KR20120041404A - Cosmetic solution from algae peptide - Google Patents

Abstract

PURPOSE: A cosmetic composition containing peptides is provided to promote anti-wrinkling, anti-aging, and whitening. CONSTITUTION: A cosmetic composition contains 0.001-10 wt% of a peptide mixture derived from microalgae as an active ingredient. The cosmetic composition is manufactured in the form of a lotion, gel, water soluble liquid, cream, essence, and oil-in-water(O/W) emulsion and water-in-oil(W/O) emulsion. The microalgae include Chlorella vulgaris, Dunaliella salina, Scenedesmus obliquus, Scenedesmus quadricauda, Spirulina maxima, or Spirulina platensis.

Description

Microalgae peptide cosmetic composition {Cosmetic solution from algae peptide}

The present invention relates to a cosmetic composition containing peptides obtained from various microalgae, and more particularly, the composition of the present invention includes a peptide mixture containing microalgae such as spirulina, chlorella, and isocrissis to improve existing skin. Compared to the cosmetics containing ingredients, the growth of fibroblasts in the skin, collagen synthesis, skin wrinkle improvement, anti-aging and whitening were improved. To this end, the present invention is characterized by culturing a variety of microalgae, the cosmetic composition prepared by using the peptide mixture obtained from the skin improvement effect.

Algae are a very diverse group of organisms known to contain about 40,000 species, and are known to produce a variety of useful substances in their natural state. In addition to the most well-known health supplements, microalgae-derived useful substances are very diverse as natural pigments, pharmaceutical raw materials, and biochemicals. Representative useful substances include vitamins, carotenoids, proteins, fabtide, amino acids, polysaccharides, etc., algae biomes are used as dietary supplements or food additives.

Among the useful materials that can be obtained from microalgae, one of the materials recently attracting attention in the field of cosmetic materials is a peptide. Peptides lower blood pressure, antioxidant, lipid metabolism. It plays an important role in immune enhancement, lowering blood cholesterol, inhibiting alcohol absorption, promoting iron and calcium absorption, inhibiting wrinkle formation of skin, and controlling various kinds of in vivo control and information, controlling hormones, cell proliferation, blood pressure, It has neurotransmission, antibiotic activity, etc. and acts as a regulatory factor in about 90% of the in vivo regulation, attracting attention as a new material in the food, cosmetics and pharmaceutical fields.

Algae are divided into microalgae and macroalgae by size, and floating algae in seawater occupy a major position as the primary producers of ecosystems, which are estimated to account for 90% of all photosynthesis on Earth. In particular, microalgae can be grown in large quantities at relatively low cost using sunlight, carbon dioxide, and the like in water.

The present invention is characterized in that the peptide composition obtained from various microalgae has a cosmetic composition containing the same and has an improvement in skin. To provide a cosmetic composition containing the peptide mixture having a proliferation and collagen synthesis, skin wrinkle improvement, anti-aging effect, whitening effect of the fibroblasts in the skin.

The cosmetic composition of the present invention is known to contain a large amount of peptides in microalga Chlorella vulgaris, Dunaliella salina, Scenedesmus obliquus, Cinedes mus quadricauda It contains a peptide mixture obtained by culturing quadricauda, Spirulina maxima, and Spirulina platensis, which improves the effect compared to cosmetics containing the skin improvement ingredients. It relates to a cosmetic composition comprising a peptide of 0.001-10% by weight in the cosmetic. The cosmetic composition is characterized in that the lotion, gel, water-soluble liquid, cream, essence, water vapor (O / W) type and water vapor (W / O) type formulation.

The microalgae-derived peptide mixture used in the present invention has an excellent moisturizing effect and proliferation of fibroblasts in the skin and collagen synthesis, which has a whitening effect by inhibiting donation and melanin production, which enhances skin wrinkles.

Six microalgae prepared peptide mixture in the present invention are Chlorella vulgaris, Dunaliella salina, Scenedesmus obliquus, Scenedesmus quadricauda, Spinullina maxima, Spirulina platensis. Containing the fabtide mixture obtained by culturing this effect was improved compared to the cosmetics containing the existing skin improvement ingredients, and relates to a cosmetic composition comprising a microalgae-derived peptide in the cosmetics 0.001-10 weight percent will be.

The cosmetic composition is characterized in that the lotion, gel, water-soluble liquid, cream, essence, water vapor (O / W) type and water vapor (W / O) type formulation.

The present invention is characterized in that at least one of the peptide mixture extracted from microalgae comprises 0.001-10% by weight relative to the total weight of the cosmetic.

In more detail the configuration of the present invention, the microalgae-derived peptide mixture of the present invention contains 0.001-10% by weight, preferably 0.01-5% by weight of the cosmetic. If the content of the microalgae-derived peptide mixture is less than 0.001% by weight, the effect of the ingredient is hardly exhibited. If the content of the peptide is more than 10% by weight, the increase in effect due to the increase of the content is insignificant, the color of the composition is not desirable, and the color is denatured. Increase the likelihood.

In addition, the cosmetic composition containing the peptide mixture derived from the microalgae of the present invention is not particularly limited in the formulation, for example, lotion, gel, water-soluble liquid, painting, essence, steam (O / W) It may have a formulation such as type and water vapor (W / O) type. In the cosmetic composition of each type, other components other than the microalgae-derived peptides may be selected and blended by those skilled in the art without difficulty according to the formulation or purpose of use of other cosmetics.

The microalgae-derived peptide mixture used in the present invention has an excellent moisturizing effect and proliferation of fibroblasts in the skin and collagen synthesis, which has a whitening effect by inhibiting donation and melanin production, which enhances skin wrinkles.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited only to the following Examples.

   Example 1

Six microalgae, Chlorella vulgaris, Dunaliella salina, Scenedesmus obliquus, Scenedesmus quadricauda, Spirulina maxima ), Spirulina platensis (Spirulina platensis) was cultured for each growth temperature and pH to prepare the culture conditions and the medium was passaged in a flask for 10-30 days.

The culture incubator was installed with a light source to facilitate photosynthesis to create a culture environment, and the flask was cultured using a carbon dioxide incubator to maintain the concentration of carbon dioxide in the culture medium.

After securing the cell number necessary for the experiment, centrifugation at 5,000rpm to remove the supernatant, and obtained only microalgae, diluted with purified water corresponding to 10 times the volume obtained, using a cell crusher to obtain a microalgae extract. . In addition, an ultrafilter was used to obtain a peptide mixture. Thus obtained was freeze-dried and used in the following Examples and Experimental Examples.

   Example 2

This example prepared a cosmetic containing the microalgae-derived peptide mixture obtained in Example 1. Cosmetics used in the experiment is in the form of an emulsion, the composition is shown in Table 1. After preliminary emulsification of the compositions listed in Table 1, the mixture is uniformly emulsified with a homomixer, and then gradually cooled to prepare an emulsion.

Raw material Example Comparative example One 2 3 4 5 6 7 8 Ethylenediaminetetra disodium acetate Quantity Quantity Quantity Quantity Quantity Quantity Quantity Quantity DL-pantenol 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 1,3-butylene glycol 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 Carboxyvinyl Polymer 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Cetearyl Alcohol / Cetearyl Glucoside 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 Fiji-20 / Methylglucoseses quisteale 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 Dimethicone 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 Isostearyl lactate 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 Cetanol 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 Triethanolamine 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Chlorella vulgaris 0.1 - - - - - 0.1 - Dunaliella salina - 0.1 - - - - 0.1 - Scenedesmus - - 0.1 - - - 0.1 - Cinedes medsquadry cauda (Scenedesmus) - - - 0.1 - - 0.1 - Spirulina maxima - - - - 0.1 - 0.1 - Sipinulina platensis
(Spirulina platensis) - - - - - 0.1 0.1 - bean - - - - - - - 0.1

<Unit in table: weight percent>

   Test Example 1: Determination of Antioxidant Effect

The active oxygen produced by xanthine and xanthine oxidase is measured by the NBT method, and the effect of the test substance removing the active oxygen, that is, the active oxygen effect is evaluated. Free radicals are produced by xanthine and xanthine oxidase.

The amount of active oxygen is measured by measuring the blue color produced by this active oxygen reacting with Nitroblue tetrazolium (NBT) at a wavelength of 560 nm.

As the reagent used

1) 0.05M (50mM) Na2CO3 buffer (MW = 105.99): A solution of 5.25g (50mM) of sodium carbonate dissolved in purified water and 50mM NaHCO3 (MW = 84.01) are mixed to pH 10.2.

2) 3mM xanthine solution: 45.6mg of xanthine (MW = 152.11) is dissolved in water to make 10ml.

3) 3mM EDTA solution: Sodium ethylenediamine tetraacetate (MW = 60.1) is dissolved in distilled water to make 3mM.

4) 0.15% BSA solution: Dissolve 15mg of BSA (Fraction V, powder, Sigma) in distilled water to make 10ml.

5) 0.75mM NBT solution: 61.32mg of Nitroblue tetrazolium (MW = 817.65) is dissolved in distilled water to make 100ml.

6) xanthine oxidase solution: Dilute xanthine oxidase (Boehringer) with distilled water about 100-fold and make the absorbance of the blank test within the range of 0.2 ~ 0.23 in the measurement operation. In other words, dilute with purified water so that the change in absorbance is A560 = 0.3 / 20 minutes.

7) 6mM CuCl2 solution: Dissolve 102.29mg of copper chloride (CuCl2-2H2O, MW = 134.45) in distilled water to make 100ml.

As a measuring method

end. 0.05M Na2CO3 -------------- 2.4ml

I. 3mM xanthine solution ------ 0.1ml

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

la. BSA solution ------------- 0.1ml

hemp. 0.72mM NBT solution ----- 0.1ml

bar. Xanthine Oxidase Solution --- 0.1ml

four. 6mM CuCl2 Solution --------- 0.1ml

(1) Add 1,2,3,4,5 to the Bayer bottle and add 0.1 ml of sample solution to it and leave at 25 ℃ for 10 minutes.

(2) Add 6, stir rapidly, and start incubation for 20 minutes at 25 ° C.

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

(4) In the blank test, absorbance Bt is measured using distilled water instead of the sample solution as described above.

(5) Again, the blank of the sample solution was operated in the same manner using distilled water instead of 6 to measure the absorbance Bo.

Result calculation of the effect was calculated by the following equation (1).

Equation 1

Inhibition Rate (%) = 1- (St-So) / (Bt-Bo) × 100

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

Bt: absorbance at 560 nm after enzymatic reaction of blank test solution

So: absorbance at 560 nm before reaction without enzyme addition of sample solution

Bo: absorbance at 560 nm before reaction without enzyme in blank test solution

The sample used in the test is shown in Table 2 the experimental results of the 0.1% by weight mixture of the peptide obtained in Example 1. As shown in Table 2, the microalgae-derived peptide mixture can be seen that the active oxygen removal effect is excellent.

division Antioxidative effect(%) Chlorella vugaris 63 Dunaliella salina 57 Scenedesmus obliquus 45 Scenedesmus quadricauda 52 Spirulina maxima 68 Spirulina platensis 63 bean 52

   Test Example 2: Measurement of Cell Activity Effect (MTT assay)

The cell line was Hs-68, a mouse-derived fibroblast, and the cells were dispensed at 2 x 10 ⁵ cells per well in a 12 well plate and cultured for 24 hours. Each sample was treated at a concentration that does not cause cytotoxicity, and after 24 hours of treatment, the medium was removed, and 500ul fresh medium and 60ul MTT solution were added to each well, followed by incubation at 37 ° C. in a CO ₂ incubator for 2 hours. Dissolve the cells and transfer 100ul of each to a 96-well plate. Measure absorbance at 565 nm with an ELISA reader. The control group was measured in the same manner as above except that no sample was added, and the absorbance was measured, and the cell proliferation rate was expressed as a comparative percentage of 100 for the control cell proliferation rate.

As shown in Table 3, the microalgae-derived peptide mixture obtained in Example 1 has an excellent cell proliferation effect on Hs-68, a mouse-derived fibroblast.

division Cell proliferation rate (%) Chlorella vugaris 128 Dunaliella salina 126 Scenedesmus obliquus 108 Scenedesmus quadricauda 110 Spirulina maxima 132 Spirulina platensis 121 bean 100

   Test Example 3: Measurement of Collagen Production Promoting Effect

The cell line used in the experiment was human fibroblast (Human Dermal Fibroblast, HDF), and ELISA (Enzyme-Linked ImmunoSorbent Assay) was used to measure the concentration of collagen, which is a major component of the extracellular matrix, after the addition of the sample. Was carried out. Samples were exchanged with added serum (serum free), incubated for 12 hours, and washed three times with HDF with PBS-T (0.02% tween-20 in PBS). Cells were fixed with 3% formaldehyde (formaldehyde) for 20 minutes and then blocked by treating with 5% skim milk for 1 hour. Primary antibody (monoclonal anti-collagen type I antybody) was reacted for 40 minutes at 37 ℃, 5% CO ₂ . After washing three times with PBS-T, the secondary antibody (anti-mouse IgG antibody) was diluted to 1 / 1,000 in PBS-T and reacted for another 40 minutes, and then PBS-T Wash 5 times with T. Alkaline phosphatase substrate solution (1 mg / ml -nitrophenyl phosphate in diethanolamine buffer) was added to the substrate for 30 minutes, and then absorbance was measured at 405 nm. The control group was measured in the same manner as above except that no sample was added, and the absorbance was measured, and the collagen production promoting effect was expressed as a comparative percentage of the collagen amount of the control group as 100.

As shown in Table 4, it can be seen that the collagen production promoting effect test for human fibroblasts using the peptide mixture derived from the microalgae obtained in Example 1 has an excellent effect.

division Collagen production rate (%) Chlorella vugaris 158 Dunaliella salina 117 Scenedesmus obliquus 105 Scenedesmus quadricauda 138 Spirulina maxima 153 Spirulina platensis 142 bean 115 Control group 100

   Test Example 4: Test of Tyrosinase Inhibitory Effect Using Tyrosinase

The test of this test example is a test to determine the whitening effect through the degree of inhibition of the function of the enzyme tyrosinase, which is a regulator of melanin biosynthesis process in the skin to confirm the whitening effect of the sample obtained in the example.

Tyrosinase is an enzyme in which the melanin acts as a regulator of the production process by promoting the oxidation process of tyrosine in vivo. In this test example, the tyrosine is oxidized to inhibit the function of this enzyme. The whitening effect was determined by applying a method of measuring the degree of suppression of the formation of the.

15ul of sample was added to a 96-well plate, 150ul of 50mM phosphate buffer (pH 6.5), 25ul of 1.5mM L-tyrosine solution were added, and 10ul of mushroom tyrosinase (1,500 units / ml, Sigma) was added and reacted at 37 ° C for 20 minutes. The inhibition rate for tyrosinase was then determined by measuring absorbance at 490 nm using a microplate reader (ELx800, USA). Inhibition rate for tyrosinase (%) was calculated by the following equation (2), IC50 value is the concentration of the sample that inhibits the tyrosinase enzyme activity by 50%.

Equation 2

% Inhibition = [(D-C)-(B-A) / (D-C)] × 100

A: Absorbance before reaction of the well to which the sample is added

B: Absorbance after reaction of the well to which the sample was added

C: Absorbance before reaction of wells without sample

D: absorbance after reaction of a well without sample

As shown in Table 5, the aqueous micropeptide-derived aqueous solution obtained in Example 1 was found to have an excellent inhibitory effect on tyrosinase.

division Tyrosinase inhibitory effect (IC 50%) Chlorella vugaris 0.37 Dunaliella salina No inhibitory effect Scenedesmus obliquus 0.21 Scenedesmus quadricauda 0.98 Spirulina maxima 1.12 Spirulina platensis No inhibitory effect bean No inhibitory effect

   Test Example 5: Melanin synthesis inhibition of melanocytes using melanocyte stimulating hormone (MSH)

The test of this test example is an experiment to confirm the melanin synthesis inhibitory effect on melanocytes in order to confirm the whitening effect of the sample of the example. The cell line used in the test was A375SM melanocyte-forming cell line, which is a human-derived cell line that does not secrete melanin under normal conditions, but binds to the cell membrane receptors of melanocytes and transmits melanin synthesis signals into melanocyte-forming melanocyte-stimulating hormones. Treating is a cell that secretes melanin. The artificial culture of these cells was treated with a-MSH, germinated black rice enzyme digested peptides obtained in Examples 1 and 2, and germinated black soybean digested peptides to compare and evaluate the degree of synthesis of melanin black pigment. The A375SM melanin forming cells used in this test example were used from KCLB (Korea Cell Line Bank, Accession No.:80004).

A375SM melanocytes were dispensed in 6-well plates at a concentration of 2 × 10 ⁶ per well and attached to the cells, followed by incubation with a-MSH (Sigma) 200nM and the sample simultaneously for 48 hours. After 48 hours of incubation, the cells were detached with trypsin-EDTA, and the cells were counted and centrifuged to recover the cells. Intracellular melanin quantification was performed by slightly modifying the method of Rotan (Cancer Res., 40,3345-3350, 1980). Cell pellets were washed once with PBS, and then 1 ml of homogenization buffer (50 mM sodium phosphate buffer, pH6.8, 1% Triton X-100, 2 mM PMSF) was added to shake cells for 5 minutes. Melanin synthesized by adding 1N sodium hydroxide solution with 10% DMSO to the cell lysate obtained by centrifugation at 3000 rpm for 10 minutes to dissolve the extracted melanin and measuring the absorbance of melanin at 405 nm with a microplate reader (ELx800, USA) After quantifying the melanin synthesis inhibition rate of the sample was measured.

Inhibition rate of melanin synthesis (%) dms of A375SM melanogenesis cells was calculated by the following equation (3), IC50 value is the concentration of the sample that inhibits melanin production by 50%.

Equation 3

% Inhibition = [(A-B) / A] × 100

A: Melanin amount in wells containing only a-MSH

B: Melanin content in the wells to which the sample and a-MSH were simultaneously applied

As shown in Table 6, the aqueous micropeptide-derived peptide solution obtained in Example 1 was found to have an excellent melanin synthesis inhibitory effect on melanocytes.

division Melanin synthesis inhibitory effect (IC 50%) Chlorella vugaris 0.28 Dunaliella salina 0.17 Scenedesmus obliquus 0.05 Scenedesmus quadricauda 0.12 Spirulina maxima 0.24 Spirulina platensis0. 0.08 bean No inhibitory effect

   Test Example 6: Measurement of skin wrinkle improvement and skin beauty effect

The cream prepared in Example 2 was applied on the right side of the face and the left side of the face for 15 consecutive experimenters (women aged 20-35 years), twice a day for 3 consecutive months.

After completion of the experiment, the skin wrinkle reduction effect is obtained by using a silicone resin copy (replica) around the facial eye tail before and after using the product for 3 months, and using the skin fine wrinkle device and skin image analyzer, The degree of skin color improvement was compared.

As shown in Table 7, it can be seen that the wrinkle-improving effect is excellent in the skin around the facial eye of the experimenter applying the emulsion prepared in Example 2.

division Average wrinkle depth reduction rate (%) Chlorella vugaris 0.37 Dunaliella salina No inhibitory effect Scenedesmus obliquus 0.21 Scenedesmus quadricauda 0.98 Spirulina maxima 1.12 Spirulina platensis No inhibitory effect bean No inhibitory effect

   Test Example 7: Measurement of Skin Moisturizing Effect

The moisturizing effect was measured using a capacitance measurement method using the Corneometer CM825. The subjects were 15 females in their 20s and 30s. 60%) for 20 minutes to keep the skin's moisture itself constant. Subsequently, the initial moisture retention amount of each sample application site was measured using the Corneo Meta CM825, and then the sample was applied. The application of the sample was applied while varying the position of the sample to be applied to each subject in order to minimize the error along the oyster side surface of the 2.0 mg / cm 2. Skin moisture content was measured at 60 minutes and 120 minutes of application.

Equation 4

Tdi: nth measured value of sample application area

Tdo: Measured value before application of sample application site (initial moisture content of test site)

NTdi: nth measured value of the uncoated area

NTdo: Measured value before application of the sample unapplied area (initial moisture content of the control part)

As shown in Table 8, the degree of skin moisture decrease with time after application of the latex oyster side of the tester coated with the emulsion prepared in Example 2, it can be seen that the skin moisture retention effect is excellent over time.

division Water content after application 60 minutes 120 minutes Chlorella vugaris 63.83 57.75 Dunaliella salina 67.24 52.67 Scenedesmus obliquus 51.89 38.35 Scenedesmus quadricauda 48.73 36.02 Spirulina maxima 73.06 65.77 Spirulina platensis 63.58 52.36 bean 53.21 39.77

Claims (3)

Cosmetic composition containing at least one of the algae-derived peptide mixture as an active ingredient
The peptide mixture of microalgae according to claim 1, characterized in that the cosmetic composition is a lotion, gel, water soluble liquid, cream, essence, water vapor (O / W) type and water vapor (W / O) type formulation. Cosmetic composition containing at least one selected from the group as an active ingredient
According to claim 1, wherein the at least one selected from the peptide mixture derived from microalgae is a cosmetic composition, characterized in that it comprises 0.001-10% by weight relative to the total weight of the cosmetic