Preparation method of anti-aging cosmetics containing exosomes

Abstract

The present invention discloses a method for preparing an anti-aging cosmetic containing exosomes, relating to the technical field of cosmetic preparation. The cosmetic comprises the following ingredients by mass: 1.01% to 5.05% liposome-encapsulated exosomes, 0.5% to 3% antioxidant, 1% to 3% Centella asiatica extract, 1% to 3% Rhodiola rosea extract, 10% to 15% moisturizer, and the remainder as a base matrix. The liposome-encapsulated exosomes are a mixture of 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. In the present invention, the antioxidant, Centella asiatica extract, and exosomes work together to improve the DPPH clearance rate of the skincare product, enhance skin brightness, and reduce roughness, achieving superior anti-aging properties. The liposome-exosome-antioxidant-Centella asiatica ternary system of the present invention works synergistically across multiple targets.

Description

Technical Field

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

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

[0003] In the cosmetics field, while traditional active ingredients (such as vitamins, peptides, and plant extracts) can improve skin problems to a certain extent, they suffer from limitations such as low transdermal absorption efficiency, poor stability, or a single target. In contrast, exosomes are considered a breakthrough ingredient due to their natural biocompatibility, efficient cell regulation, and targeted delivery properties. For example, exosomes derived from mesenchymal stem cells (MSCs) can activate fibroblast proliferation and inhibit matrix metalloproteinase (MMP) expression by delivering miRNAs and growth factors (such as EGF and TGF-β), thereby delaying skin aging and repairing photodamage. Furthermore, plant-derived exosomes (such as those extracted from grapes and ginseng) are also being used to enhance skin barrier function due to their rich antioxidant content.

[0004] However, the application of exosomes in cosmetics still faces multiple technical challenges: Preservation of activity: The biological activity of exosomes is easily affected by freeze-thaw cycles and formulation (e.g., preservatives, pH), necessitating the development of stabilization technologies such as freeze-drying or liposome encapsulation. Transdermal delivery: While their nanoscale size facilitates penetration, they must be combined with microneedles, nanocarriers, or transdermal enhancers to improve their efficiency in penetrating the stratum corneum. Safety and compliance: The source of exosomes (e.g., animal, plant, or synthetic) must comply with cosmetic regulations (e.g., EU EC 1223 / 2009). Their non-allergenicity and non-cytotoxicity must be verified, and standardized quality control methods must be established.

[0005] Currently, some companies have launched skincare products containing exosomes (such as repair serums and anti-aging creams). However, these efficacy claims are mostly based on in vitro experiments or small-sample clinical data, and their long-term safety and mechanism of action require further verification. Furthermore, the anti-aging claims made by most cosmetics are currently unsatisfactory.

[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:

[0009] On the one hand, an embodiment of the present invention provides an anti-aging cosmetic containing exosomes, the cosmetic comprising the following ingredients by mass fraction: 1.01%-5.05% liposome-encapsulated exosomes, 0.5%-3% antioxidant, 1%-3% Centella asiatica extract, 1%-3% Rhodiola rosea extract, 10%-15% moisturizer, and the remainder being a base matrix; wherein the liposome-encapsulated exosomes are a mixture of 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 ginseng.

[0010] Preferably, the liposomes are composed of dipalmitoylphosphatidylcholine DPPC and cholesterol in a mass ratio of 3:1.

[0011] Preferably, the antioxidant is any one of glutathione, niacinamide or a vitamin C derivative.

[0012] Preferably, the moisturizing agent includes one or more of glycerin, glycine, arginine, lactic acid, propylene glycol, butylene glycol, sorbitol, sodium hyaluronate, seaweed extract, and tocopherol.

[0013] 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, oil phase and emulsifier in the cosmetic are 60%-70%, 10%-15% and 2%-5%, respectively.

[0014] The second aspect of the embodiment of the present invention provides a method for preparing a cosmetic, and the preparation process is as follows:

[0015] Step 1: Prepare liposome-encapsulated exosomes: Dissolve DPPC and cholesterol in chloroform and rotary evaporate to form a thin film; then add the exosome PBS suspension, hydrate, and extrude through a 0.2 μm polycarbonate membrane to obtain liposome-encapsulated exosomes;

[0016] Step 2: Mix the liposome-encapsulated exosomes and the antioxidant at 25°C, add the base matrix preheated to 60°C; after homogenization, cool to 30°C, adjust the pH to 5.5-6.5, and obtain the cosmetics.

[0017] Preferably, the mass ratio of DPPC, cholesterol and chloroform is 3:1:7; the volume ratio of exosomes to PBS solution is 1:3.

[0018] Preferably, the homogenization is carried out at 10,000 rpm for 2 minutes.

[0019] Preferably, the prepared cosmetic is an essence, lotion or cream.

[0020] In the present invention, the Centella asiatica extract is a complex substance extracted from the plant, containing a large number of triterpenoids, such as asiaticoside, madecassoside, asiatic acid, and madecassic acid. When added to skincare products, Centella asiatica extract promotes protein synthesis, removes scars, reduces inflammation, offers antioxidant benefits, and whitens the skin, helping it maintain a youthful and healthy appearance.

[0021] The role of liposome-encapsulated exosomes:

[0022] Adipose-derived mesenchymal stem cell exosomes (ADSC-Exo): Rich in pro-regenerative factors (such as TGF-β and miR-21), they activate fibroblast collagen synthesis (type I collagen).

[0023] Ginseng-Exo: Contains ginsenoside derivatives (such as Rg3), which inhibit MMP-1 expression (reduce collagen degradation).

[0024] Liposome encapsulation advantages:

[0025] Improved transdermal penetration: DPPC / cholesterol liposomes (3:1) increased exosome transdermal penetration from 5% to 25% (fluorescence labeling experiment). Enhanced stability: Prevents exosome aggregation and inactivation in the formulation (activity retention >90% after 30 days).

[0026] Antioxidants: The effects of glutathione / nicotinamide / vitamin C derivatives: Directly scavenge free radicals: glutathione (GSH) reduces ROS; inhibit melanin transport: nicotinamide blocks the transfer of melanosomes to the stratum corneum; promote collagen regeneration: vitamin C derivatives (such as 3-O-ethyl ascorbic acid) activate prolyl hydroxylase.

[0027] Antioxidants maintain exosome membrane integrity and reduce oxidative damage; exosomes deliver miR-146a, inhibiting the NF-κB pathway and enhancing antioxidant gene expression. Centella asiatica extract enhances exosome migration to fibroblasts (scratch healing rate reaches 40%). In this invention, antioxidants, Centella asiatica extract, and exosomes work together to improve the DPPH scavenging rate of skincare products, enhance skin brightness, and reduce roughness, achieving superior anti-aging properties. The liposome-exosome-antioxidant-Centella asiatica ternary system of this invention achieves multi-target synergistic effects. DETAILED DESCRIPTION

[0028] The following will clearly and completely describe the technical solutions of the present invention in conjunction with 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 creative efforts are within the scope of protection of the present invention.

[0029] The preparation process of the fat-derived exosomes used in the present invention is as follows:

[0030] 1) Adipose-derived mesenchymal stem cells were cultured in serum-free medium for 48 h and the supernatant was collected;

[0031] 2) Exosomes were isolated by centrifugation at 3000 g for 10 min, 0.22 μm filtration, and ultracentrifugation at 100,000 g for 2 h.

[0032] 3) Purify using the ExoQuick-TC kit and store in PBS buffer (pH 7.4).

[0033] The preparation process of ginseng cell exosomes used in the present invention is as follows:

[0034] 1) Wash the ginseng and squeeze out the juice at room temperature. Filter the juice through a strainer and collect the filtered liquid. Centrifuge at 3000g for 2h, discard the precipitate, and collect the supernatant.

[0035] 2) The supernatant was centrifuged at 40,000 g for 2 h at 4°C, the precipitate was collected, and the precipitate was resuspended in phosphate buffer to obtain a resuspended secretion solution;

[0036] 3) Place the exosome resuspension in a shaker at 35°C and oscillate at 200 rpm for 1.5 h to obtain exosomes.

[0037] Example 1. This example provides a facial cream comprising the following ingredients by mass: 1.01% liposome-encapsulated exosomes, 0.5% glutathione, 3% Centella asiatica extract, 1% Rhodiola rosea extract, 10% propylene glycol, butylene glycol, sorbitol, and sodium hyaluronate, 5% PEG-40 hydrogenated castor oil, 10% squalane, and 69.49% deionized water. 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 in a mass ratio of 1.5:1.2. The liposomes are composed of dipalmitoylphosphatidylcholine (DPPC) and cholesterol in a mass ratio of 3:1.

[0038] The preparation process is as follows:

[0039] Step 1. Prepare liposome-encapsulated exosomes: dissolve DPPC and cholesterol in chloroform and rotary evaporate to form a thin 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.

[0040] Step 2: Mix the liposome-encapsulated exosomes and the antioxidant at 25°C, add the base matrix preheated to 60°C, homogenize at 10,000 rpm for 2 minutes, cool to 30°C, and adjust the pH to 5.5 to obtain the cosmetic.

[0041] Example 2. This example provides an essence comprising the following ingredients by mass: 5.05% liposome-encapsulated exosomes, 3% glutathione, 1% Centella asiatica extract, 3% Rhodiola rosea extract, 13.95% glycerol, glycine, arginine, and lactic acid combined, 2% PEG-40 hydrogenated castor oil, 12% squalane, and 60% deionized water. The glycerol, glycine, arginine, and lactic acid are present in a mass ratio of 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 in a mass ratio of 1.5:1. The liposomes are composed of dipalmitoylphosphatidylcholine (DPPC) and cholesterol in a mass ratio of 3:1.

[0042] The preparation process is as follows:

[0043] Step 1. Prepare liposome-encapsulated exosomes: dissolve DPPC and cholesterol in chloroform and rotary evaporate to form a thin 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.

[0044] Step 2: Mix the liposome-encapsulated exosomes and the antioxidant at 25°C, add the base matrix preheated to 60°C, homogenize at 10,000 rpm for 2 minutes, cool to 30°C, and adjust the pH to 6.5 to obtain the cosmetic.

[0045] Example 3. This example provides an emulsion comprising the following ingredients by mass: 3.5% liposome-encapsulated exosomes, 3% glutathione, 1.5% Centella asiatica extract, 1.5% Rhodiola rosea extract, 15% propylene glycol, butylene glycol, sorbitol, and sodium hyaluronate combined, 3% PEG-40 hydrogenated castor oil, 10.5% squalane, and 62% deionized water. 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 in a mass ratio of 1.5:1. The liposomes are composed of dipalmitoylphosphatidylcholine (DPPC) and cholesterol in a mass ratio of 3:1.

[0046] The preparation process is as follows:

[0047] Step 1. Prepare liposome-encapsulated exosomes: dissolve DPPC and cholesterol in chloroform and rotary evaporate to form a thin 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.

[0048] Step 2: Mix the liposome-encapsulated exosomes and the antioxidant at 25°C, add the base matrix preheated to 60°C, homogenize at 10,000 rpm for 2 minutes, cool to 30°C, and adjust the pH to 6.2 to obtain the cosmetic.

[0049] Comparative Example 1

[0050] Compared with Example 3, the Centella asiatica extract was removed, the content of liposome-encapsulated exosomes was increased to 4%, the content of glutathione was increased to 4%, and the other components remained unchanged.

[0051] Comparative Example 2

[0052] Compared with Example 3, the liposome-encapsulated exosomes were removed, the content of Centella asiatica extract was increased to 3%, the content of glutathione was increased to 4.5%, and the other components remained unchanged.

[0053] Comparative Example 3

[0054] Compared with Example 3, glutathione was removed, the content of liposome-encapsulated exosomes was increased to 5%, the content of Centella asiatica extract was increased to 3%, and the other components remained unchanged.

[0055] Comparative Example 4

[0056] Compared with Example 3, unencapsulated exosomes were directly added.

[0057] Comparative Example 5

[0058] Compared with Example 3, only exosomes and basic matrix are contained.

[0059] Comparative Example 6

[0060] Compared with Example 3, only ginseng exosomes were used.

[0061] Comparative Example 7

[0062] Compared with Example 3, only exosomes secreted by adipose-derived mesenchymal stem cells were used.

[0063] The 30-day active ingredient retention rate, DPPH clearance rate, and tyrosinase inhibition rate of the skin care products obtained in each example and comparative example were measured. The results are shown in Table 1.

[0064] 1. DPPH free radical scavenging rate determination

[0065] principle:

[0066] DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) is a stable free radical (purple) with strong absorption at 517 nm. When antioxidants donate hydrogen atoms to bind to it, DPPH is reduced to yellow, and the absorbance decreases. The clearance rate is calculated based on the change in absorbance.

[0067] Reagents and instruments:

[0068] DPPH solution (0.1 mM, dissolved in anhydrous ethanol)

[0069] Samples to be tested (skin care products of various embodiments and comparative examples)

[0070] UV spectrophotometer (measure absorbance at 517 nm)

[0071] Centrifuge, micropipette

[0072] step:

[0073] Sample processing:

[0074] Take 1 mL of sample solution and mix it with 1 mL of DPPH solution, and react for 30 min (25°C) in the dark.

[0075] Control group: 1 mL of anhydrous ethanol + 1 mL of DPPH solution.

[0076] Blank group: 1 mL sample + 1 mL anhydrous ethanol.

[0077] Determine absorbance:

[0078] Centrifuge (10,000 rpm, 5 min), collect the supernatant, and measure the absorbance at 517 nm.

[0079] Calculate the clearance rate:

[0080] DPPH clearance rate (%) = [1 − Acontrol (Asample − Ablank)] × 100.

[0081] 2. Tyrosinase Inhibition Rate Determination

[0082] Tyrosinase is a key enzyme in melanin synthesis, catalyzing the conversion of tyrosine to dopa to dopaquinone. The inhibitory effect of a sample on the enzyme activity was assessed by measuring the absorbance change of L-DOPA (L-DOPA) at 475 nm.

[0083] Reagents and instruments:

[0084] Tyrosinase (from mushrooms, ≥1000 U / mg)

[0085] L-DOPA solution (2 mM, pH 6.8 in phosphate buffer)

[0086] Samples to be tested

[0087] UV spectrophotometer (measure absorbance at 475 nm)

[0088] Constant temperature water bath (37°C)

[0089] step:

[0090] Reaction system (200 μL):

[0091] Control group: 50 μL tyrosinase (50 U / mL) + 100 μL L-DOPA + 50 μL buffer.

[0092] Experimental group: 50 μL tyrosinase + 100 μL L-DOPA + 50 μL sample solution.

[0093] Blank group: 50 μL buffer (without enzyme) + 100 μL L-DOPA + 50 μL sample.

[0094] Reaction conditions:

[0095] Incubate at 37°C for 20 min and immediately terminate the reaction by placing in an ice bath.

[0096] Determine absorbance:

[0097] After centrifugation, the absorbance was measured at 475 nm;

[0098] Calculate the inhibition rate:

[0099] Tyrosinase inhibition rate (%) = [1 − Acontrol (Asample − Ablank)] × 100.

[0100] Table 1 DPPH clearance rate, tyrosinase inhibition rate and transdermal rate

[0101] .

[0102] 100 female volunteers aged 20-50 years old, in good health, but with dark yellow face, dull skin, many spots, and rough skin were selected. They were randomly divided into 10 groups and, after signing a confidentiality agreement, were tested for 3 months using the skin care products of Example 3 and Comparative Examples 1-7. The skin conditions (gloss, roughness, and spot deposition) of each group of volunteers were recorded after 1 month, 2 months, and 3 months.

[0103] Skin glossiness is measured using the SkinGlossMeter (SGM2008). Higher values ​​indicate glossier skin. Skin roughness is measured using PrimosCR, which captures a 3D image of the subject's cheek skin and analyzes it using specialized analysis software. The Sa value is the arithmetic mean of the heights of each point within the measurement area. Lower values ​​indicate smoother skin.

[0104] Each group of volunteers measured the values ​​before using the skin care products and took the average; the values ​​were recorded after using the products for 3 months.

[0105] Before the experiment, the average glossiness of the corresponding volunteers in each group was between 46-48; the roughness was between 25.1-25.2.

[0106] The results are shown in Table 2;

[0107] Table 2 Records of roughness and skin glossiness of volunteers after using the product

[0108] .

[0109] As can be seen from Tables 1 and 2, the synergistic effect between the Centella asiatica extract, antioxidants, and exosomes in the present invention significantly improves the skin's radiance and antioxidant properties. At the same time, after the exosomes are encapsulated, their stability is improved, effectively preventing degradation, and increasing the content of active ingredients in the cosmetics.

[0110] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0111] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to 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 comprises the following ingredients by mass fraction: 1.01%-5.05% of liposome-encapsulated exosomes, 0.5%-3% of an antioxidant, 1%-3% of a Centella asiatica extract, 1%-3% of a Rhodiola rosea extract, 10%-15% of a moisturizer, and the remainder being a base matrix; wherein the liposome-encapsulated exosomes are a mixture of exosomes secreted by adipose-derived mesenchymal stem cells and exosomes secreted by plant cells in a mass ratio of 1.5:1.2; and the plant is ginseng.

2. The anti-aging cosmetic containing exosomes according to claim 1, characterized in that: Liposomes are 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, nicotinamide or vitamin C derivatives.

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

5. The 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, oil phase and emulsifier in the cosmetic are 60%-70%, 10%-15% and 2%-5% respectively.

6. The method for preparing the anti-aging cosmetic according to any one of claims 1 to 5, characterized in that: The preparation process is as follows: Step 1: Prepare liposome-encapsulated exosomes: Dissolve DPPC and cholesterol in chloroform and rotary evaporate to form a thin film; then add the exosome PBS suspension, hydrate, and extrude through a 0.2 μm polycarbonate membrane to obtain liposome-encapsulated exosomes; Step 2: Mix the liposome-encapsulated exosomes and the antioxidant at 25°C, add the base matrix preheated to 60°C; after homogenization, cool to 30°C, adjust the pH to 5.5-6.5, and obtain the cosmetics.

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

3.

8. The preparation method according to claim 6, characterized in that The homogenization was performed at 10,000 rpm for 2 minutes.

9. The preparation method according to claim 8, characterized in that The prepared cosmetics are essence, lotion or cream.