A water-in-oil nanoemulsion mask that can whiten and remove blemishes

Abstract

This invention discloses a water-in-oil nanoemulsion prepared by combining extracts of fragrant lotus and dodder seed with a synthesized polypeptide (Pro-Ser-Gly)2(Asn-Glu-Gly)2(Pro-Ser-Gly)2. This water-in-oil nanoemulsion primarily utilizes the abundant polysaccharides in fragrant lotus, which can form a thin film on the skin, preventing moisture evaporation. The fragrant lotus, dodder seed extract, and polypeptide work synergistically to effectively inhibit 5α-reductase activity, thereby inhibiting acne formation and achieving an acne-removing effect. The prepared mask not only has whitening and spot-removing effects but also significantly improves skin hydration. This has significant implications for dermatological research on whitening and moisturizing.

Description

Technical Field

[0001] This invention belongs to the field of dermatology, and more specifically, it relates to a water-in-oil nanoemulsion that can whiten and remove blemishes. In particular, it relates to the addition of a water-in-oil nanoemulsion prepared from extracts of fragrant lotus and dodder seed to a facial mask liquid, allowing for better absorption by the skin. Background Technology

[0002] With the healthy and sustainable development of the economy and society, people's quality of life has gradually improved, and domestic consumers' awareness of skincare has gradually increased. As a result, the consumption of face masks has continued to grow, with regional popularity rising steadily, and young consumers gradually becoming the main force in face mask consumption. The mechanism of action of face masks can be mainly divided into three points: First, when a face mask is applied to the face, it blocks the contact between the facial skin and the air, preventing excessive evaporation of facial moisture, retaining the nutrients of the facial skin, and maintaining elasticity. Second, depending on the specific function, the various nutrients contained in the face mask, including small molecules, can penetrate the skin during the application process, thereby promoting the metabolism of facial epithelial cells and achieving certain beauty effects. Third, after using the face mask, it removes dust, metabolic products, and even residual makeup that were previously attached to the skin surface, achieving a cleansing effect. Therefore, by using face masks, our facial skin can receive sufficient care and reasonable improvement, enhancing skin elasticity and improving wrinkles, dullness, and other phenomena.

[0003] Many types of face masks are currently popular on the market, with functional face masks becoming a new favorite. Functional face masks target different skin problems; one mask is essentially a solution for one skin issue. Characterized by their effectiveness, they can quickly, safely, and effectively address various skin concerns for different women. Functional face masks can be understood as a natural, safe, and professional type of mask, suitable for different skin types. They are generally safe and unlikely to cause allergic reactions, attracting considerable attention. Due to their skin type and tone, Asians tend to choose masks with whitening effects. Whitening masks contain ingredients that promote melanin metabolism, inhibit and destroy melanin production, prevent tyrosinase activation, or reduce melanin intermediates. Whitening ingredients must effectively penetrate the basal layer of the skin to be effective; ideally, the product should also contain ingredients that promote cell metabolism. Currently, there are various skin whitening masks available, such as those containing the chemical hydroquinone, and natural plant extracts like arbutin and licorice extract. While pure chemical whitening masks are more effective, they often come with side effects. Natural plant extracts, on the other hand, are increasingly favored by researchers and consumers due to their high safety profile.

[0004] In order to prepare a whitening and moisturizing facial mask containing natural extracts, this invention prepares a water-in-oil nanoemulsion containing fragrant lotus (polysaccharide) and dodder seed extract (polysaccharide and flavonoids). This nanoemulsion is added to the facial mask liquid, and the synergistic effect between the fragrant lotus extract and the dodder seed extract is utilized to make it better absorbed by the skin. Summary of the Invention

[0005] Whitening cosmetics made from natural plants and traditional Chinese medicine are favored by consumers for their gentleness and high safety. Eastern women have always admired fair skin, while Europeans and Americans, though not pursuing whitening, are keen on removing various blemishes. The factors determining skin color are pigments such as melanin, melanoidins, and carotene, as well as epidermal thickness, subcutaneous blood vessels, and light scattering. Melanin is the key factor determining skin color; when melanin increases, the skin changes from light brown to black. The mechanism of melanin formation: tyrosine in the body is oxidized by tyrosinase to become dopa, then oxidized by tyrosine oxidase to dopaquinone, further oxidized to 5,6-dihydroxyazine, and finally polymerized to form melanin. Currently, whitening cosmetics made from natural plants and traditional Chinese medicine mainly achieve whitening and blemish-removing effects by forming a thin film on the skin, preventing moisture evaporation, reducing melanin content, directly whitening, providing antioxidant protection for skin tone, and inhibiting the proliferation of melanocytes.

[0006] To inhibit the production of melanin in the human body and lock in skin moisture, this invention provides the following technical solution:

[0007] S1. Preparation of Perfume Lotus Extract: Dried perfume lotus petals were collected, and after removing insect-damaged and moldy parts, they were pulverized and passed through a 50-mesh sieve. The petals were then ultrasonically treated with distilled water and acetone solutions of 20%, 40%, 60%, 80%, and 100% (v / v) at a mass-to-volume ratio of 1 g:20 mL at 25°C for 60 min. The resulting solutions were centrifuged at 9000 r / min for 10 min, and the supernatant was filtered. This centrifugation process was repeated three times. Acetone was then removed by rotary evaporation under reduced pressure, and the mixture was freeze-dried for 24 h to obtain perfume lotus petal extract powder (polysaccharide). For analysis, this powder was reconstituted with dodder seed extract via ultrasonication to prepare a solution of the desired concentration.

[0008] S2. Preparation of Cuscuta Seed Extract: Weigh 10g of cleaned Cuscuta seed material, place it in a mortar, crush all the Cuscuta seeds, and then pack it into a bag for later use. Weigh 5g of the crushed Cuscuta seeds, place them in a crucible, and heat them with an alcohol lamp until the surface turns slightly yellow and a popping sound is heard. Remove and let cool, then seal and store for later use. Weigh 1g of the Cuscuta seeds, add 100mL of 60% ethanol, and extract twice by reflux in a water bath for 60min each time. Filter, concentrate, and evaporate to dryness to obtain Cuscuta seed extract (polysaccharides and flavonoids). For determination, mix it with water lily extract by ultrasonic reconstitution to prepare a solution of the required concentration for later use.

[0009] S3. Synthesis of protein peptides: A condensation reaction is performed after activating Fmoc-amino acids with a 4-fold excess of activator (HBTU) and a 6-fold excess of N,N-diisopropylethylamine. After each condensation reaction, the peptides are washed three times each with N,N-dimethylformamide (DMF) and dichloromethane (DCM). Then, the Fmoc protecting group is removed using 20% ​​piperidine (DMF). The condensation reaction and deprotection steps are repeated until the target peptide sequence (Pro-Ser-Gly)2 (Asn-Glu-Gly)2 (Pro-Ser-Gly)2 is synthesized. After the synthesis of n-Glu-Gly)2 (Pro-Ser-Gly)2, the N-terminus was capped with 25% acetic anhydride (DMF) solution for 20 minutes. The peptide was then cleaved from the resin using a cleavage buffer ratio of TFA:TIS:H2O = 90:3:7 for 6 hours. The peptide was then precipitated with anhydrous diethyl ether and centrifuged, discarding the supernatant and retaining the precipitate. The precipitate was washed three times with ether to obtain the target crude peptide. The crude peptide was then purified using a C18 reversed-phase high-performance liquid chromatography column, and the target product was confirmed by mass spectrometry, finally yielding peptide (Pro-Ser-Gly)2 (Asn-Glu-Gly)2 (Pro-Ser-Gly)2.

[0010] S4. Establishment of a dehydrated melanoma mouse model: The mice used in this invention were purchased from the Animal Experiment Center of Southern Medical University (animal experiment license number gdpulac2015595). The mice weighed approximately 20±2g. Before the experiment, all mice were acclimatized for one week, ensuring free access to food. All mice were placed in a dry environment and irradiated with ultraviolet light of varying intensities to enhance melanoma production in the mice.

[0011] S5. Reducing melanin in mice: A sample solution was prepared by weighing 0.3g of *Liriope muscari* extract, 0.3g of *Cuscuta chinensis* extract, 5ml of hypophosphite buffer (pH=7), and 20ml of deionized water. The solution was then extracted and emulsified. The emulsion product was combined with the polypeptide synthesized in S3 to prepare a water-in-oil nanoemulsion. Cells were taken from the skin surface of mice with dehydrated melanoma created in S4 and placed in a culture dish. After 24 hours of initial cell culture, the diluted water-in-oil nanoemulsion was added, and the cells were cultured further. The melanin content within the cells was measured periodically.

[0012] S6. Locking in moisture on mouse skin surface: A sample solution was prepared by weighing 0.3g of *Liriope muscari* extract, 0.3g of *Cuscuta chinensis* extract, 5ml of hypophosphite buffer (pH=7), and 20ml of deionized water. The solution was then extracted and emulsified. The emulsion product was combined with the peptide synthesized in S3 to prepare a water-in-oil nanoemulsion. Cells were taken from the skin surface of mice with dehydrated melanoma created in S4 and placed in a culture dish. After 24 hours of initial cell culture, the diluted water-in-oil nanoemulsion was added, and the cells were cultured further. The cell moisture retention rate was measured periodically.

[0013] Preferably, the fragrant lotus used in this invention is the fragrant lotus that is available in Zhejiang Province all year round.

[0014] Preferably, the dodder seeds used in this invention are purchased from a store.

[0015] Preferably, in S3 of the present invention, ultraviolet irradiation is used to induce melanoma transformation in mice, which is more closely related to the melanin produced in the human body by ultraviolet irradiation.

[0016] Preferably, in step S4 of the present invention, 0.3g of fragrant lotus extract and 0.3g of dodder extract are weighed and mixed with 1ml of phosphate-phosphophosphate buffer to prepare a sample solution. Attached Figure Description

[0017] Figure 1 The bar chart shows the melanin content of epidermal cells of mutagenized mice obtained in Example 1 and Comparative Examples 1-3 of this invention.

[0018] Figure 2 The bar chart shows the moisture retention rate of mouse epidermal cells obtained in Example 2 and Comparative Examples 4 and 5 of this invention.

[0019] Figure 3 The bar chart shows the moisture retention rate of mouse epidermal cells obtained in Example 3 and Comparative Examples 6 and 7 of this invention.

[0020] Figure 4 The graphs show the moisture content of Embodiment 4 and Comparative Examples 8 and 9 of this invention.

[0021] Figure 5 The molecular structure of the polypeptide (Pro-Ser-Gly)2 (Asn-Glu-Gly)2 (Pro-Ser-Gly)2 synthesized in this invention is shown below.

[0022] Figure 6 The hydrogen spectrum of the polypeptide (Pro-Ser-Gly)2 (Asn-Glu-Gly)2 (Pro-Ser-Gly)2 synthesized in this invention.

[0023] Figure 7 The carbon spectrum of the polypeptide (Pro-Ser-Gly)2 (Asn-Glu-Gly)2 (Pro-Ser-Gly)2 synthesized in this invention.

[0024] Beneficial effects:

[0025] 1. Fragrant lotus contains a lot of polysaccharides, which can form a thin film on the skin to prevent moisture evaporation.

[0026] 2. The synergistic effect of extracts from fragrant lotus and dodder seed can effectively inhibit the activity of tyrosinase, thereby inhibiting the production of melanin and achieving the effect of whitening and removing blemishes.

[0027] 3. The polypeptides synthesized in this invention are protein polypeptides, most of which are synthesized using hydrophobic amino acids as small molecule amino acids, and the repetitive sequences enhance the water-locking function of the polypeptides.

[0028] 4. The synergistic effect of extracts from fragrant lotus and dodder seed can effectively inhibit 5α-reductase activity, thereby inhibiting acne formation and achieving the effect of acne removal.

[0029] 5. By combining natural plant-based protein peptides, the synthesized water-in-oil nanoemulsion adds a water-locking function to the whitening and acne-removing ingredients. Detailed Implementation

[0030] Melanin is a key factor determining skin color; when melanin increases, the skin changes from light brown to black. The mechanism of melanin formation involves the oxidation of tyrosine in the body by tyrosinase to dopa, followed by oxidation by tyrosine oxidase to dopaquinone, then further oxidized to 5,6-dihydroxyazine, and finally polymerized into melanin. Currently, whitening cosmetics made from natural plants and traditional Chinese medicine primarily achieve whitening and spot-removing effects by promoting blood circulation to improve skin tone, reducing melanin content for direct whitening, providing antioxidant protection, and inhibiting the proliferation of melanocytes. The following specific examples illustrate the implementation of this invention. Those skilled in the art can easily understand other advantages and effects of this invention from the content disclosed in this specification. This invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of this invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other.

[0031] Example 1

[0032] S1. Preparation of Perfume Lotus Extract: Dried perfume lotus petals were collected, and after removing insect-damaged and moldy parts, they were pulverized and passed through a 50-mesh sieve. The petals were then ultrasonically treated with distilled water and acetone solutions of 20%, 40%, 60%, 80%, and 100% (v / v) at a mass-to-volume ratio of 1 g:20 mL at 25°C for 60 min. The resulting solutions were centrifuged at 9000 r / min for 10 min, and the supernatant was filtered. This centrifugation process was repeated three times. Acetone was then removed by rotary evaporation under reduced pressure, and the mixture was freeze-dried for 24 h to obtain perfume lotus petal extract powder (polysaccharide). For analysis, this powder was reconstituted with dodder seed extract via ultrasonication to prepare a solution of the desired concentration.

[0033] S2. Preparation of Cuscuta Seed Extract: Weigh 10g of cleaned Cuscuta seed material, place it in a mortar, crush all the Cuscuta seeds, and then pack it into a bag for later use. Weigh 5g of the crushed Cuscuta seeds, place them in a crucible, and heat them with an alcohol lamp until the surface turns slightly yellow and a popping sound is heard. Remove and let cool, then seal and store for later use. Weigh 1g of the Cuscuta seeds, add 100mL of 60% ethanol, and extract twice by reflux in a water bath for 60min each time. Filter, concentrate, and evaporate to dryness to obtain Cuscuta seed extract (polysaccharides and flavonoids). For determination, mix it with water lily extract by ultrasonic reconstitution to prepare a solution of the required concentration for later use.

[0034] S3. Synthesis of protein peptides: A condensation reaction is performed after activating Fmoc-amino acids with a 4-fold excess of activator (HBTU) and a 6-fold excess of N,N-diisopropylethylamine. After each condensation reaction, the peptides are washed three times each with N,N-dimethylformamide (DMF) and dichloromethane (DCM). Then, the Fmoc protecting group is removed using 20% ​​piperidine (DMF). The condensation reaction and deprotection steps are repeated until the target peptide sequence (Pro-Ser-Gly)2 (Asn-Glu-Gly)2 (Pro-Ser-Gly)2 is synthesized. After the synthesis of n-Glu-Gly)2 (Pro-Ser-Gly)2, the N-terminus was capped with 25% acetic anhydride (DMF) solution for 20 minutes. The peptide was then cleaved from the resin using a cleavage buffer ratio of TFA:TIS:H2O = 90:3:7 for 6 hours. The peptide was then precipitated with anhydrous diethyl ether and centrifuged, discarding the supernatant and retaining the precipitate. The precipitate was washed three times with ether to obtain the target crude peptide. The crude peptide was then purified using a C18 reversed-phase high-performance liquid chromatography column, and the target product was confirmed by mass spectrometry, finally yielding peptide (Pro-Ser-Gly)2 (Asn-Glu-Gly)2 (Pro-Ser-Gly)2.

[0035] S4. Establishment of a dehydrated melanoma mouse model: The mice used in this invention were purchased from the Animal Experiment Center of Southern Medical University (animal experiment license number gdpulac2015595). The mice weighed approximately 20±2g. Before the experiment, all mice were acclimatized for one week, ensuring free access to food. All mice were placed in a dry environment and irradiated with ultraviolet light of varying intensities to enhance melanoma production in the mice.

[0036] S5. Reducing melanin in mice: A sample solution was prepared by weighing 0.3g of *Liriope muscari* extract, 0.3g of *Cuscuta chinensis* extract, 5ml of hypophosphite buffer (pH=7), and 20ml of deionized water. The solution was then extracted and emulsified. The emulsion product was combined with the polypeptide synthesized in S3 to prepare a water-in-oil nanoemulsion. Cells were taken from the skin surface of mice with dehydrated melanoma created in S4 and placed in a culture dish. After 24 hours of initial cell culture, the diluted water-in-oil nanoemulsion was added, and the cells were cultured further. The melanin content within the cells was measured periodically.

[0037] S6. Culture the cells at 25℃ under 5% CO2 saturated humidity for 24 h. Discard the culture medium, wash once with hypophosphite buffer, digest with 0.25% trypsin, and then pipette the precipitated cells with hypophosphite buffer for later use. Take the prepared cell suspension, add hypophosphite buffer, and place 1 mL of the cell suspension in separate centrifuge tubes. Centrifuge at low speed for 15 min, discard the supernatant, add 2 mL of distilled water to resuspend the cells, then add 1 mL of V(ethanol):V(ether) = 1:1. Incubate at room temperature for 15 min, centrifuge at 9000 r / min for 5 min, discard the supernatant, add 1 mL of 1 mol / L NaOH solution containing 10% dimethyl sulfoxide, then incubate at 80℃ for 30 min. Finally, transfer to a 96-well plate and measure the absorbance at 470 nm using a spectrophotometer. The relative melanin mass fraction in a single cell = (A experimental group / A control group) / cell proliferation rate × 100%.

[0038] Comparative Example 1: Except for replacing the sample solution prepared in step S5 with pure ordinary culture medium, which consisted of 0.3g of fragrant lotus extract, 0.3g of dodder extract, 5ml of hypophosphite buffer (pH=7), and 20ml of deionized water, all other steps were the same as in Example 1.

[0039] Comparative Example 2 was identical to Example 1 except that the 0.3g of fragrant lotus extract in step S5 was removed.

[0040] Comparative Example 3 was identical to Example 1 except that the 0.3g of Cuscuta extract in step S5 was removed.

[0041] Table 1

[0042] Testing items Comparative Example 1 Comparative Example 2 Comparative Example 3 Implementation Column 1 Melanin content (%) 82±0.1 35±0.2 43±0.2 12±0.01

[0043] Figure 1 Table 1 shows the bar charts of melanin content in epidermal cells of mutagenized mice obtained in Example 1 and Comparative Examples 1-3 of this invention. Table 1 is a statistical table of the melanin content in epidermal cells of mutagenized mice measured five times in Example 1 and Comparative Examples 1-3 of this invention. Figure 1 As shown in Table 1, the melanin content in the mice in Example 1 was significantly lower than that in Comparative Examples 1-3. This result indicates that adding water-in-oil nanoemulsions prepared from extracts of *Liriope muscari* and *Cuscuta chinensis* to cell culture medium can effectively inhibit melanin production.

[0044] Example 2

[0045] S1. Preparation of Perfume Lotus Extract: Dried perfume lotus petals were collected, and after removing insect-damaged and moldy parts, they were pulverized and passed through a 50-mesh sieve. The petals were then ultrasonically treated with distilled water and acetone solutions of 20%, 40%, 60%, 80%, and 100% (v / v) at a mass-to-volume ratio of 1 g:20 mL at 25°C for 60 min. The resulting solutions were centrifuged at 9000 r / min for 10 min, and the supernatant was filtered. This centrifugation process was repeated three times. Acetone was then removed by rotary evaporation under reduced pressure, and the mixture was freeze-dried for 24 h to obtain perfume lotus petal extract powder. For testing, this powder was reconstituted with dodder seed extract via ultrasonication to prepare a solution of the desired concentration.

[0046] S2. Preparation of Cuscuta Seed Extract: Weigh 10g of cleaned Cuscuta seed material, place it in a mortar, crush all the Cuscuta seeds, and then pack it into a bag for later use. Weigh 5g of the crushed Cuscuta seeds, place them in a crucible, and heat them with an alcohol lamp until the surface turns slightly yellow and a popping sound is heard. Remove and let cool, then seal and store for later use. Weigh 1g of the Cuscuta seeds, add 100mL of 60% ethanol, and extract twice by reflux in a water bath for 60min each time. Filter, concentrate, and evaporate to dryness to obtain the Cuscuta seed extract. For determination, mix it with the fragrant lotus extract by ultrasonic redissolve to prepare a solution of the required concentration for later use.

[0047] S3. Synthesis of protein peptides: A condensation reaction is performed after activating Fmoc-amino acids with a 4-fold excess of activator (HBTU) and a 6-fold excess of N,N-diisopropylethylamine. After each condensation reaction, the peptides are washed three times each with N,N-dimethylformamide (DMF) and dichloromethane (DCM). Then, the Fmoc protecting group is removed using 20% ​​piperidine (DMF). The condensation reaction and deprotection steps are repeated until the target peptide sequence (Pro-Ser-Gly)2 (Asn-Glu-Gly)2 (Pro-Ser-Gly)2 is synthesized. After the synthesis of n-Glu-Gly)2 (Pro-Ser-Gly)2, the N-terminus was capped with 25% acetic anhydride (DMF) solution for 20 minutes. The peptide was then cleaved from the resin using a cleavage buffer ratio of TFA:TIS:H2O = 90:3:7 for 6 hours. The peptide was then precipitated with anhydrous diethyl ether and centrifuged, discarding the supernatant and retaining the precipitate. The precipitate was washed three times with ether to obtain the target crude peptide. The crude peptide was then purified using a C18 reversed-phase high-performance liquid chromatography column, and the target product was confirmed by mass spectrometry, finally yielding peptide (Pro-Ser-Gly)2 (Asn-Glu-Gly)2 (Pro-Ser-Gly)2.

[0048] S4. Establishment of a dehydrated melanoma mouse model: The mice used in this invention were purchased from the Animal Experiment Center of Southern Medical University (animal experiment license number gdpulac2015595). The mice weighed approximately 20±2g. Before the experiment, all mice were acclimatized for one week, ensuring free access to food. All mice were placed in a dry environment and irradiated with ultraviolet light of varying intensities to enhance melanoma production in the mice.

[0049] S5. Locking in moisture on mouse skin surface: A sample solution was prepared by weighing 0.3g of *Liriope muscari* extract, 0.3g of *Cuscuta chinensis* extract, 5ml of hypophosphite buffer (pH=7), and 20ml of deionized water. The solution was then extracted and emulsified. The emulsion product was combined with the peptide synthesized in S3 to prepare a water-in-oil nanoemulsion. Cells were taken from the skin surface of mice with dehydrated melanoma created in S4 and placed in a culture dish. After 24 hours of initial cell culture, the diluted water-in-oil nanoemulsion was added, and the cells were cultured further. The cell moisture retention rate was measured periodically.

[0050] S6. Incubate cells at 25℃ and 5% CO2 saturated humidity for 24 h. Discard the culture medium, wash once with hypophosphite buffer, digest with 0.25% trypsin, and then agitate the cells with hypophosphite buffer for further processing. Take the prepared cell suspension, add hypophosphite buffer, 10 ml of deionized water, and 3 ml of diluted water-in-oil nanoemulsion, and place in a desiccator. Weigh the samples at intervals, repeating the experiment three times. Calculate the moisture retention rate using the following formula: Moisture retention rate = Hn / Ho × 100% (where Ho is the water content of the sample before storage, and Hn is the water content of the sample after storage).

[0051] Comparative Example 4: Except for the addition of 0.2g of perfume extract in step S5 when preparing water-in-oil nanoemulsion, all other steps were the same as in Example 2.

[0052] Comparative Example 5: Except for the addition of 0.4g of perfume extract in step S5 when preparing water-in-oil nanoemulsion, all other steps were the same as in Example 2.

[0053] Table 2

[0054] Testing items Comparative Example 4 Comparative Example 5 Example 2 Moisturizing rate (%) 75±0.01 80±0.01 92±0.01

[0055] Figure 2 Table 2 shows the bar charts of the moisture retention rate of mouse epidermal cells obtained in Example 2 and Comparative Examples 4 and 5 of this invention. Table 2 is a statistical table of the moisture retention rate of epidermal cells from mutagenized mice measured five times in Example 2 and Comparative Examples 4 and 5 of this invention. Figure 2 As shown in Table 2, the moisture retention rate of mouse epidermal cells in Example 2 was higher than that in Comparative Examples 4 and 5. This result indicates that the two appropriate amounts of fragrant lotus extract provide better moisturizing effects.

[0056] Example 3

[0057] S1. Preparation of Perfume Lotus Extract: Dried perfume lotus petals were collected, and after removing insect-damaged and moldy parts, they were pulverized and passed through a 50-mesh sieve. The petals were then ultrasonically treated with distilled water and acetone solutions of 20%, 40%, 60%, 80%, and 100% (v / v) at a mass-to-volume ratio of 1 g:20 mL at 25°C for 60 min. The resulting solutions were centrifuged at 9000 r / min for 10 min, and the supernatant was filtered. This centrifugation process was repeated three times. Acetone was then removed by rotary evaporation under reduced pressure, and the mixture was freeze-dried for 24 h to obtain perfume lotus petal extract powder. For testing, this powder was reconstituted with dodder seed extract via ultrasonication to prepare a solution of the desired concentration.

[0058] S2. Preparation of Cuscuta Seed Extract: Weigh 10g of cleaned Cuscuta seed material, place it in a mortar, crush all the Cuscuta seeds, and then pack it into a bag for later use. Weigh 5g of the crushed Cuscuta seeds, place them in a crucible, and heat them with an alcohol lamp until the surface turns slightly yellow and a popping sound is heard. Remove and let cool, then seal and store for later use. Weigh 1g of the Cuscuta seeds, add 100mL of 60% ethanol, and extract twice by reflux in a water bath for 60min each time. Filter, concentrate, and evaporate to dryness to obtain the Cuscuta seed extract. For determination, mix it with the fragrant lotus extract by ultrasonic redissolve to prepare a solution of the required concentration for later use.

[0059] S3. Synthesis of protein peptides: A condensation reaction is performed after activating Fmoc-amino acids with a 4-fold excess of activator (HBTU) and a 6-fold excess of N,N-diisopropylethylamine. After each condensation reaction, the peptides are washed three times each with N,N-dimethylformamide (DMF) and dichloromethane (DCM). Then, the Fmoc protecting group is removed using 20% ​​piperidine (DMF). The condensation reaction and deprotection steps are repeated until the target peptide sequence (Pro-Ser-Gly)2 (Asn-Glu-Gly)2 (Pro-Ser-Gly)2 is synthesized. After the synthesis of n-Glu-Gly)2 (Pro-Ser-Gly)2, the N-terminus was capped with 25% acetic anhydride (DMF) solution for 20 minutes. The peptide was then cleaved from the resin using a cleavage buffer ratio of TFA:TIS:H2O = 90:3:7 for 6 hours. The peptide was then precipitated with anhydrous diethyl ether and centrifuged, discarding the supernatant and retaining the precipitate. The precipitate was washed three times with ether to obtain the target crude peptide. The crude peptide was then purified using a C18 reversed-phase high-performance liquid chromatography column, and the target product was confirmed by mass spectrometry, finally yielding peptide (Pro-Ser-Gly)2 (Asn-Glu-Gly)2 (Pro-Ser-Gly)2.

[0060] S4. Establishment of a dehydrated melanoma mouse model: The mice used in this invention were purchased from the Animal Experiment Center of Southern Medical University (animal experiment license number gdpulac2015595). The mice weighed approximately 20±2g. Before the experiment, all mice were acclimatized for one week, ensuring free access to food. All mice were placed in a dry environment and irradiated with ultraviolet light of varying intensities to enhance melanoma production in the mice.

[0061] S5. Locking in moisture on mouse skin surface: A sample solution was prepared by weighing 0.3g of *Liriope muscari* extract, 0.3g of *Cuscuta chinensis* extract, 5ml of hypophosphite buffer (pH=7), and 20ml of deionized water. The solution was then extracted and emulsified. The emulsion product was combined with 0.1g of the polypeptide synthesized in S3 to prepare a water-in-oil nanoemulsion. Cells were taken from the skin surface of mice with dehydrated melanoma created in S4 and placed in a culture dish. After 24 hours of initial cell culture, the diluted water-in-oil nanoemulsion was added, and the cells were cultured further. The cell moisture retention rate was measured periodically.

[0062] S6. Incubate cells at 25℃ and 5% CO2 saturated humidity for 24 h. Discard the culture medium, wash once with hypophosphite buffer, digest with 0.25% trypsin, and then agitate the cells with hypophosphite buffer for further processing. Take the prepared cell suspension, add hypophosphite buffer, 10 ml of deionized water, and 3 ml of diluted water-in-oil nanoemulsion, and place in a desiccator. Weigh the samples at intervals, repeating the experiment three times. Calculate the moisture retention rate using the following formula: Moisture retention rate = Hn / Ho × 100% (where Ho is the water content of the sample before storage, and Hn is the water content of the sample after storage).

[0063] Comparative Example 6: Except for step S5, in which 0.3g of Cuscuta extract was not added when preparing the water-in-oil nanoemulsion, all other steps were the same as in Example 3.

[0064] Comparative Example 7 was identical to Example 3 except that 0.3g of water-in-oil nanoemulsion was not added in step S5 when preparing the water-in-oil nanoemulsion.

[0065] Comparative Example 8: Except for step S5, in which 0.1g of the synthesized polypeptide was not added when preparing the water-in-oil nanoemulsion, all other steps were the same as in Example 3.

[0066] Table 3

[0067] Testing items Comparative Example 6 Comparative Example 7 Comparative Example 8 Example 3 Moisturizing rate (%) 72±0.01 60±0.01 40±0.01 92±0.01

[0068] Figure 3 The bar charts show the mouse epidermal cell moisture retention rates obtained in Example 3 and Comparative Examples 6 and 7 of this invention. Figure 5The present invention provides the molecular structure of the polypeptide (Pro-Ser-Gly)2 (Asn-Glu-Gly)2 (Pro-Ser-Gly)2. Figure 6 The H spectrum of its molecular structure Figure 7 The C spectrum is shown in Table 3, which is a statistical table of the moisturizing rate of epidermal cells in mutagenized mice measured five times in Examples 3 and 6 and 7 of this invention. Figure 3 As shown in Table 3, the moisturizing rate of mouse epidermal cells in Example 3 was higher than that in Comparative Examples 6 and 7. To synthesize polypeptides with specific functional amino acid sequences, it is necessary to protect the amino groups that do not participate in the formation of amide bonds—the amino and carboxyl groups of the next amino acid to be linked—that is, the carboxyl group of the previous linked amino acid. Simultaneously, it is also necessary to protect the active genes on the amino acid side chains until the reaction is complete, at which point they are removed. Alkoxycarbonyl groups are mostly used as protecting groups for α-amino groups because such protection makes it less likely for the amino acid to racemize, increasing the polypeptide synthesis yield and the whitening and spot-removing effect of the polypeptide. These results indicate that the extracts of *Liriope muscari* and *Cuscuta chinensis* have a synergistic effect on improving the moisturizing rate of mutagenic mouse epidermal cells.

[0069] Example 4

[0070] S1. Preparation of Perfume Lotus Extract: Dried perfume lotus petals were collected, and after removing insect-damaged and moldy parts, they were pulverized and passed through a 50-mesh sieve. The petals were then ultrasonically treated with distilled water and acetone solutions of 20%, 40%, 60%, 80%, and 100% (v / v) at a mass-to-volume ratio of 1 g:20 mL at 25°C for 60 min. The resulting solutions were centrifuged at 9000 r / min for 10 min, and the supernatant was filtered. This centrifugation process was repeated three times. Acetone was then removed by rotary evaporation under reduced pressure, and the mixture was freeze-dried for 24 h to obtain perfume lotus petal extract powder. For testing, this powder was reconstituted with dodder seed extract via ultrasonication to prepare a solution of the desired concentration.

[0071] S2. Preparation of Cuscuta Seed Extract: Weigh 10g of cleaned Cuscuta seed material, place it in a mortar, crush all the Cuscuta seeds, and then pack it into a bag for later use. Weigh 5g of the crushed Cuscuta seeds, place them in a crucible, and heat them with an alcohol lamp until the surface turns slightly yellow and a popping sound is heard. Remove and let cool, then seal and store for later use. Weigh 1g of the Cuscuta seeds, add 100mL of 60% ethanol, and extract twice by reflux in a water bath for 60min each time. Filter, concentrate, and evaporate to dryness to obtain the Cuscuta seed extract. For determination, mix it with the fragrant lotus extract by ultrasonic redissolve to prepare a solution of the required concentration for later use.

[0072] S3. Synthesis of protein peptides: A condensation reaction is performed after activating Fmoc-amino acids with a 4-fold excess of activator (HBTU) and a 6-fold excess of N,N-diisopropylethylamine. After each condensation reaction, the peptides are washed three times each with N,N-dimethylformamide (DMF) and dichloromethane (DCM). Then, the Fmoc protecting group is removed using 20% ​​piperidine (DMF). The condensation reaction and deprotection steps are repeated until the target peptide sequence (Pro-Ser-Gly)2 (Asn-Glu-Gly)2 (Pro-Ser-Gly)2 is synthesized. After the synthesis of n-Glu-Gly)2 (Pro-Ser-Gly)2, the N-terminus was capped with 25% acetic anhydride (DMF) solution for 20 minutes. The peptide was then cleaved from the resin using a cleavage buffer ratio of TFA:TIS:H2O = 90:3:7 for 6 hours. The peptide was then precipitated with anhydrous diethyl ether and centrifuged, discarding the supernatant and retaining the precipitate. The precipitate was washed three times with ether to obtain the target crude peptide. The crude peptide was then purified using a C18 reversed-phase high-performance liquid chromatography column, and the target product was confirmed by mass spectrometry, finally yielding peptide (Pro-Ser-Gly)2 (Asn-Glu-Gly)2 (Pro-Ser-Gly)2.

[0073] S4. Skin Test: Ten volunteers were recruited for the skin test. A sample solution was prepared by weighing 0.3g of *Liriope muscari* extract, 0.3g of *Cuscuta chinensis* extract, 5ml of hypophosphite buffer (pH=7), and 20ml of deionized water. The solution was then extracted and emulsified. The emulsion product was combined with 0.1g of the peptide synthesized in S3 to prepare a water-in-oil nanoemulsion. This water-in-oil nanoemulsion was mixed with water at a ratio of 1:10. A cotton pad was soaked in the diluted emulsion for 10 minutes and then applied to the volunteers' hands for 10 minutes. Skin moisture was tested every 10 minutes for a period of 1 hour.

[0074] Comparative Example 9: Ten volunteers were recruited to conduct skin tests. Soaking cotton pads in mineral water for 10 minutes and then applying them to the volunteers' hands for 10 minutes each time, with skin moisture measured every 10 minutes for a total of 1 hour.

[0075] Comparative ratio 10: Ten volunteers were recruited to conduct skin tests. Commercially available hyaluronic acid was mixed at a 1:10 ratio. Cotton pads were soaked in the diluted solution for 10 minutes, then applied to the volunteers' hands for 10 minutes. Skin moisture was tested every 10 minutes for a period of 1 hour.

[0076] Figure 4The graphs show the moisture content of Examples 4, 9, and 10 of this invention. As can be seen from the graphs, the skin moisture content decreased most slowly in Example 4, indicating slightly better moisturizing properties than the currently accepted hyaluronic acid. This suggests that the polysaccharides from the extracts of fragrant lotus and dodder have a synergistic effect, forming a thin film on the skin to prevent moisture evaporation.

[0077] Examples 1-4, based on different receptors and experimental protocols, comprehensively demonstrate that adding water-in-oil nanoemulsions prepared from extracts of fragrant lotus and dodder seed to a facial mask solution effectively inhibits acne formation by combining them with a facial mask solution. Fragrant lotus contains abundant polysaccharides, which can form a thin film on the skin, preventing moisture evaporation. The synergistic effect of fragrant lotus and dodder seed extracts effectively inhibits 5α-reductase activity, thereby suppressing acne formation and achieving an acne-removing effect. The prepared facial mask not only whitens and removes blemishes but also significantly improves skin hydration.

[0078] Finally, it should be noted that the above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A water-in-oil nanoemulsion for whitening and removing blemishes, comprising: fragrant lotus extract, dodder seed extract, and polypeptides, characterized in that, The specific preparation method includes the following steps: S1. Preparation of perfume lotus extract: Take dried perfume lotus petals, remove insect-damaged and moldy parts, crush them, and pass them through a 50-mesh sieve. According to the mass-to-volume ratio of 1 g:20 mL, use distilled water and acetone solutions with volume fractions of 20%, 40%, 60%, 80%, and 100% respectively. Sonicate the solutions at 25℃ for 60 min, centrifuge at 9000 r / min for 10 min, filter the supernatant, repeat the centrifugation step 3 times, remove acetone by vacuum rotary evaporation, and freeze-dry in a freeze dryer for 24 h to obtain perfume lotus petal extract; S2. Preparation of dodder seed extract: Weigh 10 g of cleaned dodder seed material, place it in a mortar, crush all the dodder seeds, and pack it in a bag for later use. Weigh 5 g of the crushed dodder seeds, place them in a crucible, heat with an alcohol lamp until the surface is slightly yellow and there is a popping sound, remove and cool, seal and store for later use. Weigh 1 g of the above-mentioned dodder seeds... g, add 100 mL of 60% ethanol, reflux twice in a water bath for 60 min each time, filter, concentrate and evaporate to dryness to obtain Cuscuta chinensis extract; S3, Polypeptide synthesis: 4 times excess Fmoc-amino acids are activated by 4 times excess activator and 6 times excess N,N-diisopropylethylamine and then subjected to condensation reaction; after each condensation reaction, wash three times each with N,N-dimethylformamide and dichloromethane; then remove the Fmoc protecting group with 20% piperidine; repeat the condensation reaction and deprotection steps until the synthesis of the target polypeptide sequence (Pro-Ser-Gly)2(Asn-Glu-Gly)2(Pro-Ser-Gly)2 is completed; after the synthesis of the target sequence (Pro-Ser-Gly)2(Asn-Glu-Gly)2(Pro-Ser-Gly)2 is completed, the N-terminus needs to be treated with 25% acetic anhydride solution. End-capping for 20 minutes; the peptide was cleaved from the resin with a cleavage solution ratio of TFA:TIS:H2O=90:3:7 and a cleavage time of 6 hours; then the peptide was precipitated with anhydrous diethyl ether and centrifuged, the supernatant was discarded and the precipitate was retained, and the peptide was washed 3 times with ether to obtain the target crude peptide; then the crude peptide was purified by C18 reversed-phase high-performance liquid chromatography column, and the target product was confirmed by mass spectrometry, finally yielding peptide (Pro-Ser-Gly)2(Asn-Glu-Gly)2(Pro-Ser-Gly)2; S4, preparation of water-in-oil nanoemulsion: 0.2g-0.4g of fragrant lotus extract, 0.3g of dodder extract, 5ml of hypophosphite buffer (pH=7), and deionized water were used to prepare a sample solution, which was then extracted and emulsified. The emulsified product was combined with 0.1g of the peptide synthesized in S3 to prepare a water-in-oil nanoemulsion.

2. The water-in-oil nanoemulsion for whitening and removing freckles according to claim 1, characterized in that: In step S1, the main component of the fragrant lotus extract is polysaccharide.

3. The water-in-oil nanoemulsion for whitening and removing freckles according to any one of claims 1 or 2, characterized in that: In step S1, the fragrant lotus extract can inhibit the activity of tyrosinase and reduce the conversion rate of melanin.

4. The water-in-oil nanoemulsion for whitening and removing freckles according to claim 1, characterized in that: In step S2, the main components of the dodder seed extract are polysaccharides and flavonoids.

Images