A whitening and anti-aging composition containing exosomes and preparation method thereof

Abstract

The present invention proposes a whitening and anti-aging composition containing exosomes and a preparation method thereof, and belongs to the technical field of cosmetics. Chinese medicine polysaccharides are prepared by water extraction and alcohol precipitation of Poria cocos, ginseng, licorice, and Dendrobium candidum, and a complex is formed with phycobiliprotein and copper salt, and loaded into milk exosomes to obtain milk exosomes loaded with the complex; quercetin, fisetin, and ginsenoside Rg1 are formed into a mixed eutectic to prepare a nanoemulsion, which is mixed with the milk exosomes loaded with the complex to prepare a whitening and anti-aging composition containing exosomes. The whitening and anti-aging composition containing exosomes prepared by the present invention has good whitening, anti-aging, and antioxidant effects, good transdermal absorption effect, and can improve microcirculation, promote skin metabolism, whiten skin, promote skin elasticity, delay skin aging, and remove facial spots and scars.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to a whitening and anti-aging composition containing exosomes and a preparation method thereof. Background Art

[0002] With the development of society and people's yearning and pursuit for a better life, beauty has become an increasingly important goal in life, making people more and more aware of skin aging. The main impact of aging skin on beauty is dry and dull skin, roughness, cracking, wrinkles, looseness and sagging, as well as a significant increase in lipofuscin and age spots. Therefore, various cosmetics and drugs are widely used, but there are also many kinds of drugs or cosmetics for skin anti-aging and whitening, and their effects are also different.

[0003] Exosomes are small vesicles with a diameter of about 30nm-150nm secreted by living cells. They have a typical lipid bilayer structure and exist in cell culture supernatants, serum, plasma, saliva, urine, amniotic fluid and other biological fluids. Exosomes contain protein rRNA and microRNA related to the cell source. Exosomes can directly activate receptor cells through cell membrane receptors, and can also transport proteins, mRNA, miRNA, lncRNA, circRNA, and even organelles into receptor cells to participate in intercellular communication. Exosomes play a key role in physiological processes such as immune response, inflammatory response, angiogenesis, apoptosis, coagulation and waste disposal. Exosomes not only play an important role in the transfer of substances and information between cells, but are also expected to become early diagnostic markers for a variety of diseases.

[0004] Stem cell exosomes contain abundant cytokines, such as VEGF and FGF, which can participate in various processes of skin tissue repair and regeneration. They promote wound healing and skin tissue regeneration by promoting the proliferation and migration of skin cells, promoting angiogenesis, and regulating immune responses. They have significant effects on skin tissue repair and anti-aging. In recent years, the application of stem cells and related components in skin anti-aging and lifting and firming has also been a hot topic of research. However, the exosome preparations prepared by the current general preparation methods have poor effects in maintaining skin firmness and anti-aging and wrinkle removal, and are prone to allergic or immune rejection reactions. Summary of the invention

[0005] The purpose of the present invention is to provide a whitening and anti-aging composition containing exosomes and a preparation method thereof, which has good whitening, anti-aging and antioxidant effects, good transdermal absorption effect, and can improve microcirculation, promote skin metabolism, whiten the skin, promote skin elasticity, delay skin aging, and remove facial spots and scars.

[0006] The technical solution of the present invention is achieved in this way:

[0007] The invention provides a method for preparing a whitening and anti-aging composition containing exosomes. The method comprises the following steps: extracting Poria cocos, ginseng, liquorice and Dendrobium officinale with water and subjecting the mixture to alcohol precipitation to obtain Chinese medicinal polysaccharides, forming a complex with phycobiliprotein and copper salt, and loading the complex into milk exosomes to obtain milk exosomes loaded with the complex; and forming a mixed eutectic with quercetin, fisetin and ginsenoside Rg1 to prepare a nanoemulsion, and mixing the nanoemulsion with the milk exosomes loaded with the complex to obtain a whitening and anti-aging composition containing exosomes.

[0008] As a further improvement of the present invention, the following steps are included:

[0009] S1. Preparation of Chinese herbal polysaccharides: Poria cocos, ginseng, licorice, and Dendrobium candidum are washed, sliced, mixed, added to water, heated to boiling for extraction, cooled to room temperature, filtered, ethanol was added to the filtrate for precipitation, filtered, washed, and dried to obtain Chinese herbal polysaccharides;

[0010] S2. Extraction of phycobiliprotein: Dry and crush Spirulina, add the dry powder to water, cool with liquid nitrogen, melt at room temperature, filter, dialyze the filtrate, and freeze-dry the dialyzate to obtain phycobiliprotein;

[0011] S3. Preparation of Chinese herbal polysaccharide-phycobiliprotein complex: adding phycobiliprotein to water, adding NHS and EDC, stirring to activate, adding Chinese herbal polysaccharide and copper salt, stirring to react, dialyzing, freeze-drying, and preparing Chinese herbal polysaccharide-phycobiliprotein complex;

[0012] S4. Preparation of milk exosomes loaded with the complex: dissolving the Chinese medicine polysaccharide-phycobiliprotein complex and milk exosomes in a PBS solution, incubating, dialyzing, and freeze-drying the dialysate to obtain milk exosomes loaded with the complex;

[0013] S5. Preparation of mixed eutectic: dissolving quercetin in acetone to obtain solution 1, dissolving fisetin in ethanol to obtain solution 2, dissolving ginsenoside Rg1 in water to obtain solution 3, mixing solution 1, solution 2, and solution 3, volatilizing to obtain eutectic, filtering, washing, and drying to obtain mixed eutectic;

[0014] S6. Preparation of nanoemulsion: Soy lecithin, Tween, Span, glycerol, 1,3-propylene glycol, isopropyl myristate were stirred and mixed uniformly, distilled water was added dropwise, and then the mixed eutectic was added, stirred and mixed to obtain a nanoemulsion;

[0015] S7. Preparation of a whitening and anti-aging composition containing exosomes: Evenly mix the milk exosomes and nanoemulsion loaded with the complex to prepare a whitening and anti-aging composition containing exosomes.

[0016] As a further improvement of the present invention, the mass ratio of Poria cocos, ginseng, liquorice and Dendrobium officinale in step S1 is 2-3:1-2:4-5:2-3, and the time for heating to boiling extraction is 3-5h.

[0017] As a further improvement of the present invention, the mass ratio of the dry powder to water in step S2 is 1:5-10, the pore size of the dialysis bag used for dialysis is 10-30 kDa, and the dialysis time is 12-24 h.

[0018] As a further improvement of the present invention, the mass ratio of phycobiliprotein, NHS, EDC, traditional Chinese medicine polysaccharide and copper salt in step S3 is 10:3-4:2-3:6-8:0.5-1, the stirring activation time is 20-30min, the stirring reaction time is 12-15h, the dialysis bag pore size used for dialysis is 30-50kDa, the dialysis time is 12-24h, and the copper salt is at least one of copper chloride, copper sulfate and copper nitrate.

[0019] As a further improvement of the present invention, the mass ratio of the traditional Chinese medicine polysaccharide-phycobiliprotein complex and the milk exosomes in step S4 is 2-3:10, the incubation conditions are 3-5°C, the incubation is 3-5h, the dialysis bag pore size used for dialysis is 40-90kDa, the dialysis time is 24-36h, the Tween is selected from at least one of Tween-20, Tween-40, Tween-60, Tween-80, and Tween-85, and the Span is selected from at least one of Span-20, Span-40, Span-60, Span-80, and Span-85.

[0020] As a further improvement of the present invention, the mass ratio of quercetin, fisetin and ginsenoside Rg1 in step S5 is 1-2:0.5-1:0.2-0.4.

[0021] As a further improvement of the present invention, the mass ratio of soybean lecithin, Tween, Span, glycerol, 1,3-propylene glycol, isopropyl myristate, distilled water, and mixed eutectic in step S6 is 3-5:0.5-1:0.5-1:2-3:1-2:1-2:50-70:1-2.

[0022] As a further improvement of the present invention, the mass ratio of the milk exosomes and the nanoemulsion loaded with the complex in step S7 is 2-3:10.

[0023] The present invention further protects a whitening and anti-aging composition containing exosomes obtained by the above-mentioned preparation method.

[0024] The present invention has the following beneficial effects:

[0025] The Chinese medicines Poria cocos, ginseng, licorice, and Dendrobium candidum are rich in polysaccharide components, which can improve skin metabolism, delay skin aging, and remove facial spots and scars. Phycobiliprotein is an important type of protein in algae, which has antioxidant and anti-aging properties and is safe and stable. The two are coupled to form a complex and chelated with copper ions. Copper ions can promote the production of collagen, help the skin heal wounds, and increase skin elasticity. The prepared Chinese medicine polysaccharide-phycobiliprotein complex can improve microcirculation, promote skin metabolism, whiten the skin, promote skin elasticity, delay skin aging, and remove facial spots and scars.

[0026] Milk is a biological fluid with a highly complex composition, containing a large number of exosomes with biologically active molecules. Milk exosomes (mEXO) have a high yield and can passively target and transport the Chinese medicine polysaccharide-phycobiliprotein complex to the interior of melanosomes through the endosomal pathway, thereby inhibiting tyrosinase and melanin production. Therefore, the milk exosomes loaded with the complex can repair and whiten the skin through mEXO on the one hand, and at the same time, promote skin elasticity and improve anti-aging properties.

[0027] Quercetin can inhibit the key transcription nodes required for the survival of senescent cells, reduce the number of senescent cells and the level of related cytokines, and achieve the purpose of anti-aging. Fisetin has strong anti-aging activity and can regulate multiple pathways to eliminate senescent cells, such as PI3K / Akt and NF-κB pathways. However, quercetin and fisetin are insoluble in water, which greatly limits their application in cosmetics. Ginseng saponins can not only inhibit the production of free radicals, but also directly counteract the damage of free radicals to tissues and cells, or directly eliminate free radicals, and can also enhance the function of the body's own antioxidant system, blocking the damage of free radicals from multiple links. Ginseng enhances the elimination of active oxidative free radicals by increasing the activity of superoxide dismutase in the body and epidermal cells of the skin, preventing the production of harmful substances in the body, improving the body's metabolism, and reducing or eliminating lipofuscin that has been produced and accumulated in the body. Ginseng also has the function of anti-ultraviolet skin care. Therefore, the present invention prepares a mixed eutectic of three components, which, on the one hand, improves its water solubility, improves its bioavailability, and promotes its transdermal absorption of energy; on the other hand, the synergistic effect of the three can significantly improve the whitening, anti-aging, and antioxidant effects of the prepared mixed eutectic.

[0028] In addition, the present invention prepares the mixed eutectic into a nanoemulsion, which is a thermodynamically stable system with a particle size of less than 100 nm and a clear and transparent appearance, formed by oil, water, an emulsifier and an emulsifier co-emulsifier. This greatly improves the transdermal absorption effect of the product and increases the transdermal absorption amount, thereby significantly improving the improvement effect on the skin.

[0029] The whitening and anti-aging composition containing exosomes prepared by the present invention has good whitening, anti-aging and antioxidant effects, good transdermal absorption effect, and can improve microcirculation, promote skin metabolism, whiten the skin, promote skin elasticity, delay skin aging, and remove facial spots and scars. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Isolation and collection of milk exosomes: The raw milk was subjected to gradient centrifugation (2000×g for 30 min, 10000×g for 30 min) to remove cell debris, and the supernatant was filtered through a 0.22μm microporous filter membrane to remove impurities; the filtrate was then centrifuged at 100000×g for 70 min, the supernatant was discarded, and the bottom of the centrifuge tube was repeatedly blown with clean 100μL DPBS (phosphate buffered saline without calcium and magnesium ions), and freeze-dried to obtain milk exosomes (mEXO).

[0032] Example 1

[0033] This embodiment provides a method for preparing a whitening and anti-aging composition containing exosomes, comprising the following steps:

[0034] S1. Preparation of Chinese herbal polysaccharides: 2g of Poria cocos, 1g of ginseng, 4g of licorice, and 2g of Dendrobium candidum were washed, sliced, mixed, added to 5 times the mass of water, heated to boiling and extracted for 3h, cooled to room temperature, filtered, ethanol was added to the filtrate until the ethanol content of the system was 80wt%, precipitated for 1h, filtered, washed, and dried to obtain Chinese herbal polysaccharides;

[0035] S2. Extraction of phycobiliprotein: Dry and crush Spirulina, add 10 g of dry powder to 50 mL of water, cool with liquid nitrogen, melt at room temperature, filter, dialyze the filtrate with a dialysis bag with a pore size of 10 kDa for 24 h, and freeze-dry the dialysate to obtain phycobiliprotein;

[0036] S3. Preparation of Chinese medicine polysaccharide-phycobiliprotein complex: 10 g of phycobiliprotein was added to 500 mL of water, 3 g of NHS and 2 g of EDC were added, and the mixture was stirred for activation for 20 min, 6 g of Chinese medicine polysaccharide and 0.5 g of copper chloride were added, and the mixture was stirred for reaction for 12 h, and the mixture was dialyzed for 24 h using a dialysis bag with a pore size of 30 kDa, and freeze-dried to obtain a Chinese medicine polysaccharide-phycobiliprotein complex;

[0037] S4. Preparation of milk exosomes loaded with the complex: 2 g of the Chinese medicine polysaccharide-phycobiliprotein complex and 10 g of milk exosomes were dissolved in 500 mL of PBS solution, incubated at 3°C ​​for 3 h, dialyzed for 36 h using a dialysis bag with a pore size of 40 kDa, and the dialyzate was freeze-dried to obtain milk exosomes loaded with the complex;

[0038] S5. Preparation of mixed eutectic: 1 g of quercetin was dissolved in 100 mL of acetone to obtain solution 1, 0.5 g of fisetin was dissolved in 100 mL of ethanol to obtain solution 2, 0.2 g of ginsenoside Rg1 was dissolved in 100 mL of water to obtain solution 3, solution 1, solution 2, and solution 3 were stirred and mixed for 5 min, evaporated for 2 d to obtain eutectic, filtered, washed, and dried to obtain mixed eutectic;

[0039] S6. Preparation of nanoemulsion: 3 g of soy lecithin, 0.5 g of Tween-40, 0.5 g of Span-40, 2 g of glycerol, 1 g of 1,3-propylene glycol, and 1 g of isopropyl myristate were stirred and mixed for 10 min, 50 mL of distilled water was added dropwise, and then 1 g of the mixed eutectic was added and stirred for 30 min to obtain a nanoemulsion;

[0040] S7. Preparation of a whitening and anti-aging composition containing exosomes: 2 g of milk exosomes loaded with the complex and 10 g of nanoemulsion were stirred and mixed for 15 min to prepare a whitening and anti-aging composition containing exosomes.

[0041] Example 2

[0042] This embodiment provides a method for preparing a whitening and anti-aging composition containing exosomes, comprising the following steps:

[0043] S1. Preparation of Chinese herbal polysaccharides: 3g of Poria cocos, 2g of ginseng, 5g of licorice, and 3g of Dendrobium candidum were washed, sliced, mixed, added to 5 times the mass of water, heated to boiling and extracted for 5h, cooled to room temperature, filtered, ethanol was added to the filtrate until the ethanol content of the system was 80wt%, precipitated for 1h, filtered, washed, and dried to obtain Chinese herbal polysaccharides;

[0044] S2. Extraction of phycobiliprotein: Dry and crush Spirulina, add 10 g of dry powder to 100 mL of water, cool with liquid nitrogen, melt at room temperature, filter, dialyze the filtrate with a dialysis bag with a pore size of 30 kDa for 12 h, and freeze-dry the dialysate to obtain phycobiliprotein;

[0045] S3. Preparation of Chinese medicine polysaccharide-phycobiliprotein complex: 10 g of phycobiliprotein was added to 500 mL of water, 4 g of NHS and 3 g of EDC were added, and the mixture was stirred for activation for 30 min, 8 g of Chinese medicine polysaccharide and 1 g of copper sulfate were added, and the mixture was stirred for reaction for 15 h, and the mixture was dialyzed for 12 h using a dialysis bag with a pore size of 50 kDa, and freeze-dried to obtain a Chinese medicine polysaccharide-phycobiliprotein complex;

[0046] S4. Preparation of milk exosomes loaded with the complex: 3 g of the Chinese medicine polysaccharide-phycobiliprotein complex and 10 g of milk exosomes were dissolved in 500 mL of PBS solution, incubated at 5°C for 5 h, dialyzed for 24 h using a dialysis bag with a pore size of 90 kDa, and the dialyzate was freeze-dried to obtain milk exosomes loaded with the complex;

[0047] S5. Preparation of mixed eutectic: 2 g of quercetin was dissolved in 100 mL of acetone to obtain solution 1, 1 g of fisetin was dissolved in 100 mL of ethanol to obtain solution 2, 0.4 g of ginsenoside Rg1 was dissolved in 100 mL of water to obtain solution 3, solution 1, solution 2, and solution 3 were stirred and mixed for 5 min, evaporated for 3 d to obtain eutectic, filtered, washed, and dried to obtain mixed eutectic;

[0048] S6. Preparation of nanoemulsion: 5 g of soy lecithin, 1 g of Tween-60, 1 g of Span-60, 3 g of glycerol, 2 g of 1,3-propylene glycol, and 2 g of isopropyl myristate were stirred and mixed for 10 min, 70 mL of distilled water was added dropwise, and then 2 g of the mixed eutectic was added and stirred for 30 min to obtain a nanoemulsion;

[0049] S7. Preparation of a whitening and anti-aging composition containing exosomes: 3 g of milk exosomes loaded with the complex and 10 g of nanoemulsion were stirred and mixed for 15 minutes to prepare a whitening and anti-aging composition containing exosomes.

[0050] Example 3

[0051] This embodiment provides a method for preparing a whitening and anti-aging composition containing exosomes, comprising the following steps:

[0052] S1. Preparation of Chinese herbal polysaccharides: 2.5 g of Poria cocos, 1.5 g of ginseng, 4.5 g of licorice, and 2.5 g of Dendrobium candidum were washed, sliced, mixed, added to 5 times the mass of water, heated to boiling and extracted for 4 h, cooled to room temperature, filtered, ethanol was added to the filtrate until the ethanol content of the system was 80 wt%, precipitated for 1 h, filtered, washed, and dried to obtain Chinese herbal polysaccharides;

[0053] S2. Extraction of phycobiliprotein: Dry and crush Spirulina, add 10 g of dry powder to 70 mL of water, cool with liquid nitrogen, melt at room temperature, filter, dialyze the filtrate with a dialysis bag with a pore size of 20 kDa for 16 h, and freeze-dry the dialysate to obtain phycobiliprotein;

[0054] S3. Preparation of Chinese medicine polysaccharide-phycobiliprotein complex: 10 g of phycobiliprotein was added to 500 mL of water, 3.5 g of NHS and 2.5 g of EDC were added, and the mixture was stirred for activation for 25 min. 7 g of Chinese medicine polysaccharide and 0.7 g of copper nitrate were added, and the mixture was stirred for reaction for 13 h. The mixture was dialyzed for 16 h using a dialysis bag with a pore size of 40 kDa, and freeze-dried to obtain a Chinese medicine polysaccharide-phycobiliprotein complex.

[0055] S4. Preparation of milk exosomes loaded with the complex: 2.5 g of the Chinese medicine polysaccharide-phycobiliprotein complex and 10 g of milk exosomes were dissolved in 500 mL of PBS solution, incubated at 4°C for 4 h, dialyzed for 30 h using a dialysis bag with a pore size of 55 kDa, and the dialyzate was freeze-dried to obtain milk exosomes loaded with the complex;

[0056] S5. Preparation of mixed eutectic: 1.5 g of quercetin was dissolved in 100 mL of acetone to obtain solution 1, 0.7 g of fisetin was dissolved in 100 mL of ethanol to obtain solution 2, 0.3 g of ginsenoside Rg1 was dissolved in 100 mL of water to obtain solution 3, solution 1, solution 2, and solution 3 were stirred and mixed for 5 min, evaporated for 3 d to obtain eutectic, filtered, washed, and dried to obtain mixed eutectic;

[0057] S6. Preparation of nanoemulsion: 4 g of soy lecithin, 0.7 g of Tween-85, 0.7 g of Span-85, 2.5 g of glycerol, 1.5 g of 1,3-propylene glycol, and 1.5 g of isopropyl myristate were stirred and mixed for 10 min, 60 mL of distilled water was added dropwise, and then 1.5 g of mixed eutectic was added, stirred and mixed for 30 min to obtain a nanoemulsion;

[0058] S7. Preparation of a whitening and anti-aging composition containing exosomes: 2.5 g of milk exosomes loaded with the complex and 10 g of nanoemulsion were stirred and mixed for 15 minutes to prepare a whitening and anti-aging composition containing exosomes.

[0059] Comparative Example 1

[0060] Compared with Example 3, the difference is that no phycobiliprotein is added in step S3.

[0061] The details are as follows:

[0062] S3. Preparation of Chinese herbal polysaccharide-phycobiliprotein complex: 17 g of Chinese herbal polysaccharide and 0.7 g of copper nitrate were stirred for 10 min, dialyzed for 16 h using a dialysis bag with a pore size of 40 kDa, and freeze-dried to obtain a Chinese herbal polysaccharide complex.

[0063] Comparative Example 2

[0064] Compared with Example 3, the difference is that no Chinese medicine polysaccharide is added in step S3.

[0065] The details are as follows:

[0066] S3. Preparation of phycobiliprotein complex: 17 g of phycobiliprotein was added to 500 mL of water, and 0.7 g of copper nitrate was added. The mixture was stirred for 10 min, dialyzed for 16 h using a dialysis bag with a pore size of 40 kDa, and freeze-dried to obtain the phycobiliprotein complex.

[0067] Comparative Example 3

[0068] Compared with Example 3, the difference is that copper nitrate is not added in step S3.

[0069] The details are as follows:

[0070] S3. Preparation of Chinese herbal polysaccharide-phycobiliprotein complex: Add 10 g of phycobiliprotein into 500 mL of water, add 3.5 g of NHS and 2.5 g of EDC, stir and activate for 25 min, add 7 g of Chinese herbal polysaccharide, stir and react for 13 h, dialyze with a dialysis bag with a pore size of 40 kDa for 16 h, freeze-dry, and obtain the Chinese herbal polysaccharide-phycobiliprotein complex.

[0071] Comparative Example 4

[0072] Compared with Example 3, the difference is that step S4 is not performed.

[0073] The details are as follows:

[0074] S1. Preparation of Chinese herbal polysaccharides: 2.5 g of Poria cocos, 1.5 g of ginseng, 4.5 g of licorice, and 2.5 g of Dendrobium candidum were washed, sliced, mixed, added to 5 times the mass of water, heated to boiling and extracted for 4 h, cooled to room temperature, filtered, ethanol was added to the filtrate until the ethanol content of the system was 80 wt%, precipitated for 1 h, filtered, washed, and dried to obtain Chinese herbal polysaccharides;

[0075] S2. Extraction of phycobiliprotein: Dry and crush Spirulina, add 10 g of dry powder to 70 mL of water, cool with liquid nitrogen, melt at room temperature, filter, dialyze the filtrate with a dialysis bag with a pore size of 20 kDa for 16 h, and freeze-dry the dialysate to obtain phycobiliprotein;

[0076] S3. Preparation of Chinese medicine polysaccharide-phycobiliprotein complex: 10 g of phycobiliprotein was added to 500 mL of water, 3.5 g of NHS and 2.5 g of EDC were added, and the mixture was stirred for activation for 25 min. 7 g of Chinese medicine polysaccharide and 0.7 g of copper nitrate were added, and the mixture was stirred for reaction for 13 h. The mixture was dialyzed for 16 h using a dialysis bag with a pore size of 40 kDa, and freeze-dried to obtain a Chinese medicine polysaccharide-phycobiliprotein complex.

[0077] S4. Preparation of mixed eutectic: 1.5 g of quercetin was dissolved in 100 mL of acetone to obtain solution 1, 0.7 g of fisetin was dissolved in 100 mL of ethanol to obtain solution 2, 0.3 g of ginsenoside Rg1 was dissolved in 100 mL of water to obtain solution 3, solution 1, solution 2, and solution 3 were stirred and mixed for 5 min, evaporated for 3 d to obtain eutectic, filtered, washed, and dried to obtain mixed eutectic;

[0078] S5. Preparation of nanoemulsion: 4 g of soy lecithin, 0.7 g of Tween-85, 0.7 g of Span-85, 2.5 g of glycerol, 1.5 g of 1,3-propylene glycol, and 1.5 g of isopropyl myristate were stirred and mixed for 10 min, 60 mL of distilled water was added dropwise, and then 1.5 g of mixed eutectic was added and stirred for 30 min to obtain a nanoemulsion;

[0079] S6. Preparation of a whitening and anti-aging composition containing exosomes: 2.5 g of a traditional Chinese medicine polysaccharide-phycobiliprotein complex and 10 g of a nanoemulsion were stirred and mixed for 15 min to prepare a whitening and anti-aging composition containing exosomes.

[0080] Comparative Example 5

[0081] Compared with Example 3, the difference is that quercetin is not added in step S5.

[0082] The details are as follows:

[0083] S5. Preparation of mixed cocrystals: dissolve 0.7 g of fisetin in 100 mL of ethanol to obtain solution 2, dissolve 0.3 g of ginsenoside Rg1 in 100 mL of water to obtain solution 3, stir and mix solution 2 and solution 3 for 5 min, evaporate for 3 d to obtain cocrystals, filter, wash, and dry to obtain mixed cocrystals.

[0084] Comparative Example 6

[0085] Compared with Example 3, the difference is that fisetin is not added in step S5.

[0086] The details are as follows:

[0087] S5. Preparation of mixed cocrystals: Dissolve 1.5 g of quercetin in 100 mL of acetone to obtain solution 1, dissolve 0.3 g of ginsenoside Rg1 in 100 mL of water to obtain solution 3, stir and mix solution 1 and solution 3 for 5 min, evaporate for 3 d to obtain cocrystals, filter, wash, and dry to obtain mixed cocrystals.

[0088] Comparative Example 7

[0089] Compared with Example 3, the difference is that ginsenoside Rg1 is not added in step S5.

[0090] The details are as follows:

[0091] S5. Preparation of mixed cocrystals: Dissolve 1.5 g of quercetin in 100 mL of acetone to obtain solution 1, and dissolve 0.7 g of fisetin in 100 mL of ethanol to obtain solution 2. Stir solution 1 and solution 2 for 5 min, evaporate for 3 d to obtain cocrystals, filter, wash, and dry to obtain mixed cocrystals.

[0092] Comparative Example 8

[0093] Compared with the embodiment 3, the difference is that step S6 is not performed.

[0094] The details are as follows:

[0095] S1. Preparation of Chinese herbal polysaccharides: 2.5 g of Poria cocos, 1.5 g of ginseng, 4.5 g of licorice, and 2.5 g of Dendrobium candidum were washed, sliced, mixed, added to 5 times the mass of water, heated to boiling and extracted for 4 h, cooled to room temperature, filtered, ethanol was added to the filtrate until the ethanol content of the system was 80 wt%, precipitated for 1 h, filtered, washed, and dried to obtain Chinese herbal polysaccharides;

[0096] S2. Extraction of phycobiliprotein: Dry and crush Spirulina, add 10 g of dry powder to 70 mL of water, cool with liquid nitrogen, melt at room temperature, filter, dialyze the filtrate with a dialysis bag with a pore size of 20 kDa for 16 h, and freeze-dry the dialysate to obtain phycobiliprotein;

[0097] S3. Preparation of Chinese medicine polysaccharide-phycobiliprotein complex: 10 g of phycobiliprotein was added to 500 mL of water, 3.5 g of NHS and 2.5 g of EDC were added, and the mixture was stirred for activation for 25 min. 7 g of Chinese medicine polysaccharide and 0.7 g of copper nitrate were added, and the mixture was stirred for reaction for 13 h. The mixture was dialyzed for 16 h using a dialysis bag with a pore size of 40 kDa, and freeze-dried to obtain a Chinese medicine polysaccharide-phycobiliprotein complex.

[0098] S4. Preparation of milk exosomes loaded with the complex: 2.5 g of the Chinese medicine polysaccharide-phycobiliprotein complex and 10 g of milk exosomes were dissolved in 500 mL of PBS solution, incubated at 4°C for 4 h, dialyzed for 30 h using a dialysis bag with a pore size of 55 kDa, and the dialyzate was freeze-dried to obtain milk exosomes loaded with the complex;

[0099] S5. Preparation of mixed eutectic: 1.5 g of quercetin was dissolved in 100 mL of acetone to obtain solution 1, 0.7 g of fisetin was dissolved in 100 mL of ethanol to obtain solution 2, 0.3 g of ginsenoside Rg1 was dissolved in 100 mL of water to obtain solution 3, solution 1, solution 2, and solution 3 were stirred and mixed for 5 min, evaporated for 3 d to obtain eutectic, filtered, washed, and dried to obtain mixed eutectic;

[0100] S6. Preparation of a whitening and anti-aging composition containing exosomes: 2.5 g of milk exosomes loaded with the complex and 10 g of the mixed eutectic were stirred and mixed for 15 minutes to prepare a whitening and anti-aging composition containing exosomes.

[0101] Test Example 1 Antioxidant Performance Test

[0102] The whitening and anti-aging composition containing exosomes prepared in Examples 1-3 or Comparative Examples 1-8 was diluted 100 times with water to prepare a test solution. Vitamin C (1 mg / mL) was used as a control.

[0103] 1. DPPH free radical scavenging ability test

[0104] Add 1 mL of 0.2 mmol / L DPPH to 1 mL of the test solution, let it stand at room temperature for 30 minutes, and then measure the absorbance change at a wavelength of 517 nm.

[0105] DPPH clearance rate (%) = [1-(A i -A j ) / A 0 ]×100%

[0106] Where: A i A is the absorbance of 1mL DPPH + 1mL test solution; j A is the absorbance of 1mL solvent + 1mL test solution; 0 The absorbance of 1mL DPPH + 1mL solvent (water)

[0107] 2. Experiment on scavenging superoxide anion free radicals

[0108] Take 2 mL of 50 mmol / L Tris-HCl buffer (pH = 8.2) and mix with 0.91 mL H 2 O, 40 μL of the test solution, mix well, add 40 μL of 10 mmol / L pyrogallol solution, record its absorbance at 320 nm, and calculate the increase in absorbance per minute within the linear range.

[0109] Superoxide anion free radical scavenging rate (%) = (△A 0 -△A) / △A 0 ×100%

[0110] Where: △A 0 is the change of the absorbance of the blank solution over time; △A is the change of the absorbance of the test solution over time.

[0111] The results are shown in Table 1.

[0112] Table 1

[0113] Group DPPH clearance rate (%) Superoxide anion free radical scavenging rate (%) Vitamin C 75.2 62.9 Example 1 89.4 93.5 Example 2 88.7 92.9 Example 3 90.2 94.7 Comparative Example 1 85.2 88.9 Comparative Example 2 82.7 85.4 Comparative Example 3 88.6 92.1 Comparative Example 4 83.9 86.4 Comparative Example 5 86.2 90.1 Comparative Example 6 84.3 87.2 Comparative Example 7 85.5 88.6 Comparative Example 8 88.9 91.7

[0114] It can be seen from the above table that the whitening and anti-aging compositions containing exosomes prepared in Examples 1-3 of the present invention have good antioxidant activity.

[0115] Test Example 2 Tyrosinase Inhibition Test

[0116] The exosome-containing whitening and anti-aging composition prepared in Example 1-3 or Comparative Example 1-8 was diluted 100 times with water to prepare a sample solution. Arbutin (1 mg / mL) was used as a control.

[0117] Phosphate buffer (pH 6.8, 0.1 mol / L), sample solution, and tyrosine solution (10 mmol / L) were added to each test tube, placed in a 37°C water bath for 10 min, and then tyrosinase (100 U / mL) solution was added, and then reacted in a 37°C water bath for 10 min, and the absorbance value was measured at 475 nm. The experimental system is shown in Table 2.

[0118] C 1 As blank control, C 2 For blank zero adjustment, T 1 is the sample tube, T 2 Used for sample zeroing.

[0119] Table 2 (Unit: mL)

[0120] Reagents C1 C2 T1 T2 PBS 2 2.5 1 1.5 Sample solution 0 0 1 1 L-Tyrosine 1 1 1 1 Tyrosinase 0.5 0 0.5 0

[0121] Tyrosinase inhibition rate (%) = (AC 1 -AT 1 ) / AC 1 ×100%

[0122] Where: AC 1 AT is the absorbance value of the blank control group; 1 is the absorbance value of the sample group.

[0123] The results are shown in Table 3.

[0124] Table 3

[0125] Group Tyrosinase inhibition rate (%) Arbutin 85.5 Example 1 90.1 Example 2 90.9 Example 3 91.4 Comparative Example 1 84.9 Comparative Example 2 81.2 Comparative Example 3 83.7 Comparative Example 4 80.1 Comparative Example 5 82.2 Comparative Example 6 81.5 Comparative Example 7 83.0 Comparative Example 8 87.8

[0126] It can be seen from the above table that the whitening and anti-aging compositions containing exosomes prepared in Examples 1-3 of the present invention have good tyrosinase inhibitory activity.

[0127] Test Example 3

[0128] 120 volunteers aged 25-45 were selected and randomly divided into Example 1-3, Comparative Example 1-8 and blank group, with 10 people in each group. Subjects with obvious facial / eye wrinkles or obvious nasolabial folds and sensitive skin were selected as test subjects. The subjects used the whitening and anti-aging composition containing exosomes prepared in Example 1-3 or Comparative Example 1-8 once in the morning and evening every day, 1 mL each time, and performed instrument tests for related items before use and on the 14th day of use at a temperature of (21±1)°C and a humidity of 50%±10%.

[0129] 1. Skin water content and transepidermal water loss

[0130] The AF200 skin water loss meter was used to measure the transepidermal water loss (TEWL) of the face and comprehensively evaluate the repair effect of the samples on human skin.

[0131] 2. Skin redness (A value)

[0132] The VISIA2010 facial image analyzer was used to measure the facial A value and comprehensively evaluate the soothing effect of the samples on human skin.

[0133] 3. Skin firmness F4, elasticity R2

[0134] CutometerdualMPA580 skin elasticity tester was used to measure the facial firmness F4 value and elasticity R2 value to evaluate the firming effect on human skin.

[0135] The results are shown in Table 4.

[0136] Table 4

[0137] Group TEWL change rate (%) A value change rate (%) F4 change rate (%) R2 change rate (%) Example 1 -20.41 -2.21 -18.25 6.82 Example 2 -21.13 -2.19 -18.13 6.95 Example 3 -21.49 -2.27 -18.97 7.04 Comparative Example 1 -16.51 -2.01 -16.78 5.56 Comparative Example 2 -18.25 -1.82 -15.22 6.02 Comparative Example 3 -19.22 -2.15 -15.86 6.10 Comparative Example 4 -14.92 -1.74 -14.28 5.10 Comparative Example 5 -16.75 -1.82 -15.24 5.62 Comparative Example 6 -15.21 -1.57 -13.92 5.31 Comparative Example 7 -15.92 -1.62 -14.27 5.49 Comparative Example 8 -14.21 -1.54 -13.88 5.05

[0138] It can be seen from the above table that the whitening and anti-aging compositions containing exosomes prepared in Examples 1-3 of the present invention have good effects on skin repair, anti-aging, increasing skin elasticity and moisturizing.

[0139] 4. Safety Assessment

[0140] After the subjects used the samples for 14 days, they completed a questionnaire survey and used scoring to evaluate whether they had any adverse reactions such as irritation or allergies:

[0141] 0 points, no adverse reaction; 1 point, slight irritation or allergy; 2 points, moderate to mild irritation or allergy; 3 points, moderate irritation or allergy; 4 points, moderate to severe irritation or allergy; 5 points, severe irritation. Only when all subjects give a score of 0 can the sample be considered mild and non-irritating and suitable for sensitive skin.

[0142] The results are shown in Table 5.

[0143] Table 5

[0144] Group Safety score Example 1 1.02 Example 2 0.99 Example 3 0.97 Comparative Example 1 1.22 Comparative Example 2 1.31 Comparative Example 3 1.19 Comparative Example 4 1.44 Comparative Example 5 1.39 Comparative Example 6 1.52 Comparative Example 7 1.55 Comparative Example 8 1.78

[0145] It can be seen from the above table that the whitening and anti-aging compositions containing exosomes prepared in Examples 1-3 of the present invention have no obvious irritation to the skin.

[0146] 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a whitening and anti-aging composition containing exosomes, characterized in that: Extracting Poria cocos, ginseng, liquorice and Dendrobium officinale with water and precipitating with alcohol to obtain Chinese medicinal polysaccharides, forming a complex with phycobiliprotein and copper salt, and loading it into milk exosomes to obtain milk exosomes loaded with the complex; forming a mixed eutectic with quercetin, fisetin and ginsenoside Rg1 to prepare a nanoemulsion, and mixing it with the milk exosomes loaded with the complex to prepare a whitening and anti-aging composition containing exosomes; comprising the following steps: S1. Preparation of Chinese medicine polysaccharide: Poria cocos, ginseng, licorice, and Dendrobium candidum are washed, sliced, mixed, added to water, heated to boiling for extraction, cooled to room temperature, filtered, ethanol was added to the filtrate for precipitation, filtered, washed, and dried to obtain Chinese medicine polysaccharide; the mass ratio of Poria cocos, ginseng, licorice, and Dendrobium candidum is 2-3:1-2:4-5:2-3; S2. Extraction of phycobiliprotein: Drying Spirulina, crushing, adding dry powder to water, cooling with liquid nitrogen, melting at room temperature, filtering, dialyzing the filtrate, and freeze-drying the dialysate to obtain phycobiliprotein; the mass ratio of the dry powder to water is 1:5-10; S3. Preparation of Chinese medicine polysaccharide-phycobiliprotein complex: adding phycobiliprotein to water, adding NHS and EDC, stirring to activate, adding Chinese medicine polysaccharide and copper salt, stirring to react, dialyzing, freeze-drying, and preparing Chinese medicine polysaccharide-phycobiliprotein complex; the mass ratio of phycobiliprotein, NHS, EDC, Chinese medicine polysaccharide and copper salt is 10:3-4:2-3:6-8:0.5-1, and the copper salt is at least one of copper chloride, copper sulfate, and copper nitrate; S4. Preparation of milk exosomes loaded with the complex: dissolving the Chinese medicine polysaccharide-phycobiliprotein complex and milk exosomes in a PBS solution, incubating, dialyzing, and freeze-drying the dialysate to obtain milk exosomes loaded with the complex; the mass ratio of the Chinese medicine polysaccharide-phycobiliprotein complex to the milk exosomes is 2-3:10; S5. Preparation of mixed eutectic: dissolving quercetin in acetone to obtain solution 1, dissolving fisetin in ethanol to obtain solution 2, dissolving ginsenoside Rg1 in water to obtain solution 3, mixing solution 1, solution 2, and solution 3, volatilizing to obtain eutectic, filtering, washing, and drying to obtain mixed eutectic; the mass ratio of quercetin, fisetin, and ginsenoside Rg1 is 1-2:0.5-1:0.2-0.4; S6. Preparation of nanoemulsion: Soy lecithin, Tween, Span, glycerol, 1,3-propylene glycol, isopropyl myristate were stirred and mixed uniformly, distilled water was added dropwise, and then the mixed eutectic was added and stirred to obtain a nanoemulsion; the mass ratio of the soy lecithin, Tween, Span, glycerol, 1,3-propylene glycol, isopropyl myristate, distilled water, and mixed eutectic was 3-5:0.5-1:0.5-1:2-3:1-2:1-2:50-70:1-2; S7. Preparation of a whitening and anti-aging composition containing exosomes: uniformly mix the milk exosomes loaded with the complex and the nanoemulsion to prepare a whitening and anti-aging composition containing exosomes; the mass ratio of the milk exosomes loaded with the complex to the nanoemulsion is 2-3:

10.

2. The preparation method according to claim 1, characterized in that: The time for heating to boiling extraction in step S1 is 3-5 hours.

3. The preparation method according to claim 1, characterized in that: The dialysis bag used in the dialysis in step S2 has a pore size of 10-30 kDa, and the dialysis time is 12-24 hours.

4. The preparation method according to claim 1, characterized in that: The stirring activation time in step S3 is 20-30 minutes, the stirring reaction time is 12-15 hours, the dialysis bag pore size used in the dialysis is 30-50 kDa, and the dialysis time is 12-24 hours.

5. The preparation method according to claim 1, characterized in that: The incubation conditions in step S4 are 3-5°C for 3-5 hours, the dialysis bag pore size used for dialysis is 40-90 kDa, the dialysis time is 24-36 hours, the Tween is selected from at least one of Tween-20, Tween-40, Tween-60, Tween-80, and Tween-85, and the Span is selected from at least one of Span-20, Span-40, Span-60, Span-80, and Span-85.

6. A whitening and anti-aging composition containing exosomes prepared by the preparation method according to any one of claims 1 to 5.