Apoptotic vesicle anti-falling hair growth composition and preparation method and application thereof

Abstract

The application discloses an apoptosis vesicle anti-hair loss composition and a preparation method and application thereof. The composition raw materials include a serum-free culture medium containing a ternary natural polyphenol inducer and bovine umbilical cord mesenchymal stem cells, and the apoptosis vesicle anti-hair loss composition includes apoptosis vesicles obtained after the bovine umbilical cord mesenchymal stem cells are induced to apoptosis in the serum-free culture medium containing the ternary natural polyphenol inducer. The composition can synergistically prevent hair loss from multiple targets such as promoting angiogenesis, antioxidation, anti-inflammation, anti-androgen and activating a Wnt pathway, all the raw materials are included in a Catalogue of Cosmetics Raw Materials (2021 Edition), and the whole chain meets the requirements of cosmetic regulations without genetic engineering modification or use of toxic inducers.

Description

Technical Field

[0001] This invention belongs to the field of biological agent technology, specifically relating to a cell apoptosis vesicle anti-hair loss and hair growth composition, its preparation method and application. Background Technology

[0002] Hair loss is a common phenomenon affecting hundreds of millions of people worldwide. Normally shed hairs are those in the telogen (resting) or catagen (transitional) phases. When hair follicle stem cell regeneration is hindered and blood supply to the hair follicle terminals decreases, the hair follicles gradually shrink and thin, leading to progressively worsening hair loss. In recent years, regenerative medicine research has revealed the important potential of apoptotic vesicles in tissue regeneration. Apoptotic cells produce a large number of apoptotic extracellular vesicles (apoEVs). A study published in Bioactive Materials in 2022 by Professor Songtao Shi and Xiaoxing Kou's team at Sun Yat-sen University (Ma L, Chen C, Liu D, Huang Z, Li J, Liu H, Kin Kwok RT, Tang B, Sui B, Zhang X, Tang J, Mao X, Huang W, Shi S, Kou X. Apoptotic extracellular vesicles are metabolized regulators nurturing the skin and hair. Bioact Mater. 2022 May) (11;19:626-641.doi:10.1016 / j.bioactmat.2022.04.022.PMID:35600968;PMCID:PMC9109130.) indicates that exogenous apoEVs can activate skin and hair follicle mesenchymal stem cells through the Wnt / β-catenin pathway, promoting wound healing and hair regeneration. This provides a scientific basis for using mesenchymal stem cell-derived vesicles to treat hair loss. Compared with direct stem cell therapy, vesicle preparations have advantages such as low cost, ease of storage and transportation, and the natural lipid bilayer membrane structure of vesicles can serve as an ideal drug carrier.

[0003] In existing technologies, Chinese patent application CN202511089969.5 discloses a "quercetin-loaded VEGF-overexpressing mesenchymal stem cell apoptotic vesicle, its preparation method, and its application in the treatment of hair loss." This technical solution involves constructing VEGF-overexpressing mesenchymal stem cells, inducing their apoptosis, and adding quercetin to obtain a quercetin-loaded apoptotic vesicle drug. However, this technical solution involves lentiviral packaging and cell transfection operations for VEGF gene overexpression, which is complex and costly. The biological risks introduced by genetic engineering increase the compliance difficulties of product commercialization. Furthermore, the use of traditional chemical inducers (such as staphylococcalin) to induce apoptosis is cytotoxic and not listed in the "Cosmetic Safety Technical Specifications" or the "Catalogue of Used Cosmetic Ingredients," posing a significant compliance risk as a cosmetic ingredient.

[0004] Providing a hair loss prevention and hair growth composition with bioactive apoptotic vesicles and multiple anti-hair loss active ingredients, as well as a technical solution that can efficiently induce mesenchymal stem cell apoptosis while avoiding the dual compliance risks of genetic engineering modification and toxicity inducers, is an effective way to promote the application of apoptotic vesicles in the cosmetics field. Summary of the Invention

[0005] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing a cell apoptosis vesicle anti-hair loss and hair growth composition, its preparation method, and its application. The composition of this invention comprises serum-free culture medium containing a ternary natural polyphenol inducer and bovine umbilical cord mesenchymal stem cells. It can induce apoptosis in bovine umbilical cord mesenchymal stem cells with high quality and efficiently generate apoptotic vesicles, while simultaneously achieving multi-dimensional synergistic prevention and treatment of hair loss. Furthermore, all raw materials comply with the regulatory requirements for cosmetic use.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: On one hand, a cell apoptosis vesicle anti-hair loss and hair growth composition is provided, the raw materials of which include serum-free culture medium containing a ternary natural polyphenol inducer and bovine umbilical cord mesenchymal stem cells. The cell apoptosis vesicle anti-hair loss and hair growth composition includes apoptotic vesicles obtained by inducing apoptosis of bovine umbilical cord mesenchymal stem cells in a serum-free culture medium containing a ternary natural polyphenol inducer; the ternary natural polyphenol inducer is composed of quercetin, resveratrol and curcumin.

[0007] On the other hand, a method for preparing the above-mentioned apoptosis vesicle anti-hair loss and hair growth composition is provided, characterized in that it includes: S1. Isolate and culture bovine umbilical cord mesenchymal stem cells; S2. Take the cultured bovine umbilical cord mesenchymal stem cells and induce apoptosis in serum-free medium containing a ternary natural polyphenol inducer. S3. Collect the supernatant of the induced system, remove intact cells by centrifugation, and obtain the apoptosis vesicle anti-hair loss and hair growth composition.

[0008] On the other hand, the present invention provides the application of the above-mentioned apoptotic vesicle anti-hair loss and hair growth composition in the preparation of anti-hair loss and / or hair growth promoting biological agents.

[0009] Compared with the prior art, the present invention has the following advantages: 1. The apoptosis-inducing vesicle composition for preventing hair loss and promoting hair growth of the present invention is based on the integrated design of inducers and active ingredients. It is the first to propose a strategy of inducing apoptosis of mesenchymal stem cells by using quercetin, resveratrol and curcumin in combination. By directly using the above three natural polyphenols as apoptosis inducers, the apoptosis regulation and active efficacy can be achieved simultaneously during the induction of bovine umbilical cord mesenchymal stem cell apoptosis. No additional active substance compounding process is required, which can effectively avoid the impact of traditional chemical toxic inducers (such as staphylococcus) on the biosafety of apoptotic vesicles.

[0010] 2. In the apoptotic vesicle anti-hair loss and hair growth composition of the present invention, the inducer is a ternary natural polyphenol, which synergistically induces, is highly efficient and safe. By precisely controlling the ratio of the three, apoptotic vesicles rich in bioactive molecules can be efficiently generated in a serum-free culture system, avoiding the process complexity and biosafety risks brought about by genetic engineering modification (such as VEGF overexpression).

[0011] 3. Compared with traditional single-ingredient (such as quercetin alone) or single-pathway anti-hair loss products, the composition of the present invention can synergistically intervene in the pathological process of hair loss from multiple dimensions such as promoting angiogenesis, anti-oxidation, anti-inflammation, anti-androgen (downregulating key genes such as FKBP5 and TGF-β2), and activating the Wnt / β-catenin pathway. It provides multi-target three-dimensional anti-hair loss and significantly improves the anti-hair loss and hair regrowth effect. Efficacy experiments show that its inhibitory effect on the expression of FKBP5 and TGF-β2 genes is better than that of the positive control minoxidil.

[0012] 4. The three polyphenol raw materials (quercetin, resveratrol, and curcumin) used in the composition of the present invention are all included in the "Catalogue of Used Cosmetic Raw Materials (2021 Edition)". The preparation process does not involve operations and substances prohibited or restricted in the cosmetic field, such as genetic engineering, lentiviral packaging, and cytotoxicity inducers. This ensures the compliance and safety of the product from the source, facilitates rapid industrial application, and complies with cosmetic regulations throughout the entire chain.

[0013] 5. The preparation method of the apoptotic vesicle anti-hair loss and hair growth composition of the present invention includes culturing bovine umbilical cord mesenchymal stem cells to a confluence of 80%~90%, inducing apoptosis culture using serum-free medium containing a ternary natural polyphenol inducer, collecting the supernatant of the cultured system and centrifuging at low temperature to obtain the apoptotic vesicle anti-hair loss and hair growth composition, and obtaining a composition containing apoptotic vesicles and a ternary natural polyphenol inducer by low-temperature centrifugation without drug withdrawal, thereby achieving a synergistic effect of multiple components on preventing hair loss and promoting hair growth.

[0014] 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

[0015] Figure 1 Microscopic images of cells induced by the combined action of three natural polyphenols at 24h and 48h. Figure 2 Fluorescence pattern for identifying apoptosis induced by the combined action of ternary natural polyphenols; Figure 3 Transmission electron microscopy image of apoptotic vesicles; Figure 4 This is a particle size distribution diagram of apoptotic vesicles; Figure 5 This is a statistical graph showing the relative expression levels of the FKBP5 gene in the samples and cells treated with minoxidil provided in the examples; # indicates the significance between the "model group" and the "control group", and # indicates P. value < 0.05, ## indicates P value < 0.01, ### indicates P value < 0.001; * indicates significance analysis between "sample group" and "model group", * indicates P value < 0.05, ** indicates P value < 0.01, *** indicates P value < 0.001; Figure 6 The graph shows the relative expression levels of the TGF-β2 gene in the samples and cells treated with minoxidil provided in the examples; # indicates the significance between the "model group" and the "control group", and # indicates P. value < 0.05, ## indicates P value < 0.01, ### indicates P value < 0.001; * indicates significance analysis between "sample group" and "model group", * indicates P value < 0.05, ** indicates P value < 0.01, *** indicates P value < 0.001; Figure 7 The results of cell viability assays after induction culture using quercetin as a single polyphenol inducer are as follows; Figure 8 The results of cell viability assays after induction culture with resveratrol as the single polyphenol inducer are as follows: Figure 9 The results of cell viability assays after induction culture using curcumin as a single polyphenol inducer are shown. Figure 10 The results of cell viability assays after induction culture in serum-free medium with different concentrations of ternary natural polyphenol inducers are shown. Detailed Implementation

[0016] The technical solution will now be clearly and completely described with reference to the embodiments of this application. Obviously, the described embodiments are only a part of the embodiments of this application, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0017] In the following description, the term "and / or" is used to describe the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, B exists alone, and A and B exist simultaneously. A and B can be singular or plural.

[0018] In the following description, the terms “including,” “containing,” “having,” and “containing” are open-ended terms, meaning that they include but are not limited to.

[0019] Those skilled in the art should understand that, in the following description of the embodiments of this application, the sequence of numbers does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0020] Those skilled in the art will understand that the numerical ranges in the embodiments of this application should be understood to specifically disclose each intermediate value between the upper and lower limits of the range. Each smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this application. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, the technical / scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. While this application describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this application. 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.

[0022] The technical principle adopted in this invention is based on the inducer, i.e. the active ingredient. By using serum-free culture medium containing a ternary natural polyphenol inducer and bovine umbilical cord mesenchymal stem cells as raw materials, apoptosis is induced to obtain a composition containing apoptotic vesicles rich in bioactive molecules, quercetin, resveratrol and curcumin, thereby achieving the synergistic effect of vesicles and the three natural polyphenols in preventing hair loss and promoting hair growth.

[0023] On one hand, a cell apoptosis vesicle anti-hair loss and hair growth composition is provided, the raw materials of which include serum-free culture medium containing a ternary natural polyphenol inducer and bovine umbilical cord mesenchymal stem cells. The cell apoptosis vesicle anti-hair loss and hair growth composition includes apoptotic vesicles obtained by inducing apoptosis of bovine umbilical cord mesenchymal stem cells in a serum-free culture medium containing a ternary natural polyphenol inducer; the ternary natural polyphenol inducer is composed of quercetin, resveratrol and curcumin.

[0024] The apoptosis-inducing vesicle hair loss prevention and hair growth composition is obtained by inducing apoptosis in bovine umbilical cord mesenchymal stem cells in a serum-free culture medium containing a ternary natural polyphenol inducer, collecting the supernatant, and then centrifuging the supernatant again. The apoptosis-inducing vesicle hair loss prevention and hair growth composition contains apoptotic vesicles and a ternary natural polyphenol inducer after apoptosis induction. Part of the ternary natural polyphenol inducer is encapsulated in the apoptotic vesicles through cellular uptake and metabolism, while the other part is in a free state. This composition can simultaneously achieve the dual effects of apoptosis regulation and activity conferring during the induction of apoptosis in bovine umbilical cord mesenchymal stem cells.

[0025] In some embodiments, the apoptotic vesicles have a particle size ranging from 30 nm to 150 nm and possess an intact lipid bilayer membrane structure.

[0026] In some embodiments, the mass ratio of quercetin, resveratrol and curcumin in the serum-free culture medium containing the ternary natural polyphenol inducer is (1~5):(1~5):1, preferably (1~2):(1~2):1, and more preferably 2:2:1.

[0027] The preferred mass ratio of quercetin, resveratrol, and curcumin in the apoptotic vesicle anti-hair loss and hair growth composition of the present invention is (1~5):(1~5):1, which can efficiently generate apoptotic vesicles rich in bioactive molecules in a serum-free culture system.

[0028] In some embodiments, in the serum-free culture medium containing the ternary natural polyphenol inducer, the final concentration of quercetin is 10–200 μg / mL, preferably 40–60 μg / mL, and more preferably 40 μg / mL; the final concentration of resveratrol is 10–200 μg / mL, preferably 40–60 μg / mL, and more preferably 40 μg / mL; and the final concentration of curcumin is 5–100 μg / mL, preferably 20–40 μg / mL, and more preferably 20 μg / mL.

[0029] During the research, the inventors discovered that the combined use of three polyphenols—quercetin, curcumin, and resveratrol—can effectively increase vesicle production and reduce impurity generation. In particular, when the final concentrations of quercetin, resveratrol, and curcumin are 40–60 μg / mL, the cell viability after apoptosis culture is moderate, with very little cell necrosis, and the product has high vesicle production and few impurities.

[0030] On the one hand, a method for preparing a hair loss prevention and hair growth composition based on apoptotic vesicles is provided, comprising the following steps: S1. Isolation and culture of bovine umbilical cord mesenchymal stem cells Bovine umbilical cord tissue was collected, repeatedly washed with PBS buffer containing penicillin and antibiotics to remove blood vessels and the outer membrane, Wharton's jelly was separated, and the tissue was cut into 1-3 mm pieces. 3 The tissue block; the PBS buffer containing the double antibody is a PBS buffer containing 1% penicillin-streptomycin; The tissue blocks were inoculated into a complete culture medium containing 10% fetal bovine serum and cultured at 37°C and 5% CO2. The complete culture medium containing 10% fetal bovine serum was DMEM / F12 complete culture medium containing 10% fetal bovine serum, 1% GlutaMAX and 1% penicillin-streptomycin. After the cells have crawled out from the edge of the tissue block, they are digested and passaged with trypsin. Bovine umbilical cord mesenchymal stem cells from generations 3 to 6 were used for subsequent preparations; S2, inducing apoptosis Bovine umbilical cord mesenchymal stem cells from passages 3-6 of step S1 were seeded into culture dishes containing complete culture medium and cultured until confluence reached 80%-90%. Then, they were placed in serum-free culture medium containing a ternary natural polyphenol inducer, composed of quercetin, resveratrol, and curcumin. The seeding density of bovine umbilical cord mesenchymal stem cells in the complete culture medium was 5.0 × 10⁻⁶. 4 cells / cm 2 ; The final concentration of quercetin is 10–200 μg / mL, preferably 40–60 μg / mL; more preferably, the final concentration of quercetin is 40 μg / mL. The final concentration of resveratrol is 10–200 μg / mL, preferably 40–60 μg / mL; more preferably, the final concentration of resveratrol is 40 μg / mL. The final concentration of curcumin is 5–100 μg / mL, preferably 20–40 μg / mL; more preferably, the final concentration of curcumin is 20 μg / mL. Cells were cultured in the above-mentioned serum-free medium containing a ternary natural polyphenol inducer at 37°C and 5% CO2 for 12-48 hours to induce apoptosis; the preferred induction time was 48 hours; during the induction process, cell morphological changes (such as cell shrinkage, appearance of membrane-like protrusions, cell budding to form apoptotic bodies, and other apoptotic characteristics) could be observed using an optical microscope. S3, Collection of the composition When the cell apoptosis rate reaches 60%~80% in step S2, the supernatant is collected and centrifuged at 4°C for 10 minutes at 200 g to remove intact cells and obtain the supernatant. The obtained supernatant is the cell apoptosis vesicle anti-hair loss and hair growth composition.

[0031] The present invention discloses a method for preparing a cell apoptosis vesicle composition for preventing hair loss and promoting hair growth. The method includes culturing isolated bovine umbilical cord mesenchymal stem cells to a confluence of 80%–90%, followed by apoptosis induction culture in a serum-free medium containing a ternary natural polyphenol inducer. The supernatant from the induced culture system is collected and centrifuged at low temperature to obtain the cell apoptosis vesicle composition for preventing hair loss and promoting hair growth. Through drug-free low-temperature centrifugation, a composition containing apoptosis vesicles rich in bioactive molecules, quercetin, resveratrol, and curcumin is obtained. This composition simultaneously achieves apoptosis regulation and bioactive efficacy during the induction of bovine umbilical cord mesenchymal stem cell apoptosis, without the need for additional active substance compounding processes, thus achieving a synergistic effect of multiple components in preventing hair loss and promoting hair growth. On the other hand, this invention provides the application of the above-mentioned apoptotic vesicle anti-hair loss and hair growth composition in the preparation of biological agents for preventing hair loss and / or promoting hair growth. The dosage forms of the biological agents include serums, emulsions, gels, lyophilized powders, liposome-encapsulated formulations, and microneedle patch formulations. When used, it is applied to the balding area of ​​the scalp through topical application or microneedle-assisted transdermal delivery.

[0032] Prior to the application for this invention, a series of experiments were conducted. Some of the experimental results are listed below to provide a more detailed description of the invention. The following is a detailed description in conjunction with the embodiments.

[0033] For any experimental steps or conditions not specified in the examples, the procedures or conditions described in the literature in this field can be followed. All reagents without specified manufacturers are commercially available standard reagent products.

[0034] Example 1 This embodiment provides a method for preparing a hair loss prevention and hair growth composition based on apoptotic vesicles, comprising: Step 1: Isolation and culture of bovine umbilical cord mesenchymal stem cells (MSCs): Step 101: Take approximately 10 cm of fresh bovine umbilical cord tissue that has passed inspection. Under aseptic conditions, wash the tissue repeatedly 3-5 times with PBS containing 1% penicillin-streptomycin to remove residual blood. Longitudinally cut open the umbilical cord, separate Wharton's jelly, remove blood vessels and outer membrane tissue, and cut the Wharton's jelly into pieces approximately 1-3 mm in size. 3 Small tissue blocks were used to obtain Wharton's colloid tissue blocks; Step 102: Evenly attach the Wharton's jelly tissue block to the bottom of a T75 culture flask, add 10 mL of DMEM / F12 complete culture medium containing 10% fetal bovine serum, 1% GlutaMAX, and 1% penicillin-streptomycin, invert the culture flask so that the attached side of the tissue block is facing upwards, and incubate in a 37°C, 5% CO2 incubator for 4-6 hours. Carefully turn the culture flask over so that the tissue block is submerged in the culture medium. Change half of the medium every 2-3 days. After about 7-10 days, spindle-shaped cells can be seen crawling out from the edge of the tissue block. When the cell confluence reaches 80%-90%, digest with 0.25% trypsin-EDTA, passage at a 1:3 ratio, and use the 4th generation bovine umbilical cord mesenchymal stem cells for subsequent apoptosis induction steps. Step 2: Tripartite natural polyphenols jointly induce apoptosis: Step 201: Take the fourth-generation bovine umbilical cord mesenchymal stem cells from Step 1, at a concentration of 5.0 × 10⁻⁶. 4 cells / cm 2 The culture medium was inoculated at a density in T175 culture flasks and cultured in DMEM / F12 complete medium until the confluence reached approximately 85%. The old medium was discarded, and the culture was washed twice with PBS. The medium was then replaced with an equal volume of serum-free medium containing a ternary natural polyphenol inducer. The serum-free medium containing the ternary natural polyphenol inducer consisted of fresh serum-free DMEM medium, quercetin, resveratrol, and curcumin. The serum-free medium containing the ternary natural polyphenol inducer contained 40 μg / ml quercetin, 40 μg / ml resveratrol, and 20 μg / ml curcumin (quercetin:resveratrol:curcumin = 2:2:1). Step 202: The culture flasks were placed at 37°C and 5% CO2 for further culture. During this period, at the 24-hour and 48-hour mark, the morphological characteristics of the cells gradually appearing as wrinkles, membrane vesicle protrusions and budding to form apoptotic bodies were observed under a microscope, confirming that the three-component polyphenol combination can effectively induce bovine umbilical cord MSCs to enter the apoptosis program under mild conditions. Step 3: Collection of the composition: After 48 hours of induction, all cell supernatant (about 20 mL) was collected and centrifuged at 4°C for 10 min at 200 g. The supernatant was collected and the cell pellet was discarded. The obtained supernatant is the cell apoptosis vesicle anti-hair loss and hair growth composition, which is aliquoted and stored at -80°C for later use.

[0035] Performance Evaluation Figure 1 Microscopic images of bovine umbilical cord mesenchymal stem cells (BUC cells) with approximately 85% confluence (before apoptosis), cultured for 24 h (24 h of apoptosis) and 48 h (48 h of apoptosis) in serum-free medium containing equal volumes of ternary natural polyphenol inducers. These images show the presence of bovine umbilical cord mesenchymal stem cells (BUC cells). Figure 1 (Left image) The cells are intact, densely packed, with clear boundaries, and a smooth cell surface, without obvious vesicle structures; apoptosis 24 h ( Figure 1 (In the middle image), the cells show obvious membrane vesicle bubbling, cytoskeleton disintegration, cell body fragmentation, and the formation of numerous spherical apoptotic vesicles of varying sizes; apoptosis occurs 48 hours later ( Figure 1 (As shown in the right figure), large apoptotic vesicles further lyse and divide, resulting in fragmented cell fragments, and the complete loss of intact cell structure.

[0036] 1. Protein concentration detection—BCA method (1) Preparation of BCA working solution: Mix 50 volumes of BCA reagent A with 1 volume of BCA reagent B (50:1) and mix thoroughly; add protein standard (0.5 mg / mL) to 96 wells at concentrations of 0, 1, 2, 4, 8, 12, 16, and 20 μL; add standard diluent to bring the total to 20 μL, so that the standard concentrations are 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, respectively. (2) The apoptosis vesicle anti-hair loss and hair growth composition of Example 1 was added to the sample wells of a 96-well plate at a rate of 2 μL per well, and three parallel samples were made. (3) Add 200 μL of BCA working solution to each well, gently shake the 96-well plate to mix, and place it at 37°C for 20-30 minutes. Measure the absorbance at wavelength A562 using an ELISA reader and calculate the protein concentration. The experimental results showed that the protein concentration of apoptotic vesicles was determined by the BCA method, and the protein standard curve for calculating the sample concentration was Y=0.0425X+0.1011 (X: sample protein content (μg), Y: sample absorbance), R2 =0.9913, and the obtained protein concentration of apoptotic vesicles was 0.54 mg / mL.

[0037] 2. Identification of apoptosis induced by ternary polyphenols Cells cultured for 24 h in step 2 of Example 1 were subjected to apoptosis identification using fluorescent staining. The most widely used and classic method, Annexin V / PI double staining (C1062L, Beyotime), was employed. In the early stages of apoptosis, phosphatidylserine residues on the inner side of the cell membrane evert to the outer side. Annexin V protein specifically binds to these everted PS residues, emitting green fluorescence after being labeled with a fluorescent dye (such as FITC). Propidium iodide, a red fluorescent nucleic acid dye, cannot penetrate intact cell membranes and can only label late-stage apoptotic or necrotic cells with compromised membrane integrity. The staining process followed the manufacturer's instructions for cell collection, washing, resuspending, and staining. After incubation, a drop of cell suspension was pipetted onto a glass slide, which was then covered with a coverslip. Observation and image acquisition were performed using a fluorescence microscope (FITC and PI channels). Results are as follows... Figure 2 As shown in the figure, the early apoptosis rate of cells was 63.3%, indicating that the combination of three polyphenols can successfully induce apoptosis.

[0038] 3. Composition of apoptotic vesicles The apoptotic vesicle anti-hair loss and hair growth composition described in step 3 of Example 1 was observed using a transmission electron microscope, and the results are as follows: Figure 3 As shown in the figure, obvious vesicle structures are present, and the particle size analysis results are as follows. Figure 4 As shown, the average particle size of apoptotic vesicles was 134.4 nm, and the concentration was 3.2 × 10⁻⁶. 10 Particles / mL.

[0039] 4. Verification of its efficacy in preventing hair loss and promoting hair growth The efficacy of the sample obtained in Example 1 was tested, and the specific steps are as follows: Mouse dermal papilla cells were seeded into 6-well plates, with a cell density of 5 × 10⁶ cells per well. 5 Each well was filled with complete culture medium and incubated at 37°C and 5% CO2 for 24 h. A control group and a sample group were set up. The drug dosage per well was 2 mL. Each group was set up with 3 replicates. The samples were incubated in the incubator (37°C and 5% CO2) for 24 h. See Table 1 below for details.

[0040] Table 1. Types of reagents added to 6-well plates Total RNA was collected from cells after treatment in each group and analyzed using quantitative real-time PCR (qRT). PCR was used to detect changes in the expression levels of FKBP5, a key gene regulating the androgen receptor, and transforming growth factor β2 (TGF-β2). The results were as follows: Figures 5-6 The results are shown.

[0041] Depend on Figure 5 It was found that the relative expression level of FKBP5 in the model group was increased compared with the control group, indicating that the model group was valid. Compared with the model group, the relative expression level of FKBP5 in the 1.25% and 0.625% sample groups was significantly decreased, with a decrease rate of 34.32% and 37.35%, respectively. Compared with the model group, the relative expression level of FKBP5 in the minoxidil-10uM group decreased, with a decrease rate of 20.87%.

[0042] Depend on Figure 6 It was found that the relative expression level of TGF-β2 in the model group was increased compared with the control group, indicating that the model group was valid. Compared with the model group, the relative expression level of TGF-β2 in the 1.25% and 0.625% sample groups was significantly decreased, with a decrease rate of 72.69% and 56.99%, respectively. Compared with the model group, the relative expression level of TGF-β2 in minoxidil-10uM was decreased, with a decrease rate of 26.97%.

[0043] 5. The effect of single natural polyphenols on promoting cell apoptosis Serum-free medium containing a single polyphenol inducer was used as the induction medium. Apoptosis induction culture was performed using the same steps as in Example 1, and cell viability was detected by the CCK8 assay after 48 h of culture. The single polyphenol inducer in the serum-free medium was quercetin, resveratrol, or curcumin, and the concentration of the single polyphenol inducer was 0 (CK), 2, 5, 10, 20, 40, 100, and 200 ug / ml. The measured cell viability is as follows: Figures 7-9 As shown, where Figure 7 The results are for cell viability assays after induction with quercetin as the sole polyphenol inducer. Figure 8 The results are for cell viability assays after induction culture using resveratrol as the sole polyphenol inducer. Figure 9 The results show the cell viability assays after induction culture using curcumin as the sole polyphenol inducer. It is evident that when the quercetin concentration in the induction medium is 2–20 μg / mL, cell viability is close to 100%, with almost no pro-apoptotic effect. At a quercetin concentration of 40 μg / mL, cell viability is approximately 90%, indicating that this concentration can mildly inhibit proliferation and gently initiate apoptosis signals. At quercetin concentrations ≥100 μg / mL, cell viability drops drastically, and large-scale cell necrosis occurs.

[0044] When the resveratrol concentration in the induction medium is 2-20 μg / mL, the cell viability is close to 100%, and the apoptosis induction ability is extremely weak. When the resveratrol concentration is 40 μg / mL, the cell viability is about 95%. When the resveratrol concentration is ≥100 μg / mL, the cell viability is <50%. The possible reason is that the strong toxicity of the induction medium leads to cell lysis and necrosis.

[0045] When curcumin is the only natural polyphenol in the induction medium, cell viability changes in stages with increasing curcumin concentration. When the curcumin concentration is 2 or 5 μg / mL, cell viability is approximately 100% with no apoptosis stimulation. When the curcumin concentration is 10 μg / mL, cell viability is approximately 95%, mildly inhibiting proliferation and gently initiating apoptosis. When the curcumin concentration is 20-40 μg / mL, cell viability is approximately 50%. When the curcumin concentration is ≥100 μg / mL, cell viability is almost zero. This may be because curcumin is highly toxic and sensitive to concentration; when the concentration is too high, the cells directly die and disintegrate.

[0046] 6. Effects of the concentration of natural polyphenol combination in serum-free culture medium containing a ternary natural polyphenol inducer on cell apoptosis. Serum-free medium containing a ternary natural polyphenol inducer was used as the induction medium. Induction culture was performed using the same steps as in Example 1, and cell viability was detected using the CCK8 assay after 48 h of culture. The ternary natural polyphenol inducers in the serum-free medium were quercetin, resveratrol, and curcumin. The concentrations of natural polyphenols in each group are shown in the table below. The measured cell viability is as follows: Figure 10 As shown.

[0047] Table 2. Concentration of natural polyphenols in different groups during cell viability testing. The results showed that the cell viability of experimental group 1 was 34%, with programmed apoptosis being the dominant mode and necrosis being extremely rare. Figure 1 of Figure 10 It can be seen that after 48 hours of apoptosis culture, the vesicle production was high and the impurities were few, indicating the best apoptosis induction effect. In experimental group 2, the cell viability was close to 60%, indicating high cell viability but low apoptotic vesicle production. This may be due to weak apoptotic stimulation, a small number of apoptotic cells, and low vesicle production. In experimental group 3, the cell viability was only 13%, with a large number of cells dying and lysing. This may be because the excessively high curcumin concentration caused extensive cell necrosis, and the necrotic cell debris contaminated the vesicles, resulting in poor purity of the apoptotic vesicles.

[0048] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the disclosure. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A hair loss prevention and hair growth composition based on apoptotic vesicles, characterized in that, The raw materials include serum-free culture medium containing a ternary natural polyphenol inducer and bovine umbilical cord mesenchymal stem cells. The apoptotic vesicle anti-hair loss and hair growth composition includes apoptotic vesicles obtained by inducing apoptosis of bovine umbilical cord mesenchymal stem cells in a serum-free culture medium containing a ternary natural polyphenol inducer. The ternary natural polyphenol inducer is composed of quercetin, resveratrol and curcumin.

2. The apoptosis-inducing vesicle composition for preventing hair loss and promoting hair growth according to claim 1, characterized in that, The apoptotic vesicles have a particle size range of 30 nm to 150 nm.

3. The apoptosis-inducing vesicle composition for preventing hair loss and promoting hair growth according to claim 1, characterized in that, In the serum-free culture medium containing the ternary natural polyphenol inducer, the mass ratio of quercetin, resveratrol, and curcumin is (1~5):(1~5):

1.

4. The apoptosis-inducing vesicle composition for preventing hair loss and promoting hair growth according to claim 1, characterized in that, In the serum-free culture medium containing the ternary natural polyphenol inducer, the final concentration of quercetin is 10–200 μg / mL, the final concentration of resveratrol is 10–200 μg / mL, and the final concentration of curcumin is 5–100 μg / mL.

5. A method for preparing the apoptosis-inducing vesicle composition for preventing hair loss and promoting hair growth as described in claim 1, characterized in that, include: S1. Isolate and culture bovine umbilical cord mesenchymal stem cells; S2. Take the cultured bovine umbilical cord mesenchymal stem cells and induce apoptosis in serum-free medium containing a ternary natural polyphenol inducer. S3. Collect the supernatant of the induced system, remove intact cells by centrifugation, and obtain the apoptosis vesicle anti-hair loss and hair growth composition.

6. The method according to claim 5, characterized in that, The bovine umbilical cord mesenchymal stem cells cultured in step S2 are bovine umbilical cord mesenchymal stem cells of the 3rd to 6th generation.

7. The method according to claim 5, characterized in that, In step S2, the bovine umbilical cord mesenchymal stem cells (BMSCs) induced to undergo apoptosis in a serum-free medium containing a ternary natural polyphenol inducer are obtained by culturing BMSCs in complete medium until the confluence reaches 80%–90%. The seeding density of the BMSCs in the complete medium is 5.0 × 10⁻⁶. 4 cells / cm 2 .

8. The method according to claim 7, characterized in that, The serum-free culture medium containing the ternary natural polyphenol inducer consists of quercetin, resveratrol, and curcumin, with a final concentration of 10–200 μg / mL for quercetin, 10–200 μg / mL for resveratrol, and 5–100 μg / mL for curcumin.

9. The method according to claim 5, characterized in that, In step S2, the culture time for apoptosis induction is 12-48 hours, and apoptosis induction is carried out at 37℃ and 5% CO2. In step S3, centrifugation is carried out at 4℃, with a centrifugation speed of 200 g and a centrifugation time of 10 min.

10. The use of the apoptotic vesicle anti-hair loss and hair growth composition as described in claim 1 in the preparation of anti-hair loss and / or hair growth promoting biological agents.

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