Preparation method of anti-aging cosmetic containing exosome

Abstract

The invention discloses a preparation method of an anti-aging cosmetic containing exosome, and relates to the technical field of cosmetic preparation, the cosmetic is prepared from the following components in percentage by mass: 1.01%-5.05% of exosome wrapped by liposome, 0.5%-3% of an antioxidant, 1%-3% of a centella asiatica extract, 1%-3% of a rhodiola rosea extract, 10%-15% of a humectant and the balance of a basic matrix; wherein the exosome wrapped by the liposome is a mixture formed by mixing an exosome secreted by adipose-derived mesenchymal stem cells and an exosome secreted by plant cells according to a mass ratio of 1.5: 1.2; the plant is ginseng. According to the skin care product, the antioxidant, the centella asiatica extract and the exosome act together to improve the DPPH clearance rate of the skin care product, the skin brightness is improved, the roughness is reduced, and the excellent anti-aging performance is achieved. The lipid exosome-antioxidant-centella asiatica ternary system disclosed by the invention has a multi-target synergistic effect.

Description

Technical Field

[0001] The present invention relates to the technical field of the preparation of anti-aging skin care products, and more particularly to a method for preparing an anti-aging cosmetic containing exosomes. Background Art

[0002] In recent years, with the rapid development of regenerative medicine and biotechnology, exosomes, as a novel bioactive component, have attracted wide attention in the fields of medicine and cosmetics. Exosomes are nanoscale (30 - 150 nm) extracellular vesicles secreted by cells, rich in bioactive substances such as proteins, lipids, nucleic acids (such as mRNA, miRNA), and growth factors, and can mediate intercellular communication and regulate the physiological functions of target cells. Research shows that exosomes have significant potential in tissue repair, anti-inflammation, antioxidant, and promoting collagen synthesis, which provides a scientific basis for their application in skin health management.

[0003] In the field of cosmetics, traditional active ingredients (such as vitamins, peptides, plant extracts, etc.) can improve skin problems to a certain extent, but have limitations such as low transdermal absorption efficiency, poor stability, or single action targets. In contrast, exosomes are regarded as breakthrough components due to their natural biocompatibility, high-efficiency cell regulation ability, and targeted delivery characteristics. For example, exosomes derived from mesenchymal stem cells (MSCs) can activate fibroblast proliferation and inhibit the expression of matrix metalloproteinases (MMPs) by transmitting miRNAs and growth factors (such as EGF, TGF-β), thereby delaying skin aging and repairing photo-damage. In addition, plant-derived exosomes (such as exosomes extracted from grapes and ginseng) are also used to enhance the skin barrier function due to their rich antioxidant substances.

[0004] However, the application of exosomes in cosmetics still faces multiple technical challenges: Active preservation: The biological activity of exosomes is easily affected by freeze-thaw cycles and formulation (such as preservatives, pH value), and stabilization technologies such as cryogenic freeze-drying or liposome encapsulation need to be developed. Transdermal delivery: Although the nanoscale size is beneficial for penetration, micro-needles, nanocarriers, or transdermal enhancers need to be combined to improve its penetration efficiency through the stratum corneum. Safety and compliance: The source of exosomes (such as animal, plant, or synthetic) needs to comply with cosmetic regulations (such as EU EC 1223 / 2009), and it is necessary to verify its non-allergenicity, non-cytotoxicity, and establish a standardized quality control method.

[0005] Currently, some companies have launched skin care products containing exosomes (such as repair essence, anti-aging cream), but their efficacy claims are mostly based on in vitro experiments or small-sample clinical data, and the safety and mechanism of action of long-term application still need to be further verified. At the same time, currently, the anti-aging effects claimed in most cosmetics are not satisfactory.

[0006] Therefore, how to provide an anti-aging cosmetic containing exosomes is a technical problem that those skilled in the art urgently need to solve. Summary of the Invention

[0007] In view of this, the present invention provides a method for preparing an anti-aging cosmetic containing exosomes.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions: On the one hand, an embodiment of the present invention provides an anti-aging cosmetic containing exosomes, and the cosmetic comprises the following components in mass fractions: 1.01%-5.05% of exosomes encapsulated by liposomes, 0.5%-3% of antioxidant, 1%-3% of centella asiatica extract, 1%-3% of rhodiola rosea extract, 10%-15% of humectant, and the balance is a base matrix; wherein, the exosomes encapsulated by liposomes are a mixture of exosomes secreted by adipose-derived mesenchymal stem cells and exosomes secreted by plant cells mixed in a mass ratio of 1.5:1.2; the plant is ginseng.

[0009] Preferably, the liposome is composed of dipalmitoyl phosphatidylcholine DPPC and cholesterol in a mass ratio of 3:1.

[0010] Preferably, the antioxidant is any one of glutathione, niacinamide or vitamin C derivative.

[0011] Preferably, the humectant includes one or more of glycerol, glycine, arginine, lactic acid, propylene glycol, butylene glycol, sorbitol, sodium hyaluronate, seaweed extract, and tocopherol.

[0012] Preferably, the base matrix includes an aqueous phase, an oil phase and an emulsifier; the aqueous phase is deionized water, the oil phase is squalane; the emulsifier is PEG-40 hydrogenated castor oil, and the final proportions of the aqueous phase, the oil phase and the emulsifier in the cosmetic are 60%-70%, 10%-15%, and 2%-5% respectively.

[0013] On the second aspect, an embodiment of the present invention provides a method for preparing the cosmetic, and the preparation process is as follows: Step 1: Prepare exosomes encapsulated by liposomes: Dissolve DPPC and cholesterol in chloroform, and rotary evaporate to form a film; then add an exosome PBS suspension, hydrate and extrude through a 0.2 μm polycarbonate membrane to obtain exosomes encapsulated by liposomes; Step 2: Mix the exosomes encapsulated by liposomes with the antioxidant at 25°C, add the base matrix preheated to 60°C; homogenize and cool to 30°C, and adjust the pH to 5.5-6.5 to obtain the cosmetic.

[0014] Preferably, the mass ratio of DPPC, cholesterol to chloroform is 3:1:7; the volume ratio of exosomes to PBS solution is 1:3.

[0015] Preferably, the homogenization is carried out at 10,000 rpm for 2 min.

[0016] Preferably, the prepared cosmetics are essence, lotion or cream.

[0017] In the present invention, the centella asiatica extract is a composite substance extracted from centella asiatica, containing a large amount of triterpenoids, such as asiaticoside, madecassoside, asiatic acid and madecassic acid, etc. Adding the centella asiatica extract to skin care products has the effects of promoting protein synthesis in the skin, removing scars, anti-inflammatory, antioxidant, whitening, etc., and can help the skin maintain a young and healthy state.

[0018] Functions of liposome-encapsulated exosomes: Adipose-derived mesenchymal stem cell exosomes (ADSC-Exo): Rich in pro-regenerative factors (such as TGF-β, miR-21), activating fibroblast collagen synthesis (type I collagen).

[0019] Ginseng exosomes (Ginseng-Exo): Containing ginsenoside derivatives (such as Rg3), inhibiting MMP-1 expression (reducing collagen degradation).

[0020] Advantages of liposome encapsulation: Improved transdermal rate: DPPC / cholesterol liposome (3:1) increases the transdermal rate of exosomes from 5% to 25% (fluorescent labeling experiment). Enhanced stability: Avoiding the aggregation and inactivation of exosomes in the formulation (activity retention > 90% after 30 days).

[0021] Antioxidants: Functions of glutathione / niacinamide / vitamin C derivatives: Directly scavenging free radicals: Glutathione (GSH) reduces ROS; Inhibiting melanin transport: Niacinamide blocks the transfer of melanosomes to the stratum corneum; Promoting collagen regeneration: Vitamin C derivatives (such as 3-O-ethyl ascorbic acid) activate prolyl hydroxylase.

[0022] Antioxidants maintain the integrity of the exosome membrane and reduce oxidative damage; Exosomes deliver miR-146a to inhibit the NF-κB pathway and enhance the expression of antioxidant genes. The centella asiatica extract enhances the migratory ability of exosomes to fibroblasts (the wound healing rate in the scratch experiment reaches 40%); In the present invention, the co-action of antioxidants, centella asiatica extract and exosomes improves the DPPH scavenging rate of skin care products, increases skin brightness and reduces roughness, achieving excellent anti-aging performance. The liposome-exosome-antioxidant-centella ternary system of the present invention acts through multi-target synergistic effects. Detailed implementation manners

[0023] In the following, in combination with the embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0024] The preparation process of the exosomes derived from fat used in the present invention is as follows: 1) Cultivate adipose-derived mesenchymal stem cells in a serum-free medium for 48 h, and collect the supernatant; 2) Separate exosomes by centrifugation at 3000 g (10 min), filtration through a 0.22-μm filter, and ultracentrifugation (100,000 g, 2 h) in sequence; 3) Purify using the ExoQuick-TC kit and store in PBS buffer (pH 7.4).

[0025] The preparation process of the ginseng extracellular vesicles used in the present invention is as follows: 1) Squeeze the juice from ginseng washed with clean water at room temperature. The squeezed juice is filtered through a filter screen, and the filtered liquid is collected; centrifuge at 3000 g for 2 h, discard the precipitate, and collect the supernatant; 2) Centrifuge the supernatant at 40000 g at 4 °C for 2 h, collect the precipitate, and resuspend the precipitate with phosphate buffer to obtain an exosome resuspension; 3) Place the exosome resuspension on a shaker at 35 °C and oscillate at 200 rpm for 1.5 h to obtain exosomes.

[0026] Example 1. This example provides a cream, which includes the following components by mass fraction: liposome-encapsulated exosomes 1.01%, glutathione 0.5%, centella asiatica extract 3%, rhodiola rosea extract 1%, the total proportion of propylene glycol, butylene glycol, sorbitol, and sodium hyaluronate is 10%, PEG-40 hydrogenated castor oil 5%, squalane 10%, deionized water 69.49%. Among them, the mass ratio of propylene glycol, butylene glycol, sorbitol, and sodium hyaluronate is 1:1:1:1; the liposome-encapsulated exosomes are a mixture of exosomes secreted by adipose-derived mesenchymal stem cells and exosomes secreted by ginseng cells mixed in a mass ratio of 1.5:1.2. The liposome is composed of dipalmitoylphosphatidylcholine DPPC and cholesterol in a mass ratio of 3:1.

[0027] The preparation process is as follows: Step 1. Preparation of liposome-encapsulated exosomes: Dissolve DPPC and cholesterol in chloroform, and rotary evaporate to form a film; then add the exosome PBS suspension, hydrate and extrude through a 0.2 μm polycarbonate membrane to obtain liposome-encapsulated exosomes; the mass ratio of DPPC, cholesterol to chloroform is 3:1:7; the volume ratio of exosomes to PBS solution is 1:3.

[0028] Step 2. Mix the liposome-encapsulated exosomes with the antioxidant at 25°C, and add the base matrix preheated to 60°C; homogenize at 10,000 rpm for 2 min and then cool to 30°C, adjust the pH to 5.5 to obtain the cosmetic.

[0029] Example 2. This example provides a serum, including the following components by mass fraction: 5.05% of liposome-encapsulated exosomes, 3% of glutathione, 1% of centella asiatica extract, 3% of rhodiola rosea extract, 13.95% in total of glycerol, glycine, arginine and lactic acid, 2% of PEG-40 hydrogenated castor oil, 12% of squalane, 60% of deionized water. Among them, the mass ratio of glycerol, glycine, arginine and lactic acid is 1:1:1:1, and the liposome-encapsulated exosomes are a mixture of exosomes secreted by adipose-derived mesenchymal stem cells and exosomes secreted by ginseng cells in a mass ratio of 1.5:1.2; the liposome is composed of dipalmitoylphosphatidylcholine DPPC and cholesterol in a mass ratio of 3:1.

[0030] The preparation process is as follows: Step 1. Preparation of liposome-encapsulated exosomes: Dissolve DPPC and cholesterol in chloroform, and rotary evaporate to form a film; then add the exosome PBS suspension, hydrate and extrude through a 0.2 μm polycarbonate membrane to obtain liposome-encapsulated exosomes; the mass ratio of DPPC, cholesterol to chloroform is 3:1:7; the volume ratio of exosomes to PBS solution is 1:3.

[0031] Step 2. Mix the liposome-encapsulated exosomes with the antioxidant at 25°C, and add the base matrix preheated to 60°C; homogenize at 10,000 rpm for 2 min and then cool to 30°C, adjust the pH to 6.5 to obtain the cosmetic.

[0032] Example 3. This example provides an emulsion, which includes the following components by mass fraction: liposome-encapsulated exosomes 3.5%, glutathione 3%, Centella asiatica extract 1.5%, Rhodiola rosea extract 1.5%, the total proportion of propylene glycol, butylene glycol, sorbitol and sodium hyaluronate 15%, PEG-40 hydrogenated castor oil 3%, squalane 10.5%, deionized water 62%. Among them, the mass ratio of propylene glycol, butylene glycol, sorbitol and sodium hyaluronate is 1:1:1:1. The liposome-encapsulated exosomes are a mixture of exosomes secreted by adipose-derived mesenchymal stem cells and exosomes secreted by ginseng cells, mixed in a mass ratio of 1.5:1.2; the liposome is composed of dipalmitoyl phosphatidylcholine DPPC and cholesterol in a mass ratio of 3:1.

[0033] The preparation process is as follows: Step 1. Prepare liposome-encapsulated exosomes: Dissolve DPPC and cholesterol in chloroform, and rotate to evaporate to form a film; then add the exosome PBS suspension, hydrate and extrude through a 0.2 μm polycarbonate membrane to obtain liposome-encapsulated exosomes; the mass ratio of DPPC, cholesterol and chloroform is 3:1:7; the volume ratio of exosomes to PBS solution is 1:3.

[0034] Step 2. Mix the liposome-encapsulated exosomes with antioxidants at 25 °C, and add the base matrix preheated to 60 °C; homogenize at 10,000 rpm for 2 min and then cool to 30 °C, and adjust the pH to 6.2 to obtain the cosmetic.

[0035] Comparative Example 1 Compared with Example 3, the Centella asiatica extract is removed, the content of liposome-encapsulated exosomes is increased to 4%, and the content of glutathione is increased to 4%, and the other components remain unchanged.

[0036] Comparative Example 2 Compared with Example 3, the liposome-encapsulated exosomes are removed, the content of Centella asiatica extract is increased to 3%, and the content of glutathione is increased to 4.5%, and the other components remain unchanged.

[0037] Comparative Example 3 Compared with Example 3, glutathione is removed, the content of liposome-encapsulated exosomes is increased to 5%, and the content of Centella asiatica extract is increased to 3%, and the other components remain unchanged.

[0038] Comparative Example 4 Compared with Example 3, unencapsulated exosomes are directly added.

[0039] Comparative Example 5 Compared with Example 3, it only contains exosomes and the base matrix.

[0040] Comparative Example 6 Compared with Example 3, only ginseng exosomes were used.

[0041] Comparative Example 7 Compared with Example 3, only exosomes secreted by adipose-derived mesenchymal stem cells were used.

[0042] The 30-day active ingredient retention rate, DPPH scavenging rate, and tyrosinase inhibition rate of the skin care products obtained in each example and each comparative example were measured. The results are shown in Table 1.

[0043] 1. Determination of DPPH free radical scavenging rate Principle: DPPH (1,1-diphenyl-2-picrylhydrazyl) is a stable free radical (purple) with a strong absorption at 517 nm. When an antioxidant provides a hydrogen atom to combine with it, DPPH is reduced to yellow, and the absorbance decreases. The scavenging rate is calculated through the change in absorbance.

[0044] Reagents and instruments: DPPH solution (0.1 mM, dissolved in absolute ethanol) Sample to be measured (skin care products of each example and comparative example) UV-visible spectrophotometer (for measuring absorbance at 517 nm) Centrifuge, micropipette Steps: Sample treatment: Take 1 mL of the sample solution and mix it with 1 mL of the DPPH solution, and react in the dark for 30 min (25 °C).

[0045] Control group: 1 mL of absolute ethanol + 1 mL of DPPH solution.

[0046] Blank group: 1 mL of sample + 1 mL of absolute ethanol.

[0047] Measurement of absorbance: Centrifuge (10,000 rpm, 5 min), take the supernatant, and measure the absorbance at 517 nm.

[0048] Calculation of scavenging rate: DPPH scavenging rate (%) = [1 - Acontrol / (Asample - Ablank)] × 100.

[0049] 2. Determination of tyrosinase inhibition rate Tyrosinase is the key enzyme for melanin synthesis, catalyzing tyrosine → dopa → dopaquinone. By detecting the change in absorbance of L-DOPA at 475 nm, the inhibitory ability of the sample on the enzyme activity is evaluated.

[0050] Reagents and instruments: Tyrosinase (from mushroom, ≥1000U / mg) L-DOPA solution (2 mM, prepared with pH 6.8 phosphate buffer) Sample to be tested UV spectrophotometer (for measuring absorbance at 475 nm) Thermostatic water bath (37 °C) Steps: Reaction system (200 μL): Control group: 50 μL tyrosinase (50 U / mL) + 100 μL L-DOPA + 50 μL buffer.

[0051] Experimental group: 50 μL tyrosinase + 100 μL L-DOPA + 50 μL sample solution.

[0052] Blank group: 50 μL buffer (enzyme-free) + 100 μL L-DOPA + 50 μL sample.

[0053] Reaction conditions: Incubate at 37 °C for 20 min, and immediately terminate the reaction by ice bath.

[0054] Measure absorbance: Measure the absorbance at 475 nm after centrifugation; Calculate the inhibition rate: Tyrosinase inhibition rate (%) = [1−Acontrol(Asample−Ablank)]×100.

[0055] Table 1 DPPH scavenging rate, tyrosinase inhibition rate and transdermal rate .

[0056] Select 100 female volunteers, aged 20 - 50 years old, in good health, but with sallow complexion, dull skin, many skin spots and rough skin. Randomly divide them into 10 groups. After signing a confidentiality agreement, they are respectively asked to try the skin care products of Example 3 and Comparative Examples 1 - 7 for 3 months, and record the skin conditions (luster, roughness, skin spot deposition) of the volunteers in each group after 1 month, 2 months and 3 months.

[0057] Among them, the skin luster is measured by SkinGlossMeter, SGM2008. The larger the value, the more lustrous the skin; the skin roughness parameter is obtained by taking a 3D image of the local skin of the subject's cheek with PrimosCR and then analyzing it with professional analysis software, and is characterized by the Sa value. The Sa value is the arithmetic mean of the heights of each point within the measured area; the lower the value of the parameter, the smoother the skin.

[0058] The average value was taken for each group of volunteers before using the skin care products; the values were recorded after 3 months of using the products.

[0059] Before the experiment, the average glossiness of the corresponding volunteers in each group was between 46 and 48; the roughness was between 25.1 and 25.2.

[0060] The results are shown in Table 2; Table 2 Record of the roughness and skin glossiness of volunteers after using the products .

[0061] As can be seen from Table 1 and Table 2, in the present invention, the synergistic effect among centella asiatica extract, antioxidant and exosome significantly improves the gloss of the skin, enhances the antioxidant performance. At the same time, after the exosome is coated, its stability is improved, effectively preventing degradation and increasing the content of active ingredients in the cosmetic.

[0062] In this specification, each embodiment is described in a progressive manner. The key point of each embodiment is to illustrate the differences from other embodiments. The same or similar parts among the embodiments can be referred to each other.

[0063] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An anti-aging cosmetic containing exosomes, characterized in that, The cosmetic product comprises the following components in mass fractions: 1.01%-5.05% of exosomes encapsulated by liposomes, 0.5%-3% of antioxidant, 1%-3% of Centella asiatica extract, 1%-3% of Rhodiola rosea extract, 10%-15% of humectant, and the balance being a base matrix; wherein, the exosomes encapsulated by liposomes are a mixture formed by mixing exosomes secreted by adipose-derived mesenchymal stem cells and exosomes secreted by plant cells in a mass ratio of 1.5:1.2; the plant is Panax ginseng.

2. The anti-aging cosmetic containing exosomes according to claim 1, characterized in that, The liposome is composed of dipalmitoylphosphatidylcholine (DPPC) and cholesterol in a mass ratio of 3:

1.

3. The anti-aging cosmetic containing exosomes according to claim 1, characterized in that, The antioxidant is any one of glutathione, niacinamide or vitamin C derivatives.

4. The preparation method of an anti-aging cosmetic containing exosomes according to claim 1, characterized in that, The humectant includes one or more of glycerol, glycine, arginine, lactic acid, propylene glycol, butylene glycol, sorbitol, sodium hyaluronate, seaweed extract, and tocopherol.

5. An anti-aging cosmetic containing exosomes according to claim 1, characterized in that, The base matrix includes an aqueous phase, an oil phase and an emulsifier; the aqueous phase is deionized water, the oil phase is squalane; the emulsifier is PEG-40 hydrogenated castor oil, and the final proportions of the aqueous phase, the oil phase and the emulsifier in the cosmetic product are 60%-70%, 10%-15%, and 2%-5% respectively.

6. The preparation method of the anti-aging cosmetic according to any one of claims 1-5, characterized in that, The preparation process is as follows: Step 1, preparing exosomes encapsulated by liposomes: dissolving DPPC and cholesterol in chloroform, and rotary evaporating to form a film; then adding an exosome PBS suspension, hydrating and extruding through a 0.2 μm polycarbonate membrane to obtain exosomes encapsulated by liposomes; Step 2, mixing the exosomes encapsulated by liposomes with the antioxidant at 25°C, and adding the preheated base matrix at 60°C; homogenizing and then cooling to 30°C, and adjusting the pH to 5.5-6.5 to obtain the cosmetic product.

7. The preparation method according to claim 6, characterized in that, The mass ratio of DPPC, cholesterol to chloroform is 3:1:7; the volume ratio of exosomes to PBS solution is 1:

3.

8. The preparation method according to claim 6, wherein The homogenization is carried out at 10,000 rpm for 2 min.

9. The preparation method according to claim 8, characterized in that, The prepared cosmetic product is essence, lotion or cream.