Whitening composition for maintaining homeostasis of skin and application thereof

Abstract

The invention relates to the technical field of cosmetics, and discloses a whitening composition for maintaining homeostasis in skin and application of the whitening composition. The whitening composition is prepared from glabridin, cordyceps militaris fermentation filtrate, marine oligosaccharide and ceramide NS, the marine oligosaccharide is a carbohydrate compound which is extracted from hydrilla and consists of 2-10 monosaccharide units. Through cooperation of melanin metabolism regulation, inflammation signal truncation, flora dynamic balance and lipid structure strengthening, the technical paradox of a traditional whitening product in whitening stimulation barrier and slow repairing and fading color can be broken through, and integrated synergism of whitening and homeostasis maintaining is achieved, so that the whitening effect of the composition is improved, and the whitening effect of the composition is improved. The skin pigmentation and erythema can be improved to a greater extent.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, and more particularly to a whitening composition for maintaining skin homeostasis and its application. Background Technology

[0002] Under the influence of current environment and lifestyle, the problem of skin microecological imbalance is becoming increasingly prominent. Environmental pollution, excessive cleansing, and antibiotic abuse can significantly disrupt the homeostasis of the skin microbiota, leading to the abnormal proliferation of opportunistic pathogens and the inhibition of beneficial bacteria (such as Staphylococcus epidermidis) colonization. Studies have shown that microbiota dysbiosis can activate inflammatory pathways, promoting the release of pro-inflammatory factors such as TNF-α and IL-6, triggering skin problems such as persistent erythema, barrier dysfunction, and pigmentation. Traditional broad-spectrum antibacterial ingredients, such as tea tree oil and capryloyl hydroxamic acid, can inhibit Propionibacterium acnes in the short term, but they exacerbate the microbiota imbalance and form a vicious cycle of "sterilization-dysregulation-inflammatory recurrence," resulting in poor whitening effects (improving erythema and pigmentation). Although the market demand for microecological balancing products has surged, existing products are mostly limited to supplementing single prebiotics (such as inulin), failing to systematically regulate the microbiota interaction network and making it difficult to achieve long-term homeostasis restoration of the skin microbiota.

[0003] Patent CN106389168A discloses a positively charged water-in-oil nano-cream with whitening and freckle-removing effects and its preparation method. It uses a compound of glycyrrhizin, ginsenosides, rose essential oil, caprylic / capric triglycerides, mixed surfactants, phytosphingosine, glycerin, cholesterol, ceramide NP, palmitic acid, tocopherol, potassium sorbate, and carbomer 940. Although it can produce a certain whitening and freckle-removing effect, it is difficult to effectively overcome the whitening irritation barrier and the technical paradox of slow repair and fading of pigmentation. Due to its poor effect on maintaining the skin's homeostasis, the whitening and freckle-removing effect is limited. Summary of the Invention

[0004] To address the technical problem of limited whitening effects caused by the poor effectiveness of existing whitening cosmetics in maintaining skin homeostasis, this invention provides a whitening composition for maintaining skin homeostasis and its application. This invention uses a compound of glycyrrhizin, Cordyceps militaris fermentation filtrate, marine oligosaccharides, and ceramide NS, which can achieve a synergistic effect of whitening and maintaining homeostasis, thereby improving the whitening effect.

[0005] The specific technical solution of this invention is as follows: In a first aspect, the present invention provides a skin whitening composition for maintaining skin homeostasis, comprising glycyrrhizin, Cordyceps militaris fermentation filtrate, marine oligosaccharides, and ceramide NS; wherein the marine oligosaccharides are saccharide compounds extracted from Elodea and composed of 2 to 10 monosaccharide units.

[0006] In the composition of this invention, an integrated pathway of "pigmentation regulation-barrier reconstruction-microecological regulation" is constructed through the multi-dimensional synergistic effects of glycyrrhizin, Cordyceps militaris fermentation filtrate, marine oligosaccharides, and ceramide NS. Specifically, glycyrrhizin is an isoflavone active ingredient isolated and extracted from the root of Glycyrrhiza glabra. It can precisely block the melanin production chain by inhibiting tyrosinase activity and possessing antioxidant capabilities, while simultaneously neutralizing free radicals and alleviating inflammatory pigmentation, laying the foundation for skin whitening. Cordyceps militaris fermentation filtrate is an active ingredient complex extracted from Cordyceps militaris using bio-fermentation technology. It can quickly soothe skin redness and acne inflammation by inhibiting the release of pro-inflammatory factors and enhancing the expression of anti-inflammatory proteins, and accelerate barrier regeneration under the action of ceramide NS. Marine oligosaccharides can directionally regulate the microecological network, selectively promoting the colonization and activity of beneficial skin bacteria (such as Staphylococcus epidermidis), enhancing the skin's ability to resist external pathogens, while inhibiting the proliferation of harmful bacteria (such as Propionibacterium acnes and Malassezia furfur), reducing inflammation recurrence and barrier disturbance through the reconstruction of microbial homeostasis. In the composition system of this invention, ceramide NS can enhance water-locking and defense functions, and optimize the lipid metabolism microenvironment, providing a stable foundation and long-lasting protection for the effects of glycyrrhizin, Cordyceps militaris fermentation filtrate, and marine oligosaccharides, thereby improving the efficacy of glycyrrhizin, Cordyceps militaris fermentation filtrate, and marine oligosaccharides. Through the above methods, the composition of this invention can achieve a quadruple synergy of melanin metabolism regulation, inflammatory signal interception, microbial dynamic balance, and lipid structure enhancement, breaking through the technical paradox of traditional whitening products that stimulate the whitening barrier and are slow to repair and fade pigmentation. It achieves an integrated synergistic effect of whitening and maintaining homeostasis, improving the whitening effect by effectively maintaining skin homeostasis.

[0007] Furthermore, compared to ceramide NP, which is commonly used in existing whitening products, this invention uses ceramide NS, which can better synergize with the other three components (glycyrrhizin, Cordyceps militaris fermentation filtrate, and marine oligosaccharides) to improve the overall whitening effect of the composition. Specifically, although ceramide NP is widely used for moisturizing and repairing, its molecular structure and function differ from those of ceramide NS. Compared to ceramide NP, the tight arrangement of non-hydroxylated fatty acid chains and sphingosine in ceramide NS can more efficiently fill the lipid gaps in the stratum corneum, and has a bidirectional consolidation effect on the physical barrier and the microenvironment of the microbial community. Therefore, it can promote the corresponding effects of glycyrrhizin, Cordyceps militaris fermentation filtrate, and marine oligosaccharides to a greater extent.

[0008] Preferably, the total mass ratio of the glycyrrhizin, Cordyceps militaris fermentation filtrate, and marine oligosaccharides to the mass ratio of ceramide NS is 50–4200:1; the mass ratio of the glycyrrhizin, Cordyceps militaris fermentation filtrate, and marine oligosaccharides is 1:50–600:0.5–60.

[0009] Furthermore, the total mass ratio of the glycyrrhizin, Cordyceps militaris fermentation filtrate, and marine oligosaccharides to the mass ratio of ceramide NS is 1100–2000:1; the mass ratio of the glycyrrhizin, Cordyceps militaris fermentation filtrate, and marine oligosaccharides is 1:150–400:15–40.

[0010] When the ratio of the total mass of glycyrrhizin, Cordyceps militaris fermentation filtrate, and marine oligosaccharides to the mass of ceramide NS is controlled within the range of 50–4200:1, a better synergistic effect can be achieved between glycyrrhizin, Cordyceps militaris fermentation filtrate, and marine oligosaccharides and ceramide NS. Ceramide NS provides a stable foundation and long-lasting protection for the effects of glycyrrhizin, Cordyceps militaris fermentation filtrate, and marine oligosaccharides, thereby reducing the content of melanin and hemoglobin in the skin to a greater extent and achieving a better whitening effect.

[0011] Based on this, when the total mass ratio of glycyrrhizin, Cordyceps militaris fermentation filtrate, and marine oligosaccharides to ceramide NS is controlled within the range of 1100–2000:1, the effect of the composition in improving skin melanin deposition can be further enhanced. Among the above four components, when the proportion of ceramide NS is relatively small, its effect in promoting the function of glycyrrhizin, Cordyceps militaris fermentation filtrate, and marine oligosaccharides is not good. When the proportion of ceramides (NS) is high, it over-strengthens the skin barrier, making the stratum corneum structure too dense. This dense "brick wall structure" slows down the renewal of keratinocytes. A healthy skin barrier means that the rate of keratinocyte shedding is kept at a normal or slightly slower level. Melanin granules are mainly found in melanocytes in the basal layer of the epidermis. After being produced, they are transferred to the surrounding keratinocytes and gradually migrate to the skin surface with the keratinocytes, eventually shedding with the dead skin cells. An overly strong barrier and slowed keratinocyte renewal mean that keratinocytes containing melanin stay on the skin surface for a longer time, causing melanin to not be metabolized and cleared in time. Visually, this manifests as dull skin tone, obvious dark spots, and poor whitening effect.

[0012] Preferably, the whitening composition further includes cholesterol and vegetable oil; the mass ratio of ceramide NS, cholesterol and vegetable oil is 1:0.5-1.5:0.5-1.5; the vegetable oil includes at least one of palmitic acid, palmitoleic acid, stearic acid, oleic acid, linolenic acid and α-linolenic acid.

[0013] Preferably, the whitening composition comprises the following raw materials: glycyrrhizin solution, Cordyceps militaris fermentation filtrate, marine oligosaccharide, and ceramide NS solution; the glycyrrhizin content in the glycyrrhizin solution is 1-50 wt%; and the ceramide NS content in the ceramide NS solution is 2-40 wt%.

[0014] Furthermore, the solvent in the glycyrrhizin solution includes 1,3-butanediol and / or water, and the glycyrrhizin solution also contains polyquaternium-51 and / or hydroxypropyl cyclodextrin; the solvent in the ceramide NS solution includes 1,3-butanediol and / or water, and the ceramide NS solution also contains at least one of polyglycerol-10 myristate, glycerol, and cholesterol.

[0015] Secondly, the present invention provides a cosmetic product comprising the aforementioned whitening composition.

[0016] Preferably, the cosmetic also includes a cosmetic base; the cosmetic base includes at least one of emulsifiers, moisturizers, solvents, skin conditioning agents, thickeners, solubilizers, fragrances, and preservatives.

[0017] Preferably, the cosmetic is a toner, lotion, serum, cream, face cream, eye cream, or facial mask.

[0018] Preferably, the whitening combination is present in the cosmetic at a concentration of 0.3–20 wt%.

[0019] Preferably, the cosmetic comprises the following components in parts by weight: 0-0.1 parts disodium ethylenediaminetetraacetate (EDTA-2Na), 2-5 parts dipropylene glycol, 0.5-3 parts glycerin, 1-3 parts 1,3-butanediol, 1-2 parts cetearyl oleate, 0-0.5 parts xanthan gum, 0-0.05 parts hydrolyzed sclerotium gum, 0-0.5 parts carbomer, 0.5-1.5 parts cocoyl oleate / caprylate, 0.5-1.5 parts hemisqualane, 0.05-0.2 parts macadamia seed oil, 0.1-0.5 parts cocoa butter, 0-0.1 parts NaOH, 5-15 parts of the whitening composition, and 0.5-1 part phenoxyethanol.

[0020] Thirdly, the present invention provides the application of the whitening composition or the cosmetic in whitening.

[0021] Preferably, the whitening process includes reducing the levels of melanin and hemoglobin in the skin.

[0022] Compared with the prior art, the present invention has the following advantages: (1) This invention uses a combination of glycyrrhizin, Cordyceps militaris fermentation filtrate, marine oligosaccharides and ceramide NS. Through the synergistic effect of melanin metabolism regulation, inflammatory signal interception, microbial dynamic balance and lipid structure enhancement, it can break through the technical paradox of traditional whitening products that stimulate the whitening barrier and repair and lighten pigmentation slowly. It achieves the integrated effect of whitening and maintaining homeostasis, thereby improving the whitening effect of the composition and enabling it to improve skin pigmentation and erythema to a greater extent.

[0023] (2) By controlling the total mass ratio of glycyrrhizin, Cordyceps militaris fermentation filtrate and marine oligosaccharides to ceramide NS at 50-4200:1, and controlling the mass ratio of glycyrrhizin, Cordyceps militaris fermentation filtrate and marine oligosaccharides at 1:50-600:0.5-60, the whitening effect of the composition can be improved to a greater extent, so that it can play a better role in reducing skin melanin content and redness. Attached Figure Description

[0024] Figure 1 The results show the effects of different samples on the release of NO from RAW264.7 macrophages stimulated by LPS.

[0025] Figure 2 The results show the effects of different samples on the expression levels of DSC3 and SPTLC2 genes in cells. Detailed Implementation

[0026] The present invention will be further described below with reference to embodiments.

[0027] General Implementation Examples First, the present invention relates to a skin whitening composition for maintaining skin homeostasis, comprising glycyrrhizin, Cordyceps militaris fermentation filtrate, marine oligosaccharides, and ceramide NS; wherein the marine oligosaccharides are saccharide compounds extracted from Elodea and composed of 2 to 10 monosaccharide units.

[0028] In some specific embodiments, the total mass ratio of glycyrrhizin, Cordyceps militaris fermentation filtrate, and marine oligosaccharides to ceramide NS is 50-4200:1 (more preferably 1100-2000:1); the mass ratio of glycyrrhizin, Cordyceps militaris fermentation filtrate, and marine oligosaccharides is 1:50-600:0.5-60 (more preferably 1:150-400:15-40).

[0029] In some specific embodiments, the whitening composition further includes cholesterol and vegetable oil; the mass ratio of ceramide NS, cholesterol, and vegetable oil is 1:0.5-1.5:0.5-1.5; the vegetable oil includes at least one of palmitic acid, palmitoleic acid, stearic acid, oleic acid, linolenic acid, and α-linolenic acid.

[0030] In some specific embodiments, the whitening composition comprises the following raw materials: glycyrrhizin solution, Cordyceps militaris fermentation filtrate, marine oligosaccharides, and ceramide NS solution; the content of glycyrrhizin in the glycyrrhizin solution is 1-50 wt%; the content of ceramide NS in the ceramide NS solution is 2-40 wt%. Optionally or preferably, the solvent in the glycyrrhizin solution includes 1,3-butanediol and / or water, and the glycyrrhizin solution further contains polyquaternium-51 and / or hydroxypropyl cyclodextrin; the solvent in the ceramide NS solution includes 1,3-butanediol and / or water, and the ceramide NS solution further contains at least one of polyglycerol-10 myristate, glycerol, and cholesterol.

[0031] Second, the present invention relates to a cosmetic product comprising the aforementioned whitening composition.

[0032] In some specific embodiments, the cosmetic also includes a cosmetic matrix; the cosmetic matrix includes at least one of emulsifiers, moisturizers, solvents, skin conditioning agents, thickeners, solubilizers, fragrances, and preservatives.

[0033] In some specific embodiments, the cosmetic is a toner, lotion, serum, cream, face cream, eye cream, or facial mask liquid.

[0034] In some specific embodiments, the whitening combination is present in the cosmetic at a concentration of 0.3–20 wt%.

[0035] In some specific embodiments, the cosmetic comprises the following components in parts by weight: 0-0.1 parts disodium ethylenediaminetetraacetate (EDTA-2Na), 2-5 parts dipropylene glycol, 0.5-3 parts glycerin, 1-3 parts 1,3-butanediol, 1-2 parts cetearyl oleate, 0-0.5 parts xanthan gum, 0-0.05 parts hydrolyzed sclerotium gum, 0-0.5 parts carbomer, 0.5-1.5 parts cocoyl oleate / caprylate, 0.5-1.5 parts hemisqualane, 0.05-0.2 parts macadamia seed oil, 0.1-0.5 parts cocoa butter, 0-0.1 parts NaOH, 5-15 parts of the whitening composition, and 0.5-1 part phenoxyethanol.

[0036] Third, the present invention relates to the application of the whitening composition or the cosmetic in whitening.

[0037] In some specific embodiments, the skin whitening includes reducing the content of melanin and hemoglobin in the skin. Specific Implementation The present invention will now be described through specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention, and the scope of protection of the present invention is defined by the appended claims and any equivalents thereof.

[0039] In the following examples, comparative examples, and test cases, the Cordyceps militaris fermentation filtrate used was purchased from Bloomage Biotechnology Co., Ltd., and its product name is BioyouthTM-FCM Cordyceps militaris fermentation filtrate, which contains two active substances: lactic acid bacteria fermentation products and Cordyceps militaris extract; the marine oligosaccharide was purchased from Qingdao Haida Marine Oligosaccharide Technology Co., Ltd., and its product name is marine oligosaccharide ZnPro-II, which is a sugar compound extracted from Elodea and composed of 2 to 10 monosaccharide units.

[0040] Examples 1-6 and Comparative Examples 1-11: Formulation and preparation methods of whitening compositions and cosmetic emulsions According to the raw material formula in Table 1 (where the amount of each raw material is expressed in parts by weight), the whitening compositions of each example and comparative example were prepared. The preparation method is as follows: the components are thoroughly stirred and mixed at room temperature until a homogeneous and stable emulsion composition is formed.

[0041] Table 1 Whitening Composition Formulation The whitening compositions from Examples 1-6 and Comparative Examples 1-11 were used to prepare corresponding cosmetic emulsions. The formulations of the cosmetic emulsions are shown in Table 2. The preparation methods are as follows: Phase A and Phase B were heated to 85°C and completely dissolved. Phase B was then added to Phase A and homogenized for 2 minutes, followed by Phase C and homogenization for 2 minutes. After stirring and cooling to 40°C, Phase D was added and stirred until homogeneous. Then Phase E was added and stirred until homogeneous.

[0042] Table 2 Cosmetic Emulsion Formulations Test Example 1: Effect of inhibiting melanin production The whitening compositions of Example 1, Comparative Example 10, and Comparative Example 11, along with Vitamin C and glycyrrhizin, were used to test their effects on inhibiting melanin production. The method was as follows: Cells of the same generation in the logarithmic growth phase were selected, routinely digested with trypsin, and then pipetted into single-cell suspensions. Cells were counted and quantitatively seeded into 96-well plates. After 24 hours, the supernatant was discarded, and culture medium containing the experimental materials shown in Table 3 was added. The cells were cultured at 37°C under saturated humidity with 5% CO2 (v / v). RPMI 1640 culture medium was added to the control wells. The cells were cultured for three days, with the culture medium changed once in between. The supernatant was discarded, and after routine trypsin digestion, the cell density was counted. The cell pellet was centrifuged, washed twice with PBS, centrifuged again, and the supernatant was discarded. A quantitative amount of 1N NaOH solution containing 10% DMSO was added, and the cells were incubated at 80°C for 50 minutes. The absorbance was then measured at 405 nm using a microplate reader. The melanin production inhibition rate is obtained by calculating the ratio between the absorbance of each test material and the absorbance of the control group (i.e., absorbance of each test material / absorbance of the control group).

[0043] The test results for the effect of inhibiting melanin production are shown in Table 3.

[0044] Table 3 Experimental design and results of the effect of inhibiting melanin production. The experimental results in Table 3 show that: (1) In the composition system of the present invention, compared with the use of ascorbic acid (experimental group 2), the use of glycyrrhizin (experimental group 1) can show a more significant efficacy advantage in inhibiting melanin production.

[0045] (2) Compared to experimental group 1, experimental group 3 replaced ceramide NS in the composition with ceramide NP, and the inhibition rate of melanin production was significantly lower than that of experimental group 1. This indicates that in the system of the present invention, when ceramide NS is used, the effect of inhibiting melanin production is better than that of ceramide NP.

[0046] (3) The comparative analysis of experimental groups 1, 4 and 5 further confirmed that, compared with single whitening ingredients, the composition of the present invention exhibits a more superior comprehensive effect in inhibiting melanin production.

[0047] Test Example 2: Effect of inhibiting tyrosinase activity In the biosynthesis of melanin in the skin, tyrosinase is a key enzyme that acts on dopa to form dopaquinone, which then spontaneously undergoes a series of reactions to finally form melanin. Therefore, in this test, the whitening compositions of Example 1, Comparative Example 10, and Comparative Example 11, as well as vitamin C and glycyrrhizin, were used to test their inhibitory effects on tyrosinase activity. The test principle is as follows: Tyrosinase catalyzes the conversion of dopa to dopaquinone in a phosphoric acid solution at pH 6.8, and the absorbance can be measured at 475 nm using a spectrophotometer. Raw materials with tyrosinase-inhibiting activity can reduce the conversion of dopa to dopaquinone, thereby lowering the absorbance. Based on the change in absorbance, the inhibitory effect of the analyte on tyrosinase activity is evaluated. The specific testing method is as follows: Tyrosinase was dissolved in phosphate buffer (pH 6.8) to prepare a 120 U / mL tyrosinase solution; L-tyrosine was dissolved in phosphate buffer (pH 6.8) to prepare a 1.6 mol / L L-tyrosine solution; the experimental materials shown in Table 4 were added to phosphate buffer (pH 6.8) to prepare a 0.15 mg / mL test sample solution (i.e., the concentration of each experimental material shown in Table 4 in the test sample solution is 0.15 mg / mL); 0.5 mL of the test sample solution was added to 0.5 mL of tyrosinase solution and 1.5 mL of phosphate buffer (pH 6.8), preheated at 37°C for 15 min, then 0.5 mL of L-tyrosine solution was added and reacted at 37°C for 15 min. The absorbance was measured at 475 nm. Three groups of the same test sample solution were tested, and the average value was taken. The absorbance values ​​of the L-tyrosine, test sample, and tyrosinase system are recorded as D1, the absorbance values ​​of the test sample, tyrosinase, and phosphate buffer system are recorded as D2, the absorbance values ​​of the L-tyrosine, tyrosinase, and phosphate buffer system are recorded as D3, and the absorbance values ​​of the tyrosinase and phosphate buffer system are recorded as D4. The tyrosinase inhibition rate is obtained by calculating 1-(D1-D2) / (D3-D4).

[0048] The results of the test on the effect of inhibiting tyrosinase activity are shown in Table 4.

[0049] Table 4. Experimental design and results of the effect of inhibiting tyrosinase activity. Group Experimental materials Tyrosinase inhibition rate / % Experimental group 1 0.5wt% Example 1 66.83(±0.13) Experimental group 2 0.5wt% Comparative Example 10 54.35(±0.44) Experimental group 3 0.5wt% Comparative Example 11 33.23(±0.04) Experimental group 4 0.001wt% VC 22.47(±0.14) Experimental group 5 0.001wt% glycyrrhizin 47.25(±0.07) The experimental results in Table 4 show that: (1) In terms of inhibiting tyrosinase activity, compared with ascorbic acid (experimental group 2), when glycyrrhizin (experimental group 1) is used, it can form a better complementary and synergistic effect with other raw materials in the composition of the present invention, and inhibit tyrosinase activity to a greater extent.

[0050] (2) The experimental results of experimental groups 1 and 3 show that when ceramide NS (experimental group 1) is used in the composition of the present invention, the inhibitory effect on tyrosinase activity is significantly better than that of experimental group 3 using ceramide NP.

[0051] (3) By comparing the results of experimental groups 1, 4 and 5, it was further confirmed that the composition of the present invention is significantly better than the use of a single whitening ingredient in inhibiting tyrosinase activity.

[0052] Test Example 3: Anti-inflammatory effect The whitening composition of Example 1 and the fermentation filtrate of Cordyceps militaris were taken and their effects on the release of NO from RAW264.7 macrophages stimulated by lipopolysaccharide (LPS) were tested, as follows: (1) Reagent preparation: Preparation of PBS: Weigh 8.00g NaCl, 0.20g KCl, 0.24g anhydrous KH2PO4, and 3.60g Na2HPO4·12H2O, add 1L of ultrapure water, mix well, and then autoclave.

[0053] Preparation of test sample solutions: Under aseptic conditions, test sample solutions were prepared with PBS, and each test sample solution was set to a concentration of 10 μmol / mL.

[0054] Culture medium preparation: Add 1% penicillin and streptomycin to DMEM medium and mix thoroughly. When using, add 10% fetal bovine serum (FBS) to prepare a complete culture medium.

[0055] Preparation of LPS solution: Weigh 1.0 mg LPS and dissolve it in 1 mL PBS. After complete dissolution, the resulting stock solution has a mass concentration of 1.0 mg / mL. Store it at -20℃ for later use. Prepare the solution according to the required mass concentration when needed.

[0056] Preparation of Griss reagent: This solution should be prepared in the dark. Add H3PO4 to 250mL of distilled water at a ratio of 1:40, then add 0.5g of p-aminobenzenesulfonamide and 0.5g of N-1-naphthylethylenediamine hydrochloride respectively. After complete dissolution, store at 4℃.

[0057] Preparation of colorless 1640 culture medium: Add 1% penicillin and streptomycin to the colorless 1640 culture medium and mix well. Add 10% FBS before use to prepare the culture medium.

[0058] (2) Cell culture: RAW264.7 macrophage cells were cultured in DMEM complete medium under the following incubator conditions: 5% CO2, 37°C, and saturated humidity.

[0059] (3) Detection of NO release in RAW264.7 cells: To count the number of cells, RAW264.7 cells were diluted to 5 × 10⁻⁶ cells using prepared colorless 1640 medium. 5 Cell suspension of 160 μL / mL was added to each well and cultured at 37°C until cells were fully adhered. For the sample group, 20 μL of LPS (10 μg / mL) and 20 μL of the test sample solution (10 μg / mL) were added; for the model group, 20 μL of LPS and 20 μL of PBS were added; and for the negative control group, 40 μL of PBS was added. After 48 h of culture at 37°C, 100 μL of the supernatant was added to 50 μL of Griess reagent, and the reaction was allowed to proceed for 10 min. The OD value was measured at 543 nm and substituted into the standard curve to calculate the NO release. The results are shown in […]. Figure 1 .

[0060] from Figure 1 The experimental results show that: When the LPS concentration was 10 μg / mL, the NO release in the model group was significantly higher than that in the negative control group, indicating that the inflammation model was successfully constructed. Compared with the model group, the composition of Example 1 significantly reduced the release of NO from RAW264.7 stimulated by LPS, and the effect was better than that of using Cordyceps militaris fermentation filtrate alone, which shows that the composition of the present invention has a better anti-inflammatory effect.

[0061] Test Example 4: Effects of regulating skin microecological balance The whitening compositions of Example 1 and Comparative Example 11, along with marine oligosaccharides, were used to test their effects on Staphylococcus epidermidis, Malassezia furfur, and Propionibacterium acnes, respectively, using the following methods: (1) Preparation of Oxford cup double-layer plates: Pour the heated and melted sterile culture medium into a sterile petri dish, let it stand until it solidifies, and then vertically and properly place it into a sterilized Oxford cup.

[0062] (2) Preparation of bacterial suspension: Rinse the bacterial slant of the experimental strain with sterile physiological saline, and place the rinsing solution into a sterile test tube; take an appropriate amount of bacterial suspension (the final concentration of bacterial suspension in the culture medium is 10). 6 Add (CFU / mL) to the cooled sterile culture medium, mix well, and prepare a culture medium containing indicator bacteria. Transfer an appropriate amount to a petri dish, avoiding foaming. After complete solidification, use sterile forceps to remove the Oxford cup to obtain the test plate.

[0063] (3) Preparation of test sample solution: Weigh 1g of test sample and add 9g of distilled water. Stir at 55℃ for 5min until uniformly dispersed to prepare a 10% test sample solution.

[0064] (4) Addition of test samples: Use a disposable pipette to draw up the test sample solution cooled to 25°C and add it aseptically into the well of the test plate. Cover the plate and incubate it in a constant temperature incubator for 18 hours for Staphylococcus epidermidis, 48 ​​hours for Malassezia furfur, and 24 hours for Propionibacterium acnes.

[0065] (5) Measurement of inhibition zone diameter: The diameter (including pore size) of the inhibition zone was measured using a vernier caliper or an automated colony analyzer and recorded. When measuring the inhibition zone, a uniform and completely sterile inhibition zone should be selected. The measured diameters of the inhibition zones are shown in Table 5. An inhibition zone diameter greater than 7 mm is considered to have an inhibitory effect; an inhibition zone diameter less than or equal to 7 mm is considered to have no inhibitory effect.

[0066] Table 5. Experimental design and results of the effect of regulating skin microecological balance. The experimental results in Table 5 show that: (1) The compositions of Example 1 and Comparative Example 11, as well as the marine oligosaccharide, effectively inhibited the growth of Malassezia furfur and Propionibacterium acnes, while having no significant inhibitory effect on Staphylococcus epidermidis, demonstrating the characteristic of maintaining the skin microecological balance.

[0067] (2) Compared with the use of marine oligosaccharides alone (experimental group 3), the combination of marine oligosaccharides with glycyrrhizin, Cordyceps militaris fermentation filtrate, ceramides and other ingredients (experimental groups 1 and 2) is more effective in regulating the skin microecological balance and can inhibit the growth of Malassezia furfur and Propionibacterium acnes to a greater extent. The combination using ceramide NS (experimental group 1) is more effective than the combination using ceramide NP (experimental group 2).

[0068] Test Example 5: Effects of Promoting DSC3 and SPTLC2 Gene Expression. The protein encoded by the DSC3 (desmosome core glycoprotein 3) gene is a component of desmosomes. Desmosomes are intercellular junction structures, mainly found between epithelial cells, and play a crucial role in cell adhesion. In the skin, DSC3 helps to tightly connect keratinocytes, enhancing the skin's mechanical stability. When DSC3 expression is normal, keratinocytes can be tightly packed, reducing water loss and maintaining the skin's barrier function.

[0069] The protein encoded by the SPTLC2 (serine palmitoyltransferase long-chain base subunit 2) gene is a subunit of the serine palmitoyltransferase (SPT) complex. The SPT complex (including SPTLC2) plays a central role in the initiation steps of sphingolipid biosynthesis. This complex is primarily responsible for catalyzing the first step of sphingolipid synthesis, converting serine and palmitoyl-CoA into 3-keto-dihydrosphingosine. This reaction is a crucial upstream step in ceramide synthesis. Ceramides belong to the sphingolipid family, so this initiation reaction involving SPTLC2 provides the necessary precursors for ceramide synthesis. When the SPTLC2 gene is normally expressed, sufficient 3-keto-dihydrosphingosine synthesis is ensured, providing ample raw materials for subsequent ceramide production and allowing ceramide synthesis to proceed smoothly.

[0070] Therefore, in this test case, the whitening compositions of Example 1 and Comparative Example 11, as well as ceramide NS and ceramide NP, were used to detect their effects on the expression levels of DSC3 and SPTLC2 genes in cells, respectively. The methods are as follows: (1) HaCat cells in the logarithmic growth phase were divided into groups of 3 × 10⁻⁶ cells. 5 ~5×10 5 Cells were seeded at a density of [number] cells / mL in 6-well plates and cultured for an initial 24 hours. After good cell adhesion, the control group received no additional substances, while four experimental groups were established, each receiving one of the following substances: ceramide NS (0.0311%); ceramide NP (0.0311%); the whitening composition of Example 1 (0.25%); and the whitening composition of Comparative Example 1 (0.25%). The culture medium was changed, and the cells were cultured for another 48 hours before proceeding to the next experimental step.

[0071] (2) After rinsing HaCat cells twice with PBS, add 1 mL of Trizol lysis buffer to each well to lyse the cells. Then add 200 μL of chloroform, mix thoroughly, centrifuge to obtain the supernatant, and transfer it to a 1.5 mL centrifuge tube. Add an equal volume of isopropanol, mix, and centrifuge again. A trace amount of RNA precipitate will be visible at the bottom of the tube. Carefully discard the supernatant. Add 1 mL of freshly prepared 75% anhydrous ethanol to each tube, gently invert to wash the precipitate, centrifuge, discard the supernatant, add 1 mL of anhydrous ethanol again, centrifuge, and discard the supernatant. Invert the centrifuge tubes to dry on absorbent paper. Add DEPC water (approximately 10–25 μL) to dissolve the RNA precipitate, depending on the amount of white precipitate. Detect the RNA concentration using a Nano Drop instrument. Once the concentration and purity meet the standard, proceed to the next step.

[0072] (3) The HiScript III RT SuperMix kit was used for reverse transcription, with the mRNA as a template, and the reverse transcriptase was used to reverse transcribe it into cDNA; the SYBR qPCR Master Mix kit was used for RT-qPCR, with the cDNA as a template and primers combined for PCR amplification, and the expression of the target gene was obtained.

[0073] (4) GAPDH was selected as the internal reference gene for control. The relative expression levels of the target gene in each treatment group were calculated using the 2-ΔΔCt method to obtain quantitative results. The detection results are shown in […]. Figure 2 .

[0074] from Figure 2 The experimental results show that: When the four samples (the whitening compositions of Example 1 and Comparative Example 11, as well as ceramides NS and ceramides NP) were applied to HaCat cells, the expression of DSC3 and SPTLC2 increased. Among them, the expression levels of DSC3 and SPTLC2 were increased more significantly when using the composition of Example 1 compared with the use of ceramides NS and ceramides NP alone, indicating that the composition of Example 1 has a strong effect on moisturizing and promoting ceramide synthesis, and can achieve better skin barrier repair function. Compared with the composition of Comparative Example 1, the effect of increasing the expression levels of DSC3 and SPTLC2 was better when using the sample of Example 1, indicating that the ceramide NS used in this invention can enhance the skin barrier repair function of the composition.

[0075] Test Example 6: Safety Test of Cosmetic Lotions In accordance with the relevant requirements of the "Cosmetic Safety Technical Specifications" (2015 edition), patch safety tests were conducted on the cosmetic emulsions of Examples 1-6 and Comparative Examples 1-11. Specifically, 30 healthy volunteers were selected, and the cosmetic compositions of this invention were applied to filter paper pads (20 μL / pad) and placed on healthy skin areas of the back for 48 hours. Skin reactions were observed at 30 minutes, 24 hours, and 48 hours after removal, and scored according to the ICDRG standard (0-4 points): 0 points for no reaction, 1 point for mild erythema, 2 points for moderate erythema (with or without edema), 3 points for severe erythema (with edema or papules), and 4 points for severe erythema (with vesicles or erosions). A reaction score of 2 or higher at any time point was considered positive; no positive reaction was considered a pass. The positive reaction rate was statistically analyzed, and inter-group comparisons were performed to verify the skin safety of each cosmetic emulsion.

[0076] Test results: No positive reactions were observed in any of the subjects, indicating that the cosmetic lotions in each example and comparative example posed no safety risk.

[0077] Test Example 7: Whitening Effect Seventeen volunteers were randomly selected from each group (17 groups in total). Each group applied the cosmetic lotion prepared in Examples 1-6 and Comparative Examples 1-11 evenly to their cheeks daily. The lotion was used twice daily, once in the morning and once in the evening, with 2 mL applied each time, for 30 days. A cosmetic lotion without the added whitening composition was used as a control group. The following tests were performed on the volunteers in each group: (1) Melanin Index (MI) test: The melanin content (MI value) of each group of volunteers was measured using the MX18 skin melanin and hemoglobin tester from Guangzhou Walker Trading Co., Ltd. The MI value is a quantitative indicator of the melanin content in the skin. The higher the MI value, the higher the melanin content.

[0078] (2) Erythema Index (EI) test: The skin heme and heme tester with product number MX18 from Guangzhou Walker Trading Co., Ltd. was used to test the skin heme content (EI value) of each group of volunteers. EI is a quantitative indicator that reflects the erythema on the skin surface (caused by inflammation, vasodilation or sensitivity reaction). The higher the measured EI value, the higher the heme content.

[0079] The average test results of each group of volunteers are recorded in Tables 6 and 7.

[0080] Table 6 MI Value Detection Results Table 7 EI value detection results The experimental results in Tables 6 and 7 show that: (1) Compared with Comparative Examples 3-4, Examples 1-4 showed a greater reduction in MI and EI values, indicating that glycyrrhizin, Cordyceps militaris fermentation filtrate and marine oligosaccharides can produce a synergistic effect with ceramide NS in improving melanin deposition and redness reduction.

[0081] (2) Compared with Examples 1-4, the reduction rates of MI and EI values ​​in Comparative Examples 1 and 2 were significantly smaller, even lower than those in Comparative Examples 3 and 4. This indicates that the ratio of the total mass of glycyrrhizin, Cordyceps militaris fermentation filtrate, and marine oligosaccharides to the mass of ceramide NS affects their synergistic effect. When this ratio is controlled within the range of 50-4200:1, a better synergistic effect can be achieved in improving melanin deposition and fading redness. In Examples 1-4, the reduction rates of MI values ​​in Examples 2 and 3 were lower than those in Examples 1 and 4, indicating that when the ratio of the total mass of glycyrrhizin, Cordyceps militaris fermentation filtrate, and marine oligosaccharides to the mass of ceramide NS is controlled within the range of 1100-2000:1, melanin deposition can be improved to a greater extent.

[0082] (3) Compared with comparative examples 7-9, the reduction rates of MI and EI values ​​in Examples 1, 5 and 6 were greater, indicating that glycyrrhizin, Cordyceps militaris fermentation filtrate and marine oligosaccharides could produce a synergistic effect in improving melanin deposition and redness reduction.

[0083] (4) Compared with Comparative Examples 5 and 6, Examples 1, 5 and 6 showed a larger reduction rate in MI value and a larger reduction rate in EI value at 30 days, indicating that the ratio between glycyrrhizin, Cordyceps militaris fermentation filtrate and marine oligosaccharides affects their synergistic effect. When the mass ratio of the three is 1:50-600:0.5-60, a better whitening effect can be achieved.

[0084] (5) In Examples 1 and 10, glycyrrhizin and ascorbic acid were used in the whitening compositions, respectively. After 30 days of use, the reduction rate of MI and EI values ​​of the cosmetic emulsions in Example 1 was significantly higher than that in Comparative Example 10. This phenomenon indicates that, in the whitening composition system of the present invention, the use of glycyrrhizin can achieve a better whitening effect than ascorbic acid.

[0085] (6) The comparison results between Example 1 and Comparative Example 11 show that when ceramide NS in the whitening composition is replaced with ceramide NP, the effect of improving melanin deposition and redness reduction is significantly reduced. This phenomenon indicates that ceramide NS can give the composition of the present invention a better whitening effect than ceramide NP.

[0086] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Unless otherwise specified, the raw materials and equipment used in this invention are conventional in the art and can be obtained through conventional commercial means; unless otherwise specified, the methods used in this invention are conventional methods in the art.

[0087] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A skin-whitening composition for maintaining skin homeostasis, characterized in that, It includes glycyrrhizin, Cordyceps militaris fermentation filtrate, marine oligosaccharides, and ceramide NS; the marine oligosaccharides are carbohydrate compounds extracted from Elodea and composed of 2 to 10 monosaccharide units.

2. The whitening composition according to claim 1, characterized in that, The total mass ratio of glycyrrhizin, Cordyceps militaris fermentation filtrate, and marine oligosaccharides to ceramide NS is 50~4200:1; the mass ratio of glycyrrhizin, Cordyceps militaris fermentation filtrate, and marine oligosaccharides is 1:50~600:0.5~60.

3. The whitening composition according to claim 2, characterized in that, The total mass ratio of glycyrrhizin, Cordyceps militaris fermentation filtrate, and marine oligosaccharides to ceramide NS is 1100~2000:1; the mass ratio of glycyrrhizin, Cordyceps militaris fermentation filtrate, and marine oligosaccharides is 1:150~400:15~40.

4. The whitening composition according to claim 1, characterized in that, It also includes cholesterol and vegetable oil; the mass ratio of ceramide NS, cholesterol and vegetable oil is 1:0.5~1.5:0.5~1.5; the vegetable oil includes at least one of palmitic acid, palmitoleic acid, stearic acid, oleic acid, linolenic acid and α-linolenic acid.

5. The whitening composition according to any one of claims 1 to 3, characterized in that, The product includes the following raw materials: glycyrrhizin solution, Cordyceps militaris fermentation filtrate, marine oligosaccharides, and ceramide NS solution; the glycyrrhizin content in the glycyrrhizin solution is 1~50 wt%; and the ceramide NS content in the ceramide NS solution is 2~40 wt%.

6. A cosmetic product, characterized in that, Including the whitening composition according to any one of claims 1 to 5.

7. The cosmetic product according to claim 6, characterized in that: The cosmetic also includes a cosmetic base, which comprises at least one of emulsifiers, moisturizers, solvents, skin conditioning agents, thickeners, solubilizers, fragrances, and preservatives; and / or The cosmetics mentioned are toners, lotions, serums, creams, face creams, eye creams, or facial masks.

8. The cosmetic product according to claim 6 or 7, characterized in that, The whitening combination is present in the cosmetic at a concentration of 0.3-20 wt%.

9. The cosmetic product according to claim 6, characterized in that, The product comprises the following components in parts by weight: 0-0.1 parts disodium EDTA, 2-5 parts dipropylene glycol, 0.5-3 parts glycerin, 1-3 parts 1,3-butanediol, 1-2 parts cetearyl oleate, 0-0.5 parts xanthan gum, 0-0.05 parts hydrolyzed sclerotium gum, 0-0.5 parts carbomer, 0.5-1.5 parts cocoyl oleate / caprylate, 0.5-1.5 parts hemisqualane, 0.05-0.2 parts macadamia seed oil, 0.1-0.5 parts cocoa butter, 0-0.1 parts NaOH, 5-15 parts of the whitening composition, and 0.5-1 part phenoxyethanol.

10. The use of the whitening composition according to any one of claims 1 to 5 or the cosmetic according to any one of claims 6 to 9 in whitening.

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