Plant exosome composition for beautifying and anti-aging and preparation method of plant exosome composition

Abstract

The invention belongs to the technical field of beauty and skin care, and discloses a beauty and anti-aging plant exosome composition and a preparation method thereof.The composition is formed by compounding fresh ginger exosome, ginseng exosome and brown algae exosome according to the mass ratio of 1: 1.5-2: 4. The preparation process comprises the steps of raw material pretreatment, two-stage ultracentrifugation, ultrafiltration concentration, ion chromatography membrane purification, mixed sterilization and low-temperature preservation. According to the plant exosome composition for beautifying and resisting aging and the preparation method of the plant exosome composition, natural plants serve as exosome sources, and triple effects of resisting aging and wrinkles, repairing skin barriers and relieving sensitive skin and resisting inflammation are achieved through the synergistic effect of the plant exosome compositions. Meanwhile, the preparation process is optimized, so that the exosome is high in purity and stable in activity, the efficacy shows definite dose dependence, and a safe and efficient brand-new technical scheme is provided for research and development of functional skincare products.

Description

Technical Field

[0001] This invention belongs to the field of beauty and skincare technology, and in particular relates to a plant exosome composition for beauty and anti-aging and its preparation method. Background Technology

[0002] Skin aging is determined by both internal and external factors. Internal aging is genetically determined; as we age, the activity of fibroblasts in the skin declines, collagen decreases by about 1%-3% annually, and the structure of elastic fibers is damaged, ultimately leading to dull, rough skin, sagging facial contours, depressions, and wrinkles. External aging refers to skin aging caused by photoaging, environmental pollution, and unhealthy lifestyle habits. For example, UV rays penetrate the dermis, causing age spots; PM2.5 and other particulate matter adhere to the skin surface, triggering inflammatory responses and oxidative damage; and staying up late and consuming high-sugar diets inhibit collagen synthesis and accelerate skin glycation.

[0003] Exosomes are nanoscale vesicles, approximately 30-150 nm in diameter, actively secreted by cells. They serve as core carriers for intercellular information transmission and substance exchange, carrying essential substances such as nucleic acids, proteins, lipids, and bioactive factors. They can precisely target cells, regulating physiological activities such as cell growth, repair, and metabolism. In recent years, with the rapid development of biotechnology and cosmetic science, exosomes, as an emerging ingredient, have shown broad application prospects in the fields of medicine, skincare, and regenerative medicine.

[0004] Currently, there are few exosome-based products used in the beauty and skincare field. Most existing exosome products rely on animal stem cell sources, which not only incurs high production costs but also raises ethical and safety concerns. Furthermore, these products often have limited ingredient variety, failing to address multiple skincare needs such as anti-aging, barrier repair, and soothing / anti-inflammatory effects for sensitive skin. Additionally, they suffer from cumbersome, time-consuming preparation processes and inconsistent purity. Therefore, developing an exosome-based skincare composition that is safe in origin, has a well-balanced formula, offers comprehensive efficacy, and utilizes an optimized preparation process has become a pressing technical challenge in this field. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a plant exosome composition for beauty and anti-aging and its preparation method. This method overcomes the limitations of traditional cell therapy, exerting its effects in a "cell-free" form, avoiding the high costs and safety issues associated with relying on animal stem cell preparation. By utilizing the natural activity and skincare value of various plant exosomes, a beauty and skincare product with multiple effects such as anti-aging, barrier repair, soothing and anti-inflammatory properties, and high safety can be prepared.

[0006] To achieve the above objectives, the present invention provides a plant exosome composition comprising ginger exosomes, ginseng exosomes and brown algae exosomes, wherein the mass ratio of ginger exosomes, ginseng exosomes and brown algae exosomes is 1:1.5-2:4.

[0007] A method for preparing a plant exosome composition is also provided, comprising the following steps: 1) Pretreatment: After washing ginger and ginseng with brown algae, juice them, filter them, then centrifuge them separately to remove precipitates and cell debris, and filter the supernatant through a filter membrane. 2) Ultracentrifugation: After the supernatant of ginger, ginseng and brown algae pretreated in step 1) is centrifuged for the first time, the precipitate is taken and resuspended in PBS buffer to obtain the initial sample. Then, the samples are centrifuged for the second time and the precipitate is taken and resuspended in PBS buffer to obtain the final sample. 3) Ultrafiltration concentration: The final samples were concentrated by ultrafiltration using hollow fiber columns, and the concentrate was collected; 4) Purification: The concentrated solution obtained in step 3) is purified by ion chromatography membrane, and the eluent is collected to obtain ginger exosomes, ginseng exosomes and brown algae exosomes, respectively. 5) Mixed preservation: Ginger exosomes, ginseng exosomes and brown algae exosomes are mixed at a mass ratio of 1:1.5-2:4, filtered after sterilization, and then preserved.

[0008] Preferably, in step 1), the screen used for filtration has a mesh size of 80; the centrifugation is carried out at 2-5°C, first at 1000-1200×g for 8-12 min, then at 2500-3000×g for 10-30 min, and finally at 10000-12000×g for 20-40 min; the filter membrane has a size of 0.22 μm.

[0009] Preferably, in step 2), the first centrifugation is performed at 3-5℃ for 80-100 min at 140,000-160,000×g; the second centrifugation is performed at 3-5℃ for 40-50 min at 170,000-190,000×g.

[0010] Preferably, in step 3), the hollow fiber column has a molecular weight cutoff of 300 kDa, a liquid feed rate of 3-5 L / mm, and a concentration factor of 5-10 times.

[0011] Preferably, in step 4), the ion chromatography membrane is made of polypropylene and has a volume of 4 mL. The purification process includes: combining the concentrate with the sample membrane, loading the sample at a flow rate of 10-30 mL / min, setting the column pressure to 0.1-0.5 MPa, equilibrating 20-40 membrane volumes with an equilibration buffer containing 1.0-1.3 M KAC-HAC and 0.02-0.05 M NaCl at a flow rate of 10-30 mL / min, and then eluting with an elution buffer containing 0.1-0.2 M Tris-HCl and 0.5-0.8 M NaCl at a flow rate of 10-30 mL / min, and collecting the eluent.

[0012] Preferably, step 5) uses a 0.22μm PVDF sterilization filter and is stored at a temperature of -80℃.

[0013] It also provides the application of plant exosome compositions in the preparation of beauty and skin care products, which have the effects of anti-aging and anti-wrinkle, skin barrier repair, and soothing and anti-inflammatory effects on sensitive skin.

[0014] Compared with the prior art, the present invention has the following advantages and technical effects: 1) This invention combines ginger exosomes, ginseng exosomes, and brown algae exosomes. Based on their unique skin-care activities, these three components synergistically enhance each other, forming a triple-effect system of anti-aging, repair, and anti-inflammation. Ginger exosomes can induce the expression of antioxidant and anti-inflammatory factors, specifically alleviating skin inflammation. Ginseng exosomes reverse UV oxidative damage through the Nrf2 / Keap1 pathway, promoting collagen synthesis and combating the core issues of photoaging and intrinsic aging. Brown algae exosomes are rich in active ingredients, enhancing skin hydration, soothing sensitivity, and promoting barrier repair. This fills the gaps in moisturizing and barrier repair offered by single exosomes, resulting in a composition that significantly improves anti-aging, wrinkle reduction, barrier repair, and soothing / anti-inflammatory effects, meeting consumers' core needs for multifunctional skincare products.

[0015] 2) This invention abandons the existing preparation route of exosome products that relies on animal stem cells. It uses commercially available ginger, 6-year-old fresh ginseng from Jilin, and commercially available kelp (brown algae) as raw materials. These materials are widely available, easy to obtain, and cost-controllable. At the same time, plant exosomes naturally have good biocompatibility and do not have the safety hazards such as immune rejection or pathogen residues that may be caused by animal-derived components, which significantly improves the safety of the product and its market acceptance.

[0016] 3) The preparation process of this invention can be scaled up, the raw material cost is controllable, and it is suitable for industrial production. The plant exosome composition can be directly applied to the formulation of various beauty and skin care products such as face cream, serum, and mask. It can achieve efficient skin care without adding too many irritating ingredients. It provides a new technical path for the research and development of functional skin care products, and helps to promote the beauty and skin care industry to upgrade towards "natural, safe and efficient". It has broad market application prospects and significant economic value.

[0017] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0018] Figure 1 The image shows a comparison of the skin appearance of nude mice after 28 days of intervention. In the image, A represents the solvent group, B represents the medium-dose group of the test sample, and C represents the high-dose group of the test sample. Figure 2 The distribution of collagen fibers in the skin of experimental animals using Masson's trichrome staining method (magnification 100×) is shown in the figure. A represents the solvent group, B represents the medium-dose group of the test sample, and C represents the high-dose group of the test sample. Figure 3 Figure 1 shows the results of transdermal water loss and stratum corneum moisture content detection in the solvent group, medium-dose group, and high-dose group of the test samples. In the figure, A represents the transdermal water loss detection result, and B represents the stratum corneum moisture content detection result. Here, ns represents P>0.05. This means P < 0.01. This represents P < 0.001. This represents P < 0.0001; Figure 4 This image shows the results of detecting inflammatory factor levels in tissue extracts from the solvent group, the medium-dose group of the test sample, and the high-dose group of the test sample. In the image, A represents inflammatory factor TNF-α, B represents inflammatory factor IL-1β, and C represents inflammatory factor IL-6. This means P < 0.01. Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0021] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards.

[0022] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0023] Unless otherwise specified, all experimental instruments, equipment, and reagents used in the following examples are commercially available raw materials. The ginger selected in this invention is commercially available ginger; the ginseng is commercially available 6-year-old fresh ginseng from Jilin; and the brown algae is commercially available kelp.

[0024] Example 1 Preparation of plant exosome compositions.

[0025] S1. Pretreatment: Ginger and ginseng were washed and juiced after being washed with brown algae. The juices were then filtered through an 80-mesh sieve. The filtrate was centrifuged at 1000×g for 10 min at 4℃, then at 2500×g for 20 min, and finally at 10000×g for 30 min to remove precipitates and cell debris. The supernatants of ginger, ginseng and brown algae were then filtered through a 0.22μm microporous membrane.

[0026] S2. Ultracentrifugation: The pretreated ginger, ginseng, and brown algae supernatant were subjected to ultracentrifugation under the following conditions: 1) Perform ultracentrifugation at 4℃, 150000×g, for 90 min. After centrifugation, collect the precipitate and resuspend the precipitate in PBS buffer to obtain the initial sample. 2) The initial sample obtained in step 1) was subjected to ultracentrifugation at 4°C, 180,000×g, for 45 min. After centrifugation, the precipitate was collected and resuspended with PBS buffer to obtain the final samples of ginger, ginseng and brown algae.

[0027] S3. Ultrafiltration Concentration: The final samples obtained in step S2 were concentrated by ultrafiltration using hollow fiber columns. The hollow fiber columns had a molecular weight cutoff of 300 kDa, a feed rate of 3 L / mm, and a concentration factor of 5. After concentration, all the liquid in the pipeline was discharged and collected, yielding concentrated samples of ginger, ginseng, and brown algae, respectively.

[0028] S4. Purification: The concentrated sample obtained in step S3 is purified using ion chromatography membranes. The membranes are made of polypropylene and have a volume of 4 mL. The specific steps are as follows: 1) Concentrated sample membrane binding: The sample loading flow rate is 10 mL / min, and the column inlet pressure is 0.3 MPa; 2) Post-membrane equilibration: The membrane was equilibrated using a membrane-specific equilibration buffer at a flow rate of 10 mL / min and an equilibration volume of 30 membrane volumes. The buffer contained 1.3 M KAC-HAC and 0.05 M NaCl. 3) Sample elution: The sample was eluted using a membrane-specific elution buffer at a flow rate of 10 mL / min. The eluted sample was collected. The buffer contained 0.1 M Tris-HCl and 0.8 M NaCl. The collected sample yielded ginger exosomes, ginseng exosomes, and brown algae exosomes, respectively.

[0029] S5. The purified sample was mixed with ginger exosomes, ginseng exosomes, and brown algae exosomes at a mass ratio of 1:1.5:4, and then sterilized and filtered using a 0.22μm PVDF sterilizing filter to obtain a plant exosome composition of ginger exosomes, ginseng exosomes, and brown algae exosomes. This plant exosome composition was stored at -80℃.

[0030] Example 2 Validation of anti-aging and barrier repair efficacy.

[0031] S1. Experimental Preparation: Nine healthy SPF-grade (specific pathogen-free) male BALB / c nude mice, 7 weeks old and weighing between 22-25g, were selected (the experimental animals were purchased from Liaoning Changsheng Biotechnology Co., Ltd.). After acclimatization under SPF conditions for 3 days, the experiment was conducted. During the experiment, the room temperature was 25±1℃, the relative humidity was 40-60%, and the mice had free access to food and water, and were fed standard feed.

[0032] S2. Modeling: A skin aging and barrier damage model was constructed by irradiating the backs of nude mice with UVB ultraviolet light (wavelength 280-320 nm) in stages. The irradiation process lasted for 8 weeks (150-300 Lux, 12 h light / dark cycle), during which the UV irradiation intensity was increased from 60 mJ / cm². 2 Gradually increase to 240 mJ / cm 2 The skin was irradiated three times a week to simulate the characteristics of skin aging and barrier damage (irradiation parameters are shown in Table 1).

[0033] Table 1 UV Irradiation Parameters

[0034] Before the first experiment, the transepidermal water loss (TEWL) value and skin moisture content (measured three times and averaged) of the dorsal skin of all nude mice were measured. During the experiment, dermoscopy was performed every two weeks, and to reduce error, the TEWL value and skin moisture content of the same site of the nude mice were measured.

[0035] After an 8-week experimental period, the results showed that the skin of nude mice in the UVB model group was thickened, and the area and relative depth of wrinkles were significantly increased. Statistical analysis of TEWL (as shown in Table 2) revealed that long-term ultraviolet radiation can lead to a significant increase in transepidermal water loss and a decrease in the water content of the stratum corneum, resulting in impaired skin barrier function.

[0036] Table 2. Transepidermal water loss (TEWL) and stratum corneum water content (x±s) before and after the experiment.

[0037] S3. Group intervention: The above 9 nude mice after modeling were randomly divided into 3 groups of 3 mice each, namely the solvent group, the medium dose group of the test sample, and the high dose group of the test sample; the test sample was a plant exosome composition of ginger exosomes, ginseng exosomes and brown algae exosomes.

[0038] Solvent group: 1 mL of physiological saline was applied to the modeling area on the back of nude mice after modeling was completed, once a day for 28 consecutive days. In the test sample, the plant exosome composition was applied to the modeling area on the back of nude mice after modeling was completed, once a day at a dose of 1 mL, for 28 consecutive days. High-dose group of test samples: Plant exosome composition was applied to the modeling area on the back of nude mice after modeling was completed, once a day at a dose of 2 mL, for 28 consecutive days.

[0039] S4. Indicator Detection: After 28 days of intervention, the solvent group, the medium-dose group of the test samples, and the high-dose group of the test samples were tested to comprehensively verify the anti-aging and repair effects.

[0040] 1) In vitro characteristics: After 28 days of intervention with the test sample in nude mice, the skin condition of the mice was observed. The wrinkle area and relative depth of the mice in the medium-dose and high-dose test sample groups were significantly lower than those in the solvent group, and the wrinkle improvement in the high-dose test sample group was better than that in the medium-dose test sample group (e.g., Figure 1 (As shown).

[0041] 2) Masson's trichrome staining method: After the experiment, skin tissue from the back of nude mice was taken, and the distribution of collagen fibers and muscle fibers in the skin tissue was detected using Masson's trichrome staining method. The results are as follows: Figure 2As shown, compared to the solvent group, both the medium-dose and high-dose groups of the tested samples exhibited increased collagen fiber thickness in the nude mouse skin, with a denser and more organized tissue distribution. This demonstrates that the tested samples can achieve anti-aging effects such as skin wrinkling and firming.

[0042] 3) Detection of skin barrier repair related indicators: During the 28-day intervention in nude mice with the test sample, the TEWL and skin water content of the same area of ​​the nude mice were measured every 7 days. Statistical comparison revealed (as shown in Table 3) Figure 3 As shown in the figure, the skin water content of nude mice in the medium-dose group and the high-dose group of the test sample was significantly higher than that in the solvent group, and the skin water content of nude mice in the high-dose group of the test sample was significantly higher than that in the medium-dose group of the test sample.

[0043] Table 3. TWEL and stratum corneum water content in nude mice after intervention (x±s)

[0044] Example 3 Verification of its soothing and anti-inflammatory effects.

[0045] S1. Experimental Preparation: Nine healthy SPF-grade male BALB / c nude nude mice, 7 weeks old and weighing between 22-25g, were selected (the experimental animals were purchased from Liaoning Changsheng Biotechnology Co., Ltd.). After acclimatization under SPF conditions for 3 days, the experiment was conducted. During the experiment, the room temperature was 25±1℃, the relative humidity was 40-60%, and the mice had free access to food and water, and were fed standard feed.

[0046] S2. Constructing a skin sensitivity and inflammation model: Sensitization and continuous stimulation were induced on the backs of nude mice by supplementing them with a low concentration of the irritant 2,4-dinitrofluorobenzene (DNFB), thus constructing a skin sensitivity and inflammation model that closely matches the actual state of skin sensitivity and inflammation.

[0047] S3. Group intervention: Nine nude mice after modeling were randomly divided into three groups of three mice each: solvent group, medium dose test sample group, and high dose test sample group. The test sample was a plant exosome composition of ginger exosomes, ginseng exosomes, and brown algae.

[0048] Solvent group: 1 mL of physiological saline was applied to the modeling area on the back of nude mice after modeling was completed, once a day for 7 consecutive days; In the test sample, the dosage group was treated by applying a plant exosome composition to the modeling area on the back of nude mice after modeling was completed, once daily at a dose of 1 mL, for 7 consecutive days.

[0049] High-dose group of test samples: Plant exosome composition was applied to the modeling area on the back of nude mice after modeling was completed, once a day at a dose of 2 mL, for 7 consecutive days.

[0050] S4. Detection of skin soothing related indicators: The levels of inflammatory factors in skin tissue were detected using TNF-α, IL-1β and IL-6 ELISA kits to determine the soothing effect of the test samples on skin inflammation.

[0051] S5. Result: As shown in the following example. Figure 4 As shown, the levels of TNF-α, IL-1β, and IL-6 inflammatory factors in the skin tissue of the test sample group were significantly downregulated, and the anti-inflammatory effect of the high-dose group was better than that of the medium-dose group, confirming that the composition has clear soothing and anti-inflammatory activity.

[0052] In summary, after intervention with the test sample, the depth of wrinkles and roughness of the skin in the experimental nude mice decreased, and the expression level of collagen in the skin tissue was increased, confirming that the plant exosome composition has a clear anti-aging effect. Further comparison of the differences between dosage groups showed that the high-dose group of the test sample had a better anti-aging effect than the medium-dose group. Simultaneously, the test sample effectively improved transdermal water loss in the experimental nude mice and increased skin hydration, demonstrating that the sample has a good repair effect on the damaged skin barrier, and its repair effect showed a clear dose-dependent relationship, with the high-dose group showing a better repair effect than the medium-dose group. Intervention with the test sample significantly downregulated the levels of inflammatory factors in the skin tissue of the experimental nude mice, effectively inhibited local skin inflammation, and alleviated sensitivity, verifying that the test sample has a clear soothing and anti-inflammatory activity; dose comparison analysis showed that the high-dose group of the test sample had a better soothing and anti-inflammatory effect than the medium-dose group.

[0053] It is evident that the plant exosome composition can effectively improve skin aging, repair damaged skin barrier, and reduce skin inflammation. The three effects work synergistically and complement each other, fully verifying the effectiveness and application value of the product of this invention, and providing a brand-new technical solution for the research and development and application of functional skin care products.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A plant exosome composition, characterized in that, The plant exosome composition consists of ginger exosomes, ginseng exosomes and brown algae exosomes, with a mass ratio of ginger exosomes, ginseng exosomes and brown algae exosomes of 1:1.5-2:

4.

2. A method for preparing the plant exosome composition according to claim 1, characterized in that, Includes the following steps: 1) Pretreatment: After washing ginger and ginseng with brown algae, juice them, filter them, then centrifuge them separately to remove precipitates and cell debris, and filter the supernatant through a membrane. 2) Ultracentrifugation: After the supernatant of ginger, ginseng and brown algae pretreated in step 1) is centrifuged for the first time, the precipitate is taken and resuspended in PBS buffer to obtain the initial sample. Then, the samples are centrifuged for the second time and the precipitate is taken and resuspended in PBS buffer to obtain the final sample. 3) Ultrafiltration concentration: The final samples were concentrated by ultrafiltration using hollow fiber columns, and the concentrate was collected; 4) Purification: The concentrated solution obtained in step 3) is purified by ion chromatography membrane, and the eluent is collected to obtain ginger exosomes, ginseng exosomes and brown algae exosomes, respectively. 5) Mixed preservation: Ginger exosomes, ginseng exosomes and brown algae exosomes are mixed at a mass ratio of 1:1.5-2:4, filtered after sterilization, and then preserved.

3. The preparation method according to claim 2, characterized in that, Step 1) The filter screen has a mesh size of 80; the centrifugation is carried out at 2-5℃, first at 1000-1200×g for 8-12 min, then at 2500-3000×g for 10-30 min, and finally at 10000-12000×g for 20-40 min; the filter membrane has a mesh size of 0.22μm.

4. The preparation method according to claim 2, characterized in that, Step 2) The first centrifugation is performed at 3-5℃ for 80-100 min at 140,000-160,000×g; the second centrifugation is performed at 3-5℃ for 40-50 min at 170,000-190,000×g.

5. The preparation method according to claim 2, characterized in that, Step 3) The hollow fiber column has a molecular weight cutoff of 300 kDa, a liquid feed rate of 3-5 L / mm, and a concentration factor of 5-10 times.

6. The preparation method according to claim 2, characterized in that, Step 4) The ion chromatography membrane is made of polypropylene and has a volume of 4 mL. The purification includes: combining the concentrate with the sample membrane, loading the sample at a flow rate of 10-30 mL / min, setting the column pressure to 0.1-0.5 MPa, equilibrating 20-40 membrane volumes with an equilibration buffer containing 1.0-1.3 M KAC-HAC and 0.02-0.05 M NaCl at a flow rate of 10-30 mL / min, and then eluting with an elution buffer containing 0.1-0.2 M Tris-HCl and 0.5-0.8 M NaCl at a flow rate of 10-30 mL / min, and collecting the eluent.

7. The preparation method according to claim 2, characterized in that, Step 5) The sterilization filtration uses a 0.22μm PVDF sterilization filter and is stored at a temperature of -80℃.

8. The application of the plant exosome composition according to claim 1 in the preparation of cosmetic skincare products, characterized in that, The beauty and skincare products described have anti-aging and anti-wrinkle effects, skin barrier repair, and soothing and anti-inflammatory effects for sensitive skin.

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