Dunaliella salina extract, composition containing Dunaliella salina extract and application of Dunaliella salina extract

Abstract

The invention discloses a dunaliella salina extract, a composition containing the dunaliella salina extract and application of the dunaliella salina extract and the composition, and mainly relates to application of the combination of the dunaliella salina extract and myophotide C in preparation of cosmetics or skin care products with repairing effects, light aging resistance, aging resistance and oxidation resistance. Tests prove that the composition disclosed by the invention can remarkably improve the anti-oxidation and anti-photoaging capabilities of cells, increase the skin elasticity, strengthen the skin barrier, play an anti-oxidation role and enable the skin to look brighter and brighter.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and particularly relates to a Dunaliella salina extract, a composition containing the same, and uses thereof. Background Art

[0002] Dunaliella salina, also known as Dunaliella salina, is a halophilic green microalga belonging to the Chlorophyceae Volvocales order and is widely distributed in high-salinity waters such as salt lakes, salt pans, and seawater evaporation ponds. Dunaliella salina is an alga with very strong antioxidant ability and has received extensive attention due to its strong salt tolerance and rich carotenoids (mainly β-carotene).

[0003] Research over the years has shown that Dunaliella salina has the following characteristics:

[0004] 1. Extreme environmental adaptability: It can survive under harsh conditions such as high-salt environments (salt concentration can reach 30% NaCl), strong light, and high temperatures (above 40°C).

[0005] 2. Rich in β-carotene: When growing in high-salt and high-light environments, it will synthesize a large amount of β-carotene, making it appear red-orange and being used for natural pigment extraction.

[0006] 3. Rich in glycerol: In order to maintain cell osmotic balance in a high-osmotic pressure environment, Dunaliella salina synthesizes and accumulates a large amount of glycerol, which has moisturizing and antioxidant effects.

[0007] Dunaliella salina is a microalgae resource with great potential and has been widely used in many fields such as food, health products, medicine, cosmetics, bioenergy, and aquaculture. Especially its high content of β-carotene and glycerol makes it an important ingredient for antioxidant, skin care, and health care. With the improvement of biotechnology and industrialization level, the development and utilization prospects of Dunaliella salina will be even broader.

[0008] L-Ascorbic acid is a natural water-soluble antioxidant and whitening agent, also known as vitamin C, and is widely used as an anti-aging ingredient in cosmetics. However, its sensitivity to light and oxygen and unstable structure limit its application in the cosmetics industry. International Patent Application WO2022 / 228475A1 discloses an ascorbic acid polypeptide derivative, which has a novel structure and can solve the stability problem of the traditional VC structure. Therefore, the content about this novel structure ascorbic acid polypeptide derivative will be incorporated into the present invention in its entirety. The above application prepares an active molecule - C-PhytoPeptide raw material with excellent stability and efficacy using the ascorbic acid polypeptide derivative as the core ingredient and commercially sells this raw material (this C-PhytoPeptide contains 1000 ppm EAC-L-Carnosine).

[0009] Although both Dunaliella salina and myoxinol C play important roles in the field of cosmetics, their mechanisms are different, and each has its own advantages and disadvantages when used alone. Although there are already many antioxidant and anti-photoaging products on the market, they basically stay at the simple superposition of components with the same mechanism, and the advantages are not obvious compared with single components, or there is a lack of clear synergistic effect. Moreover, how to extract the important components from Dunaliella salina and use them in combination with myoxinol C to exert the maximum effect is an urgent problem to be solved in this field. Summary of the Invention

[0010] To solve the defects of the prior art, the present invention provides a Dunaliella salina extract, a composition containing the same, and uses thereof. By combining the Dunaliella salina extract and myoxinol C, the present invention can significantly improve the antioxidant and anti-photoaging abilities of cells, increase skin elasticity, strengthen the skin barrier, play an antioxidant role, and make the skin look smoother and brighter.

[0011] In a first aspect, the present invention provides the use of a combination of Dunaliella salina extract and myoxinol C in the preparation of cosmetics or skin care products having repair effects, anti-photoaging, anti-aging, and antioxidant properties.

[0012] Further, the structural formula and preparation method of the myoxinol C are recorded in the international patent application WO2022 / 228475A1, and the following is the specific citation content:

[0013] The present disclosure provides an ascorbic acid polypeptide derivative, a compound represented by the following general formula (1) or a salt thereof:

[0014]

[0015] Wherein, m is 0 and n is 1, or, m is 1 and n is 0;

[0016] X1 and X2 are independently C1-C6 alkyl, C1-C6 alkoxy or halogen group;

[0017] Q1 and Q2 are independently C1-C6 alkyl chains;

[0018] a, b, and c are independently selected from integers from 1 to 10;

[0019] e is 0 and d is an integer from 1 to 10, or, e is 1 and d is 0.

[0020] Further, the ascorbic acid polypeptide derivative is a compound represented by the following general formula (3):

[0021]

[0022] Further, the ascorbic acid polypeptide derivative is a compound represented by the following general formula (4):

[0023]

[0024] Further, the ascorbic acid polypeptide derivative is a compound represented by the following general formula (6):

[0025]

[0026] The present disclosure provides a method for preparing the above ascorbic acid polypeptide derivative, including: first, carboxyl activation is performed on raw material A having an amino protecting group, and then it reacts with raw material B, and after deprotecting the protecting group, the ascorbic acid polypeptide derivative is obtained;

[0027] Among them, the raw material A is a compound represented by the following general formula (7):

[0028]

[0029] a and b independently selected from integers from 1 to 10;

[0030] The raw material B is a compound represented by the following general formula (8):

[0031]

[0032] k is selected from an integer from 1 to 10; Q is an alkyl chain of C1-C6;

[0033] Y is an alkyl group of C1-C6, an alkoxy group of C1-C6 or a halogen group.

[0034] Further, the raw material A is carnosine having an amino protecting group; the raw material B is 3-O-ethyl ascorbic acid;

[0035] Further, the ascorbic acid polypeptide derivative is a compound represented by general formula (3), formula (4), and formula (6);

[0036] Further, the compounds represented by the above (3), (4), and (6) are EAC-L-Carnosine. The applicant of international patent application WO2022 / 228475A1 uses EAC-L-Carnosine (a mixture of the compounds represented by formula (3), (4), and (6) in any proportion) as the raw material of myophotope C, and commercially sells this myophotope C (this myophotope C contains 1000 ppm EAC-L-Carnosine).

[0037] Further, the Dunaliella salina extract is obtained by the following extraction steps:

[0038] 1) Prepare a 1,3 - butanediol solution with a mass fraction of 10%, using deionized water as the solvent;

[0039] 2) According to the ratio of Dunaliella salina powder to the 1,3 - butanediol solution in step 1) of 1:20 (W / V), dissolve the Dunaliella salina powder in the 1,3 - butanediol solution, stir and mix evenly to obtain a Dunaliella salina alcohol solution;

[0040] 3) Ultrasonically treat the Dunaliella salina alcohol solution;

[0041] 4) After the ultrasonic treatment, place the solution at 4°C and let it stand for 1 - 2 hours or centrifuge it in a centrifuge at a speed of 4000 rpm for 15 minutes. Then take the supernatant and filter the supernatant with a 0.45 μm filter membrane;

[0042] 5) Filter the filtered filtrate through a 0.22 μm sterile filter, and then add a preservative with a mass fraction of 2% to the filtrate to obtain a Dunaliella salina extract.

[0043] Furthermore, the conditions for ultrasonic treatment are: standing, ultrasonic temperature 50°C, ultrasonic time 60 minutes, ultrasonic power 360W.

[0044] Furthermore, the preservative is Cosmecare 583.

[0045] In a second aspect, the present invention provides a method for extracting a Dunaliella salina extract, and the extraction steps include:

[0046] 1) Prepare a 1,3 - butanediol solution with a mass fraction of 10%, using deionized water as the solvent;

[0047] 2) According to the ratio of Dunaliella salina powder to the 1,3 - butanediol solution in step 1) of 1:20 (W / V), dissolve the Dunaliella salina powder in the 1,3 - butanediol solution, stir and mix evenly to obtain a Dunaliella salina alcohol solution;

[0048] 3) Ultrasonically treat the Dunaliella salina alcohol solution;

[0049] 4) After the ultrasonic treatment, place the solution at 4°C and let it stand for 1 - 2 hours or centrifuge it in a centrifuge at a speed of 4000 rpm for 15 minutes. Then take the supernatant and filter the supernatant with a 0.45 μm filter membrane;

[0050] 5) Filter the filtered filtrate through a 0.22 μm sterile filter, and then add a preservative with a mass fraction of 2% to the filtrate to obtain a Dunaliella salina extract.

[0051] Further, the conditions for ultrasonic treatment are: standing still, ultrasonic temperature of 50°C, ultrasonic time of 60 minutes, and ultrasonic power of 360W.

[0052] Further, the preservative is Cosmecare 583.

[0053] In a third aspect, the present invention provides a Dunaliella salina extract obtained by the extraction method described in the second aspect.

[0054] In a fourth aspect, the present invention provides a composition containing 0.1% ascorbic acid polypeptide derivative and 0.1% of the Dunaliella salina extract described in the third aspect, based on the total mass of the composition.

[0055] In a fifth aspect, the present invention provides a composition containing 0.25% ascorbic acid polypeptide derivative and 0.25% of the Dunaliella salina extract described in the third aspect, based on the total mass of the composition.

[0056] In a sixth aspect, the present invention provides a composition containing 0.1% myristoyl pentapeptide-17 and 0.1% of the Dunaliella salina extract described in the third aspect, based on the total mass of the composition.

[0057] In a seventh aspect, the present invention provides a composition containing 0.25% myristoyl pentapeptide-17 and 0.25% of the Dunaliella salina extract described in the third aspect, based on the total mass of the composition.

[0058] In an eighth aspect, the present invention provides a composition containing myristoyl pentapeptide-17 and the Dunaliella salina extract described in the third aspect, wherein the mass ratio of myristoyl pentapeptide-17 to the Dunaliella salina extract is 1:1.

[0059] In a ninth aspect, the present invention provides the use of the composition described in the sixth aspect and the composition described in the seventh aspect in the preparation of cosmetics or skin care products with repair efficacy, anti-photoaging, anti-aging, and antioxidant properties.

[0060] Compared with the prior art, the present invention has the following beneficial effects:

[0061] The inventors of the present application unexpectedly found that the combined use of L-ascorbic acid derivatives (such as myristoyl pentapeptide-17) and Dunaliella salina extract can significantly improve cell viability after SLS damage, inhibit ROS generation induced by UVB, enhance the inhibitory effect of UVA-induced Col I, increase the content of collagen, significantly improve the antioxidant and anti-photoaging abilities of cells, achieve multi-faceted whitening, reach a significant whitening effect, and have an effect significantly higher than that of using L-ascorbic acid derivatives or Dunaliella salina extract alone, demonstrating a clear and significant synergistic effect. Description of the Drawings

[0062] Figure 1 These are the experimental results of keratinocyte activity test.

[0063] Figure 2 These are the experimental results of the anti - peroxidation ability of keratinocytes. The composition sample is composed of 0.25% Dunaliella salina extract and 0.25% meiolanin C compounded.

[0064] Figure 3 These are the experimental results of the anti - peroxidation ability of keratinocytes. The composition sample is composed of 0.1% Dunaliella salina extract and 0.1% meiolanin C compounded.

[0065] Figure 4 These are the experimental results of the Col I content of fibroblasts. Detailed implementation mode

[0066] Although the numerical ranges and parameter approximations shown in the broad scope of the present invention, the numerical values shown in the specific embodiments are recorded as accurately as possible. However, any numerical value inherently necessarily contains certain errors, which are caused by the standard deviations existing in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all sub - ranges contained therein. For example, the range of "1 to 10" should be considered to include any and all sub - ranges between the minimum value of 1 and the maximum value of 10 (including the endpoints); that is to say, all sub - ranges starting from the minimum value of 1 or greater, such as 1 to 6.1, and all sub - ranges ending with the maximum value of 10 or less, such as 5.5 to 10. In addition, any reference cited as "incorporated herein" should be understood to be incorporated in its entirety.

[0067] Example 1: Extraction process of Dunaliella salina extract

[0068] 1) Prepare a 1,3 - butanediol solution with a mass fraction of 10%, using deionized water as the solvent;

[0069] 2) According to the ratio of Dunaliella salina powder to the 1,3 - butanediol solution in step 1) of 1:20 (W / V), dissolve the Dunaliella salina powder in the 1,3 - butanediol solution, stir and mix evenly to obtain a Dunaliella salina alcohol solution;

[0070] 3) Ultrasonically treat the Dunaliella salina alcohol solution;

[0071] 4) After the ultrasonic treatment is completed, place the solution at 4°C and let it stand for 1 - 2 hours, then take the supernatant and filter the supernatant with a 0.45μm filter membrane;

[0072] 5) Filter the filtered filtrate through a 0.22μm sterile filter, and then add a preservative with a mass fraction of 2% to the filtrate to obtain Dunaliella salina extract.

[0073] Example 2: Experiment on the efficacy of cell damage repair

[0074] 2.1 Keratinocyte activity test

[0075] Experimental materials: HaCaT cells (induced by SLS: 1%)

[0076] Experimental method: CCK-8 (After the cells were plated for 24 h, they were induced with complete medium containing 1% SLS and the corresponding concentration of the test substance for 24 h, and then the cell activity was detected by the CCK-8 method).

[0077] Test groups: 0.5% Dunaliella salina extract; 0.5% Vitapep C; 0.25% Vitapep C + 0.25% Dunaliella salina extract (the above components are mass fractions), and Vitapep C is also known as: Vitapep Purchased from Hemei Biotechnology (Zhejiang) Co., Ltd.

[0078] Effect calculation formula:

[0079] Upregulation rate (%) = (T / C - 1) * 100%

[0080] Where: T - Average cell activity of the test substance (sample group)

[0081] C - Average cell activity of the negative group (Model, only induced)

[0082] Table 1 Results of the experiment on the efficacy of cell damage repair

[0083]

[0084] The experimental results show (see Figure 1 and Table 1): Under the experimental conditions of this experiment, compared with the negative control group, both 0.5% Dunaliella salina extract and 0.5% Vitapep C have the effect of improving the cell activity after SLS damage, while the combination of 0.25% Dunaliella salina extract and 0.25% Vitapep C has a significant synergistic effect.

[0085] 2.2 Keratinocyte ROS test

[0086] Experimental materials: HaCaT cells (induced by UVB: 40 mJ / cm 2 ).

[0087] Experimental method: Fluorescence method (After the cells were plated for 24 h, they were induced by UVB, and after adding the corresponding concentration of the sample for 24 h, the ROS content in the cells was detected by the fluorescence probe method).

[0088] Test groups: 0.5% Dunaliella salina extract; 0.5% cyclo(His - Pro); 0.25% cyclo(His - Pro) + 0.25% Dunaliella salina extract (the above components are mass fractions).

[0089] Effect calculation formula:

[0090] Inhibition rate (%) = (1 - T / C) * 100%

[0091] Where: T—average ROS content of the test substance (sample group)

[0092] C—average ROS content of the negative group (Model, only induction)

[0093] Table 2 Experimental results of anti - peroxidative damage of keratinocytes

[0094]

[0095] The experimental results show that ( Figure 2 and Table 2): Under the experimental conditions of this experiment, compared with the negative control group, both 0.5% Dunaliella salina extract and 0.5% cyclo(His - Pro) have the effect of inhibiting the generation of ROS induced by UV, while the combination of 0.25% Dunaliella salina extract and 0.25% cyclo(His - Pro) has a synergistic effect.

[0096] In addition, the applicant conducted relevant experiments on another group of test groups (0.2% cyclo(His - Pro); 0.2% Dunaliella salina extract; 0.1% cyclo(His - Pro) + 0.1% Dunaliella salina extract (the above components are mass fractions)), and the results are as Figure 3 shown in Table 3:

[0097] Under the experimental conditions of this experiment, compared with the negative control group, both 0.2% Dunaliella salina extract and 0.2% cyclo(His - Pro) have the effect of inhibiting the generation of ROS induced by UVB, while the combination of 0.1% Dunaliella salina extract and 0.1% cyclo(His - Pro) has a significant synergistic effect and achieves unexpected technical effects.

[0098] Table 3 Experimental results of anti - peroxidative damage of keratinocytes

[0099]

[0100] Example 3: Experimental results of fibroblast Col I test

[0101] Experimental materials: HSF cells (UVA induction: 40 mJ / cm 2 )

[0102] Experimental method: ELISA (UVA induction 24 h after cell seeding, adding samples at corresponding concentrations, collecting the supernatant 24 h later, and detecting the cell Col I content by ELISA).

[0103] Test groups: 0.2% Glutathione C; 0.2% Dunaliella salina extract; 0.1% Glutathione C + 0.1% Dunaliella salina extract (the above components are mass fractions).

[0104] Effect calculation formula:

[0105] Up-regulation rate (%) = (T / C - 1) * 100%

[0106] Where: T—the average content of type I collagen in the test substance (sample group)

[0107] C—the average content of type I collagen in the negative group (Model, only induction)

[0108] Table 4 Results of fibroblast Col I test experiment

[0109]

[0110] The experimental results show (see Figure 4 and Table 4): Under the conditions of this experiment, compared with the negative control group, 0.2% Dunaliella salina extract has improved the inhibitory effect of UVA-induced Col I and increased the content of collagen. Moreover, the combined use of 0.1% Glutathione C and 0.1% Dunaliella salina extract has a significant synergistic effect and achieves unexpected technical effects.

[0111] The above description only shows the preferred implementation embodiments, which are only examples and do not limit the combination of essential features for implementing the present invention. The provided titles do not intend to limit the various implementation embodiments of the present invention. Terms such as "comprising", "containing" and "including" are not intended to be limiting. In addition, unless otherwise specified, when not modified by a numeral, it includes the plural form, and "or", "either...or" means "and / or". Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0112] The above embodiments are preferred embodiments of the present invention, but the implementation embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of a combination of Dunaliella salina extract and myo-phototripeptide C in the preparation of a cosmetic or skin care product having repair efficacy, anti-photoaging, anti-aging and antioxidant properties.

2. The use according to claim 1, characterized in that, The Dunaliella salina extract is obtained by the following extraction steps: 1) Prepare a 1,3-butanediol solution with a mass fraction of 10%, using deionized water as the solvent; 2) Dissolve Dunaliella salina powder in the 1,3-butanediol solution prepared in step 1) at a mass-to-volume ratio of 1:20 (W / V), stir and mix well to obtain a Dunaliella salina alcohol solution; 3) Subject the Dunaliella salina alcohol solution to ultrasonic treatment; 4) After the ultrasonic treatment is completed, let the solution stand at 4°C for 1 - 2 hours or centrifuge it at a speed of 4000 rpm for 15 minutes, then take the supernatant and filter the supernatant with a 0.45 μm filter membrane; 5) Filter the filtered filtrate through a 0.22 μm sterile filter, and then add a preservative with a mass fraction of 2% to the filtrate to obtain the Dunaliella salina extract.

3. The use according to claim 2, characterized in that, The conditions for ultrasonic treatment are: standing, ultrasonic temperature 50°C, ultrasonic time 60 minutes, ultrasonic power 360W.

4. The use according to any one of claims 2-3, characterized in that The preservative is Cosmecare 583.

5. A method for extracting Dunaliella salina extract, characterized in that, The extraction steps include: 1) Prepare a 1,3-butanediol solution with a mass fraction of 10%, using deionized water as the solvent; 2) Dissolve Dunaliella salina powder in the 1,3-butanediol solution prepared in step 1) at a mass-to-volume ratio of 1:20 (W / V), stir and mix well to obtain a Dunaliella salina alcohol solution; 3) Subject the Dunaliella salina alcohol solution to ultrasonic treatment; 4) After the ultrasonic treatment is completed, let the solution stand at 4°C for 1 - 2 hours or centrifuge it at a speed of 4000 rpm for 15 minutes, then take the supernatant and filter the supernatant with a 0.45 μm filter membrane; 5) Filter the filtered filtrate through a 0.22 μm sterile filter, and then add a preservative with a mass fraction of 2% to the filtrate to obtain the Dunaliella salina extract.

6. The extraction method according to claim 5, wherein The conditions for ultrasonic treatment are: standing, ultrasonic temperature 50°C, ultrasonic time 60 minutes, ultrasonic power 360W.

7. The extraction method according to any one of claims 5-6, characterized in that, The preservative is Cosmecare583.

8. A Dunaliella salina extract, characterized in that, Obtained by the extraction method according to any one of claims 5 - 7.

9. A composition, characterized in that, The composition contains 0.1% myo-phototripeptide C and 0.1% of the Dunaliella salina extract according to claim 8, based on the total mass of the composition.

10. A composition, characterized in that, The composition contains 0.25% myo-phototripeptide C and 0.25% of the Dunaliella salina extract according to claim 8, based on the total mass of the composition.

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