Use of plant-derived extracellular vesicles in the preparation of products for the prevention and treatment of photoaging and topical application compositions

Abstract

The application discloses application of plant-derived extracellular vesicles in preparation of products for preventing and treating photoaging and a local application composition. The application takes Panax notoginseng as raw material to extract and purify the plant-derived extracellular vesicles, and the results show that the extracellular vesicles derived from Panax notoginseng can significantly treat or improve photoaging. The application explores the practical application of extracellular vesicles of fresh Panax notoginseng based on traditional Chinese medicine theory, and clarifies the molecular mechanism of the extracellular vesicles. In general, the extracellular vesicles derived from Panax notoginseng have wide application prospects in treatment of skin aging.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, and in particular relates to the application of plant-derived extracellular vesicles in the preparation of products for preventing and treating photoaging, as well as topical application compositions. Background Technology

[0002] Aging is defined as a progressive process of physiological decline that can lead to aging and age-related diseases, ultimately resulting in death. Aging is a major contributing factor to cancer, cardiovascular disease, diabetes, and neurodegenerative diseases, and targeting the aging process can treat many age-related diseases.

[0003] Skin aging is the most direct manifestation of aging, characterized by increased epidermal thickness, collagen degradation, subcutaneous fat loss, sagging skin, and increased wrinkles. Aging skin exhibits reduced structural integrity and impaired barrier function, increasing the risk of skin diseases and skin cancer. Ultraviolet (UV) radiation is a significant environmental factor that can induce health hazards. Currently, there are no effective cures for photoaging-induced skin damage; therefore, exploring the prevention and treatment of UVB-induced photoaging damage is a hot topic in dermatological research. DNA damage and oxidative stress are the main pathological events causing skin cell damage due to UVB irradiation. Supplementing with antioxidants to enhance the antioxidant capacity of skin cells is an effective strategy for preventing UVB-induced oxidative stress and skin damage.

[0004] Currently, clinically used techniques for treating skin aging mainly include chemical peels, hyaluronic acid fillers, radiofrequency ablation, and laser therapy. However, these treatments do not truly provide physiological relief for skin aging and also have drawbacks such as pain, bruising, and complications. In recent years, cell therapy and gene modification technologies have flourished, and cell therapy derivatives have gradually become a research hotspot in the pharmaceutical field. While traditional Chinese herbal medicines are widely used in anti-aging research, there is still a need to develop plant-based cell therapy derivatives to meet the requirements of higher safety, biocompatibility, physicochemical stability, low cost, and wider application scenarios. Summary of the Invention

[0005] To address at least some of the problems in the prior art, the inventors obtained extracellular vesicles derived from Panax notoginseng through separation and purification. Animal experiments showed that the extracellular vesicles derived from Panax notoginseng prepared in this invention can significantly improve photoaging. Specifically, this invention includes the following:

[0006] In a first aspect, the present invention provides a topical composition for preventing photoaging, comprising plant-derived extracellular vesicles and excipients, wherein the plant-derived extracellular vesicles are derived from pretreated Panax notoginseng, and the photoaging includes UVB-induced skin damage. The preparation of the Panax notoginseng-derived extracellular vesicles includes the following steps:

[0007] (1) In the later stage of the growth of Panax notoginseng plants, the pretreatment is carried out by injecting a stress agent solution into the root zone soil or the root-stem junction, the stress agent solution containing methyl jasmonate and NaCl.

[0008] (2) Take the roots and rhizomes of Panax notoginseng with a moisture content of not less than 70% after pretreatment, homogenize them, and then obtain crude extract by gradient ultracentrifugation.

[0009] (3) The crude extract is enriched with 1 M-2 M sucrose or with 45%-60% sucrose to obtain the plant-derived extracellular vesicles.

[0010] In some embodiments, the topical composition for preventing photoaging according to the present invention contains plant-derived extracellular vesicles with a particle size of 200-250 nm and a zeta potential range of -40 mV to -10 mV.

[0011] In some embodiments, the topical application composition for preventing photoaging according to the present invention includes a buffer as an excipient.

[0012] In some embodiments, the topical application composition for preventing photoaging according to the present invention includes Tris-HCl as an excipient.

[0013] In some embodiments, the topical composition for preventing photoaging according to the present invention contains Tris-HCl at a concentration of 0.5-50 mM.

[0014] A second aspect of the present invention provides the use of plant-derived extracellular vesicles in the preparation of products for preventing and treating photoaging, wherein the photoaging includes skin damage caused by UVB, and the plant-derived extracellular vesicles are derived from pretreated Panax notoginseng, and their preparation includes the following steps:

[0015] (1) In the later stage of the growth of Panax notoginseng plants, the pretreatment is carried out by injecting a stress agent solution into the root zone soil or the root-stem junction, the stress agent solution containing methyl jasmonate and NaCl.

[0016] (2) Take the roots and rhizomes of Panax notoginseng with a moisture content of not less than 70% after pretreatment, homogenize them, and then obtain crude extract by gradient ultracentrifugation.

[0017] (3) The crude extract is enriched with 1 M-2 M sucrose or with 45%-60% sucrose to obtain the plant-derived extracellular vesicles.

[0018] In some embodiments, according to the application described in the present invention, the conditions for the gradient ultracentrifugation include: after gradient centrifugation at 400-10000 g, ultracentrifugation is performed at a rate of not less than 100000 g.

[0019] In some embodiments, according to the application described in the present invention, the product includes cosmetics, medical aesthetic products, or pharmaceuticals.

[0020] In some embodiments, according to the application described in the present invention, the cosmetic includes a serum, lotion, essence, cream, toner, lotion, face cream, mask, primer, eye cream, spray, sunscreen, or foundation.

[0021] In some embodiments, according to the application described in the present invention, the medical aesthetic product includes a serum, dressing, cooling patch, gel, sponge, membrane, hyaluronic acid injection, implant, lotion, cream, or ointment.

[0022] This invention utilizes Panax notoginseng as a raw material to extract and purify plant-derived extracellular vesicles. Results show that the Panax notoginseng-derived extracellular vesicles of this invention can significantly treat or improve UVB-induced photoaging. Based on traditional Chinese medicine theory, this invention explores the practical application of fresh Panax notoginseng extracellular vesicles and elucidates their molecular mechanism of action. In summary, the Panax notoginseng-derived extracellular vesicles of this invention have higher safety and biocompatibility, stable physicochemical properties, and low preparation cost, thus showing broad application prospects in the treatment of skin aging. Attached Figure Description

[0023] Figure 1 To investigate the effects of different sources of Panax notoginseng extracellular vesicles on the viability of BJ1 cells and the repair effects of different concentrations after UVB damage.

[0024] Figure 2 The effect of different exposure times (UVB) on the viability of BJ1 cells.

[0025] Figure 3 This is for the uptake of extracellular vesicles derived from Panax notoginseng by cells.

[0026] Figure 4 The results are from fluorescence detection of reactive oxygen species.

[0027] Figure 5 This is the quantitative result of reactive oxygen species fluorescence detection.

[0028] Figure 6 The result is the fluorescence detection result of superoxide anion.

[0029] Figure 7 This is the quantitative result of superoxide anion fluorescence detection.

[0030] Figure 8 This study investigated the inhibitory effect of extracellular vesicles derived from Panax notoginseng on the accumulation of β-galactosidase.

[0031] Figure 9 Extracellular vesicles derived from Panax notoginseng exert their antioxidant effects by activating the Nrf2 / HO-1 signaling pathway. In this study, A represents the Western blot results of Nrf2 / HO-1 protein, B represents the quantitative results of HO-1 protein, and C represents the quantitative results of Nrf2.

[0032] Figure 10 To verify the effect of Panax notoginseng-derived extracellular vesicles on UVB-induced photoaging in an in vivo model.

[0033] Figure 11 The therapeutic effect of extracellular vesicles derived from Panax notoginseng on UVB irradiation was detected by HE assay. In this study, A was the model group, B was the positive control group, C was the high-dose group, D was the medium-dose group, and E was the low-dose group.

[0034] Figure 12 This is a schematic diagram of the extraction of extracellular vesicles from Panax notoginseng.

[0035] Figure 13 The stability of extracellular vesicles derived from fresh Panax notoginseng is shown in Figure A, where A represents the stability of extracellular vesicles derived from Panax notoginseng in PBS and B represents the stability of extracellular vesicles derived from Panax notoginseng in Tris-HCl.

[0036] Figure 14 Images of extracellular vesicles derived from Panax notoginseng observed under various extraction conditions using transmission electron microscopy. In the images, A represents extracellular vesicles obtained from fresh Panax notoginseng, B represents extracellular vesicles obtained from dried Panax notoginseng powder, C represents the upper layer of fresh Panax notoginseng enriched with 1 M sucrose, D represents the extraction layer of fresh Panax notoginseng enriched with 1-2 M sucrose, E represents the extraction layer of fresh Panax notoginseng enriched with 30-45% sucrose, and F represents the extraction layer of fresh Panax notoginseng enriched with 45-60% sucrose.

[0037] Figure 15 The values ​​represent the number of particles enriched in different layers as detected by NTA. Among them, A represents the number of particles in Panax notoginseng powder, B represents the number of particles in the 30%-45% sucrose purification layer of fresh Panax notoginseng, C represents the number of particles in the 45%-60% sucrose purification layer of fresh Panax notoginseng, D represents the number of particles in the 1M sucrose purification layer of fresh Panax notoginseng, and E represents the number of particles in the 1M-2M sucrose purification layer of fresh Panax notoginseng.

[0038] Figure 16The results show the particle size and potential of extracellular vesicles derived from Panax notoginseng under various extraction conditions. In the figures, A represents the particle size of extracellular vesicles enriched from 1 M fresh Panax notoginseng, B represents the particle size of extracellular vesicles enriched from 1-2 M sucrose, C represents the potential of extracellular vesicles enriched from 1 M fresh Panax notoginseng, and D represents the potential of extracellular vesicles enriched from 1-2 M sucrose. M represents the potential of extracellular vesicles derived from Panax notoginseng enriched with sucrose; E represents the particle size of extracellular vesicles from Panax notoginseng powder (unpurified); F represents the particle size of extracellular vesicles from fresh Panax notoginseng (unpurified); G represents the potential of extracellular vesicles from Panax notoginseng powder (unpurified); H represents the potential of extracellular vesicles from fresh Panax notoginseng (unpurified); I represents the particle size of extracellular vesicles derived from Panax notoginseng enriched with 30-45% sucrose; J represents the particle size of extracellular vesicles derived from Panax notoginseng enriched with 45-60% sucrose; K represents the potential of extracellular vesicles derived from Panax notoginseng enriched with 30-45% sucrose; L represents the potential of extracellular vesicles derived from Panax notoginseng enriched with 45-60% sucrose.

[0039] Figure 17 Precipitation potential of 60% sucrose purified extracellular vesicles from fresh Panax notoginseng.

[0040] Figure 18 The effect of lipid components in extracellular vesicles derived from Panax notoginseng on cell viability.

[0041] Figure 19 The effect of protein components in extracellular vesicles derived from Panax notoginseng on cell viability.

[0042] Figure 20 The effect of miRNA components in extracellular vesicles derived from Panax notoginseng on cell viability.

[0043] Figure 21 The results are the results of the transfection efficiency determination in Example 3.

[0044] Figure 22 The effect of miR-21-5p in extracellular vesicles derived from Panax notoginseng on reactive oxygen species (ROS).

[0045] Figure 23 The effect of miR-21-5p in extracellular vesicles derived from Panax notoginseng on cell viability.

[0046] Figure 24 The results of the skin cytotoxicity experiment of the optimized Panax notoginseng-derived extracellular vesicles (YH-SQ Evs).

[0047] Figure 25 The results of ROS activity assay for the optimized Panax notoginseng-derived extracellular vesicles (YH-SQ Evs) are shown.

[0048] Figure 26 The results of DHE activity assay for optimized Panax notoginseng-derived extracellular vesicles (YH-SQ Evs) are presented.

[0049] Figure 27 The study aimed to investigate the inhibitory effect of optimized Panax notoginseng-derived extracellular vesicles (YH-SQ Evs) on UVB-induced accumulation of intracellular β-galactosidase. Detailed Implementation

[0050] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0051] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0052] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0053] Topical application composition

[0054] One aspect of the present invention provides a topical composition for preventing photoaging, wherein the plant-derived extracellular vesicles are extracellular vesicles derived from Panax notoginseng. Panax notoginseng ( Panax notoginseng Panax notoginseng (Burk.) FHChen is a plant belonging to the genus Panax notoginseng in the family Araliaceae. The inventors have discovered that extracellular vesicles derived from Panax notoginseng, obtained through appropriate extraction and purification methods, can improve or treat photoaging, particularly photoaging or skin damage caused by UVB radiation or irradiation. Extracellular vesicles (EVs) are mostly reported from animal sources. Due to the presence of plant cell walls and the unique characteristics of plant vesicles, it is necessary to conduct in-depth research on extraction and purification methods for plant-derived extracellular vesicles (PEVs) to obtain Panax notoginseng-derived extracellular vesicles with excellent therapeutic effects.

[0055] In a preferred embodiment, the preparation of extracellular vesicles derived from Panax notoginseng includes the following steps:

[0056] (1) Take the roots and rhizomes of Panax notoginseng with a water content of not less than 70%, homogenize them, and then obtain crude extract by gradient ultracentrifugation.

[0057] (2) The crude extract is enriched with 1 M-2 M sucrose or with 45%-60% sucrose to obtain the plant-derived extracellular vesicles.

[0058] In step (1) of this invention, the roots and rhizomes of Panax notoginseng are first sliced ​​or cut into pieces and homogenized at low temperature in a buffer solution. Unless otherwise specified, the low temperature in this invention refers to 0-10°C, for example, 0, 2, 4, 6, 8, or 10°C. Subsequently, the homogenized liquid is filtered, and a protein inhibitor is added to adjust the pH to neutral (preferably 6-8, for example, 6, 6.5, 7, 7.5, or 8). Then, a low-temperature gradient centrifugation is performed at 400-10000 g. Here, gradient centrifugation means centrifuging at speeds of 400, 800, and 10000 g sequentially for 5-40 min, preferably 10-30 min, for example, 10, 15, 20, 25, or 30 min. Collect the supernatant and then perform low-temperature ultracentrifugation at a speed of not less than 100,000 g (e.g., not less than 110,000, 120,000, 130,000, 140,000, 150,000, or even greater than 150,000 g), and collect the precipitate to obtain the crude extract. Further dissolve the crude extract with excipients, preferably in a buffer, to obtain the crude extract solution.

[0059] In step (1) of the present invention, the buffer is preferably Tris-HCl, more preferably 0.5-50 mM Tris-HCl, and even more preferably 1-40 mM (e.g. 5-40 mM, 10-30 mM, 15-25 mM, 18-22 mM) Tris-HCl, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 mM.

[0060] In step (2) of this invention, the crude extract (or crude extract liquid) can be enriched (purified) with 1 M-2 M sucrose or with 45%-60% sucrose to obtain the plant-derived extracellular vesicles. The inventors have studied the purification steps and found that specific purification methods can yield Panax notoginseng-derived extracellular vesicles with excellent therapeutic effects. This may be attributed to the specific particle size, potential, particle number, and integrity of the extracellular vesicles after specific purification treatment, as well as the substances contained in the extracellular vesicles (including but not limited to proteins, nucleic acids, secondary metabolites, and different lipid structures).

[0061] In step (2) of the present invention, enriching the crude extract with 1 M-2 M sucrose means performing low-temperature high-speed centrifugation (preferably 4°C, not less than 100,000 g) together with 1 M sucrose and 2 M sucrose for 0.1-2 h, preferably 0.5-1.5 h, for example 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 h, and taking the intermediate liquid after centrifugation. Alternatively, enrichment with 45%-60% sucrose can be achieved. This means purifying the crude extract of Panax notoginseng with a 45% and 60% sucrose concentration gradient, collecting liquids from different sucrose layers, washing the sucrose with the aforementioned concentration of Tris-HCl, and then centrifuging at low temperature and high speed (preferably 4°C, not less than 100,000 g) for 0.1-2 h, preferably 0.5-1.5 h, for example 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, and 1.5 h.

[0062] In this invention, the raw material of Panax notoginseng is Panax notoginseng with a moisture content of not less than 70%, rather than dried Panax notoginseng. The specific criteria for determining whether Panax notoginseng has a moisture content of not less than 70% or dried Panax notoginseng are known in the art. For example, the moisture content can be determined by drying according to the moisture determination method (General Rule 0832, Method II) in the Chinese Pharmacopoeia (2020 edition). Specifically, Panax notoginseng with a moisture content of not less than 70% refers to Panax notoginseng that, after being sampled and pulverized, is dried at 105°C to constant weight, and the measured moisture content is not less than 70%, preferably 70%-85%, and even more preferably 75%-80% (w / w). Dried Panax notoginseng powder refers to Panax notoginseng that, after being processed by a drying process (such as vacuum freeze-drying, hot air drying, or microwave drying), has a moisture content that is significantly reduced to ≤10% (w / w), preferably 5%-8% (w / w), or even less than 1% or substantially free of moisture.

[0063] In a preferred embodiment, to obtain plant-derived extracellular vesicles with significant therapeutic effects, the extracellular vesicles of the present invention are derived from pretreated roots and rhizomes of Panax notoginseng. Research has shown that the ratio of active ingredients (protein, lipid, and small RNA) in natural plant-derived extracellular vesicles (i.e., directly extracted from the roots and rhizomes of Panax notoginseng in their natural growth state) is approximately 44:48:1. Further investigation revealed that the small RNA contains a high level of miR-21-5p, which has a limited effect on improving photoaging. However, the ratio of protein, lipid, and small RNA in the pretreated extracellular vesicles changes, and a significantly improved photoaging effect is observed. In a preferred embodiment, the pretreatment refers to injecting a stress agent solution into the root zone soil or the root-rhizome junction during the later stage of Panax notoginseng plant growth (or at least before vesicle extraction) to achieve localized, high-intensity induction. The stress agent solution contains methyl jasmonate solution and NaCl, preferably less than 5 mM, more preferably less than 1 mM, more preferably 0.1-0.5 mM of methyl jasmonate solution, and less than 500 mM, preferably less than 200 mM, more preferably less than 100 mM, more preferably 50-100 mM of NaCl.

[0064] In this invention, the induction time is not particularly limited and can vary from 30 minutes to 96 hours, preferably 1-48 hours, and more preferably 1-24 hours.

[0065] In a preferred embodiment, the optimized extracellular vesicles have a total protein:total lipid:total small RNA mass content ratio in the range of (10-30):(50-80):(2-15), more preferably (20-30):(50-70):(5-10), even more preferably (20-30):(50-60):(6-10), and even more preferably (20-30):(50-60):9.

[0066] In a preferred embodiment, in the optimized extracellular vesicles, based on the mass of total protein, total lipids, and total small RNA, the mass percentage of total small RNA is at least 5%, preferably at least 6%, even more preferably at least 8%, and most preferably at least 9%. In a preferred embodiment, in the optimized extracellular vesicles, based on the mass of total protein, total lipids, and total small RNA, the mass percentage of total lipids is at least 50%, preferably at least 15%, and even more preferably at least 20%. In a preferred embodiment, in the optimized extracellular vesicles, based on the mass of total protein, total lipids, and total small RNA, the mass percentage of total protein is at most 40%, preferably at most 35%, and even more preferably at most 30%.

[0067] The method of this invention may further include a step of identifying plant-derived extracellular vesicles. Identification of plant-derived extracellular vesicles is well-known in the art; for example, electron microscopy (TEM or SEM) and nanoparticle tracking analysis (NTA) can be used to determine the morphology, integrity, quantity, particle size, and electrical potential of the obtained plant-derived extracellular vesicles. Alternatively, surface markers can be detected. Furthermore, lipid composition analysis can be performed, for example, to determine whether the isolated and purified plant extracellular vesicles contain specific lipids, such as phosphatidic acid. In this invention, the identification of plant-derived extracellular vesicles also includes the detection of the bioactivity of the plant-derived extracellular vesicles. Bioactivity detection includes, but is not limited to: determining the toxic effects of the obtained plant-derived extracellular vesicles on cells, whether cells effectively take them up, whether they inhibit UVB-induced oxidative stress, whether they can inhibit the accumulation of β-galactosidase, whether they alleviate UVB-induced oxidative stress by activating the Nrf2 / HO-1 signaling pathway, and whether they can alleviate UVB-induced photoaging.

[0068] In one specific embodiment, the plant-derived extracellular vesicles obtained by the present invention have a distinct double-membrane structure on the outside, and are generally in the form of saucer-like or hemispherical structures of varying sizes, with an intact structure (containing no or substantially no fragments), a yield of not less than 480 mg / kg, a particle size of 200-250 nm (preferably 200-240 nm, more preferably 200-230 nm, even more preferably 210-230 nm, for example 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230 nm), and a zeta potential range of -40 mV to -10 mV (preferably -35 mV to -15 mV, even more preferably -32 mV to -14 mV). mV, for example -32, -31, -30, -29, -28, -27, -26, -25, -24, -23, -22, -21, -20, -19, -18, -17, -16, -15, -14 mV).

[0069] In another specific embodiment, the plant-derived extracellular vesicles obtained by the present invention possess excellent biological activity, including but not limited to: having no or substantially no toxicity to cells (e.g., maintaining at least 80%, preferably 85%, even more preferably 90%, or even 95%, such as 96%, 97%, 98%, 99%, 99.99%), being able to effectively take up plant-derived extracellular vesicles by cells, having a significant inhibitory effect on UVB-induced oxidative stress (inhibition rate reaching at least 80%, preferably 85%, even more preferably 90%, or even 95%, such as 96%, 97%, 98%, 99%, 99.99%), and being able to significantly inhibit the accumulation of β-galactosidase (inhibition rate reaching...). At least 80%, preferably 85%, even more preferably 90%, or even 95%, such as 96%, 97%, 98%, 99%, 99.99%), can alleviate UVB-induced oxidative stress by activating the Nrf2 / HO-1 signaling pathway, and can alleviate UVB-induced photoaging (including but not limited to treating or improving skin damage caused by photoaging, such as scaling, erythema, wrinkles, ulceration, crusting, leathery feel, epidermal acanthosis, acanthosis and dissolution of acanthosis cells, acanthosis cells appearing as unstructured eosinophilic material, uneven thickness of stratum corneum and acanthosis, dermal connective tissue hyperplasia, dermal lymphocyte infiltration, reduced number of skin appendages such as hair follicles, hair follicle dilation, flattening of epithelial cells, and increased keratinization within hair follicles).

[0070] As a topical application composition, the inventors also investigated the excipients (buffers) and found that not all buffers can form a stable, homogeneous, and non-stratified solution with the extracellular vesicles derived from Panax notoginseng. Preferably, the buffer is Tris-HCl, more preferably 0.5-50 mM Tris-HCl, and even more preferably 1-40 mM (e.g., 5-40 mM, 10-30 mM, 15-25 mM, 18-22 mM) Tris-HCl, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 mM.

[0071] The compositions of the present invention may further include cosmetically acceptable additives or carriers. The term "cosmetically acceptable" refers to a component that is compatible with and harmless to other components in the product. Such additives or carriers are known in the art, including but not limited to viscosity control agents (e.g., coconut oil fatty acid monoethanolamide, sodium chloride, carbomer), pH adjusters (e.g., citric acid), preservatives (e.g., parabens, phenoxyethanol, methylisothiazolinone), nutritional additives (e.g., vitamin E, vitamin B2, panthenol), antioxidants (e.g., p-hydroxyacetophenone), emulsifiers (e.g., cetearyl alcohol), and conditioning agents (e.g., cationic...). The product contains at least one of the following: guar gum, emulsified silicone oil, propylene glycol, keratin, salicylic acid, adhesives (e.g., polyvinyl alcohol), lubricants (e.g., terminal polydimethylsiloxane, dimethyl silicone oil), thickeners (e.g., sodium carboxymethyl cellulose), penetration enhancers (e.g., propylene glycol, vitamin B5), humectants (e.g., glycerin, ethylhexylglycerin, sorbitol, propylene glycol), pigments, fragrances (e.g., lemon fragrance, strawberry fragrance, and aloe fragrance), solvents (e.g., water, ethanol, ethylene glycol, glycerin), surfactants (e.g., sodium fatty alcohol polyoxyethylene ether sulfate, coconut oil diethanolamide, betaine, ammonium dodecyl sulfate), or excipients (e.g., dimethyl silicone oil).

[0072] In one embodiment, the composition is a topical skin preparation selected from at least one of sprays, aerosols, ointments, liquids, lotions, patches, powders for application, oils, creams, and gels. "Topical application" means applying the composition (preferably by coating or spraying) to a site on the skin of a subject. In this invention, "topical" specifically refers to skin and / or keratinized tissue. "Keratinized tissue" includes the outermost protective covering of mammals (containing the keratin layer) and includes, but is not limited to, lips, skin, hair, and nails.

[0073] Furthermore, those skilled in the art are familiar with the excipients added when preparing topical skin preparations, such as sprays, aerosols, ointments, liquids, emulsions, patches, powders for application, oils, creams, and gels, and there are no particular limitations on this.

[0074] In this invention, the composition or topical skin preparation may contain 1-100 parts by weight of extracellular vesicles derived from Panax notoginseng and 1-100 parts by weight of a buffer, as well as optional additives or carriers. When there are two or more additives or carriers in combination, the proportions between the components can be adjusted as needed to obtain the desired effect of preventing and treating skin damage caused by photoaging. Preferably, the composition or topical skin preparation comprises 1-90 parts by weight, 1-80 parts by weight, 1-70 parts by weight, 1-60 parts by weight, or 1-50 parts by weight, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 parts by weight of extracellular vesicles derived from Panax notoginseng and 1-90 parts by weight, 1-80 parts by weight, 1-70 parts by weight, 1-60 parts by weight, or 1-50 parts by weight, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 parts by weight of a buffer.

[0075] In this invention, the composition or topical skin preparation may contain extracellular vesicles derived from Panax notoginseng and a buffer in a volume ratio (v / v) of 1:1-100. Preferably, the composition or topical skin preparation contains extracellular vesicles derived from Panax notoginseng and a buffer in a volume ratio (v / v) of 1:1-90, 1:1-80, 1:1-70, 1:1-60, 1:1-50, such as 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50.

[0076] In this invention, the composition or topical skin preparation may contain extracellular vesicles of Panax notoginseng and a buffer in a weight ratio of 1:1-100. Preferably, the composition or topical skin preparation contains extracellular vesicles of Panax notoginseng and a buffer in a weight ratio of 1:1-90, 1:1-80, 1:1-70, 1:1-60, 1:1-50, such as 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50.

[0077] application

[0078] This invention also provides the use of plant-derived extracellular vesicles in the preparation of products for preventing and treating skin damage caused by photoaging, wherein prevention and treatment are achieved by administering a therapeutically effective amount of the composition to a subject in need, wherein the skin damage caused by photoaging includes skin damage caused by UVB. Preferably, the prevention and treatment means that the product can achieve at least one of the following:

[0079] (1) It has a significant inhibitory effect on UVB-induced oxidative stress (inhibition rate reaches at least 80%, preferably 85%, even more preferably 90%, or even 95%, such as 96%, 97%, 98%, 99%, 99.99%).

[0080] (2) Significantly inhibits the accumulation of β-galactosidase (inhibition rate reaches at least 80%, preferably 85%, even more preferably 90%, or even 95%, such as 96%, 97%, 98%, 99%, 99.99%);

[0081] (3) Reduce UVB-induced oxidative stress by activating the Nrf2 / HO-1 signaling pathway;

[0082] (4) It can alleviate photoaging, especially skin damage caused by UVB (including but not limited to treating or improving scaling, erythema, wrinkles, ulceration, crusting, leathery feel, epidermal acanthosis, necrosis and dissolution of spinous cells, unstructured eosinophilic substances in spinous cells, uneven thickness of stratum corneum and spinous layer, dermal connective tissue hyperplasia, dermal lymphocyte infiltration, reduced number of skin appendages such as hair follicles, hair follicle dilation, flattening of epithelial cells, and increased keratinization in hair follicles).

[0083] Example 1

[0084] I. Experimental Methods

[0085] 1. Isolation and purification of extracellular vesicles derived from Panax notoginseng

[0086] Take an appropriate amount of Panax notoginseng with a moisture content of not less than 70%, wash it clean with distilled water, and air dry it. Cut the Panax notoginseng into slices 3-5 cm thick. Homogenize it in a homogenizer with 1*PBS (calcium and magnesium-free). The temperature is controlled at 4℃ throughout the homogenization process. Pass the homogenate through a silk cloth to obtain Panax notoginseng homogenate. Immediately add protease inhibitors (leucopeptide, PMSF, sodium azide). Then adjust the pH of the Panax notoginseng homogenate to 7.0 with 1 M Tris-HCl. Centrifuge at 4℃, 400 g, 800 g, and 10000 g for 20 min in sequence and collect the supernatant. Ultracentrifuge at 4℃ for 100000 g and collect the precipitate after 1 h. Dissolve the precipitate with 20 mM Tris-HCl to obtain crude Panax notoginseng extract.

[0087] Two purification methods were compared: (1) the crude extract of Panax notoginseng was centrifuged with 1 M sucrose and 2 M sucrose (4℃, 100,000 g, 1 h) to obtain the intermediate layer liquid; (2) the crude extract of Panax notoginseng was purified with a sucrose concentration gradient of 15%, 30%, 45%, and 60%, and the liquid from different sucrose layers was aspirated, then the sucrose was washed with 20 mM Tris-HCl and centrifuged (4℃, 100,000 g, 1 h) to obtain purified Panax notoginseng-derived extracellular vesicles, which were stored at -80℃. The concentration of Panax notoginseng-derived extracellular vesicles was detected using a BCA protein concentration assay kit.

[0088] Weigh an appropriate amount of Panax notoginseng powder, pass it through a 100-mesh sieve, and weigh it to 80 g. The extraction method is the same as that for fresh product extraction.

[0089] 2. Skin cell toxicity test

[0090] The concentration of extracellular vesicle proteins derived from Panax notoginseng was determined using a BCA assay kit. BJ1 cells in logarithmic growth phase were used at a concentration of 6 × 10⁻⁶ cells / cells. 4 Cells were seeded per well in 96-well culture plates and cultured for 24 h. Extracellular vesicles of Panax notoginseng from different sources (from dried Panax notoginseng powder and Panax notoginseng with a water content of not less than 70%), extracellular vesicles of Panax notoginseng with different water contents, and extracellular vesicles of Panax notoginseng with different concentrations were added and incubated together for 24 h. CCK-8 solution was added at a ratio of 10:1 and incubated in a cell culture incubator for 1 h. The absorbance was measured at 450 nm and the relative growth rate of cells in each group was calculated.

[0091] Cell viability = (absorbance of drug-treated group - absorbance of blank group) / (absorbance of blank control group wells - absorbance of blank group) × 100% (Drug-treated group: cell culture with different concentrations of Panax notoginseng-derived extracellular vesicles; blank control group: cell culture without Panax notoginseng-derived extracellular vesicles; blank group: no cell culture, only culture medium).

[0092] 3. Reactive Oxygen Species Experiment

[0093] BJ1 cells 8×10 4 Each cell / well was seeded in a 6-well plate and cultured for 24 h. The cells were divided into 5 groups: a blank group, a model group, and a UVB irradiation group (1200 mJ / cm²). 2 The cells were co-incubated with the Panax notoginseng-derived extracellular vesicle groups (5, 10, and 20 μg / ml) for 24 h. The ROS levels in BJ1 cells were detected using a reactive oxygen species (ROS) detection kit. The fluorescent probe DCFH-DA can penetrate the cell membrane and be oxidized by intracellular ROS into fluorescent DCF, which was then detected using a fluorescence microscope.

[0094] In situ probe loading: Dilute DCFH-DA 1:1000 with blank culture medium, discard the original culture medium in each well, add 1 ml of diluted DCFH-DA to each well, place in a 37℃ constant temperature incubator for 30 min, discard DCFH-DA, wash three times with PBS, add 2 ml of serum-free culture medium to each well, and take pictures under a fluorescence microscope in the dark for detection.

[0095] 4. DHE Experiment

[0096] Cell treatment followed the same method as the in situ loading of reactive oxygen species probes. DHE was diluted to 5 μM with serum-free medium, the original culture medium in the 6-well plate was discarded, 1 ml of diluted DHE was added to each well, and the plate was placed in a 37°C incubator for 30 min. The DHE was then discarded, and the cells were washed three times with PBS. 2 ml of serum-free medium was added to each well. DHE can directly label live cells. The higher the level of superoxide anion in the cells, the stronger the fluorescence. The cells were photographed and observed under a fluorescence microscope in the dark.

[0097] 5. Intake experiment

[0098] In vitro absorption assay: BJ1 cells were incubated at 7 × 10⁻⁶ cells / day. 4 Extracellular vesicles of Panax notoginseng labeled with PKH26 were seeded in confocal microplates. After co-culturing with cells for 24 h, the cell nuclei were stained with DAPI, and cell uptake was observed under a laser confocal microscope. PKH26 is a specialized membrane labeling probe that can stably insert into the lipid region of the cell membrane. Extracellular vesicles of Panax notoginseng labeled with PKH26 appear red under a laser confocal microscope.

[0099] 6. β-galactose experiment

[0100] The cell treatment method is the same as the in situ loading of probes with reactive oxygen species. Cell senescence β-galactosidase staining kit is used to detect cell senescence. Using X-Gal as a substrate, a deep blue product is generated under the catalysis of senescence-specific β-galactosidase, so cells expressing β-galactosidase can be observed to turn blue under an optical microscope.

[0101] For adherent cells: Aspirate the cell culture medium, wash once with PBS, add 1 ml of β-galactosidase staining and fixing solution, and fix at room temperature for 15 min. Aspirate the cell fixative, wash three times with PBS, 3 min each time. Aspirate the PBS, and add 1 ml of the prepared staining working solution to each well. Incubate at 37℃ for 48 h, and observe under a regular light microscope.

[0102] 7. Immunoblotting to detect the levels of related proteins in cells

[0103] BJ1 cells were fed at 11 × 10 4Cells were seeded in small dishes at a density of 1 cell / well and cultured for 24 h. Different concentrations of Panax notoginseng-derived extracellular vesicles were added, and after 24 h, cells were collected and total protein was extracted using RIPA lysis buffer. Protein concentration was determined using a BCA kit and adjusted to a consistent level. Samples were subjected to SDS-PAGE gel electrophoresis, transferred to nitrocellulose (NC) membranes, and placed in PBS buffer containing 5% skim milk powder. Blocking was performed at room temperature for 1 h, followed by incubation at 4°C overnight with the corresponding primary antibody (all diluted 1:1000). Incubation was then performed at room temperature for 1 h with HRP-labeled secondary antibody (all diluted 1:1000). Finally, the cells were developed using ECL chemiluminescence solution in a gel imaging system.

[0104] 8. Animal experiments

[0105] Animal modeling: After one week of acclimatization, 25 BALB / c mice were randomly divided into 5 groups: model group, positive control drug (VE) group, and Panax notoginseng-derived extracellular vesicle administration groups (low-dose, medium-dose, and high-dose groups), with 5 mice in each group. All mice underwent dorsal hair removal. All groups were irradiated with UVB (irradiation dose 12000 mJ / cm²). 2 Models were created using a narrow-band UVB lamp with an emission wavelength between 306 nm and 316 nm, and the radiation intensity was measured using an ultraviolet radiometer.

[0106] Animal administration: One hour after UVB irradiation, mice were given drug treatment (different concentrations of Panax notoginseng-derived extracellular vesicles were applied to the bare skin on the back of mice). The model group was given distilled water, and the positive control group was given vitamin E soft capsules. The procedure was performed once a day for 30 days.

[0107] Fresh mouse skin was fixed in 4% paraformaldehyde and embedded in paraffin. The paraffin-embedded tissue was stained with H&E according to standard procedures. The tissue was then photographed and observed using an optical microscope.

[0108] 9. Statistical methods

[0109] All data represent at least three independent experiments and are expressed as mean ± SEM. Statistical computations were performed using GraphPad Prism 6.01 (GraphPad Software, San Diego, CA, USA). Statistical comparisons were performed using one-way ANOVA and Dunnett's post-hoc test. P <0.05 indicates a statistically significant difference.

[0110] II. Experimental Results

[0111] 1. The toxic effects of Panax notoginseng-derived extracellular vesicles on BJ1 cells

[0112] First, extracellular vesicles were extracted from Panax notoginseng with a moisture content of 75%-80%. Extracellular vesicles were also extracted from Panax notoginseng powder and Panax notoginseng with a moisture content of 40%-50%. These were then administered to BJ1 cells at the same concentration gradient. The results showed that extracellular vesicles of Panax notoginseng (75%-80%) at concentrations of 2.5 μg / ml-20 μg / ml were not toxic to the cells. Subsequent experiments were conducted at concentrations of 5 μg / ml, 10 μg / ml, and 20 μg / ml. All concentrations of extracellular vesicles from Panax notoginseng powder and Panax notoginseng (40%-50%) had some effect on the cells. After UVB damage, extracellular vesicles from Panax notoginseng (75%-80%) showed a certain repair effect on UVB damage. A comparison was made between the repair effects of extracellular vesicles from Panax notoginseng powder and those from Panax notoginseng (40%-50%). The results showed that extracellular vesicles from Panax notoginseng (40%-50%) at a concentration of 20 μg / ml had a weak repair effect on UVB damage, while extracellular vesicles from Panax notoginseng powder did not show a significant repair effect on UVB damage. Figure 1 Based on the results of this study, subsequent experiments all used extracellular vesicles of Panax notoginseng (75%-80%) for further experiments.

[0113] Then, different concentrations of Panax notoginseng-derived extracellular vesicles were co-cultured with BJ1 cells for 24 h to detect their cytotoxic effects on BJ1 cells. The results showed that different concentrations of the drug did not exhibit cytotoxicity within a certain range. Specifically, Figure 2 The effects of UVB irradiation at different times on BJ1 cell viability were demonstrated. Subsequently, BJ1 cells were co-cultured with UVB irradiation at different times for 24 h to determine the optimal time for an in vivo UVB damage model. The results showed that UVB irradiation at different times all had a damaging effect on BJ1 cells, with significant differences starting from 5 min. The optimal irradiation time was finally determined to be 10 min, with an intensity of 2 mw / cm². 2 .

[0114] 2. Cellular uptake of extracellular vesicles derived from Panax notoginseng

[0115] To investigate whether extracellular vesicles derived from Panax notoginseng could be taken up by BJ1 cells, PKH26-labeled extracellular vesicles from Panax notoginseng were co-cultured with BJ1 cells for 24 h and observed and photographed under a fluorescence microscope. The results showed that extracellular vesicles derived from Panax notoginseng could be taken up by BJ1 cells. Figure 3 ).

[0116] 3. Extracellular vesicles derived from Panax notoginseng can inhibit UVB-induced oxidative stress in vitro.

[0117] ROS and DHE are both products of oxidative stress in the body. To investigate the effects of Panax notoginseng-derived extracellular vesicles on UVB-induced oxidative stress, the activities of ROS and DHE in BJ1 cells were examined. The results are as follows: Figure 4-7 As shown, the results indicate that UVB significantly increased the levels of ROS and DHE in BJ1 cells, while treatment with Panax notoginseng-derived extracellular vesicles reduced the increase in ROS and DHE. These data suggest that Panax notoginseng-derived extracellular vesicles have a certain inhibitory effect on UVB-induced oxidative stress.

[0118] 4. The inhibitory effect of Panax notoginseng-derived extracellular vesicles on the accumulation of β-galactosidase.

[0119] β-galactosidase staining was used to detect senescent cells. The substrate X-ga1 hydrolyzes to produce a deep blue product, causing senescent cells to appear blue. The activity of β-galactosidase in cells was detected using a kit. Results showed that the β-galactosidase content increased in the model group, as shown by blue-green dots in the image. The β-galactosidase content decreased after treatment with Panax notoginseng-derived extracellular vesicles, indicating that Panax notoginseng-derived extracellular vesicles can reduce UVB-induced accumulation of intracellular β-galactosidase. Figure 8 ).

[0120] 5. Extracellular vesicles derived from Panax notoginseng alleviate UVB-induced oxidative stress in vitro by activating the Nrf2 / HO-1 signaling pathway.

[0121] The Nrf2 / HO-1 signaling pathway, an essential signaling pathway in oxidative stress responses, participates in processes such as anti-inflammation, anti-oxidation, and apoptosis. This study investigated the protein regulatory effect of Panax notoginseng-derived extracellular vesicles on the Nrf2 / HO-1 signaling pathway in UVB-treated BJ1 cells. Western blot data showed that Panax notoginseng-derived extracellular vesicles ameliorated the downregulation of Nrf2 and its downstream factor HO-1 caused by UVB damage, demonstrating that the antioxidant effect is achieved by activating the Nrf2 / HO-1 signaling pathway (e.g., Figure 9 (As shown in A, B, and C).

[0122] 6. Validating the effect of Panax notoginseng-derived extracellular vesicles on UVB-induced skin damage in an in vivo model.

[0123] One day before the irradiation experiment, the backs of mice were shaved. The irradiated areas on the backs of the mice showed extensive scaling, erythema, wrinkles, and even localized ulceration, scabs, and a leathery texture, indicating that the mouse photodermal injury model was successfully established. Macroscopic observation revealed that the low, medium, and high dose groups showed significantly better recovery compared to the model group, and the positive control group was less effective than the group treated with Panax notoginseng-derived extracellular vesicles. Figure 10 ).

[0124] 7. HE detection of the therapeutic effect of Panax notoginseng-derived extracellular vesicles on UVB irradiation

[0125] The skin morphology of mice was observed using H&E staining, and the results are as follows: Figure 11 As shown in A, B, C, D, and E, the model group's skin tissue shows small-scale epidermal acanthosis (black arrow), a small number of dermal ridges extending downwards, occasional necrosis and dissolution of acanthosis cells (brown arrow), presenting as unstructured eosinophilic material, and uneven thickness of the stratum corneum and stratum spinosum; a small amount of connective tissue hyperplasia in the dermis (orange arrow), accompanied by a small amount of lymphocyte infiltration (red arrow), occasional neovascularization (purple arrow), a small number of skin appendages such as hair follicles, occasional hair follicle dilation, flattened epithelial cells, and a small amount of keratinized material within the hair follicles. In the positive control group and the high-dose administration group of Panax notoginseng-derived extracellular vesicles, the epidermis of the skin tissue was intact, the structure of each layer was clear, the thickness was relatively uniform, and no obvious abnormalities were observed. The collagen fibers in the dermis were coarse and cord-like, tightly arranged and irregular, with occasional connective tissue hyperplasia (orange arrow) and occasional lymphocyte infiltration (red arrow). Skin appendages such as hair follicles and sebaceous glands were abundant and scattered, with rare hair follicle dilation (blue arrow), irregular shape, flattened epithelial cells, and a small amount of keratinized material visible in the hair follicles. The subcutaneous tissue, located below the dermis, was composed of loose connective tissue, adipose tissue and muscle layer, and no obvious abnormalities were observed. In the medium-dose group of Panax notoginseng-derived extracellular vesicles, local epidermal acanthosis was observed in the skin tissue, with a small number of dermal ridges extending downwards and a few vacuoles of varying sizes (black arrows). The stratum corneum and stratum spinosum were uneven in thickness, and hemorrhage was occasionally observed (green arrows). Lymphocytic infiltration was occasionally observed in the dermis (red arrows). Hair follicles and other skin appendages were abundant and scattered, with occasional hair follicle dilation and irregular shapes. Epithelial cells were flattened, and a small amount of keratinized material was observed within the hair follicles. In the low-dose group of Panax notoginseng-derived extracellular vesicles, epidermal necrosis was occasionally observed in the skin tissue (brown arrows), with occasional necrotic cell fragments and unstructured eosinophilic material, accompanied by a small amount of parakeratosis (black arrows). The collagen fibers in the dermis were coarse, tightly arranged, and irregular. Skin appendages such as hair follicles and sebaceous glands were abundant and scattered, with a small number of lymphocytes and granulocytes scattered infiltrating or infiltrating in focal areas (red arrows). H&E staining showed that the therapeutic effect of Panax notoginseng-derived extracellular vesicles on UVB damage was in the order of high dose > medium dose > low dose.

[0126] Example 2

[0127] To investigate the differences in therapeutic effects brought about by extracellular vesicles from different sources of Panax notoginseng and different purification processes, this embodiment further studies the above-mentioned extracellular vesicles, as detailed below.

[0128] 1. Qualitative and quantitative results of extracellular vesicles derived from Panax notoginseng under various extraction conditions

[0129] This embodiment uses Panax notoginseng with a water content of not less than 70% as raw material to extract and purify extracellular vesicles derived from Panax notoginseng (e.g. Figure 12As shown in the figure, purified Panax notoginseng-derived extracellular vesicles were dissolved in two buffers, PBS and Tris-HCl, respectively. The results showed that the Panax notoginseng-derived extracellular vesicles dissolved in Tris-HCl buffer were more uniformly dispersed and stable, and did not separate into layers after prolonged standing. In contrast, the Panax notoginseng-derived extracellular vesicles dissolved in PBS showed obvious layering and poor uniformity after standing (e.g., ...). Figure 13 As shown in the figure, Tris-HCl was therefore chosen as the final buffer solution for delivery of Panax notoginseng-derived extracellular vesicles in subsequent experiments.

[0130] The morphology of extracellular vesicles derived from Panax notoginseng was observed using transmission electron microscopy. They exhibited uniform morphology, a distinct double-membrane structure, and generally resembled saucers or hemispherical structures of varying sizes, consistent with the microscopic identification characteristics of extracellular vesicles. Comparison of extracellular vesicles extracted from dried Panax notoginseng powder and Panax notoginseng with a moisture content of at least 70% revealed that, compared to extracellular vesicles from Panax notoginseng with a moisture content of at least 70%, the extracellular vesicles from dried Panax notoginseng powder showed more fragmentation and a lower content of extracellular vesicles under electron microscopy. Figure 14 (AB). In the experiment of separating extracellular vesicles from fresh Panax notoginseng using a two-layer sucrose slurry, the number of extracellular vesicles in the 1 M upper layer was less and the size of the extracellular vesicles was smaller, while the 1 M-2 M sucrose slurry contained more enriched extracellular vesicles. Figure 14 (CD). In the experiment of isolating extracellular vesicles from fresh Panax notoginseng in four layers of sucrose, the size and number of extracellular vesicles enriched in the 30-45% and 45-60% layers showed little difference. Figure 14 EF).

[0131] Next, in this embodiment, NTA was used to detect the number of particles enriched in different layers. The results are as follows: Figure 15 As shown, Figure 15 A in the figure indicates the particle number of Panax notoginseng powder detected by NTA (2.46*10⁻⁶). 12 Particles / mL), Figure 15 B in the figure indicates the number of particles (2.18*10) of Panax notoginseng with a moisture content of not less than 70% as detected by NTA after purification with 30%-45% sucrose. 12 Particles / mL), Figure 15 The 'C' in the figure indicates the number of particles (2.15*10^6) of fresh Panax notoginseng purified with 45%-60% sucrose by NTA. 12 Particles / mL), Figure 15 The D value indicates the number of particles (3.66*10⁻⁶) of Panax notoginseng with a moisture content of not less than 70% as detected by NTA after purification by 1 M sucrose supernatant. 12 Particles / mL), Figure 15 E in the figure indicates the number of particles (5*10) of Panax notoginseng with a moisture content of not less than 70% as detected by NTA after purification with 1-2 M sucrose. 12(particles / mL). A comparison was made between dried Panax notoginseng powder and NTA containing Panax notoginseng with a moisture content of not less than 70%. The results showed that the size and number of nanoparticles obtained from both were basically the same. In addition, the nanoparticles obtained from Panax notoginseng with a moisture content of not less than 70% under different concentrations of sucrose purification were of consistent size, but the number of particles purified at a concentration of 2 sucrose was twice that at a concentration of 4 sucrose.

[0132] Meanwhile, the yield under various extraction conditions was measured in this embodiment. The results showed that compared with Panax notoginseng with a moisture content of not less than 70%, the yield of extracellular vesicles obtained from dried Panax notoginseng powder was lower, with an extracellular vesicle yield of 123.63 mg / kg for dried Panax notoginseng powder and 483.84 mg / kg for Panax notoginseng with a moisture content of 75%-80%. During purification at four concentrations, the protein concentration was 3.87 mg / ml for 30-45% moisture content and 3.77 mg / ml for 45-60% moisture content. For both concentrations, the protein concentration in the 1 M layer was 5.87 mg / ml, and the protein concentration in the 1-2 M layer was 5.02 mg / ml.

[0133] Subsequently, the particle size and potential of extracellular vesicles derived from Panax notoginseng were measured, and the results are as follows: Figure 16-17 As shown, the average diameter of unpurified extracellular vesicles from dried Panax notoginseng powder was 116.6 nm, with a Zeta potential range of -12.4 ± 6.83 mV. The average diameter of unpurified Panax notoginseng with 75%-80% moisture content was 281.2 nm, with a Zeta potential range of -24.3 ± 7 mV. The particle size of the extracted dried Panax notoginseng was smaller compared to that of Panax notoginseng with 75%-80% moisture content. The average diameter of the upper layer of extracellular vesicles from 75%-80% moisture content Panax notoginseng purified with 1 M sucrose concentration was 141.8 nm, with a Zeta potential range of -24.8 ± 7.2 mV; the average diameter of the 1 M-2 M sucrose layer was 213 nm, with a Zeta potential range of -22.0 ± 8.04 mV. After purification with four sucrose concentrations, the average diameter of extracellular vesicles derived from Panax notoginseng with a moisture content of 75%-80% and a 30%-45% sucrose concentration gradient was 209.5 nm, with a Zeta potential range of -26.3 ± 6.6 mV. The average diameter with a 45%-60% sucrose concentration gradient was 217.6 nm, with a Zeta potential range of -25.2 ± 5.41 mV. The identification results of extracellular vesicles derived from Panax notoginseng purified with a 45%-60% sucrose concentration gradient were consistent with those purified with a 1 M-2 M sucrose concentration gradient. In comparison, the 1 M and 2 M purification methods are simpler and more convenient, with less extracellular vesicle loss, thus optimizing the extraction process.

[0134] Based on the measured results regarding particle size, potential, particle number, and extracellular vesicle integrity, the extracellular vesicles extracted from the dry powder are not suitable for subsequent purification, cell and animal experiments, and their impact on the therapeutic effect of UVB-induced skin damage is expected to be minimal. Furthermore, the extracellular vesicles extracted using other extraction methods in this embodiment (extracellular vesicles enriched with 30%-45%, 45%-60%, and less than 1 M or more than 2 M sucrose) are also unsuitable for subsequent cell and animal experiments compared to extracellular vesicles enriched with 1-2 M sucrose, in terms of particle size, potential, particle number, and extracellular vesicle integrity, and their impact on the therapeutic effect of UVB-induced skin damage is expected to be minimal.

[0135] Example 3

[0136] Based on previous research, this invention found that extracellular vesicles obtained from fresh Panax notoginseng roots and rhizomes do indeed exhibit effects in in vitro inhibition of UVB-induced oxidative stress, inhibition of β-galactosidase accumulation, and in vivo skin damage recovery, but there is still considerable room for improvement. Considering that the contents of extracellular vesicles are key to these effects, this embodiment first extracts and analyzes the contents of extracellular vesicles obtained from fresh Panax notoginseng roots and rhizomes, as detailed below:

[0137] 1. Extraction of active ingredients

[0138] 1.1 Extraction of lipid components from extracellular vesicles of Panax notoginseng cells based on the Bligh-Dyer method

[0139] Take 12.5 mL of methanol, 6.25 mL of chloroform and Panax notoginseng extracellular vesicles and vortex. Then add 6.25 mL of chloroform and RO water respectively and vortex. Centrifuge at 2000 rpm at room temperature for 10 minutes to separate the upper, middle and lower layers. Take the lower layer of solution and place it in an evaporating dish. Evaporate the chloroform in a 55℃ water bath, weigh it, resuspend it in 1 mL of PBS, and store at 4℃.

[0140] 1.2 Protein components were extracted using a commercially available protein extraction kit.

[0141] Extraction preparation: Add 2 μl of protease inhibitor mixture to every 500 μl of cold protein extract, mix well, and place on ice. Add 50 μl of cold protein extract to the Panax notoginseng extracellular vesicle sample, mix by pipetting, and vortex at 4°C for 20-30 minutes. Centrifuge at 12000×g for 10 minutes at 4°C, and aspirate the supernatant into another pre-chilled clean centrifuge tube to obtain the total protein from the extracellular vesicles. Quantify the above protein extract, aliquot, and store at -80°C for later use. Determine the protein concentration using a BCA protein concentration assay kit (Beyotime, P0011).

[0142] 1.3 miRNA was extracted using a commercially available microRNA rapid extraction kit.

[0143] Using the SPARKEASY Plant MICRO RNA Rapid Extraction Kit AC1502, the resuspended extracellular vesicle suspension was transferred to an RNase-free centrifuge tube, and at least 3 volumes of lysis buffer were added. Immediately, the mixture was vigorously pipetted for at least 15 seconds to ensure complete membrane lysis. The lysate was transferred to a centrifuge tube, vigorously shaken for 15 seconds, and centrifuged at 12500 rpm for 5-10 minutes to precipitate unlysed fragments and PLANTspark bound to polysaccharides and polyphenols. The supernatant of the lysate was transferred to a new centrifuge tube, and half the volume of anhydrous ethanol was added. The mixture was immediately pipetted and mixed, but not centrifuged. The mixture was then added to a genomic clearance column, which was placed in a collection tube and centrifuged at 12500 rpm for 2 minutes. The filtrate (containing microRNA) was retained. The volume of the filtrate was accurately estimated using a micropipette, and an equal volume of anhydrous ethanol was added. The mixture was vortexed or pipetted thoroughly, but not centrifuged. Immediately add the mixture to an adsorption column RA, place the column in a collection tube, centrifuge at 12500 rpm for 2 min, and discard the waste liquid. Add 700 μL WashSolution 1, centrifuge at 12000 rpm for 30 s, and discard the waste liquid. Add 500 μL WashSolution 2 / 3, centrifuge at 12000 rpm for 30 s, and discard the waste liquid. Repeat this step. Place the adsorption column RA back into the empty collection tube, centrifuge at 12500 rpm for 2 min to remove as much wash liquid as possible, remove the adsorption column RA, place it in an RNase-Free centrifuge tube, add 30-50 μL of RNase-Free H2O to the middle of the adsorption membrane according to the expected RNA yield, incubate at room temperature for 1 min, centrifuge at 12000 rpm for 1 min, and store the obtained microRNA at low temperature as soon as possible. Measure the nano value using a micro spectrophotometer.

[0144] 2. In vitro cell experiments of extracellular vesicles

[0145] The extracted components were co-incubated with BJ1 cells, and cell viability was measured under UVB induction. The results of lipid composition are as follows: Figure 18 As shown, the results for protein composition are as follows: Figure 19 As shown, the results for miRNA components are as follows: Figure 20 As shown, the inventors unexpectedly discovered that using each component alone did not significantly improve the cell survival rate induced by UVB. They speculated that the anti-photoaging effect observed in the extracellular vesicles of Panax notoginseng was due to the synergistic effect of multiple components.

[0146] Based on in vitro cell experiments, this embodiment further quantitatively analyzed each component. The results showed that the content ratio of protein:lipid:small RNA was approximately 44:48:1. It is worth noting that the content of small RNA in the extracellular vesicles of Panax notoginseng in its natural state is relatively low. At present, more and more evidence suggests that small RNA performs important biological functions, so it is necessary to conduct in-depth research on this component.

[0147] 3. Sequencing analysis and cell function studies of small RNAs

[0148] RNA was extracted from the extracellular vesicles of Panax notoginseng cells, followed by the construction and sequencing of a small RNA library. Biological analysis revealed that small RNAs from these vesicles were screened, with miR-21-5p showing high levels. Further investigation was conducted by artificially synthesizing miRNA mimics and inhibitors, forming complexes through transfection reagents, which were then taken up by cells. This allowed for the overexpression or inhibition of specific miRNAs within the cells, thereby studying their effects on cellular function. Details are as follows:

[0149] The transfection experiments were divided into the following groups: untreated group (Control), control group (lipo2000), negative control group (NC), mimic group, and inhibitor group. Cell suspensions were seeded into 24-well cell culture plates at an appropriate density.

[0150] Diluting miRNA: A certain amount of miRNA stock solution was diluted with 50 μL of serum-free Opti-MEM medium in a sterile 1.5 mL EP tube. Preliminary concentration gradient experiments were conducted to optimize the mimic / inhibitor concentrations of 50 and 100 nM.

[0151] Diluting the transfection reagent: In another sterile 1.5 mL EP tube, dilute an appropriate amount of transfection reagent with 50 μL of serum-free culture medium. Following the manufacturer's recommended range, Lipofectamine 2000 is commonly used at 1-2 μL in a 24-well plate; use 1, 1.5, or 2 μL for transfection.

[0152] Preparation of the transfection complex: Add the entire diluted miRNA solution (solution A) to the diluted transfection reagent (solution B). Gently mix with a pipette and let stand at room temperature for 15-20 minutes to allow the miRNA-transfection reagent complex to form.

[0153] Transfection: Remove the cell culture plate from the incubator and aspirate the old culture medium. Gently wash the cells 1-2 times with preheated PBS to remove residual serum. Add 400 μL of Opti-MEM medium to each well. Add 100 μL of the prepared transfection complex evenly to the corresponding cell culture well and gently shake the culture plate to mix.

[0154] Post-transfection processing and detection: 4-6 hours after transfection, aspirate the culture medium containing the transfection complex. Add 500 μL of fresh, complete culture medium containing serum and antibiotics. Return the cells to a 37°C, 5% CO2 incubator and continue culturing for 24-48 hours.

[0155] Transfection efficiency monitoring: The transfection efficiency was estimated by observing under a fluorescence microscope approximately 24 hours after transfection.

[0156] Results: The optimal transfection efficiency was achieved by using 1 μL of Lipofectamine 2000 transfection complex with 100 nM mimic / inhibitor and transfection for 6 h. Figure 21 ).

[0157] The effects of miR-21-5p on UVB-induced reactive oxygen species and cell viability were further investigated, and the results are as follows: Figure 22 and Figure 23 As shown, the results indicate that miR-21-5p has a certain ameliorative effect on UVB-induced oxidative stress and cell survival (P<0.05), but there is still great potential for improvement.

[0158] Example 4

[0159] Based on the research results of Example 3 and the differences in the anti-photoaging effects of various components, this example uses a series of optimization methods to determine a pretreatment method that can change the proportion of each component in the extracellular vesicles of Panax notoginseng under natural conditions, thereby significantly improving the anti-photoaging effect of Panax notoginseng extracellular vesicles. Specifically, during the later stage of Panax notoginseng plant growth, a stress agent solution is injected into the root zone soil or the root-stem junction using a micro-syringe to achieve localized, high-intensity induction. After the stress is completed, Panax notoginseng extracellular vesicles (named YH-SQ EVs) are extracted from the roots. The stress agent solution contains a low concentration (0.1-0.5 mM) of methyl jasmonate solution and 50-100 mM NaCl. The extraction method uses the extraction conditions determined in the aforementioned examples.

[0160] After extracting Panax notoginseng extracellular vesicles, the component content was first determined using the method in Example 3. The results showed that the ratio of protein:lipid:small RNA was approximately 26:55:9. In this example, the determination of the content of each component showed that the relative content of protein decreased, while the content of lipid components increased slightly. Further determination of miR-21-5p content showed that the content of miR-21-5p after pretreatment was nearly 7 times higher than that of untreated Panax notoginseng extracellular vesicles.

[0161] Next, this embodiment conducted skin cell toxicity experiments, reactive oxygen species experiments, and β-galactose experiments on YH-SQ Evs to evaluate the inhibitory effect of the optimized Panax notoginseng extracellular vesicles (YH-SQ Evs) on UVB-induced oxidative stress and the inhibitory effect on the accumulation of β-galactosidase. For specific experimental procedures, please refer to the above embodiment, which will not be repeated here.

[0162] Skin cell toxicity test results as follows Figure 24 As shown, both YH-SQ Evs and the unoptimized Panax notoginseng extracellular vesicles have certain effects on cells. After UVB damage, YH-SQ Evs was found to have a significant repair effect on UVB damage, and it showed a significant difference compared to the unoptimized Panax notoginseng extracellular vesicles.

[0163] The activity detection results of ROS and DHE are as follows: Figure 25 , 26 As shown, the results indicate that UVB significantly increased the levels of ROS and DHE in BJ1 cells, while treatment with YH-SQ Evs and the unoptimized Panax notoginseng extracellular vesicles reduced the increase in ROS and DHE, with significant differences compared to the unoptimized Panax notoginseng extracellular vesicles. These data suggest that, compared to the unoptimized Panax notoginseng extracellular vesicles, the optimized Panax notoginseng-derived extracellular vesicles can significantly inhibit UVB-induced oxidative stress.

[0164] The results of β-galactosidase-positive cell rate detection showed that the β-galactosidase-positive cell rate increased in the model group, while the β-galactosidase-positive cell rate was significantly reduced after treatment with YH-SQ Evs and the unoptimized Panax notoginseng extracellular vesicles, and the difference was statistically significant compared with the unoptimized Panax notoginseng extracellular vesicles. These data indicate that, compared with the unoptimized Panax notoginseng extracellular vesicles, the optimized Panax notoginseng-derived extracellular vesicles can reduce UVB-induced intracellular β-galactosidase accumulation. Figure 27 ).

[0165] 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 the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of plant-derived extracellular vesicles in the preparation of a product for the prevention and treatment of skin damage caused by UVB, characterized in that, The plant-derived extracellular vesicles are derived from pretreated Panax notoginseng, with a particle size of 200-250 nm. The mass ratio of total protein:total lipids:total small RNA in the extracellular vesicles ranges from (10-30):(50-80):(2-15). The preparation of the extracellular vesicles includes the following steps: (1) In the later stage of the growth of Panax notoginseng plants, the pretreatment is carried out by injecting a stress agent solution into the root zone soil or the root-stem junction, the stress agent solution containing methyl jasmonate and NaCl. (2) Take the roots and rhizomes of Panax notoginseng with a moisture content of not less than 70% after pretreatment, homogenize them, and then obtain crude extract by gradient ultracentrifugation. (3) The crude extract is enriched with 1 M-2 M sucrose or with 45%-60% sucrose to obtain the plant-derived extracellular vesicles.

2. Use according to claim 1, characterized in that, The conditions for gradient ultracentrifugation include: after gradient centrifugation at 400-10000 g, ultracentrifugation is performed at a speed of not less than 100000 g.

3. Use according to claim 1, characterized in that, The products mentioned include cosmetics, medical aesthetic products, or drugs.

4. Use according to claim 3, characterized in that, The cosmetics include serums, lotions, essences, creams, toners, lotions, face creams, masks, sunscreens, eye creams, sprays, sunscreens, or foundations.

5. Use according to claim 3, characterized in that, The medical aesthetic products include serums, dressings, cooling patches, gels, sponges, films, hyaluronic acid injections, implants, lotions, creams, or ointments.

6. A topical composition for the prevention and treatment of UVB-induced skin damage, characterized in that, The product comprises plant-derived extracellular vesicles and excipients. The plant-derived extracellular vesicles are derived from pretreated Panax notoginseng. The particle size of the plant-derived extracellular vesicles is 200-250 nm. The mass ratio of total protein:total lipids:total small RNA in the extracellular vesicles ranges from (10-30):(50-80):(2-15). The preparation of the extracellular vesicles includes the following steps: (1) In the later stage of the growth of Panax notoginseng plants, the pretreatment is carried out by injecting a stress agent solution into the root zone soil or the root-stem junction, the stress agent solution containing methyl jasmonate and NaCl. (2) Take the roots and rhizomes of Panax notoginseng with a moisture content of not less than 70% after pretreatment, homogenize them, and then obtain crude extract by gradient ultracentrifugation. (3) The crude extract is enriched with 1 M-2 M sucrose or with 45%-60% sucrose to obtain the plant-derived extracellular vesicles.

7. The topical composition for preventing UVB-induced skin damage according to claim 6, characterized in that, The zeta potential ranges from -40 mV to -10 mV.

8. The topical composition for preventing and treating skin damage caused by UVB according to claim 6, wherein The excipients include a buffer, wherein the buffer includes Tris-HCl.

9. The topical composition for preventing and treating skin damage caused by UVB according to claim 6, characterized by, The concentration of Tris-HCl is 0.5-50 mM.

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