Ginseng exosome, extraction method thereof and application of ginseng exosome in preparation of anti-photoaging skin care product

Abstract

The invention discloses a ginseng exosome, an extraction method thereof and application of the ginseng exosome in preparation of anti-photoaging skin care products, and belongs to the technical field of biological medicines. In order to solve the technical blank of ginseng exosomes in the aspect of light aging resistance in the prior art and solve the technical problems of complicated steps, long time consumption and small treatment capacity of exosome extraction by an ultracentrifugation method in the prior art, the invention provides an extraction method of ginseng exosomes. Therefore, the extraction rate of the ginseng exosome is improved, the treatment steps are simplified, the time is saved, and the purpose of producing the ginseng exosome in a large scale is achieved. The ginseng exosome obtained through the extraction method can be applied to preparation of anti-photoaging skin care products, and clinical application of the exosome is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to ginseng exosomes and an extraction method thereof, and application thereof in the preparation of anti-photoaging skin care products. Background Art

[0002] Skin photoaging is primarily caused by ultraviolet (UVB)-induced oxidative stress, collagen degradation, and fibroblast dysfunction. UVB can lead to skin sensitivity, pigmentation, sagging, and the deepening of wrinkles. Traditional treatments, such as antioxidants (vitamin C, coenzyme Q10) or laser therapy, have the following limitations: antioxidants have low transdermal absorption (<5%) and cannot reach the dermis; laser therapy can easily induce post-inflammatory hyperpigmentation and has a high recurrence rate.

[0003] Ginseng is a traditional herb widely used in Traditional Chinese Medicine (TCM) and is believed to have numerous health benefits, including immune system enhancement, fatigue resistance, anti-aging, antioxidant activity, anti-inflammatory properties, and blood sugar regulation. Exosomes are a type of nanoscale membrane vesicle secreted by cells, typically ranging from 30 to 200 nm in diameter. They carry intracellular molecules such as proteins, lipids, and RNA, and are capable of transmitting signals between cells. In recent years, exosomes have garnered attention as a key mediator of intercellular communication due to their diverse biological activities.

[0004] Ginseng exosomes are nanoscale membrane vesicles secreted from ginseng cells. Research has shown that they possess immunomodulatory, anti-inflammatory, and anti-tumor properties; however, their application in anti-photoaging has not been fully explored. Furthermore, the current ultracentrifugation method for extracting ginseng exosomes is cumbersome, making ginseng juice difficult to clarify and prone to clogging filter membranes. This results in a time-consuming process, low throughput, and difficulty in achieving large-scale production. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a ginseng exosome and an extraction method thereof and an application thereof in the preparation of anti-photoaging skin care products.

[0006] One of the purposes of the present invention is to provide a method for extracting ginseng exosomes, the extraction method comprising the following steps:

[0007] S1: Using distilled water, remove the dirt from the surface of ginseng until the surface is clean and free of dirt, cut the ginseng into small pieces, and squeeze the juice with a juicer to obtain crude ginseng juice; the ginseng is five-year-old garden ginseng or fifteen-year-old or older wild ginseng;

[0008] S2: The crude ginseng juice obtained in S1 is centrifuged in a centrifuge pre-cooled to 4°C to obtain ginseng stock solution;

[0009] S3: Add chitosan solution to the ginseng stock solution obtained in S2, heat and stir, let stand at room temperature for 2 hours, centrifuge at 10,000×g for 10-30 minutes, discard the precipitate, and obtain ginseng clarified solution;

[0010] S4: Using a tangential flow ultrafiltration system, a hollow fiber column was selected to ultrafilter and concentrate the ginseng clarified liquid obtained in S3 to obtain ginseng exosomes.

[0011] In a preferred embodiment of the present invention, the conditions for the centrifugal treatment in S2 are: 200×g, 10 min; 2000×g, 20 min; 10000×g, 30 min.

[0012] In a preferred embodiment of the present invention, the chitosan solution in S3 is obtained by dissolving chitosan powder in 1% acetic acid solution and stirring until completely dissolved; the final concentration of chitosan in the ginseng clarified solution is 0.2-2% (w / v).

[0013] In a preferred embodiment of the present invention, the processing conditions of the tangential flow ultrafiltration system in S4 are: transmembrane pressure of 0.3-1.0 bar, and flow rate of 50-200 mL / min.

[0014] In a preferred embodiment of the present invention, the molecular weight cut-off of the hollow fiber column in the tangential flow ultrafiltration system in S4 is 300 kDa.

[0015] A second object of the present invention is to provide ginseng exosomes, which are obtained by the above-mentioned extraction method.

[0016] The third object of the present invention is to provide a ginseng exosome freeze-dried powder, which is prepared by uniformly mixing the above-mentioned ginseng exosomes with trehalose having a final concentration of 1%-2% (w / v), freezing the mixture at -80°C overnight, and freeze-drying the mixture using a freeze dryer to obtain the ginseng exosome freeze-dried powder.

[0017] A fourth object of the present invention is to provide a ginseng exosome transdermal delivery system, which is composed of a ginseng exosome freeze-dried powder solvent, a microneedle array, an iontophoresis instrument, and the above-mentioned ginseng exosome freeze-dried powder.

[0018] In a preferred embodiment of the present invention, the ginseng exosome freeze-dried powder solvent is composed of 1%-5% glycerol, 0.1%-0.5% oligopeptide-1, 0.1%-0.5% sodium hyaluronate, 0.5-1% vitamin C ethyl ether, 0.1%-0.5% phenoxyethanol and water; the length of the microneedle array is 200 μm and the needle density is 50 needles / cm 2 ; The voltage of the ion introduction instrument is 5V and the frequency is 10kHz.

[0019] A fifth object of the present invention is to provide the use of the above-mentioned ginseng exosomes, ginseng exosome freeze-dried powder or ginseng exosome transdermal delivery system in the preparation of anti-photoaging skin care products.

[0020] Beneficial Effects of the Invention: The present invention provides a method for extracting ginseng exosomes. By adding chitosan and a tangential flow ultrafiltration system, the method simplifies the existing ultracentrifugation exosome extraction process. Chitosan is a natural high-molecular-weight polysaccharide that carries a positive charge under acidic conditions. By adding a 0.2-2% chitosan solution dissolved in acetic acid, the chitosan binds to negatively charged macromolecules (such as proteins and some polysaccharides) in ginseng juice through electrostatic interactions. These substances aggregate to form larger particles, which then precipitate. Combined with heating and stirring under certain temperature conditions, the ginseng juice is clarified. The present invention utilizes a tangential flow ultrafiltration system, which flows tangentially through the filtration membrane, reducing membrane clogging and enabling rapid processing of large volumes of clarified ginseng liquid, avoiding the time constraints associated with multiple centrifugation cycles in traditional ultracentrifugation methods. Furthermore, the tangential design of the tangential flow system reduces mechanical shear forces, preventing damage to the exosome membrane structure. This improves the extraction rate of ginseng exosomes, simplifies processing steps, and saves time, ultimately achieving large-scale production of ginseng exosomes.

[0021] The ginseng exosomes obtained by the extraction method of the present invention, the ginseng exosome freeze-dried powder and the ginseng exosome transdermal delivery system can all be used in the preparation of anti-photoaging skin care products, filling the lack of ginseng exosome applications in anti-photoaging in the existing technology and realizing the clinical application of ginseng exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a transmission electron micrograph of ginseng exosomes in Example 1;

[0023] Figure 2 This is a diagram showing the particle size of ginseng exosomes in Example 1;

[0024] Figure 3 This is a transmission electron micrograph of the ginseng exosome freeze-dried powder after reconstitution in the solvent solution in Example 3;

[0025] Figure 4 This is a graph showing the particle size of the ginseng exosome freeze-dried powder after reconstitution with the solvent solution in Example 3;

[0026] Figure 5 These are the efficacy evaluation effect diagrams in Example 4; A is a sensitivity comparison diagram, B is a pigmentation comparison diagram, C is a skin quality comparison diagram, and D is an aging degree comparison diagram. DETAILED DESCRIPTION

[0027] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant persons can modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0028] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments. The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained from commercial channels by those skilled in the art.

[0029] Example 1: Preparation of ginseng exosomes

[0030] S1: Use distilled water to remove the dirt on the surface of ginseng until the surface is clean and free of dirt, cut the ginseng into small pieces, and squeeze the juice with a juicer to obtain ginseng juice;

[0031] S2: The crude ginseng juice obtained in S1 is subjected to three-step centrifugation in a centrifuge precooled to 4°C to obtain ginseng stock solution; the three-step centrifugation conditions are: 200×g, 10 min; 2000×g, 20 min; 10000×g, 30 min;

[0032] S3: Add 0.2% chitosan (w / v) to the ginseng stock solution obtained in S2, stir at 45-65°C for 0.5-1 hour, let stand at room temperature for 2 hours, centrifuge at 10,000 × g for 10-30 minutes, discard the precipitate, and obtain a ginseng clarified solution;

[0033] S4: Using a tangential flow ultrafiltration system, a hollow fiber column with a molecular weight cutoff of 300 kDa was selected to ultrafilter and concentrate the ginseng clarified liquid obtained in S3 to obtain ginseng exosomes; the processing conditions of the tangential flow ultrafiltration system were: a transmembrane pressure of 0.3-1.0 bar and a flow rate of 50-200 mL / min.

[0034] In this example, ginseng exosomes extracted by tangential flow ultrafiltration system were observed by transmission electron microscopy. Figure 1 As shown in the figure, the ginseng exosomes extracted by the tangential flow ultrafiltration system have a saucer-like structure and a phospholipid bilayer structure.

[0035] In this example, nanoflow cytometry was used to detect the ginseng exosomes. The results are as follows: Figure 2As shown, the particle size of ginseng exosomes is mainly distributed between 50nm-200nm, with a particle size of 74.5nm and a particle concentration of 1.14+E12particle / mL.

[0036] Example 2: Preparation of ginseng exosome freeze-dried powder

[0037] In this example, the ginseng exosomes prepared in Example 1 were evenly mixed with trehalose having a final concentration of 2% (w / v), frozen overnight at -80°C, and freeze-dried using a freeze dryer to obtain ginseng exosome freeze-dried powder.

[0038] Example 3: A ginseng exosome transdermal delivery system

[0039] The ginseng exosome transdermal delivery system is composed of the ginseng exosomes prepared in Example 1, the ginseng exosome freeze-dried powder prepared in Example 2, a ginseng exosome freeze-dried powder solvent, a microneedle array, and an iontophoresis instrument; the ginseng exosome freeze-dried powder solvent is composed of 1%-5% glycerol, 0.1%-0.5% oligopeptide-1, 0.1%-0.5% sodium hyaluronate, 0.5-1% vitamin C ethyl ether, 0.1%-0.5% phenoxyethanol, and water; the microneedle array has a length of 200 μm and a needle density of 50 needles / cm 2 ; The voltage of the ion introduction instrument is 5V and the frequency is 10kHz.

[0040] In this example, transmission electron microscopy was performed on ginseng exosomes after reconstitution of ginseng exosome freeze-dried powder in solvent. Figure 3 As shown, it was found that the ginseng exosomes also had a saucer-like structure and a phospholipid bilayer.

[0041] Nanoflow cytometry results Figure 4 As shown, the particle size of ginseng exosomes is mainly distributed between 50nm-200nm, with a particle size of 74.2nm and a particle concentration of 1.06+E12particle / mL.

[0042] Example 4: Application of ginseng exosomes in the preparation of anti-photoaging skin care products

[0043] (1) Detection of DPPH free radical scavenging rate

[0044] The ginseng exosome freeze-dried powder prepared in Example 2 at different concentrations (50, 100 and 200 μg / mL) was dissolved in 5 mL of 70% ethanol to prepare the sample solutions to be tested; 5 mg of DPPH was weighed and placed in a 100 mL volumetric flask, and anhydrous ethanol was added until completely dissolved, and the volume was adjusted to obtain a 100 ug / mL DPPH solution, which was diluted 10 times with anhydrous ethanol before use; 2.5 mL of the diluted DPPH solution and 1.5 mL of anhydrous ethanol were added to an EP tube in sequence, mixed, and placed for 30 minutes before being transferred to a cuvette. The absorbance at a wavelength of 517 nm was measured using a spectrophotometer, which was recorded as D0; the above DPPH solutions were added to the EP tube in sequence. 2.5 mL of the diluted DPPH solution and 1.5 mL of the ginseng exosome sample solution were mixed, and after standing for 30 minutes, the mixture was transferred to a cuvette. The absorbance at a wavelength of 517 nm was measured using a spectrophotometer, which was recorded as Da. 2.5 mL of anhydrous ethanol and 1.5 mL of the ginseng exosome sample solution were added to the EP tube in sequence, and the mixture was mixed. After standing for 30 minutes, the mixture was transferred to a cuvette. The absorbance at a wavelength of 517 nm was measured using a spectrophotometer, which was recorded as Dr. The results were repeated three times and the average value was taken. The DPPH free radical scavenging rate was:

[0045] DPPH free radical scavenging rate % = [1-(Da-Dr) / D0] × 100.

[0046] The results are shown in Table 1. The ginseng exosome freeze-dried powder provided by the present invention has a high DPPH free radical scavenging rate, indicating that the anti-photoaging skin care product prepared using the above-mentioned ginseng exosome freeze-dried powder can effectively neutralize free radicals, alleviate the damage of oxidative stress to the skin, and help delay photoaging and cell damage.

[0047] Table 1

[0048] Sample concentration DPPH free radical scavenging rate (%) 50 μg / mL 23.5％ 100 μg / mL 36.7％ 200 μg / mL 52.4％

[0049] (2) Evaluation of the efficacy of anti-photoaging skin care products

[0050] In this example, 30 valid subjects aged 32 to 55 were selected for efficacy evaluation to test the nourishing, moisturizing, repairing, soothing, brightening, anti-wrinkle and firming effects of the anti-photoaging skin care product prepared with ginseng exosomes, as well as its suitability for sensitive skin.

[0051] Usage: Redissolve the ginseng exosome freeze-dried powder prepared in Example 2 with the ginseng exosome freeze-dried powder solvent, and apply it to the facial skin using the ginseng exosome transdermal delivery system. Use it once in the morning and evening for a period of 28 days.

[0052] Evaluation period: before using the product (D0), 28 days after using the product (D28);

[0053] Evaluation parameters: The facial images of the subjects are collected using a skin detector and the skin condition is analyzed to obtain sensitivity scores, pigmentation scores, skin quality scores, and aging scores;

[0054] Data statistics: SPSS 28.0 was used for statistics, and the evaluation data were statistically analyzed using the T-test method. The statistical significance level was p < 0.05.

[0055] The results are shown in Table 2 and Figure 5 As shown, the evaluation results of 30 sensitive skin subjects using the product continuously for 28 days showed that the ginseng exosomes obtained by the extraction method provided by the present invention, combined with the ginseng exosome freeze-dried powder solvent solution re-dissolution and the ginseng exosome transdermal delivery system provided by the present invention, make the prepared anti-photoaging skin care product have good anti-sensitivity, brightening and anti-aging effects.

[0056] Table 2

[0057]

[0058]

[0059] Any matters not described in detail in this specification are well known to those skilled in the art. Although the present invention has been disclosed above with reference to preferred embodiments, these are not intended to limit the present invention. Anyone skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A method for extracting ginseng exosomes, characterized in that: The extraction method comprises the following steps: S1: Using distilled water, remove the dirt from the surface of ginseng until the surface is clean and free of dirt, cut the ginseng into small pieces, and squeeze the juice with a juicer to obtain crude ginseng juice; the ginseng is five-year-old garden ginseng or fifteen-year-old or older wild ginseng; S2: The crude ginseng juice obtained in S1 is centrifuged in a centrifuge pre-cooled to 4°C to obtain ginseng stock solution; S3: Add chitosan solution to the ginseng stock solution obtained in S2, heat and stir, let stand at room temperature for 2 hours, centrifuge at 10,000×g for 10-30 minutes, discard the precipitate, and obtain ginseng clarified solution; S4: Using a tangential flow ultrafiltration system, a hollow fiber column was selected to ultrafilter and concentrate the ginseng clarified liquid obtained in S3 to obtain ginseng exosomes.

2. The extraction method according to claim 1, wherein The centrifugal treatment conditions in S2 are: 200×g, 10 min; 2000×g, 20 min; 10000×g, 30 min.

3. The extraction method according to claim 1, wherein The chitosan solution in S3 is obtained by dissolving chitosan powder in 1% acetic acid solution and stirring until completely dissolved; the final concentration of chitosan in the ginseng clarified solution is 0.2-2% (w / v).

4. The extraction method according to claim 1, wherein The processing conditions of the tangential flow ultrafiltration system described in S4 are: transmembrane pressure of 0.3-1.0 bar, and flow rate of 50-200 mL / min.

5. The extraction method according to claim 1, wherein The molecular weight cut-off of the hollow fiber column in the tangential flow ultrafiltration system described in S4 is 300 kDa.

6. A ginseng exosome, characterized in that: The ginseng exosomes are obtained by the extraction method according to any one of claims 1 to 5.

7. A ginseng exosome freeze-dried powder, characterized in that: The ginseng exosome freeze-dried powder is prepared by uniformly mixing the ginseng exosomes according to claim 6 with trehalose having a final concentration of 1%-2% (w / v), freezing the mixture at -80°C overnight, and freeze-drying the mixture using a freeze dryer to obtain the ginseng exosome freeze-dried powder.

8. A ginseng exosome transdermal delivery system, characterized in that: The ginseng exosome transdermal delivery system is composed of a ginseng exosome freeze-dried powder solvent, a microneedle array, an iontophoresis instrument, and the ginseng exosome freeze-dried powder according to claim 7.

9. The ginseng exosome transdermal delivery system according to claim 8, characterized in that The ginseng exosome freeze-dried powder solvent is composed of 1%-5% glycerol, 0.1%-0.5% oligopeptide-1, 0.1%-0.5% sodium hyaluronate, 0.5-1% vitamin C ethyl ether, 0.1%-0.5% phenoxyethanol and water; the length of the microneedle array is 200 μm and the needle density is 50 needles / cm 2 ; The voltage of the ion introduction instrument is 5V and the frequency is 10kHz.

10. Use of the ginseng exosomes according to claim 6, the ginseng exosome freeze-dried powder according to claim 7, and the ginseng exosome transdermal delivery system according to claim 8 in the preparation of anti-photoaging skin care products.

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