Desert algae extract, method for preparing the same, and use thereof in the preparation of skin whitening agents

Abstract

The application discloses a desert algae extract, a preparation method thereof and application of the desert algae extract in preparation of a skin whitening agent, relates to the technical field of desert algae application, and provides the preparation method of the desert algae extract, which is simple, can effectively extract flavonoids in halosarcia banksii as the desert algae extract, and the obtained desert algae extract has very strong in-vitro tyrosinase activity inhibition capacity, can reduce melanin production of B16 cells, has development value as a skin whitening agent, and simultaneously, the selected desert algae extract can significantly reduce the content of skin melanin, and can be widely applied to skin care products or cosmetics as a whitening agent. The skin care product provided by the application takes the desert algae skin care raw material as the skin whitening agent, reduces tyrosinase activity, and inhibits the production of skin melanin.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of desert algae application, in particular to a desert algae extract, a preparation method thereof and application of the desert algae extract in preparing a skin whitening agent. BACKGROUND

[0002] At present, there are more than 300,000 species of microalgae, of which 30,000 species are recorded, and only about 15 species are used for animal or human nutrition and commercialization in the pharmaceutical field, and the potential for application and development is huge. The development and application of microalgae in China dates back to the 1950s and 1960s of last century. After half a century of development, the trend of spirulina, chlorella and haematococcus pluvialis is developing, and other microalgae industries are gradually emerging, showing a pattern of diversified development. A variety of microalgae has been listed as new resource food and general food in China.

[0003] Microalgae are diverse in species, and microalgae cells contain high-value nutrients and chemical raw materials such as proteins, lipids, algal polysaccharides, β-carotene, and various inorganic elements (such as Cu, Fe, Se, Mn, Zn, etc.). The protein content of microalgae is very high, which is an important source of single-cell protein. The vitamins A, E, thiamine, riboflavin, pyridoxol, vitamin B12, vitamin C, biotin, inositol, folic acid, pantothenic acid calcium, and niacin contained in microalgae increase its value as SCP. The content of carotenoids in algae is high, and the content of β-carotene is as high as 14%, which has coloring and nutritional effects, and can be used to prevent cancer, resist radiation, delay aging, and enhance the body's immune function. The content of glycerol in algal cells is high, which is a high-quality cosmetic raw material and an organic intermediate widely used in chemical, light industry, and pharmaceutical industry. In recent years, the research and application of microalgae in desert areas have attracted widespread attention and attention. Desert algae belongs to an ancient single-cell species, and grows in extremely harsh dry environments, strong sunlight, and large temperature differences, so its cell activity is stronger, and its self-protection ability and evolution ability are stronger than other organisms. Among microalgae resources, desert algae is considered a very promising biological resource, and its development and application research needs to be strengthened.

[0004] Haxiphytospira is one of the desert algae species distributed in desert soil. Haxiphytospira cells contain rich polysaccharide components, and are a kind of excellent high-yield algal polysaccharide microalgae resources. A glycosylated lipopeptide is isolated from the cyanobacteria of Haxiphytospira, which has very strong antifungal activity.

[0005] The growth environment of desert algae (extreme drought, large temperature difference, strong sunlight, and poor soil) determines the special nutritional value of Haxiphytospira, improves the activity of cell stress components, and evolves the survival ability and self-protection form of cells. The development and utilization of Haxiphytospira show an attractive prospect.

[0006] However, there are few reports on the efficacy of the use of the Haematococcus pluvialis outside the skin, resulting in limited application of the Haematococcus pluvialis in cosmetics.

[0007] In view of this, the present application is proposed. SUMMARY

[0008] The present application aims to provide a Haematococcus pluvialis extract, a preparation method thereof, and an application thereof in preparing a skin whitening agent.

[0009] The present application is implemented as follows:

[0010] In a first aspect, the present application provides a preparation method of a Haematococcus pluvialis extract, comprising:

[0011] The Haematococcus pluvialis is cultured and harvested, and then freeze-dried and powdered to obtain Haematococcus pluvialis powder;

[0012] Anhydrous ethanol is added to the Haematococcus pluvialis powder, and a shaking table is shaken, and then filtered to obtain a filtrate, and this step is repeated 2-4 times;

[0013] The filtrates are combined, and vacuum distillation is performed on a vacuum rotary evaporator to obtain a Haematococcus pluvialis crude extract;

[0014] The Haematococcus pluvialis crude extract is redissolved in water and filtered, and passed through a HZ 803 macroporous adsorption resin column to remove pigments and odors, to obtain Haematococcus pluvialis flavonoids, which is the Haematococcus pluvialis extract.

[0015] In an optional embodiment, the ratio of the Haematococcus pluvialis powder to the anhydrous ethanol is 1:20-30.

[0016] In an optional embodiment, the vacuum distillation is performed at a temperature of 40-45°C and a pressure of-0.1 Mpa to 0.08 Mpa.

[0017] In a second aspect, the present application provides a Haematococcus pluvialis extract prepared by the preparation method of the Haematococcus pluvialis extract according to any one of the preceding embodiments.

[0018] In a third aspect, the present application provides an application of the Haematococcus pluvialis extract according to the preceding embodiments in preparing a tyrosinase activity inhibitor.

[0019] In a fourth aspect, the present application provides an application of the Haematococcus pluvialis extract according to the preceding embodiments in preparing a reagent for reducing the content of skin melanin.

[0020] In a fifth aspect, the present application provides an application of the Haematococcus pluvialis extract according to the preceding embodiments in preparing a skin whitening agent, which is a reagent for inhibiting the activity of tyrosinase and reducing the content of skin melanin in vitro.

[0021] In a fifth aspect, the present application provides a skin care product, which comprises, by mass percentage, 0.01-25% of a moisturizer, 0.02-0.8% of a thickening agent, 0.01-1% of a PH regulator, 0.01-5% of a soothing agent, 0.01-1.5% of a preservative, 0.01-5% of a skin conditioning agent, 0.01-0.5% of a solubilizer, 0.01-1% of a chelating agent, 0.005-0.5% of a fragrance, 0.01-20% of a skin whitening agent, and the balance of water, wherein the skin whitening agent is the desert algae extract as described in the foregoing embodiments.

[0022] In an optional embodiment, the skin care product comprises a water agent, an emulsion, a cream, or a serum.

[0023] In a sixth aspect, the present application provides a whitening serum, which comprises an A phase, a B phase, a C phase, a D phase, and an F phase, by mass percentage:

[0024] The A phase comprises part of water, 4%-6% of 1,3-propanediol, 1.6%-2.4% of part of glycerol, 0.16%-0.24% of hydroxybenzoic acid methyl ester, 0.24%-0.36% of betaine, 0.12%-0.18% of carbomer, 0.04%-0.06% of disodium EDTA, 0.024%-0.036% of hydrolyzed sclerotium gum, 0.016%-0.024% of sodium hyaluronate, and 0.008%-0.012% of β-glucan;

[0025] The B phase comprises the balance of water and 0.04%-0.06% of potassium hydroxide.

[0026] The C phase comprises 0.8%-1.2% of 1,2-pentanediol and 0.28%-0.42% of phenoxyethanol.

[0027] The D phase comprises 1.6%-2.4% of glycerol polyether-26, 1.6%-2.4% of PEG / PPG-17 / 6 copolymer, 0.96%-1.44% of PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerol, 0.64%-0.96% of glycerol polyacrylate, the balance of glycerol, and part of water.

[0028] The E phase comprises 0.00008%-0.00012% of PEG-35 castor oil, 0.04% of PEG-50 hydrogenated castor oil, 0.04%-0.06% of nonylphenol polyether-12, 0.006%-0.0084% of propylene glycol, part of water, and 0.0024%-0.0036% of a fragrance.

[0029] The F phase comprises 0.5%-15% of the desert algae extract as described in claim 4, 0.8%-1.2% of the calendula extract, the balance being water, 0.0004%-0.0006% of butylene glycol, and 0.00024%-0.00036% of hydroxypropyl tetrahydropyranotriol;

[0030] The preparation method of the whitening essence water includes:

[0031] Add phase A to the emulsification pot, stir and heat to 80-85℃, homogenize for 2-3 minutes, keep warm and stir for 10-20 minutes, and then cool to 58-62℃.

[0032] Add phase B to the emulsification pot, stir evenly, and then cool to 42-48℃;

[0033] The C phase is pre-dissolved, added to an emulsifying pot, and stirred until homogeneous;

[0034] The D phase is pre-dissolved, added to an emulsification pot, stirred evenly, and then cooled to 32-38℃.

[0035] Add the E phase and the F phase to the emulsification pot and stir until homogeneous;

[0036] After the material is discharged and left to stand for 24 hours, it is inspected and qualified before being repackaged, inspected again, and the finished product is put into storage.

[0037] The present invention has the following beneficial effects:

[0038] The method for preparing the desert algae extract provided in this application extracts flavonoids from *Haemaphysalis*, which maximizes the preservation of active substances without damage. Simultaneously, the extract has low pigment and odor, making it suitable for cosmetic applications. Various experiments were conducted using the desert algae extract obtained through the above extraction as a raw material. Experiments have demonstrated that the desert algae extract in this application possesses a very strong in vitro tyrosinase activity inhibitory ability, which can reduce melanin production in B16 cells. It has development value for use as a skin whitening agent. Furthermore, the desert algae extract selected in this invention can significantly reduce the melanin content of the skin and can be widely used as a whitening agent in skincare or cosmetic products. By adding desert algae extract to skincare products, the inhibitory effect of the desert algae extract on tyrosinase can effectively reduce the melanin content of the skin, achieving the purpose of skin whitening. Attached Figure Description

[0039] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0040] Figure 1 This is a schematic diagram showing the test results of different whitening cosmetic additive samples inhibiting tyrosinase activity in Example 3;

[0041] Figure 2 This is a schematic diagram of the test results of different whitening cosmetic additive samples inhibiting melanin production in B16 cells in Example 4;

[0042] Figure 3 This is a schematic diagram of the test results of the product examples and product comparison examples in Example 5, showing the inhibition of skin melanin production. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0044] This application provides a method for preparing a desert algae extract, comprising:

[0045] (1) Cultivation.

[0046] Spirulina platensis, isolated from arid desert regions of China, was cultured, harvested, freeze-dried, and powdered to obtain Spirulina platensis powder.

[0047] The BG11 culture medium for *Hassaela spp.* consists of: 1.5 g / L NaNO3, 0.04 g / L K2HPO4, 0.075 g / L MgSO4·7H2O, 0.0284 g / L CaCl2, 0.006 g / L citric acid, 0.006 g / L ferric ammonium citrate, 0.001 g / L EDTA-Na2, 0.02 g / L Na2CO3, and 1 mL / L A5 solution. *Hassaela spp.* is inoculated into a 50 L plastic container, with CO2 supplemented to maintain the pH between 7.0 and 8.0. The medium is stirred regularly daily for a 4-week cultivation period, with an average temperature of 25°C and a light exposure of 1000-2000 Lux, set to 12 hours of light / 12 hours of darkness. After harvesting, *Hassaela spp.* is freeze-dried to prepare powder for later use. Low-temperature drying maximizes the preservation of active substances within the algae.

[0048] (2) Crude extraction.

[0049] Add anhydrous ethanol to the *Hasarum heterotropoides* powder, with a powder-to-ethanol ratio of 1:20-30, and shake on a shaker (45℃, 180 rpm overnight). Then filter to obtain the filtrate. Repeat this step 2-4 times. Combine the filtrates and distill under reduced pressure in a vacuum rotary evaporator. The conditions for reduced pressure distillation are: temperature 40℃-45℃, pressure -0.1 MPa to 0.08 MPa, to obtain crude *Hasarum heterotropoides* extract.

[0050] Flavonoids are generally readily soluble in highly polar solvents such as water, ethanol, and methanol, and ethanol has low toxicity and can be recycled. Therefore, this application uses anhydrous ethanol to extract *Haemaphysalis* powder. After extraction, the mixture is filtered, and the resulting residue is extracted 2-4 times. The filtrates are combined and then vacuum distilled to obtain crude *Haemaphysalis* extract. Vacuum distillation lowers the boiling point of ethanol, accelerates its volatilization at a lower temperature, and maximizes the preservation of active substances.

[0051] (3) Purification.

[0052] The crude extract of *Hasa salsa* was redissolved in water and filtered, then passed through an HZ 803 macroporous adsorption resin column to remove pigments and odors, yielding *Hasa salsa* flavonoids, which is the desert algae extract.

[0053] The HZ 803 macroporous resin adsorption method is used to decolorize aqueous solutions of crude desert algae extracts, solving the problems of poor adsorbent regeneration and low yield of active ingredients. This preparation method significantly reduces pigments and odors in the extract, making it more suitable for use in cosmetics and ensuring product quality.

[0054] Fair, rosy, and radiant skin has been a long-held aspiration and pursuit for women in East Asia throughout history, and it has become a fashionable trend among Chinese women in recent years. Influenced by this, the market for skin-whitening and blemish-removing cosmetics has become increasingly active, with demand growing daily, making it one of the mainstream products in functional skincare. Skin whitening has become a consistently popular topic of discussion, and the skin whitening market continues to expand, showing a clear positive growth trend. At the same time, consumers' demand for whitening skincare products is gradually shifting towards higher quality.

[0055] In recent years, the search for natural tyrosinase inhibitors in algae has attracted considerable attention. This application uses the desert algae extract obtained above as a raw material for various experiments. Experiments have demonstrated that the desert algae extract of this application possesses a very strong in vitro tyrosinase activity inhibitory ability, which can reduce melanin production in B16 cells. It has development value as a skin whitening agent. Furthermore, the desert algae extract selected in this invention can significantly reduce the melanin content in the skin and can be widely used as a whitening agent in skincare or cosmetic products.

[0056] By adding desert algae extract to skincare products, the inhibitory effect of desert algae extract on tyrosinase can effectively reduce skin melanin content, achieving skin whitening. Therefore, this application proposes the use of desert algae extract in the preparation of tyrosinase activity inhibitors and reagents for reducing skin melanin content. Alternatively, it proposes the use of desert algae extract in the preparation of skin whitening agents, which are reagents that inhibit tyrosinase activity and reduce skin melanin content in vitro.

[0057] In addition, this application can also add desert algae extract to skin care or cosmetic products to enhance the skin whitening effect of the skin care or cosmetic products.

[0058] Specifically, this application provides a skin care product, whose components, by mass percentage, include: 0.01-25% moisturizer, 0.02-0.8% thickener, 0.01-1% pH adjuster, 0.01-5% soothing agent; 0.01-1.5% preservative, 0.01-5% skin conditioning agent, 0.01-0.5% solubilizer, 0.01-1% chelating agent, 0.005-0.5% fragrance, 0.01-20% skin whitening agent, and the balance being water, wherein the skin whitening agent is the aforementioned desert algae extract.

[0059] In this embodiment, the skin whitening agent used is desert algae extract.

[0060] In one embodiment, the humectant includes one or more of the following: dipropylene glycol, betaine, dipropylene glycol, panthenol, 1,3-propanediol, butylene glycol, 1,2-pentanediol, 1,2-hexanediol, sorbitol, panthenol, glycerin, PEG / PPG-17 / 6 copolymer, PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerin, glyceryl polyacrylate, glyceryl polyether-26, and sodium hyaluronate.

[0061] In one embodiment, the thickener includes one or more of the following: acrylic / C10-30 alkanol acrylate crosspolymer, hydroxyethyl cellulose, xanthan gum, hydroxyethyl acrylate / sodium acryloyldimethyl taurate copolymer, hydrolyzed sclerotium gum, carbomer, and ammonium acryloyldimethyl taurate / VP copolymer.

[0062] In one embodiment, the pH adjuster includes one or more of aminomethylpropanol, citric acid, sodium citrate, potassium hydroxide, sodium hydroxide, arginine, etc.

[0063] In one embodiment, the soothing agent includes one or more of the following: chamomile extract, calendula extract, bisabolol, ginger root extract, aloe vera extract, centella asiatica extract, asiaticoside, purslane extract, witch hazel flower water, etc.

[0064] In one embodiment, the amount of preservative added is 0.01-1.5%, and the preservative may include one or a combination of two or more of phenoxyethanol, methylparaben, benzoic acid and its salts, sorbic acid and its salts, chlorphenesin, and propylparaben.

[0065] In one embodiment, the skin conditioning agent includes one or more of the following: allantoin, hydrolyzed collagen, ceramide NP, β-glucan, *Syzygium buergerianum* extract, hydroxypropyl tetrahydropyrantriol, trehalose, squalane, brown algae extract, *Spirulina bonata* extract, palmitoyl tripeptide-5, dipeptide diaminobutyryl benzyl amide diacetate, *Bifida ferment lysate* fermentation product, yeast fermentation product filtrate, and *Lactobacillus* / soybean fermentation product extract.

[0066] In one embodiment, the solubilizer comprises one or more of the following: polysorbate-20, PEG-40 hydrogenated castor oil, nonylphenol polyether-12, PEG-50 hydrogenated castor oil, PEG-35 castor oil, PPG-26-butanol polyether-26, a complex of PEG-100 stearate and glyceryl stearate, polyglycerol-3-methylglucose distearate, a complex of polyacrylamide and lauryl ether-7 and C13-14 isoparaffins, and glyceryl alcohol ether-25PCA isostearate.

[0067] In one embodiment, the chelating agent may be EDTA-2Na and / or EDTA-4Na, etc.

[0068] In one embodiment, the fragrance agent may be a fragrance or the like.

[0069] In one embodiment, the amount of the added skin-whitening agent, desert algae extract, is 0.01-20%, preferably 0.5-15%, and even more preferably 0.5-10%. The skin-whitening performance increases with increasing dosage, but the rate of increase slows down after reaching 10%. Considering both cost and effectiveness, an added desert algae extract content of 0.5-10% is the preferred option.

[0070] The skin care products of this invention can be in the form of water, lotion, cream, gel, etc., and use the selected desert algae extract as a skin whitening agent, which can effectively reduce the melanin content of the skin and achieve the effect of whitening the skin.

[0071] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0072] Example 1: Preparation of *Hasarum heterotropoides* extract

[0073] (1) The cultivation method of *Hassaela spp.* is as follows:

[0074] *Hassa harsabina* (labeled FACHB-2021, purchased from the Freshwater Algae Culture Bank of the Chinese Academy of Sciences), isolated from arid desert regions, was cultured. The BG11 medium for *Hassa harsabina* consisted of: 1.5 g / L NaNO3, 0.04 g / L K2HPO4, 0.075 g / L MgSO4·7H2O, 0.0284 g / L CaCl2, 0.006 g / L citric acid, 0.006 g / L ferric ammonium citrate, 0.001 g / L EDTA-Na2, 0.02 g / L Na2CO3, and 1 mL / L A5 solution.

[0075] The *Hassa stylosa* was inoculated into a 50L plastic container, and CO2 was added to maintain the pH of the culture medium between 7.0 and 8.0. The medium was stirred regularly daily for a 4-week cultivation period, with an average temperature of 25℃ and a light intensity of 1000-2000 Lux, set to 12 hours of light / 12 hours of darkness. After harvesting, the *Hassa stylosa* was freeze-dried to prepare powder for later use.

[0076] (2) Extraction:

[0077] Weigh 20g of *Hasarum heterotropoides* powder, add 500ml of anhydrous ethanol at a material-to-liquid ratio of 1:25, shake overnight at 45℃ and 180rpm, filter and collect the filtrate the next day, and repeat the above steps 3 times with the algae; combine the filtrates obtained from the above three times, and distill under reduced pressure in a vacuum rotary evaporator at 45℃ and a pressure of -0.1Mpa to 0.08Mpa to obtain crude extract of *Hasarum heterotropoides*.

[0078] (3) Purification:

[0079] The crude extract was redissolved in water to obtain an aqueous solution. This solution was then added to a pretreated and activated HZ 803 macroporous resin column, and this step was repeated until the resulting liquid had no obvious color or odor. This process significantly reduces the pigment and odor of the extract, which can be used for skin whitening tests.

[0080] Example 2: Cytotoxicity Test

[0081] MTT stock solution: Prepare MTT to 5 mg / mL with PBS, filter (0.22 μm microporous membrane), and store at 2℃-8℃ protected from light.

[0082] MTT working solution: Dilute the MTT stock solution to 1 mg / mL with phenol red-free medium and prepare fresh before use.

[0083] The immortalized human keratinocyte HaCaT cell line (purchased from the National Biomedical Experimental Cell Resource Bank) used in the experiment was passaged at least once before the test. 100 μL of the cell line was seeded into 96-well plates one day before the experiment. Cell confluence was measured at 50%-70% to determine the cytotoxicity of the test substance. The culture medium in the 96-well plates was discarded, and different concentrations of the test substance solution were added according to Table 1. A blank control group (NC) was also set up at 100 μL / well, with six replicates for each concentration. After the test, the cell plate was capped, sealed, and incubated in a CO2 incubator for 24 h.

[0084] After culture, discard the culture medium, wash 1-2 times with PBS, add 50 μL of MTT working solution per well, and continue incubation in a CO2 incubator for 2-6 hours. Discard the culture medium, add 150 μL of DMSO to each well, shake for 5-10 minutes, and measure the absorbance at 570 nm using a microplate reader. Calculate the relative cell activity based on the results (average of cell activity from 6 replicates). When the cell viability at the corresponding concentration in a 96-well plate is ≥90% (Table 1), and there is no significant morphological difference between the sample group and the control group, it indicates that the sample has no significant cytotoxicity at that concentration.

[0085] Table 1. Cytotoxicity test results

[0086] Sample Final concentration (mg / mL) Absorbance (OD) Cell relative viability (%) Blank group 0 1.6202 100 H. zollingeri extract 1.25 1.6413 101.4 H. zollingeri extract 2.5 1.6151 99.0 H. zollingeri extract 5 1.4927 91.3 H. zollingeri extract 10 1.0170 62.8 H. zollingeri extract 20 0.8732 53.7 H. zollingeri crude extract 5 1.4875 91.8

[0087] Example 3: In vitro tyrosinase inhibition test

[0088] The cell-whitening properties of *Hassarum heterotropoides* extract were tested using an in vitro tyrosinase activity assay. Tyrosinase activity was measured by the activity of tyrosinase catalyzing the conversion of L-DOPA to dopaquinone. Specifically:

[0089] Add 0.4 mL (1.0 mg / mL) of substrate L-DOPA solution to a test tube, and add 2.4 mL of phosphate buffer at pH 6.8. Incubate in a water bath at 30 °C for 10 min, and then add 0.2 mL (250 U / mL) of tyrosinase to obtain the control group reaction solution.

[0090] Add 2.8 mL of phosphate buffer (pH 6.8) to a test tube, incubate at 30°C for 10 min, and then add 0.2 mL of tyrosinase (250 U / mL) to obtain the blank reaction solution.

[0091] Add 0.4 mL (1.0 mg / mL) of substrate L-DOPA solution to a test tube, then add 2.4 mL of test solution containing the final concentration shown in Table 2, and add phosphate buffer with pH 6.8. After incubating in a water bath at 30°C for 10 min, add 0.2 mL (250 U / mL) of tyrosinase to obtain the experimental reaction solution.

[0092] An enzymatic reaction converts L-DOPA to the red product dopaquinone, which exhibits maximum absorption at 475 nm. The absorbance A at 475 nm was read 1 minute after mixing. 475 The rate of the enzyme-catalyzed reaction is expressed in terms of A per minute. 475 The increase is expressed as a percentage. The absorbance (A) of the control group, blank group, and experimental group reaction solutions was measured at 475 nm 1 minute after the start of the reaction, and the inhibition rate of tyrosinase activity was calculated using the following formula. Note that the mass concentrations of the whitening cosmetic additive samples in the table represent the final mass concentrations of different whitening cosmetic additive samples in the test system.

[0093] Inhibition rate = [1 - (A3 - A2) / (A1 - A2)] × 100%

[0094] In the formula: A1 is the absorbance of the control group reaction solution, which contains only the substrate and no whitening cosmetic additives.

[0095] A2 represents the absorbance of the blank reaction solution, which does not contain any whitening cosmetic additives or substrates.

[0096] A3 represents the absorbance of the experimental group reaction solution, which contains whitening cosmetic additive samples and substrates.

[0097] Among them, the crude extract of *Haloxylon ammodendron* in Table 2 is the crude extract of *Haloxylon ammodendron* obtained in step (2) of Example 1; the extraction method of *Haloxylon ammodendron* polysaccharide extract is water extraction and alcohol precipitation; the extraction method of *Haloxylon ammodendron* protein extract is water extraction and salt precipitation; the extraction method of nitrogen-fixing *Anabaena* is alcohol extraction; and the extraction method of *Pseudobranchia javanica* is alcohol extraction.

[0098] After testing, the inhibition rates of tyrosinase on different samples in Table 2 were calculated. The results are shown in Table 2 and... Figure 1 As shown.

[0099] Table 2. Results of Tyrosinase Activity Test

[0100]

[0101] As shown in Table 2, *Hydrocotyle spp.*, *Anabaena nigra*, and *Pseudocladus javanica* are all desert algae belonging to the genus *Cyanobacteria*. However, the table also shows that not all desert algae extracts exhibit tyrosinase inhibition, especially *Anabaena nigra*, whose tyrosinase inhibition rate is only 0.4%. In this application, the flavonoid extract of *Hydrocotyle spp.* was used as the desert algae extract, which showed strong inhibition of tyrosinase in a dose-dependent manner, with better inhibition observed at concentrations above 5%. Other extracts of *Hydrocotyle spp.*, such as polysaccharide or protein extracts, did not show tyrosinase inhibition. Therefore, this application selected a specific flavonoid extract of *Hydrocotyle spp.* as the desert algae extract, which can significantly inhibit tyrosinase.

[0102] Example 4: Melanin Synthesis Inhibition Test

[0103] The cell whitening properties of the extract of Alternaria harzianum were tested using an in vitro cell melanin production test.

[0104] Melanoma B16 cells were diluted to 2.5*102.5 4 Cells / ml were transferred to 9cm culture dishes, and 100μL of cell suspension was added to each well of a 96-well plate using a multichannel pipette. The culture plate was placed in a 5% CO2 incubator and incubated at 37°C for 24 hours. Then, the whitening cosmetic additive sample (according to the mass concentration in Table 3) was added. After 4 hours of incubation, 1*103 -6 Cells were inoculated with α-MSH at 1 mol / L, and the culture medium was aspirated after 48 hours. The cells were then digested with 0.25% trypsin, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. Cells were added to 200 μL of NaOH (1 mol / L) solution containing 10% DMSO, and the melanin granules were completely dissolved in a 65°C water bath. The absorbance was measured at 490 nm.

[0105] Melanin change rate (%) = [(OD 实验组 -OD 空白组 ) / (OD 对照组 -OD 空白组 )-1]×100%

[0106] 1. The experimental group was treated with a whitening cosmetic additive sample plus α-MSH;

[0107] 2. The control group was supplemented with α-MSH;

[0108] 3. Add phosphate buffer to the blank group.

[0109] Table 3. Inhibition effect of B16 cell melanin production

[0110]

[0111]

[0112] Through Table 3 and Figure 2 The results showed that *Hassaela spp.* extract could block melanin production induced by α-MSH in a dose-dependent manner. Based on this, *Hassaela spp.* extract could inhibit tyrosinase activity and α-MSH-induced melanin production in cells. The inhibitory effect increased with increasing concentration of *Hassaela spp.* extract, and the purified extract showed a higher inhibitory effect than the crude extract. Given that the cell viability of 10 mg / mL *Hassaela spp.* extract was less than 90%, the concentration of the extract was not further increased in this application. Furthermore, other extracts of *Hassaela spp.*, such as polysaccharide or protein extracts, did not show any melanin-inhibiting effect. Therefore, this application selected specific flavonoid extracts of *Hassaela spp.* as desert algae extracts, which can significantly inhibit melanin production.

[0113] Example 5: Skin care products

[0114] This invention provides a skincare product, formulated as shown in Table 4 for Product Examples 1-4 and Product Comparative Example 1. In Product Examples 1-4, different amounts of the *Haloxylon ammodendron* extract of this invention are added as a skin whitening agent, while in Product Comparative Example 1, no *Haloxylon ammodendron* extract is added.

[0115] Table 4: Proportion of Skincare Product Formulas

[0116]

[0117]

[0118] The skincare product preparation process of the above formula in this embodiment is as follows:

[0119] The essence water was prepared according to the content (mass percentage) of each component in the whitening essence water formula in Table 4 above, and following the production process steps below. The production process steps are as follows:

[0120] 1. Add phase A to the emulsifying pot, stir and heat to 80-85℃, homogenize for 2 minutes, keep warm and stir for 15 minutes, and then cool to 60℃;

[0121] 2. Add phase B to the emulsifying pot, stir well, and then cool to 45°C;

[0122] 3. Pre-dissolve phase C, add it to the emulsifying pot, and stir until homogeneous;

[0123] 4. After pre-dissolving phase D, add it to the emulsification pot, stir evenly, and then cool to 35℃;

[0124] 5. Add phase E and phase F to the emulsifying pot and stir until well mixed;

[0125] 6. Discharge the material, let it stand for 24 hours, and after passing the inspection, repackage and pack it. Then inspect it again and put the finished product into the warehouse.

[0126] Note: Phases A, B, C, D, E, and F in the process are respectively:

[0127] Phase A includes some water, 1,3-propanediol, some glycerol, methylparaben, betaine, carbomer, disodium EDTA, hydrolyzed sclerotium gum, sodium hyaluronate, and β-glucan.

[0128] Phase B includes the remainder of water and potassium hydroxide;

[0129] Phase C includes 1,2-pentanediol and phenoxyethanol;

[0130] Phase D comprises glycerol polyether-26, PEG / PPG-17 / 6 copolymer, PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerol, glycerol polyacrylate, the balance of glycerol, and some water;

[0131] Phase E includes PEG-35 castor oil, PEG-50 hydrogenated castor oil, nonylphenol polyether-12, propylene glycol, some water, and fragrance;

[0132] Phase F includes the *Hasarum heterotropoides* extract, *Calendula officinalis* extract, water, butylene glycol, and hydroxypropyltetrahydropyranotriol from Example 1.

[0133] Among them, 1,3-propanediol, 1,2-pentanediol, betaine, propylene glycol, butylene glycol, sodium hyaluronate, glycerin, glyceryl polyether-26, PEG / PPG-17 / 6 copolymer, PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerin, and glyceryl polyacrylate are moisturizers; carbomer and hydrolyzed sclerotium gum are thickeners; *Haloxylon ammodendron* extract is a skin whitening agent; PEG-35 castor oil, nonylphenol polyether-12, and PEG-50 hydrogenated castor oil are solubilizers; β-glucan and hydroxypropyl tetrahydropyranotriol are skin conditioning agents; calendula extract is a soothing agent; phenoxyethanol and methylparaben are preservatives; disodium EDTA is a chelating agent; potassium hydroxide is a pH adjuster; and fragrance is an aromatic agent.

[0134] In this embodiment, the skin melanin content of the obtained whitening essence water was tested.

[0135] Methods for testing the rate of change of skin melanin: The melanin content in the skin is determined by measuring the amount of light reflected from the skin after a specific wavelength of light shines on it. The transmitter of the probe emits light with wavelengths of 568nm, 660nm, and 880nm, which shines on the skin surface. The receiver measures the light reflected from the skin and the amount of light absorbed by the skin, thereby determining the rate of change of skin melanin.

[0136] The rate of change in skin melanin was tested using a pigment analyzer from CK (Germany). The MX 18 skin melanin testing probe consists of a light source emitter and a receiver, with a spring to maintain constant pressure on the skin during testing. The probe's measurement range is 0-999; a higher measurement value indicates a higher melanin content in the skin.

[0137] The study involved 33 participants over a 4-week period. Whitening essence waters from Examples 1-4 and Comparative Example 1 were used. These essence waters were applied to different areas of the inner forearm morning and evening. The skin reflectance of the tested areas was measured at the initial value (Day 0) and at the end of the first, second, third, and fourth weeks of use. The reflectance of light at a specific wavelength was used to determine the melanin content in the skin, thus characterizing the rate of melanin change. The specific results of the melanin change rate are shown in Table 5. Figure 3 As shown.

[0138] Table 5. Change rate of skin melanin content

[0139]

[0140] Melanin change rate (%) = (Melanin value in week X - initial value) / initial value * 100.

[0141] X represents 1, 2, 3, 4.

[0142] Therefore, adding *Hassa salsa* extract to skincare products can reduce melanin content in the skin, thus achieving a skin-whitening effect. Furthermore, the effect increases with the dosage of *Hassa salsa* extract; however, when the dosage exceeds 10%, the increase in melanin reduction becomes less significant. Therefore, considering both cost and effectiveness, adding 0.5-10% *Hassa salsa* extract is the optimal implementation method.

[0143] In summary, the preparation method of the desert algae extract provided in this application, by extracting flavonoids from *Algae halophila*, can retain the active substances to the maximum extent without damage. Simultaneously, the extract has low pigment and odor, making it suitable for application in cosmetics. In this application, the desert algae extract obtained by the above extraction was used as a raw material for various experiments. Experiments have demonstrated that the desert algae extract of this application has a very strong in vitro tyrosinase activity inhibitory ability, which can reduce melanin production in B16 cells. It has development value as a skin whitening agent. Furthermore, the desert algae extract selected in this invention can also significantly reduce the melanin content of the skin and can be widely used as a whitening agent in skincare or cosmetic products. By adding desert algae extract to skincare products, the inhibitory effect of desert algae extract on tyrosinase can be utilized to effectively reduce the melanin content of the skin, achieving the purpose of skin whitening.

[0144] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method of preparing a desert algae extract, characterized by, It comprises: The haxiang algae is cultured, harvested, freeze-dried and powdered to obtain haxiang algae powder; Anhydrous ethanol is added to the haxiang algae powder, and the mixture is shaken on a shaking table, and then filtered to obtain a filtrate, and this step is repeated 2-4 times; The filtrates are combined and distilled under reduced pressure in a vacuum rotary evaporator to obtain a haxiang algae crude extract; The haxiang algae crude extract is redissolved in water and filtered, and passed through a HZ 803 macroporous adsorption resin column to remove pigments and odors, thereby obtaining a haxiang algae flavonoid substance, which is the desert algae extract. The ratio of the haxiang algae powder to the anhydrous ethanol is 1:20-30. The conditions for the reduced pressure distillation are a temperature of 40-45°C and a pressure of-0.1 Mpa to-0.08 Mpa.

2. A desert algae extract, characterized in that, It is prepared by the method of claim 1.

3. Use of the desert algae extract of claim 2 in the preparation of a reagent for reducing the content of skin melanin.

4. Use of the desert algae extract according to claim 2 for the preparation of a skin whitening agent, characterized in that, The skin whitening agent is a reagent that inhibits the activity of tyrosinase and reduces the content of skin melanin in vitro.

5. A skin care product, characterized by, The components include, by mass percentage, a humectant 0.01-25%, a thickening agent 0.02-0.8%, a PH regulator 0.01-1%, a soothing agent 0.01-5%, a preservative 0.01-1.5%, a skin conditioner 0.01-5%, a solubilizer 0.01-0.5%, a chelating agent 0.01-1%, a fragrance 0.005-0.5%, a skin whitening agent 0.01-20%, and the balance of water, wherein the skin whitening agent is the desert algae extract of claim 2.

6. A whitening serum, characterized by, The components include phase A, phase B, phase C, phase D, phase E, and phase F, by mass percentage: The phase A includes part of water, 1,3-propanediol 4%-6%, part of glycerol 1.6%-2.4%, hydroxybenzoic acid methyl ester 0.16%-0.24%, betaine 0.24%-0.36%, carbomer 0.12%-0.18%, disodium EDTA 0.04%-0.06%, hydrolyzed arthrobotrys gum 0.024%-0.036%, sodium hyaluronate 0.016%-0.024%, and β-glucan 0.008%-0.012%; The phase B includes the balance of water and potassium hydroxide 0.04%-0.06%; The phase C includes 1,2-pentanediol 0.8%-1.2% and phenoxyethanol 0.28%-0.42%; The phase D includes glycerol polyether-26 1.6%-2.4%, PEG / PPG-17 / 6 copolymer 1.6%-2.4%, PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerol 0.96%-1.44%, glycerol polyacrylate 0.64%-0.96%, the balance of glycerol, and part of water; The phase E includes PEG-35 castor oil 0.00008%-0.00012%, PEG-50 hydrogenated castor oil 0.32%-0.048%, nonylphenol polyether-12 0.04%-0.06%, propylene glycol 0.006%-0.0084%, part of water, and fragrance 0.0024%-0.0036%. The F phase comprises 0.5%-15% of desert algae extract, 0.8%-1.2% of calendula extract, the balance of water, 0.0004%-0.0006% of butanediol, and 0.00024%-0.00036% of hydroxypropyl tetrahydro pyran triol as claimed in claim 2; The preparation method of the whitening essence water comprises: The A phase is added into an emulsifying kettle, stirred and heated to 80-85 DEG C, homogenized for 2-3 min, cooled to 58-62 DEG C after being stirred and kept for 10-20 min; The B phase is added into the emulsifying kettle, stirred and cooled to 42-48 DEG C; The C phase is pre-dissolved and added into the emulsifying kettle, stirred and uniformly mixed; The D phase is pre-dissolved and added into the emulsifying kettle, stirred and uniformly mixed, and cooled to 32-38 DEG C; The E phase and the F phase are added into the emulsifying kettle, stirred and uniformly mixed; The product is discharged, placed for 24 hours, reexamined, and then packed and reexamined before being put into a warehouse.

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