A milk nanocapsule loading material with high transdermal performance and its preparation method and application

Abstract

The present invention proposes a milk nano-vesicle loading material with high transdermal performance and its preparation method and application. The milk is adjusted to pH and centrifuged to obtain protein bodies and supernatant. The supernatant is ultrafiltered to obtain milk extracellular vesicles. The milk nano-vesicles are heated and irradiated to obtain milk nano-vesicles. The protein bodies are reacted with phytic acid and β-cyclodextrin to obtain β-cyclodextrin / phytic acid / milk nano-vesicle complexes. The protein bodies are reacted with alkylated beauty peptides to obtain peptide-loaded protein bodies. The protein bodies are embedded with β-cyclodextrin / phytic acid / milk nano-vesicle complexes, vitamin E, and vitamin C to obtain a milk nano-vesicle loading material with high transdermal performance. The milk nano-vesicle loading material with high transdermal performance has good stability, a high loading capacity for protein peptides, and a good transdermal absorption-promoting effect. It can effectively resist aging, improve skin elasticity, reduce fine lines, and promote skin repair, regeneration, and protection.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to a milk nanocapsule loading material with high transdermal performance, a preparation method thereof, and an application thereof. Background Art

[0002] Peptides and proteins are amino acid polymers. Peptides are short amino acid chains. Naturally occurring human peptides are used for cellular communication, such as protein regulation, cell proliferation, cell migration, inflammation, angiogenesis, and melanogenesis, contributing to a wide variety of physiological processes, including defense, immunity, stress, growth, homeostasis, and reproduction. In the late 1980s, the first copper peptide was incorporated into skincare products. It wasn't until the early 2000s that palmitoyl pentapeptide-4 was developed. Since then, research and industry have developed numerous short, stable synthetic peptides that play roles in extracellular matrix synthesis, pigmentation, innate immunity, and inflammation. These peptides are used for collagen stimulation, wound healing, "botulinum-like" wrinkle smoothing, and for antioxidant, antimicrobial, and whitening effects.

[0003] Beauty peptides are a highly effective class of active cosmetic ingredients, but their transdermal penetration is often limited. Previous studies have shown that only peptides that meet relatively stringent criteria have a reasonable degree of transdermal penetration: a molecular weight below 500 Da, an octanol / water partition coefficient between 1 and 3, a melting point below 200°C, a water solubility >1 mg / mL, and a molecular structure largely devoid of polar centers. However, most peptides fail to meet these requirements. Furthermore, anti-aging and whitening effects require the active molecules to penetrate deeply into the dermis to be effective. The poor transdermal penetration of peptide products has significantly limited their application in cosmetics and resulted in waste of raw materials. Summary of the Invention

[0004] The purpose of the present invention is to propose a milk nanocapsule loading material with high transdermal performance and its preparation method and application. It has good stability, a high loading capacity for protein peptides, and has a good transdermal absorption promoting effect. It can play a good anti-aging role, improve skin elasticity, reduce fine lines, promote skin repair, regeneration and protection, and has broad application prospects.

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

[0006] The present invention provides a method for preparing a milk nano-vesicle loading material with high transdermal performance. The method comprises the following steps: adjusting the pH of milk and statically centrifuging the milk to obtain protein bodies and a supernatant; ultrafiltration of the supernatant to obtain milk extracellular vesicles; heating and irradiation treatment; and reacting the supernatant with phytic acid and β-cyclodextrin to obtain a β-cyclodextrin / phytic acid / milk nano-vesicle complex; reacting the protein bodies with an alkylated beauty peptide to obtain a peptide-loaded protein body; and encapsulating the protein body with the β-cyclodextrin / phytic acid / milk nano-vesicle complex, vitamin E, and vitamin C to obtain a milk nano-vesicle loading material with high transdermal performance.

[0007] As a further improvement of the present invention, the following steps are included:

[0008] S1. The milk is then pH-adjusted, allowed to stand, and centrifuged to collect the solid and supernatant, where the solid is the protein body. The supernatant is ultrafiltered, and the permeate is collected. The permeate is purified by anion exchange chromatography, cation exchange chromatography, and molecular sieve chromatography to obtain a flow-through solution, which is the purified extracellular vesicle solution. The solution is then freeze-dried to produce milk extracellular vesicles.

[0009] S2. The milk extracellular vesicles were added to water, heated, and then irradiated with cobalt 60 and freeze-dried to obtain milk nanovesicles;

[0010] S3. Add the protein body to water, add EDC and NHS, stir to activate, add the alkylated beauty peptide, stir to react, then adjust the pH, let stand, centrifuge, collect the solid, wash, and freeze-dry to produce the peptide-loaded protein body;

[0011] S4. Milk nanovesicles, phytic acid, β-cyclodextrin, and potassium dihydrogen phosphate were added to water, heated and stirred, and a hydrothermal reaction was performed. The product was added to PBS buffer, stirred, centrifuged, washed, and freeze-dried to obtain a β-cyclodextrin / phytic acid / milk nanovesicle complex;

[0012] S5. Dissolve soybean phosphatidylcholine and lecithin in an ethanol solution, add β-cyclodextrin / phytic acid / bovine milk nanovesicle complex, peptide-loaded proteosomes, vitamin E, vitamin C, and water, stir and mix thoroughly, 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, the pH value is adjusted to 4.5-4.8 in step S1, the centrifugation conditions are 3000-8000g, the time is 3-5min, the ligand of the filler of 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 quaternary ammonium group, diethylaminoethyl group, and diethylaminopropyl group, the filler particle size range is 15-300μm, and the pore size range is 20-150nm.

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

[0015] As a further improvement of the present invention, the mass ratio of the proteosome, EDC, NHS and alkylated beauty peptide in step S3 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 pH value is adjusted to 4.5-5, and the alkylated beauty peptide is selected from 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 at least one of 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, with a mass ratio of 10:5-8.

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

[0018] As a further improvement of the present invention, the mass ratio of soybean phosphatidylcholine, lecithin, β-cyclodextrin / phytic acid / milk nanovesicle complex, peptide-loaded proteosome, vitamin E, and vitamin C in step S5 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 nanocapsule loading material with high transdermal performance obtained by the above preparation method.

[0020] The present invention further protects the use of the above-mentioned milk nanocapsule loading material with high skin transdermal performance in cosmetics.

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

[0022] The present invention adjusts the pH of milk to 4.5-4.8, the isoelectric point of protein, and collects the protein bodies after standing, preserving the fat globule structure. This method reduces mechanical damage to the fat globules. Ultrafiltration is also used to separate the fat globules, and purified extracellular vesicles are obtained through anion exchange chromatography and molecular sieve chromatography.

[0023] Heat treatment of the prepared milk extracellular vesicles denatures the milk proteins. Heating disrupts the spatial structure of the protein molecules, loosening them and exposing more hydrophobic and polar groups. This facilitates the formation of lipid microspheres, as the hydrophobic groups interact with lipids, promoting their aggregation and stability. Lipid oxidation reactions induced by irradiation can cross-link lipid molecules, forming a more stable network structure. This cross-linking enhances the mechanical strength and stability of the lipid microspheres, preventing aggregation or rupture during storage and use, resulting in more stable lipid microspheres.

[0024] In the present invention, the prepared protein body is loaded with alkylated beauty peptide through dehydration condensation of amino and carboxyl groups. At the same time, the alkyl group on the alkylated beauty peptide improves its compatibility in liposomes.

[0025] The present invention reacts the prepared milk nanocapsules with phytic acid and β-cyclodextrin. The cavity of β-cyclodextrin can effectively form a complex with the peptide-loaded protein body, thereby increasing the loading capacity of the peptide-loaded protein body. The body is then encapsulated in liposomes, thereby improving the drug loading capacity and biocompatibility of the prepared milk nanocapsule with high transdermal performance, promoting the transdermal absorption of the beauty protein peptide, and being able to penetrate the phospholipid bilayer of the skin well and enter the epidermal cells, thereby 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 the synthesis of collagen and making the skin firmer and more 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 the synthesis of collagen, increase the elasticity and firmness of the skin, break down collagen that causes skin aging and wrinkle formation, promote the proliferation of fibroblasts, help repair damaged skin tissue, 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. The two work together to more effectively increase collagen content, making the skin firmer and more elastic. At the same time, they can inhibit the activity of MMPs, thereby reducing the decomposition of collagen, repairing and protecting the skin, and more comprehensively promoting skin repair, regeneration and protection, achieving better synergistic beauty effects.

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

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0031] Figure 1 This is a TEM image of the milk extracellular vesicles prepared in Example 1;

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

[0033] Figure 3 This is a characterization diagram of the particle size distribution of the milk nanocapsule with high transdermal performance prepared in Example 1;

[0034] Figure 4 This is a characterization diagram of the number of particles loaded into the prepared milk nanovesicles with high transdermal performance;

[0035] Figure 5 This is a characteristic diagram of the zeta potential of the milk nanocapsule-loaded material with high transdermal performance prepared in Example 1;

[0036] Figure 6 This is a comparison chart of the human body experimental results of the milk nanocapsule-loaded substance with high transdermal performance prepared in Example 1. DETAILED DESCRIPTION

[0037] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are 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 nanocapsule loading material with high transdermal performance, comprising the following steps:

[0041] S1. Adjust the pH of raw milk to 4.5, let it stand, and centrifuge at 3000g for 5 minutes. Collect the solid and supernatant. The solid is the protein body. Ultrafilter the supernatant and collect the permeate. After anion exchange chromatography and molecular sieve chromatography, the flow-through obtained is the purified extracellular vesicle solution. Figure 1 This is the TEM image of the prepared milk extracellular vesicles, showing a typical saucer shape;

[0042] The exchange ligand of the filler of the anion exchange chromatography is diethylaminoethyl;

[0043] S2. 1g of milk extracellular vesicles was added to 50mL of water, heated to 75°C for 35min, and then irradiated with cobalt-60 at a dose of 5KGy to obtain milk nanovesicles. Figure 2 This is the TEM image of the prepared milk nanovesicles, whose morphology is not much different from that of milk extracellular vesicles;

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

[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. 10 g of milk nanovesicles, 3 g of phytic acid, 4 g of β-cyclodextrin, and 0.2 g of potassium dihydrogen phosphate were added to 200 mL of water, heated to 50°C, and stirred for 1 hour. The product was then added to 200 mL of PBS (pH 8), stirred, centrifuged, washed, and freeze-dried to prepare a β-cyclodextrin / phytic acid / milk nanovesicle complex.

[0047] S5. 8 g of soy phosphatidylcholine and 3 g of lecithin were dissolved in 100 mL of ethanol solution. 1 g of β-cyclodextrin / phytic acid / milk nanovesicle complex, 0.1 g of peptide-loaded proteosomes, 0.05 g of vitamin E, 0.03 g of vitamin C, and 200 mL of water were added. The mixture was stirred until uniform. The ethanol was removed under reduced pressure, and the mixture was freeze-dried to obtain a milk nanovesicle-loaded material with high transdermal performance. Figure 3 This is a characterization diagram of the particle size distribution of the milk nanocapsule loading material with high transdermal performance. The median particle size is 83 nm. Figure 4 This is a characterization diagram of the particle number of the milk nanovesicles loaded with high transdermal performance. Figure 5This is the Zeta potential characteristic diagram of the prepared milk nanovesicle loading with high transdermal performance, and its Zeta potential is -17.25 mV.

[0048] The prepared milk nanocapsule with high transdermal performance was used for a 52-year-old woman with mild fine lines around the eyes. The cycle was 7 days. The milk nanocapsule with high transdermal performance was made into a 0.01mg / mL sample solution and compared with similar products on the market. Massage until absorbed. During the experiment, other anti-wrinkle skin care products were stopped. The skin around the eyes of the test area was applied with the product after cleansing every morning and evening. Gently press to promote product absorption. Use once in the morning and evening. Results are shown in the figure. Figure 6 As can be seen from the figure, after using the milk nanocapsule loading material with high transdermal performance of the present invention, the wrinkles around the corners of the woman's eyes were significantly reduced, which is significantly better than similar products on the market.

[0049] Example 2

[0050] This embodiment provides a method for preparing a milk nanocapsule loading material with high transdermal performance, comprising the following steps:

[0051] S1. Raw milk was adjusted to pH 4.8, allowed to stand, and centrifuged at 8000 g for 3 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.

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

[0053] S2. 1 g of milk extracellular vesicles was added to 50 mL of water, heated to 85°C for 25 minutes, and then irradiated with cobalt-60 at a dose of 15 kGy to obtain milk nanovesicles.

[0054] S3. 1.5 g of protein body was added to 100 mL of water, followed by 0.5 g of EDC and 0.5 g of NHS. The mixture was stirred and activated for 30 minutes. 0.7 g of alkylated beauty peptide was added and stirred for 12 hours. The pH was then adjusted to 5, the mixture was allowed to stand, centrifuged, and the solid was collected and washed to obtain peptide-loaded protein body.

[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 soybean 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 proteosomes, 0.07 g of vitamin E, 0.04 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.

[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 no acid precipitation treatment is performed in step S1.

[0073] The details are as follows:

[0074] S1. The raw milk was centrifuged at 5000 g for 4 minutes, and the solid and supernatant were collected. The solid was the protein body, and the supernatant was ultrafiltered. The permeate was collected and subjected to anion exchange chromatography and molecular sieve chromatography. The obtained flow-through was the purified extracellular vesicle solution;

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

[0076] Comparative Example 2

[0077] Compared with Example 3, the difference is that no heating treatment is performed in step S2.

[0078] The details are as follows:

[0079] S2. 1 g of milk extracellular vesicles was added to 50 mL of water and irradiated with cobalt-60 at a dose of 10 kGy to obtain milk nanovesicles.

[0080] Comparative Example 3

[0081] Compared with Example 3, the difference is that no irradiation treatment is performed in step S2.

[0082] The details are as follows:

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

[0084] Comparative Example 4

[0085] Compared with embodiment 3, the difference is that step S2 is not performed.

[0086] The details are as follows:

[0087] 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.

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

[0089] S2. 1.2 g of protein body was added to 100 mL of water, along with 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.

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

[0091] S3. 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 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.

[0092] S4. Dissolve 9 g of soybean 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 proteosomes, 0.07 g of vitamin E, 0.04 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.

[0093] Comparative Example 5

[0094] Compared with embodiment 3, the difference is that step S3 is not performed.

[0095] The details are as follows:

[0096] 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.

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

[0098] 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.

[0099] S3. 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.

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

[0101] Comparative Example 6

[0102] Compared with embodiment 3, the difference is that step S4 is not performed.

[0103] The details are as follows:

[0104] 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.

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

[0106] 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.

[0107] 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.

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

[0109] S4. Dissolve 9g of soy phosphatidylcholine and 4g of lecithin in 100mL of ethanol solution. Add 1.2g of milk nanovesicles, 0.15g of peptide-loaded proteosomes, 0.07g of vitamin E, 0.04g of vitamin C, and 200mL of water. Stir and mix thoroughly. 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 the liposome embedding in step S5 is not performed.

[0112] The details are as follows:

[0113] 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.

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

[0115] 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.

[0116] 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.

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

[0118] 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.

[0119] S5. 1.2 g of β-cyclodextrin / phytic acid / milk nanovesicle complex, 0.15 g of peptide-loaded proteosomes, 0.07 g of vitamin E, and 0.04 g of vitamin C were stirred and mixed for 10 min to prepare a milk nanovesicle loading with high transdermal performance.

[0120] Test Example 1 Antioxidant Test

[0121] Test samples: The milk nanovesicles with high transdermal performance prepared in Examples 1-5 and Comparative Examples 1-7 were prepared into 0.01 mg / mL solutions. Vitamin C of the same concentration was used as a positive control.

[0122] Experimental Method: Add 200 μL of the test substance to 1 mL of DPPH (2,2-diphenyl-1-picrylhydrazyl) working solution (0.0082 g of DPPH powder dissolved in 100 mL of ethanol) and incubate in the dark for 30 minutes. The absorbance reading at 517 nm is A1. Add 200 μL of distilled water to 1 mL of DPPH working solution and incubate in the dark for 30 minutes. The absorbance reading at 517 nm is A2. Add 200 μL of the test substance to 1 mL of ethanol and incubate in the dark for 30 minutes. The absorbance reading at 517 nm is A3.

[0123] Clearance 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 nanocapsules loaded with high transdermal performance prepared in Examples 1-3 of the present invention have good antioxidant properties.

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

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

[0130] Experimental Methods: Subjects aged 22-54 years were randomly divided into 12 groups, each consisting of 8 participants. A 5 cm x 5 cm area was marked on the flexor side of the right forearm and sterilized with medical disinfectant. A 36-pin microneedle was inserted into the sample solution for 30 seconds. Residual liquid was gently wiped with a cotton ball soaked in saline, and the marked area was freeze-dried. The test sample was placed in the wells of a spot tester, size 1-12, at a volume of 10-20 μL. Bathing was prohibited during the test, and a negative reaction was detected in the normal range. The sample solution was distributed to the subject and applied to the face, neck, and behind the ear. After 10 minutes, the skin condition of the subject was observed. The results are shown in Table 2.

[0131] Table 2

[0132]

[0133] As can be seen from the above table, the milk nanocapsule loading material with high transdermal performance prepared in the present invention is safe to use and has no allergic reaction.

[0134] Test Example 3 Experiment on Promoting Synthesis of Type III Collagen

[0135] Test sample: The milk nanovesicles loaded with high transdermal performance prepared in Examples 1-5 and Comparative Examples 1-7 were prepared into 0.01 mg / mL sample solutions.

[0136] Experimental methods: Human fibroblasts (Merck Life Sciences) cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS (fetal bovine serum) were seeded in 96-well plates. After cell attachment, the medium was exchanged with DMEM containing 0.2 wt% FBS and 250 μmol / L magnesium ascorbyl phosphate. 100 μL of sample solution (100 μL of deionized water was added to the control group) was added to each well and mixed thoroughly. After 72 hours of culture, the supernatant was collected and centrifuged. The type III collagen content in the supernatant was determined using a kit, and the type III collagen synthesis promotion rate was calculated. The results are shown in Table 3.

[0137] Type III collagen synthesis promotion rate (%) = (A1-A0) / A0×100%

[0138] A1: Amount of type III collagen produced after adding sample solution;

[0139] A0: The amount of type III collagen produced when no sample was added to the solution.

[0140] Table 3

[0141]

[0142] As can be seen from the above table, the milk nanocapsules loaded with high transdermal performance prepared in Examples 1-3 of the present invention have a significant effect of promoting the production of type III collagen.

[0143] Test Example 4

[0144] Healthy female volunteers aged 38-54 years with mild or greater fine lines around the eyes were selected as subjects. Subject exclusion criteria met the inclusion and exclusion criteria of the "Diagnostic Criteria and Treatment Principles for Cosmetic Contact Dermatitis." After cleansing their faces every morning and evening, 1 mL of the highly transdermal milk nanocapsules prepared in Examples 1-5 and Comparative Examples 1-7 were used to prepare a 0.01 mg / mL sample solution, along with a commercially available similar product. Massage until absorbed. Other anti-wrinkle skincare products were discontinued during the experiment. The skin around the eyes of the test site was applied after cleansing the face every morning and evening. Gently press to promote product absorption. Each group had 10 participants. Any adverse skin reactions such as erythema, itching, stinging, and burning that occurred during the test evaluation were recorded, and serious adverse reactions were reported promptly. During the experimental test, the test site was cleaned and placed in a constant temperature and humidity environment at 25±1.0°C and 55%±5%RH for 30 minutes before data collection. The cycle was 28 days, with skin tests performed on day 0 and day 28.

[0145] (1) Skin moisture content

[0146] The skin moisture content of the test area was measured using a skin moisture meter for three times and the average value was taken. The changes in skin moisture content before and after product use and between the test and control sides were compared.

[0147] (2) Skin elasticity

[0148] The skin elasticity of the test and evaluation parts of the subjects was measured using a skin elasticity meter for 3 times, and the average value was taken.

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

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

[0151] Crow's feet change rate = (test value on day 28 - test value on day 0) / test value on day 0 × 100%

[0152] The results are shown in Table 4.

[0153] Table 4

[0154]

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

[0156] Test Example 5

[0157] With reference to the transdermal test method of GBT27818-2011, an in vitro skin transdermal test was conducted on the milk nanocapsule-loaded materials with high transdermal performance prepared in Examples 1-3 and Comparative Examples 1-4, 6-7.

[0158] Using pig dorsal skin as a test tool, equal amounts of milk nanovesicles with high transdermal performance and free palmitoyl tripeptide-1 were added to the supply pool for a 24-hour transdermal experiment. The skin retention rate of palmitoyl tripeptide-1 was measured 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 nanovesicles 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 better than palmitoyl tripeptide-1 itself. The milk nanovesicles 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 description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a milk nanocapsule-loaded material with high transdermal performance, characterized in that: The pH of the milk is adjusted and centrifuged to obtain protein bodies and supernatant. The supernatant is ultrafiltrated to obtain milk extracellular vesicles, which are heated and irradiated, and reacted with phytic acid and β-cyclodextrin to obtain β-cyclodextrin / phytic acid / milk nanovesicle complexes; the protein bodies are reacted with alkylated beauty peptides to obtain peptide-loaded protein bodies, which are encapsulated with β-cyclodextrin / phytic acid / milk nanovesicle complexes, vitamin E, and vitamin C to obtain milk nanovesicle loading with high transdermal performance.

2. The preparation method according to claim 1, characterized in that The following steps are involved: S1. The pH of the milk is adjusted, the milk is allowed to stand, and the supernatant is collected. The solid is the protein body. The supernatant is ultrafiltered, and the permeate is collected. The permeate is subjected to anion exchange chromatography and molecular sieve chromatography to obtain a purified extracellular vesicle solution. The flow-through is freeze-dried to produce milk extracellular vesicles. S2. The milk extracellular vesicles were added to water, heated, and then irradiated with cobalt 60 and freeze-dried to obtain milk nanovesicles; S3. Add the protein body to water, add EDC and NHS, stir to activate, add the alkylated beauty peptide, stir to react, then adjust the pH, let stand, centrifuge, collect the solid, wash, and freeze-dry to produce the peptide-loaded protein body; S4. Milk nanovesicles, phytic acid, β-cyclodextrin, and potassium dihydrogen phosphate were added to water, heated and stirred, and the product was added to PBS buffer, stirred, centrifuged, washed, and freeze-dried to obtain a β-cyclodextrin / phytic acid / milk nanovesicle complex; S5. Dissolve soybean phosphatidylcholine and lecithin in an ethanol solution, add β-cyclodextrin / phytic acid / bovine milk nanovesicle complex, peptide-loaded proteosomes, vitamin E, vitamin C, and water, stir and mix thoroughly, 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 pH value is adjusted to 4.5-4.8, the centrifugation conditions are 3000-8000g, and the time is 3-5min. The ligand of the filler 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 quaternary ammonium, diethylaminoethyl, and diethylaminopropyl. The filler particle size range is 15-300μm, and the pore size range is 20-150nm.

4. The preparation method according to claim 2, characterized in that The temperature of the heat treatment in step S2 is 75-85° C., the time is 25-35 min, and the irradiation dose is 5-15 KGy.

5. The preparation method according to claim 2, characterized in that The mass ratio of the proteosome, EDC, NHS and alkylated beauty peptide in step S3 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 peptide is selected from 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 at least one of 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, with a mass ratio of 10:5-8.

7. The preparation method according to claim 2, characterized in that In step S4, the mass ratio of milk nanovesicles, phytic acid, β-cyclodextrin, and potassium dihydrogen phosphate is 10:3-4:4-6:0.2-0.3, the heating and stirring temperature is 50-60°C, the time is 1-2h, and the time is 8-10h, and the pH of the PBS buffer is 8-8.

5.

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

05.

9. A milk nanocapsule loading material with high transdermal performance obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the milk nanocapsule loading material with high transdermal performance as claimed in claim 9 in cosmetics.

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