Preparation method of active peptide and stem cell exosome mixed hair restorer

Abstract

The invention provides a preparation method of a mixed pilatory of active peptide and stem cell exosome, and belongs to the technical field of pilatory preparation. The preparation method comprises the following steps: extracting the stem cell exosome; extracting an active extract; preparing an exosome entrapped active extract; preparing the liposome loaded active peptide; preparing the liposome loaded active peptide modified by the gallic acid modified chitosan; and preparing the mixed hair restorer. The prepared mixed hair restorer can provide a suitable growth environment for hair follicles and promote hair growth, the synergistic effect of the active peptide and the exosome can provide abundant signal molecules and enhance the stimulation effect on hair follicle cells, and the active peptide and the exosome can promote angiogenesis and increase the blood flow volume of scalp, so that the hair follicle can be effectively promoted. More nutrition and oxygen are provided for hair follicles, and the growth of hair is effectively promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of hair growth agent preparation, and in particular to a method for preparing a mixed hair growth agent of active peptides and stem cell exosomes. Background Art

[0002] Hair loss has always been a topic of widespread concern around the world, affecting the quality of life of millions or even tens of millions of people. Hair loss not only affects the appearance, but may also bring psychological burden and social adaptation problems to patients. Currently, the solutions to hair loss problems on the market mainly include drug treatment, hair transplant surgery, and various hair growth agents. However, existing hair growth agents still have many shortcomings in terms of effectiveness and safety.

[0003] The effects of most hair growth agents are not significant enough, and long-term use is required to see the effects. The effects are also unstable, and some hair growth agents contain highly irritating chemical ingredients that may cause scalp allergies or other adverse reactions. Some hair growth agents have complex ingredients, and it is difficult to ensure that all ingredients are safe and harmless to the human body. At the same time, existing hair growth agents often lack in-depth research on the growth mechanism of hair follicle cells and cannot accurately promote hair growth. Moreover, the bioavailability of single-ingredient hair growth agents is low, and they cannot fully exert their due efficacy, resulting in poor hair growth effects. Summary of the invention

[0004] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for preparing a mixed hair growth agent of active peptides and stem cell exosomes.

[0005] A method for preparing a mixed hair growth agent of active peptides and stem cell exosomes comprises the following steps:

[0006] S1: Extraction of stem cell exosomes

[0007] Stem cell exosomes were extracted from adipose tissue near the groin and testicles of rats;

[0008] S2: Extraction of active extracts

[0009] The saw palmetto medicinal materials, ginger and Platycladus orientalis leaves were subjected to ethanol hot extraction respectively, and the extracts were mixed to obtain the active extract;

[0010] S3: Preparation of exosomes encapsulating active extracts

[0011] The active extract is introduced into exosomes by ultrasonic treatment to prepare exosome-encapsulated active extract;

[0012] S4: Preparation of liposome-loaded active peptides

[0013] Myristoyl pentapeptide-4, copper peptide and acetyl tetrapeptide-3 were loaded by soybean lecithin and cholesterol to prepare liposome loaded active peptides;

[0014] S5: Preparation of gallic acid-modified chitosan-modified liposomes loaded with active peptides

[0015] After chitosan is modified by gallic acid, the liposome loaded with active peptide is modified to obtain gallic acid-modified chitosan and then modified liposome loaded with active peptide;

[0016] S6: Preparation of mixed hair growth agent

[0017] The aldehyde-modified hyaluronic acid solution, the silk fibroin solution, starch, the exosome-encapsulated active extract and the gallic acid-modified chitosan-modified liposome-loaded active peptide are mixed to prepare a mixed hair growth agent.

[0018] Furthermore, step S1 of extracting stem cell exosomes specifically comprises the following steps:

[0019] S1.1: Use surgical scissors to aseptically cut the adipose tissue near the groin and testicles of the rat, wash the tissue surface with pre-cooled sterile PBS buffer, cut 10-20 parts by weight of the processed adipose tissue into pieces and transfer them into a centrifuge tube, add 10-20 parts by weight of 0.1-0.2wt% type I collagenase, and digest in a water bath at 37-38°C for 50-60 minutes. After the digestion is completed, add 1-2 parts by weight of DMEM / F12 culture medium and fetal bovine serum to terminate the digestion, and obtain the digested adipose tissue;

[0020] S1.2: The digested adipose tissue was centrifuged at 1100-1200 r / min for 10-12 min, the solution was resuspended and passed through a 200-300 mesh cell filter into a new centrifuge tube, and centrifuged again at 1100-1200 r / min for 10-12 min. The centrifuged cells were resuspended in DMEM / F12 medium and then transferred to a 6-8 cm culture dish for culture. After culturing for 24-26 hours, the medium was half changed, and then the medium was changed every 2-3 days. When the cells grew to a density of 80-85%, they were digested and subcultured with 0.25-0.3wt% trypsin to obtain adipose mesenchymal stem cells;

[0021] S1.3: The 3rd-4th generation adipose-derived mesenchymal stem cells were expanded and cultured. When the cell density reached 80-85%, the culture medium was removed and washed with PBS buffer, and serum-free culture medium was added. After culturing for 48-50 hours, the cell supernatant was collected;

[0022] S1.4: Centrifuge the cell supernatant at 4-5°C, 1500-2000r / min for 10-12min, collect the supernatant and pass it through a 0.22-0.24μm filter membrane, transfer the filtered cell supernatant into an ultracentrifuge tube, centrifuge at 4-5°C, 10000-20000r / min for 70-80min, remove the supernatant, and obtain stem cell exosomes.

[0023] Furthermore, step S2 of extracting the active extract specifically comprises the following steps:

[0024] S2.1: extracting 100-200 parts by weight of saw palmetto medicinal material with 300-500 parts by weight of 85-90% ethanol by hot soaking for 2-3 times, each time for 2-3 hours, combining the extracts after hot soaking, filtering, recovering ethanol from the filtrate, and concentrating the filtrate to 1 / 10-1 / 8 of the original volume to obtain a saw palmetto extract;

[0025] S2.2: extracting 100-200 parts by weight of ginger with 300-500 parts by weight of 85-90% ethanol by hot soaking for 2-3 times, each time for 2-3 hours, combining the extracts after hot soaking, filtering, recovering ethanol from the filtrate, and concentrating the filtrate to 1 / 10-1 / 8 of the original volume to obtain a ginger extract;

[0026] S2.3: 100-200 parts by weight of Platycladus orientalis leaves are hot-extracted with 300-500 parts by weight of 85-90% ethanol for 2-3 times, each time for 2-3 hours, the extracts after hot-extraction are combined and filtered, ethanol is recovered from the filtrate, and the filtrate is concentrated to 1 / 10-1 / 8 of the original volume to obtain a Platycladus orientalis leaf extract;

[0027] S2.4: 2-3 parts by weight of saw palmetto extract, 3-5 parts by weight of Platycladus orientalis leaf extract and 5-8 parts by weight of ginger extract are mixed and stirred to obtain an active extract.

[0028] Furthermore, step S3 of preparing the exosome-encapsulated active extract specifically comprises the following steps:

[0029] S3.1: Prepare 20-30 parts by weight of the active extract into a 1-2 mg / mL active extract standard solution with PBS buffer, add stem cell exosomes into PBS buffer to prepare a 4-5 mg / mL exosome solution for later use;

[0030] S3.2: 20-30 parts by weight of the active extract standard solution and 20-30 parts by weight of the live exosome solution are mixed and then subjected to ultrasonic treatment in an ultrasonic cell disruptor at 4-5°C, with an amplitude of 20-30%, 6-8 cycles, 4-5s on, 2-3s off, and the mixed solution is cooled on ice for 2-3 minutes between each cycle to obtain a treated mixed solution;

[0031] S3.3: Transfer the treated mixed solution to a LEP tube and incubate it in a 4-5℃ refrigerator for 3-5h. Then centrifuge it at 4-5℃ and 1500-2000r / min for 10-12min, and then centrifuge it at 4-5℃ and 10000-20000r / min for 70-80min to obtain exosome-encapsulated active extract.

[0032] Furthermore, step S4, the preparation of liposome-loaded active peptides, specifically comprises the following steps:

[0033] S4.1: Add 2-3 parts by weight of myristoyl pentapeptide-4 to 3-5 parts by weight of propylene glycol, and dissolve at 45-50° C. to obtain solution I, mix 2-3 parts by weight of copper peptide, 2-3 parts by weight of acetyl tetrapeptide-3, and 5-8 parts by weight of deionized water, and dissolve at 45-50° C. to obtain solution II, and stir and mix solution I and solution II to obtain an active peptide mixed solution;

[0034] S4.2: 3-5 parts by weight of soybean lecithin and 2-3 parts by weight of cholesterol are added to 40-50 parts by weight of anhydrous ethanol to dissolve, and then rotary evaporated under reduced pressure at 40-45°C to obtain a film, and the film is placed in an active peptide mixture and hydrated at 37-38°C for 1-2 hours to obtain a hydrated solution;

[0035] S4.3: The hydrated solution was sonicated in an ice bath using an ultrasonic cell crusher for 5-8 minutes, with an ultrasonic power of 300-350 W, a working time of 3-5 s, and an interval time of 2-3 s. After the sonication, a membrane extruder was used to pass the sonicated solution through polycarbonate membranes with pore sizes of 0.45 μm and 0.22 μm in turn to obtain liposome-loaded active peptides.

[0036] Furthermore, step S5 of preparing gallic acid-modified chitosan and then modified liposomes loaded with active peptides specifically comprises the following steps:

[0037] S5.1: 0.5-0.6 parts by weight of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride and 0.3-0.4 parts by weight of N-hydroxysuccinimide are dissolved in a 1:1 mixture of 50-60 parts by weight of ethanol and water to obtain a mixed solution;

[0038] S5.2: dehydrogenating 10-20 parts by weight of a 1.5-2 wt% chitosan aqueous solution under a nitrogen atmosphere at 55-60° C. for 10-12 min, then adding 0.12-0.15 parts by weight of gallic acid to obtain a mixed solution, adding the mixed solution to the mixed solution, reacting for 12-14 h while keeping the pH constant, and drying after the reaction to obtain gallic acid-modified chitosan;

[0039] S5.3: Prepare 1-2% glacial acetic acid solution with deionized water, then add 0.1-0.2 weight parts of gallic acid modified chitosan, stir to dissolve, and adjust to pH 6-6.5, then slowly add liposome-loaded active peptide in a volume ratio of 1:1, stir at a speed of 700-800r / min for 1-2h to obtain gallic acid modified chitosan and then modified liposome-loaded active peptide.

[0040] Furthermore, the preparation of the mixed hair growth agent in step S6 specifically includes the following steps:

[0041] S6.1: Under light-proof conditions, 1-2 parts by weight of hyaluronic acid is dissolved in 120-130 parts by weight of deionized water, stirred evenly, and then 1-2 parts by weight of sodium periodate is added, reacted for 4-5 hours, then 3-4 parts by weight of ethylene glycol is added, stirred for 2-3 hours, dialyzed in deionized water for 4-5 days, dried to obtain aldehyde-modified hyaluronic acid, and deionized water is used to prepare a 4% aldehyde-modified hyaluronic acid solution;

[0042] S6.2: Add 10-12 parts by weight of anhydrous calcium chloride to 20-30 parts by weight of distilled water, stir evenly, then add 5-8 parts by weight of starch, stir at 70-80° C. for 1-2 hours, then add 5-8 parts by weight of 7% silk fibroin solution, stir and mix to obtain a mixed solution;

[0043] S6.3: Add 2-3 parts by weight of exosome-encapsulated active extract and 3-5 parts by weight of gallic acid-modified chitosan-modified liposome-loaded active peptide to 10-12 parts by weight of aldehyded hyaluronic acid solution, mix evenly with ultrasound, and then oscillate and mix with the mixed solution at a volume ratio of 1:1. After mixing evenly, a mixed hair growth agent is obtained.

[0044] Furthermore, the PBS buffer is 1×PBS buffer.

[0045] Furthermore, in step S1.1, the amount of fetal bovine serum added is 10-12% of the mass of the DMEM / F12 culture medium.

[0046] Furthermore, the starch in step S6.2 is high-amylose corn starch G70.

[0047] Compared with the prior art, the present invention has at least the following beneficial effects:

[0048] 1. The present invention encapsulates the active extract through exosomes. The encapsulation of exosomes can provide a relatively stable microenvironment for the active extract, so that the active extract maintains its chemical stability during the storage and use of the hair growth agent. Exosomes have a natural homing ability and can more accurately transport the encapsulated active extract to target sites such as hair follicle cells, thereby increasing the concentration of active ingredients in hair growth-related cells, thereby enhancing the hair growth effect. In addition, the membrane structure of exosomes helps to fuse with the cell membrane of hair follicle cells, promotes the cell's uptake of the active extract, makes it easier for the active ingredients to enter the cells to exert their effects, and further improves the hair growth effect.

[0049] 2. The present invention loads the active peptides on liposomes. The liposomes have a phospholipid bilayer structure similar to the skin cell membrane and can fuse well with the skin. The active peptides loaded on the liposomes can make the active peptides reach the hair follicle cells more effectively and play their role in promoting hair growth. The liposomes can achieve a sustained and slow release of the active peptides and maintain the effective concentration of the active peptides in the hair follicles for a long time, thereby continuously stimulating the growth and development of the hair follicle cells. In addition, the liposomes have good biocompatibility and will not cause obvious allergic reactions. The safety of the active peptides can be improved, making it easier for patients to accept treatment.

[0050] 3. The present invention modifies liposomes by modifying chitosan with gallic acid. The surface modification of chitosan with gallic acid improves the stability of liposomes. Chitosan modified with gallic acid can reduce the oxidative damage of free radicals to hair follicle cells. Chitosan modified with gallic acid can interact with receptors on the surface of hair follicle cells and activate signal pathways in cells, thereby promoting the proliferation of hair follicle cells and effectively promoting hair growth.

[0051] 4. The mixed hair growth agent prepared by the present invention can provide a suitable growth environment for hair follicles and promote hair growth. Aldehydated hyaluronic acid has better biocompatibility and tissue adhesion, which helps the active ingredients in the mixed hair growth agent to adhere to and continue to act on the scalp, while inhibiting the apoptosis of hair follicle cells and maintaining the number of hair follicle cells. Starch can play a thickening role, which helps the hair growth agent to adhere to and stay on the scalp. Silk fibroin is a natural protein with good biocompatibility and certain nutritional effects. It may provide nutritional support for hair follicle cells and promote cell metabolism, thereby helping hair growth. Active peptides and exosomes can provide rich signal molecules to enhance the stimulation of hair follicle cells. Active peptides and exosomes can promote angiogenesis, increase blood flow to the scalp, provide more nutrition and oxygen to hair follicles, and effectively promote hair growth.

[0052] 5. The present invention prepares the active extract by mixing saw palmetto extract, Platycladus orientalis leaf extract and ginger extract. The ginger extract contains biologically active ingredients such as gingerol, has a certain irritation, can promote blood circulation of the scalp, and enhance the vitality of hair follicles. The ginger extract has a certain improvement effect on alopecia areata and other types of hair loss; Platycladus orientalis leaves contain volatile oils, flavonoids and other ingredients, have the effects of cooling blood, stopping bleeding, and promoting hair growth, can improve the living environment of hair follicles by regulating the oil secretion of the scalp, and can also promote the metabolism of hair follicle cells, which is conducive to the growth of hair. Platycladus orientalis leaf extract has a certain improvement effect on alopecia areata and other types of hair loss. It has a certain therapeutic effect on seborrheic alopecia; saw palmetto extract can inhibit the activity of 5-α reductase and reduce the production of dihydrotestosterone. By reducing the production of dihydrotestosterone, it can reduce its damage to hair follicles, thereby protecting hair follicles and promoting hair growth. Saw palmetto extract has a certain therapeutic effect on androgenic alopecia. Therefore, after the mixed active extracts are encapsulated into hair follicle cells and other target sites through exosomes, they can interact with each other, protect hair follicles and promote hair growth from multiple angles, so that the prepared hair growth agent can be used for a variety of hair loss problems, which is more economical. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The accompanying drawings, which are incorporated herein and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, further serve to explain the principles of the invention and to enable those skilled in the relevant art to make and use the invention.

[0054] Figure 1 This is a flow chart of a method for preparing a mixed hair growth agent of active peptides and stem cell exosomes used in an embodiment of the present invention. DETAILED DESCRIPTION

[0055] The following is a detailed description of the preparation method of a mixed hair growth agent of active peptides and stem cell exosomes provided by the present invention in combination with the accompanying drawings and specific examples. At the same time, it is explained here that in order to make the embodiments more detailed, the following embodiments are the best and preferred embodiments, and those skilled in the art may also adopt other alternative methods for some known technologies; and the accompanying drawings are only for more specific description of the embodiments, and are not intended to specifically limit the present invention.

[0056] Example 1

[0057] A method for preparing a mixed hair growth agent of active peptides and stem cell exosomes, such as Figure 1 As shown, the following steps are included:

[0058] S1: Extraction of stem cell exosomes

[0059] S1.1: Use surgical scissors to aseptically cut the adipose tissue near the groin and testicles of the rat, wash the tissue surface with pre-cooled sterile 1× PBS buffer, cut 10 parts by weight of the processed adipose tissue into pieces and transfer them into a centrifuge tube, add 10 parts by weight of 0.1wt% type I collagenase, and digest in a water bath at 37°C for 50 minutes. After the digestion is completed, add 1 part by weight of DMEM / F12 culture medium and 10% fetal bovine serum to terminate the digestion, and obtain the digested adipose tissue;

[0060] S1.2: The digested adipose tissue was centrifuged at 1100 r / min for 10 min, the solution was resuspended and passed through a 200-mesh cell filter into a new centrifuge tube, and centrifuged again at 1100 r / min for 10 min. The centrifuged cells were resuspended in DMEM / F12 medium and then transferred to a 6 cm culture dish for culture. After 24 h of culture, the medium was half changed, and then the medium was changed every 2 days. When the cells grew to 80% density, they were digested and subcultured with 0.25wt% trypsin to obtain adipose mesenchymal stem cells;

[0061] S1.3: The third generation of adipose-derived mesenchymal stem cells were expanded and cultured. When the cell density reached 80%, the culture medium was removed and washed with 1× PBS buffer, and serum-free culture medium was added. After culturing for 48 hours, the cell supernatant was collected;

[0062] S1.4: The cell supernatant was centrifuged at 4°C, 1500 r / min for 10 min, the supernatant was collected and filtered through a 0.22 μm filter membrane, the filtered cell supernatant was transferred into an ultracentrifuge tube, and centrifuged at 4°C, 10000 r / min for 70 min, the supernatant was removed, and stem cell exosomes were obtained;

[0063] S2: Extraction of active extracts

[0064] S2.1: 100 parts by weight of saw palmetto medicinal material is hot-extracted with 300 parts by weight of 85% ethanol twice, each time for 2 hours, the extracts after hot-extraction are combined and filtered, ethanol is recovered from the filtrate, and the filtrate is concentrated to 1 / 10 of the original volume to obtain a saw palmetto extract;

[0065] S2.2: 100 parts by weight of ginger is hot-extracted with 300 parts by weight of 85% ethanol twice, each time for 2 hours, the extracts after hot-extraction are combined and filtered, the filtrate is used to recover ethanol, and the filtrate is concentrated to 1 / 10 of the original volume to obtain ginger extract;

[0066] S2.3: 100 parts by weight of Platycladus orientalis leaves are hot-extracted with 300 parts by weight of 85% ethanol twice, each time for 2 hours, the extracts after hot-extraction are combined and filtered, ethanol is recovered from the filtrate, and the filtrate is concentrated to 1 / 10 of the original volume to obtain a Platycladus orientalis leaf extract;

[0067] S2.4: 2 parts by weight of saw palmetto extract, 3 parts by weight of Platycladus orientalis leaf extract and 5 parts by weight of ginger extract are mixed and stirred to obtain an active extract;

[0068] S3: Preparation of exosomes encapsulating active extracts

[0069] S3.1: 30 parts by weight of the active extract is prepared into a 1 mg / mL active extract standard solution with 1×PBS buffer, and the stem cell exosomes are added into 1×PBS buffer to prepare a 4 mg / mL exosome solution for later use;

[0070] S3.2: 20 parts by weight of the active extract standard solution and 20 parts by weight of the live exosome solution were mixed and then subjected to ultrasonic treatment in an ultrasonic cell disruptor at 4°C, with an amplitude of 20%, 6 cycles, 4s on and 2s off, and the mixed solution was cooled on ice for 2 minutes between each cycle to obtain a treated mixed solution;

[0071] S3.3: The treated mixed solution was transferred to a LEP tube, incubated in a 4°C refrigerator for 3 h, then centrifuged at 4°C, 1500 rpm for 10 min, and then centrifuged at 4°C, 10000 rpm for 70 min to obtain the exosome-encapsulated active extract;

[0072] S4: Preparation of liposome-loaded active peptides

[0073] S4.1: Add 2 parts by weight of myristoyl pentapeptide-4 to 3 parts by weight of propylene glycol and dissolve at 45°C to obtain solution I, mix 2 parts by weight of copper peptide, 2 parts by weight of acetyl tetrapeptide-3 and 5 parts by weight of deionized water and dissolve at 45°C to obtain solution II, and stir and mix solution I and solution II to obtain an active peptide mixed solution;

[0074] S4.2: 3 parts by weight of soybean lecithin and 2 parts by weight of cholesterol are added to 40 parts by weight of anhydrous ethanol to dissolve, and then rotary evaporated under reduced pressure at 40°C to obtain a film, and the film is placed in an active peptide mixture and hydrated at 37°C for 1 hour to obtain a hydrated solution;

[0075] S4.3: The hydrated solution was sonicated for 5 min using an ultrasonic cell disruptor in an ice bath, with an ultrasonic power of 300 W, a working time of 3 s, and an interval time of 2 s. After the sonication, a membrane extruder was used to pass the sonicated solution through polycarbonate membranes with pore sizes of 0.45 μm and 0.22 μm in sequence to obtain liposome-loaded active peptides;

[0076] S5: Preparation of gallic acid-modified chitosan-modified liposomes loaded with active peptides

[0077] S5.1: 0.5 parts by weight of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride and 0.3 parts by weight of N-hydroxysuccinimide are dissolved in a 1:1 mixture of 50 parts by weight of ethanol and water to obtain a mixed solution;

[0078] S5.2: dehydrogenating 10 parts by weight of a 1.5 wt% chitosan aqueous solution at 55° C. in a nitrogen atmosphere for 10 min, then adding 0.12 parts by weight of gallic acid to obtain a mixed solution, adding the mixed solution to the mixed solution, reacting for 12 h while keeping the pH constant, and drying after the reaction to obtain gallic acid-modified chitosan;

[0079] S5.3: Prepare 1% glacial acetic acid solution with deionized water, then add 0.1 weight part of gallic acid modified chitosan, stir to dissolve, and adjust to pH 6, then slowly add liposome loaded active peptide in a volume ratio of 1:1, stir at a speed of 700r / min for 1h, to obtain gallic acid modified chitosan and then modified liposome loaded active peptide;

[0080] S6: Preparation of mixed hair growth agent

[0081] S6.1: Under light-proof conditions, 1 part by weight of hyaluronic acid was dissolved in 120 parts by weight of deionized water, and after stirring evenly, 2 parts by weight of sodium periodate was added, and the mixture was reacted for 4 hours, and then 3 parts by weight of ethylene glycol was added, and the mixture was stirred for 2 hours and dialyzed in deionized water for 4 days, and dried to obtain aldehyde-modified hyaluronic acid, and deionized water was used to prepare a 4% aldehyde-modified hyaluronic acid solution;

[0082] S6.2: 10 parts by weight of anhydrous calcium chloride is added to 20 parts by weight of distilled water, and after stirring evenly, 5 parts by weight of high amylose corn starch G70 is added, and the mixture is stirred at 70° C. for 1 hour, and then 5 parts by weight of 7% silk fibroin solution is added, and the mixture is stirred to obtain a mixed solution;

[0083] S6.3: Add 2 parts by weight of exosome-encapsulated active extract and 3 parts by weight of gallic acid-modified chitosan-modified liposome-loaded active peptide to 10 parts by weight of aldehyded hyaluronic acid solution, mix evenly with ultrasound, and then oscillate and mix with the mixed solution at a volume ratio of 1:1. After mixing evenly, a mixed hair growth agent is obtained.

[0084] Example 2

[0085] A method for preparing a mixed hair growth agent of active peptides and stem cell exosomes, such as Figure 1 As shown, the following steps are included:

[0086] S1: Extraction of stem cell exosomes

[0087] S1.1: Use surgical scissors to aseptically cut the adipose tissue near the groin and testicles of the rat, wash the tissue surface with pre-cooled sterile 1× PBS buffer, cut 10 parts by weight of the processed adipose tissue into pieces and transfer them into a centrifuge tube, add 10 parts by weight of 0.1wt% type I collagenase, and incubate in a water bath at 38°C for 60 minutes. After the digestion is completed, add 1 part by weight of DMEM / F12 culture medium and 10% fetal bovine serum to terminate the digestion, and obtain the digested adipose tissue;

[0088] S1.2: The digested adipose tissue was centrifuged at 1200 r / min for 12 min, the solution was resuspended and passed through a 300-mesh cell filter into a new centrifuge tube, and centrifuged again at 1200 r / min for 12 min. The centrifuged cells were resuspended in DMEM / F12 medium and transferred to an 8 cm culture dish for culture. After 26 h of culture, the medium was half changed, and then the medium was changed every 3 days. When the cells grew to 80% of the density, they were digested and subcultured with 0.25 wt% trypsin to obtain adipose-derived mesenchymal stem cells.

[0089] S1.3: The third generation of adipose-derived mesenchymal stem cells were expanded and cultured. When the cell density reached 80%, the culture medium was removed and washed with 1× PBS buffer, and serum-free culture medium was added. After culturing for 50 hours, the cell supernatant was collected;

[0090] S1.4: The cell supernatant was centrifuged at 5°C, 2000 r / min for 12 min, the supernatant was collected and filtered through a 0.24 μm filter membrane, the filtered cell supernatant was transferred into an ultracentrifuge tube, and centrifuged at 5°C, 20,000 r / min for 80 min, the supernatant was removed, and stem cell exosomes were obtained;

[0091] S2: Extraction of active extracts

[0092] S2.1: 100 parts by weight of saw palmetto medicinal material is hot-extracted with 300 parts by weight of 85% ethanol for 3 times, each time for 3 hours, the extracts after hot-extraction are combined and filtered, ethanol is recovered from the filtrate, and the filtrate is concentrated to 1 / 10 of the original volume to obtain a saw palmetto extract;

[0093] S2.2: 100 parts by weight of ginger is hot-extracted with 300 parts by weight of 85% ethanol for 3 times, each time for 3 hours, the extracts after hot-extraction are combined and filtered, the filtrate is recovered with ethanol, and the filtrate is concentrated to 1 / 10 of the original volume to obtain a ginger extract;

[0094] S2.3: 100 parts by weight of Platycladus orientalis leaves are hot-extracted with 300 parts by weight of -90% ethanol for 3 times, each time for 3 hours, the extracts after hot-extraction are combined and filtered, ethanol is recovered from the filtrate, and the filtrate is concentrated to 1 / 10 of the original volume to obtain Platycladus orientalis leaf extract;

[0095] S2.4: 2 parts by weight of saw palmetto extract, 3 parts by weight of Platycladus orientalis leaf extract and 5 parts by weight of ginger extract are mixed and stirred to obtain an active extract;

[0096] S3: Preparation of exosomes encapsulating active extracts

[0097] S3.1: 20 parts by weight of the active extract is prepared into a 1 mg / mL active extract standard solution with 1×PBS buffer, and the stem cell exosomes are added into 1×PBS buffer to prepare a 4 mg / mL exosome solution for later use;

[0098] S3.2: 20 parts by weight of the active extract standard solution and 20 parts by weight of the live exosome solution were mixed and then subjected to ultrasonic treatment in an ultrasonic cell disruptor at 5°C, with an amplitude of 30%, 8 cycles, 5s on and 3s off, and the mixed solution was cooled on ice for 3 minutes between each cycle to obtain a treated mixed solution;

[0099] S3.3: The treated mixed solution was transferred to a LEP tube, placed in a 5°C refrigerator for incubation for 5 h, then centrifuged at 5°C, 2000 r / min for 12 min, and then centrifuged at 5°C, 20000 r / min for 80 min to obtain the exosome-encapsulated active extract;

[0100] S4: Preparation of liposome-loaded active peptides

[0101] S4.1: Add 2 parts by weight of myristoyl pentapeptide-4 to 3 parts by weight of propylene glycol and dissolve at 50°C to obtain solution I, mix 2 parts by weight of copper peptide, 2 parts by weight of acetyl tetrapeptide-3 and 5 parts by weight of deionized water and dissolve at 50°C to obtain solution II, and stir and mix solution I and solution II to obtain an active peptide mixed solution;

[0102] S4.2: 3 parts by weight of soybean lecithin and 2 parts by weight of cholesterol are added to 40 parts by weight of anhydrous ethanol to dissolve, and then rotary evaporated under reduced pressure at 45°C to obtain a film, and the film is placed in an active peptide mixture and hydrated at 38°C for 2 hours to obtain a hydrated solution;

[0103] S4.3: The hydrated solution was sonicated for 8 min using an ultrasonic cell crusher in an ice bath, with an ultrasonic power of 350 W, a working time of 5 s, and an interval time of 3 s. After the sonication, a membrane extruder was used to pass the sonicated solution through polycarbonate membranes with pore sizes of 0.45 μm and 0.22 μm in sequence to obtain liposome-loaded active peptides;

[0104] S5: Preparation of gallic acid-modified chitosan-modified liposomes loaded with active peptides

[0105] S5.1: 0.5 parts by weight of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride and 0.3 parts by weight of N-hydroxysuccinimide are dissolved in a 1:1 mixture of 50 parts by weight of ethanol and water to obtain a mixed solution;

[0106] S5.2: dehydrogenating 10 parts by weight of a 1.5 wt% chitosan aqueous solution at 60° C. in a nitrogen atmosphere for 12 min, then adding 0.12 parts by weight of gallic acid to obtain a mixed solution, adding the mixed solution to the mixed solution, reacting for 14 h while keeping the pH constant, and drying after the reaction to obtain gallic acid-modified chitosan;

[0107] S5.3: Prepare 1% glacial acetic acid solution with deionized water, then add 0.1 weight part of gallic acid modified chitosan, stir to dissolve, and adjust to pH 6, then slowly add liposome loaded active peptide in a volume ratio of 1:1, stir at a speed of 800r / min for 2h, to obtain gallic acid modified chitosan and then modified liposome loaded active peptide;

[0108] S6: Preparation of mixed hair growth agent

[0109] S6.1: Under light-proof conditions, 1 part by weight of hyaluronic acid was dissolved in 120 parts by weight of deionized water, and after stirring evenly, 1 part by weight of sodium periodate was added, and the mixture was reacted for 5 hours, and then 3 parts by weight of ethylene glycol was added, and the mixture was stirred for 3 hours and dialyzed in deionized water for 5 days, and dried to obtain aldehyde-modified hyaluronic acid, and deionized water was used to prepare a 4% aldehyde-modified hyaluronic acid solution;

[0110] S6.2: 10 parts by weight of anhydrous calcium chloride are added to 20 parts by weight of distilled water, and after stirring evenly, 5 parts by weight of high-amylose corn starch G70 are added, and the mixture is incubated at 80° C. for 2 h, and then 5 parts by weight of 7% silk fibroin solution is added, and the mixture is stirred and mixed to obtain a mixed solution;

[0111] S6.3: Add 2 parts by weight of exosome-encapsulated active extract and 3 parts by weight of gallic acid-modified chitosan-modified liposome-loaded active peptide to 10 parts by weight of aldehyded hyaluronic acid solution, mix evenly with ultrasound, and then oscillate and mix with the mixed solution at a volume ratio of 1:1. After mixing evenly, a mixed hair growth agent is obtained.

[0112] Example 3

[0113] A method for preparing a mixed hair growth agent of active peptides and stem cell exosomes, such as Figure 1 As shown, the following steps are included:

[0114] S1: Extraction of stem cell exosomes

[0115] S1.1: Use surgical scissors to aseptically cut the adipose tissue near the groin and testicles of the rat, wash the tissue surface with pre-cooled sterile 1× PBS buffer, cut 20 parts by weight of the processed adipose tissue into pieces and transfer them into a centrifuge tube, add 20 parts by weight of 0.2wt% type I collagenase, and digest in a water bath at 37°C for 50 minutes. After the digestion is completed, 2 parts by weight of DMEM / F12 culture medium and 2% fetal bovine serum are added to terminate the digestion to obtain the digested adipose tissue;

[0116] S1.2: The digested adipose tissue was centrifuged at 1100 r / min for 10 min, the solution was resuspended and passed through a 200-mesh cell filter into a new centrifuge tube, and centrifuged again at 1100 r / min for 10 min. The centrifuged cells were resuspended in DMEM / F12 medium and then transferred to a 6 cm culture dish for culture. After 24 h of culture, the medium was half changed, and then the medium was changed every 2 days. When the cells grew to 80% of the density, they were digested and subcultured with 0.3 wt% trypsin to obtain adipose mesenchymal stem cells;

[0117] S1.3: The third generation of adipose-derived mesenchymal stem cells were expanded and cultured. When the cell density reached 85%, the culture medium was removed and washed with 1× PBS buffer, and serum-free culture medium was added. After culturing for 48 hours, the cell supernatant was collected;

[0118] S1.4: The cell supernatant was centrifuged at 4°C, 1500 r / min for 10 min, the supernatant was collected and filtered through a 0.22 μm filter membrane, the filtered cell supernatant was transferred into an ultracentrifuge tube, and centrifuged at 4°C, 10000 r / min for 70 min, the supernatant was removed, and stem cell exosomes were obtained;

[0119] S2: Extraction of active extracts

[0120] S2.1: 200 parts by weight of saw palmetto medicinal material are hot-extracted with 500 parts by weight of 90% ethanol for 2 times, each time for 2 hours, the extracts after hot-extraction are combined and filtered, ethanol is recovered from the filtrate, and the filtrate is concentrated to 1 / 8 of the original volume to obtain a saw palmetto extract;

[0121] S2.2: 200 parts by weight of ginger are hot-extracted with 500 parts by weight of 90% ethanol twice, each time for 2 hours, the extracts after hot-extraction are combined and filtered, ethanol is recovered from the filtrate, and the filtrate is concentrated to 1 / 8 of the original volume to obtain a ginger extract;

[0122] S2.3: 200 parts by weight of Platycladus orientalis leaves are hot-extracted with 500 parts by weight of 90% ethanol for 2 times, each time for 2 hours, the extracts after hot-extraction are combined and filtered, ethanol is recovered from the filtrate, and the filtrate is concentrated to 1 / 8 of the original volume to obtain Platycladus orientalis leaf extract;

[0123] S2.4: 3 parts by weight of saw palmetto extract, 5 parts by weight of Platycladus orientalis leaf extract and 8 parts by weight of ginger extract are mixed and stirred to obtain an active extract;

[0124] S3: Preparation of exosomes encapsulating active extracts

[0125] S3.1: 30 parts by weight of the active extract is prepared into a 2 mg / mL active extract standard solution with 1×PBS buffer, and the stem cell exosomes are added into 1×PBS buffer to prepare a 5 mg / mL exosome solution for later use;

[0126] S3.2: 30 parts by weight of the active extract standard solution and 30 parts by weight of the live exosome solution were mixed and then subjected to ultrasonic treatment in an ultrasonic cell disruptor at 4°C, with an amplitude of 20%, 6 cycles, 4 seconds on and 2 seconds off, and the mixed solution was cooled on ice for 2 minutes between each cycle to obtain a treated mixed solution;

[0127] S3.3: The treated mixed solution was transferred to a LEP tube, incubated in a 4°C refrigerator for 3 h, then centrifuged at 4°C, 1500 rpm for 10 min, and then centrifuged at 4°C, 10000 rpm for 70 min to obtain the exosome-encapsulated active extract;

[0128] S4: Preparation of liposome-loaded active peptides

[0129] S4.1: 3 parts by weight of myristoyl pentapeptide-4 are added to 5 parts by weight of propylene glycol and dissolved at 45° C. to obtain solution I, 3 parts by weight of copper peptide, 3 parts by weight of acetyl tetrapeptide-3 and 8 parts by weight of deionized water are mixed and dissolved at 45° C. to obtain solution II, and solution I and solution II are stirred and mixed to obtain an active peptide mixed solution;

[0130] S4.2: 5 parts by weight of soybean lecithin and 3 parts by weight of cholesterol are added to 50 parts by weight of anhydrous ethanol to dissolve, and then rotary evaporated under reduced pressure at 40°C to obtain a film, and the film is placed in an active peptide mixture and hydrated at 37°C for 1 hour to obtain a hydrated solution;

[0131] S4.3: The hydrated solution was sonicated for 5 min using an ultrasonic cell disruptor in an ice bath, with an ultrasonic power of 300 W, a working time of 3 s, and an interval time of 2 s. After the sonication, a membrane extruder was used to pass the sonicated solution through polycarbonate membranes with pore sizes of 0.45 μm and 0.22 μm in sequence to obtain liposome-loaded active peptides;

[0132] S5: Preparation of gallic acid-modified chitosan-modified liposomes loaded with active peptides

[0133] S5.1: 0.6 parts by weight of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride and 0.4 parts by weight of N-hydroxysuccinimide are dissolved in a 1:1 mixture of 60 parts by weight of ethanol and water to obtain a mixed solution;

[0134] S5.2: Dehydrogenate 20 parts by weight of a 2 wt% chitosan aqueous solution at 55° C. in a nitrogen atmosphere for 10 min, then add 0.15 parts by weight of gallic acid to obtain a mixed solution, add the mixed solution to the mixed solution, react for 12 h while keeping the pH constant, and dry after the reaction to obtain gallic acid-modified chitosan;

[0135] S5.3: Prepare a 2% glacial acetic acid solution with deionized water, then add 0.2 parts by weight of gallic acid-modified chitosan, stir to dissolve, and adjust to pH 6.5, then slowly add liposome-loaded active peptide in a volume ratio of 1:1, stir at a speed of 700 r / min for 1 hour, and obtain gallic acid-modified chitosan and then modified liposome-loaded active peptide;

[0136] S6: Preparation of mixed hair growth agent

[0137] S6.1: Under light-proof conditions, 2 parts by weight of hyaluronic acid was dissolved in 130 parts by weight of deionized water, and then 2 parts by weight of sodium periodate was added after being stirred evenly, and the mixture was reacted for 4 hours, and then 4 parts by weight of ethylene glycol was added, and the mixture was stirred for 2 hours and then dialyzed in deionized water for 4 days, and dried to obtain aldehyde-modified hyaluronic acid, and deionized water was used to prepare a 4% aldehyde-modified hyaluronic acid solution;

[0138] S6.2: 12 parts by weight of anhydrous calcium chloride are added to 30 parts by weight of distilled water, and after stirring evenly, 8 parts by weight of high amylose corn starch G70 are added, and the mixture is stirred at 70° C. for 1 hour, and then 8 parts by weight of 7% silk fibroin solution are added, and the mixture is stirred and mixed to obtain a mixed solution;

[0139] S6.3: Add 3 parts by weight of exosome-encapsulated active extract and 5 parts by weight of gallic acid-modified chitosan-modified liposome-loaded active peptide to 12 parts by weight of aldehyded hyaluronic acid solution, mix evenly by ultrasonication, and then oscillate and mix with the mixed solution at a volume ratio of 1:1. After mixing evenly, a mixed hair growth agent is obtained.

[0140] Animal Experimentation:

[0141] 40 male B6CBAF1 / J mice were subcutaneously injected with testosterone propionate on the back of the mice to establish the model. After the model was established successfully, they were randomly divided into 4 groups, with 10 mice in each group. The corresponding mixed hair growth agents of Example 1, Example 2 and Example 3 were applied externally, twice a day, and massaged for five minutes;

[0142] The blank group was not given any medication.

[0143] After 4 weeks of the experiment, the hair growth of the four groups of experimental animals was observed. The hair removal area on the back of the mice was taken with a sample area of ​​1×1 cm, deep into the fascia, to explore the effect on the number of hair follicles in the mice. The average value was taken. The results are shown in Table 1.

[0144] Table 1 The growth of hair in experimental animals and the effect on the number of hair follicles in mice

[0145] Hair length(mm) Number of hair follicles Example 1 8.45 18 Example 2 8.24 17 Example 3 8.37 18 Blank Group 5.53 5

[0146] According to the data in Table 1, compared with the blank group, the number of hair and hair follicles of mice in the mixed hair growth agent group increased significantly, indicating that the mixed hair growth agent prepared in the present application has a significant hair growth effect.

[0147] Comparative Example 1

[0148] Compared with Example 1, the difference of Comparative Example 1 is that, in Comparative Example 1, step S3 is removed, and the exosome-encapsulated active extract in step S6.3 is replaced with equal weight portions of stem cell exosomes and active extract, and the other steps remain unchanged to prepare a mixed hair growth agent, which is recorded as Comparative Example 1.

[0149] The data of Example 1 and the blank group were used, and the mixed hair growth agent prepared in Comparative Example 1 was subjected to an animal experiment to measure the hair growth of the experimental animals and the effect on the number of hair follicles of mice. The average values ​​were taken. The results are shown in Table 2.

[0150] Table 2 The growth of hair in experimental animals of comparative example 1 and its effect on the number of hair follicles in mice

[0151] Hair length(mm) Number of hair follicles Example 1 8.45 18 Comparative Example 1 6.77 13 Blank Group 5.53 5

[0152] Comparative Example 2

[0153] Compared with Example 1, the difference of Comparative Example 2 is that Comparative Example 2 removes steps S4.2-S4.3 and S5, replaces the gallic acid-modified chitosan-modified liposome-loaded active peptide in step S6.3 with an equal weight portion of active peptide mixed solution, and the other steps remain unchanged to prepare a mixed hair growth agent, which is recorded as Comparative Example 2.

[0154] The data of Example 1 and the blank group were used, and the mixed hair growth agent prepared in Comparative Example 2 was subjected to an animal experiment to measure the growth of hair in the experimental animals and the effect on the number of hair follicles in mice. The average values ​​were taken. The results are shown in Table 3.

[0155] Table 3 The growth of hair in experimental animals of comparative example 2 and the effect on the number of hair follicles in mice

[0156]

[0157]

[0158] Comparative Example 3

[0159] Compared with Example 1, the difference of Comparative Example 3 is that, in Comparative Example 3, steps S5.1-S5.2 are removed, the gallic acid-modified chitosan in step S5.3 is replaced with an equal weight portion of chitosan, and the other steps remain unchanged to prepare a mixed hair growth agent, which is recorded as Comparative Example 3.

[0160] The data of Example 1 and the blank group were used, and the mixed hair growth agent prepared in Comparative Example 3 was subjected to an animal experiment to measure the growth of hair of the experimental animals and the effect on the number of hair follicles of mice. The average values ​​were taken. The results are shown in Table 4.

[0161] Table 4 Comparative Example 3: Hair growth of experimental animals and its effect on the number of hair follicles in mice

[0162] Hair length(mm) Number of hair follicles Example 1 8.45 18 Comparative Example 3 7.67 14 Blank Group 5.53 5

[0163] Comparative Example 4

[0164] Compared with Example 1, the difference of Comparative Example 4 is that, in Comparative Example 4, step S5 is removed, and the gallic acid-modified chitosan-modified liposome-loaded active peptide in step S6.3 is replaced with an equal weight portion of liposome-loaded active peptide, and the other steps remain unchanged to prepare a mixed hair growth agent, which is recorded as Comparative Example 4.

[0165] The data of Example 1 and the blank group were used, and the mixed hair growth agent prepared in Comparative Example 4 was subjected to an animal experiment to measure the hair growth of the experimental animals and the effect on the number of hair follicles of mice. The average values ​​were taken. The results are shown in Table 5.

[0166] Table 5 The growth of hair in experimental animals of comparative example 4 and the effect on the number of hair follicles in mice

[0167] Hair length(mm) Number of hair follicles Example 1 8.45 18 Comparative Example 4 6.92 11 Blank Group 5.53 5

[0168] According to the data in Tables 2 to 5, it can be seen that the data of Comparative Example 1 is lower than that of the embodiment, but higher than that of the blank group, indicating that encapsulating the active extract by exosomes can increase the concentration of the active ingredient in the hair growth-related cells, thereby enhancing the hair growth effect; the data of Comparative Examples 2-4 are lower than that of the embodiment, but higher than that of the blank group, indicating that loading the active peptide on the liposome can make the active peptide reach the hair follicle cells more effectively and play its role in promoting hair growth, and modifying the liposome by modifying chitosan with gallic acid can promote the proliferation of hair follicle cells, thereby effectively promoting hair growth.

[0169] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A method for preparing a mixed hair growth agent of active peptides and stem cell exosomes, It is characterized in that The steps include: S1: Extraction of stem cell exosomes Stem cell exosomes were extracted from adipose tissue near the groin and testicles of rats; S2: Extraction of active extracts The saw palmetto medicinal materials, ginger and Platycladus orientalis leaves were subjected to ethanol hot extraction respectively, and the extracts were mixed to obtain the active extract; S3: Preparation of exosomes encapsulating active extracts The active extract is introduced into exosomes by ultrasonic treatment to prepare exosome-encapsulated active extract; S4: Preparation of liposome-loaded active peptides Myristoyl pentapeptide-4, copper peptide and acetyl tetrapeptide-3 were loaded by soybean lecithin and cholesterol to prepare liposome loaded active peptides; S5: Preparation of gallic acid-modified chitosan-modified liposomes loaded with active peptides After chitosan is modified by gallic acid, the liposome loaded with active peptide is modified to obtain gallic acid-modified chitosan and then modified liposome loaded with active peptide; S6: Preparation of mixed hair growth agent The aldehyde-modified hyaluronic acid solution, the silk fibroin solution, starch, the exosome-encapsulated active extract and the gallic acid-modified chitosan-modified liposome-loaded active peptide are mixed to prepare a mixed hair growth agent.

2. A method for preparing a mixed hair growth agent of active peptides and stem cell exosomes according to claim 1, It is characterized in that Step S1: Extraction of stem cell exosomes, specifically comprising the following steps: S1.1: Use surgical scissors to aseptically cut the adipose tissue near the groin and testicles of the rat, wash the tissue surface with pre-cooled sterile PBS buffer, cut 10-20 parts by weight of the processed adipose tissue into pieces and transfer them into a centrifuge tube, add 10-20 parts by weight of 0.1-0.2wt% type I collagenase, and digest in a water bath at 37-38°C for 50-60 minutes. After the digestion is completed, add 1-2 parts by weight of DMEM / F12 culture medium and fetal bovine serum to terminate the digestion, and obtain the digested adipose tissue; S1.2: The digested adipose tissue was centrifuged at 1100-1200 r / min for 10-12 min, the solution was resuspended and passed through a 200-300 mesh cell filter into a new centrifuge tube, and centrifuged again at 1100-1200 r / min for 10-12 min. The centrifuged cells were resuspended in DMEM / F12 medium and then transferred to a 6-8 cm culture dish for culture. After culturing for 24-26 hours, the medium was half changed, and then the medium was changed every 2-3 days. When the cells grew to a density of 80-85%, they were digested and subcultured with 0.25-0.3wt% trypsin to obtain adipose mesenchymal stem cells; S1.3: The 3rd-4th generation adipose-derived mesenchymal stem cells were expanded and cultured. When the cell density reached 80-85%, the culture medium was removed and washed with PBS buffer, and serum-free culture medium was added. After culturing for 48-50 hours, the cell supernatant was collected; S1.4: Centrifuge the cell supernatant at 4-5°C, 1500-2000r / min for 10-12min, collect the supernatant and pass it through a 0.22-0.24μm filter membrane, transfer the filtered cell supernatant into an ultracentrifuge tube, centrifuge at 4-5°C, 10000-20000r / min for 70-80min, remove the supernatant, and obtain stem cell exosomes.

3. A method for preparing a mixed hair growth agent of active peptides and stem cell exosomes according to claim 2, It is characterized in that Step S2: Extraction of active extract, specifically comprising the following steps: S2.1: extracting 100-200 parts by weight of saw palmetto medicinal material with 300-500 parts by weight of 85-90% ethanol by hot soaking for 2-3 times, each time for 2-3 hours, combining the extracts after hot soaking, filtering, recovering ethanol from the filtrate, and concentrating the filtrate to 1 / 10-1 / 8 of the original volume to obtain a saw palmetto extract; S2.2: extracting 100-200 parts by weight of ginger with 300-500 parts by weight of 85-90% ethanol by hot soaking for 2-3 times, each time for 2-3 hours, combining the extracts after hot soaking, filtering, recovering ethanol from the filtrate, and concentrating the filtrate to 1 / 10-1 / 8 of the original volume to obtain a ginger extract; S2.3: 100-200 parts by weight of Platycladus orientalis leaves are hot-extracted with 300-500 parts by weight of 85-90% ethanol for 2-3 times, each time for 2-3 hours, the extracts after hot-extraction are combined and filtered, ethanol is recovered from the filtrate, and the filtrate is concentrated to 1 / 10-1 / 8 of the original volume to obtain a Platycladus orientalis leaf extract; S2.4: 2-3 parts by weight of saw palmetto extract, 3-5 parts by weight of Platycladus orientalis leaf extract and 5-8 parts by weight of ginger extract are mixed and stirred to obtain an active extract.

4. A method for preparing a mixed hair growth agent of active peptides and stem cell exosomes according to claim 3, It is characterized in that Step S3: Preparation of exosome-encapsulated active extract, specifically comprising the following steps: S3.1: Prepare 20-30 parts by weight of the active extract into a 1-2 mg / mL active extract standard solution with PBS buffer, add stem cell exosomes into PBS buffer to prepare a 4-5 mg / mL exosome solution for later use; S3.2: 20-30 parts by weight of the active extract standard solution and 20-30 parts by weight of the live exosome solution are mixed and then subjected to ultrasonic treatment in an ultrasonic cell disruptor at 4-5°C, with an amplitude of 20-30%, 6-8 cycles, 4-5s on, 2-3s off, and the mixed solution is cooled on ice for 2-3 minutes between each cycle to obtain a treated mixed solution; S3.3: Transfer the treated mixed solution to a LEP tube and incubate it in a 4-5℃ refrigerator for 3-5h. Then centrifuge it at 4-5℃ and 1500-2000r / min for 10-12min, and then centrifuge it at 4-5℃ and 10000-20000r / min for 70-80min to obtain exosome-encapsulated active extract.

5. A method for preparing a mixed hair growth agent of active peptides and stem cell exosomes according to claim 4, It is characterized in that Step S4: Preparation of liposome-loaded active peptides, specifically comprising the following steps: S4.1: Add 2-3 parts by weight of myristoyl pentapeptide-4 to 3-5 parts by weight of propylene glycol, and dissolve at 45-50° C. to obtain solution I, mix 2-3 parts by weight of copper peptide, 2-3 parts by weight of acetyl tetrapeptide-3, and 5-8 parts by weight of deionized water, and dissolve at 45-50° C. to obtain solution II, and stir and mix solution I and solution II to obtain an active peptide mixed solution; S4.2: 3-5 parts by weight of soybean lecithin and 2-3 parts by weight of cholesterol are added to 40-50 parts by weight of anhydrous ethanol to dissolve, and then rotary evaporated under reduced pressure at 40-45°C to obtain a film, and the film is placed in an active peptide mixture and hydrated at 37-38°C for 1-2 hours to obtain a hydrated solution; S4.3: The hydrated solution was sonicated in an ice bath using an ultrasonic cell crusher for 5-8 minutes, with an ultrasonic power of 300-350 W, a working time of 3-5 s, and an interval time of 2-3 s. After the sonication, a membrane extruder was used to pass the sonicated solution through polycarbonate membranes with pore sizes of 0.45 μm and 0.22 μm in turn to obtain liposome-loaded active peptides.

6. A method for preparing a mixed hair growth agent of active peptides and stem cell exosomes according to claim 5, It is characterized in that Step S5 is the preparation of gallic acid-modified chitosan and then modified liposomes loaded with active peptides, specifically comprising the following steps: S5.1: 0.5-0.6 parts by weight of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride and 0.3-0.4 parts by weight of N-hydroxysuccinimide are dissolved in a 1:1 mixture of 50-60 parts by weight of ethanol and water to obtain a mixed solution; S5.2: dehydrogenating 10-20 parts by weight of a 1.5-2 wt% chitosan aqueous solution under a nitrogen atmosphere at 55-60° C. for 10-12 min, then adding 0.12-0.15 parts by weight of gallic acid to obtain a mixed solution, adding the mixed solution to the mixed solution, reacting for 12-14 h while keeping the pH constant, and drying after the reaction to obtain gallic acid-modified chitosan; S5.3: Prepare 1-2% glacial acetic acid solution with deionized water, then add 0.1-0.2 weight parts of gallic acid modified chitosan, stir to dissolve, and adjust to pH 6-6.5, then slowly add liposome-loaded active peptide in a volume ratio of 1:1, stir at a speed of 700-800r / min for 1-2h to obtain gallic acid modified chitosan and then modified liposome-loaded active peptide.

7. The method for preparing a mixed hair growth agent of active peptides and stem cell exosomes according to claim 6, It is characterized in that Step S6: Preparation of the mixed hair growth agent, specifically comprising the following steps: S6.1: Under light-proof conditions, 1-2 parts by weight of hyaluronic acid is dissolved in 120-130 parts by weight of deionized water, stirred evenly, and then 1-2 parts by weight of sodium periodate is added, reacted for 4-5 hours, then 3-4 parts by weight of ethylene glycol is added, stirred for 2-3 hours, dialyzed in deionized water for 4-5 days, dried to obtain aldehyde-modified hyaluronic acid, and deionized water is used to prepare a 4% aldehyde-modified hyaluronic acid solution; S6.2: Add 10-12 parts by weight of anhydrous calcium chloride to 20-30 parts by weight of distilled water, stir evenly, then add 5-8 parts by weight of starch, stir at 70-80° C. for 1-2 hours, then add 5-8 parts by weight of 7% silk fibroin solution, stir and mix to obtain a mixed solution; S6.3: Add 2-3 parts by weight of exosome-encapsulated active extract and 3-5 parts by weight of gallic acid-modified chitosan-modified liposome-loaded active peptide to 10-12 parts by weight of aldehyded hyaluronic acid solution, mix evenly with ultrasound, and then oscillate and mix with the mixed solution at a volume ratio of 1:

1. After mixing evenly, a mixed hair growth agent is obtained.

8. The method for preparing a mixed hair growth agent of active peptides and stem cell exosomes according to claim 7, It is characterized in that The PBS buffer was 1× PBS buffer.

9. The method for preparing a mixed hair growth agent of active peptides and stem cell exosomes according to claim 2, It is characterized in that In step S1.1, the amount of fetal bovine serum added is 10-12% of the mass of DMEM / F12 culture medium.

10. The method for preparing a mixed hair growth agent of active peptides and stem cell exosomes according to claim 7, It is characterized in that The starch in step S6.2 is high amylose corn starch G70.

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