Stem cell extract composition for promoting hair growth and preparation method thereof

Abstract

The invention provides a stem cell extract composition for promoting hair growth and a preparation method thereof, and belongs to the technical field of cosmetics. Fetal calf serum, dexamethasone, penicillin, streptomycin, interleukin-2 and magnetic composite particles are added into a DMEM culture medium to prepare the culture medium, the culture medium is used for in-vitro culture of stem cells, during culture, a stem cell solution is prepared by combining a low-frequency magnetic field with low-energy laser culture, exosomes are extracted from an extracting solution, the exosomes and the stem cells are mixed and embedded to prepare embedded microspheres, and the embedded microspheres are used for in-vitro culture of the stem cells. The stem cell extract composition for promoting hair growth is prepared by uniformly mixing the stem cell extract with extracts extracted from fresh ginger, ginseng and polygonum multiflorum, the storage stability of stem cells and exosomes is obviously improved, hair growth is promoted, hair follicle development is promoted, mouse hair is induced and maintained in a growth period, and the stem cell extract composition is free of hormone addition, safe and non-irritant, and has a good application prospect. Wide application prospects are realized.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to a stem cell extract composition for promoting hair growth and a preparation method thereof. Background Art

[0002] Hair, as one of the important appendages of the skin organ, is a unique feature of mammals. The main functions of hair include protecting the skin from mechanical damage, heat preservation, sensory functions, etc. A normal person loses about 50-100 hairs a day, and hair loss is defined as hair loss when the number of hair loss is greater than 100 / day. Alopecia (hair falling off the scalp) occurs due to various reasons, such as internal inducements (such as genetic composition and the effects of male hormones); mental stress in daily life; and external inducements (such as the accumulation of lipid peroxides in the scalp). It is known that the symptoms of hair loss are caused by a very complex process. In order to improve this hair loss, various types of hair growth liquids are commercially available.

[0003] At present, there are only two drugs approved by the U.S. Food and Drug Administration (FDA), such as minoxidil and finasteride, which were announced in 1998 (about 15 years ago). Therefore, alopecia of various causes cannot be completely cured with only these two drugs. Since the hair growth liquid on the market has the side effects caused by the application of hormone drugs and is only effective for the part where the hair root is activated, they are effective in preventing hair loss, but have no significant effect on the growth of hair in a long-term resting phase, and must be given or applied continuously. Therefore, it is necessary to develop an economical and stable technology for alopecia or thinning hair. In Korea, compared with developed countries, the research and development of drugs for alopecia is technically insufficient, and the research and development related to the prevention of hair loss, stimulation of hair growth and hair regeneration is a very urgent problem. However, regarding research related to alopecia, documents related to hair root and hair regeneration have been reported since 1950, but due to lack of reproducibility, hair regeneration has been considered impossible in the past 50 years.

[0004] Stem cells are cells that have the ability to differentiate into various types of cells that constitute the human body and the ability to self-replicate due to the environment and stimulation inside and outside the human body. There are three known types of stem cells, such as embryonic stem cells (ES cells) isolated from early embryos, embryonic germ cells (EG cells) isolated from primordial germ cells in the embryonic period, and multipotent adult progenitor cells (MAPC cells) isolated from adult bone marrow. In the prior art, there are few studies on the application of stem cells in hair promotion. Therefore, it is of great significance to develop a safe and effective stem cell-based hair growth promotion product. Summary of the invention

[0005] The purpose of the present invention is to propose a stem cell extract composition for promoting hair growth and a preparation method thereof, which can significantly improve the storage stability of stem cells and exosomes and extend the storage period. The composition prepared by mixing with the extract can significantly promote hair growth, promote hair follicle development, induce and maintain mouse hair in the growth phase, and has no added hormones, is safe and non-irritating, and has broad application prospects.

[0006] The technical solution of the present invention is achieved in this way: The invention provides a method for preparing a stem cell extract composition for promoting hair growth. Fetal bovine serum, dexamethasone, penicillin, streptomycin, interleukin-2 and magnetic composite particles are added to a DMEM culture medium to prepare a culture medium for in vitro stem cell culture. During the culture, a low-frequency magnetic field combined with a low-energy laser culture is used to prepare a stem cell solution. Exosomes are extracted from the extract, mixed and embedded with the stem cells to prepare embedded microspheres, and evenly mixed with extracts extracted from ginger, ginseng and Polygonum multiflorum to prepare a stem cell extract composition for promoting hair growth.

[0007] As a further improvement of the present invention, the following steps are included: S1. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, interleukin-2, and magnetic composite particles were added to DMEM culture medium to prepare a culture medium; S2. In vitro culture of stem cells: The excised umbilical cord tissue is washed with tissue protection solution to remove residual fat, connective tissue and blood, and then disinfected with medical alcohol. The tissue is then digested with digestive solution, centrifuged, and the supernatant is discarded. The digested tissue is collected, washed, and added to the culture medium. After low-frequency magnetic field culture, low-energy laser culture is used to remove tissue blocks. After the cells adhere to the wall and grow to 80-90% fusion, the magnetic material is separated by a magnet, the residue is centrifuged, the supernatant is collected, the solid is washed with D-Hank's solution, digested, resuspended in DMEM culture medium, and frozen to obtain a stem cell solution; S3. Extraction of exosomes: The supernatant in step S2 is extracted with ultracentrifugation combined with ultrafiltration tube method to extract exosomes; S4. Preparation of Chinese herbal medicine extract: Ginger, ginseng and Polygonum multiflorum are mixed and crushed, heated and boiled for extraction, the extraction is repeated 2-3 times, filtered, the filtrate is combined, concentrated, and dried to obtain an extract; S5. Preparation of embedded microspheres: After the stem cell solution in step S2 is revived, the exosomes prepared in step S3 are added, sodium alginate and an emulsifier are added, and the mixture is stirred and mixed evenly, and then added dropwise to the fish oil, emulsified, and calcium chloride solution is added dropwise, solidified at room temperature, centrifuged, washed, and dried to prepare embedded microspheres; S6. Preparation of a stem cell extract composition for promoting hair growth: Evenly mix the embedded microspheres and the extract to prepare a stem cell extract composition for promoting hair growth.

[0008] As a further improvement of the present invention, in step S1, the content of fetal bovine serum in the culture medium is 9-11wt%, the content of dexamethasone is 5-15nmol / L, the content of penicillin is 100-120U / mL, the content of streptomycin is 100-120μg / mL, the content of interleukin-2 is 5-15μg / mL, and the content of magnetic composite particles is 400-500μg / mL; the preparation method of the magnetic composite particles is as follows: T1. Preparation of magnetic oxide: ball-mill zirconium oxide ceramic powder, add it into water, add ferric chloride and ferrous chloride, add ammonia water under the protection of inert gas, heat and stir to react, centrifuge, wash, dry, and calcine to obtain magnetic oxide; T2. Modification: Add magnetic oxide into water, add tannic acid and catalyst, heat and stir to react, separate by magnet, wash and dry to obtain modified magnetic oxide; T3. Loading: Add the modified magnetic oxide into water, add NHS and EDC, stir to activate, add transforming growth factor-β and glutathione, stir to react, separate with a magnet, wash, and dry to obtain magnetic composite particles.

[0009] As a further improvement of the present invention, the ball milling time in step T1 is 0.5-1h, the mass ratio of the zirconium oxide, ferric chloride and ferrous chloride is 10:3.24:1.26, the temperature of the heating and stirring reaction is 80-90°C, the time is 3-5h, the temperature of the calcination is 400-600°C, and the time is 1-2h; the mass ratio of the magnetic oxide, tannic acid and catalyst in step T2 is 10:2-3:0.1-0.5, the catalyst is a Tris-HCl solution with a pH of 8.5-9.5, the temperature of the heating and stirring reaction is 40-50°C, and the time is 2-4h.

[0010] As a further improvement of the present invention, in step T3, the mass ratio of the modified magnetic oxide, NHS, EDC, transforming growth factor-β, and glutathione is 12-15:2-3:1.5-2.5:3-4:2-3, the stirring activation time is 20-40 minutes, and the stirring reaction time is 10-12 hours.

[0011] As a further improvement of the present invention, the digestion solution in step S2 is a PBS buffer solution with a pH of 7.5-7.7 containing 1-2wt% trypsin and 0.5-1wt% alkaline protease; the culture conditions are 36-38°C, the CO2 concentration is 3-6v / v%, the low-frequency magnetic field culture time is 5-7d, the low-energy laser culture time is 3-5d, the frequency of the low-frequency magnetic field is 40-50Hz, the stimulation time is 30-40min / d, and the energy density of the low-energy laser is 3-7J / cm 2 , wavelength is 600-800nm, total irradiation time is 3-5min / d, and each irradiation time is 30-60s.

[0012] As a further improvement of the present invention, the ultrahigh-speed centrifugation method combined with the ultrafiltration tube method in step S3 is as follows: the cell supernatant is centrifuged at 4°C, 500-1000r / min for 10-20min to remove cells, and then centrifuged at 4°C, 3000-4000r / min for 15-25min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, 4°C, 12000-15000r / min centrifuged for 20-30min to remove large vesicles and proteins, the supernatant is filtered with a 0.22μm filter membrane, and finally 4°C, 30000-35000r / min centrifuged for 50-70min, pH=6.7-7 PBS resuspended precipitate, 4°C, 30000-35000r / min centrifuged again for 50-70min, the obtained precipitate is exosomes, resuspended with PBS with pH=6.7-7, protein quantification is performed according to the BCA kit steps to control the protein concentration to 80-100mg / L, and -80°C refrigerator is frozen.

[0013] As a further improvement of the present invention, in step S4, the mass ratio of ginger, ginseng and Polygonum multiflorum is 7-10:1-2:5-7, and the time for heating and boiling the extract is 2-4 hours.

[0014] As a further improvement of the present invention, the mass ratio of the stem cell solution, exosomes, and sodium alginate in step S5 is 15-30:2-3:7-10.

[0015] As a further improvement of the present invention, the mass ratio of the embedded microspheres to the extract in step S6 is 12-15:7-10.

[0016] The present invention further protects a stem cell extract composition for promoting hair growth obtained by the above preparation method.

[0017] The present invention has the following beneficial effects: The present invention prepares magnetic composite particles, using zirconium oxide ceramic powder, a ceramic component that releases far-infrared, as a matrix, and depositing magnetic ferrosoferric oxide on the surface to obtain magnetic oxides. After the surface is modified with tannic acid, it can couple transforming growth factor-β and glutathione under the activation of NHS and EDC. These two substances can significantly synergistically promote the growth of stem cells, have important regulatory effects on cell growth, differentiation and immune function, and promote stem cells to have more active paracrine functions, greatly increase the secretion of exosomes, and simultaneously promote the proliferation of stem cells.

[0018] Low-frequency electromagnetic fields can act on certain components in cell culture fluid, thereby affecting cell proliferation. They can increase the permeability of small molecules by affecting the structure of cell membranes and cell membrane-related enzymes, thereby changing cell behavior with the help of second messengers. At the same time, they can control the opening of voltage-gated calcium channels, causing calcium ion influx and an increase in intracellular calcium ion concentration. In addition, under the action of low-frequency electromagnetic fields, magnetic composite particles can effectively bind to protein receptors on the cell membrane, such as iron-sulfur poly compounds, causing the opening of ion channels and changes in membrane tension, thereby depolarizing the cell membrane and generating action potentials, thereby promoting the entry of transforming growth factor-β and glutathione loaded into the cell and regulating cell growth. At the same time, iron-sulfur poly compounds also have the property of responding to magnetic fields. This property may form a magnetosome-like structure, which can then be constrained or controlled to promote cell growth and development.

[0019] In addition, low-frequency electromagnetic fields can also affect the differentiation direction of stem cells. At the same time, studies have shown that low-frequency electromagnetic fields promote the expression of NF-kB and the activation of the MAPK signaling pathway, thereby enhancing the expression of C-Mys and cydinD1, and inhibit the activity of glycogen synthase kinase by activating PI3K (phosphatidylinositol 3-kinase), which is beneficial to the transcription of downstream genes, and can affect multiple links in the intracellular Wnt signaling pathway, thereby promoting the growth of stem cells.

[0020] In addition, the zirconium oxide ceramic powder of the present invention can also release far-infrared waves, which can locally increase the temperature of cells, thereby increasing the activity of cells, further increasing the activity of cells, promoting cells to secrete a large number of highly active exosomes, and making cells proliferate faster. The present invention can promote cell repair and regeneration during low-energy laser culture and enhance the activity of cells in vitro.

[0021] The prepared stem cells and exosomes are embedded in sodium alginate. In the sodium alginate-embedded microspheres, sodium alginate can replace water molecules to form hydration bonds with hydrophilic groups on cell membranes and proteins, thereby avoiding structural changes of cell membranes and protein molecules and maintaining the stability of their structures and functions, so that stem cells can be preserved for a long time, thereby improving their storage stability, and also improving the storage stability of exosomes.

[0022] The stem cell extract composition for promoting hair growth prepared by the present invention can significantly improve the storage stability of stem cells and exosomes, prolong the storage period, and the composition prepared by mixing with the extract can significantly promote hair growth, promote hair follicle development, induce and maintain mouse hair in the growth phase, and has no added hormones, is safe and non-irritating, and has broad application prospects. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Zirconia ceramic powder, particle size D50<5μm, was purchased from Jiaozuo Zhongcheng New Materials Co., Ltd.; transforming growth factor-β, TGF-β1, molecular weight about 42.3KDa, was purchased from Nanjing Oukai Biotechnology Co., Ltd.; trypsin, 200,000 U / g, alkaline protease, 200,000 U / g, were purchased from Xiasheng (Beijing) Enzyme Biotechnology Development Co., Ltd.

[0025] NHS, N-hydroxysuccinimide; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.

[0026] Preparation Example 1 Preparation of magnetic composite particles Here’s how: T1. Preparation of magnetic oxide: 10g zirconium oxide ceramic powder was ball-milled for 0.5h, added to 500mL water, 3.24g ferric chloride and 1.26g ferrous chloride were added, 20mL ammonia water was added under nitrogen protection, heated to 80℃, stirred for 3h, centrifuged, washed, dried, and calcined at 400℃ for 1h to obtain magnetic oxide; T2. Modification: Add 10g of magnetic oxide to 200mL of water, add 2g of tannic acid and 0.1g of catalyst, heat to 40°C, stir and react for 2h, separate with a magnet, wash, and dry to obtain a modified magnetic oxide; The catalyst is a Tris-HCl solution with a pH of 8.5; T3. Loading: Add 12 g of modified magnetic oxide into 500 mL of water, add 2 g of NHS and 1.5 g of EDC, stir and activate for 20 min, add 3 g of transforming growth factor-β and 2 g of glutathione, stir and react for 10 h, separate with a magnet, wash, and dry to obtain magnetic composite particles.

[0027] Preparation Example 2 Preparation of magnetic composite particles Here’s how: T1. Preparation of magnetic oxide: 10g zirconium oxide ceramic powder was ball-milled for 1h, added to 500mL water, 3.24g ferric chloride and 1.26g ferrous chloride were added, 20mL ammonia water was added under nitrogen protection, heated to 90℃, stirred for 5h, centrifuged, washed, dried, and calcined at 600℃ for 2h to obtain magnetic oxide; T2. Modification: 10 g of magnetic oxide was added to 200 mL of water, 3 g of tannic acid and 0.5 g of catalyst were added, heated to 50 ° C, stirred for 4 h, separated by magnet, washed and dried to obtain modified magnetic oxide; The catalyst is a Tris-HCl solution with a pH of 9.5; T3. Loading: Add 15 g of modified magnetic oxide into 500 mL of water, add 3 g of NHS and 2.5 g of EDC, stir and activate for 40 min, add 4 g of transforming growth factor-β and 3 g of glutathione, stir and react for 12 h, separate with a magnet, wash, and dry to obtain magnetic composite particles.

[0028] Preparation Example 3 Preparation of magnetic composite particles Here’s how: T1. Preparation of magnetic oxide: 10g zirconium oxide ceramic powder was ball-milled for 1h, added to 500mL water, 3.24g ferric chloride and 1.26g ferrous chloride were added, 20mL ammonia water was added under nitrogen protection, heated to 85℃, stirred for 4h, centrifuged, washed, dried, and calcined at 500℃ for 1.5h to obtain magnetic oxide; T2. Modification: 10 g of magnetic oxide was added to 200 mL of water, 2.5 g of tannic acid and 0.3 g of catalyst were added, heated to 45 ° C, stirred for 3 h, separated by magnet, washed and dried to obtain modified magnetic oxide; The catalyst is a Tris-HCl solution with a pH of 9; T3. Loading: Add 13 g of modified magnetic oxide into 500 mL of water, add 2.5 g of NHS and 2 g of EDC, stir and activate for 30 min, add 3.5 g of transforming growth factor-β and 2.5 g of glutathione, stir and react for 11 h, separate with a magnet, wash, and dry to obtain magnetic composite particles.

[0029] Comparative Preparation Example 1 Compared with Preparation Example 3, the difference is that no zirconium oxide ceramic powder is added in step T1.

[0030] The details are as follows: T1. Preparation of magnetic oxide: 10.44 g ferric chloride and 4.06 g ferrous chloride were added to 500 mL water, and 20 mL ammonia water was added under nitrogen protection, and the mixture was heated to 85 ° C, stirred for 4 h, centrifuged, washed, dried, and calcined at 500 ° C for 1.5 h to obtain magnetic oxide; T2. Modification: 10 g of magnetic oxide was added to 200 mL of water, 2.5 g of tannic acid and 0.3 g of catalyst were added, heated to 45 ° C, stirred for 3 h, separated by magnet, washed and dried to obtain modified magnetic oxide; The catalyst is a Tris-HCl solution with a pH of 9; T3. Loading: Add 13 g of modified magnetic oxide into 500 mL of water, add 2.5 g of NHS and 2 g of EDC, stir and activate for 30 min, add 3.5 g of transforming growth factor-β and 2.5 g of glutathione, stir and react for 11 h, separate with a magnet, wash, and dry to obtain magnetic composite particles.

[0031] Comparative Preparation Example 2 Compared with Preparation Example 3, the difference is that ferric chloride and ferrous chloride are not added in step T1.

[0032] The details are as follows: T1. Preparation of ceramic powder: 14.5 g of zirconia ceramic powder was ball-milled for 1 h to obtain ceramic powder; T2. Modification: Add 10 g of ceramic powder to 200 mL of water, add 2.5 g of tannic acid and 0.3 g of catalyst, heat to 45 ° C, stir and react for 3 h, centrifuge, wash, and dry to obtain modified ceramic powder; The catalyst is a Tris-HCl solution with a pH of 9; T3. Loading: 13 g of modified ceramic powder was added to 500 mL of water, 2.5 g of NHS and 2 g of EDC were added, and the mixture was stirred for activation for 30 min. 3.5 g of transforming growth factor-β and 2.5 g of glutathione were added, and the mixture was stirred for reaction for 11 h. The mixture was centrifuged, washed, and dried to obtain composite particles.

[0033] Comparative Preparation Example 3 Compared with Preparation Example 3, the difference is that transforming growth factor-β is not added in step T3.

[0034] The details are as follows: T1. Preparation of magnetic oxide: 10g zirconium oxide ceramic powder was ball-milled for 1h, added to 500mL water, 3.24g ferric chloride and 1.26g ferrous chloride were added, 20mL ammonia water was added under nitrogen protection, heated to 85℃, stirred for 4h, centrifuged, washed, dried, and calcined at 500℃ for 1.5h to obtain magnetic oxide; T2. Modification: 10 g of magnetic oxide was added to 200 mL of water, 2.5 g of tannic acid and 0.3 g of catalyst were added, heated to 45 ° C, stirred for 3 h, separated by magnet, washed and dried to obtain modified magnetic oxide; The catalyst is a Tris-HCl solution with a pH of 9; T3. Loading: 13 g of modified magnetic oxide was added to 500 mL of water, 2.5 g of NHS and 2 g of EDC were added, and the mixture was stirred for activation for 30 min. 6 g of glutathione was added, and the mixture was stirred for reaction for 11 h. The mixture was separated by a magnet, washed, and dried to obtain magnetic composite particles.

[0035] Comparative Preparation Example 4 Compared with Preparation Example 3, the difference is that glutathione is not added in step T3.

[0036] The details are as follows: T1. Preparation of magnetic oxide: 10g zirconium oxide ceramic powder was ball-milled for 1h, added to 500mL water, 3.24g ferric chloride and 1.26g ferrous chloride were added, 20mL ammonia water was added under nitrogen protection, heated to 85℃, stirred for 4h, centrifuged, washed, dried, and calcined at 500℃ for 1.5h to obtain magnetic oxide; T2. Modification: 10 g of magnetic oxide was added to 200 mL of water, 2.5 g of tannic acid and 0.3 g of catalyst were added, heated to 45 ° C, stirred for 3 h, separated by magnet, washed and dried to obtain modified magnetic oxide; The catalyst is a Tris-HCl solution with a pH of 9; T3. Loading: Add 13 g of modified magnetic oxide into 500 mL of water, add 2.5 g of NHS and 2 g of EDC, stir and activate for 30 min, add 6 g of transforming growth factor-β, stir and react for 11 h, separate with a magnet, wash, and dry to obtain magnetic composite particles.

[0037] Example 1: This example provides a method for preparing a stem cell extract composition for promoting hair growth, comprising the following steps: S1. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, interleukin-2, and the magnetic composite particles prepared in Preparation Example 1 were added to DMEM culture medium to prepare a culture medium; The content of fetal bovine serum in the culture medium was 9wt%, the content of dexamethasone was 5nmol / L, the content of penicillin was 100U / mL, the content of streptomycin was 100μg / mL, the content of interleukin-2 was 5μg / mL, and the content of the magnetic composite particles prepared in Preparation Example 1 was 400μg / mL; S2. In vitro culture of stem cells: The excised umbilical cord tissue is washed with tissue protection solution to remove residual fat, connective tissue and blood, and then disinfected with medical alcohol. The tissue is then digested with digestive solution, centrifuged, and the supernatant is discarded. The digested tissue is collected, washed, and added to the culture medium. After culturing at 36°C and a CO2 concentration of 3v / v%, low-frequency magnetic field culture is used, and low-energy laser culture is used. Tissue blocks are removed, and after the cells adhere to the wall and grow to 80-90% fusion, the magnetic material is separated with a magnet, the residue is centrifuged, and the supernatant is collected. The solid is washed with D-Hank's solution, digested, resuspended in DMEM culture medium, and frozen to obtain a stem cell solution; The digestion solution is a PBS buffer with a pH of 7.5 containing 1wt% trypsin and 0.5wt% alkaline protease; The low-frequency magnetic field culture time is 5 days, the low-energy laser culture time is 3 days, the frequency of the low-frequency magnetic field is 40 Hz, and the stimulation time is 30 min / d; The energy density of the low energy laser is 3J / cm 2 , wavelength is 600nm, total irradiation time is 3min / d, each irradiation time is 30s; S3. Extraction of exosomes: The supernatant in step S2 is extracted with ultracentrifugation combined with ultrafiltration tube method to extract exosomes; The ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell supernatant is centrifuged at 4°C and 1000 r / min for 15 min to remove cells, and then centrifuged at 4°C and 3500 r / min for 20 min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, and centrifuged at 4°C and 13000 r / min for 25 min to remove large vesicles and proteins, the supernatant is filtered with a 0.22 μm filter membrane, and finally centrifuged at 4°C and 32000 r / min for 60 min, and the precipitate is resuspended in PBS with pH=6.8, and centrifuged again at 4°C and 32000 r / min for 60 min. The obtained precipitate is exosomes, which is resuspended in PBS with pH=6.8, and the protein concentration is controlled to be 90 mg / L according to the steps of the BCA kit, and the protein is frozen in a -80°C refrigerator; S4. Preparation of Chinese herbal extract: 7 g ginger, 1 g ginseng, 5 g Polygonum multiflorum were mixed and crushed, heated and boiled for 2 h, and the extraction was repeated twice, filtered, the filtrate was combined, concentrated, and dried to obtain an extract; S5. Preparation of embedded microspheres: After resuscitation of 15 g of the stem cell solution in step S2, add 2 g of the exosomes prepared in step S3, add 7 g of sodium alginate and 0.5 g of lecithin, stir and mix for 15 min, drop into 200 mL of fish oil, emulsify at 5000 r / min for 15 min, add 10 mL of 2 wt% calcium chloride solution, solidify at room temperature for 30 min, centrifuge, wash, and dry to obtain embedded microspheres; S6. Preparation of a stem cell extract composition for promoting hair growth: 12 g of embedded microspheres and 7 g of the extract were mixed for 10 min to prepare a stem cell extract composition for promoting hair growth.

[0038] Example 2: This example provides a method for preparing a stem cell extract composition for promoting hair growth, comprising the following steps: S1. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, interleukin-2, and the magnetic composite particles prepared in Preparation Example 2 were added to DMEM culture medium to prepare a culture medium; The content of fetal bovine serum in the culture medium was 11wt%, the content of dexamethasone was 15nmol / L, the content of penicillin was 120U / mL, the content of streptomycin was 120μg / mL, the content of interleukin-2 was 15μg / mL, and the content of the magnetic composite particles prepared in Preparation Example 2 was 500μg / mL; S2. In vitro culture of stem cells: The excised umbilical cord tissue is washed with tissue protection solution to remove residual fat, connective tissue and blood, and then disinfected with medical alcohol. The tissue is then digested with digestive solution, centrifuged, and the supernatant is discarded. The digested tissue is collected, washed, and added to the culture medium. After culturing at 38°C and a CO2 concentration of 6v / v%, low-frequency magnetic field culture is used, and low-energy laser culture is used. Tissue blocks are removed, and after the cells adhere to the wall and grow to 80-90% fusion, the magnetic material is separated with a magnet, the residue is centrifuged, and the supernatant is collected. The solid is washed with D-Hank's solution, digested, resuspended in DMEM culture medium, and frozen to obtain a stem cell solution; The digestion solution is a PBS buffer solution with a pH of 7.7 containing 2wt% trypsin and 1wt% alkaline protease; The low-frequency magnetic field culture time is 7 days, the low-energy laser culture time is 5 days, the frequency of the low-frequency magnetic field is 50 Hz, and the stimulation time is 40 min / d; The energy density of the low energy laser is 7 J / cm 2 , wavelength is 800nm, total irradiation time is 5min / d, and each irradiation time is 60s; S3. Extraction of exosomes: The supernatant in step S2 is extracted with ultracentrifugation combined with ultrafiltration tube method to extract exosomes; The ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell supernatant is centrifuged at 4°C and 1000 r / min for 15 min to remove cells, and then centrifuged at 4°C and 3500 r / min for 20 min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, and centrifuged at 4°C and 13000 r / min for 25 min to remove large vesicles and proteins, the supernatant is filtered with a 0.22 μm filter membrane, and finally centrifuged at 4°C and 32000 r / min for 60 min, and the precipitate is resuspended in PBS with pH=6.8, and centrifuged again at 4°C and 32000 r / min for 60 min. The obtained precipitate is exosomes, which is resuspended in PBS with pH=6.8, and the protein concentration is controlled to be 90 mg / L according to the steps of the BCA kit, and the protein is frozen in a -80°C refrigerator; S4. Preparation of Chinese herbal extract: 10 g ginger, 2 g ginseng, 7 g Polygonum multiflorum were mixed and crushed, heated and boiled for 4 h, and the extraction was repeated 3 times, filtered, the filtrate was combined, concentrated, and dried to obtain an extract; S5. Preparation of embedded microspheres: After 30 g of the stem cell solution in step S2 was resuscitated, 3 g of the exosomes prepared in step S3 were added, 10 g of sodium alginate and 0.5 g of lecithin were added, and the mixture was stirred for 15 min, and then dropped into 200 mL of fish oil, emulsified at 5000 r / min for 15 min, and 10 mL of 2 wt% calcium chloride solution was added, and the mixture was cured at room temperature for 30 min, centrifuged, washed, and dried to obtain embedded microspheres; S6. Preparation of a stem cell extract composition for promoting hair growth: 15 g of embedded microspheres and 10 g of the extract were mixed for 10 min to prepare a stem cell extract composition for promoting hair growth.

[0039] Example 3: This example provides a method for preparing a stem cell extract composition for promoting hair growth, comprising the following steps: S1. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, interleukin-2, and the magnetic composite particles prepared in Preparation Example 3 were added to DMEM culture medium to prepare a culture medium; The content of fetal bovine serum in the culture medium was 10wt%, the content of dexamethasone was 10nmol / L, the content of penicillin was 110U / mL, the content of streptomycin was 110μg / mL, the content of interleukin-2 was 10μg / mL, and the content of the magnetic composite particles prepared in Preparation Example 3 was 450μg / mL; S2. In vitro culture of stem cells: The excised umbilical cord tissue is washed with tissue protection solution to remove residual fat, connective tissue and blood, and then disinfected with medical alcohol. The tissue is then digested with digestive solution, centrifuged, and the supernatant is discarded. The digested tissue is collected, washed, and added to the culture medium. The culture is carried out at 37°C with a CO2 concentration of 5v / v%, and then cultured with a low-frequency magnetic field and a low-energy laser. The tissue blocks are removed, and after the cells adhere to the wall and grow to 80-90% fusion, the magnetic material is separated with a magnet, the residue is centrifuged, and the supernatant is collected. The solid is washed with D-Hank's solution, digested, resuspended in DMEM culture medium, and frozen to obtain a stem cell solution. The digestion solution is a PBS buffer with a pH of 7.6 containing 1.5wt% trypsin and 0.7wt% alkaline protease; The low-frequency magnetic field culture time is 6 days, the low-energy laser culture time is 4 days, the frequency of the low-frequency magnetic field is 45 Hz, and the stimulation time is 35 min / d; The energy density of the low energy laser is 5 J / cm 2 , wavelength is 700nm, total irradiation time is 4min / d, and each irradiation time is 45s; S3. Extraction of exosomes: The supernatant in step S2 is extracted with ultracentrifugation combined with ultrafiltration tube method to extract exosomes; The ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell supernatant is centrifuged at 4°C and 1000 r / min for 15 min to remove cells, and then centrifuged at 4°C and 3500 r / min for 20 min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, and centrifuged at 4°C and 13000 r / min for 25 min to remove large vesicles and proteins, the supernatant is filtered with a 0.22 μm filter membrane, and finally centrifuged at 4°C and 32000 r / min for 60 min, and the precipitate is resuspended in PBS with pH=6.8, and centrifuged again at 4°C and 32000 r / min for 60 min. The obtained precipitate is exosomes, which is resuspended in PBS with pH=6.8, and the protein concentration is controlled to be 90 mg / L according to the steps of the BCA kit, and the protein is frozen in a -80°C refrigerator; S4. Preparation of Chinese herbal extract: 8 g ginger, 1.5 g ginseng, and 6 g Polygonum multiflorum were mixed and crushed, heated to boiling and extracted for 3 h, the extraction was repeated 3 times, filtered, the filtrate was combined, concentrated, and dried to obtain an extract; S5. Preparation of embedded microspheres: After resuscitation of 22 g of the stem cell solution in step S2, add 2.5 g of the exosomes prepared in step S3, add 8 g of sodium alginate and 0.5 g of lecithin, stir and mix for 15 min, drop into 200 mL of fish oil, emulsify at 5000 r / min for 15 min, add 10 mL of 2 wt% calcium chloride solution, solidify at room temperature for 30 min, centrifuge, wash, and dry to obtain embedded microspheres; S6. Preparation of a stem cell extract composition for promoting hair growth: 13 g of embedded microspheres and 8 g of the extract were mixed for 10 min to prepare a stem cell extract composition for promoting hair growth.

[0040] Comparative Examples 1-4 Compared with Example 3, the difference is that the magnetic composite particles are prepared by Comparative Preparation Examples 1-4.

[0041] Comparative Example 5 The difference compared with Example 3 is that no magnetic composite particles are added in step S1.

[0042] The details are as follows: S1. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, and interleukin-2 were added to DMEM medium to prepare a culture medium; The content of fetal bovine serum in the culture medium was 10wt%, the content of dexamethasone was 10nmol / L, the content of penicillin was 110U / mL, the content of streptomycin was 110μg / mL, and the content of interleukin-2 was 10μg / mL.

[0043] Comparative Example 6 Compared with Example 3, the difference is that interleukin-2 is not added in step S1.

[0044] The details are as follows: S1. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, and the magnetic composite particles prepared in Preparation Example 3 were added to DMEM culture medium to prepare a culture medium; The content of fetal bovine serum in the culture medium was 10wt%, the content of dexamethasone was 10nmol / L, the content of penicillin was 110U / mL, the content of streptomycin was 110μg / mL, and the content of the magnetic composite particles prepared in Preparation Example 3 was 450μg / mL.

[0045] Comparative Example 7 Compared with Example 3, the difference is that low-frequency magnetic field cultivation is not performed in step S2.

[0046] The details are as follows: S2. In vitro culture of stem cells: The excised umbilical cord tissue was washed with tissue protection solution to remove residual fat, connective tissue and blood, and then disinfected with medical alcohol. The tissue was digested with digestive solution, centrifuged, and the supernatant was discarded. The digested tissue was collected, washed, and added to the culture medium. The culture was carried out at 37°C and the CO2 concentration was 5v / v%. Low-energy laser was used for 10 days to remove the tissue blocks. After the cells adhered to the wall and grew to 80-90% fusion, the magnetic substances were separated by magnets, the residues were centrifuged, and the supernatant was collected. The solid was washed with D-Hank's solution, digested, resuspended in DMEM culture medium, and frozen to obtain a stem cell solution. The digestion solution is a PBS buffer with a pH of 7.6 containing 1.5wt% trypsin and 0.7wt% alkaline protease; The energy density of the low energy laser is 5 J / cm 2 , wavelength is 700nm, total irradiation time is 4min / d, and each irradiation time is 45s.

[0047] Comparative Example 8 Compared with Example 3, the difference is that low-energy laser cultivation is not performed in step S2.

[0048] The details are as follows: S2. In vitro culture of stem cells: The excised umbilical cord tissue is washed with tissue protection solution to remove residual fat, connective tissue and blood, and then disinfected with medical alcohol. The tissue is then digested with digestive solution, centrifuged, and the supernatant is discarded. The digested tissue is collected, washed, and added to the culture medium. The culture is carried out at 37°C, with a CO2 concentration of 5v / v%, and a low-frequency magnetic field for 10 days. The tissue blocks are removed, and after the cells adhere to the wall and grow to 80-90% fusion, the magnetic material is separated with a magnet, the residue is centrifuged, and the supernatant is collected. The solid is washed with D-Hank's solution, digested, resuspended in DMEM culture medium, and frozen to obtain a stem cell solution; The digestion solution is a PBS buffer with a pH of 7.6 containing 1.5wt% trypsin and 0.7wt% alkaline protease; The frequency of the low-frequency magnetic field is 45 Hz, and the stimulation time is 35 min / d.

[0049] Comparative Example 9 Compared with Example 3, the difference is that in step S2, no low-frequency magnetic field culture and low-energy laser culture are performed, and the cells are directly cultured at 37° C. and a CO2 concentration of 5 v / v% for 10 days.

[0050] The details are as follows: S2. In vitro culture of stem cells: The excised umbilical cord tissue was washed with tissue protection solution to remove residual fat, connective tissue and blood, and then disinfected with medical alcohol. The tissue was digested with digestive solution, centrifuged, and the supernatant was discarded. The digested tissue was collected, washed, and added to the culture medium. The culture was carried out at 37°C and the CO2 concentration was 5v / v% for 10 days. The tissue blocks were removed. After the cells adhered to the wall and grew to 80-90% fusion, the magnetic substances were separated by magnets, the residues were centrifuged, and the supernatant was collected. The solid was washed with D-Hank's solution, digested, resuspended in DMEM culture medium, and frozen to obtain a stem cell solution. The digestion solution is a PBS buffer solution with a pH of 7.6 and containing 1.5 wt % trypsin and 0.7 wt % alkaline protease.

[0051] Comparative Example 10 Compared with Embodiment 3, the difference is that step S5 is not performed.

[0052] The details are as follows: S1. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, interleukin-2, and the magnetic composite particles prepared in Preparation Example 3 were added to DMEM culture medium to prepare a culture medium; The content of fetal bovine serum in the culture medium was 10wt%, the content of dexamethasone was 10nmol / L, the content of penicillin was 110U / mL, the content of streptomycin was 110μg / mL, the content of interleukin-2 was 10μg / mL, and the content of the magnetic composite particles prepared in Preparation Example 3 was 450μg / mL; S2. In vitro culture of stem cells: The excised umbilical cord tissue is washed with tissue protection solution to remove residual fat, connective tissue and blood, and then disinfected with medical alcohol. The tissue is then digested with digestive solution, centrifuged, and the supernatant is discarded. The digested tissue is collected, washed, and added to the culture medium. The culture is carried out at 37°C with a CO2 concentration of 5v / v%, and then cultured with a low-frequency magnetic field and a low-energy laser. The tissue blocks are removed, and after the cells adhere to the wall and grow to 80-90% fusion, the magnetic material is separated with a magnet, the residue is centrifuged, and the supernatant is collected. The solid is washed with D-Hank's solution, digested, resuspended in DMEM culture medium, and frozen to obtain a stem cell solution. The digestion solution is a PBS buffer with a pH of 7.6 containing 1.5wt% trypsin and 0.7wt% alkaline protease; The low-frequency magnetic field culture time is 6 days, the low-energy laser culture time is 4 days, the frequency of the low-frequency magnetic field is 45 Hz, and the stimulation time is 35 min / d; The energy density of the low energy laser is 5 J / cm 2 , wavelength is 700nm, total irradiation time is 4min / d, and each irradiation time is 45s; S3. Extraction of exosomes: The supernatant in step S2 is extracted with ultracentrifugation combined with ultrafiltration tube method to extract exosomes; The ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell supernatant is centrifuged at 4°C and 1000 r / min for 15 min to remove cells, and then centrifuged at 4°C and 3500 r / min for 20 min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, and centrifuged at 4°C and 13000 r / min for 25 min to remove large vesicles and proteins, the supernatant is filtered with a 0.22 μm filter membrane, and finally centrifuged at 4°C and 32000 r / min for 60 min, and the precipitate is resuspended in PBS with pH=6.8, and centrifuged again at 4°C and 32000 r / min for 60 min. The obtained precipitate is exosomes, which is resuspended in PBS with pH=6.8, and the protein concentration is controlled to be 90 mg / L according to the steps of the BCA kit, and the protein is frozen in a -80°C refrigerator; S4. Preparation of Chinese herbal extract: 8 g ginger, 1.5 g ginseng, and 6 g Polygonum multiflorum were mixed and crushed, heated to boiling and extracted for 3 h, the extraction was repeated 3 times, filtered, the filtrate was combined, concentrated, and dried to obtain an extract; S5. Preparation of a mixture: After resuscitation of 22 g of the stem cell solution in step S2, 2.5 g of the exosomes prepared in step S3 were added, mixed evenly, and freeze-dried to obtain a mixture; S6. Preparation of a stem cell extract composition for promoting hair growth: 13 g of the mixture and 8 g of the extract were mixed for 10 min to prepare a stem cell extract composition for promoting hair growth.

[0053] Test Example 1 The stem cell extract composition for promoting hair growth was prepared into a 1 mg / mL sample solution with physiological saline.

[0054] Use an electric hair clipper and depilatory cream to remove about 4cm×4cm of hair on the back of the mouse, and establish a depilatory cream depilatory mouse model. The depilatory mice were randomly divided into a blank group (normal saline, 200μL), Example 1-3 and Comparative Example 1-10 groups (sample solution, 200μL) and a positive control group (5% minoxidil, 200μL), with 10 mice in each group. Apply the above solution to the depilatory area once a day. Observe and photograph the color changes of the epidermis of each mouse every day with a digital camera, record the number of mice in each group that enter the hair growth period at different time points, record the length of the new hair of each group of mice after 14 days of intervention, and record the quality of the new hair of each group of mice after 14 days of intervention.

[0055] The results are shown in Table 1.

[0056] Table 1

[0057] Notes: Compared with the blank group, P<0.05.

[0058] It can be seen from the above table that the stem cell extract composition for promoting hair growth prepared in Examples 1-3 of the present invention has a good hair growth promoting effect on the depilatory cream method hair removal mouse model.

[0059] Test Example 2 The stem cell extract composition for promoting hair growth was prepared into a 1 mg / mL sample solution with physiological saline.

[0060] Testosterone propionate is diluted with olive oil, and after ultrasonic mixing, a solution of 5mg / mL is prepared, and mice are subcutaneously injected (5mg / kg) on ​​the back, and the male hormone alopecia model is established. After 3 weeks of modeling, the hair on the back of mice begins to lose its luster, and as the injection time is extended, the hair on the back of mice falls off to varying degrees. After 6 weeks of modeling, the hair on the back of mice falls off more and more, and part of the hair is fused into a piece, and the hair shaft is converted from the thick and dark black of the terminal hair to the fine and non-pigmented vellus hair, and the number of the terminal hair is reduced, and the number of vellus hair increases, which meets the pathological changes of androgenic alopecia, and prompts modeling success. Take 110 mice with successful modeling, and they are randomly divided into model group, embodiment 1-3 and comparative example 1-10 groups, 10 in each group, and 10 healthy mice not injected with androgen (testosterone propionate) are taken as blank group. Experimental group mice are smeared with sample solution 0.2mL at depilation, once a day, for 21d in a row, and model group and blank group are treated the same with 0.2mL normal saline. On the last day of drug administration, the length of new hair at the depilatory site of mice in each group was randomly measured with a ruler.

[0061] The results are shown in Table 2.

[0062] Table 2

[0063] Notes: Compared with the blank group, P < 0.05; # compared with the model group, P < 0.05.

[0064] It can be seen from the above table that the stem cell extract composition for promoting hair growth prepared in Examples 1-3 of the present invention has a good hair growth promoting effect on the androgenic alopecia mouse model.

[0065] Detection of T and DHT levels in mouse serum and skin tissue: After the administration, the mice were fasted overnight, blood was collected from the eyeballs, the blood was allowed to stand and then centrifuged to collect the upper serum, the back hair of the mice was removed, the back skin was taken, homogenized, and the supernatant was collected after centrifugation. According to the instructions of the corresponding kit, the ELISA method was used to detect the levels of T (testosterone) and DHT (androgen) in serum and skin tissue homogenate.

[0066] The results are shown in Table 3.

[0067] Table 3

[0068] Notes: Compared with the blank group, P < 0.05; # compared with the model group, P < 0.05.

[0069] It can be seen from the above table that the stem cell extract composition for promoting hair growth prepared in Examples 1-3 of the present invention can significantly improve the hair growth of mice.

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a stem cell extract composition for promoting hair growth, characterized in that: Fetal bovine serum, dexamethasone, penicillin, streptomycin, interleukin-2 and magnetic composite particles are added to DMEM culture medium to prepare a culture medium for in vitro stem cell culture. During the culture, a low-frequency magnetic field combined with low-energy laser culture is used to prepare a stem cell solution, and exosomes are extracted from the extract, mixed with the stem cells for embedding to prepare embedded microspheres, which are evenly mixed with extracts obtained from ginger, ginseng and Polygonum multiflorum to prepare a stem cell extract composition for promoting hair growth.

2. The preparation method according to claim 1, characterized in that: The following steps are involved: S1. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, interleukin-2, and magnetic composite particles were added to DMEM culture medium to prepare a culture medium; S2. In vitro culture of stem cells: The excised umbilical cord tissue is washed with tissue protection solution to remove residual fat, connective tissue and blood, and then disinfected with medical alcohol. The tissue is then digested with digestive solution, centrifuged, and the supernatant is discarded. The digested tissue is collected, washed, and added to the culture medium. After low-frequency magnetic field culture, low-energy laser culture is used to remove tissue blocks. After the cells adhere to the wall and grow to 80-90% fusion, the magnetic material is separated by a magnet, the residue is centrifuged, the supernatant is collected, the solid is washed with D-Hank's solution, digested, resuspended in DMEM culture medium, and frozen to obtain a stem cell solution; S3. Extraction of exosomes: The supernatant in step S2 is extracted with ultracentrifugation combined with ultrafiltration tube method to extract exosomes; S4. Preparation of Chinese herbal medicine extract: Ginger, ginseng and Polygonum multiflorum are mixed and crushed, heated and boiled for extraction, the extraction is repeated 2-3 times, filtered, the filtrate is combined, concentrated, and dried to obtain an extract; S5. Preparation of embedded microspheres: After the stem cell solution in step S2 is revived, the exosomes prepared in step S3 are added, sodium alginate and an emulsifier are added, and the mixture is stirred and mixed evenly, and then added dropwise to the fish oil, emulsified, and calcium chloride solution is added dropwise, solidified at room temperature, centrifuged, washed, and dried to prepare embedded microspheres; S6. Preparation of a stem cell extract composition for promoting hair growth: Evenly mix the embedded microspheres and the extract to prepare a stem cell extract composition for promoting hair growth.

3. The preparation method according to claim 2, characterized in that: In step S1, the content of fetal bovine serum in the culture medium is 9-11wt%, the content of dexamethasone is 5-15nmol / L, the content of penicillin is 100-120U / mL, the content of streptomycin is 100-120μg / mL, the content of interleukin-2 is 5-15μg / mL, and the content of magnetic composite particles is 400-500μg / mL; the preparation method of the magnetic composite particles is as follows: T1. Preparation of magnetic oxide: ball-mill zirconium oxide ceramic powder, add it into water, add ferric chloride and ferrous chloride, add ammonia water under the protection of inert gas, heat and stir to react, centrifuge, wash, dry, and calcine to obtain magnetic oxide; T2. Modification: Add magnetic oxide into water, add tannic acid and catalyst, heat and stir to react, separate by magnet, wash and dry to obtain modified magnetic oxide; T3. Loading: Add the modified magnetic oxide into water, add NHS and EDC, stir to activate, add transforming growth factor-β and glutathione, stir to react, separate with a magnet, wash, and dry to obtain magnetic composite particles.

4. The preparation method according to claim 3, characterized in that: The ball milling time in step T1 is 0.5-1h, the mass ratio of the zirconium oxide, ferric chloride and ferrous chloride is 10:3.24:1.26, the temperature of the heating and stirring reaction is 80-90°C, the time is 3-5h, and the temperature of the calcination is 400-600°C, and the time is 1-2h; the mass ratio of the magnetic oxide, tannic acid and catalyst in step T2 is 10:2-3:0.1-0.5, the catalyst is a Tris-HCl solution with a pH of 8.5-9.5, the temperature of the heating and stirring reaction is 40-50°C, and the time is 2-4h.

5. The preparation method according to claim 2, characterized in that: In step T3, the mass ratio of the modified magnetic oxide, NHS, EDC, transforming growth factor-β, and glutathione is 12-15:2-3:1.5-2.5:3-4:2-3, the stirring activation time is 20-40 minutes, and the stirring reaction time is 10-12 hours.

6. The preparation method according to claim 2, characterized in that: The digestion solution in step S2 is a PBS buffer with a pH of 7.5-7.7 containing 1-2wt% trypsin and 0.5-1wt% alkaline protease; the culture conditions are 36-38°C, CO2 concentration is 3-6v / v%, the low-frequency magnetic field culture time is 5-7d, the low-energy laser culture time is 3-5d, the frequency of the low-frequency magnetic field is 40-50Hz, the stimulation time is 30-40min / d, and the energy density of the low-energy laser is 3-7J / cm 2 , wavelength is 600-800nm, total irradiation time is 3-5min / d, and each irradiation time is 30-60s.

7. The preparation method according to claim 2, characterized in that: In step S4, the mass ratio of ginger, ginseng and Polygonum multiflorum is 7-10:1-2:5-7, and the time for heating and boiling the extract is 2-4 hours.

8. The preparation method according to claim 2, characterized in that: The mass ratio of the stem cell solution, exosomes, and sodium alginate in step S5 is 15-30:2-3:7-10.

9. The preparation method according to claim 2, characterized in that: The mass ratio of the embedded microspheres to the extract in step S6 is 12-15:7-10.

10. A stem cell extract composition for promoting hair growth obtained by the preparation method according to any one of claims 1 to 9.

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