Cow milk nano-vesicle loading material with high transdermal performance as well as preparation method and application of cow milk nano-vesicle loading material

Abstract

The invention provides a cow milk nano-vesicle load with high transdermal performance and a preparation method and application thereof.The preparation method comprises the steps that cow milk is adjusted in pH, left to stand and centrifuged to obtain a protein body and supernate, the supernate is subjected to ultrafiltration to obtain cow milk extracellular vesicles, the cow milk extracellular vesicles are subjected to heating and irradiation treatment to obtain cow milk nano-vesicles, and then the cow milk nano-vesicles are loaded into the cow milk nano-vesicles. Reacting with phytic acid and beta-cyclodextrin to prepare a beta-cyclodextrin / phytic acid / cow milk nano-vesicle compound; the protein body reacts with alkylated cosmetic peptide to prepare a peptide-loaded protein body, and the peptide-loaded protein body is embedded with a beta-cyclodextrin / phytic acid / cow milk nano-vesicle compound, vitamin E and vitamin C to prepare the cow milk nano-vesicle loading material with high transdermal performance. The cow milk nano-vesicle loading material with high transdermal performance has relatively good stability, is high in loading capacity of protein peptide, has a very good transdermal absorption promoting effect, and can well achieve the effects of resisting aging, improving skin elasticity, reducing fine wrinkles and promoting skin repair, regeneration and protection.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and particularly relates to a milk-derived nanovesicle loading with high transdermal performance, a preparation method thereof, and an application thereof. Background Art

[0002] Polypeptides and proteins are polymers of amino acids. A peptide is a short chain of amino acids. Naturally occurring human polypeptides are used for cell communication, such as protein regulation, cell proliferation, cell migration, inflammation, angiogenesis, and melanogenesis, which lead to a variety of physiological processes, including defense, immunity, stress, growth, homeostasis, and reproduction. In the late 1980s, the first copper peptide was incorporated into skin care products. Until the early 2000s, palmitoyl pentapeptide-4 was developed, and since then, many short, stable synthetic peptides have been studied and industrially developed, which play roles in extracellular matrix synthesis, pigmentation, innate immunity, and inflammation. These peptides are used for collagen stimulation, wound healing, "botulinum-like" smoothing of wrinkles, as well as antioxidant, antibacterial, and whitening effects.

[0003] Beauty peptides are a class of highly effective cosmetic active ingredients, but their transdermal ability is often limited. Past studies have shown that only beauty peptides that meet relatively stringent conditions have a certain transdermal ability: a molecular weight below 500 Da, a logarithm of the octanol / water partition coefficient between 1 and 3, a melting point below 200 °C, a water solubility > 1 mg / mL, and basically no polar centers in the molecular structure. Most peptides do not meet the above conditions. On the other hand, anti-aging, whitening, and other effects require active molecules to penetrate deep into the dermis to take effect. The poor transdermal effect of peptide products greatly limits their application in cosmetics and causes waste of raw materials. Summary of the Invention

[0004] The object of the present invention is to provide a milk-derived nanovesicle loading with high transdermal performance, a preparation method thereof, and an application thereof, which has good stability, a high loading capacity for protein peptides, and a good transdermal absorption promoting effect, can effectively play the roles of anti-aging, improving skin elasticity, reducing fine lines, promoting skin repair, regeneration, and protection, and has broad application prospects.

[0005] The technical solution of the present invention is realized as follows:

[0006] The present invention provides a preparation method of a milk nanovesicle loading with high transdermal performance. The pH of milk is adjusted and then it is allowed to stand and centrifuged to obtain protein bodies and supernatant. The supernatant is ultrafiltered to obtain milk extracellular vesicles, which are then subjected to heating and irradiation treatments and reacted with phytic acid and β-cyclodextrin to prepare a β-cyclodextrin / phytic acid / milk nanovesicle complex. The protein bodies are reacted with alkylated beauty peptides to prepare peptide-loaded protein bodies, and are embedded with the β-cyclodextrin / phytic acid / milk nanovesicle complex, vitamin E, and vitamin C to prepare a milk nanovesicle loading with high transdermal performance.

[0007] As a further improvement of the present invention, it includes the following steps:

[0008] S1. Adjust the pH value of milk, allow it to stand, centrifuge, collect the solid and the supernatant. The solid is the protein body. The supernatant is ultrafiltered, and the permeate is collected. After anion exchange chromatography, cation exchange chromatography, and molecular sieve chromatography, the flow-through obtained is a purified extracellular vesicle solution, which is freeze-dried to obtain milk extracellular vesicles.

[0009] S2. Add the milk extracellular vesicles to water, perform a heating treatment, and then perform a cobalt-60 irradiation treatment on it, and freeze-dry to obtain milk nanovesicles.

[0010] S3. Add the protein bodies to water, add EDC and NHS, stir and activate, add alkylated beauty peptides, stir and react, then adjust the pH value, allow it to stand, centrifuge, collect the solid, wash, and freeze-dry to obtain peptide-loaded protein bodies.

[0011] S4. Add the milk nanovesicles, phytic acid, β-cyclodextrin, and potassium dihydrogen phosphate to water, heat and stir, perform a hydrothermal reaction, add the product to a PBS buffer solution, stir, centrifuge, wash, and freeze-dry to obtain a β-cyclodextrin / phytic acid / milk nanovesicle complex.

[0012] S5. Dissolve soy phosphatidylcholine and lecithin in an ethanol solution, add the β-cyclodextrin / phytic acid / milk nanovesicle complex, peptide-loaded protein bodies, vitamin E, vitamin C, and water, stir and mix evenly, remove ethanol under reduced pressure, and freeze-dry to obtain a milk nanovesicle loading with high transdermal performance.

[0013] As a further improvement of the present invention, in step S1, the adjusted pH value is 4.5 - 4.8, the centrifugation conditions are 3000 - 8000g, the time is 3 - 5 min, and the ligand of the packing material for the anion exchange chromatography is a strong anion exchange ligand or a weak anion exchange ligand; the anion exchange ligand is one or a combination of several of quaternary ammonium groups, diethylaminoethyl, and diethylaminopropyl, the particle size range of the packing material is 15 - 300 μm, and the pore size range is 20 - 150 nm.

[0014] As a further improvement of the present invention, in step S2, the temperature of the heat treatment is 75 - 85 °C, the time is 25 - 35 min, and the dose of the irradiation is 5 - 15 KGy.

[0015] As a further improvement of the present invention, in step S3, the mass ratio of the proteosome, EDC, NHS, and the alkylated beauty peptide is 10 - 15:3 - 5:3 - 5:4 - 7, the time for stirring activation is 20 - 30 min, the time for the stirring reaction is 10 - 12 h, the adjusted pH value is 4.5 - 5, and the alkylated beauty peptide is selected from at least one of palmitoyl tripeptide - 1, palmitoyl pentapeptide - 4, palmitoyl tetrapeptide - 7, palmitoyl hexapeptide - 12, myristoyl hexapeptide - 5, palmitoyl hexapeptide - 15, palmitoyl dipeptide - 7, palmitoyl hexapeptide - 14, palmitoyl tripeptide - 5, palmitoyl tripeptide - 8, palmitoyl tetrapeptide - 10, palmitoyl tetrapeptide - 5, palmitoyl pentapeptide - 5, myristoyl pentapeptide - 4.

[0016] As a further improvement of the present invention, the alkylated beauty peptide is a mixture of palmitoyl tripeptide - 1 and palmitoyl hexapeptide - 14, and the mass ratio is 10:5 - 8.

[0017] As a further improvement of the present invention, in step S4, the mass ratio of the milk protein nanovesicles, phytic acid, β - cyclodextrin, and potassium dihydrogen phosphate is 10:3 - 4:4 - 6:0.2 - 0.3, the temperature for heating and stirring is 50 - 60 °C, the time is 1 - 2 h, the time is 8 - 10 h, and the pH of the PBS buffer is 8 - 8.5.

[0018] As a further improvement of the present invention, in step S5, the mass ratio of soy phosphatidylcholine, lecithin, β - cyclodextrin / phytic acid / milk protein nanovesicle complex, peptide - loaded proteosome, vitamin E, and vitamin C is 8 - 10:3 - 5:1 - 2:0.1 - 0.2:0.05 - 0.1:0.03 - 0.05.

[0019] The present invention further protects a milk protein nanovesicle loading product with high transdermal performance prepared by the above - mentioned preparation method.

[0020] The present invention further protects the application of the above - mentioned milk protein nanovesicle loading product with high transdermal performance in cosmetics.

[0021] The present invention has the following beneficial effects:

[0022] The present invention adjusts the pH of milk to the isoelectric point of proteins at 4.5 - 4.8, and after standing, the proteosome is collected, retaining the fat globule structure. This method reduces the mechanical damage to the fat globules. In addition, ultrafiltration separation is used to remove the fat globules, and purified extracellular vesicles are obtained through anion - exchange chromatography and molecular sieve chromatography.

[0023] The prepared extracellular vesicles of milk are subjected to heat treatment, which can denature the proteins in milk. Under heating, the spatial structure of protein molecules is destroyed and becomes loose, exposing more hydrophobic groups and polar groups, which is beneficial to the formation of lipid microspheres because the hydrophobic groups can interact with lipids and promote the aggregation and stability of lipid microspheres. The lipid oxidation reaction caused by irradiation can crosslink lipid molecules to form a more stable network structure. This crosslinking effect can enhance the mechanical strength and stability of lipid microspheres, prevent the microspheres from aggregating or rupturing during storage and use, and thus the prepared lipid microspheres have better stability.

[0024] In addition, the prepared proteosomes of the present invention are loaded with alkylated beauty peptides through dehydration condensation of amino groups and carboxyl groups. At the same time, the alkyl groups carried on the alkylated beauty peptides improve their compatibility in liposomes.

[0025] The present invention reacts the prepared milk nanovesicles with phytic acid and β-cyclodextrin. The cavity of β-cyclodextrin can effectively form a complex with the peptide-loaded proteosomes, improving the loading amount of the peptide-loaded proteosomes, and then embedding them in liposomes, improving the drug-loading capacity and biocompatibility of the prepared milk nanovesicle loading with high transdermal performance, promoting the transdermal absorption of beauty protein peptides, and being able to penetrate the phospholipid bilayer of the skin well and enter epidermal cells, thus exerting a better beauty effect.

[0026] Palmitoyl tripeptide-1 can stimulate skin fibroblasts to synthesize extracellular matrix components such as collagen, fibronectin, and hyaluronic acid. By mimicking the function of natural signal peptides, it activates the signal transduction pathway of skin cells, thereby promoting collagen synthesis, making the skin more firm and elastic. At the same time, it has good antioxidant effects, reduces the appearance of wrinkles and fine lines, and enhances the skin's barrier function.

[0027] Palmitoyl hexapeptide-14 can stimulate collagen synthesis, increase skin elasticity and firmness, break down collagen, cause skin aging and wrinkle formation, promote the proliferation of fibroblasts, help repair damaged skin tissues, and accelerate the wound healing process.

[0028] Palmitoyl tripeptide-1 activates skin cells by mimicking natural signal peptides, while palmitoyl hexapeptide-14 stimulates collagen synthesis by binding to specific receptors. Their combined action can more effectively increase the content of collagen, make the skin more firm and elastic. At the same time, both can inhibit the activity of MMPs, thereby reducing the breakdown of collagen, repairing and protecting the skin, and more comprehensively promoting skin repair, regeneration, and protection, achieving a better synergistic beauty effect.

[0029] The milk-derived nanovesicle loading preparation with high transdermal performance prepared by the present invention has good stability, a high loading capacity for protein peptides, and excellent transdermal absorption promoting effects. It can effectively play roles in anti-aging, improving skin elasticity, reducing fine lines, and promoting skin repair, regeneration and protection, and thus has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0031] Figure 1 TEM image of the milk-derived extracellular vesicles prepared in Example 1;

[0032] Figure 2 TEM image of the milk-derived nanovesicles prepared in Example 1;

[0033] Figure 3 Particle size distribution characterization diagram of the milk-derived nanovesicle loading preparation with high transdermal performance prepared in Example 1;

[0034] Figure 4 Particle number characterization diagram of the milk-derived nanovesicle loading preparation with high transdermal performance;

[0035] Figure 5 Zeta potential characteristic diagram of the milk-derived nanovesicle loading preparation with high transdermal performance prepared in Example 1;

[0036] Figure 6 Comparison diagram of the human experiment results of the milk-derived nanovesicle loading preparation with high transdermal performance prepared in Example 1. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] The following will clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some, rather than all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0038] EDC, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; NHS, N-hydroxysuccinimide.

[0039] Example 1

[0040] This embodiment provides a method for preparing a milk-derived nanovesicle loading with high transdermal performance, which includes the following steps:

[0041] S1. Adjust the pH value of raw milk to 4.5, let it stand, centrifuge at 3000g for 5 min, collect the solid and the supernatant. The solid is the proteosome, ultrafilter the supernatant, collect the permeate, and obtain the purified extracellular vesicle solution as the flow-through fraction through anion exchange chromatography and molecular sieve chromatography. Figure 1 Figure 5 shows the TEM image of the milk-derived extracellular vesicles prepared, presenting a typical saucer shape.

[0042] The exchange ligand of the packing material for the anion exchange chromatography is diethylaminoethyl.

[0043] S2. Add 1 g of milk-derived extracellular vesicles to 50 mL of water, heat to 75 °C, treat for 35 min, and then perform cobalt-60 irradiation treatment with a dose of 5 kGy to obtain milk-derived nanovesicles. Figure 2 Figure 13 shows the TEM image of the milk-derived nanovesicles prepared, and their morphology is not much different from that of the milk-derived extracellular vesicles.

[0044] S3. Add 1 g of proteosome to 100 mL of water, add 0.3 g of EDC and 0.3 g of NHS, stir and activate for 20 min, add 0.4 g of alkylated beauty peptide, stir and react for 10 h, then adjust the pH value to 4.5, let it stand, centrifuge, collect the solid, and wash to obtain the peptide-loaded proteosome.

[0045] The alkylated beauty peptide is a mixture of palmitoyl tripeptide-1 and palmitoyl hexapeptide-14 with a mass ratio of 10:5.

[0046] S4. Add 10 g of milk-derived nanovesicles, 3 g of phytic acid, 4 g of β-cyclodextrin, and 0.2 g of potassium dihydrogen phosphate to 200 mL of water, heat to 50 °C, stir for 1 h, add the product to 200 mL of PBS solution with pH = 8, stir, centrifuge, wash, and freeze-dry to obtain the β-cyclodextrin / phytic acid / milk-derived nanovesicle complex.

[0047] S5. Dissolve 8 g of soy phosphatidylcholine and 3 g of lecithin in 100 mL of ethanol solution, add 1 g of β-cyclodextrin / phytic acid / milk-derived nanovesicle complex, 0.1 g of peptide-loaded proteosome, 0.05 g of vitamin E, 0.03 g of vitamin C, and 200 mL of water, stir and mix evenly, remove ethanol under reduced pressure, and freeze-dry to obtain the milk-derived nanovesicle loading with high transdermal performance. Figure 3 Figure 27 shows the particle size distribution characterization diagram of the milk-derived nanovesicle loading with high transdermal performance, and its median particle size is 83 nm. Figure 4 Figure 29 shows the particle number characterization diagram of the milk-derived nanovesicle loading with high transdermal performance. Figure 5Zeta potential characteristic diagram of the prepared milk-derived nanovesicle loading with high transdermal performance, with its Zeta potential being -17.25 mV respectively.

[0048] The prepared milk-derived nanovesicle loading with high transdermal performance was used for a 52-year-old female with fine lines at the corners of the eyes above mild degree for a period of 7 days. The milk-derived nanovesicle loading with high transdermal performance was made into a sample solution of 0.01 mg / mL and compared with commercially available similar products for trial use. It was massaged until absorbed. Other anti-wrinkle skin care products were discontinued during the experiment. The test site was the skin around the eyes. After cleansing the face every morning and evening, the product was taken and applied to the skin around the eyes on the test side, and gently pressed to promote the absorption of the product. It was used once in the morning and once in the evening. The results are shown in Figure 6 , as can be seen from the figure, after using the milk-derived nanovesicle loading with high transdermal performance of the present invention, the wrinkles around the corners of the eyes of this female were significantly lightened, which was significantly better than commercially available similar products.

[0049] Example 2

[0050] This example provides a preparation method of a milk-derived nanovesicle loading with high transdermal performance, including the following steps:

[0051] S1. Adjust the pH value of raw milk to 4.8, let it stand, centrifuge at 8000 g for 3 min, collect the solid and the supernatant. The solid is the proteosome, and the supernatant is ultrafiltered. The permeate is collected and, through anion exchange chromatography and molecular sieve chromatography, the flow-through obtained is the purified extracellular vesicle solution;

[0052] The exchange ligand of the filler for the anion exchange chromatography is diethylaminopropyl;

[0053] S2. Add 50 mL of water to 1 g of milk extracellular vesicles, heat to 85 °C, treat for 25 min, and then perform cobalt-60 irradiation treatment on it with a dose of 15 kGy to obtain milk-derived nanovesicles;

[0054] S3. Add 1.5 g of proteosome to 100 mL of water, add 0.5 g of EDC and 0.5 g of NHS, stir and activate for 30 min, add 0.7 g of alkylated beauty peptide, stir and react for 12 h, then adjust the pH value to 5, let it stand, centrifuge, collect the solid, and wash to obtain the peptide-loaded proteosome;

[0055] The alkylated beauty peptide is a mixture of palmitoyl tripeptide-1 and palmitoyl hexapeptide-14, with a mass ratio of 10:8;

[0056] S4. 10 g of milk nanovesicles, 4 g of phytic acid, 6 g of β-cyclodextrin, and 0.3 g of potassium dihydrogen phosphate were added to 200 mL of water, heated to 60°C, and stirred for 2 h. The product was then added to 200 mL of PBS (pH 8.5), stirred, centrifuged, washed, and freeze-dried to prepare a β-cyclodextrin / phytic acid / milk nanovesicle complex.

[0057] S5. Dissolve 10 g of soybean phosphatidylcholine and 5 g of lecithin in 100 mL of ethanol solution, add 2 g of β-cyclodextrin / phytic acid / milk nanovesicle complex, 0.2 g of peptide-loaded proteosomes, 0.1 g of vitamin E, 0.05 g of vitamin C, and 200 mL of water, stir and mix thoroughly, remove ethanol under reduced pressure, and freeze-dry to obtain a milk nanovesicle loading with high transdermal performance.

[0058] Example 3

[0059] This embodiment provides a method for preparing a milk nanocapsule loading material with high transdermal performance, comprising the following steps:

[0060] S1. Raw milk was adjusted to a pH of 4.6, allowed to stand, and centrifuged at 5000 g for 4 minutes. The solid and supernatant were collected. The solid was the protein bodies. The supernatant was ultrafiltered, and the permeate was collected. The permeate was subjected to anion exchange chromatography and molecular sieve chromatography to obtain the purified extracellular vesicle solution.

[0061] The exchange ligand of the filler of the anion exchange chromatography is a quaternary ammonium group;

[0062] S2. 1 g of milk extracellular vesicles was added to 50 mL of water, heated to 80°C for 30 minutes, and then irradiated with cobalt-60 at a dose of 10 kGy to obtain milk nanovesicles.

[0063] S3. 1.2 g of protein body was added to 100 mL of water, followed by 0.4 g of EDC and 0.4 g of NHS. The mixture was stirred and activated for 25 minutes. 0.55 g of alkylated beauty peptide was added and stirred for 11 hours. The pH was then adjusted to 4.7, the mixture was allowed to stand, centrifuged, and the solid was collected and washed to obtain peptide-loaded protein body.

[0064] The alkylated beauty peptide is a mixture of palmitoyl tripeptide-1 and palmitoyl hexapeptide-14, with a mass ratio of 10:7;

[0065] S4. 10 g of milk nanovesicles, 3.5 g of phytic acid, 5 g of β-cyclodextrin, and 0.25 g of potassium dihydrogen phosphate were added to 200 mL of water, heated to 55°C, and stirred for 1.5 h. The product was then added to 200 mL of PBS (pH 8.2), stirred, centrifuged, washed, and freeze-dried to prepare a β-cyclodextrin / phytic acid / milk nanovesicle complex.

[0066] S5. Dissolve 9 g of soy phosphatidylcholine and 4 g of lecithin in 100 mL of ethanol solution, add 1.2 g of β-cyclodextrin / phytic acid / milk nanovesicle complex, 0.15 g of peptide carrier, 0.07 g of vitamin E, 0.04 g of vitamin C and 200 mL of water, stir and mix evenly, remove ethanol under reduced pressure, and freeze-dry to obtain a milk nanovesicle loading with high transdermal performance.

[0067] Example 4

[0068] Compared with Example 3, the difference is that the alkylated beauty peptide is palmitoyl tripeptide-1.

[0069] Example 5

[0070] Compared with Example 3, the difference is that the alkylated beauty peptide is palmitoyl hexapeptide-14.

[0071] Comparative Example 1

[0072] Compared with Example 3, the difference is that the acid precipitation treatment was not carried out in step S1.

[0073] Specifically as follows:

[0074] S1. Centrifuge 5000 g of raw milk for 4 min, collect the solid and the supernatant. The solid is the protein body, and the supernatant is ultrafiltered. Collect the permeate, and through anion exchange chromatography and molecular sieve chromatography, the flow-through obtained is the purified extracellular vesicle solution;

[0075] The exchange ligand of the packing material for the anion exchange chromatography is a quaternary ammonium group.

[0076] Comparative Example 2

[0077] Compared with Example 3, the difference is that the heating treatment was not carried out in step S2.

[0078] Specifically as follows:

[0079] S2. Add 1 g of milk extracellular vesicles to 50 mL of water, and perform cobalt-60 irradiation treatment on it with a dose of 10 KGy to obtain milk nanovesicles.

[0080] Comparative Example 3

[0081] Compared with Example 3, the difference is that the irradiation treatment was not carried out in step S2.

[0082] Specifically as follows:

[0083] S2. Add 1 g of milk extracellular vesicles to 50 mL of water, heat to 80 °C, treat for 30 min, and freeze-dry to obtain milk nanovesicles.

[0084] Comparative Example 4

[0085] Compared with Example 3, the difference lies in that step S2 was not carried out.

[0086] Specifically as follows:

[0087] S1. Adjust the pH value of raw milk to 4.6, let it stand, centrifuge at 5000g for 4 min, collect the solid and the supernatant. The solid is the proteosome, and the supernatant is ultrafiltered. Collect the permeate, and through anion exchange chromatography and molecular sieve chromatography, the flow-through obtained is the purified extracellular vesicle solution;

[0088] The exchange ligand of the packing material for the anion exchange chromatography is a quaternary ammonium group;

[0089] S2. Add 1.2 g of proteosome to 100 mL of water, add 0.4 g of EDC and 0.4 g of NHS, stir and activate for 25 min, add 0.55 g of alkylated beauty peptide, stir and react for 11 h, then adjust the pH value to 4.7, let it stand, centrifuge, collect the solid, wash it, and obtain the peptide-loaded proteosome;

[0090] The alkylated beauty peptide is a mixture of palmitoyl tripeptide-1 and palmitoyl hexapeptide-14, and the mass ratio is 10:7;

[0091] S3. Add 10 g of milk extracellular vesicles, 3.5 g of phytic acid, 5 g of β-cyclodextrin, and 0.25 g of potassium dihydrogen phosphate to 200 mL of water, heat to 55 °C, stir for 1.5 h, add the product to 200 mL of PBS solution with pH = 8.2, stir, centrifuge, wash, and freeze-dry to obtain the β-cyclodextrin / phytic acid / milk nanovesicle complex;

[0092] S4. Dissolve 9 g of soy phosphatidylcholine and 4 g of lecithin in 100 mL of ethanol solution, add 1.2 g of β-cyclodextrin / phytic acid / milk nanovesicle complex, 0.15 g of peptide-loaded proteosome, 0.07 g of vitamin E, 0.04 g of vitamin C, and 200 mL of water, stir and mix evenly, remove ethanol under reduced pressure, and freeze-dry to obtain the milk nanovesicle loading with high transdermal performance.

[0093] Comparative Example 5

[0094] Compared with Example 3, the difference lies in that step S3 was not carried out.

[0095] Specifically as follows:

[0096] S1. Adjust the pH value of raw milk to 4.6, let it stand, centrifuge at 5000 g for 4 min, collect the solid and the supernatant. The solid is the protein body, ultrafilter the supernatant, collect the permeate, and obtain the purified extracellular vesicle solution through anion exchange chromatography and molecular sieve chromatography;

[0097] The exchange ligand of the packing material for the anion exchange chromatography is a quaternary ammonium group;

[0098] S2. Add 50 mL of water to 1 g of milk extracellular vesicles, heat to 80 °C, treat for 30 min, and then perform cobalt-60 irradiation treatment with a dose of 10 kGy to obtain milk nanovesicles;

[0099] S3. Add 10 g of milk nanovesicles, 3.5 g of phytic acid, 5 g of β-cyclodextrin, and 0.25 g of potassium dihydrogen phosphate to 200 mL of water, heat to 55 °C, stir for 1.5 h, add the product to 200 mL of PBS solution with pH = 8.2, stir, centrifuge, wash, and freeze-dry to prepare the β-cyclodextrin / phytic acid / milk nanovesicle complex;

[0100] S4. Dissolve 9 g of soy phosphatidylcholine and 4 g of lecithin in 100 mL of ethanol solution, add 1.2 g of β-cyclodextrin / phytic acid / milk nanovesicle complex, 0.15 g of protein body, 0.07 g of vitamin E, 0.04 g of vitamin C, and 200 mL of water, stir and mix evenly, remove ethanol under reduced pressure, and freeze-dry to prepare a milk nanovesicle loading with high transdermal performance.

[0101] Comparative Example 6

[0102] Compared with Example 3, the difference is that step S4 is not carried out.

[0103] Specifically as follows:

[0104] S1. Adjust the pH value of raw milk to 4.6, let it stand, centrifuge at 5000 g for 4 min, collect the solid and the supernatant. The solid is the protein body, ultrafilter the supernatant, collect the permeate, and obtain the purified extracellular vesicle solution through anion exchange chromatography and molecular sieve chromatography;

[0105] The exchange ligand of the packing material for the anion exchange chromatography is a quaternary ammonium group;

[0106] S2. Add 50 mL of water to 1 g of milk extracellular vesicles, heat to 80 °C, treat for 30 min, and then perform cobalt-60 irradiation treatment with a dose of 10 kGy to obtain milk nanovesicles;

[0107] S3. Add 1.2 g of proteosome to 100 mL of water, add 0.4 g of EDC and 0.4 g of NHS, stir and activate for 25 min, add 0.55 g of alkylated beauty peptide, stir and react for 11 h, then adjust the pH value to 4.7, let stand, centrifuge, collect the solid, wash, and obtain the peptide-loaded proteosome;

[0108] The alkylated beauty peptide is a mixture of palmitoyl tripeptide-1 and palmitoyl hexapeptide-14, and the mass ratio is 10:7;

[0109] S4. Dissolve 9 g of soy phosphatidylcholine and 4 g of lecithin in 100 mL of ethanol solution, add 1.2 g of milk nanovesicles, 0.15 g of peptide-loaded proteosome, 0.07 g of vitamin E, 0.04 g of vitamin C and 200 mL of water, stir and mix evenly, remove ethanol under reduced pressure, and freeze-dry to obtain a milk nanovesicle loading with high transdermal performance.

[0110] Comparative Example 7

[0111] Compared with Example 3, the difference is that liposome embedding in step S5 is not carried out.

[0112] Specifically as follows:

[0113] S1. Adjust the pH value of raw milk to 4.6, let stand, centrifuge at 5000 g for 4 min, collect the solid and the supernatant. The solid is proteosome, and the supernatant is ultrafiltered. Collect the permeate, and after anion exchange chromatography and molecular sieve chromatography, the flow-through solution obtained is the purified extracellular vesicle solution;

[0114] The exchange ligand of the filler for the anion exchange chromatography is quaternary ammonium group;

[0115] S2. Add 1 g of milk extracellular vesicles to 50 mL of water, heat to 80 °C, treat for 30 min, and then perform cobalt-60 irradiation treatment on it with a dose of 10 kGy to obtain milk nanovesicles;

[0116] S3. Add 1.2 g of proteosome to 100 mL of water, add 0.4 g of EDC and 0.4 g of NHS, stir and activate for 25 min, add 0.55 g of alkylated beauty peptide, stir and react for 11 h, then adjust the pH value to 4.7, let stand, centrifuge, collect the solid, wash, and obtain the peptide-loaded proteosome;

[0117] The alkylated beauty peptide is a mixture of palmitoyl tripeptide-1 and palmitoyl hexapeptide-14, and the mass ratio is 10:7;

[0118] S4. Add 10 g of milk nanovesicles, 3.5 g of phytic acid, 5 g of β-cyclodextrin, and 0.25 g of potassium dihydrogen phosphate into 200 mL of water, heat to 55 °C, stir for 1.5 h, add the product into 200 mL of PBS solution with pH = 8.2, stir, centrifuge, wash, and freeze-dry to obtain the β-cyclodextrin / phytic acid / milk nanovesicle complex;

[0119] S5. Stir and mix 1.2 g of β-cyclodextrin / phytic acid / milk nanovesicle complex, 0.15 g of peptide-loaded proteosome, 0.07 g of vitamin E, and 0.04 g of vitamin C for 10 min to obtain the milk nanovesicle loading with high transdermal performance.

[0120] Test Example 1 Antioxidant Test

[0121] Test samples: Prepare the milk nanovesicle loading with high transdermal performance obtained in Examples 1-5 and Comparative Examples 1-7 into a solution of 0.01 mg / mL. Use vitamin C at the same concentration as the positive control.

[0122] Experimental method: Take 1 mL of DPPH (2,2-diphenyl-1-picrylhydrazyl) working solution (0.0082 g of DPPH powder dissolved in 100 mL of ethanol), add 200 μL of the test substance, react in the dark for 30 min, and the absorbance reading at a wavelength of 517 nm is A1. Take 1 mL of DPPH working solution, add 200 μL of distilled water, react in the dark for 30 min, and the absorbance reading at a wavelength of 517 nm is A2. Take 1 mL of ethanol, add 200 μL of the test substance, react in the dark for 30 min, and the absorbance reading at a wavelength of 517 nm is A3.

[0123] Scavenging rate (%) = [(A2 + A3 - A1) / A2] × 100%

[0124] The results are shown in Table 1.

[0125] Table 1

[0126]

[0127] As can be seen from the above table, the milk nanovesicle loading with high transdermal performance obtained in Examples 1-3 of the present invention has good antioxidant performance.

[0128] Test Example 2 Human skin patch and allergy experiment

[0129] Test samples: The milk nanovesicle loading with high transdermal performance obtained in Examples 1-5 and Comparative Examples 1-7 of the present invention is prepared into a sample solution of 1 mg / mL.

[0130] Experimental method: Subjects aged 22 - 54 were selected. Among them, they were randomly divided into 12 groups with 8 people in each group. Among them, a 5 cm × 5 cm area on the flexor side of the right forearm of the subjects was marked and disinfected medically. The marked area was traumatized, 36 micro - needles were inserted into the sample solution for 30 s, and the residual liquid was gently wiped with a cotton ball moistened with normal saline, and the marked area was kept freeze - dried; the test samples were respectively placed into the wells of the patch test device numbered 1 - 12, with a dosage of 10 - 20 μL. Bathing was prohibited during the test period, and a normal reaction was a negative reaction. The sample solution was distributed to the subjects and applied to the face, neck and back of the ears of the subjects. After 10 min, the skin condition of the subjects was observed. The experimental results are shown in Table 2.

[0131] Table 2

[0132]

[0133] As can be seen from the above table, the milk - derived nanovesicle loading with high transdermal performance prepared by the present invention is safe to use and has no allergic reactions.

[0134] Test Example 3: Experiment on promoting the synthesis of type III collagen

[0135] Test samples: The milk - derived nanovesicle loading with high transdermal performance prepared in Examples 1 - 5 and Comparative Examples 1 - 7 were made into a sample solution with a concentration of 0.01 mg / mL.

[0136] Experimental method: Human fibroblasts (Merck Life) cultured in DMEM (Dulbecco's Modified Eagle Medium) medium containing 10% FBS (fetal bovine serum) were inoculated into 96 - well plates. After the cells adhered, the medium was exchanged with DMEM medium containing 0.2 wt% FBS and 250 μmol / L magnesium ascorbyl phosphate. 100 μL of the sample solution was added respectively (100 μL of deionized water was added to the control group), and after mixing evenly, the supernatant was collected after culturing for 72 h, centrifuged, and the content of type III collagen in the obtained supernatant was measured using a kit, and the promotion rate of type III collagen synthesis was calculated. The results are shown in Table 3.

[0137] Promotion rate of type III collagen synthesis (%) = (A1 - A0) / A0 × 100%

[0138] A1: The amount of type III collagen produced after adding the sample solution;

[0139] A0: The amount of type III collagen produced without adding the sample solution.

[0140] Table 3

[0141]

[0142] As can be seen from the above table, the milk-derived nanovesicle loading with high transdermal performance prepared in Examples 1-3 of the present invention has an obvious effect of promoting the generation of type III collagen.

[0143] Test Example 4

[0144] Healthy female volunteers were selected as subjects, aged 38-54 years old, with fine lines at the corners of the eyes above mild degree. The exclusion criteria for the subjects met the inclusion and exclusion criteria of the "Diagnostic Criteria and Treatment Principles for Cosmetic Contact Dermatitis". After cleansing the face every morning and evening, 1 mL of the milk-derived nanovesicle loading with high transdermal performance prepared in Examples 1-5 and Comparative Examples 1-7 was used each time to prepare a sample solution of 0.01 mg / mL and a commercially available similar product, and massaged until absorbed. Other anti-wrinkle skin care products were discontinued during the experiment. The test site was the skin around the eyes. After cleansing the face every morning and evening, the product was applied to the skin around the eyes on the test side, and gently pressed to promote the absorption of the product, and used once in the morning and once in the evening. There were 10 people in each group. Skin adverse reactions such as skin erythema, itching, stinging, burning, etc. occurring during the test and evaluation period were recorded, and serious adverse reactions should be reported in a timely manner. During the experimental test, the test site was cleaned, and after sitting still for 30 minutes in a constant temperature and humidity environment at 25±1.0°C and relative humidity 55%±5%RH, data was collected. The cycle was 28 days, and skin tests were performed on the 0th day and the 28th day respectively.

[0145] (1)Skin moisture content

[0146] A skin moisture meter was used to measure the skin moisture content of the test and evaluation sites of the subjects, and it was measured 3 times in total, and the average value was taken. By comparing the changes in the skin moisture content values before and after product use and between the test side and the control side.

[0147] (2)Skin elasticity

[0148] A skin elasticity meter was used to measure the skin elasticity of the test and evaluation sites of the subjects, and it was measured 3 times in total, and the average value was taken.

[0149] (3)Analysis of the area of crow's feet

[0150] A facial image analyzer was used to collect and analyze the area of crow's feet on the face, and the anti-wrinkle efficacy of the product was evaluated by comparing the changes in the area of crow's feet before and after product use.

[0151] Change rate of crow's feet = (test value on the 28th day - test value on the 0th day) / test value on the 0th day × 100%

[0152] The results are shown in Table 4.

[0153] Table 4

[0154]

[0155] As can be seen from the above table, the milk-derived nanovesicle-loaded formulations with high transdermal performance prepared in Examples 1-3 of the present invention can significantly improve skin elasticity, reduce crow's feet, and enhance skin moisturizing ability.

[0156] Test Example 5

[0157] An in vitro skin transdermal experiment was conducted on the milk-derived nanovesicle-loaded formulations with high transdermal performance prepared in Examples 1-3 and Comparative Examples 1-4, 6-7 according to the transdermal experiment method of GBT27818-2011.

[0158] Using the back skin of domestic pigs as the tool, equal amounts of the milk-derived nanovesicle-loaded formulations with high transdermal performance and free palmitoyl tripeptide-1 were added to the supply pool respectively, and a 24-hour transdermal experiment was carried out. The retention rate of palmitoyl tripeptide-1 in the skin was detected by liquid chromatography-mass spectrometry.

[0159] The results are shown in Table 5.

[0160] Table 5

[0161]

[0162] As can be seen from the above table, the milk-derived nanovesicle-loaded formulations with high transdermal performance prepared in Examples 1-3 of the present invention can significantly improve the transdermal absorption ability of palmitoyl tripeptide-1, which is superior to palmitoyl tripeptide-1 itself. The milk-derived nanovesicle-loaded formulations with high transdermal performance can significantly enhance its ability to penetrate the stratum corneum, thereby improving the bioavailability of palmitoyl tripeptide-1.

[0163] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of a milk-derived nanovesicle loading with high transdermal performance, characterized in that, Adjust the pH of milk, let it stand, and centrifuge to obtain protein bodies and supernatant. Ultrafilter the supernatant to obtain extracellular vesicles of milk. Then, perform heating and irradiation treatments, and react with phytic acid and β-cyclodextrin to prepare β-cyclodextrin / phytic acid / milk nanovesicle complex; react the protein bodies with alkylated beauty peptides to prepare peptide-loaded protein bodies, and encapsulate them with β-cyclodextrin / phytic acid / milk nanovesicle complex, vitamin E, and vitamin C to obtain a milk nanovesicle loading with high transdermal performance.

2. The preparation method according to claim 1, characterized in that, It includes the following steps: S1. Adjust the pH value of milk, let it stand, centrifuge, and collect the solid and supernatant. The solid is protein bodies. Ultrafilter the supernatant and collect the permeate. After anion exchange chromatography and molecular sieve chromatography, the flow-through obtained is a purified extracellular vesicle solution, which is freeze-dried to obtain extracellular vesicles of milk. S2. Add water to the extracellular vesicles of milk, perform a heating treatment, and then perform a cobalt-60 irradiation treatment on it, and freeze-dry to obtain milk nanovesicles. S3. Add protein bodies to water, add EDC and NHS, stir to activate, add alkylated beauty peptides, stir and react, then adjust the pH value, let it stand, centrifuge, collect the solid, wash, and freeze-dry to obtain peptide-loaded protein bodies. S4. Add milk nanovesicles, phytic acid, β-cyclodextrin, and potassium dihydrogen phosphate to water, heat and stir. Add the product to PBS buffer, stir, centrifuge, wash, and freeze-dry to obtain β-cyclodextrin / phytic acid / milk nanovesicle complex. S5. Dissolve soy phosphatidylcholine and lecithin in an ethanol solution, add β-cyclodextrin / phytic acid / milk nanovesicle complex, peptide-loaded protein bodies, vitamin E, vitamin C, and water, stir and mix evenly, remove ethanol under reduced pressure, and freeze-dry to obtain a milk nanovesicle loading with high transdermal performance.

3. The preparation method according to claim 2, characterized in that, In step S1, the adjusted pH value is 4.5 - 4.8, the centrifugation conditions are 3000 - 8000 g for 3 - 5 min, and the ligand of the filler for anion exchange chromatography is a strong anion exchange ligand or a weak anion exchange ligand; the anion exchange ligand is one or a combination of quaternary ammonium groups, diethylaminoethyl, and diethylaminopropyl. The filler particle size range is 15 - 300 μm, and the pore size range is 20 - 150 nm.

4. The preparation method according to claim 2, wherein In step S2, the temperature of the heating treatment is 75 - 85 °C for 25 - 35 min, and the irradiation dose is 5 - 15 KGy.

5. The preparation method according to claim 2, wherein, In step S3, the mass ratio of the protein bodies, EDC, NHS, and alkylated beauty peptides is 10 - 15:3 - 5:3 - 5:4 - 7. The stirring activation time is 20 - 30 min, the stirring reaction time is 10 - 12 h, the adjusted pH value is 4.5 - 5, and the alkylated beauty peptides are selected from at least one of palmitoyl tripeptide-1, palmitoyl pentapeptide-4, palmitoyl tetrapeptide-7, palmitoyl hexapeptide-12, myristoyl hexapeptide-5, palmitoyl hexapeptide-15, palmitoyl dipeptide-7, palmitoyl hexapeptide-14, palmitoyl tripeptide-5, palmitoyl tripeptide-8, palmitoyl tetrapeptide-10, palmitoyl tetrapeptide-5, palmitoyl pentapeptide-5, and myristoyl pentapeptide-4.

6. The preparation method according to claim 5, characterized in that, The alkylated beauty peptide is a mixture of palmitoyl tripeptide-1 and palmitoyl hexapeptide-14, and the mass ratio is 10:5-8.

7. The preparation method according to claim 2, characterized in that, In step S4, the mass ratio of the milk nanovesicles, phytic acid, β-cyclodextrin, and potassium dihydrogen phosphate is 10:3-4:4-6:0.2-0.

3. The temperature of the heating and stirring is 50-60°C, the time is 1-2h, and the time is 8-10h. The pH of the PBS buffer solution is 8-8.

5.

8. The preparation method according to claim 2, characterized in that, In step S5, the mass ratio of soy lecithin, lecithin, β-cyclodextrin / phytic acid / milk nanovesicle complex, peptide-loaded proteosome, vitamin E, and vitamin C is 8-10:3-5:1-2:0.1-0.2:0.05-0.1:0.03-0.

05.

9. A milk nanovesicle loading with high transdermal performance prepared by the preparation method according to any one of claims 1-8.

10. Use of a milk nanovesicle loading with high transdermal performance according to claim 9 in cosmetics.

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