of confirming the antioxidant effect of the mutant.
(A) The DPPH radical scavenging activity was measured by ESR. (B) ABTS radical scavenging ability of various mutant microalgae.
Figure 3 shows the results of various A. platensis . The result of measuring the inhibitory effect of the mutant on the elastase activity.
The inhibitory activity in each sample was determined by measuring the production of p-nitroanilide at a wavelength of 410 nm using an ELISA reader. Each measurement is expressed as the mean ± standard deviation for three experiments. ^{The ae} value was significantly (p <0.05) significant according to Duncan's multiple range test.
Figure 4 shows the results of measurement of cytotoxicity of various A. platensis mutants in HDF (Human Dermal Fibroblast) cells.
Cells were treated with mutagenic microalgae at various concentrations (50, 100, 200, 500 μg / mL) for 24 h (A: UV mutant microalgae, B: electron beam mutant microalgae). Each measurement is expressed as the mean ± standard deviation for three experiments.
Figure 5 shows the effect of A. platensis on UVB -induced MMP-1 expression in HDF cells. This is the result of measuring the effect of the mutant.
HDF cells were irradiated at 90 mJ / cm ² and treated with 100 μg / mL of various A. platensis mutants for 72 h. The amount of MMP-1 protein in culture supernatant was measured by ELISA. Each measurement represents the mean ± standard deviation of the three test results. ^AJ values were significantly (p <0.05) significant according to the Duncan's multiple range test.
FIG. 6 is a graph showing the effect of UVB- induced procollagen type-I synthesis on A. platensis . This is the result of measuring the effect of the mutant.
HDF cells were irradiated at 90 mJ / cm ² and treated with 100 μg / mL of various A. platensis mutants for 72 h. The amount of procollagen type-I protein in the culture supernatant was measured by ELISA. Each measurement represents the mean ± standard deviation of the three test results. ^AJ values were significantly (p <0.05) significant according to the Duncan's multiple range test.
FIG. 7 shows the results of measuring the effect of UV1-2 on the activation of MAPK in UVB irradiated HDF cells.
1 × 10 ⁶ cells per 100 π dish are cultured and treated with various concentrations of UV1-2 and 10 μM retinoic acid before UVB irradiation (90 mJ / cm ² ). After incubation for 30 minutes, cell lysates were obtained, electrophoresed on SDS-PAGE and then Western blot performed.
8 shows the results of measuring the effect of UV1-2 on NF-κB translocation p-IκBα and IκBα in UVB-irradiated HDF cells.
1 × 10 ⁶ cells per 100 π dish are cultured and treated with various concentrations of UV1-2 and 10 μM retinoic acid before UVB irradiation (90 mJ / cm ² ). After incubation for 30 minutes, cell lysates were obtained, electrophoresed on SDS-PAGE and then Western blot performed. β-Actin is used as a control.
FIG. 9 shows the results of measuring the effect of UV1-2 on the AP-1 family in UVB irradiated HDF cells.
1 × 10 ⁶ cells per 100 π dish are cultured and treated with various concentrations of UV1-2 and 10 μM retinoic acid before UVB irradiation (90 mJ / cm ² ). After incubation for 24 hours, cell lysates were obtained, electrophoresed on SDS-PAGE and then Western blot performed. β-Actin is used as a control.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1: Establishment of optimum extraction conditions of Spirulina mutant

In order to utilize Spirulina as a functional cosmetic material, 11 kinds of mutant strains capable of increasing the activity of existing Spirulina through ultraviolet irradiation and electron beam irradiation were obtained and studied (Table 1).

Spirulina mutants induced by ultraviolet and electron beam irradiation UV Electron beam Variant name Induction conditions (254 nm, 15 W) Variant name 200 kV, 1.2 mA UV1-2 UV irradiation 1 minute 240-1 Irradiation dose 240 kGy UV3-2 UV irradiation 3 minutes 240-5 Irradiation dose 240 kGy UV3-3 UV irradiation 3 minutes 240-6 Irradiation dose 240 kGy UV5-9 UV irradiation 5 minutes 240-29 Irradiation dose 240 kGy UV10-5 UV irradiation 10 minutes 384-5 Irradiation dose 384 kGy - - 384-6 Irradiation dose 384 kGy

1-1. Establish extraction condition

The contents of chlorophyll a and b were determined according to methanol concentration and temperature of spirulina. 30 ml of each of 40%, 60%, 80% and 100% solutions of methanol was added to 1 g of dried spirulina, and then the temperature was extracted at room temperature, 40 ° C, 50 ° C and 60 ° C for 24 hours. The extracted solution was filtered using a 45 μm filter and the absorbance was measured at 650 nm and 665 nm using an ELISA reader (Infinite 200 pro, Tecan), and the chlorophyll content was measured according to the following equation (FIG. 1 ).

Chlorophyll a (Chl a) = (15.65 x A ₆₆₅ ) - (7.34 x A ₆₅₀ )

Chlorophyll b (Chl b) = (27.05 x A ₆₅₀₎ - (11.21 x A ₆₆₅ )

As shown in FIG. 1, the extraction efficiency of spirulina by the temperature and the methanol concentration was examined based on the content of chlorophyll. As a result, it was confirmed that the extraction efficiency was significantly increased as the concentration of methanol increased . Chlorophyll a and b were extracted at 0.053 and 0.061 mg / g, respectively, compared to the dry mutant weight at 40% methanol at room temperature, but 10.9 and 4.4 mg / g were found for methanol 100%, respectively. In case of chlorophyll a, the highest extraction efficiency was obtained at room temperature, while in chlorophyll b, it was the highest at 60 ℃ and 7.47 mg / g. Therefore, the extraction of the functional material of the mutant strains was performed at 100% methanol, and the difference in extraction efficiency by temperature was not large.

1-2. Spirulina mutant extraction yield

The extraction yields of nine spirulina mutants were determined by extraction conditions established in previous experiments (MeOH 100%, room temperature, 24 hours). After 30 times (W / V) methanol was added to the dried Spirulina mutant, the mixture was extracted at room temperature for 24 hours with stirring and then centrifuged (5,000 rpm, 5 minutes) to obtain supernatant. The supernatant was then dried using a speed- The weight was measured. Most extracts of Spirulina and its varieties were obtained at a yield of about 15%, and it was confirmed that even if the mutants were induced by UV or electron beam, no specific amount of extract was changed under the same extraction conditions. The thus obtained extract was dissolved in DMSO 10% solution, stored at -20 ° C, and then used for antioxidant and cell model experiments.

Extraction Yield of Spirulina and Spirulina Variants sample Sample dry weight before extraction (mg) Dry weight of extract (mg) yield(%) Wild type 398 61 15.33 UV1-2 190 30 15.79 UV3-2 204 33 16.18 UV3-3 199 28 14.07 UV5-9 205 28 13.66 UV10-5 202 31 15.31 240-1 203 33 16.18 240-5 196 30 15.31 240-6 209 34 16.21 240-29 199 32 16.02 384-5 197 32 16.19 384-6 204 27 13.22

Extraction conditions: MeOH 100%, room temperature, 24 hours, sample / solvent = 1: 30 (W / V)

Example 2: Antioxidant and wrinkle-improving effect of Spirulina mutant

2-1. DPPH radical scavenging ability and ABTS measurement using ESR (Electron spin resonance)

The radical scavenging activity was measured by mixing 30 μL of 60 μM DPPH dissolved in methanol and 30 μL of the sample prepared at 1 mg / mL. After stirring for 10 seconds, the sample was allowed to stand at room temperature for 2 minutes, transferred to a capillary tube and analyzed by ESR spectrometer (Jeol Co., was measured in Japan), the measurement conditions are central field: 3475 G, modulation frequency : 100 kHz, modulation amplitude: 2 G, microwave power: 5 mW, gain: 6.3 × 10 5, temperature: was 298K. The antioxidant activity of ABTS radicals was measured by using ABTS free radicals produced by the reaction with potassium persulfate, which were removed by antioxidants in the extract and decolorized the cyan color of the radicals. To the solution, 2.4 mM of potassium sulfide was added, diluted well, and reacted for 12-16 hours in a dark room. After adjusting the solution to 414 nm with distilled water to give an absorbance of 1.5, 1.0 mL of the diluted solution was added, and 0.1 mL of the sample was added. The solution was reacted at room temperature for 10 minutes, and absorbance was measured at 414 nm.

As shown in FIG. 2, the DPPH scavenging activity of 9 kinds of microalgae mutants at a concentration of 1.0 mg / mL was found to be as low as 5% or less in Spirulina wild type (WT) and most mutant strains. However, in the ABTS radical scavenging experiment, it was found that 26.5% of the wild type scavenging ability was extinguished, whereas most spirulina mutant scavenging ability was increased compared to the wild type. Especially, in case of UV 3-2 and 240-1 mutant strains, it was confirmed that the mutagenicity was increased by more than 70% compared with wild type in 35.85% and 36.0%, respectively. This ABTS radical scavenging activity is believed to be due to its ability to measure both hydrogen-donating and chain-breaking antioxidant effects as compared to the DPPH method.

2-2. Elastase inhibitory activity of microalgae mutant

Elastase is an enzyme that breaks down skin elasticity fibers and decomposes collagen such as elastin, which plays an important role in skin elasticity and inhibition of skin wrinkle formation. Through the inhibition of the elastase activity, it is possible to suppress the elasticity maintenance of the skin and the formation of the wrinkles of the skin. To measure the elastase activity, 10 μl of 3.3 nM Succ-Ala-Ala-Ala-p-nitroanilide and 10 μl of microalgae extract were added to 70 μL of 0.2 M Tris-HCl buffer (pH 8.0) , 10 μl of PPE (porcine pancreatic elastase) diluted to 1 μg / ml was added and reacted for 10 minutes. The amount of liberated pn-nitroanilide was confirmed by measuring the absorbance at 410 nm using an ELISA reader. As shown in Fig. 3, arbutin showed no inhibitory activity, while spirulina mutant increased or suppressed the activity. UV1-2, UV10-5 and 384-6 inhibited elastase by 39.5%, 47.0% and 53.4%, respectively.

2-3. Cell growth promotion effect on HDF (Human dermal fibrobrast) cells

In order to examine the wrinkle-removing effect of the microalgae mutant extract, HDF, a human fibroblast sarcoma cell, was used to examine cell growth, MMP-1 expression and pro-collagen synthesis. HDF cells were purchased from Korean Cell Line Bank. For the cell culture, DMEM medium was supplemented with 10% FBS, 1% penicillin, and streptomycin. The cells were incubated at 37 ° C with 5% CO ₂ .

All treatments for cell culture and preparation of samples were carried out on aseptic tables and all the test instruments used in the tests were used under sterilization with a high pressure sterilizer. To measure the toxicity and cell growth promoting effect of each microalgae to HDF cells, the samples were divided into 24 well plates at 5 × 10 ⁴ cells / well and stabilized for 24 hours. After that, the samples were treated with the concentration of each strain of microalgae and cultured for 24 hours. After that, MTT reagent at 5 μg / mL was added and incubated for 4 hours. After incubation, the supernatant was removed, washed with PBS, and DMSO was added to dissolve the cells. Then, the formazan produced by MTT reduction in intracellular mitochondria was analyzed with an ELISA microplate reader (Infinite 200 PRO, TECAN Group Ltd., Switzerland) Absorbance was measured at 570 nm.

As shown in FIG. 4, the microalgae extracts induced by ultraviolet and electron beams did not show any cytotoxicity up to 100 μg / ml. However, in the UV-induced mutants UV1-2 and 3-2 at a concentration of 200 μg / Cell growth by 50% and inhibited cell growth by 80% at 500 μg / ml. In addition, the extracts of 240-1 mutants promoted the growth of HDF cells at a concentration of 50 μg / ml by 21.3%, but at a concentration of 100 μg / ml 7%. Promotion of HDF cell growth ultimately promotes intracellular collagen production, thereby inducing the effect of inhibiting wrinkling. Therefore, in the subsequent experiments, the wrinkle-reducing effect of the microalgae mutant extract was observed to be less than 100 μg / ml.

2-4. Inhibition of MMP-1 (human) expression in HDF cells using an ELISA kit

The main cause of skin aging is ultraviolet rays. Exposure of skin to ultraviolet rays causes appearance changes such as melanin pigmentation and wrinkle formation, and it can cause cancer in severe cases. Ultraviolet light permeates the skin and activates reactive oxygen species, which leads to inflammation and subsequent inflammatory mediators that increase matrix metalloproteinases (MMPs) -1 expression and inhibit procollagen synthesis, thereby promoting skin wrinkle formation. Thus, the inhibitory effect of the microalgae mutant extract on MMP-1 expression was confirmed in UVB-irradiated HDF cells. HDF cells were plated on a 24-well plate at a density of 1 × 10 ⁵ cells / well, cultured for 24 hours, treated with each microalgae concentration, and incubated for 1 hour. Then UVB (90 mJ / cm ² ) was irradiated and cultured for 72 hours. After that, the amount of MMP-1 contained in the culture broth was measured by a manufacturer's analysis method using an MMP-1 ELISA kit (AnaSpec, Inc.).

As shown in FIG. 5, when UVB was irradiated to HDF, it was confirmed that MMP-1 expressed 5.6 ng / ml, which is about twice that of the normal state. The wild type extract was treated with 100 μg / ml of HDF cells exposed to UVB And 29.4% more than the untreated cells. In the case of UV1-2, UV10-5 and 240-1, 67.8%, 67.7% and 73.8% of the microalgae mutants, respectively, showed more than 2-fold inhibitory effect compared to WT. Especially, in 240-1, MMP-1 expression And 1.3 ng / ml, respectively, which was lower than 2.7 ng / ml of normal cells. These results suggest that the production of physiologically active components such as antioxidants is increased in the microalgae mutants as compared with the wild type, which is expected to suppress active oxygen species and inflammatory mediators present in normal cells.

2-5. Promoting effect of pro-collagen type I production in HDF cells

HDF cells were plated on a 24-well plate at a density of 1 × 10 ⁵ cells / well, cultured for 24 hours, treated with each microalgae concentration, and incubated for 1 hour. Then UVB (90 mJ / cm ² ) was irradiated and cultured for 72 hours. Then, after obtaining the culture solution, the amount of pro-collagen type I contained in the culture solution was measured by a manufacturer's analysis method using a kit (TAKARA, Inc.). 6 shows that 39.4% inhibition of procollagen synthesis after UVB irradiation on HDF cells promotes the formation of wrinkles in the skin. However, when treated with 100 μg / ml of spirulina and mutant extracts, the amount of procollagen produced after 72 hours was found to increase by 37.4% in UVB-inhibited 37.4% , The amount of procollagen production was increased by 48%. In the case of the UV1-2 mutant extract, the inhibition of MMP-1 expression by 67.8% and the inhibitory activity of elastase were higher in the previous experiment, thus exhibiting the best anti-wrinkle effect of all the mutants tested. It is expected that this cause increased antioxidant and inflammation inhibitory effect and cell growth by increasing the expression of physiologically active substance due to oxidative stress generated during microalgae mutant induction process.

2-6. Effect of MAPK signaling on UV-induced HDF cells

Proteins involved in intracellular signal transduction by UVB in UV1-2 samples selected through cell proliferation, MMP-1 inhibition, and pro-collagen proliferation were screened by Western blotting with a positive control, retinoic acid (10 μM) I looked at it. MAPKs signaling is involved in the overexpression of MMP-1 protein by UVB. As shown in FIG. 7, the effect of the UV1-2 sample on the expression of phosphorylated MAPK proteins (JNK, ERK and p38) produced in HDF cells was examined. It was confirmed that the expression of the control group was increased by UVB (90 mJ / cm ² ) compared to the untreated group. The increased phosphorylated proteins JNK, ERK and p38 all showed a tendency to decrease by UV1-2. In contrast to the positive control group, retinoic acid (10 μM), the UV1-2 concentration of 200 μg / It is found that it is remarkably reduced. In conclusion, it was confirmed that the UV1-2 sample inhibited the phosphorylation of MAPK proteins (JNK, ERK and p38) and inhibited the expression of MMP-1.

2-7. Influence of activation of NF-κB protein on UV-induced HDF cells

As shown in FIG. 7, it was confirmed that the sample of UV1-2 decreased the expression of the phosphorylated MAPK protein. Western blot analysis was performed to determine the amount of protein expression of NF-κB and IκBα. As can be seen from FIG. 8, it was confirmed that the expression of IκBα protein and NF-κB protein phosphorylated by UVB (90 mJ / cm ² ) was increased in the control group and that the non-phosphorylated IκBα protein was decreased in the control group . The results of treatment with UV1-2 showed that the expression of IκBα, which is an increased phosphorylated protein, was suppressed and the overexpressed NF-κB protein was also decreased by UV1-2. The positive control group, retinoic acid (10 μM ), It was found that the concentration of UV1-2 decreased to a similar tendency at a concentration of 200 μg / mL.

2-8. Influence of activation of AP-1 protein on UV-induced HDF cells

In the previous results, it was confirmed that the UV1-2 sample inhibited the expression of the MAPK pathway and NF-κB protein overexpressed by UVB. MAPK signal transduction causes AP-1 to migrate into the nucleus and result in an increase in MMP-1 enzyme. In FIG. 9, expression amounts of AP-1 family proteins c-FOS and cJUN protein were confirmed by Western blotting. As can be seen from FIG. 9, the expression of c-FOS and c-JUN protein was increased by UVB (90 mJ / cm ² ) in the control group compared to the untreated group. The UV1-2 sample inhibited the expression of the increased c-FOS and c-JUN protein, and at a concentration of 200 μg / mL of the UV1-2 sample in comparison to the positive control, retinoic acid (10 μM) Which is a significant decrease.

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. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Korea Biotechnology Research Institute KCTC18254P 20130719

Claims (8)

Arthrospira platensis UV1-2 mutant strains (KCTC 18254P) having an effect of improving skin condition include the above-mentioned skin condition improvement, wrinkle improvement, skin regeneration, skin whitening, skin elasticity improvement, skin aging prevention, skin moisturization improvement, Lt; / RTI &gt; strain.
delete The spirulina mutant strain according to claim 1, wherein the mutant strain has an elastase inhibitory activity which is increased by 20% to 45% as compared to wild type Spirulina strain.
The spirulina mutant strain according to claim 1, wherein the mutant strain has a 1.8 to 2.5-fold increase in inhibition of MMPs (matrix metalloproteinases) -1 expression as compared to the wild-type Spirulina strain.
The spirulina mutant strain according to claim 1, wherein the mutant strain increases the synthesis of pro-collagen by 15% to 35% as compared to the wild-type spirulina strain.
A cosmetic composition for improving skin condition comprising the Arthrospira platensis UV1-2 mutant strain (KCTC 18254P) according to any one of claims 1 to 5, a culture of the strain or an extract of the strain, Characterized in that the improvement is wrinkle improvement, skin whitening, skin elasticity improvement, skin aging prevention, skin moisturizing improvement or black spot removal. delete delete

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