Preparation method and application of nannochloropsis extract for after-sun repair for people with dark skin

Abstract

The application belongs to the technical field of cosmetic preparation, and discloses a micro-nannochloropsis extract for after-sun repair of deep-skinned people, a preparation method and application thereof. The method comprises the following steps: defatting and decolorizing filtration of micro-nannochloropsis powder, enzyme hydrolysis of the filter residue after addition of cellulase liquid, and then water extraction under acidic conditions with pH of 1-3 and at 80 DEG C, cooling and filtration, pH of 8-10 alkaline condition and 80 DEG C extraction of the obtained filter residue, cooling and filtration, combined filtration of the filtrates obtained by the two times of filtration, ultrafiltration treatment, 95% ethanol precipitation, centrifugation to obtain supernatant, reduced pressure concentration, dilution of the obtained concentrated solution, and then loading onto a pre-activated SPE column, elution with 60%, 70% and 80% ethanol in sequence, combined elution, and reduced pressure concentration to obtain the micro-nannochloropsis extract. The extract has the effects of enhancing skin repair and anti-inflammation, is especially suitable for after-sun care of deep-skinned people, limits melanin production, slows down the secretion speed of oil, and repairs the skin barrier.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cosmetic preparation, and particularly relates to a Nannochloropsis oculata extract for after-sun repair of deep-skinned people, a preparation method and application thereof. BACKGROUND

[0002] With the increase of global ultraviolet radiation intensity, the problem of sunburn has become a growing concern for more and more people. Especially for deep-skinned people, due to the high content of melanin in their skin, although they have a certain natural sun protection effect, they still suffer from post-sun inflammation, pigmentation and decreased elasticity. The existing after-sun repair products mainly use moisturizing and antioxidant ingredients for repair, but often ignore the unique reaction mechanism and repair needs of deep-skinned people's skin.

[0003] Nannochloropsis oculata is a eukaryotic alga of the family of Monoraphidaceae in the class of Eustigmatophyceae in the phylum of Brown algae, with spherical cells, high photosynthetic efficiency and short growth cycle, and is an important economic microalgae that is easy to cultivate. Nannochloropsis oculata is rich in protein, carbohydrate and mineral substances, and is currently mainly used in the aquaculture industry as feed, resulting in a large amount of resource waste. In recent years, algal proteins have been welcomed as a new source of plant proteins. As a kind of efficient microalgae, it is rich in various bioactive substances such as oligopeptides, polyphenols and minerals, and has been applied in the field of cosmetics.

[0004] A skin care composition containing Chlorella minutissima extract, its preparation method and application are disclosed in Chinese Patent Publication No. CN118319799A, which includes the following steps: 1) mixing emulsifiers, moisturizers, emollients, preservatives, and deionized water, and obtaining a vesicle solution after high-pressure microjet treatment; 2) mixing the vesicle solution obtained in step 1) with Chlorella minutissima extract, alginate, squalane, Nannochloropsis oculata extract, and Haematococcus pluvialis extract, and obtaining the skin care composition containing Chlorella minutissima extract after high-pressure microjet treatment. Chinese Patent Publication No. CN113584102B discloses a preparation method of Chlorella minutissima polysaccharide with antioxidant activity, which includes the following steps: 1) ultrasonic-microwave extraction after dissolving Chlorella minutissima in water; 2) enzymatic hydrolysis of the extract obtained in step 1) with a complex enzyme; 3) centrifugation after enzyme inactivation of the enzymatic hydrolysate in step 2); 4) vacuum distillation of the supernatant obtained in step 3), and alcohol precipitation by adding anhydrous ethanol; 5) centrifugation of the alcohol precipitation mixture in step 4), freezing the centrifugation precipitate to the eutectic point, and vacuum freeze-drying to obtain Chlorella minutissima polysaccharide crude extract; 6) dissolving the polysaccharide crude extract and adding a decolorizing agent, and placing it in a shaking bed for shock decolorization, and obtaining a treated liquid after decolorization and centrifugation; 7) adding Sevage reagent to the treated liquid and mixing to remove proteins, and obtaining a supernatant after centrifugation, and vacuum freeze-drying treatment to obtain Chlorella minutissima polysaccharide purified product; 8) preparing a Chlorella minutissima polysaccharide purified product solution, and modifying Chlorella minutissima polysaccharide by adding α-amylase, and obtaining enzyme-modified Chlorella minutissima polysaccharide after high-temperature enzyme inactivation.

[0005] Many current after-sun repair products are targeted at white-skinned and sensitive-skinned people, mainly providing calming and soothing effects, but there are few after-sun repair products for dark-skinned people. Dark-skinned people have more melanin in their skin, and after sun exposure, melanin is more likely to settle, oil secretion is faster, and the skin barrier is more easily damaged. Existing technologies show that Chlorella minutissima extract can promote skin anti-aging and skin tightening, but its application in after-sun repair for dark-skinned people has not been fully explored.

[0006] Currently, many after-sun repair products on the market are not optimized for the special needs of dark-skinned people. The skin of dark-skinned people has different melanin distribution and structural characteristics from that of light-skinned people, and their response to UV rays and repair mechanisms are also different. Existing repair products are mostly based on general ingredients and technologies, ignoring the unique needs of dark-skinned skin, resulting in unsatisfactory repair effects and even potentially causing new skin problems such as fast melanin production and slow metabolism. SUMMARY

[0007] To overcome the shortcomings and deficiencies in the prior art, the primary purpose of the present application is to provide a preparation method of Chlorella minutissima extract for after-sun repair of dark-skinned people.

[0008] Another object of the present application is to provide a Nannochloropsis extract for post-sun repair of deep skin color people prepared by the above method; the Nannochloropsis extract has the effects of enhancing skin repair and anti-inflammation, is particularly suitable for post-sun care of deep skin color people, limits melanin production, slows down the secretion speed of oil, and repairs the skin barrier.

[0009] Still another object of the present application is to provide the application of the above Nannochloropsis extract for post-sun repair of deep skin color people; the use of the Nannochloropsis extract in cosmetic products ensures that it has the effect of repairing post-sun skin, can effectively reduce the pigment deposition caused by ultraviolet rays, avoids the problem of pigment rebound of deep skin color skin during post-sun repair, and thus provides a solution for post-sun melanin activity of deep skin color people.

[0010] The object of the present application is achieved by the following technical solutions:

[0011] A preparation method of a Nannochloropsis extract for post-sun repair of deep skin color people, comprising the following operation steps:

[0012] (1) The dried Nannochloropsis powder is coarsely crushed by a crusher and sieved to obtain a powder with uniform particle size; 95% ethanol by volume is added to the powder, and the mixture is stirred at 60°C for 30 minutes twice to remove fat and color, filtered, and the filter residue I is collected;

[0013] (2) Water and 0.05% cellulase solution by weight of the filter residue I are added to the filter residue I obtained in step (1) at a solid-liquid ratio of 1g:20mL, and the cellulase solution has an activity of 10000U / g; the mixture is enzymatically hydrolyzed at 40-60°C for 3-5h;

[0014] (3) The filter residue I after enzymatic hydrolysis in step (2) is added with water as an extraction solvent at a solid-liquid ratio of 1g:40-100mL, and the mixture is extracted at an acid pH of 1-3 and a temperature of 80°C for 1-3h; the mixture is cooled and filtered, and the filtrate is collected to obtain filter residue II; the filter residue II is extracted at an alkaline pH of 8-10 and a temperature of 80°C for 1-3h, cooled and filtered, and the filtrate is collected to obtain filter residue III; the filtrates obtained by the two times of filtration are combined; the combined filtrate is subjected to ultrafiltration treatment using an ultrafiltration membrane with a molecular weight cutoff value of 10KDa, then alcohol precipitation is performed using 95% ethanol by volume, centrifugation is performed to obtain the supernatant, and the supernatant is concentrated under reduced pressure to obtain a concentrated solution;

[0015] (4) The pH value of the concentrated solution is adjusted to 5, and the solution is diluted and loaded onto a pre-activated SPE column; the column is eluted with 60%, 70%, and 80% ethanol by volume in sequence, and the eluates obtained by the three times of elution are combined and concentrated under reduced pressure to obtain the Nannochloropsis extract.

[0016] Preferably, the sieving in step (1) is a 30-mesh analytical sieve; and the ethanol with a volume fraction of 95% is added to the powder at a ratio of 1g:20mL.

[0017] Preferably, the ratio of the material to the liquid in step (3) is 1g:100mL.

[0018] Preferably, the temperature of the enzymatic hydrolysis in step (2) is 50℃, and the enzymatic hydrolysis time is 5h.

[0019] Preferably, the acidic condition in step (3) is pH 2, the basic condition is pH 8, and the leaching time is 2h.

[0020] Preferably, the weight of the ethanol with a volume fraction of 95% in step (3) is 3 times the weight of the combined filtrate.

[0021] Preferably, the dilution in step (4) is dilution of the concentrated solution to 10 times the volume; and the reduced-pressure concentration is concentration to a weight equal to the weight of the dried microalgae powder in step (1).

[0022] A microalgae extract for post-sun repair for people with deep skin color prepared by the above preparation method, the microalgae extract has the effects of repairing skin barrier, limiting melanin production and slowing down the secretion rate of sebum.

[0023] The above-mentioned microalgae extract for post-sun repair for people with deep skin color is applied to the preparation of post-sun repair cosmetics.

[0024] The above-mentioned microalgae extract for post-sun repair for people with deep skin color is applied to the preparation of post-sun repair cosmetics for people with deep skin color.

[0025] A composition for post-sun repair for people with deep skin color, which is prepared from the following raw materials: 20 parts by mass of the above-mentioned microalgae extract, 20 parts by mass of an emulsifier, 1 part by mass of a thickening agent, and 0.5 parts by mass of a preservative.

[0026] The emulsifier is composed of polyglycerol fatty acid ester, glyceryl laurate, and fatty acid polyoxyethylene ester at a mass ratio of 1:1:1.

[0027] The thickening agent is sodium chloride.

[0028] The preservative is p-hydroxyacetophenone.

[0029] The present application has the following advantages and beneficial effects relative to the prior art:

[0030] (1) The present application aims to overcome the deficiencies of existing cosmetic products for after-sun repair for deep-skinned people, extract a Nannochloropsis extract, and through a specific formula, the after-sun repair effect for deep-skinned people is widened, and the application of the Nannochloropsis extract in cosmetics for deep-skinned people is widened.

[0031] (2) The Nannochloropsis extract of the present application is mild and non-irritating, suitable for various skin types, especially for sunburned skin, effectively avoiding allergic and irritating problems, and is particularly suitable for deep-skinned people and sensitive skin.

[0032] (3) The present application uses a natural source of Nannochloropsis extract, avoiding the use of chemical synthetic additives, and not only can bring significant repair effect, but also can ensure skin health, and long-term use will not cause burden or dependence on the skin; its natural after-sun repair effect ensures the health and comfort of the skin, especially for deep-skinned people, providing a safe and mild repair option.

[0033] (4) The present application optimizes the preparation steps and process parameters of the Nannochloropsis extract, as well as the selection and amount ratio of the raw materials of the specific formula, to obtain a Nannochloropsis extract and its composition with the remarkable effects of limiting melanin production, slowing down the secretion speed of sebum, and repairing the skin barrier. DETAILED DESCRIPTION

[0034] The content of the present application will be further illustrated in conjunction with specific examples, but should not be understood as a limitation of the present application.

[0035] Example 1

[0036] A Nannochloropsis extract is prepared by the following method:

[0037] S1. The dried Nannochloropsis powder is pretreated, which is first coarsely crushed by a pulverizer and passed through a 30-mesh analytical sieve to obtain a powder with uniform particle size; 95% ethanol by volume is added to the powder at a solid-liquid ratio of 1g:20mL, and the mixture is stirred at 60℃ for 30 minutes twice, and then filtered and the filter residue I is collected.

[0038] S2. To the filter residue I obtained in step S1, water and cellulase liquid accounting for 0.05% of the weight of the filter residue I are added at a solid-liquid ratio of 1g:20mL, and the activity of the cellulase liquid is 10000U / g, and the mixture is enzymatically hydrolyzed at 50℃ for 5h.

[0039] S3. The filter residue I after enzymatic hydrolysis in step S2 was added with water as extraction solvent at a solid-liquid ratio of 1 g: 100 mL, and then extracted at an acidic condition of pH 2 and a temperature of 80°C for 2 h. After cooling and filtration, the filtrate was collected to obtain filter residue II. Then, the filter residue II was extracted at an alkaline condition of pH 8 and a temperature of 80°C for 2 h. After cooling and filtration, the filtrate was collected to obtain filter residue III. The filtrates obtained in the two extractions were combined. The combined filtrate was subjected to ultrafiltration treatment using an ultrafiltration membrane with a molecular weight cutoff of 10 KDa. Then, the combined filtrate was subjected to alcohol precipitation using ethanol with a volume fraction of 95% (the weight of the ethanol with a volume fraction of 95% was 3 times the weight of the combined filtrate), and the supernatant was obtained by centrifugation. Finally, the supernatant was concentrated under reduced pressure to obtain a concentrated solution.

[0040] S4. The pH value of the concentrated solution was adjusted to 5, and then the concentrated solution was diluted by 10 times in volume and loaded onto a pre-activated SPE column. Then, the SPE column was eluted with ethanol with a volume fraction of 60%, 70% and 80% in sequence. The eluates obtained in the three elutions were combined, and then concentrated under reduced pressure to a weight equal to that of the dried N. salina powder in step S1 to obtain a N. salina extract.

[0041] Example 2

[0042] A N. salina extract was prepared by the following method:

[0043] The other steps were the same as in Example 1, except that the solid-liquid ratio in S3 was different. Specifically, S3 in this example was as follows:

[0044] S3. The filter residue I after enzymatic hydrolysis was added with water as extraction solvent at a solid-liquid ratio of 1 g: 40 mL, and then extracted at an acidic condition of pH 2 and a temperature of 80°C for 2 h. After cooling and filtration, the filtrate was collected to obtain filter residue II. Then, the filter residue II was extracted at an alkaline condition of pH 8 and a temperature of 80°C for 2 h. After cooling and filtration, the filtrate was collected to obtain filter residue III. The filtrates obtained in the two extractions were combined. The combined filtrate was subjected to ultrafiltration treatment using an ultrafiltration membrane with a molecular weight cutoff of 10 KDa. Then, the combined filtrate was subjected to alcohol precipitation using ethanol with a volume fraction of 95% (the weight of the ethanol with a volume fraction of 95% was 3 times the weight of the combined filtrate), and the supernatant was obtained by centrifugation. Finally, the supernatant was concentrated under reduced pressure to obtain a concentrated solution.

[0045] Example 3

[0046] A N. salina extract was prepared by the following method:

[0047] The other steps were the same as in Example 1, except that the pH value of the acidic condition in S3 was different. Specifically, S3 in this example was as follows:

[0048] S3. The filter residue I after enzymatic hydrolysis in step S2 was added with water as extraction solvent at a solid-liquid ratio of 1 g: 100 mL, and then extracted under acidic conditions at pH 1 and at a temperature of 80°C for 2 h. After cooling and filtration, the filtrate was collected to obtain filter residue II. Then, the filter residue II was extracted under alkaline conditions at pH 8 and at a temperature of 80°C for 2 h. After cooling and filtration, the filtrate was collected to obtain filter residue III. The filtrates obtained in the two extractions were combined. The combined filtrate was subjected to ultrafiltration treatment using an ultrafiltration membrane with a molecular weight cutoff value of 10 KDa. Then, the combined filtrate was subjected to alcohol precipitation using ethanol with a volume fraction of 95% (the weight of the ethanol with a volume fraction of 95% was 3 times the weight of the combined filtrate), and the supernatant was obtained by centrifugation. The supernatant was concentrated under reduced pressure to obtain a concentrated solution.

[0049] Example 4

[0050] The other steps were the same as in Example 1, except that the pH value of the alkaline conditions in S3 was different, i.e., S3 in this example was as follows:

[0051] S3. The filter residue I after enzymatic hydrolysis in step S2 was added with water as extraction solvent at a solid-liquid ratio of 1 g: 100 mL, and then extracted under acidic conditions at pH 2 and at a temperature of 80°C for 2 h. After cooling and filtration, the filtrate was collected to obtain filter residue II. Then, the filter residue II was extracted under alkaline conditions at pH 10 and at a temperature of 80°C for 2 h. After cooling and filtration, the filtrate was collected to obtain filter residue III. The filtrates obtained in the two extractions were combined. The combined filtrate was subjected to ultrafiltration treatment using an ultrafiltration membrane with a molecular weight cutoff value of 10 KDa. Then, the combined filtrate was subjected to alcohol precipitation using ethanol with a volume fraction of 95% (the weight of the ethanol with a volume fraction of 95% was 3 times the weight of the combined filtrate), and the supernatant was obtained by centrifugation. The supernatant was concentrated under reduced pressure to obtain a concentrated solution.

[0052] Example 5

[0053] A microalgae Nannochloropsis extract was prepared by the following method:

[0054] The other steps were the same as in Example 1, except that the extraction time under acidic conditions in S3 was different, i.e., S3 in this example was as follows:

[0055] S3. The filter residue I after enzymatic hydrolysis in step S2 was added with water as extraction solvent at a solid-liquid ratio of 1 g: 100 mL, and then extracted under acidic conditions at pH 2 and at a temperature of 80°C for 2 h. After cooling and filtration, the filtrate was collected to obtain filter residue II. Then, the filter residue II was extracted under alkaline conditions at pH 10 and at a temperature of 80°C for 2 h. After cooling and filtration, the filtrate was collected to obtain filter residue III. The filtrates obtained in the two extractions were combined. The combined filtrate was subjected to ultrafiltration treatment using an ultrafiltration membrane with a molecular weight cutoff value of 10 KDa. Then, the combined filtrate was subjected to alcohol precipitation using ethanol with a volume fraction of 95% (the weight of the ethanol with a volume fraction of 95% was 3 times the weight of the combined filtrate), and the supernatant was obtained by centrifugation. The supernatant was concentrated under reduced pressure to obtain a concentrated solution.

[0056] Example 6

[0057] A microalgae extract is prepared by the following method:

[0058] Other steps are the same as in Example 1, except that the extraction time under alkaline conditions in S3 is different, i.e. S3 in this example is as follows:

[0059] S3. The filter residue I after enzymatic hydrolysis in step S2 is added with water as extraction solvent at a solid-liquid ratio of 1 g: 100 mL, and is extracted under acidic conditions at pH 2 and at a temperature of 80°C for 2 h, cooled and filtered, and the filtrate is collected to obtain filter residue II; the filter residue II is extracted under alkaline conditions at pH 8 and at a temperature of 80°C for 1 h, cooled and filtered, and the filtrate is collected to obtain filter residue III, and the filtrates obtained in the two extractions are combined; the combined filtrate is subjected to ultrafiltration treatment using an ultrafiltration membrane with a molecular weight cutoff value of 10 KDa, and is then subjected to alcohol precipitation using ethanol with a volume fraction of 95% (the weight of ethanol with a volume fraction of 95% is 3 times the weight of the combined filtrate), centrifuged to obtain the supernatant, and concentrated under reduced pressure to obtain a concentrated solution.

[0060] Comparative Example 1

[0061] Other steps are the same as in Example 1, except that the enzymatic hydrolysis time in step S2 is different, i.e. S2 in this comparative example is as follows:

[0062] S2. Water and cellulase liquid accounting for 0.05% of the weight of the filter residue I are added to the filter residue I obtained in step S1 at a solid-liquid ratio of 1 g: 20 mL, and the cellulase liquid has an activity of 10000 U / g, and enzymatic hydrolysis is carried out at 50°C for 1 h.

[0063] Comparative Example 2

[0064] Other steps are the same as in Example 1, except that the enzymatic hydrolysis temperature in step S2 is different, i.e. S2 in this comparative example is as follows:

[0065] S2. Water and cellulase liquid accounting for 0.05% of the weight of the filter residue I are added to the filter residue I obtained in step S1 at a solid-liquid ratio of 1 g: 20 mL, and the cellulase liquid has an activity of 10000 U / g, and enzymatic hydrolysis is carried out at 20°C for 5 h.

[0066] Comparative Example 3

[0067] Other steps are the same as in Example 1, except that the solid-liquid ratio in step S3 is different, i.e. S3 in this comparative example is as follows:

[0068] S3. The residue I after enzymatic hydrolysis in step S2 was added with water as extraction solvent at a solid-liquid ratio of 1 g: 200 mL, and then extracted under acidic condition at pH 2 and 80°C for 2 h. The filtrate was collected after cooling and filtration. The residue II was extracted under alkaline condition at pH 8 and 80°C for 2 h. The filtrate was collected after cooling and filtration. The filtrates obtained in the two extractions were combined. The combined filtrate was subjected to ultrafiltration treatment using an ultrafiltration membrane with a molecular weight cut-off of 10 KDa. The ultrafiltration-treated filtrate was then subjected to alcohol precipitation using 95% (v / v) ethanol (the weight of 95% (v / v) ethanol was 3 times the weight of the combined filtrate), and the supernatant was obtained by centrifugation. The supernatant was concentrated under reduced pressure to obtain a concentrated solution.

[0069] Comparative Example 4

[0070] The other steps were the same as in Example 1, except that the extraction time under acidic condition in step S3 was different, i.e., S3 in the present comparative example was as follows:

[0071] S3. The residue I after enzymatic hydrolysis in step S2 was added with water as extraction solvent at a solid-liquid ratio of 1 g: 200 mL, and then extracted under acidic condition at pH 2 and 80°C for 2 h. The filtrate was collected after cooling and filtration. The residue II was extracted under alkaline condition at pH 8 and 80°C for 2 h. The filtrate was collected after cooling and filtration. The filtrates obtained in the two extractions were combined. The combined filtrate was subjected to ultrafiltration treatment using an ultrafiltration membrane with a molecular weight cut-off of 10 KDa. The ultrafiltration-treated filtrate was then subjected to alcohol precipitation using 95% (v / v) ethanol (the weight of 95% (v / v) ethanol was 3 times the weight of the combined filtrate), and the supernatant was obtained by centrifugation. The supernatant was concentrated under reduced pressure to obtain a concentrated solution.

[0072] Comparative Example 5

[0073] The other steps were the same as in Example 1, except that the extraction time under alkaline condition in step S3 was different, i.e., S3 in the present comparative example was as follows:

[0074] S3. The residue I after enzymatic hydrolysis in step S2 was added with water as extraction solvent at a solid-liquid ratio of 1 g: 200 mL, and then extracted under acidic condition at pH 2 and 80°C for 2 h. The filtrate was collected after cooling and filtration. The residue II was extracted under alkaline condition at pH 8 and 80°C for 2 h. The filtrate was collected after cooling and filtration. The filtrates obtained in the two extractions were combined. The combined filtrate was subjected to ultrafiltration treatment using an ultrafiltration membrane with a molecular weight cut-off of 10 KDa. The ultrafiltration-treated filtrate was then subjected to alcohol precipitation using 95% (v / v) ethanol (the weight of 95% (v / v) ethanol was 3 times the weight of the combined filtrate), and the supernatant was obtained by centrifugation. The supernatant was concentrated under reduced pressure to obtain a concentrated solution.

[0075] Comparative Example 6

[0076] Other steps are the same as example 1, the difference is that the step S3 acid pH value is different, namely the S3 of the present example is as follows:

[0077] S3. The filter residue I after step S2 enzymolysis is added with water as extraction solvent at a material-liquid ratio of 1 g: 100 mL, and then extracted under an acid condition at pH 5 and at a temperature of 80°C for 2 h. After cooling and filtration, the filtrate is collected to obtain filter residue II. Then the filter residue II is extracted under an alkaline condition at pH 8 and at a temperature of 80°C for 2 h. After cooling and filtration, the filtrate is collected to obtain filter residue III. The filtrates obtained in the two extractions are combined. The combined filtrate is subjected to ultrafiltration treatment using an ultrafiltration membrane with a molecular weight cutoff value of 10 KDa. Then the combined filtrate is subjected to alcohol precipitation using ethanol with a volume fraction of 95% (the weight of the ethanol with a volume fraction of 95% is 3 times the weight of the combined filtrate), centrifugation to obtain supernatant, and vacuum concentration to obtain a concentrated solution.

[0078] Comparative Example 7

[0079] Other steps are the same as example 1, the difference is that the step S3 alkaline pH value is different, namely the S3 of the present example is as follows:

[0080] S3. The filter residue I after step S2 enzymolysis is added with water as extraction solvent at a material-liquid ratio of 1 g: 100 mL, and then extracted under an acid condition at pH 2 and at a temperature of 80°C for 2 h. After cooling and filtration, the filtrate is collected to obtain filter residue II. Then the filter residue II is extracted under an alkaline condition at pH 11 and at a temperature of 80°C for 2 h. After cooling and filtration, the filtrate is collected to obtain filter residue III. The filtrates obtained in the two extractions are combined. The combined filtrate is subjected to ultrafiltration treatment using an ultrafiltration membrane with a molecular weight cutoff value of 10 KDa. Then the combined filtrate is subjected to alcohol precipitation using ethanol with a volume fraction of 95% (the weight of the ethanol with a volume fraction of 95% is 3 times the weight of the combined filtrate), centrifugation to obtain supernatant, and vacuum concentration to obtain a concentrated solution.

[0081] Comparative Example 8

[0082] Other steps are the same as example 1, the difference is that the step S3 alkaline pH value is different, namely the S3 of the present example is as follows:

[0083] S3. The filter residue I after step S2 enzymolysis is added with water as extraction solvent at a material-liquid ratio of 1 g: 100 mL, and then extracted under an acid condition at pH 2 and at a temperature of 80°C for 2 h. After cooling and filtration, the filtrate is collected to obtain filter residue II. Then the filter residue II is extracted under an alkaline condition at pH 11 and at a temperature of 80°C for 2 h. After cooling and filtration, the filtrate is collected to obtain filter residue III. The filtrates obtained in the two extractions are combined. The combined filtrate is subjected to ultrafiltration treatment using an ultrafiltration membrane with a molecular weight cutoff value of 10 KDa. Then the combined filtrate is subjected to alcohol precipitation using ethanol with a volume fraction of 95% (the weight of the ethanol with a volume fraction of 95% is 3 times the weight of the combined filtrate), centrifugation to obtain supernatant, and vacuum concentration to obtain a concentrated solution.

[0084] Test Example 1: After-sun repair test

[0085] 1. The post-sun repair composition is prepared by using the Nannochloropsis extract obtained in Examples 1-6 and Comparative Examples 1-8, respectively:

[0086] The post-sun repair composition containing the Nannochloropsis extract is prepared from the following raw materials in parts by mass: emulsifier 20 parts, thickening agent 1 part, preservative 0.5 part, and Nannochloropsis extract 20 parts; the emulsifier, thickening agent, and Nannochloropsis extract are stirred uniformly, then the preservative is added, and the post-sun repair composition is obtained after standing and defoaming;

[0087] The emulsifier is composed of polyglyceryl fatty acid ester, glyceryl laurate, and fatty acid polyoxyethylene ester in a mass ratio of 1:1:1;

[0088] The thickening agent is sodium chloride;

[0089] The preservative is p-hydroxyacetophenone.

[0090] 2. Test subjects: ① Volunteers with skin types V and VI according to the Fitzpatrick scale are selected; ② Their ages are all within 18-35 years old, and they are in good health without a history of cosmetic allergy; ③ They are willing to comply with the test requirements to complete the entire test and can complete the follow-up as required.

[0091] 3. Transdermal water loss test

[0092] Test method: 160 volunteers are randomly divided into test groups 1-16, 10 people in each group. After being sunburned to the extent that the skin appears obvious burning and swelling, the test products are used respectively. Test groups 1-6 use the post-sun repair compositions prepared from the Nannochloropsis extract obtained in Examples 1-6, respectively. Test groups 7-14 use the post-sun repair compositions prepared from the Nannochloropsis extract obtained in Comparative Examples 1-8, respectively. Test groups 15-16 use two kinds of commercially available post-sun repair products (the main component of commercially available product ① is aloe extract, and the main component of commercially available product ② is Centella asiatica extract). The volunteers use the test products twice a day on the bare forehead after sunburn, for 15 consecutive days. The transdermal water loss test is performed before use, after 5 days of use, and after 15 days of use, and the test results are comprehensively analyzed. The test results are shown in Table 1 below:

[0093] Table 1 Test results of transdermal water loss

[0094]

[0095]

[0096] The test results of Table 1 show that the after-sun repair composition prepared from the Nannochloropsis extract of Examples 1-6 has a more obvious barrier effect on the after-sun repair of deep-skinned people than the after-sun repair composition prepared from the Nannochloropsis extract of Comparative Examples 1-8 and the commercially available product. Therefore, the after-sun repair product prepared from the Nannochloropsis extract of Examples 1-6 has a more obvious barrier effect on the after-sun repair of deep-skinned people.

[0097] The enzymolysis time, enzymolysis temperature, extraction pH value, extraction time and acid-base sequential extraction process parameters mentioned in the present application determine the extraction efficiency and structural integrity of the oil control related active substances in the Nannochloropsis cells. Deviation from the optimal conditions will result in insufficient extraction components or reduced activity, thereby reducing the repair barrier effect of the product.

[0098] Firstly, the appropriate enzymolysis time (such as 5 hours) and temperature (such as 50℃) can ensure that the cell wall is fully lysed, while avoiding the thermal denaturation of functional components such as proteins, so that a large amount of barrier repair active substances such as polypeptides, polysaccharides, oligopeptides, oligosaccharides and amino acids can be fully released. In contrast, if the enzymolysis time is insufficient or the temperature is too low (such as Comparative Examples 1 and 2), the cell wall is not fully broken, which affects the release of components.

[0099] Secondly, acid-base sequential extraction is one of the key processes. Acidic conditions (such as pH = 2) are conducive to the release of antioxidant small molecules, which can help to scavenge free radicals induced by ultraviolet light; alkaline conditions (such as pH = 8) can further extract polysaccharides, polypeptides, oligosaccharides and oligopeptides with intact structures, which can effectively enhance the structure of the stratum corneum and promote the generation of ceramides, thereby improving the integrity of the skin barrier. If any of the steps is missing (such as omitting the alkaline extraction in Comparative Example 8), the types of extracted components are single, and the synergistic repair effect cannot be achieved.

[0100] In addition, the extraction pH value and time also have a great influence on the stability of the components. Deviation from the optimal range (such as acidic pH = 5 or alkaline pH = 11) will lead to partial degradation or aggregation of the active ingredients, thereby reducing the bioavailability. Insufficient extraction time (such as 0.5h in Comparative Examples 4 and 5) will also result in incomplete extraction, which affects the barrier repair ability.

[0101] In summary, the optimal parameter combination used in the present application significantly improves the extraction efficiency and activity integrity of the barrier repair components such as polysaccharides, polypeptides, oligopeptides, oligosaccharides and amino acids in Nannochloropsis, thereby exhibiting superior after-sun skin barrier repair effect in practical applications, especially for deep-skinned people with fragile barrier function after sunburn and other skin conditions.

[0102] 4. Oil control effect test

[0103] Test method: Using the same batch of subjects as above, after the above sunburn and continuous use of the test product for 15 days, the oil content was measured before and after 15 days of use using an oil meter, and the test results were comprehensively analyzed, as shown in Table 2 below:

[0104] Table 2 Test results of oil control effect

[0105]

[0106]

[0107] As can be seen from the test results in Table 2, the post-sun repair composition prepared from the Nannochloropsis extract obtained in Examples 1-6 has a more obvious effect on limiting oil secretion after sunburn for deep-skinned people than the post-sun repair composition prepared from the Nannochloropsis extract obtained in Comparative Examples 1-8 and the commercially available product. Therefore, the post-sun repair product prepared from the Nannochloropsis extract obtained in Examples 1-6 has a more obvious effect on limiting oil secretion after sunburn for deep-skinned people.

[0108] The enzymolysis time, enzymolysis temperature, extraction pH value, extraction time, and acid-base sequential extraction process parameters mentioned in the present application determine the extraction efficiency and structural integrity of the oil control related active substances in the Nannochloropsis cells. Deviating from the optimal conditions will result in insufficient extraction of components or reduced activity, thereby reducing the oil control ability of the product.

[0109] Among them, the appropriate enzymolysis temperature (such as 50℃) and enzymolysis time (such as 5 hours) can ensure that the cell wall of Nannochloropsis is fully ruptured, releasing a large amount of functional polypeptides and polysaccharides, which have a significant effect on regulating sebaceous gland activity and maintaining water and oil balance. If the enzymolysis is insufficient (such as too short time or too low temperature), the amount of effective components extracted will be insufficient, and the oil control effect will be significantly reduced.

[0110] At the same time, the design of acid-base sequential extraction can maximize the extraction of active ingredients of different polarity. Acidic conditions are beneficial to the dissolution of antioxidant small molecule polyphenols, which can inhibit inflammatory oil metabolism disorder caused by sunburn; alkaline conditions are conducive to the extraction of polysaccharides, polypeptides, oligosaccharides and oligopeptides, etc. These components can enhance the skin barrier and reduce the sebaceous secretion rate. If any step is missing (such as omitting alkaline extraction in Comparative Example 8), the types and concentrations of active ingredients will be limited, resulting in a significant reduction in oil control effect.

[0111] In addition, the extraction pH value and time also need to be accurately controlled. Too low or too high pH value may damage the structure of effective components, and too short or too long extraction time may respectively result in incomplete extraction or component degradation. The extraction liquid obtained under the optimized conditions is rich in synergistic oil control components, thereby achieving more significant and more mild oil secretion regulation effect after sunburn for deep-skinned people.

[0112] In summary, the difference in oil control effect is essentially derived from the influence of process conditions on the extraction efficiency and integrity of active ingredients.

[0113] 5. Melanin change rate test

[0114] Test method: Using the same batch of subjects as described above, after the sunburn and continuous use of the test product for 15 days, the melanin content was tested before use, 5 days after use and 15 days after use with a skin melanin tester (Hexameter MX18), and the test results were comprehensively analyzed. The melanin change rate was calculated according to the following formula, and the test results are shown in Table 3 below.

[0115] Melanin change rate = (melanin content before use - melanin content after use) / melanin content before use x 100%

[0116] Table 3 Melanin change rate test results

[0117]

[0118]

[0119] As can be seen from the test results in Table 3, the after-sun repair composition prepared from the Nannochloropsis extract obtained in Examples 1-6 has a more obvious effect on limiting melanin production after sunburn than the after-sun repair composition prepared from the Nannochloropsis extract obtained in Comparative Examples 1-8 and the commercially available product, so it can be seen that the after-sun repair product prepared from the Nannochloropsis extract obtained in Examples 1-6 has a more obvious effect on after-sun repair for deep-skinned people.

[0120] The process parameters mentioned in the present application, such as enzymatic hydrolysis time, enzymatic hydrolysis temperature, extraction pH value, extraction time and sequential extraction with acid and alkali, determine the extraction efficiency and structural integrity of the oil control related active substances in Nannochloropsis cells. Deviating from the optimal conditions will result in insufficient extraction of components or reduced activity, thereby weakening the effect of the product on inhibiting melanin production.

[0121] Among them, the control of enzymatic hydrolysis time and temperature ensures the effective wall breaking of Nannochloropsis cells, while retaining sensitive intracellular antioxidants and melanin inhibiting active peptides such as glutathione, anti-tyrosinase polypeptides, etc. Insufficient enzymatic hydrolysis or low temperature (such as Comparative Examples 1 and 2) will result in insufficient release of these melanin inhibiting components, thereby weakening the effect of inhibiting melanin production.

[0122] Secondly, the acid-base sequential extraction process significantly improves the comprehensiveness and synergy of ingredient extraction. The acidic stage (e.g., pH = 2) helps to dissolve polyphenols, antioxidant small molecules, etc., which can neutralize the oxygen free radicals generated under ultraviolet light stimulation, indirectly inhibiting tyrosinase activity; while the alkaline stage (e.g., pH = 8) can extract polypeptides, polysaccharides, oligosaccharides, and oligopeptides, which can directly participate in the regulation of the melanin production pathway or regulate the metabolic transport of intracellular pigment granules. If any extraction step is omitted or the optimal pH value is deviated (e.g., Comparative Examples 6, 7, and 8), the extraction of functional ingredients will be incomplete, and the melanin change rate will significantly decrease.

[0123] In addition, the extraction time directly affects the dissolution amount of active ingredients. Too short a time (e.g., 0.5 h) cannot fully release functional ingredients; while too long a time may cause oxidation or degradation, affecting the melanin inhibition effect. The extract obtained by the process conditions of the present application can simultaneously exert anti-inflammatory, antioxidant, tyrosinase inhibition, and metabolic promotion effects in the regulation of melanin metabolism, thereby achieving a more significant effect of lightening color deposition.

[0124] In summary, the difference in melanin change rate is due to the control of the extraction process on the extraction efficiency and integrity of active ingredients. The optimized process combination makes the product of the present application exhibit a more superior and safer melanin inhibition effect in deep-skinned people, significantly reducing the risk of post-sun pigment deposition and rebound.

[0125] In summary, the microalgae extract prepared by the method of the present application helps post-sun repair in deep-skinned people, is mild and non-irritating, can limit melanin production, slow down the secretion speed of sebum, and repair the skin barrier.

[0126] The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. A method for preparing a Nannochloropsis algae extract for sun-repair for dark-skinned people, characterized in that The following steps are included: (1) The dried Nannochloropsis algae powder was coarsely crushed with a grinder and sieved to obtain a powder with uniform particle size; 95% by volume ethanol was added to the powder, and the mixture was stirred at 60° C. for degreasing and decolorization twice, each time for 30 minutes, and the residue was filtered and collected; (2) adding water and 0.05% by weight of a cellulase solution having an activity of 10,000 U / g to the filter residue I obtained in step (1) at a material-liquid ratio of 1 g:20 mL, and performing enzymatic hydrolysis at 40-60° C. for 3-5 h; (3) adding water as an extraction solvent to the residue I after enzymatic hydrolysis in step (2) at a solid-liquid ratio of 1 g:100 mL, extracting under acidic conditions of pH 2 and a temperature of 80°C for 2 h, cooling and filtering, and collecting the filtrate to obtain residue II; The residue II was then extracted under alkaline conditions of pH 8 and 80°C for 2 hours, cooled and filtered, and the filtrate was collected to obtain the residue III. The filtrates obtained from the two filtrations were combined; the combined filtrates were ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and then precipitated with 95% by volume ethanol, centrifuged, and the supernatant was concentrated under reduced pressure to obtain the concentrated solution I. (4) The pH value of the concentrated solution I was adjusted to 5, diluted and loaded onto a pre-activated SPE column, and eluted with 60%, 70% and 80% ethanol in sequence. The three eluates were combined and concentrated under reduced pressure to obtain the Nannochloropsis algae extract.

2. The method for preparing a Nannochloropsis algae extract for after-sun repair for dark-skinned people according to claim 1, characterized in that: The sieving in step (1) is through a 30-mesh analytical sieve; and 95% by volume ethanol is added to the powder, wherein the material-liquid ratio is 1 g:20 mL.

3. The method for preparing a Nannochloropsis algae extract for after-sun repair for dark-skinned people according to claim 1, characterized in that: The weight of the 95% ethanol by volume in step (3) is 3 times the weight of the combined filtrate.

4. The method for preparing a Nannochloropsis algae extract for after-sun repair for dark-skinned people according to claim 1, characterized in that: The enzymatic hydrolysis temperature in step (2) is 50° C., and the enzymatic hydrolysis time is 5 h.

5. The method for preparing a Nannochloropsis algae extract for after-sun repair for dark-skinned people according to claim 1, characterized in that: The dilution in step (4) is to dilute the concentrated solution to 10 times its volume; the reduced pressure concentration is to concentrate the solution to a weight equal to the weight of the dried Chlorella powder in step (1).

6. A Nannochloropsis algae extract for sunburn repair for dark-skinned people prepared by the preparation method according to any one of claims 1 to 5, characterized in that: The Nannochloropsis algae extract has the effects of repairing the skin barrier, limiting the generation of melanin and slowing down the secretion of oil.

7. Use of the Nannochloropsis algae extract for after-sun repair of dark-skinned people according to claim 6 in the preparation of after-sun repair cosmetics.

8. Use of the Nannochloropsis extract for after-sun repair of dark-skinned people according to claim 6 in the preparation of after-sun repair cosmetics for dark-skinned people.

9. A composition for after-sun repair for dark-skinned people, characterized by: The composition is prepared from the following raw materials: 20 parts by mass of the Nannochloropsis algae extract for after-sun repair of dark-skinned people according to claim 6, 20 parts by mass of an emulsifier, 1 part by mass of a thickener, and 0.5 parts by mass of a preservative; The emulsifier is composed of polyglycerol fatty acid ester, monolaurin and fatty acid polyoxyethylene ester in a mass ratio of 1:1:1; The thickener is sodium chloride; The preservative is p-hydroxyacetophenone.

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