Whitening emulsion based on oxyresveratrol-loaded exosome as well as preparation method and application of whitening emulsion

Abstract

The invention provides a whitening emulsion based on oxyresveratrol-loaded exosome as well as a preparation method and application thereof, and belongs to the field of cosmetics. The oxyresveratrol-loaded exosome disclosed by the invention is prepared from the following components in parts by weight: 0.01 to 1 part of oxyresveratrol and 0.05 to 20 parts of exosome, wherein the oxyresveratrol is wrapped in the exosome; the weight ratio of the oxyresveratrol to the exosome is (1: 5)-(1: 20). When the oxyresveratrol-loaded exosome disclosed by the invention is prepared, when the weight ratio of the oxyresveratrol to the exosome is 1: (5-20), the loading capacity of the oxyresveratrol in the exosome can be greatly improved, so that the effects of the whitening and soothing composition and the whitening emulsion are ensured.

Description

Technical Field

[0001] The present invention belongs to the field of cosmetics, and particularly relates to a whitening lotion based on exosomes loaded with resveratrol oxide, a preparation method thereof, and an application thereof. Background Art

[0002] Current research in the field of whitening focuses on multi-target regulation of melanin metabolism. The mainstream technologies revolve around tyrosinase inhibitors (such as phenethyl resorcinol), melanin transport blockers (such as niacinamide), and antioxidants (such as glutathione). At the same time, nanocarrier technologies (such as liposomes, polymer micelles) are widely used to improve the stability of ingredients and transdermal efficiency. In recent years, resveratrol oxide has been explored for whitening due to its inhibitory effect on tyrosinase activity and antioxidant properties, but its solubility and stability are poor, making it difficult to be effectively delivered in conventional formulations and easily degraded and inactivated; although exosomes have been tried to interfere with melanin production signals by delivering bioactive ingredients, the existing applications are still limited to single-mechanism regulation and do not fully exert their potential for multi-target synergistic delivery.

[0003] The existing technologies generally have defects such as poor permeability of ingredients (such as the transdermal rate of kojic acid is less than 5%), single action target (such as arbutin only inhibits tyrosinase), strong irritation (such as hydroquinone causing contact dermatitis), and insufficient long-term effect (such as vitamin C being easily oxidized and inactivated), especially for sensitive skin, it is easy to cause barrier damage and aggravation of post-inflammatory hyperpigmentation; the traditional water-oil formulation system is difficult to balance the dissolution of active ingredients and the affinity for the stratum corneum, resulting in multi-pathway whitening ingredients being difficult to be delivered synchronously and accurately act on each layer of the epidermis, and lacking comprehensive regulation of the upstream signal of melanin production (such as the cAMP / MC1R / MITF pathway) and the downstream skin microenvironment (such as the balance of water-lipid film), and it is impossible to achieve a whitening effect with low irritation, fast effect and no rebound. Summary of the Invention

[0004] In view of this, the present invention mainly provides a whitening lotion based on exosomes loaded with resveratrol oxide, a preparation method thereof, and an application thereof.

[0005] The present invention first proposes an exosome loaded with resveratrol oxide, and then combines Lithothamnion calcareum and ceramide NP to form a whitening and soothing composition. This composition forms a "trinity" full-path whitening link by blocking the upstream pro-melanogenic signal transduction, inhibiting the middle-stream melanin production, and blocking the downstream melanin transport. Combining the exosome encapsulation technology and the water-oil balance system, it effectively enhances the solubility, stability and transdermal absorption rate of the components, and reduces skin irritation, thereby achieving the effects of whitening, brightening and soothing, and the effects are rapid, long-lasting and non-rebound, and it is suitable for people with sensitive skin. Finally, the whitening and soothing composition is combined with a thickening agent, a moisturizing agent, a pH regulator, an emulsifier, and a preservative to prepare a whitening lotion.

[0006] To solve the above technical problems, the present invention is realized through the following technical solutions:

[0007] An exosome loaded with resveratrol oxide, comprising the following components in parts by weight:

[0008] 0.01 to 1 part by weight of resveratrol oxide, 0.05 to 20 parts by weight of exosomes, and the resveratrol oxide is encapsulated within the exosomes; the weight ratio of the resveratrol oxide to the exosomes is 1:5 to 1:20.

[0009] All the exosomes are dissolved in a sufficient amount of PBS buffer and formulated into a 5 mg / mL solution, and all the resveratrol oxide is dissolved in a sufficient amount of alcohol and formulated into a 5 mg / mL solution. After mixing the two, the resveratrol oxide is loaded onto the exosomes by the low-temperature repeated freeze-thaw method. Preferably, the exosomes are at least one of milk exosomes and goat milk exosomes;

[0010] The alcohol is selected from methanol and absolute ethanol.

[0011] Preferably, an exosome loaded with resveratrol oxide of the present invention is prepared by the repeated freeze-thaw method. When prepared by the repeated freeze-thaw method, the specific preparation method includes the following steps: Dissolve all the exosomes in a sufficient amount of PBS buffer to obtain a 5 mg / mL solution, dissolve all the resveratrol oxide in a sufficient amount of alcohol to obtain a 5 mg / mL solution (reaching saturation), mix the two, and load the resveratrol oxide onto the exosomes by low-temperature repeated freeze-thaw. After thawing, ultracentrifuge, collect the precipitate, and obtain the exosome loaded with resveratrol oxide;

[0012] Among them, the temperature for repeated freeze-thaw is -20°C to -80°C, the thawing temperature is 4°C to 25°C, and the number of freeze-thaw cycles is 3 to 5 times; preferably, the temperature for repeated freeze-thaw is -80°C, the thawing temperature is 4°C, and the number of freeze-thaw cycles is 3 times;

[0013] The speed of ultracentrifugation is 30000 to 120000 g, and the centrifugation time is 10 to 60 min; preferably, the speed of ultracentrifugation is 100000 g, and the centrifugation time is 30 min.

[0014] A whitening and soothing composition, comprising the following components in parts by weight:

[0015] 0.01 to 1 part of resveratrol oxide, 0.05 to 20 parts of exosomes, 0.01 to 5 parts of Lithothamnion calcareum, 0.01 to 5 parts of ceramide NP, wherein,

[0016] The weight ratio of the resveratrol oxide to the exosomes is 1:5 to 1:20, preferably 1:10. And the exosomes are loaded with resveratrol oxide to obtain an exosome loaded with resveratrol oxide, and then mixed with Lithothamnion calcareum and ceramide NP.

[0017] The exosomes loaded with resveratrol oxide were prepared by the repeated freezing and thawing method, including the following steps: dissolving the exosomes with PBS buffer and preparing a solution with a concentration of 5 mg / mL, dissolving resveratrol oxide with alcohol and preparing a solution with a concentration of 5 mg / mL, mixing the two solutions, and then loading resveratrol oxide into the exosomes by repeated freezing and thawing at low temperature. After thawing, ultracentrifugation was carried out to collect the precipitate, and the exosomes loaded with resveratrol oxide were obtained.

[0018] Preferably, the exosomes are at least one of milk exosomes and goat milk exosomes.

[0019] Resveratrol oxide is a stilbene compound of natural origin, which has a significant effect of inhibiting tyrosinase activity, directly reducing melanin synthesis, and at the same time inhibiting the transport of melanin from melanocytes to epidermal cells.

[0020] Exosomes are cell-secreted vesicles with a diameter of 30 - 150 nm, carrying signal molecules such as miRNA, regulating melanin metabolism by blocking the conduction of melanocyte-stimulating hormone, and promoting collagen production and improving skin microcirculation.

[0021] Lithothamnion calcareum is a coral algae extract rich in calcium carbonate, which activates endogenous ceramide synthase by balancing the epidermal calcium ion gradient and maintains the water-oil balance of the skin.

[0022] Ceramide NP, as the core lipid of the stratum corneum, reduces inflammatory pigment deposition by repairing the barrier function and improves the transdermal absorption efficiency of other whitening components by enhancing epidermal cohesion.

[0023] Therefore, in the whitening and soothing composition of the present invention, resveratrol oxide, exosomes, Lithothamnion calcareum, and ceramide NP act on the synthesis, transport, signal conduction, and barrier defense links of melanin production respectively. Through the exosome encapsulation technology, the stable delivery of resveratrol oxide components is realized, forming a whole-pathway synergistic whitening system from source blocking to metabolic regulation, while taking into account soothing sensitivity and long-term moisturization.

[0024] A whitening lotion, comprising the following components in percentage by content:

[0025] Phase A: thickener 0.1% - 0.3%, humectant 0.3% - 1.0%, pH regulator 0.3% - 0.8%;

[0026] Phase B: emulsifier 1% - 5%;

[0027] Phase C: preservative 0.5% - 1.5%;

[0028] Whitening and soothing composition 1% - 5%;

[0029] pH regulator: 0.05% - 0.3%;

[0030] Water: the balance.

[0031] Preferably, the whitening lotion of the present invention comprises the following components in percentage by content:

[0032] Phase A: thickener 0.2%, humectant 0.6%, pH regulator 0.52%;

[0033] Phase B: emulsifier 3.5%;

[0034] Phase C: preservative 1.1%;

[0035] Whitening and soothing composition 1 - 5%;

[0036] pH regulator 0.15%;

[0037] Water: the balance.

[0038] In Phase A, the thickener includes at least one of polyacrylate cross - polymer - 6, carbomer, carrageenan, gellan gum, xanthan gum, microcrystalline cellulose, cellulose gum, ethyl cellulose, tara gum, guar gum, acryloyldimethyltaurate / VP copolymer, and acrylate copolymer. The humectant includes at least one of glycerin, D - panthenol, vitamin B5, 1,3 - butanediol, 1,3 - propanediol, sodium hyaluronate, tremella polysaccharide, trehalose, betaine, allantoin, low - molecular - weight sodium hyaluronate, sodium polyacrylate, and hydrogenated lecithin. The pH regulator includes at least one of arginine, disodium ethylenediaminetetraacetate, tromethamine, and EDTA - disodium.

[0039] In Phase B, the emulsifier includes at least one of triglyceride caprylate / caprate, C14 - 22 alcohol, C12 - 20 alkyl glucoside, cetearyl glucoside, isononyl isononanoate, pentaerythrityl tetraester, polydimethylsiloxane, stearyl alcohol, hydroxystearic acid, polymethylsilsesquioxane, pentaerythrityl distearate, and sucrose stearate.

[0040] In Phase C, the preservative includes at least one of p - hydroxyacetophenone and polyol preservatives. The polyol preservatives include at least one of common alcohols such as 1,2 - hexanediol, 1,2 - pentanediol, ethylhexylglycerin, and phenoxyethanol.

[0041] The pH regulator includes at least one of arginine, disodium ethylenediaminetetraacetate, tromethamine, and EDTA - disodium.

[0042] When preparing the exosomes loaded with resveratrol oxide of the present invention, the dosage ratio of resveratrol oxide and exosomes is controlled and formulated into solutions with different concentrations. After mixing, the low-temperature repeated freezing and thawing method is used for preparation, which can greatly increase the loading amount of resveratrol oxide in exosomes, thereby ensuring the effects of the whitening and soothing composition and the whitening lotion. Detailed implementation manners

[0043] To make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to specific embodiments.

[0044] Example 1

[0045] A whitening and soothing composition comprises 0.2 parts by weight of resveratrol oxide, 2 parts by weight of milk exosomes, 0.5 part by weight of Lithothamnion calcareum, and 0.2 part by weight of ceramide NP.

[0046] In this example, the weight ratio of resveratrol oxide to milk exosomes is 1:10.

[0047] Among them, the resveratrol oxide and exosomes are first dissolved in absolute ethanol and PBS buffer solution respectively to prepare a 5 mg / ml solution. After the two solutions are mixed, the repeated freezing and thawing method is used to load resveratrol oxide into the exosomes to obtain the exosomes loaded with resveratrol oxide, and then they are mixed with Lithothamnion calcareum and ceramide NP to obtain the whitening and soothing composition.

[0048] Specifically, the preparation method of the exosomes loaded with resveratrol oxide includes the following steps: dissolving milk exosomes in PBS buffer solution to form a 5 mg / ml solution, dissolving resveratrol oxide in absolute ethanol to form a 5 mg / ml solution, mixing the two, and using low-temperature repeated freezing and thawing to load resveratrol oxide in milk exosomes. After thawing, ultracentrifugation is carried out, and the precipitate is collected to obtain the exosomes loaded with resveratrol oxide;

[0049] Among them, the temperature of repeated freezing and thawing can be -20°C to -80°C, the thawing temperature can be 4°C to 25°C, and the number of freeze-thaw cycles can be 3 to 5 times; in this example, the temperature of repeated freezing and thawing is -80°C, the thawing temperature is 4°C, and the number of freeze-thaw cycles is 3 times;

[0050] The rotational speed of ultracentrifugation can be 30000 to 120000 g, and the centrifugation time can be 10 to 60 min; in this example, the rotational speed of ultracentrifugation is 100000 g, and the centrifugation time is 30 min.

[0051] Example 2

[0052] A whitening and soothing composition comprises 0.1 part of resveratrol oxide, 1 part of milk exosomes, 1 part of Lithothamnion calcareum, and 0.5 part of ceramide NP.

[0053] In this example, the weight ratio of resveratrol oxide to milk exosomes is 1:10.

[0054] The rest is the same as in Example 1.

[0055] Example 3

[0056] A whitening and soothing composition comprises 0.5 parts of resveratrol oxide, 5 parts of milk exosomes, 0.1 part of Lithothamnion calcareum, and 0.1 part of ceramide NP.

[0057] In this example, the weight ratio of resveratrol oxide to milk exosomes is 1:10.

[0058] The rest is the same as in Example 1.

[0059] Example 4

[0060] A whitening and soothing composition comprises 0.01 part of resveratrol oxide, 0.05 part of milk exosomes, 5 parts of Lithothamnion calcareum, and 5 parts of ceramide NP.

[0061] In this example, the weight ratio of resveratrol oxide to milk exosomes is 1:5.

[0062] The rest is the same as in Example 1.

[0063] Example 5

[0064] A whitening and soothing composition comprises 1 part of resveratrol oxide, 20 parts of milk exosomes, 0.01 part of Lithothamnion calcareum, and 0.01 part of ceramide NP.

[0065] In this example, the weight ratio of resveratrol oxide to milk exosomes is 1:20.

[0066] The rest is the same as in Example 1.

[0067] Example 6

[0068] A whitening and soothing composition comprises 0.5 part of resveratrol oxide, 10 parts of milk exosomes, 1 part of Lithothamnion calcareum, and 1 part of ceramide NP.

[0069] In this example, the weight ratio of resveratrol oxide to milk exosomes is 1:20.

[0070] The rest is the same as in Example 1.

[0071] Example 7

[0072] A whitening and soothing composition comprises 0.1 part of resveratrol oxide, 0.5 part of milk exosomes, 5 parts of Lithothamnion calcareum, and 5 parts of ceramide NP.

[0073] In this example, the weight ratio of resveratrol oxide to milk exosomes is 1:5.

[0074] The rest is the same as in Example 1.

[0075] Example 8

[0076] A whitening and soothing composition comprises 1 part of resveratrol oxide, 5 parts of milk exosomes, 2 parts of Lithothamnion calcareum, and 3 parts of ceramide NP.

[0077] In this example, the weight ratio of resveratrol oxide to milk exosomes is 1:5.

[0078] The rest is the same as in Example 1.

[0079] Comparative Example 1

[0080] A whitening and soothing composition comprises 0.05 part of resveratrol oxide, 2 parts of milk exosomes, 0.5 part of Lithothamnion calcareum, and 0.2 part of ceramide NP.

[0081] In this comparative example, the weight ratio of resveratrol oxide to milk exosomes is 1:40.

[0082] The rest is the same as in Example 1.

[0083] Comparative Example 2

[0084] A whitening and soothing composition comprises 0.02 part of resveratrol oxide, 1 part of milk exosomes, 1 part of Lithothamnion calcareum, and 0.5 part of ceramide NP.

[0085] In this comparative example, the weight ratio of resveratrol oxide to milk exosomes is 1:50.

[0086] The rest is the same as in Example 1.

[0087] Comparative Example 3

[0088] A whitening and soothing composition comprises 1.5 parts of resveratrol oxide, 5 parts of milk exosomes, 0.1 part of Lithothamnion calcareum, and 0.1 part of ceramide NP.

[0089] In this comparative example, the weight ratio of resveratrol oxide to milk exosomes is 1:3.3.

[0090] The rest is the same as in Example 1.

[0091] Comparative Example 4

[0092] A whitening and soothing composition comprises 0.01 part of resveratrol oxide, 0.04 part of milk exosomes, 5 parts of Lithothamnion calcareum, and 5 parts of ceramide NP.

[0093] In this comparative example, the weight ratio of resveratrol oxide to milk exosomes is 1:4.

[0094] The rest is the same as in Example 1.

[0095] Comparative Example 5

[0096] A whitening and soothing composition comprises 1 part of resveratrol oxide, 25 parts of milk exosomes, 0.01 part of Lithothamnion calcareum, and 0.01 part of ceramide NP.

[0097] In this comparative example, the weight ratio of resveratrol oxide to milk exosomes is 1:25.

[0098] The rest is the same as in Example 1.

[0099] Comparative Example 6

[0100] A whitening and soothing composition comprises 0.5 part of resveratrol oxide, 12 parts of milk exosomes, 1 part of Lithothamnion calcareum, and 1 part of ceramide NP.

[0101] In this comparative example, the weight ratio of resveratrol oxide to milk exosomes is 1:24.

[0102] The rest is the same as in Example 1.

[0103] Comparative Example 7

[0104] A whitening and soothing composition comprises 0.1 part of resveratrol oxide, 0.4 part of milk exosomes, 5 parts of Lithothamnion calcareum, and 5 parts of ceramide NP.

[0105] In this comparative example, the weight ratio of resveratrol oxide to milk exosomes is 1:4.

[0106] The rest is the same as in Example 1.

[0107] Comparative Example 8

[0108] A whitening and soothing composition comprises 1 part of resveratrol oxide, 4 parts of milk exosomes, 5 parts of Lithothamnion calcareum, and 5 parts of ceramide NP.

[0109] In this comparative example, the weight ratio of resveratrol oxide to milk exosomes is 1:4.

[0110] The rest is the same as in Example 1.

[0111] Comparative Example 9

[0112] A whitening and soothing composition comprises 0.2 parts by weight of resveratrol oxide, 2 parts by weight of milk exosomes, 0.5 parts by weight of Lithothamnion calcareum, and 0.2 parts by weight of ceramide NP.

[0113] In this comparative example, the weight ratio of resveratrol oxide to milk exosomes is 1:10.

[0114] The difference from Example 1 is that resveratrol oxide, milk exosomes, Lithothamnion calcareum, and ceramide NP are directly mixed, and resveratrol oxide and milk exosomes are not fusion-loaded.

[0115] Table 1 Components and dosages (parts by weight) of Examples 1-8 and Comparative Examples 1-9

[0116]

[0117] A whitening lotion of the present invention comprises components in the following percentage contents:

[0118] Phase A: thickener 0.1% - 0.3%, humectant 0.3% - 1.0%, pH regulator 0.3% - 0.8%; Phase B: emulsifier 1% - 5%;

[0119] Phase C: preservative 0.5% - 1.5%;

[0120] Whitening and soothing composition 1% - 5%;

[0121] pH regulator 0.05% - 0.3%;

[0122] Water: the balance.

[0123] Preferably, the whitening lotion of the present invention comprises components in the following percentage contents:

[0124] Phase A: thickener 0.2%, humectant 0.6%, pH regulator 0.52%;

[0125] Phase B: emulsifier 3.5%;

[0126] Phase C: preservative 1.1%;

[0127] Whitening and soothing composition 1 - 5%;

[0128] pH regulator 0.15%;

[0129] Water: the balance.

[0130] The thickener includes at least one of polyacrylate cross - polymer - 6, carbomer, carrageenan, gellan gum, xanthan gum, microcrystalline cellulose, cellulose gum, ethyl cellulose, Caesalpinia spinosa gum, guar gum, acryloyldimethyltaurate / VP copolymer, and acrylate copolymer. The humectant includes at least one of glycerin, D - panthenol, vitamin B5, 1,3 - butanediol, 1,3 - propanediol, sodium hyaluronate, tremella polysaccharide, trehalose, betaine, allantoin, low - molecular - weight sodium hyaluronate, sodium polyacrylate, and hydrogenated lecithin. The pH regulator includes at least one of arginine, disodium ethylenediaminetetraacetate, tromethamine, and EDTA - disodium.

[0131] The emulsifier includes at least one of triglyceride caprylate / caprate, C14 - 22 alcohol, C12 - 20 alkyl glucoside, cetearyl glucoside, isononyl isononanoate, pentaerythritol tetraester, polydimethylsiloxane, stearyl alcohol, hydroxystearic acid, polymethylsilsesquioxane, pentaerythritol distearate, and sucrose stearate.

[0132] The preservative includes at least one of p - hydroxyacetophenone and polyol preservatives. The polyol preservatives include at least one of common alcohols such as 1,2 - hexanediol, 1,2 - pentanediol, ethylhexylglycerin, and phenoxyethanol.

[0133] Application Examples

[0134] The application examples and comparative application examples of the present invention each provide a whitening lotion, and the components (mass percentages) of the lotion are shown in Table 2; among them, the whitening and soothing compositions used in Application Examples 1 - 8 are the whitening and soothing compositions prepared in Examples 1 - 8 respectively, and the whitening and soothing compositions used in Application Examples 9 - 10 are the whitening and soothing composition prepared in Example 1, but the dosage is adjusted relative to Application Examples 1 - 8; the whitening and soothing compositions used in Comparative Application Examples 1 - 9 are the whitening and soothing compositions prepared in Comparative Examples 1 - 9 respectively; in addition, a blank application example is set, and the only difference between the blank application example and Application Example 1 is that the whitening and soothing composition is not added.

[0135] Table 2:

[0136]

[0137] This example provides a preparation method of a whitening lotion, and the preparation method includes the following steps:

[0138] (1) Mix the humectant, thickener, and pH regulator (EDTA - disodium) with water and stir. After heating to 85 °C, homogenize at a speed of 1300 rpm for 4 min. After the homogenization is completed, keep it warm for standby to obtain the pre - prepared Phase A;

[0139] (2) Mix the emulsifier with water, heat to 75 °C, and homogenize at a speed of 1300 rpm for 4 min. After homogenization, keep it warm for standby to obtain the prefabricated Phase B.

[0140] (3) Mix the preservative with water and heat to 60 °C to melt it to obtain the prefabricated Phase C.

[0141] (4) Heat the prefabricated Phase A to 80 °C and add the prefabricated Phase B at a speed of 300 rpm, then stir and mix.

[0142] (5) Then cool down to 60 °C, add the prefabricated Phase C and stir to mix at a speed of 300 rpm. Subsequently, cool down to below 45 °C, add the whitening and soothing composition and continue to stir for 8 min. Finally, add the pH regulator (arginine) to adjust the pH to 5.5 - 7.0, then stop stirring and discharge to obtain the emulsion.

[0143] Effect experiment

[0144] 1. Detection of the stability of resveratrol oxide in the whitening and soothing compositions of the examples and comparative examples by high performance liquid chromatography

[0145] Stability test: Accurately weigh 0.1 g of the whitening and soothing compositions prepared in Examples 1 - 8 and Comparative Examples 1 - 9 respectively, dissolve them in 10 mL of methanol, filter through a 0.22 μm filter membrane, and then use high performance liquid chromatography to determine the content of resveratrol oxide in the extracts. Use the resveratrol oxide standard product (purchased from Yuanye Bio, B21467) to prepare standard solutions with gradient concentrations of 0.01, 0.025, 0.05, 0.10, 0.25, 0.50, 1.00, 2.50 mg / mL, and draw a standard curve. Calculate the content of resveratrol oxide M0 in each sample according to the standard curve formula. The chromatographic column used is a C18 column (100 mm × 2.1 mm, 3 μM), the mobile phase is 60% methanol, the flow rate is 0.5 mL / min, the sample injection volume is 10 μL, and the detection is carried out at a wavelength of 305 nm. Weigh 5 g of the whitening and soothing compositions prepared in the examples and comparative examples respectively into 10 mL transparent sealed PE bottles, place them at temperatures of 4 °C, 25 °C and 50 °C respectively, and irradiate them with a 15 W light. Then detect the mass M1 of resveratrol oxide on the 7th day. The detection method is the same as M0. Subsequently, calculate the retention rate of resveratrol oxide. The retention rate of resveratrol oxide = M1 / M0 × 100%.

[0146] The results are shown in Table 3:

[0147] Table 3

[0148]

[0149]

[0150] As can be seen from Table 3, the content of resveratrol oxide in Examples 1-8 far exceeds that in Comparative Examples 1-9. In particular, from the data of the content of resveratrol oxide in Example 1 and Comparative Example 9, it can be known that the content of resveratrol oxide can be greatly retained after fusion loading with milk exosomes. In addition, too high or too low ratio of resveratrol oxide to milk exosomes will affect the content of resveratrol oxide. After analysis, this is because when exosomes load resveratrol oxide, the exosome membrane needs to go through a process of temporary disruption and membrane restoration. When there are too many exosomes and too little resveratrol oxide, the density of exosomes is large, and the distance between adjacent exosomes is too close, which affects the normal disruption of the exosome membrane during the freeze-thaw process, and adjacent exosomes affect more resveratrol oxide from entering the exosomes with ruptured membranes. Therefore, even if exosomes and resveratrol oxide are added in sufficient amounts, it will affect the loading rate of resveratrol oxide in exosomes. When there are too few exosomes and too much resveratrol oxide, not enough resveratrol oxide can be loaded by exosomes, resulting in a large amount of resveratrol oxide being retained outside the exosomes, and finally the amount of exosomes loaded with resveratrol oxide is insufficient, thus affecting the retention rate.

[0151] 2. Skin Penetration Test of Whitening and Soothing Composition

[0152] The present invention explores the skin permeability of the whitening and soothing composition, specifically including the following steps:

[0153] Experimental method: In vitro pig skin test was used, and the exposed skin area in the diffusion cell was 1.5 cm 2 , and the volume of the receiving chamber was 5.0 mL. A phosphate buffer solution with a pH of 5.8 and a volume ratio of 50:50 of phosphate buffer solution to absolute ethanol was used as the receiving solution. 20 μL of the corresponding whitening and soothing compositions prepared in Examples 1-8 and Comparative Examples 1-9 were respectively added to 180 μL of deionized water and mixed evenly, with a total volume of 200 μL. They were respectively evenly applied to the test pig skin and covered with a film to prevent evaporation. The content of resveratrol oxide M0 in the sample was measured by HPLC, and the transdermal absorption amount M1 of resveratrol oxide after 24 h was measured. The 24 h transdermal absorption rate of resveratrol oxide was calculated by the following formula:

[0154] 24 h transdermal absorption rate = M1 / M0 * 100%.

[0155] The greater the transdermal absorption rate, the better the skin permeability of the composition. The results are shown in Table 4 below.

[0156] Table 4

[0157]

[0158]

[0159] From the data of Examples 1-8 in Table 4, it can be seen that the loading of resveratrol oxide in exosomes can greatly improve the transdermal penetration rate of resveratrol oxide. However, due to the effect of the skin barrier, the transdermal absorption amount of resveratrol oxide cannot increase infinitely. According to the data of Comparative Examples 1-9, the dosage ratio of exosomes to resveratrol oxide affects the loading rate of resveratrol oxide, and thus further affects the transdermal absorption rate of resveratrol oxide. Loading resveratrol oxide in exosomes can greatly improve the transdermal absorption of resveratrol oxide.

[0160] 3. Test on the inhibition of melanin production and transport by the whitening lotion

[0161] Experimental method: The cell line used was mouse melanoma cell B16F10 (Wuhan Sai'ao Si Biotechnology Co., Ltd.). The test conditions were: the temperature in the incubator was 37 ± 1°C, the saturated humidity, and the carbon dioxide was 5 ± 1%; cell culture and treatment were carried out according to the grouping, and then the test was carried out. The specific test was the determination of melanin content, and the test method was as follows:

[0162] (1) Inoculate the cell suspension in a 6-well plate at a culture density of 1×10 6 cells / mL and culture with DMEM culture medium (Gibco) for 24 hours;

[0163] (2) The blank control group was added with DMEM culture medium containing the lotion prepared from the blank application example at 0.1%. The sample groups were respectively added with DMEM culture medium containing the lotions prepared from the application examples and comparative application examples at 0.1%. After culturing for 24 hours, the cells were collected;

[0164] (3) Determination of cell melanin content: Lyse the cells at 80°C, and then use an enzyme-labeled instrument to measure the total amount of melanin in each lysate at a wavelength of 405 nm. Calculate the total protein concentration and adjust the melanin content (OD value). Normalize the melanin content of the blank group (solvent control), and compare the sample groups with it. Use the following formula to calculate the reduction rate of cell melanin content in each example and comparative example:

[0165] Reduction rate of cell melanin content (%) = (OD value of blank group - OD value of sample) / OD value of blank group * 100%.

[0166] (4) Determination of extracellular melanin content: Collect the cell culture medium, centrifuge at 4000 g for 10 min, take the supernatant, and then use an enzyme-labeled instrument to measure the total amount of melanin in each cell supernatant at a wavelength of 405 nm. Calculate the total protein concentration and adjust the melanin content. Normalize the melanin content of the blank group (solvent control), and compare the sample groups with it. Use the following formula to calculate the reduction rate of extracellular melanin content in each example and comparative example:

[0167] Extracellular melanin content reduction rate (%) = (OD value of blank group - OD value of sample) / OD value of blank group * 100%.

[0168] The ability to inhibit melanin production is represented by the reduction rate of cellular melanin content, and the ability to inhibit melanin transport is represented by the reduction rate of extracellular melanin content.

[0169] The results are shown in Table 5.

[0170] Table 5

[0171]

[0172]

[0173] 4. Human skin patch test.

[0174] Recruit 30 volunteers, 15 males and 15 females, aged 20 - 50 years old. Using the closed patch test method, place an equal amount (0.020 mL - 0.025 mL) of the test samples (emulsions prepared from application examples, comparative application examples, and blank application examples) in a specific patch tester, apply it to the volunteers' arms with a low-sensitization tape, gently press to make it evenly adhere to the skin, and leave it for 24 h; the blank control group is distilled water, and the blank application example is an emulsion without the whitening and soothing composition. After 24 h, remove the patch tester, and observe the skin reaction at 0.5 h, 24 h, and 48 h, and record the results. The adverse reaction grades of the skin are shown in Table 6 below.

[0175] Table 6

[0176]

[0177] After testing, the emulsions prepared from the application examples, comparative application examples, and blank application examples of the present invention are all negative reactions after human patch testing, and they are safe and non-irritating to human skin.

[0178] 5. Human efficacy tests for whitening, soothing, and moisturizing

[0179] Experimental method: Select Asian adult testers with sensitive skin aged 18 - 60 years old, randomly divide them into 20 groups, with 6 people in each group. The volunteers use the samples (emulsions prepared from Application Examples 1 - 10, Comparative Application Examples 1 - 9, and Blank Application Example) on the whole face once in the morning and once in the evening every day. Data are collected when they visit on the 0th day (T0), 14th day (T14), and 28th day (T28);

[0180] After the volunteers arrived, they washed their faces with facial cleanser and sat still for 30 minutes in an air-conditioned room at a temperature of 21±1°C and a humidity of 50±10%. The ITA° value of the skin color on the cheeks was measured using a spectrophotometer CM-26d, the redness a* value of the cheeks was measured using a CK skin color test probe CL400, and the skin moisture content of the cheekbones was measured using a skin moisture test probe Corneometer.

[0181] The skin whitening ability is represented by the improvement of the ITA° value, the soothing ability is represented by the improvement of the a* value, and the moisturizing ability is represented by the improvement of the skin moisture content. The formulas are as follows:

[0182] Improvement rate of ITA° value = (T0 - Tx) / T0 * 100%;

[0183] Improvement rate of a* value = (T0 - Tx) / T0 * 100%;

[0184] Improvement rate of skin moisture content = (Tx - T0) / T0 * 100%;

[0185] The results obtained are shown in Table 7;

[0186] Table 7

[0187]

[0188]

[0189] Note: After testing, the skin whitening ability, soothing ability, and moisturizing ability of the comparative application example were extremely poor during use.

[0190] Based on the result data summarized in Table 7, the whitening lotion loaded with resveratrol oxide exosomes of the present invention has achieved excellent effects in skin whitening ability, soothing ability, and moisturizing ability. In the comparative application example, even though it contains resveratrol oxide exosomes, the ratio of resveratrol oxide to exosomes exceeds the range of 1:(5 - 20) of the whitening lotion, which affects the ability of exosomes to load resveratrol oxide, and further affects the function of the whitening lotion.

[0191] The above is the preferred implementation mode of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An exosome loaded with resveratrol oxide, characterized in that, Comprising the following components in parts by weight: 0.01 to 1 part by weight of resveratrol oxide, 0.05 to 20 parts by weight of exosomes, and the resveratrol oxide is encapsulated in the exosomes; Among them, the weight ratio of the resveratrol oxide to the exosomes is 1:5 to 1:20, preferably 1:

10. All the exosomes are dissolved in a sufficient amount of PBS buffer and formulated into a solution with a concentration of 5 mg / mL, and all the resveratrol oxide is dissolved in a sufficient amount of alcohol and formulated into a solution with a concentration of 5 mg / mL. After mixing the two, the resveratrol oxide is loaded into the exosomes by the method of repeated freezing and thawing at low temperature.

2. The exosomes loaded with resveratrol oxide according to claim 1, wherein The exosomes are at least one of milk exosomes and goat milk exosomes; The alcohol is selected from methanol and absolute ethanol.

3. The exosomes loaded with resveratrol as claimed in claim 1 or 2, wherein Prepared by the method of repeated freezing and thawing, including the following steps: Dissolve the exosomes in PBS buffer and formulate into a solution with a concentration of 5 mg / mL, dissolve the resveratrol oxide in alcohol and formulate into a solution with a concentration of 5 mg / mL. After mixing the two, load the resveratrol oxide into the exosomes by repeated freezing and thawing at low temperature. After thawing, perform ultracentrifugation, collect the precipitate, and obtain the exosomes loaded with resveratrol oxide; Among them, the temperature for repeated freezing and thawing is -20°C to -80°C, the thawing temperature is 4°C to 25°C, and the number of freeze-thaw cycles is 3 to 5 times; The rotational speed of ultracentrifugation is 30000 to 120000 g, and the centrifugation time is 10 to 60 min.

4. The preparation method of an exosome loaded with resveratrol as described in claim 3, characterized in that, The temperature for repeated freezing and thawing is -80°C, the thawing temperature is 4°C, and the number of freeze-thaw cycles is 3 times; The rotational speed of ultracentrifugation is 100000 g, and the centrifugation time is 30 min.

5. A whitening and soothing composition, characterized in that, Comprising the following components in parts by weight: 0.01 to 1 part of resveratrol oxide, 0.05 to 20 parts of exosomes, 0.01 to 5 parts of Lithothamnion glaciale, 0.01 to 5 parts of ceramide NP, among which, The weight ratio of the resveratrol oxide to the exosomes is 1:5 to 1:20, preferably 1:

10. The resveratrol oxide is encapsulated in the exosomes. All the exosomes are dissolved in a sufficient amount of PBS buffer and formulated into a solution with a concentration of 5 mg / mL, and all the resveratrol oxide is dissolved in a sufficient amount of absolute ethanol and formulated into a solution with a concentration of 5 mg / mL. After mixing the two, load the resveratrol oxide into the exosomes by the method of repeated freezing and thawing at low temperature.

6. The whitening and soothing composition according to claim 5, wherein, The exosomes are at least one of milk exosomes and goat milk exosomes.

7. A whitening lotion, characterized in that, Comprising the following components in percentage by content: Phase A: 0.1% - 0.3% of thickener, 0.3% - 1.0% of humectant, 0.3% - 0.8% of pH regulator; Phase B: 1% - 5% of emulsifier; Phase C: 0.5% - 1.5% of preservative; 1% - 5% of the whitening and soothing composition according to claim 5 or 6; 0.05% - 0.3% of pH regulator; Water: the balance.

8. A whitening lotion according to claim 7, characterized in that, Comprising the following components in percentage by content: Phase A: 0.2% of thickener, 0.6% of humectant, 0.52% of pH regulator; Phase B: 3.5% of emulsifier; Phase C: 1.1% of preservative; 5% of the whitening and soothing composition; 0.15% of pH regulator; Water: the balance.

9. A whitening lotion according to claim 7 or 8, characterized in that In Phase A, the thickener includes at least one of polyacrylate cross-linked polymer-6, carbomer, carrageenan, gellan gum, xanthan gum, microcrystalline cellulose, cellulose gum, ethyl cellulose, tara gum, guar gum, acryloyldimethyltaurate / VP copolymer, acrylic acid (ester) copolymer; the humectant includes at least one of glycerin, D-panthenol, vitamin B5, 1,3-butanediol, 1,3-propanediol, sodium hyaluronate, tremella polysaccharide, trehalose, betaine, allantoin, low molecular weight sodium hyaluronate, sodium polyacrylate, hydrogenated lecithin; In Phase B, the emulsifier includes at least one of glyceryl caprylate / caprate, C14-22 alcohol, C12-20 alkyl glucoside, cetearyl glucoside, isononyl isononanoate, pentaerythrityl tetraester, polydimethylsiloxane, stearyl alcohol, hydroxystearic acid, polymethylsilsesquioxane, pentaerythrityl distearate, sucrose stearate; In Phase C, the preservative includes at least one of p-hydroxyacetophenone, polyol preservatives; The polyol preservatives include at least one of common alcohols such as 1,2-hexanediol, 1,2-pentanediol, ethylhexylglycerin, phenoxyethanol.

10. A whitening lotion according to claim 7 or 8, characterized in that, The pH regulator includes at least one of arginine, disodium ethylenediaminetetraacetate, tromethamine, EDTA disodium.

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