Anti-shedding hair solidifying exosome composition

Abstract

The application belongs to the technical field of exosome application, and discloses an exosome composition for preventing hair loss and fixing hair. The exosome composition is obtained by adding plant exosomes in animal stem cell culture, and it is found that the plant exosomes can significantly promote the secretion of animal stem cell exosomes. On this basis, a scheme for constructing the exosome composition for preventing hair loss and fixing hair is constructed. The exosome composition is obtained by mixing fat stem cell exosomes secreted by fat stem cells treated by curcuma exosomes, urine source epithelial stem cell exosomes secreted by urine source epithelial stem cells treated by olea europaea leaf exosomes and biota leaf exosomes according to weight proportions of 1-2:2-5:2-3. The exosome composition can significantly promote hair papilla cell proliferation, has a significant effect on preventing hair loss and fixing hair, has a large number of plant exosomes, can be regenerated, can significantly reduce the use amount of animal stem cell exosomes, and has a better effect after combination, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of exosome application technology, and more specifically, relates to an exosome composition for preventing hair loss and strengthening hair. Background Technology

[0002] Hair loss has become a common health and beauty challenge affecting many people worldwide, giving rise to a huge market for anti-hair loss and hair-strengthening products. However, most mainstream products on the market rely on chemically synthesized substances such as minoxidil and finasteride. While these ingredients have been clinically proven to promote hair growth, their side effects cannot be ignored. Long-term or improper use may lead to local reactions such as scalp irritation, dryness, itching, and redness. Some ingredients may also cause systemic effects such as hormonal fluctuations and cardiovascular discomfort. Furthermore, the potency of chemical formulas is often accompanied by interference with the scalp microenvironment, potentially disrupting the scalp barrier balance and leading to problems such as sensitivity and dependence. Consumers' increasing focus on health, safety, and natural ingredients has led to growing skepticism and challenges for current anti-hair loss and hair-strengthening products that primarily rely on chemical substances. Therefore, developing gentler, less irritating alternative solutions that combine efficacy and safety has become a crucial research direction urgently needing breakthroughs in the field of hair health.

[0003] In recent years, with the rapid development of regenerative medicine and biotechnology, the rise and promotion of exosome application technology has opened up revolutionary new perspectives and possibilities for solving the traditional problem of hair loss prevention and strengthening. Exosomes are nanoscale vesicles actively secreted by cells. As natural intercellular messengers, they can carry abundant bioactive substances such as proteins, nucleic acids (e.g., mRNA, miRNA), and lipids, precisely regulating the physiological functions and signaling pathways of target cells. Research in the field of hair has shown that exosomes derived from specific cells such as mesenchymal stem cells can effectively penetrate the hair follicle microenvironment and improve hair follicle health and vitality from the root through multiple mechanisms, including transmitting growth-promoting signals, regulating the hair follicle cycle (e.g., prolonging the anagen phase and alleviating the telogen phase), enhancing the activity of dermal papilla cells, inhibiting inflammation around the hair follicle, and promoting angiogenesis. Compared with traditional chemical drugs, exosome therapy shows significant advantages such as strong targeting, high biocompatibility, multidimensional mechanisms of action, and lower risk of side effects, and is expected to become a more precise, safe, and physiologically sound intervention strategy. Therefore, applying exosome technology to the field of hair loss prevention and strengthening not only represents an important transformation of cutting-edge biotechnology into the consumer health market, but also a highly promising breakthrough direction to address the limitations of existing chemical solutions, attracting widespread attention and active exploration from academia and industry.

[0004] Despite the enormous potential of stem cell exosomes in the field of hair loss prevention and treatment, the further development and clinical translation of this technology face a fundamental bottleneck: under the current production system, the yield of exosomes is severely insufficient, and the delivery and utilization efficiency during application is low. From the production side, the in vitro culture conditions of stem cells are stringent, their proliferation rate is limited, and the number of naturally secreted exosomes is scarce. Furthermore, existing separation and extraction technologies (such as ultracentrifugation) suffer from problems such as complex processes, lengthy time consumption, high costs, and unstable yields, making it difficult to meet the needs of large-scale, standardized applications. From the application side, extracted stem cell exosomes are prone to inactivation and degradation during storage and delivery. These limitations collectively mean that extremely high doses of stem cell exosomes are often required to observe effects in practical applications, further amplifying the supply gap and driving up treatment costs.

[0005] Against this backdrop, the discovery and research of plant-derived exosomes offer a highly attractive potential solution to the aforementioned bottlenecks. Recent studies have shown that plant cells, during growth and stress resistance, also secrete nanoscale vesicles rich in bioactive molecules (such as lipids, proteins, small RNAs, and natural metabolites), known as plant exosomes. Compared to exosomes derived from animal stem cells, plant exosomes exhibit significant advantages in large-scale production and cost: their raw material sources are widely available (such as plant tissues from fruits, vegetables, and traditional Chinese medicines), enabling large-scale, renewable, and standardized production through agricultural biotechnology such as hydroponics and tissue culture. Furthermore, the extraction process is relatively simple, potentially significantly reducing preparation costs and ensuring a stable supply. In addition, plant exosomes generally possess good biocompatibility and low immunogenicity, and their naturally loaded plant active ingredients (such as antioxidants and anti-inflammatory substances) may synergistically act on the scalp microenvironment, providing multi-target support for hair health. Therefore, exploring plant-derived exosomes as novel, scalable functional carriers not only holds promise for overcoming the limitations of animal cell exosomes in terms of yield and cost, but also opens up a promising innovative path for developing a new generation of efficient, safe, and widely accessible anti-hair loss biological formulations, and is becoming a rapidly growing research hotspot in this field. Summary of the Invention

[0006] In view of the above-mentioned defects in the existing technology, the present invention firstly provides an exosome composition for preventing hair loss and strengthening hair.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] This invention first discovered that by adding plant exosomes to animal stem cell culture, plant exosomes can significantly promote the secretion of exosomes from animal stem cells. Based on this, a scheme for an anti-hair loss and hair-strengthening exosome composition was constructed.

[0009] Therefore, this invention first protects the application of turmeric exosomes in promoting the secretion of exosomes from adipose stem cells.

[0010] Preferably, the preparation of the turmeric exosomes includes the following steps:

[0011] (1) Take a fresh sample of turmeric root, crush it, and filter it coarsely through gauze;

[0012] (2) Centrifuge at 3000-5000 r / min for 30-50 min; take the supernatant and discard the precipitate;

[0013] (3) Centrifuge at 10000-12000 r / min for 30-50 min; take the supernatant and discard the precipitate;

[0014] (4) Centrifuge at 10000-12000 r / min for 30-50 min, take the supernatant and discard the precipitate; repeat this step 2-3 times until there is no obvious precipitate;

[0015] (5) The microporous membrane is used for filtration in sequence to obtain the supernatant;

[0016] (6) Add the filtered supernatant to the exosome extraction kit and extract turmeric exosomes.

[0017] This invention also protects the use of olive leaf exosomes in promoting the secretion of exosomes from urinary epithelial stem cells.

[0018] Preferably, the preparation of the olive leaf exosomes includes the following steps:

[0019] (1) Wash the olive leaves and juice them to obtain liquid juice;

[0020] (2) Filter the liquid juice and centrifuge the filtered juice at 1000-2000×g for 10-15 min at low temperature;

[0021] (3) The first filtrate is obtained by filtration through a microporous membrane;

[0022] (4) Centrifuge the first filtrate at 1000-2000×g for 10-15min and filter it through a microporous membrane to obtain the second filtrate;

[0023] (5) Centrifuge the second filtrate at 10000-12000×g for 10-15 min for the first time, and at 20000×g for the second time for 10-15 min, and then recover the supernatant;

[0024] (6) Centrifuge the supernatant at 100,000 × g for 70-80 min at low temperature to obtain a precipitate. Resuspend the obtained precipitate in PBS to obtain olive leaf exosomes.

[0025] This invention also protects an exosome composition for preventing hair loss and strengthening hair, which is obtained by mixing adipose stem cell exosomes secreted by adipose stem cells cultured after being treated with turmeric exosomes, urinary epithelial stem cell exosomes secreted by urinary epithelial stem cells cultured after being treated with olive leaf exosomes, and arborvitae leaf exosomes in a weight ratio of 1-2:2-5:2-3.

[0026] Preferably, the adipose-derived stem cell exosomes secreted by adipose-derived stem cells treated with turmeric exosomes are obtained by adding turmeric exosomes to adipose-derived stem cells, culturing for 24-72 hours, and then collecting the supernatant for separation; the urinary epithelial stem cell exosomes secreted by urinary epithelial stem cells treated with olive leaf exosomes are obtained by adding olive leaf exosomes to urinary epithelial stem cell culture medium, culturing for 24-72 hours, and then collecting the supernatant for separation.

[0027] The exosome composition obtained by this invention can significantly promote the proliferation of dermal papilla cells and has a significant effect on preventing hair loss and strengthening hair. Therefore, this invention also protects the use of the exosome composition described above in promoting the proliferation of dermal papilla cells.

[0028] This invention also protects the use of the exosome composition in the preparation of hair loss prevention and hair strengthening products.

[0029] This invention also protects a hair loss prevention and hair strengthening product containing the aforementioned exosome composition.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] This invention, by adding plant exosomes to animal stem cell cultures, discovered that plant exosomes significantly promote the secretion of animal stem cell exosomes. Based on this, a formula for an anti-hair loss and hair-strengthening exosome composition was developed. The composition is obtained by mixing adipose-derived stem cell exosomes secreted by adipose-derived stem cells cultured with turmeric exosomes, urinary epithelial stem cell exosomes secreted by urinary epithelial stem cells cultured with olive leaf exosomes, and Platycladus orientalis leaf exosomes in a weight ratio of 1-2:2-5:2-3. This exosome composition significantly promotes the proliferation of dermal papilla cells, exhibiting a significant effect on preventing hair loss and strengthening hair. The plant exosomes are abundant, regenerative, and can significantly reduce the amount of animal stem cell exosomes used. Furthermore, the combined effect is better, showing broad application prospects. Attached Figure Description

[0032] Figure 1 Electron micrograph of turmeric exosomes;

[0033] Figure 2Electron micrograph of olive leaf exosomes;

[0034] Figure 3 Electron micrograph of exosomes from Platycladus orientalis leaves;

[0035] Figure 4 Electron micrograph of exosomes from adipose-derived stem cells;

[0036] Figure 5 Electron micrograph of exosomes from urinary epithelial stem cells;

[0037] Figure 6 The effect of plant exosomes on the number of exosomes secreted by adipose-derived stem cells;

[0038] Figure 7 The effect of plant exosomes on the expression level of CD81, a molecular marker on the surface of adipose-derived stem cell exosomes;

[0039] Figure 8 The effect of plant exosomes on the number of exosomes secreted by urinary epithelial stem cells;

[0040] Figure 9 The effect of plant exosomes on the expression level of CD81, a molecular marker on the surface of exosomes from urinary epithelial stem cells;

[0041] Figure 10 Microscopic images of hair before and after application to volunteers (magnification 40x); Figure A: Subject before application; Figure B: Minoxidil user after 60 days; Figure C: Exosome composition of the present invention after 60 days of use. Detailed Implementation

[0042] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific drawings and embodiments. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0043] The exosome extraction and purification kit (cell supernatant) (Umibio: UR52121) was purchased from Shanghai Yumeibo Biotechnology Co., Ltd.

[0044] Example 1: Preparation of exosomes

[0045] I. Preparation and Characterization of Curcuma Exosomes

[0046] Includes the following steps:

[0047] (1) Take fresh turmeric root samples and wash them 3 times with ddH2O; crush them in a juicer (add 1×PBS buffer as appropriate during the process), and filter them coarsely with gauze;

[0048] (2) Centrifuge at 3000 r / min for 30 min; take the supernatant and discard the precipitate;

[0049] (3) Centrifuge at 10000 r / min for 30 min; take the supernatant and discard the precipitate;

[0050] (4) Centrifuge at 12000 r / min for 30 min, collect the supernatant and discard the precipitate. Repeat this step 2-3 times until no obvious precipitate remains;

[0051] (5) Filter sequentially through a 0.22 μm microporous membrane to obtain the supernatant;

[0052] (6) Add 5 mL of ECS (Exosome Concentration Solution) to 20 mL of filtered supernatant and let stand at 4℃ for 4 h;

[0053] (7) Centrifuge at 12000 r / min for 60 min, discard the supernatant, and resuspend the precipitate in 1×PBS (add 1~2 mL).

[0054] (8) Transfer the resuspension to a 1.5 mL centrifuge tube, centrifuge at 12000 r / min for 10 min, and collect the supernatant (if there is a lot of precipitate, centrifuge multiple times to collect the supernatant).

[0055] (9) Transfer the supernatant to an EPF (Exosome Purification Filter) column, centrifuge at 12000 r / min for 2 min, and filter to the bottom of the EPF column to obtain turmeric exosomes. After extraction, proceed directly to subsequent experiments or store at -80℃.

[0056] The obtained turmeric exosomes were analyzed by a nanoparticle size analyzer. The particle size of the turmeric exosomes was concentrated at 155.3 ± 25.3 nm. Their transmission electron microscopy images are shown below. Figure 1 As shown, the purified turmeric exosomes exhibit a distinct double-membrane structure, resembling a saucer, which is consistent with the microscopic identification characteristics of exosomes.

[0057] The protein concentration of turmeric exosomes was determined to be 7.53 μg / μL using the BCA protein quantification method.

[0058] II. Preparation and Characterization of Olive Leaf Exosomes

[0059] Includes the following steps:

[0060] (1) Wash the olive leaves, cut the leaves and stems into pieces no more than 5cm, and use a low-speed screw press at a stirring speed of 40rpm to extract the juice;

[0061] (2) The juice was filtered through a 400-mesh screen to remove large suspended matter. The filtered juice was centrifuged at 10,000 × g for 10 min at 4°C to remove proteins and cell walls;

[0062] (3) The supernatant obtained by the above operation is filtered through a filter membrane with 0.22 μm pores to obtain the first filtrate;

[0063] (4) Centrifuge the first filtrate at 2000×g for 10 min and filter it through a filter membrane with 0.22μm pores to obtain the second filtrate;

[0064] (5) Centrifuge the second filtrate at 10000×g for 10 min for the first time and at 20000×g for 10 min for the second time, and then recover the supernatant;

[0065] (6) The supernatant was centrifuged at 100,000 × g for 70 min at 4 °C to obtain a precipitate. The obtained precipitate was resuspended in PBS to obtain olive leaf exosomes.

[0066] The obtained olive leaf exosomes were analyzed by a nanoparticle size analyzer. The particle size of the olive leaf exosomes was concentrated at 128.5 ± 10.7 nm. Their transmission electron microscopy images are shown below. Figure 2 As shown, the purified olive leaf exosomes are typical biconcave disc-shaped or cup-shaped vesicles, consistent with the microscopic identification characteristics of exosomes.

[0067] The protein concentration of olive leaf exosomes was determined to be 10.16 μg / μL using the BCA protein quantification method.

[0068] III. Preparation and Characterization of Platycladus orientalis Leaf Exosomes

[0069] The leaves of Platycladus orientalis were ground using a tissue homogenizer, dissolved in PBS at a ratio of 1:100, filtered through gauze, and centrifuged at 9000 rpm for 60 min. The supernatant was collected and transferred to an ultracentrifuge tube. After precise balancing with an error of less than 0.001 g, the tube was placed in a pre-chilled ultracentrifuge and centrifuged at 4 °C and 9500 × g for 70 min. The supernatant was discarded, and the precipitate was retained and resuspended in PBS. After balancing, the precipitate was centrifuged again at 4 °C and 9500 × g for 70 min. The precipitate was collected, resuspended in a small amount of PBS, and stored in an ultra-low temperature freezer.

[0070] The obtained Platycladus orientalis leaf exosomes were analyzed by a nanoparticle size analyzer. The particle size of the Platycladus orientalis leaf exosomes was concentrated at 116.3 ± 8.1 nm. Their transmission electron microscopy images are shown below. Figure 3 As shown, the purified Platycladus orientalis leaf exosomes are cup-shaped vesicles, consistent with the microscopic identification characteristics of exosomes.

[0071] The protein concentration of Platycladus orientalis leaf exosomes was determined to be 6.85 μg / μL using the BCA protein quantification method.

[0072] IV. Preparation and Characterization of Adipose-Derived Stem Cell Exosomes

[0073] 1. Isolation of exosomes from adipose-derived stem cells, including the following steps:

[0074] (1) Isolate and culture human adipose stem cells to the 3rd-5th generation; when the cell confluence reaches 70-80%, replace the original culture medium with a complete culture medium that has been pre-removed from bovine exosomes, and continue culturing for 24-48 hours;

[0075] (2) Collect the supernatant containing exosomes secreted by ADSC (i.e., conditioned medium), and immediately centrifuge at 300 ×g for 10 min at 4℃ to remove floating cells;

[0076] (3) Centrifuge the supernatant sequentially at 4℃: 2000 × g, 10 min; 10000 × g, 30 min;

[0077] (4) Transfer the supernatant to an ultracentrifuge tube and centrifuge at 100,000 × g for 90 min at 4 °C;

[0078] (5) Carefully discard the supernatant, resuspend the precipitate with a large amount of pre-cooled phosphate buffer (PBS), and centrifuge again at 100,000×g for 90 min;

[0079] (6) Finally, the exosome precipitate was resuspended in a small amount of PBS to obtain an adipose stem cell exosome suspension.

[0080] The obtained adipose-derived stem cell exosomes were analyzed using a nanoparticle size analyzer. The particle size of the adipose-derived stem cell exosomes was concentrated at 103.8 ± 10.2 nm, as shown in the transmission electron microscopy image below. Figure 4 As shown.

[0081] The protein concentration of adipose stem cell exosomes was determined to be 12.76 μg / μL using the BCA protein quantification method.

[0082] V. Preparation and Characterization of Urinary Epithelial Stem Cell Exosomes

[0083] (1) Collect 50 mL of fresh midstream urine, place it immediately on ice, and add antibiotics (such as penicillin / streptomycin) to prevent bacterial growth. Centrifuge at 400 × g for 10 min at 4°C;

[0084] (2) Centrifuge the supernatant again at 4°C at 2,000 × g for 20 min and collect the precipitate;

[0085] (3) The cell pellet was resuspended in DMEM / F12 medium and seeded into culture flasks. Urinary epithelial stem cells were obtained by morphological (paving stone-like) and surface marker (such as positive CD44, CD73, CD90, and negative CD34) identification.

[0086] (4) After culturing to the 3rd-5th generation, when the cells are stable and pure, they are used to prepare conditioned medium;

[0087] (5) When the cell confluence reaches 70-80%, replace the old culture medium with complete culture medium prepared from fetal bovine serum with exosomes removed. Continue culturing for 24-48 hours, then collect the supernatant (i.e., conditioned medium).

[0088] (6) Centrifuge at 300×g for 10 min at 4℃ to remove floating cells, then centrifuge at 2000×g for 20 min and 10000×g for 30 min to gradually remove dead cells, cell debris and large organelles.

[0089] (7) Transfer the pretreated supernatant to an ultracentrifuge tube. Centrifuge at 100,000 × g for 90 min at 4 °C. Carefully discard the supernatant;

[0090] (8) Gently resuspend the precipitate with a large amount of pre-cooled PBS (phosphate buffer), and then centrifuge again at 100,000×g for 90 min under the same conditions to remove co-precipitated proteins.

[0091] (10) Finally, the exosome precipitate was resuspended in 100 μL of sterile PBS to obtain urinary epithelial stem cell exosomes.

[0092] The obtained urinary epithelial stem cell exosomes were analyzed by a nanoparticle size analyzer. The particle size of the urinary epithelial stem cell exosomes was concentrated at 105.4±9.6 nm, as shown in the transmission electron microscopy image. Figure 5 As shown.

[0093] The protein concentration of urinary epithelial stem cell exosomes was determined to be 13.69 μg / μL using the BCA protein quantification method.

[0094] Example 2: Effects of plant exosomes on stem cell exosome secretion

[0095] I. Influence of the number of exosomes on adipose-derived stem cells

[0096] Human adipose-derived stem cells were suspended in DMEM medium containing 10% fetal bovine serum (FBS), 100 µg / mL penicillin, and 100 µg / mL streptomycin. They were then seeded into flasks and cultured in an incubator at 37°C with 5% CO2.

[0097] Low, medium, and high concentration treatment groups of turmeric exosomes were set up, and 10 μL, 30 μL, and 50 μL of turmeric exosomes were added to the culture medium.

[0098] Low, medium, and high concentration treatment groups of olive leaf exosomes were set up, with 10 μL, 30 μL, and 50 μL of turmeric exosomes added to the culture medium.

[0099] When cells reached 80% or higher confluence, they were washed with PBS, and the culture medium was subsequently replaced with DMEM containing 100 µg / mL penicillin and 100 µg / mL streptomycin. Human adipose-derived stem cells from the untreated control group and the low, medium, and high concentrations of plant exosomes treated groups were then cultured for 24 to 72 hours. Culture supernatants from each group were collected. After supernatant collection, particle size and concentration were measured by nanoparticle tracking analysis (NTA).

[0100] The results are as follows Figure 6 As shown, compared with the untreated control, the number of exosome particles in the supernatant collected in the turmeric exosome treatment group increased by 32% in the low-concentration treatment group, 51% in the medium-concentration treatment group, and 105% in the high-concentration treatment group. However, the number of exosome particles in the olive leaf exosome treatment group did not increase significantly.

[0101] II. Investigating the effect of CD81, a marker on the surface of adipose-derived stem cell exosomes.

[0102] Since CD81 is a representative positive marker for ectosomes, CD81 levels are linearly proportional to ectosome levels. Increased CD81 levels indicate a proportional increase in exosome content. Here, we analyzed the exosome content (i.e., exosome secretion) in supernatants collected from the untreated control group and the low, medium, and high concentrations of plant exosomes treated groups. For this purpose, an exosome-human CD81 flow cytometry assay was used. Each supernatant was mixed with the exosome-human CD81 flow cytometry assay overnight, and then each mixture was reacted with PE mouse anti-human CD81 for 1 hour. After the reaction, the mean fluorescence intensity (MFI) of the samples was measured by flow cytometry.

[0103] Simultaneously, linear regression analysis was performed using the average fluorescence intensity of PE to calculate CD81 content. Specifically, linear regression analysis was performed using the CD81 protein concentrations at consecutive dilutions and their corresponding MFI values. Standard quantitative analysis plots generated from the linear regression analysis were used to determine the exosome content (CD81 content) in each supernatant. Results are as follows: Figure 7 As shown, the comparison of CD81 content determined that when human adipose-derived stem cells were cultured in a medium containing curcumin exosomes, the exosome content (CD81 content) in the supernatant was increased compared with the untreated control.

[0104] This confirms that when human adipose-derived stem cells are cultured in a medium containing curcumin exosomes, the secretion of exosomes by adipose-derived stem cells increases.

[0105] III. Effects of the number and secretion of exosomes from urinary epithelial stem cells

[0106] Following the above method, the effects of plant exosomes on urinary epithelial stem cell exosomes were investigated, and the results are as follows: Figure 8 As shown, the number of exosome particles in the supernatant collected from the olive leaf exosome treatment group increased by 41% in the low concentration treatment group, 77% in the medium concentration treatment group, and 136% in the high concentration treatment group. However, the number of exosome particles from urinary epithelial stem cells did not increase significantly in the turmeric exosome treatment group.

[0107] Following the above method, the exosome content (i.e., exosome secretion) in the supernatants from the untreated control group and the low, medium, and high concentration plant exosome treatment groups was examined. An exosome-human CD81 flow cytometry assay was used. Each supernatant was mixed with the exosome-human CD81 flow cytometry assay overnight, and then each mixture was reacted with PE mouse anti-human CD81 for 1 hour. After the reaction, the mean fluorescence intensity (MFI) of the samples was measured by flow cytometry. The results are as follows: Figure 9 As shown, the comparison of CD81 content determined that when urinary epithelial stem cells were cultured in a medium containing olive leaf exosomes, the exosome content (CD81 content) in the supernatant was increased compared with the untreated control.

[0108] This confirms that when urinary epithelial stem cells are cultured in a medium containing olive leaf exosomes, the secretion of urinary epithelial stem cell oosomes increases.

[0109] Example 3: Effect of plant exosome-treated and cultured exosome composition on the proliferation of human dermal papilla cells.

[0110] Rat dermal papilla cells (generations 3-5) were seeded into 96-well plates (5000 cells / well) and incubated overnight at 37°C with 5% CO2 to allow adhesion. The culture medium was discarded, and the cells were washed three times with PBS. Then, exosome compositions from different experimental groups were added and incubated for 24 hours. Before detection, CCK8 reagent was added to DMEM basal medium at a 1:10 ratio, mixed well, and then added to each well. The cells were incubated at 37°C for 1 hour. The absorbance at OD450 nm after 24 hours was measured using a microplate reader to observe dermal papilla cell proliferation.

[0111] The exosome composition for experimental group 1 was obtained by combining adipose-derived stem cell exosomes secreted by cultured adipose-derived stem cells treated with turmeric exosomes, urinary epithelial stem cell exosomes secreted by cultured urinary epithelial stem cells treated with olive leaf exosomes, and Platycladus orientalis leaf exosomes in a weight ratio of 1:2:2. The turmeric exosomes and olive leaf exosomes were cultured according to Example 2, with 50 μL of turmeric exosomes and 50 μL of olive leaf exosomes added.

[0112] The exosome composition of experimental group 2 was obtained by combining adipose stem cell exosomes, urinary epithelial stem cell exosomes and Platycladus orientalis leaf exosomes in a weight ratio of 1:2:2.

[0113] Experimental Group 3 Exosome Composition: Adipose stem cell exosomes secreted by cultured adipose stem cells treated with turmeric exosomes, urinary epithelial stem cell exosomes, and Platycladus orientalis leaf exosomes were combined in a weight ratio of 1:2:2; wherein, the turmeric exosome treatment and culture process was as described in Example 2, and the amount of turmeric exosomes added was 50 μL.

[0114] Experimental Group 4 Exosome Composition: Adipose-derived stem cell exosomes, urinary epithelial stem cell exosomes secreted by urinary epithelial stem cells treated with olive leaf exosomes and arborvitae leaf exosomes were combined in a weight ratio of 1:2:2; wherein, the olive leaf exosome treatment and culture process was as described in Example 2, and the amount of olive leaf exosomes added was 50 μL.

[0115] The exosome composition of experimental group 5 was obtained by combining adipose stem cell exosomes, urinary epithelial stem cell exosomes, Platycladus orientalis leaf exosomes, turmeric exosomes and olive leaf exosomes, wherein the weight ratio of adipose stem cell exosomes, urinary epithelial stem cell exosomes and Platycladus orientalis leaf exosomes was 1:2:2; the amount of turmeric exosomes added to the exosome composition in this embodiment was 50 μL, and the amount of olive leaf exosomes added to the exosome composition in this embodiment was 50 μL.

[0116] Control group: No exosome composition added.

[0117] Table 1

[0118] As shown in Table 1, animal stem cells cultured after treatment with plant exosomes secreted a greater effective amount of exosomes, which significantly promoted the proliferation of human dermal papilla cells. For experimental group 5, the exosome composition obtained by directly mixing plant exosomes and animal stem cell exosomes was easily affected by different animal stem cells. The secretion-promoting effect of plant exosomes was not significant, and the animal stem cell exosomes were easily influenced by plant exosomes, resulting in a poor effect on promoting the proliferation of human dermal papilla cells.

[0119] Example 4: Effect of exosome composition ratio on human dermal papilla cell proliferation

[0120] The experimental procedure was the same as in Example 4, except that the exosome composition used was set as follows:

[0121] Experimental Group 1: Adipose-derived stem cell exosomes secreted by cultured adipose-derived stem cells treated with turmeric exosomes, urinary epithelial stem cell exosomes secreted by cultured urinary epithelial stem cells treated with olive leaf exosomes, and Platycladus orientalis leaf exosomes were combined in a weight ratio of 1:2:2. The turmeric exosomes and olive leaf exosomes were cultured according to Example 2, with 50 μL of turmeric exosomes and 50 μL of olive leaf exosomes added.

[0122] Experimental Group 2: Adipose-derived stem cell exosomes secreted by cultured adipose-derived stem cells treated with turmeric exosomes, urinary epithelial stem cell exosomes secreted by cultured urinary epithelial stem cells treated with olive leaf exosomes, and Platycladus orientalis leaf exosomes were combined in a weight ratio of 1:2:3, with the rest being the same as Experimental Group 1.

[0123] Experimental Group 3: Adipose-derived stem cell exosomes secreted by cultured adipose-derived stem cells treated with turmeric exosomes, urinary epithelial stem cell exosomes secreted by cultured urinary epithelial stem cells treated with olive leaf exosomes, and Platycladus orientalis leaf exosomes were combined in a weight ratio of 2:4:3, with the rest being the same as Experimental Group 1.

[0124] Experimental Group 4: Adipose-derived stem cell exosomes secreted by cultured adipose-derived stem cells treated with turmeric exosomes, urinary epithelial stem cell exosomes secreted by cultured urinary epithelial stem cells treated with olive leaf exosomes, and Platycladus orientalis leaf exosomes were combined in a weight ratio of 1:5:3, with the rest being the same as Experimental Group 1.

[0125] Experimental Group 5: Adipose-derived stem cell exosomes secreted by cultured adipose-derived stem cells treated with turmeric exosomes, urinary epithelial stem cell exosomes secreted by cultured urinary epithelial stem cells treated with olive leaf exosomes, and Platycladus orientalis leaf exosomes were combined in a weight ratio of 2:2:3, with the rest being the same as Experimental Group 1.

[0126] Comparative Example 1: Adipose-derived stem cell exosomes secreted by cultured adipose-derived stem cells treated with turmeric exosomes, urinary epithelial stem cell exosomes secreted by cultured urinary epithelial stem cells treated with olive leaf exosomes, and Platycladus orientalis leaf exosomes were combined in a weight ratio of 1:1:1, with the rest being the same as Experimental Group 1.

[0127] Comparative Example 2: Adipose-derived stem cell exosomes secreted by cultured adipose-derived stem cells treated with turmeric exosomes, urinary epithelial stem cell exosomes secreted by cultured urinary epithelial stem cells treated with olive leaf exosomes, and Platycladus orientalis leaf exosomes were combined in a weight ratio of 1:2:5, with the rest being the same as Experimental Group 1.

[0128] Comparative Example 3: Adipose-derived stem cell exosomes secreted by cultured adipose-derived stem cells treated with turmeric exosomes, urinary epithelial stem cell exosomes secreted by cultured urinary epithelial stem cells treated with olive leaf exosomes, and Platycladus orientalis leaf exosomes were combined in a weight ratio of 1:1:2, with the rest being the same as Experimental Group 1.

[0129] Comparative Example 4: Adipose-derived stem cell exosomes secreted by cultured adipose-derived stem cells treated with turmeric exosomes, urinary epithelial stem cell exosomes secreted by cultured urinary epithelial stem cells treated with olive leaf exosomes, and Platycladus orientalis leaf exosomes were combined in a weight ratio of 1:6:2, with the rest being the same as experimental group 1.

[0130] Comparative Example 5: Adipose-derived stem cell exosomes secreted by cultured adipose-derived stem cells treated with turmeric exosomes, urinary epithelial stem cell exosomes secreted by cultured urinary epithelial stem cells treated with olive leaf exosomes, and Platycladus orientalis leaf exosomes were combined in a weight ratio of 3:2:2, with the rest being the same as Experimental Group 1.

[0131] Table 2

[0132] As can be seen from the results in Table 2, the ratio of exosomes in the exosome composition has a significant effect on the proliferation of human dermal papilla cells. In this invention, the weight ratio of the three exosomes is in the range of 1-2:2-5:2-3, which has a significant promoting effect on the proliferation of human dermal papilla cells.

[0133] Example 5: Hair Loss Prevention and Strengthening Experiment

[0134] I. Anti-hair loss experiment

[0135] In Example 4, the exosome compositions of all experimental groups and comparative examples were prepared into drug solutions, and volunteers were recruited to conduct anti-hair loss experiments. The drug solution preparation method was as follows: (1) Deionized water, 1,3-propanediol, butanediol and phenoxyethanol were put into a stirring vessel, heated to 60°C, stirred at 120 r / min for 10 min, and mixed evenly to obtain a premix. (2) The premix was cooled to 35°C, and the exosome compositions were added separately and stirred evenly to obtain the drug solution.

[0136] Several male volunteers aged 30-40 years old with some degree of hair loss were recruited. Volunteers were randomly divided into groups of 10. Each group used a test shampoo containing the same solution used in the examples and comparative studies. Each time, 5 mL of the test shampoo was applied evenly to the hair, massaged for 3 minutes, and then rinsed thoroughly. No other hair care products were used during the experiment. All wastewater was collected after each shampoo, and the shed hairs were filtered out. After drying, the amount of hair lost during the shampooing process was calculated, and the average value was taken. Volunteers washed their hair every two days, and the amount of hair lost on the 1st, 7th, and 15th shampoo sessions was recorded.

[0137] Preparation of the shampoo to be tested: Sodium lauryl ether sulfate, sodium lauroyl amphoteric acid, sodium chloride, cocoamide MEA, disodium EDTA, the drug solution, and deionized water were mixed evenly to obtain the shampoo to be tested.

[0138] Table 3

[0139] According to the data comparison in Table 3, the combination of adipose stem cell exosomes, urinary epithelial stem cell exosomes and Platycladus orientalis exosomes in each experimental group can better improve hair follicle activity, and the anti-hair loss effect is faster and better. The comparative ratio destroys the synergistic effect of adipose stem cell exosomes, urinary epithelial stem cell exosomes and Platycladus orientalis exosomes, resulting in a decrease in the anti-hair loss effect.

[0140] II. Hair Loss Experiment

[0141] According to the Hamilton Depression Rating Scale, 100 male subjects who met the Hamilton Depression Rating Scale II-IV were selected and divided into two groups. One group used the drug solution containing the exosome composition of Experimental Group 3 prepared above in this invention, and the other group used minoxidil tincture. There were no significant differences in age and Hamilton Depression Rating Scale between the two groups.

[0142] The method of use is as follows: After washing and drying hair, dilute the medication with water 5 times, then spray 1.2ml of the diluted medication evenly onto the balding areas of the scalp. After spraying, gently massage the scalp with your fingertips for 5 minutes, twice a day, morning and evening. The method of use for minoxidil solution is as follows: After washing and drying hair, apply 1mL of minoxidil solution to the balding areas of the scalp. After application, gently massage the scalp with your fingertips for 5 minutes, twice a day, morning and evening.

[0143] Efficacy criteria: Cured: Hair growth in the bald areas returns to normal, and the density and thickness of the hair are basically the same as before the hair loss; Significantly effective: Hair increases significantly, with density increasing by more than 50%, or hair diameter increasing by more than 50%, and a large number of new terminal hairs growing; Effective: Hair loss is controlled to a certain extent, the thinning of hair is improved, density increases by 15%-50%, or hair diameter increases by 15%-50%, and a certain number of new vellus hairs or a small number of terminal hairs grow; Ineffective: Hair loss is not significantly improved, hair density does not increase significantly, large amounts of hair loss continue, or only a small number of vellus hairs grow and fall out quickly, and the overall appearance is basically the same as before treatment.

[0144] After two months of use, the efficacy of each group was statistically analyzed, as shown in Table 4. Figure 10 Microscopic comparison images of effective subjects before and after 2 months of use are also provided. Table 4 and Figure 10 As can be seen, the exosome composition of the present invention has a better anti-hair loss effect than minoxidil.

[0145] Table 4

Claims

1. Application of turmeric exosomes in promoting exosome secretion from adipose stem cells.

2. The application according to claim 1, characterized in that, The preparation of the turmeric exosomes includes the following steps: (1) Take a fresh sample of turmeric root, crush it, and filter it coarsely through gauze; (2) Centrifuge at 3000-5000 r / min for 30-50 min; take the supernatant and discard the precipitate; (3) Centrifuge at 10000-12000 r / min for 30-50 min; take the supernatant and discard the precipitate; (4) Centrifuge at 10000-12000 r / min for 30-50 min, take the supernatant and discard the precipitate; repeat this step 2-3 times until there is no obvious precipitate. (5) The microporous membrane is used for filtration in sequence to obtain the supernatant; (6) Add the filtered supernatant to the exosome extraction kit and extract turmeric exosomes.

3. Application of olive leaf exosomes in promoting the secretion of exosomes from urinary epithelial stem cells.

4. The application according to claim 3, characterized in that, The preparation of the olive leaf exosomes includes the following steps: (1) Wash the olive leaves and juice them to obtain liquid juice; (2) Filter the liquid juice and centrifuge the filtered juice at 1000-2000×g for 10-15 min at low temperature; (3) The first filtrate is obtained by filtration through a microporous membrane; (4) Centrifuge the first filtrate at 1000-2000×g for 10-15min and filter it through a microporous membrane to obtain the second filtrate; (5) Centrifuge the second filtrate at 10000-12000×g for 10-15 min for the first time, and at 20000×g for the second time for 10-15 min, and then recover the supernatant; (6) Centrifuge the supernatant at 100,000 × g for 70-80 min at low temperature to obtain a precipitate. Resuspend the obtained precipitate in PBS to obtain olive leaf exosomes.

5. An exosome composition for preventing hair loss and strengthening hair, characterized in that, The adipose stem cell exosomes secreted by adipose stem cells cultured and treated with turmeric exosomes, the urinary epithelial stem cell exosomes secreted by urinary epithelial stem cells cultured and treated with olive leaf exosomes, and the arborvitae leaf exosomes were mixed in a weight ratio of 1-2:2-5:2-3.

6. The exosome composition for preventing hair loss and strengthening hair as described in claim 5, characterized in that, The adipose-derived stem cell exosomes secreted by adipose-derived stem cells treated with turmeric exosomes are obtained by adding turmeric exosomes to adipose-derived stem cells, culturing for 24-72 hours, and then collecting the supernatant for separation. The urinary epithelial stem cell exosomes secreted by urinary epithelial stem cells treated with olive leaf exosomes are obtained by adding olive leaf exosomes to urinary epithelial stem cell culture medium, culturing for 24-72 hours, and then collecting the supernatant for separation.

7. The use of the exosome composition of claim 5 in promoting the proliferation of dermal papilla cells.

8. The use of the exosome composition of claim 5 in the preparation of hair loss prevention and hair strengthening products.

9. A hair loss prevention and hair strengthening product, characterized in that, The composition contains the exosome composition of claim 5.

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