Nannochloropsis oculata extracting solution for after-sun repair of crowd with deep skin color as well as preparation method and application of nannochloropsis oculata extracting solution

Abstract

The invention belongs to the technical field of cosmetic preparation, and discloses a nannochloropsis oculata extracting solution for after-sun repair of people with deep skin color as well as a preparation method and application of the nannochloropsis oculata extracting solution. The method comprises the following steps: degreasing, decolorizing and filtering nannochloropsis oculata powder, adding cellulase liquid for enzymolysis, extracting filter residues with water under the acidic condition of pH being 1-3 and at 80 DEG C, cooling and filtering, extracting the obtained filter residues under the alkaline condition of pH being 8-10 and at 80 DEG C, cooling and filtering, merging filtrates obtained by two times of filtration, and performing ultrafiltration treatment to obtain the nannochloropsis oculata powder. And performing ethanol precipitation with 95% ethanol, performing centrifugation to take supernate, performing vacuum concentration, diluting the obtained concentrated solution, loading the diluted concentrated solution into a pre-activated SPE column, performing elution with 60% ethanol, 70% ethanol and 80% ethanol in sequence, combining the eluents, and performing vacuum concentration to obtain the nannochloropsis oculata extracting solution. The extracting solution has the effects of enhancing skin repair and resisting inflammation, is particularly suitable for after-sun care of people with deep skin color, limits melanogenesis, slows down grease secretion speed and repairs skin barrier.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetic preparation, and in particular relates to a Nannochloropsis algae extract for after-sun repair of people with dark skin, as well as a preparation method and application thereof. Background Art

[0002] With the global increase in ultraviolet radiation intensity, sunburn has become a growing concern for many people. This is especially true for people with darker skin tones. While their skin's high melanin content provides some natural sun protection, they can still experience post-sun inflammation, dark spots, and decreased elasticity. Existing after-sun repair products primarily utilize moisturizing and antioxidant ingredients, but often overlook the unique skin reaction mechanisms and repair needs of darker skin types.

[0003] Nannochloropsis oculata is a eukaryotic algae belonging to the family Euophyceae, class Phaeophyceae. Its spherical cells exhibit advantages such as high photosynthetic efficiency and a short growth cycle, making it an important economic microalgae that is easy to cultivate. Nannochloropsis oculata is rich in protein, carbohydrates, and minerals, but is currently primarily used as feed in the aquaculture industry, resulting in significant resource waste. In recent years, algal protein has gained popularity as a new source of plant protein. As a highly efficient microalgae, it is rich in various bioactive substances, such as oligopeptides, polyphenols, and minerals, and has been used in cosmetics.

[0004] Chinese Patent with Publication No. CN118319799A discloses a skin care composition containing extracts of Chlorella minutissima, its preparation method and application, including the following steps: 1) Mix an emulsifier, a humectant, an emollient, a preservative and deionized water, and obtain a vesicle solution after high-pressure microfluidization treatment; 2) Mix the vesicle solution obtained in step 1) with extracts of Chlorella minutissima, seaweed polysaccharide, squalane, extracts of Nannochloropsis oculata, and extracts of Haematococcus pluvialis, and obtain the skin care composition containing extracts of Chlorella minutissima after high-pressure microfluidization treatment. Chinese Patent with Publication No. CN118319829A discloses a preparation method of Nannochloropsis polysaccharide with antioxidant activity, including the following steps: 1) Dissolve Nannochloropsis in water and perform ultrasonic-microwave extraction; 2) Enzymatically hydrolyze the extract obtained in step 1) with a complex enzyme; 3) Inactivate the enzyme in the enzymatic hydrolysate obtained in step 2) and then centrifuge; 4) Distill the supernatant obtained in step 3) under reduced pressure, add absolute ethanol for alcohol precipitation; 5) Centrifuge the alcohol precipitation mixture obtained in step 4), freeze the centrifuged precipitate to the eutectic point, and perform vacuum freeze-drying to obtain a crude extract of Nannochloropsis polysaccharide powder; 6) Dissolve the crude polysaccharide extract and add a decolorizing agent, place it in a shaker for shaking decolorization, and centrifuge after decolorization to obtain a treated solution; 7) Add Sevage reagent to the treated solution and mix well to remove proteins, and perform vacuum freeze-drying treatment on the supernatant obtained after centrifugation to obtain a purified Nannochloropsis polysaccharide; 8) Prepare a solution of the purified Nannochloropsis polysaccharide, add α-amylase to modify the Nannochloropsis polysaccharide, and obtain enzyme-modified Nannochloropsis polysaccharide after inactivating the enzyme at high temperature.

[0005] Currently, many post-sun repair products are targeted at people with fair skin and sensitive skin, mainly playing a role of calming and soothing, but there are few post-sun repair products targeted at people with dark skin. People with dark skin contain more melanin in their skin. After sun exposure, melanin is more likely to deposit, the sebum secretion rate is faster, and the skin barrier is easily damaged. Existing technologies show that the extract of Nannochloropsis can promote skin anti-aging, firm the skin, etc., but its application in the post-sun repair of people with dark skin has not been fully explored.

[0006] At present, many post-sun repair products on the market have not been optimized for the special needs of people with dark skin. The skin of people with dark skin has different melanin distribution and structural characteristics from that of people with light skin, and the response and repair mechanisms to ultraviolet rays are also different. Existing repair products are mostly based on general ingredients and technologies, ignoring the unique needs of dark skin, resulting in unsatisfactory repair effects and even possibly causing new skin problems, such as fast melanin production and slow metabolism. Summary of the Invention

[0007] In order to overcome the above-mentioned drawbacks and deficiencies in the prior art, the primary object of the present invention is to provide a preparation method of an extract of Nannochloropsis for post-sun repair of people with dark skin.

[0008] Another object of the present invention is to provide a Nannochloropsis extract for post - sun repair of dark - skinned people prepared by the above - mentioned preparation method; this Nannochloropsis extract has the effects of enhancing skin repair and anti - inflammation, is particularly suitable for post - sun care of dark - skinned people, restricts melanin production, slows down the sebum secretion rate and repairs the skin barrier.

[0009] Another object of the present invention is to provide the application of the above - mentioned Nannochloropsis extract for post - sun repair of dark - skinned people; when the Nannochloropsis extract is used in cosmetic products, while ensuring its ability to repair post - sun skin, it can effectively reduce pigment deposition caused by ultraviolet rays and avoid the problem of pigment rebound in dark - skinned skin during the post - sun repair process, thus providing a solution for active melanin in dark - skinned people after sun exposure.

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

[0011] A preparation method of a Nannochloropsis extract for post - sun repair of dark - skinned people includes the following operating steps:

[0012] (1) The dried Nannochloropsis powder is coarsely pulverized by a pulverizer and sieved to obtain a powder with uniform particle size; 95% ethanol by volume is added to the powder, and it is stirred for defatting and decolorization twice at 60 °C for 30 minutes each time, then filtered and the residue I is collected;

[0013] (2) Water is added to the residue I obtained in step (1) at a material - to - liquid ratio of 1 g:20 mL, and a cellulase solution accounting for 0.05% of the weight of the residue I is added. The activity of the cellulase solution is 10000 U / g, and enzymatic hydrolysis is carried out at 40 - 60 °C for 3 - 5 h;

[0014] (3) The residue I after enzymatic hydrolysis in step (2) is added with water as an extraction solvent at a material - to - liquid ratio of 1 g:40 - 100 mL, and extracted at an acidic condition with a pH of 1 - 3 and a temperature of 80 °C for 1 - 3 h, cooled and filtered, and the filtrate is collected to obtain residue II; then the residue II is extracted at an alkaline condition with a pH of 8 - 10 and a temperature of 80 °C for 1 - 3 h, cooled and filtered, and the filtrate is collected to obtain residue III, and the filtrates obtained from the two filtrations are combined; the combined filtrate is ultrafiltered with a ultrafiltration membrane with a molecular weight cut - off value of 10 KDa, then precipitated with 95% ethanol by volume, centrifuged to take the supernatant, and concentrated under reduced pressure to obtain a concentrated solution;

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

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

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

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

[0019] Preferably, the acidic condition in step (3) is pH 2; the alkaline condition is pH 8; the extraction time is 2 h for both.

[0020] Preferably, the weight of 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 to dilute the concentrated solution to 10 times its volume; the vacuum concentration is to concentrate to a weight equal to the weight of the Nannochloropsis powder after drying treatment in step (1).

[0022] A Nannochloropsis extract for post-sun repair for dark skin tone people prepared by the above preparation method, and the Nannochloropsis extract has the effects of repairing the skin barrier, restricting melanin production, and slowing down the sebum secretion rate.

[0023] Application of the above Nannochloropsis extract for post-sun repair for dark skin tone people in the preparation of post-sun repair cosmetics.

[0024] Application of the above Nannochloropsis extract for post-sun repair for dark skin tone people in the preparation of post-sun repair cosmetics for dark skin tone people.

[0025] A composition for post-sun repair for dark skin tone people, which is prepared from the following raw materials: 20 parts by mass of the above Nannochloropsis extract, 20 parts by mass of an emulsifier, 1 part by mass of a thickener, and 0.5 part by mass of a preservative;

[0026] The emulsifier is composed of polyglycerol fatty acid ester, glycerol monolaurate, and fatty acid polyoxyethylene ester with a mass ratio of 1:1:1;

[0027] The thickener is sodium chloride;

[0028] The preservative is p-hydroxyacetophenone.

[0029] The present invention has the following advantages and beneficial effects compared with the prior art:

[0030] (1) The present invention aims to overcome the deficiencies in the post - sun repair for people with dark skin tones in existing cosmetic products, extract and prepare a Nannochloropsis extract, and through a specific formula, target the post - sun repair effect for people with dark skin tones, thus broadening the application of Nannochloropsis extract in cosmetics for people with dark skin tones.

[0031] (2) The Nannochloropsis extract of the present invention is mild and non - irritating, suitable for various skin types, especially very mild to sunburned skin, effectively avoiding allergic and irritation problems, and is particularly suitable for people with dark skin tones and sensitive skin types.

[0032] (3) The present invention uses a Nannochloropsis extract from natural sources, avoiding the use of chemically synthesized additives. After use, it can not only bring significant repair effects but also ensure skin health, and long - term use will not cause burden or dependence on the skin; its natural post - sun repair effect ensures skin health and comfort, especially for people with dark skin tones, providing a safe and mild repair option.

[0033] (4) The present invention optimizes the preparation steps and process parameters of the Nannochloropsis extract, as well as the raw material selection and dosage ratio of the specific formula, to obtain a Nannochloropsis extract and its composition with the significant effects of restricting melanin production, slowing down the sebum secretion rate, and repairing the skin barrier. Detailed Embodiments

[0034] The following further illustrates the content of the present invention with specific embodiments, but should not be construed as a limitation to the present invention.

[0035] Example 1

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

[0037] S1. Pretreat the dried Nannochloropsis powder. Specifically, first coarsely crush it with a pulverizer and pass through a 30 - mesh analytical sieve to obtain a powder with uniform particle size; add ethanol with a volume fraction of 95% to the powder at a material - to - liquid ratio of 1 g:20 mL, stir for defatting and decolorization twice at 60 °C, each time for 30 minutes, filter and collect the filter residue Ⅰ.

[0038] S2. Add water and a cellulase solution accounting for 0.05% of the weight of the filter residue Ⅰ to the filter residue Ⅰ obtained in step S1 at a material - to - liquid ratio of 1 g:20 mL. The activity of the cellulase solution is 10000 U / g, and enzymolysis is carried out at 50 °C for 5 h.

[0039] S3. Add the residue I after enzymatic hydrolysis in step S2 to water as the extraction solvent at a material-liquid ratio of 1 g: 100 mL, extract for 2 h under acidic conditions with a pH of 2 and a temperature of 80 °C, cool and filter, collect the filtrate to obtain residue II; then extract residue II for 2 h under alkaline conditions with a pH of 8 and a temperature of 80 °C, cool and filter, collect the filtrate to obtain residue III, and combine the filtrates obtained from the two filtrations; subject the combined filtrate to ultrafiltration treatment using an ultrafiltration membrane with a molecular weight cut-off value of 10 KDa, then perform alcohol precipitation with 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), centrifuge to take the supernatant, and concentrate under reduced pressure to obtain a concentrated solution.

[0040] S4. Adjust the pH value of the concentrated solution to 5, dilute it by 10 times in volume and then load it onto a pre-activated SPE column, elute successively with ethanol with a volume fraction of 60%, 70% and 80%, combine the three eluates, and concentrate under reduced pressure to a weight equal to the weight of the Nannochloropsis powder after drying treatment in step S1 to obtain the Nannochloropsis extract.

[0041] Example 2

[0042] A Nannochloropsis extract is prepared by the following method:

[0043] Other steps are the same as in Example 1, the difference lies in the different material-liquid ratio in S3, specifically, S3 in this example is as follows:

[0044] S3. Add the residue I after enzymatic hydrolysis to water as the extraction solvent at a material-liquid ratio of 1 g: 40 mL, extract for 2 h under acidic conditions with a pH of 2 and a temperature of 80 °C, cool and filter, collect the filtrate to obtain residue II; then extract residue II for 2 h under alkaline conditions with a pH of 8 and a temperature of 80 °C, cool and filter, collect the filtrate to obtain residue III, and combine the filtrates obtained from the two filtrations; subject the combined filtrate to ultrafiltration treatment using an ultrafiltration membrane with a molecular weight cut-off value of 10 KDa, then perform alcohol precipitation with 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), centrifuge to take the supernatant, and concentrate under reduced pressure to obtain a concentrated solution.

[0045] Example 3

[0046] A Nannochloropsis extract is prepared by the following method:

[0047] Other steps are the same as in Example 1, the difference lies in the different pH value of the acidic conditions in S3, specifically, S3 in this example is as follows:

[0048] S3. Add the residue I after enzymatic hydrolysis in step S2 to water as the extraction solvent at a solid-liquid ratio of 1 g: 100 mL, extract at 80 °C under acidic conditions with a pH of 1 for 2 h, cool and filter, collect the filtrate to obtain residue II; then extract residue II at 80 °C under alkaline conditions with a pH of 8 for 2 h, cool and filter, collect the filtrate to obtain residue III, and combine the filtrates obtained from the two filtrations; subject the combined filtrate to ultrafiltration using an ultrafiltration membrane with a molecular weight cut-off value of 10 KDa, then perform alcohol precipitation with ethanol at a volume fraction of 95% (the weight of ethanol with a volume fraction of 95% is 3 times the weight of the combined filtrate), centrifuge to obtain the supernatant, and concentrate under reduced pressure to obtain the concentrated solution.

[0049] Example 4

[0050] Other steps are the same as in Example 1, except that the pH value of the alkaline condition in S3 is different, that is, S3 in this example is specifically as follows:

[0051] S3. Add the residue I after enzymatic hydrolysis in step S2 to water as the extraction solvent at a solid-liquid ratio of 1 g: 100 mL, extract at 80 °C under acidic conditions with a pH of 2 for 2 h, cool and filter, collect the filtrate to obtain residue II; then extract residue II at 80 °C under alkaline conditions with a pH of 10 for 2 h, cool and filter, collect the filtrate to obtain residue III, and combine the filtrates obtained from the two filtrations; subject the combined filtrate to ultrafiltration using an ultrafiltration membrane with a molecular weight cut-off value of 10 KDa, then perform alcohol precipitation with ethanol at a volume fraction of 95% (the weight of ethanol with a volume fraction of 95% is 3 times the weight of the combined filtrate), centrifuge to obtain the supernatant, and concentrate under reduced pressure to obtain the concentrated solution.

[0052] Example 5

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

[0054] Other steps are the same as in Example 1, except that the extraction time under acidic conditions in S3 is different, that is, S3 in this example is specifically as follows:

[0055] S3. Add the residue I after enzymatic hydrolysis in step S2 to water as the extraction solvent at a solid-liquid ratio of 1 g: 100 mL, extract at 80 °C under acidic conditions with a pH of 2 for 1 h, cool and filter, collect the filtrate to obtain residue II; then extract residue II at 80 °C under alkaline conditions with a pH of 8 for 2 h, cool and filter, collect the filtrate to obtain residue III, and combine the filtrates obtained from the two filtrations; subject the combined filtrate to ultrafiltration using an ultrafiltration membrane with a molecular weight cut-off value of 10 KDa, then perform alcohol precipitation with ethanol at a volume fraction of 95% (the weight of ethanol with a volume fraction of 95% is 3 times the weight of the combined filtrate), centrifuge to obtain the supernatant, and concentrate under reduced pressure to obtain the concentrated solution.

[0056] Example 6

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

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

[0059] S3. Add the residue I after enzymatic hydrolysis in step S2 to water as the extraction solvent at a solid-liquid ratio of 1 g: 100 mL, extract at an acidic condition with a pH of 2 and a temperature of 80 °C for 2 h, cool and filter, collect the filtrate to obtain residue II; then extract residue II at an alkaline condition with a pH of 8 and a temperature of 80 °C for 1 h, cool and filter, collect the filtrate to obtain residue III, and combine the filtrates obtained from the two filtrations; ultrafilter the combined filtrate with an ultrafiltration membrane with a molecular weight cut-off value of 10 KDa, then perform alcohol precipitation with 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), centrifuge to take the supernatant, and concentrate under reduced pressure to obtain a concentrated solution.

[0060] Comparative Example 1

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

[0062] S2. Add water and 0.05% (by weight of residue I) of cellulase solution with an activity of 10,000 U / g to the residue I obtained in step S1 at a solid-liquid ratio of 1 g: 20 mL, and perform enzymatic hydrolysis at 50 °C for 1 h.

[0063] Comparative Example 2

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

[0065] S2. Add water and 0.05% (by weight of residue I) of cellulase solution with an activity of 10,000 U / g to the residue I obtained in step S1 at a solid-liquid ratio of 1 g: 20 mL, and perform enzymatic hydrolysis at 20 °C for 5 h.

[0066] Comparative Example 3

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

[0068] S3. Add the residue I after enzymatic hydrolysis in step S2 to water as the extraction solvent at a material-liquid ratio of 1 g: 200 mL, extract at an acidic condition with a pH of 2 and a temperature of 80 °C for 2 h, cool and filter, collect the filtrate to obtain residue II; then extract residue II at an alkaline condition with a pH of 8 and a temperature of 80 °C for 2 h, cool and filter, collect the filtrate to obtain residue III, and combine the filtrates obtained from the two filtrations; subject the combined filtrate to ultrafiltration using an ultrafiltration membrane with a molecular weight cut-off value of 10 KDa, then perform alcohol precipitation with ethanol at a volume fraction of 95% (the weight of ethanol at a volume fraction of 95% is 3 times the weight of the combined filtrate), centrifuge to take the supernatant, and concentrate under reduced pressure to obtain the concentrated solution.

[0069] Comparative Example 4

[0070] Other steps are the same as those in Example 1, the difference lies in the extraction time under acidic conditions in step S3, specifically, S3 in this comparative example is as follows:

[0071] S3. Add the residue I after enzymatic hydrolysis in step S2 to water as the extraction solvent at a material-liquid ratio of 1 g: 100 mL, extract at an acidic condition with a pH of 2 and a temperature of 80 °C for 0.5 h, cool and filter, collect the filtrate to obtain residue II; then extract residue II at an alkaline condition with a pH of 8 and a temperature of 80 °C for 2 h, cool and filter, collect the filtrate to obtain residue III, and combine the filtrates obtained from the two filtrations; subject the combined filtrate to ultrafiltration using an ultrafiltration membrane with a molecular weight cut-off value of 10 KDa, then perform alcohol precipitation with ethanol at a volume fraction of 95% (the weight of ethanol at a volume fraction of 95% is 3 times the weight of the combined filtrate), centrifuge to take the supernatant, and concentrate under reduced pressure to obtain the concentrated solution.

[0072] Comparative Example 5

[0073] Other steps are the same as those in Example 1, the difference lies in the extraction time under alkaline conditions in step S3, specifically, S3 in this comparative example is as follows:

[0074] S3. Add the residue I after enzymatic hydrolysis in step S2 to water as the extraction solvent at a material-liquid ratio of 1 g: 100 mL, extract at an acidic condition with a pH of 2 and a temperature of 80 °C for 2 h, cool and filter, collect the filtrate to obtain residue II; then extract residue II at an alkaline condition with a pH of 8 and a temperature of 80 °C for 0.5 h, cool and filter, collect the filtrate to obtain residue III, and combine the filtrates obtained from the two filtrations; subject the combined filtrate to ultrafiltration using an ultrafiltration membrane with a molecular weight cut-off value of 10 KDa, then perform alcohol precipitation with ethanol at a volume fraction of 95% (the weight of ethanol at a volume fraction of 95% is 3 times the weight of the combined filtrate), centrifuge to take the supernatant, and concentrate under reduced pressure to obtain the concentrated solution.

[0075] Comparative Example 6

[0076] The other steps are the same as those in Example 1, except that the acidic pH value in step S3 is different. Specifically, S3 in this comparative example is as follows:

[0077] S3. Add the residue I after enzymatic hydrolysis in step S2 to water as the extraction solvent at a material-liquid ratio of 1 g: 100 mL, extract at an acidic condition with a pH of 5 and a temperature of 80 °C for 2 h, cool and filter, collect the filtrate to obtain residue II; then extract residue II at a basic condition with a pH of 8 and a temperature of 80 °C for 2 h, cool and filter, collect the filtrate to obtain residue III, and combine the filtrates obtained from the two filtrations; subject the combined filtrate to ultrafiltration treatment using an ultrafiltration membrane with a molecular weight cut-off value of 10 KDa, then perform alcohol precipitation with ethanol at a volume fraction of 95% (the weight of ethanol with a volume fraction of 95% is 3 times the weight of the combined filtrate), centrifuge to take the supernatant, and concentrate under reduced pressure to obtain a concentrated solution.

[0078] Comparative Example 7

[0079] The other steps are the same as those in Example 1, except that the basic pH value in step S3 is different. Specifically, S3 in this comparative example is as follows:

[0080] S3. Add the residue I after enzymatic hydrolysis in step S2 to water as the extraction solvent at a material-liquid ratio of 1 g: 100 mL, extract at an acidic condition with a pH of 2 and a temperature of 80 °C for 2 h, cool and filter, collect the filtrate to obtain residue II; then extract residue II at a basic condition with a pH of 11 and a temperature of 80 °C for 2 h, cool and filter, collect the filtrate to obtain residue III, and combine the filtrates obtained from the two filtrations; subject the combined filtrate to ultrafiltration treatment using an ultrafiltration membrane with a molecular weight cut-off value of 10 KDa, then perform alcohol precipitation with ethanol at a volume fraction of 95% (the weight of ethanol with a volume fraction of 95% is 3 times the weight of the combined filtrate), centrifuge to take the supernatant, and concentrate under reduced pressure to obtain a concentrated solution.

[0081] Comparative Example 8

[0082] The other steps are the same as those in Example 1, except that the extraction step under basic conditions in step S3 is omitted. Specifically, S3 in this comparative example is as follows:

[0083] S3. Add the residue I after enzymatic hydrolysis in step S2 to water as the extraction solvent at a material-liquid ratio of 1 g: 100 mL, extract at an acidic condition with a pH of 2 and a temperature of 80 °C for 2 h, cool and filter, collect the filtrate to obtain residue II; subject the filtrate to ultrafiltration treatment using an ultrafiltration membrane with a molecular weight cut-off value of 10 KDa, then perform alcohol precipitation with ethanol at a volume fraction of 95% (the weight of ethanol with a volume fraction of 95% is 3 times the weight of the combined filtrate), centrifuge to take the supernatant, and concentrate under reduced pressure to obtain a concentrated solution.

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

[0085] 1. Prepare after-sun repair compositions using the Nannochloropsis algae extracts obtained in Examples 1-6 and Comparative Examples 1-8, respectively:

[0086] The after-sun repair composition containing Nannochloropsis extract is prepared from the following raw materials, calculated by weight: 20 parts of an emulsifier, 1 part of a thickener, 0.5 parts of a preservative, and 20 parts of the Nannochloropsis extract. The emulsifier, thickener, and Nannochloropsis extract are uniformly stirred, and then the preservative is added. The mixture is allowed to stand for defoaming to obtain the after-sun repair composition.

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

[0088] The thickener is sodium chloride;

[0089] The preservative is p-hydroxyacetophenone.

[0090] 2. Subjects: ① Volunteers with skin types V and VI according to the Fitzpatrick skin scale were selected; ② They were aged between 18 and 35 years, in good health, and had no history of cosmetic allergies; ③ They were willing to comply with the trial requirements, complete the entire trial, and be able to complete follow-up visits as required.

[0091] 3. Transepidermal water loss test

[0092] Test method: 160 volunteers were randomly divided into test groups 1 to 16, with 10 people in each group. After the skin was exposed to the sun until obvious burning sensation and redness and swelling appeared, the test products were used respectively. Test groups 1 to 6 used the after-sun repair composition prepared by the extract of Nannochloropsis algae obtained in Examples 1 to 6, test groups 7 to 14 used the after-sun repair composition prepared by the extract of Nannochloropsis algae obtained in Comparative Examples 1 to 8, and test groups 15 to 16 used two commercially available after-sun repair products (commercially available products) respectively. The main ingredient is aloe vera extract, and commercially available products The main ingredient is Centella asiatica extract. After sunburn, the test product was applied twice daily to the exposed forehead of volunteers for 15 consecutive days. Transepidermal water loss (TEWL) was measured before, after 5 days, and after 15 days of use. The test results were comprehensively analyzed and are shown in Table 1 below:

[0093] Table 1 Transepidermal water loss test results

[0094]

[0095] From the test results in Table 1, it can be seen that the post-sun repair compositions prepared from the Nannochloropsis extracts obtained in Examples 1-6 have significantly better barrier repair effects for dark-skinned people after sun exposure than the post-sun repair compositions prepared from the Nannochloropsis extracts obtained in Comparative Examples 1-8 and commercial products. Thus, it can be seen that the post-sun repair products prepared from the Nannochloropsis extracts obtained in Examples 1-6 of the present invention have a more obvious effect on repairing the skin barrier for dark-skinned people after sunburn.

[0096] The process parameters such as the enzymatic hydrolysis time, enzymatic hydrolysis temperature, extraction pH value, extraction time, and sequential acid-base extraction mentioned in the present invention determine the extraction efficiency and structural integrity of the oil-control related active substances in Nannochloropsis cells. Deviating from the optimal conditions will lead to insufficient extraction components or reduced activity, thereby decreasing the barrier repair effect of the product.

[0097] First of all, an appropriate enzymatic hydrolysis time (such as 5 hours) and temperature (such as 50 °C) can ensure sufficient lysis of the cell wall, while avoiding thermal denaturation of functional components such as proteins, so that a large number of barrier repair active substances such as polypeptides, polysaccharides, oligopeptides, oligosaccharides, and amino acids can be fully released. In contrast, if the enzymatic hydrolysis time is insufficient or the temperature is too low (such as in Comparative Examples 1 and 2), the cell wall breaking will be insufficient, affecting the release of components.

[0098] Secondly, sequential acid-base extraction is one of the key processes. Acidic conditions (such as pH = 2) are beneficial to the release of antioxidant small molecules, which helps to scavenge free radicals induced by ultraviolet rays; alkaline conditions (such as pH = 8) can further extract polysaccharides, polypeptides, oligosaccharides, and oligopeptides with intact structures, and these components can effectively enhance the stratum corneum structure and promote the generation of ceramides, thereby improving the integrity of the skin barrier. If any step is missing (such as omitting the alkali extraction in Comparative Example 8), the types of extracted components will be single, and the synergistic repair effect cannot be achieved.

[0099] In addition, the extraction pH value and time also have a great impact on the stability of components. Deviating from the optimal range (such as acidic pH = 5 or alkaline pH = 11) will cause partial degradation or aggregation inactivation of the active components, thereby reducing the bioavailability. Insufficient extraction time (such as 0.5 h in Comparative Examples 4 and 5) will also result in incomplete extraction, affecting the barrier repair ability.

[0100] In summary, the optimal parameter combination adopted in the present invention significantly improves the extraction efficiency and activity integrity of barrier repair components such as polysaccharides, polypeptides, oligopeptides, oligosaccharides, and amino acids in Nannochloropsis, thereby showing excellent post-sun skin barrier repair effects in practical applications, and is particularly suitable for skin conditions such as the fragile barrier function of dark-skinned people after sunburn.

[0101] 4. Oil Control Effect Test

[0102] Test method: Using the same batch of subjects as above, after the above-mentioned sunburn and continuous use of the test product for 15 days, the oil content was measured using an oil meter before use and 15 days after use, and the test results were comprehensively analyzed. The test results are shown in Table 2 below:

[0103] Table 2 Test results of oil control effect

[0104] From the test results in Table 2, it can be seen that the post-sun repair compositions prepared from the Nannochloropsis extracts obtained in Examples 1-6 have a significantly better effect on restricting oil secretion after sunburn in people with dark skin tones than the post-sun repair compositions prepared from the Nannochloropsis extracts obtained in Comparative Examples 1-8 and commercial products. Thus, it can be seen that the post-sun repair products prepared from the Nannochloropsis extracts obtained in Examples 1-6 of the present invention have a more obvious effect on restricting oil secretion after sunburn in people with dark skin tones.

[0105] The process parameters such as the enzymatic hydrolysis time, enzymatic hydrolysis temperature, extraction pH value, extraction time, and sequential acid-base extraction mentioned in the present invention determine the extraction efficiency and structural integrity of the oil control-related active substances in Nannochloropsis cells. Deviating from the optimal conditions will lead to insufficient extraction components or reduced activity, thereby decreasing the oil control ability of the product.

[0106] Among them, appropriate enzymatic hydrolysis temperature (such as 50°C) and enzymatic hydrolysis time (such as 5 hours) can ensure that the cell walls of Nannochloropsis are fully broken, releasing a large amount of functional polypeptides and polysaccharides, which play a significant role in regulating the activity of sebaceous glands and maintaining the water-oil balance. If the enzymatic hydrolysis is insufficient (such as too short time or too low temperature), the extraction amount of active ingredients will be insufficient, and the oil control effect will decrease significantly.

[0107] At the same time, the design of sequential acid-base extraction can extract active ingredients with different polarities to the greatest extent. The acidic condition is conducive to the dissolution of antioxidant small molecule polyphenolic substances, which can inhibit the inflammatory oil metabolism disorder caused by sunburn; the alkaline condition helps to extract substances such as polysaccharides, polypeptides, oligosaccharides, and oligopeptides, which can enhance the skin barrier and reduce the sebum secretion rate. If any step is missing (such as Comparative Example 8 omitting alkaline extraction), the types and concentrations of active ingredients will be limited, resulting in a significant reduction in the oil control effect.

[0108] In addition, the extraction pH value and time also need to be precisely controlled. Too low or too high pH value may damage the structure of active ingredients, while too short or too long extraction time will respectively lead to incomplete extraction or ingredient degradation. The extraction solution obtained under optimized conditions is rich in synergistic oil control components, thereby achieving a more significant and milder oil secretion regulation effect on people with dark skin tones after sunburn.

[0109] In summary, the differences in oil control effects essentially stem from the influence of process conditions on the extraction efficiency and integrity of active ingredients.

[0110] 5. Melanin change rate test

[0111] Test method: Using the same batch of subjects as above, after the above-mentioned sunburn and continuous use of the test product for 15 days, the melanin content was measured with a skin melanin tester (Hexameter MX18) before use, 5 days after use, and 15 days after use respectively, 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.

[0112] Melanin change rate = (melanin content before use - melanin content after use) / melanin content before use × 100%

[0113] Table 3 Melanin change rate test results

[0114]

[0115] It can be seen from the test results in Table 3 that the post-sun repair compositions prepared from the Nannochloropsis extracts obtained in Examples 1-6 are significantly better than the post-sun repair compositions prepared from the Nannochloropsis extracts obtained in Comparative Examples 1-8 and the commercial products in terms of restricting melanin production after sunburn for people with dark skin tones. Thus, it can be seen that the post-sun repair products prepared from the Nannochloropsis extracts obtained in Examples 1-6 of the present invention have a more obvious post-sun repair effect on people with dark skin tones.

[0116] The process parameters such as the enzymatic hydrolysis time, enzymatic hydrolysis temperature, extraction pH value, extraction time, and sequential acid-base extraction mentioned in the present invention 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 components or reduced activity, thereby weakening the effect of the product in inhibiting melanin production.

[0117] Among them, the control of the enzymatic hydrolysis time and temperature ensures the effective disruption of Nannochloropsis cells while retaining the intracellular sensitive antioxidants and melanin-inhibiting active peptides, such as glutathione, anti-tyrosinase polypeptides, etc. Insufficient enzymatic hydrolysis or a low temperature (such as in Comparative Examples 1 and 2) will lead to insufficient release of these melanin-inhibiting components, and thus the effect of inhibiting melanin production will be weakened.

[0118] Secondly, the sequential acid-base extraction process significantly enhances the comprehensiveness and synergistic effect of component extraction. The acidic stage (e.g., pH = 2) helps dissolve polyphenols, antioxidant small molecules, etc. These components can neutralize oxygen free radicals generated under ultraviolet stimulation and indirectly inhibit the activity of tyrosinase; while the alkaline stage (e.g., pH = 8) can extract polypeptides, polysaccharides, oligosaccharides, and oligopeptides, and these components can directly participate in the regulation of the melanin generation pathway or regulate the metabolic transport of pigment granules in cells. If any one of the extraction steps is omitted or the optimal pH value is deviated from (such as Comparative Examples 6, 7, and 8), the extraction of functional components is incomplete and the melanin change rate decreases significantly.

[0119] In addition, the extraction time directly affects the dissolution amount of active ingredients. If the time is too short (e.g., 0.5 h), the functional components cannot be fully released; while if the time is too long, it may cause oxidation or degradation, affecting the melanin inhibition effect. The extract obtained by the process conditions of the present invention can simultaneously play multiple roles such as anti-inflammatory, antioxidant, tyrosinase inhibition, and metabolism promotion in the regulation of melanin metabolism, thereby achieving a more significant effect of fading skin pigmentation.

[0120] In summary, the difference in the melanin change rate stems from the control of the extraction efficiency and integrity of active ingredients by the extraction process. The optimized process combination enables the product of the present invention to exhibit a more superior and safer melanin inhibition effect in dark-skinned people, significantly reducing the risk of post-sun pigment deposition and rebound.

[0121] In conclusion, the Nannochloropsis extract prepared by the method of the present invention is helpful for the post-sun repair of dark-skinned people, is mild and non-irritating, can limit melanin production, slow down the sebum secretion rate, and repair the skin barrier.

[0122] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to 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. A preparation method of a Nannochloropsis extract for post-sun repair of people with dark skin tones, characterized in that It includes the following operation steps: (1) Coarsely crush the dried Nannochloropsis powder using a pulverizer and sieve it to obtain a powder with uniform particle size; add ethanol with a volume fraction of 95% to the powder, stir for defatting and decolorization twice at 60 °C, each time for 30 minutes, filter and collect the filter residue I; (2) Add water to the filter residue I obtained in step (1) at a material-liquid ratio of 1 g: 20 mL and add cellulase solution accounting for 0.05% of the weight of the filter residue I. The activity of the cellulase solution is 10,000 U / g, and enzymolysis is carried out at 40 - 60 °C for 3 - 5 h; (3) Add water as an extraction solvent to the filter residue I after enzymolysis in step (2) at a material-liquid ratio of 1 g: 40 - 100 mL, carry out extraction at an acidic condition with a pH of 1 - 3 and a temperature of 80 °C for 1 - 3 h, cool and filter, collect the filtrate to obtain filter residue II; Then carry out extraction on filter residue II at an alkaline condition with a pH of 8 - 10 and a temperature of 80 °C for 1 - 3 h, cool and filter, collect the filtrate to obtain filter residue III, and combine the filtrates obtained from the two filtrations; carry out ultrafiltration treatment on the combined filtrate using an ultrafiltration membrane with a molecular weight cut-off value of 10 KDa, then carry out ethanol precipitation with ethanol with a volume fraction of 95%, centrifuge and take the supernatant, and concentrate under reduced pressure to obtain concentrated solution I; (4) Adjust the pH value of concentrated solution I to 5, dilute it and load it onto a pre-activated SPE column, elute successively with ethanol with a volume fraction of 60%, 70% and 80%, combine the three eluates, and concentrate under reduced pressure to obtain the Nannochloropsis extract.

2. The preparation method of a Nannochloropsis extract for post-sun repair of dark skin people according to claim 1, wherein: The sieving in step (1) is through a 30-mesh analytical sieve; when adding ethanol with a volume fraction of 95% to the powder, the material-liquid ratio is 1 g: 20 mL.

3. The preparation method of a Nannochloropsis extract for post-sun repair of dark skin people according to claim 1, characterized in that: The material-liquid ratio in step (3) is 1 g: 100 mL; the weight of the ethanol with a volume fraction of 95% is 3 times the weight of the combined filtrate.

4. The preparation method of a Nannochloropsis extract for post-sun repair of people with dark skin according to claim 1, characterized in that: The temperature of the enzymolysis in step (2) is 50 °C, and the time of enzymolysis is 5 h.

5. The preparation method of the extract of Nannochloropsis for post-sun repair of dark skin people according to claim 1, characterized in that: The acidic condition in step (3) is a pH of 2; the alkaline condition is a pH of 8; the extraction time is 2 h for both.

6. The preparation method of the extract of Nannochloropsis for post-sun repair of people with dark skin according to claim 1, characterized in that: The dilution in step (4) is to dilute the concentrated solution to 10 times its volume; the concentration under reduced pressure is to concentrate to a weight equal to the weight of the dried Nannochloropsis powder in step (1).

7. A Nannochloropsis extract for post - sun repair for dark - skinned people prepared by the preparation method according to any one of claims 1 to 6, characterized in that: The Nannochloropsis extract has the effects of repairing the skin barrier, restricting melanin production and slowing down the sebum secretion rate.

8. Use of the Nannochloropsis extract for post-sun repair for dark skin tone people according to claim 7 in the preparation of post-sun repair cosmetics.

9. Use of the Nannochloropsis extract for post-sun repair for dark skin tone people according to claim 7 in the preparation of post-sun repair cosmetics for dark skin tone people.

10. A composition for post-sun repair for people with dark skin, characterized in that: The composition is prepared from the following raw materials: 20 parts by mass of the Nannochloropsis extract for post-sun repair for dark skin tone people according to claim 7, 20 parts by mass of an emulsifier, 1 part by mass of a thickener and 0.5 part by mass of a preservative; The emulsifier is composed of polyglycerol fatty acid ester, glycerol monolaurate and fatty acid polyoxyethylene ester with a mass ratio of 1: 1: 1; The thickener is sodium chloride; The preservative is p-hydroxyacetophenone.

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