Hair follicle repair protein composition, method for producing the same, and its use

Abstract

The hair follicle repair protein composition is prepared by adding 20 U / mL to 35 U / mL of either one or a combination of nuclease and ultranuclease to a cell protein extract, enzymatically decomposing the extract for 15 to 40 minutes at 37°C±1°C, and isolating and purifying the enzymatic decomposition solution. The obtained cell protein extract and cell protein composition have a repairing effect on damaged hair follicle cells, efficiently repair damaged hair follicles, and significantly improve the activity of hair follicles. They also have the advantages of being highly pure, stable, safe, and effective, and effectively solving the problems that viable cells require refrigeration and that their activity is limited to the cell viability time.

Description

[Technical Field]

[0001] This invention belongs to the field of biomedical technology and specifically relates to a hair follicle repair protein composition, a method for producing the same, and its use. [Background technology]

[0002] Abundant hair volume is an important indicator of overall health and hair health. Hair follicle health is the foundation of hair health. The key to maintaining hair follicle health is activating the stromal cells within the scalp system. Hair follicles experience a cyclical growth cycle consisting of growth, regression, and resting phases. The cyclical changes in hair follicles are closely related to the cyclical division, proliferation, and differentiation of stromal cells. Highly active stromal cells continuously release large amounts of stromal growth factors, promoting a healthy cycle of self-repair, growth, division, and hair growth.

[0003] Modern people are affected by factors such as busy lifestyles, work stress, and environmental pollution, leading to problems such as easily broken hair, premature aging, severe hair loss, and hair regeneration disorders. These issues have a serious impact on personal image and can even lead to psychological disorders. According to data from the National Health Commission of China, there are over 260 million people with hair loss in China, with men accounting for approximately 63%, and hair loss mainly being due to male hormones. The trend of hair loss occurring at younger ages is evident, with 60% showing symptoms of hair loss around the age of 25. Among the "post-90s" generation (a term used in China for young people born between 1990 and 2000), the proportion of people with hair loss is high at 84%, and 46.7% experience severe hair loss.

[0004] Hair disorders include physiological or pathological symptoms such as sparse hair, excessive hair, abnormal hair distribution, abnormal hair density, abnormal hair diameter, abnormal hair pigmentation, and abnormal hair shaft. Hair loss includes physiological hair loss and pathological hair loss. Physiological hair loss includes spontaneous hair loss, seasonal hair loss, infant hair loss, senile hair loss, and postpartum hair loss. Pathological hair loss includes non-scarring hair loss and scarring hair loss. Non-scarring hair loss includes male hormone-induced hair loss (approximately 95%), alopecia areata (approximately 4%), syphilitic hair loss, trichotillomania, telogen effluvium, anagen effluvium, traction hair loss, physical hair loss, alopecia folliculitis, perifolliculitis capitis abscedens et suffodiens, and congenital hypotrichosis. Hair regrowth occurs when the pathogenic factor of non-scarring hair loss is removed. Scarring alopecia permanently destroys hair follicles, resulting in permanent hair loss. Causes include trauma, infection, inflammatory skin diseases (e.g., lupus erythema discoide, lichen planus), scalp lichen planus, lupus erythema discoide, radiation alopecia, female fibrous alopecia, and scalp burns.

[0005] In patients with pathological alopecia, the amount of hair is noticeably reduced due to hair abnormalities and excessive shedding. Common etiologies of alopecia include excess male hormones, genetics, excessive stress, pregnancy, disease, medication, and injury. Changes in hormone levels in the body can worsen hair loss due to diseases such as autoimmune diseases, thyroid diseases, lupus erythematosus, diabetes, iron deficiency, eating disorders, and anemia. Temporary hair loss can be caused by antitumor drugs, anticoagulants, antihypertensive drugs, contraceptives, burns, various injuries, and X-ray radiation.

[0006] Seborrheic alopecia (androgenetic alopecia, AGA) is a hair disorder characterized by excessive oil loss from the scalp, leading to gradually thinner, softer, and less abundant hair. It may also be accompanied by scalp itching, dandruff, and other symptoms. It is a progressive hair follicle lesion with genetic involvement and dependence on the effects of male hormones. The total number of AGA patients in China exceeds 100 million, and the prevalence is higher in men (20.2%) than in women (5.1%).

[0007] Cells are the basic units of life and the foundation of a living organism's health. When the oxidation-reduction balance of a living organism is disrupted, it leads to a disruption of oxidation-reduction signals and control, inducing oxidative stress damage and various diseases. Studies have already shown that factors such as the number and activity of hair follicle stem cells and the regenerative capacity of hair follicles are directly related to hair loss. A decrease in the number and activity of hair follicle stem cells weakens the differentiation ability of hair follicle cells, affecting the hair regeneration cycle, which in turn causes hair follicle blockage and leads to permanent hair loss. Major methods for treating hair loss include hair washing care, drug therapy, laser hair regrowth, and hair transplantation. Hair washing care mainly involves stimulating the scalp with topical products such as hair regrowth liquids or hair regrowth agents to promote blood circulation in the scalp, thereby promoting blood supply to the head, nourishment of the head, and hair growth. However, it has drawbacks such as being ineffective for hair regrowth and hair loss prevention, and being ineffective against damaged or dormant hair follicles. Drug therapy includes oral medications (e.g., finasteride, spironolactone, vitamin B2, vitamin B6, tancinon capsules, glycyrrhizin tablets, white peony sugar capsules, etc.) and topical medications (e.g., minoxidil solution, halcinonide solution, selenium sulfide preparations, etc.), but these have drawbacks such as poor efficacy in treating hair loss, significant side effects, and drug dependence. Laser hair regrowth utilizes low-energy laser irradiation with a wavelength of 670nm to enhance cell activity, promote microcirculation of blood, increase blood supply and nutrients to the scalp, promote the absorption of nutrients into the hair, improve the activity of hair matrix cells and hair follicle stem cells, accelerate protein synthesis, and activate hair follicles. However, it has drawbacks such as slow hair regrowth and high cost. Hair transplantation requires surgery, in which hair follicles from the back and sides of the patient's head are selected as hair sources. After separating the harvested hair follicles into single or multiple follicles, a single or multiple follicle is transplanted to the area of ​​hair loss that needs transplantation using precise microsurgery. This ensures that the newly transplanted single or multiple follicles survive and grow in the new transplant site, further restoring the local hair distribution and density.While hair transplantation can solve the problem of completely blocked hair follicles, it is limited by the availability of transplantable sites and hair follicle resources. Furthermore, hair transplantation surgery is painful, carries a risk of infection, does not guarantee the survival rate of newly transplanted hair follicles, fails to solve the problem of repairing damaged hair follicles, leaves scars at the transplant site affecting aesthetic appearance, and is expensive. For these reasons, there is an urgent need in clinical practice for a hair growth composition that is more effective in promoting hair growth and preventing hair loss, has reliable therapeutic effects, is safe, and is effective.

[0008] When cells and microorganisms are subjected to stress induction by external stimuli and exogenous stressors (including cold, heat, acid, alkali, electric current, radiation, chemical substances, etc.), they induce a stress response that causes them to produce stress proteins. Reference 1 (New limonophyllines AC from the stem of Atalantia monophylla and cytotoxicity against cholangiocarcinoma and HepG2 cell lines, Arch. Pharm. Res. (2018) 41:431~437) discloses that compounds 1-16, extracted from the Rutaceae plant (Atalantia monophylla), have activity that inhibits the proliferation of tumor cells, etc.

[0009] Mesenchymal stem cells (MSCs) possess self-renewal and multidirectional differentiation capabilities, are widely present in tissues such as bone marrow, fat, synovial membrane, dental pulp, amniotic fluid, placenta, umbilical cord, embryo, umbilical cord blood, amniotic membrane, peripheral blood, muscle, and urine, have a wide supply source, do not require matching, have a low infection rate, exhibit strong differentiation potential, strong proliferative capacity, and are easy to collect. They can produce active factors such as stem cell growth factor (SCF), nerve growth factor (NGF), interleukin-6 (IL-6), interleukin-7 (IL-7), tumor necrosis factor (TNF), and interferon (IFN), and are involved in regulating processes such as cell proliferation, apoptosis, cell differentiation, antiviral activity, and immunomaturation. They can be used for immunomodulation, tissue repair, and treatment of diseases such as acute lung injury, severe pneumonia, and acute respiratory distress syndrome. However, MSC products require refrigeration during production, storage, transportation, and use, and their cellular activity can only be maintained for less than 12 hours, thus limiting their therapeutic use. Therefore, there is a need to develop safe and effective skin repair agents to meet clinical needs. [Overview of the project] [Problems that the invention aims to solve]

[0010] The object of the present invention is to provide a hair follicle repair protein composition. [Means for solving the problem]

[0011] A hair follicle repair protein composition, the preparation of which is Step (1) involves adding 20 U / mL to 35 U / mL of either a nuclease or an ultranuclease, or a combination thereof, to the cell protein extract of the present invention, and then performing enzymatic decomposition at 37°C ± 1°C for 15 min to 40 min to prepare an enzymatic decomposition solution. Under the condition of 2°C to 8°C, the enzymatic hydrolysis solution prepared in step (1) is formulated to 5 to 15 mg / ml with an elution solvent containing 300 mmol / L sodium chloride in 50 mmol / L phosphate buffer (pH 6.8), passed through a chromatography column at an elution flow rate of 0.1 to 1 ml / min, and the elution site with an ultraviolet wavelength of 280 nm is monitored and collected in step (2).

[0012] In a preferred technical solution of the present invention, the preparation of the protein extract is as follows Density is 5.0×10 6 cells / mL to 1.0×10 7 mesenchymal passage cells / mL are placed in a medium containing 40 to 50% DMEM / F12, 40 to 50% RPMI1640, bovine serum protein (BSA) 0.1 to 2%, epidermal growth factor (EGF) 1 to 15 μg / mL, fibroblast growth factor (FGF) 1 to 15 μg / mL, insulin transferrin 1 to 15 μg / mL, amino acid compound (18AA) 0.01 to 0.1%, and a stress substance selected from any one or a combination of compounds 1 to 16 at 2 to 10 μmol / L. After culturing for 10 to 60 min at 37.0°C ± 0.5°C and 5% ± 1.0% CO2 conditions, they are separated, washed, and the cells are collected in step S-1.

Chemical formula

Chemical formula

[0013] In a preferred technical solution of the present invention, the medium in step S-1 contains 42-45% DMEM / F12, 42-45% RPMI1640, 0.5-1.5% bovine serum protein (BSA), 5-10 μg / mL epidermal growth factor (EGF), 5-10 μg / mL fibroblast growth factor (FGF), 5-10 μg / mL insulin transferrin, 0.02-0.05% amino acid compound (18AA), and 3-8 μmol / L stress substance.

[0014] In a preferred technical solution of the present invention, the medium in step S-1 contains 45% DMEM / F12, 45% RPMI1640, 0.5% bovine serum protein (BSA), 10 μg / mL epidermal growth factor (EGF), 10 μg / mL fibroblast growth factor (FGF), 10 μg / mL insulin transferrin, 0.05% amino acid compound (18AA), and 4-6 μmol / L stress substance.

[0015] In a preferred technical solution of the present invention, the density of mesenchymal passage cells in step S-1 is 6.0×10 6 ~2.0×10 7 cells / mL, preferably 8.0×10 6 ~1.0×10 7 cells / mL.

[0016] In a preferred technical solution of the present invention, the mesenchymal passage cells in step S-1 are cultured in the medium for 15-50 min, preferably 20-40 min.

[0017] In a preferred technical solution of the present invention, the solvent for washing the cells in step S-1 is any one or a combination selected from physiological saline, 5% glucose solution, phosphate buffer solution (PBS), TBPS buffer solution, TBST buffer solution, and Tris buffer solution. The number of times of washing the cells is 2-5 times, preferably 3-4 times.

[0018] In a preferred technical application of the present invention, the separation described in step S-1 is one or a combination selected from centrifugation and filtration, where the centrifugation conditions are 1000-2000 rpm * 3-15 min, preferably 1200 rpm-1500 rpm * 5-10 min.

[0019] In a preferred technical application of the present invention, the ultrasonic conditions in step S-2 are 2°C to 8°C, 25kHz, and 360W, and ultrasonic treatment is performed for 1 to 5 minutes, with a 3-second interval between operations.

[0020] In a preferred technical application of the present invention, the separation described in step S-3 is one or a combination selected from centrifugation at 2000-8000 rpm for 10-30 minutes, multi-stage centrifugation, and multi-stage filtration, preferably at 3000-7000 rpm for 15-25 minutes.

[0021] In a preferred technical application of the present invention, the multi-stage centrifugal separation in step S-3 is performed sequentially at 3000-4000 rpm for 3-5 minutes, 5000-6000 rpm for 3-5 minutes, and 7000 rpm for 5-8 minutes.

[0022] In a preferred technical application of the present invention, the pore size of the filtration membrane for the multi-stage filtration is one selected from 80 μm, 50 μm, 30 μm, 10 μm, and 5 μm.

[0023] In a preferred technical application of the present invention, the cell protein extract prepared in step S-3 is enzymatically degraded using either a nuclease or an ultranuclease, and then separated and purified.

[0024] In a preferred technical application of the present invention, the nuclease is one selected from RNA nucleases and DNA nucleases, or a combination thereof.

[0025] In a preferred technical application of the present invention, the molecular weight of the hair follicle repair protein composition is 20 kDa to 300 kDa, preferably 50 kDa to 200 kDa.

[0026] In a preferred technical application of the present invention, the cell protein extract prepared in step S-3 or the protein composition prepared in step (2) is frozen and stored, preferably at -40°C to -20°C.

[0027] In a preferred technical application of the present invention, the cell protein extract prepared in step S-3 or the protein composition prepared in step (2) is mixed with mannitol, sorbitol, dextran, glycerol, sucrose, trehalose, glucose, lactose, maltose, glucan, tricaprylin (HES), and polyethylene glycol. , A lyophilization protective agent, which is one or a combination of phosphate, acetate, citrate, sorbitol, or starch, is added and lyophilized to produce a lyophilized cell protein extract preparation or a lyophilized protein composition preparation.

[0028] In a preferred technical application of the present invention, the lyophilized cell protein extract or protein composition formulation contains 0.5 to 8%, preferably 1 to 5%, of the lyophilized protective agent by mass.

[0029] In a preferred technical application of the present invention, a protein stabilizer, selected from albumin, zinc salts, and aluminum salts, is optionally added to the cell protein extract prepared in step S-3 or the protein composition prepared in step (2).

[0030] In a preferred technical application of the present invention, the lyophilized cell protein extract preparation or the lyophilized protein composition preparation has a pH of 6 to 8, preferably 7 to 7.5.

[0031] In a preferred technical example of the present invention, the protein composition in the hair follicle repair protein composition is as shown in Figure 2.

[0032] In a preferred technical solution of the present invention, immediately before using the freeze-dried preparation, it is redissolved in an isotonic solution which is any one selected from physiological saline, 5% glucose solution, phosphate buffer (PBS), TBPS buffer, TBST buffer, Tris buffer or a combination thereof, and then used by any one or a combination of the methods of smearing, rolling needle, micro needle, massage, intravenous injection, intramuscular injection, subcutaneous injection, acupoint injection, lumbar puncture.

[0033] In a preferred technical solution of the present invention, the culture of the primary mesenchymal stem cells is a culture method in the art.

[0034] In a preferred technical solution of the present invention, the culture of the mesenchymal passage stem cells is to add the primary mesenchymal stem cells to a passage medium at an initial density of 5.0×10 5 ~5.0×10 6 cells / ml, and then culture it at 37.0°C±0.5°C and 5%±1.0% CO2 for 10 to 15 days. Observe the yellowing of the passage medium every 2 to 3 days, and then replace half of the passage medium. Here, the passage medium is a DMEM / F12 medium containing 10% FBS, 100 U / ml penicillin and 100 μg / ml streptomycin, including steps.

[0035] In a preferred technical solution of the present invention, the culture of the primary mesenchymal stem cells is After washing and disinfecting the umbilical cord, dissect the tissue, take the Wharton's jelly layer tissue, cut it into small pieces of 3 mm 3 , centrifuge and wash to collect tissue pieces, put them into a DMEM / F12 medium containing 10% fetal bovine serum FBS, 100 μg / ml penicillin, and 100 μg / ml streptomycin, and then culture it at 37.0°C±0.5°C and 5%±1.0% CO2. Replace half of the medium every 2 to 3 days and culture until cells emerge from the tissue pieces, step A; Step B includes shaking, collecting the lower layer cells and washing with PBS, then adding 0.25% trypsin and digesting for 2-3 minutes, adding the same volume of trypsin stop solution to stop digestion, gently blowing and tapping with a pipette, centrifugation at 1200-1500 rpm / min for 5-8 minutes, and then collecting the cells.

[0036] The object of the present invention is to provide a method for producing a cell protein extract having a hair follicle repair effect, and this method is Density is 5.0 × 10 6 pieces / mL~1.0×10 7 Step S-1 involves placing mesenchymal passaged cells / mL into a culture medium containing 40-50% DMEM / F12, 40-50% RPMI1640, 0.1-2% bovine serum protein (BSA), 1-15 ug / mL epidermal growth factor (EGF), 1-15 ug / mL fibroblast growth factor (FGF), 1-15 ug / mL insulin transferrin, 0.01-0.1% amino acid compound (18AA), and one or a combination of 1-16 compounds selected from 2-10 μmol / L. The medium is then cultured at 37.0°C ± 0.5°C and 5% ± 1.0% CO2 for 20-40 minutes, after which the cells are separated, washed, and collected. The collected cells were 5.0 × 10 6 pieces / mL~5.0×10 7 Step S-2 involves dispersing the cells in one or a combination of physiological saline, 5% glucose solution, phosphate buffer (PBS), TBPS buffer, TBST buffer, or Tris buffer to a density of cells / mL, and then sonicating the mixture at 2°C to 8°C to prepare a cell lysate. Step S-3 includes separating the cell lysate prepared in step S-2, and then sequentially filtering the resulting separation through 0.45 μm and 0.22 μm filtration membranes.

[0037] In a preferred technical application of the present invention, the culture medium in step S-1 contains 42-45% DMEM / F12, 42-45% RPMI1640, 0.5-1.5% bovine serum protein (BSA), 5-10 ug / mL epidermal growth factor (EGF), 5-10 ug / mL fibroblast growth factor (FGF), 5-10 ug / mL insulin transferrin, 0.02-0.05% amino acid compound (18AA), and 3-8 μmol / L of stress substance.

[0038] In a preferred technical application of the present invention, the culture medium in step S-1 contains 45% DMEM / F12, 45% RPMI1640, 0.5% bovine serum protein (BSA), 10 ug / mL epidermal growth factor (EGF), 10 ug / mL fibroblast growth factor (FGF), 10 ug / mL insulin transferrin, 0.05% amino acid compound (18AA), and 4-6 μmol / L of stress substance.

[0039] In a preferred technical application of the present invention, the mesenchymal cell density in step S-1 is 6.0 × 10⁶ 6 ~2.0×10 7 The concentration is 10 cells / mL, preferably 8.0 × 10 6 ~1.0×10 7 The concentration is cells / mL.

[0040] In a preferred technical method of the present invention, the mesenchymal subculture cells from step S-1 are cultured in culture medium for 15 to 50 minutes, preferably 20 to 40 minutes.

[0041] In a preferred technical application of the present invention, the solvent used to wash the cells in step S-1 is one or a combination selected from physiological saline, 5% glucose solution, phosphate buffer (PBS), TBPS buffer, TBST buffer, and Tris buffer, and the number of cell washes is 2 to 5 times, preferably 3 to 4 times.

[0042] In a preferred technical application of the present invention, the separation described in step S-1 is one or a combination selected from centrifugation and filtration, where the centrifugation conditions are 1000-2000 rpm * 3-15 min, preferably 1200 rpm-1500 rpm * 5-10 min.

[0043] In a preferred technical application of the present invention, the ultrasonic conditions in step S-2 are 2°C to 8°C, 25kHz, and 360W, and ultrasonic treatment is performed for 1 to 5 minutes, with a 3-second interval between operations.

[0044] In a preferred technical application of the present invention, the separation described in step S-3 is one or a combination selected from centrifugation at 2000-8000 rpm for 10-30 minutes, multi-stage centrifugation, and multi-stage filtration, preferably at 3000-7000 rpm for 15-25 minutes.

[0045] In a preferred technical application of the present invention, the multi-stage centrifugal separation in step S-3 is performed sequentially at 3000-4000 rpm for 3-5 minutes, 5000-6000 rpm for 3-5 minutes, and 7000 rpm for 5-8 minutes.

[0046] In a preferred technical application of the present invention, the pore size of the filtration membrane for the multi-stage filtration is one selected from 80 μm, 50 μm, 30 μm, 10 μm, and 5 μm.

[0047] In a preferred technical application of the present invention, the cell protein extract prepared in step S-3 is frozen and stored, preferably at -40°C to -20°C.

[0048] In a preferred technical application of the present invention, the cell protein extract prepared in step S-3 is enzymatically degraded using either a nuclease or an ultranuclease, and then separated and purified.

[0049] In a preferred technical application of the present invention, the culture of the primary mesenchymal stem cells is a culture method of the present art.

[0050] In a preferred technical application of the present invention, the culture of the mesenchymal subcultivated stem cells is performed using primary mesenchymal stem cells in a 5.0 × 10⁻¹⁶ 5 ~5.0×10 6 The step includes adding the cells / ml to the subculture medium to achieve a density of cells / ml, then culturing it at 37.0°C ± 0.5°C and 5% ± 1.0% CO2 for 10 to 15 days, observing the yellowing of the subculture medium every 2 to 3 days, and then replacing half of the subculture medium, where the subculture medium is DMEM / F12 medium containing 10% FBS, 100 U / ml penicillin, and 100 U / ml streptomycin.

[0051] In a preferred technical application of the present invention, the culture of primary mesenchymal stem cells is performed. After cleaning and disinfecting the umbilical cord, the tissue was dissected, and a 3mm sample of Wharton's jelly layer tissue was taken. 3 Step A involves cutting the tissue into small pieces, centrifuging and washing them to collect the tissue fragments, adding them to DMEM / F12 medium containing 10% fetal bovine serum (FBS), 100 ug / ml penicillin, and 100 ug / ml streptomycin, and then culturing them at 37.0°C ± 0.5°C and 5% ± 1.0% CO2, changing half of the medium every 2-3 days, and culturing until cells emerge from the tissue fragments. Step B includes shaking, collecting the lower layer cells and washing with PBS, then adding 0.25% trypsin and digesting for 2-3 minutes, adding the same volume of trypsin stop solution to stop digestion, gently blowing and tapping with a pipette, centrifugation at 1200-1500 rpm / min for 5-8 minutes, and then collecting the cells.

[0052] The object of the present invention is to provide a method for producing a hair follicle repair protein composition, and this method is Step (1) involves adding 20 U / mL to 35 U / mL of either a nuclease or an ultranuclease, or a combination thereof, to the cell protein extract of the present invention, and then performing enzymatic decomposition at 37°C ± 1°C for 15 min to 40 min to prepare an enzymatic decomposition solution. Step (2) includes preparing the enzyme hydrolysate prepared in step (1) at 2°C to 8°C, mixing it to 5 to 15 mg / ml with an elution solvent consisting of 50 mmol / L phosphate buffer (pH 6.8) containing 300 mmol / L sodium chloride, passing it through a chromatography column at an elution flow rate of 0.1 to 1 ml / min, and monitoring and collecting the elution site at an ultraviolet wavelength of 280 nm.

[0053] In a preferred technical application of the present invention, the nuclease is one selected from RNA nucleases and DNA nucleases, or a combination thereof.

[0054] In a preferred technical application of the present invention, the molecular weight of the hair follicle repair protein composition is 20 kDa to 300 kDa, preferably 50 kDa to 200 kDa.

[0055] In a preferred technical application of the present invention, the protein composition prepared in step (2) is frozen and stored, preferably at -40°C to -20°C.

[0056] In a preferred technical application of the present invention, the protein composition prepared in step (2) contains mannitol, sorbitol, dextran, glycerol, sucrose, trehalose, glucose, lactose, maltose, glucan, tricaprylin (HES), and polyethylene glycol. , A freeze-drying protective agent, which is one or a combination of nitrate, acetate, citrate, sorbitol, and starch, is added and freeze-dried to obtain the product.

[0057] In a preferred technical application of the present invention, the freeze-dried formulation contains 0.5 to 8%, preferably 1 to 5%, of the freeze-dried protective agent by mass.

[0058] In a preferred technical application of the present invention, a protein stabilizer is optionally added to the protein composition prepared in step (2), wherein the protein stabilizer is selected from albumin, zinc salts, and aluminum salts.

[0059] In a preferred technical application of the present invention, the freeze-dried protein composition formulation has a pH of 6 to 8, preferably 7 to 7.5.

[0060] In a preferred technical example of the present invention, the protein composition in the hair follicle repair protein composition is as shown in Figure 2.

[0061] In a preferred technical application of the present invention, the lyophilized preparation is redissolved immediately before use in an isotonic solution selected from physiological saline, 5% glucose solution, phosphate buffer (PBS), TBPS buffer, TBST buffer, and Tris buffer, or a combination thereof, and then used by one or a combination thereof via smearing, rolling needle, microneedling, massage, intravenous injection, intramuscular injection, subcutaneous injection, acupuncture injection, or lumbar puncture.

[0062] Another object of the present invention is to provide a hair follicle repair composition comprising one or a combination thereof of a cell protein extract or a hair follicle repair protein composition having the hair follicle repair effect of the present invention and a pharmaceutically acceptable carrier.

[0063] The pharmaceutically acceptable dose or type of carrier of the present invention is determined by factors such as the physicochemical properties and content of the active ingredient in the composition, the type of formulation, the solubility and bioavailability of the formulation.

[0064] The compositions of the present invention can be in dosage forms of the art and can be prepared using pharmaceutical techniques of the art.

[0065] In a preferred technical application of the present invention, the composition is one selected from a freeze-drying agent, a gelling agent, a nasal spray, an ointment, a cream, an emulsion, a liquid dressing, an injection, and a suppository.

[0066] In a preferred technical application of the present invention, the method of administering the composition is one or a combination selected from smearing, rolling needles, microneedling, and massage.

[0067] Another object of the present invention is to provide the use of the cell protein extract, hair follicle repair protein composition, or composition thereof having a hair follicle repair effect as described in the present invention in the production of cell repair or hair follicle repair products.

[0068] In a preferred technical application of the present invention, the product is selected from any one of the following: a hair growth product, a product for preventing and treating hair diseases, a product for preventing and treating pathological hair loss, a product for delaying physiological hair loss, and a product for improving the scalp environment, wherein the hair disease is selected from any one or a combination thereof from thinning hair, excessive hair, abnormal distribution, abnormal hair density, abnormal hair diameter, abnormal hair pigment, and hair shaft; the pathological hair loss is selected from any one of the following: androgenous hair loss, alopecia areata, syphilitic hair loss, trichotillomania, physical hair loss, alopecia folliculitis, perifolliculitis capitis abscedens et suffodiens, congenital hypotrichosis, scalp lichen planus, hair lichen planus, scalp lupus discoid erythematous, radioactive hair loss, female fibrous hair loss, and scalp burns; and the physiological hair loss is selected from any one of the following: spontaneous hair loss, seasonal hair loss, infantile hair loss, senile hair loss, and postpartum hair loss. Another object of the present invention is to provide the use of the hair growth product of the present invention in a dosage regimen for hair growth treatment and prevention of hair loss, wherein the subject receives a treatment in which the product is administered once every two to four weeks, with three administrations forming one treatment course, by puncturing the hair loss area with either a microneedle or a rolling needle, then applying the product topically by smearing, and massaging until completely absorbed.

[0069] In a preferred technical application of the present invention, the treatment site is locally anesthetized before treatment.

[0070] Another object of the present invention is to provide the use of compounds 1 to 16 in stress induction of stem cells to produce functional proteins having a reparative effect.

[0071] In a preferred technical application of the present invention, the repair is one of the following: cell repair, hair follicle repair, joint repair, or nerve repair.

[0072] Unless otherwise specified, when the present invention relates to a percentage of liquids, the percentage is volume / volume percentage; when the present invention relates to a percentage of liquids and solids, the percentage is volume / weight percentage; when the present invention relates to a percentage of solids and liquids, the percentage is weight / volume percentage; and for the remainder, it is weight / weight percentage.

[0073] Unless otherwise specified, the identification of mesenchymal stem cells (MSCs) in this invention is based on "Standards for the culture and quality control of umbilical cord mesenchymal stromal cells for neurorestorative clinical application." [Effects of the Invention]

[0074] Compared to the conventional technology, the present invention has the following beneficial effects.

[0075] 1. In this invention, a culture medium containing stress substances is scientifically screened to induce stress in mesenchymal stem cells to produce functional proteins that have the effect of cell repair and hair follicle repair. The culture medium also contains various growth factors and proteins that have the function of regulating cell growth and differentiation, cell repair and cell nutrition, and the resulting cell protein extract, cell protein composition, or composition thereof has a repair effect on cells and damaged hair follicle cells. After puncturing the hair loss area using either a microneedle or a rolling needle, the hair follicle repair protein composition of this invention is introduced locally and percutaneously, and the root of the hair follicle is on the surface of the scalp. It provides a direct and effective administration route, efficiently repairs damaged hair follicles, significantly improves hair follicle activity, and offers advantages such as high purity, good stability, safety and effectiveness, effectively solving the problem of viable cells requiring refrigeration and their activity being limited by the cell's viability time. It promotes the repair of damaged hair follicles and the production of collagen protein, promotes hair regeneration, significantly improves accompanying symptoms such as scalp itchiness, excessive oil loss, and dandruff, and improves patient medication compliance. It provides a reliable therapeutic effect, high absorption rate, no scalp irritation, is safe and effective, and offers a simple and rapid treatment method for clinical practice.

[0076] 2. The manufacturing method of the present invention has advantages such as being easy to operate, environmentally friendly, cost-effective, and suitable for industrial production. [Brief explanation of the drawing]

[0077] [Figure 1] This shows the electrophoretic separation results of the hair follicle repair protein composition of the present invention. [Figure 2] This is the result of high-performance liquid chromatography detection of the hair follicle repair protein composition of the present invention. [Figure 3] This study investigates the reparative effect of the cell protein extract of the present invention on oxidatively damaged skin. [Figure 4] These are the results of studies on the hair follicle repair effect of the hair follicle repair protein composition of the present invention in a mouse model of AGA, comparing the test group and the model group. [Figure 5]These are the H&E staining results for the test group and model group in a study of the hair follicle repair effect of the hair follicle repair protein composition of the present invention on an AGA mouse model (scale bar: 200 μm). [Figure 6] This shows the quantitative analysis of HF in the test group and model group mice in a study of the hair follicle repair effect of the hair follicle repair protein composition of the present invention on an AGA mouse model, where *** indicates p<0.005. [Modes for carrying out the invention]

[0078] The details of the present invention will be further interpreted and explained below with reference to specific examples, but the scope of protection of the present invention is not limited thereto.

[0079] 1. Culture of primary mesenchymal stem cells The culture of primary mesenchymal stem cells is After cleaning and disinfecting the umbilical cord, the tissue was dissected, and a 3mm sample of Wharton's jelly layer tissue was taken. 3 Step 1) involves cutting the tissue into small pieces, centrifuging and washing them, collecting the tissue pieces, placing them in a culture flask, adding DMEM / F12 medium containing 10% fetal bovine serum (FBS), 100 ug / ml penicillin, and 100 ug / ml streptomycin, then culturing at 37°C and 5% CO2 to promote adhesion to the flask wall, and every 2-3 days, after observing the yellowing of the medium, replacing half of the medium and culturing for 10-12 days until cells are visible emerging from the edges of the tissue pieces. Step 2) involves gently shaking to dislodge the tissue fragments, collecting the tissue fragments and lower layer cells separately, and then reattaching the collected tissue fragments to the wall and culturing them. After washing the collected low-level cells with PBS, an appropriate amount of 0.25% trypsin is added and digested for 2-3 minutes, then the digestion is stopped by adding the same volume of trypsin stop solution, the bottom of the flask is lightly blown and tapped with a pipette, and the cells are centrifuged at 1500 rpm / min for 5 minutes, after which the cells are collected, and the obtained step 3) is included.

[0080] 2. Subculture of primary mesenchymal stem cells Subculture of primary mesenchymal stem cells: Primary mesenchymal stem cells are cultured in 1.0 × 10⁻¹⁶ cultures. 5 ~6.0×10 5 Add 10% FBS, 100 U / ml penicillin, and 100 ug / ml streptomycin to DMEM / F12 medium to achieve an initial density of cells / ml. Then, culture at 37.0°C ± 0.5°C and 5% ± 1.0% CO2 for 10 to 15 days, and replace half of the medium every 2 to 3 days after observing the yellowing of the medium.

[0081] 3. For the preparation of compounds 1-16, refer to reference 1 (New limonophyllines AC from the stem of Atalantia monophylla and cytotoxicity against cholangiocarcinoma and HepG2 cell lines, Arch. Pharm. Res. (2018) 41:431-437).

[0082] (Example 1) Preparation of a cell protein extract having hair follicle repair effect according to the present invention The present invention provides a method for producing a cell protein extract having a hair follicle repair effect. Mesenchymal passaged cells 5.0 × 10 6 Step (1) involves adding a culture medium containing 45% DMEM / F12, 45% RPMI1640, 0.5% bovine serum protein (BSA), 10 ug / mL epidermal growth factor (EGF), 10 ug / mL fibroblast growth factor (FGF), 10 ug / mL insulin transferrin, 0.05% amino acid compound (18AA), and 5 μmol / L of compound 16 to achieve a density of cells / mL, then culturing it at 37°C under 5% CO2 conditions for 30 min, centrifugation at 1200 rpm for 5 min, washing three times with PBS, and then collecting the cells. The cells collected in step (1) were 8 × 10 6 Step (2) involves dispersing the cells in physiological saline to a density of cells / mL, and then performing sonication for 2 minutes at 2-8°C, 25kHz, and 360W, with 3 seconds of operation followed by a 1-second interval, to prepare a cell lysate. The cell lysate prepared in step (2) is centrifuged at 7000 rpm for 20 min, and the resulting centrifugal is sequentially filtered through 0.45 μm and 0.22 μm filtration membranes to obtain step (3).

[0083] (Example 2) Preparation of the hair follicle repair protein composition of the present invention The preparation of the hair follicle repair protein composition of the present invention is Step (1) involves adding 25 U / mL of ultranuclease (UCF.ME UltraNuclease) to the cell protein extract prepared in Example 1, performing enzymatic decomposition at 37°C for 30 minutes, and then preparing the enzymatic decomposition solution. Step (2) involves preparing the enzyme hydrolysate prepared in step (1) at 2°C to 8°C, mixing it to 10 mg / ml with an elution solvent consisting of 50 mmol / L phosphate buffer (pH 6.8) containing 300 mmol / L sodium chloride, passing it sequentially through a high-purity silica gel liquid chromatography guard column (WondaGuard C18, 4.6 × 5 mm) and a high-purity silica gel liquid chromatography preparation column (SHIMSEN Ankylo C18, 5 μm, 4.6 × 250 mm), monitoring and collecting the elution site at an elution rate of 0.1 to 1 ml / min and an ultraviolet wavelength of 280 nm, and obtaining the result.

[0084] (Example 3) Preparation of a freeze-dried hair follicle repair protein composition according to the present invention The cell protein composition prepared in Example 2 is mixed with the required amount of mannitol, stirred to ensure uniformity, and then freeze-dried. The resulting freeze-dried formulation contains 2% mannitol (m / m).

[0085] (Example 4) Preparation of cell protein extract having hair follicle repair effect according to the present invention The present invention provides a method for producing a cell protein extract having a hair follicle repair effect. Mesenchymal passaged cells 8.0 × 10 6Step (1) involves adding a culture medium containing 45% DMEM / F12, 45% RPMI1640, 0.5% bovine serum protein (BSA), 10 ug / mL epidermal growth factor (EGF), 10 ug / mL fibroblast growth factor (FGF), 10 ug / mL insulin transferrin, 0.05% amino acid compound (18AA), and 5 μmol / L of compound 13 to achieve a density of cells / mL, then culturing it at 37°C under 5% CO2 conditions for 30 minutes, centrifuging it at 1200 rpm for 5 minutes, washing it three times with PBS, and then collecting the cells. The cells collected in step (1) were 1.0 × 10 7 Step (2) involves dispersing the cells in physiological saline to a density of cells / mL, and then performing sonication for 2 minutes at 2-8°C, 25kHz, and 360W, with 3 seconds of operation followed by a 1-second interval, to prepare a cell lysate. The cell lysate prepared in step (2) is centrifuged at 7000 rpm for 20 min, and the resulting centrifugal is sequentially filtered through 0.45 μm and 0.22 μm filtration membranes to obtain step (3).

[0086] (Example 5) Preparation of the hair follicle repair protein composition of the present invention The preparation of the hair follicle repair protein composition of the present invention is Step (1) involves adding 20 U / mL of ultranuclease (UCF.ME UltraNuclease) to the cell protein extract prepared in Example 4, performing enzymatic decomposition at 37°C for 30 minutes, and then preparing the enzymatic decomposition solution. Step (2) involves preparing the enzyme hydrolysate prepared in step (1) at 2°C to 8°C, mixing it to 10 mg / ml with an elution solvent consisting of 50 mmol / L phosphate buffer (pH 6.8) containing 300 mmol / L sodium chloride, passing it sequentially through a high-purity silica gel liquid chromatography guard column (WondaGuard C18, 4.6 × 5 mm) and a high-purity silica gel liquid chromatography preparation column (SHIMSEN Ankylo C18, 5 μm, 4.6 × 250 mm), monitoring and collecting the elution site at an elution rate of 0.1 to 1 ml / min and an ultraviolet wavelength of 280 nm, and obtaining the result.

[0087] (Example 6) Preparation of a freeze-dried hair follicle repair protein composition according to the present invention Sorbitol is added to the cell protein composition prepared in Example 5, stirred, and mixed uniformly, then freeze-dried, and the resulting freeze-dried formulation contains 5% sorbitol (m / m).

[0088] (Example 7) Preparation of cell protein extract having hair follicle repair effect according to the present invention The present invention provides a method for producing a cell protein extract having a hair follicle repair effect. Mesenchymal passaged cells 1.0 × 10 7 Step (1) involves adding 45% DMEM / F12, 45% RPMI1640, 0.5% bovine serum protein (BSA), 10 ug / mL epidermal growth factor (EGF), 10 ug / mL fibroblast growth factor (FGF), 10 ug / mL insulin transferrin, 0.05% amino acid compound (18AA), and 8 μmol / L compound 14 to a medium to achieve a density of cells / mL, then culturing it at 37°C under 5% CO2 conditions for 30 minutes, centrifugating it at 1200 rpm for 5 minutes, washing it three times with PBS, and then collecting the cells. The cells collected in step (1) were 5.0 × 10 7 Step (2) involves dispersing the cells in physiological saline to a density of cells / mL, and then performing sonication for 2 minutes at 2-8°C, 25kHz, and 360W, with 3 seconds of operation followed by a 1-second interval, to prepare a cell lysate. The cell lysate prepared in step (2) is centrifuged at 7000 rpm for 20 min, and the resulting centrifugal is sequentially filtered through 0.45 μm and 0.22 μm filtration membranes to obtain step (3).

[0089] (Example 8) Preparation of the hair follicle repair protein composition of the present invention The preparation of the hair follicle repair protein composition of the present invention is Step (1) involves adding 30 U / mL of ultranuclease (UCF.ME UltraNuclease) to the cell protein extract prepared in Example 7, performing enzymatic decomposition at 37°C for 30 minutes, and then preparing the enzymatic decomposition solution. Step (2) involves preparing the enzyme hydrolysate prepared in step (1) at 2°C to 8°C, mixing it to 10 mg / ml with an elution solvent consisting of 50 mmol / L phosphate buffer (pH 6.8) containing 300 mmol / L sodium chloride, passing it sequentially through a high-purity silica gel liquid chromatography guard column (WondaGuard C18, 4.6 × 5 mm) and a high-purity silica gel liquid chromatography preparation column (SHIMSEN Ankylo C18, 5 μm, 4.6 × 250 mm), monitoring and collecting the elution site at an elution rate of 0.1 to 1 ml / min and an ultraviolet wavelength of 280 nm, and obtaining the result.

[0090] (Example 9) Preparation of a freeze-dried hair follicle repair protein composition according to the present invention Mannitol is added to the cell protein composition prepared in Example 8, stirred, and mixed uniformly, then freeze-dried, and the resulting freeze-dried formulation contains 5% mannitol (m / m).

[0091] (Example 10) Preparation of cell protein extract having hair follicle repair effect according to the present invention The present invention provides a method for producing a cell protein extract having a hair follicle repair effect. Mesenchymal passaged cells 5.0 × 10 6 Step (1) involves adding 45% DMEM / F12, 45% RPMI1640, 0.5% bovine serum protein (BSA), 10 ug / mL epidermal growth factor (EGF), 10 ug / mL fibroblast growth factor (FGF), 10 ug / mL insulin transferrin, 0.05% amino acid compound (18AA), and 2 μmol / L compound 15 to a medium to achieve a density of cells / mL, then culturing it at 37°C under 5% CO2 conditions for 30 minutes, centrifugating it at 1200 rpm for 5 minutes, washing it three times with PBS, and then collecting the cells. The cells collected in step (1) were 8.0 × 10 6Step (2) involves dispersing the cells in physiological saline to a density of cells / mL, and then performing sonication for 2 minutes at 2-8°C, 25kHz, and 360W, with 3 seconds of operation followed by a 1-second interval, to prepare a cell lysate. The cell lysate prepared in step (2) is centrifuged at 7000 rpm for 20 min, and the resulting centrifugal is sequentially filtered through 0.45 μm and 0.22 μm filtration membranes to obtain step (3).

[0092] (Example 11) Preparation of the hair follicle repair protein composition of the present invention The preparation of the hair follicle repair protein composition of the present invention is Step (1) involves adding 30 U / mL of ultranuclease (UCF.ME UltraNuclease) to the cell protein extract prepared in Example 10, performing enzymatic decomposition at 37°C for 30 minutes, and then preparing the enzymatic decomposition solution. Step (2) involves preparing the enzyme hydrolysate prepared in step (1) at 2°C to 8°C, mixing it to 10 mg / ml with an elution solvent consisting of 50 mmol / L phosphate buffer (pH 6.8) containing 300 mmol / L sodium chloride, passing it sequentially through a high-purity silica gel liquid chromatography guard column (WondaGuard C18, 4.6 × 5 mm) and a high-purity silica gel liquid chromatography preparation column (SHIMSEN Ankylo C18, 5 μm, 4.6 × 250 mm), monitoring and collecting the elution site at an elution rate of 0.1 to 1 ml / min and an ultraviolet wavelength of 280 nm, and obtaining the result.

[0093] (Example 12) Preparation of a freeze-dried hair follicle repair protein composition according to the present invention Dextran was added to the cell protein composition prepared in Example 11, stirred, and mixed uniformly, then freeze-dried to obtain a lyophilized formulation containing 1% dextran (m / m).

[0094] (Example 13) Preparation of the lyophilized cell protein extract formulation of the present invention The cell protein extract prepared in Example 1 is mixed with the required amount of mannitol, stirred, and then freeze-dried to obtain a freeze-dried formulation containing 2% mannitol (m / m).

[0095] (Example 14) Detection of the molecular weight distribution of the hair follicle repair protein composition of the present invention The molecular weight distribution of the nerve repair protein composition of the present invention was detected using standard molecular weight polyacrylamide gel electrophoresis. Step 1 involves selecting a glass plate with a thickness of 1.5 mm and placing it horizontally, then sequentially applying the prepared gel solution (15% lower layer gel, 4% separation gel) onto the glass plate as shown in Table 1, and inserting a comb vertically into the lower layer gel. [Table 1] Step 2 involves thawing markers (20kDa~245kDa) that have been frozen at -40°C, then mixing the markers and the lyophilized powders of the hair follicle repair protein compositions prepared in Examples 3, 6, 9, and 12 in PBS at a concentration of 10ug / ul for each test sample, adding 20ul of the test sample to the loading well, performing electrophoresis under 60~80V conditions until relatively clear bands appear, then adjusting the voltage to 100~120V until marker separation is complete, placing the PAGE gel in Coomassie brilliant blue staining solution, stopping the staining when clear bands appear on the gel, washing with pure water, and then destaining with a 10% acetic acid solution, stopping the destaining when the gel becomes clear. The results are shown in Figure 1, where band A is from Example 3, band B is from Example 6, band C is from Example 9, and band D is from Example 12.

[0096] (Example 15) High-performance liquid chromatography detection of the hair follicle repair protein composition of the present invention The freeze-dried powder of the hair follicle repair protein composition from Example 3 is dissolved in deionized water to prepare a 10 mg / ml test solution. Chromatography column: SHIMSEN Ankylo (300mm*4.6mm, D., 3um; P / N: 380-01215-05) Shimadzu Mobile phase: 50 mmol / L phosphate buffer (pH=6.8) containing 300 mmol / L sodium chloride, flow rate 0.3 mL / min, injection volume 10 μl, column temperature 25°C, detection wavelength 280 nm, isocratic elution, and collection for 30 min. See Figure 2 for results.

[0097] (Test Example 1) Study on the repair effect of the cellular protein extract having hair follicle repair effect of the present invention on oxidative damage. 3D Skin Model: EpiKutis (R) I purchased it from a retailer called EpiKutis. SLS Working Solution: 0.0080 g of SLS was weighed, dissolved in 2 mL of PBS solution, and filtered through 0.22 μm to prepare a 0.4% SLS mother liquor. 0.5 mL of the SLS mother liquor was aspirated, 0.5 mL of PBS was added, and a 0.2% SLS working solution was prepared. WY14643 Working Solution: 10 mg of WY14643 (PPARα stimulant) was weighed and dissolved in 1 mL of DMSO, and then mixed into a 30 mM WY14643 mother liquor. Subsequently, 10 μL of the WY14643 mother liquor (30 mM) was added to 6 mL of model culture medium and mixed into a 50 μM WY14643 working solution. Test sample: The lyophilized powder of the cell protein extract prepared in Example 13 was dissolved in physiological saline to obtain a 1% lyophilized cell protein extract solution. Model culture medium: DMEM basal medium. 0.9 mL of model culture medium was added to a 6-well plate, the 3D skin model was transferred to the 6-well plate, and the test number was labeled. Blank control (BC): The skin model was left untreated and incubated in a CO2 incubator (37°C, 5% CO2) for 24 hours. Negative control (NC): 25 μL of 0.2% SLS working solution was added to the surface of a skin model and incubated in a CO2 incubator (37°C, 5% CO2) for 24 hours. Positive control (PC): 25 μL of 0.2% SLS working solution was added to the surface of a skin model, and it was incubated in a CO2 incubator (37°C, 5% CO2) for 24 hours. Then, 25 μL of 50 μM WY14643 working solution was added, and it was incubated in a CO2 incubator (37°C, 5% CO2) for another 24 hours.

[0098] Test group: 25 μL of 0.2% SLS working solution was added to the surface of a skin model, incubated in a CO2 incubator (37°C, 5% CO2) for 24 hours, then 25 μL of 1% cell protein extract solution was added, and incubated again in a CO2 incubator (37°C, 5% CO2) for 24 hours. After incubation for 24 hours, the skin model was washed with PBS solution to remove any remaining fluid inside and outside the model. After fixing with 4% paraformaldehyde for 24 hours, the model was removed by circular excision, subjected to H&E staining, and then imaged and observed under a microscope. See Figure 3 for the results. The cell protein extract of the present invention significantly repairs oxidatively damaged skin.

[0099] (Test Example 2) Consideration of the hair growth effect of the hair follicle repair protein composition of the present invention Eleven C57BL / 6J male mice weighing 20-25g were selected and randomly divided into a model group and a drug group, with 5 mice in the model group and 6 in the drug group. An AGA mouse model was constructed by applying testosterone solution topically to the test animals daily. First, the dorsal skin of the test mice was depilated using animal scissors and depilatory cream, and then 0.1 mL / cm³ of 0.5% testosterone ethanol solution (w / v) (prepared by dissolving an appropriate amount of testosterone in a 50% ethanol solution (v / v)) was applied. 2The test mice were smeared daily with the testosterone ethanol solution to the bald skin of the test mice, and the test mice were also smeared with the testosterone ethanol solution for 33 consecutive days after baldness. For the test group, from day 8 to day 24 after baldness, the mice were smeared twice daily with the freeze-dried powder of the hair follicle repair protein composition of Example 3 of the present invention (2 mg dissolved in 200 µl of PBS) to a dose of 2 mg / smear / mouse. The hair growth of the mice was observed on days 0, 8, 16, and 24 after baldness. On day 25 after administration to the test animals, the mice were disposed of, and the skin from the back of the mice was taken and stained with HE (scale bar: 200 μm) to observe the hair growth and hair follicle regeneration of the bald skin. Three fields of view were randomly selected and the number of HFs was calculated. See Figures 4 to 6 for the results. In the model group of mice, the hair loss areas remained pink, showing little to no new hair growth or hair follicle regeneration. More than half of the HFs were in the resting phase, and the miniaturized HFs were located entirely within the dermis, resulting in incomplete hair tissue and skin morphology. In the test group of mice, the hair loss areas showed clear new hair growth and significantly more regenerated hair follicles, as well as the formation of hypertrophied bulbs and endroot sheaths located in the subcutaneous tissue. A large proportion of HFs had already entered the growth phase, and dense hair growth continued on the dorsal skin of the mice up to 33 days after hair loss. The hair tissue and skin morphology were complete, promoting hair follicle growth, maintaining skin morphology, and preserving the physiological function of the skin. The hair follicle repair protein composition of the present invention significantly promotes the repair of damaged hair follicles and hair regeneration, as well as activating HF stem cells (p<0.05).

[0100] The above-described description of specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art can make various changes or modifications based on the present invention, all of which should fall within the scope of protection of the claims of the present invention, as long as they do not deviate from the spirit of the invention.

Claims

1. A hair follicle repair protein composition, the preparation of which is Step (1) involves adding 20 U / mL to 35 U / mL of nuclease to a cell protein extract and performing enzymatic decomposition at 37°C ± 1°C for 15 min to 40 min to prepare an enzymatic decomposition solution. Step (2) includes preparing the enzyme hydrolysate prepared in step (1) at 2°C to 8°C, mixing it to a concentration of 5 to 15 mg / ml with an elution solvent consisting of 300 mmol / L sodium chloride in 50 mmol / L phosphate buffer at pH 6.8, passing it through a chromatography column at an elution flow rate of 0.1 to 1 ml / min, and monitoring and collecting the elution site at an ultraviolet wavelength of 280 nm. The preparation of the aforementioned cell protein extract is as follows: Step S-1 involves placing mesenchymal passaged cells with a density of 5.0 × 10⁶ cells / mL to 1.0 × 10⁷ cells / mL into a culture medium containing 40–50% DMEM / F12, 40–50% RPMI1640, 0.1–2% bovine serum protein (BSA), 1–15 ug / mL epidermal growth factor (EGF), 1–15 ug / mL fibroblast growth factor (FGF), 1–15 ug / mL insulin transferrin, 0.01–0.1% amino acid compound 18AA, and 2–10 μmol / L of a stress substance represented by the following formula. After culturing at 37.0°C ± 0.5°C and 5% ± 1.0% CO₂ for 10–60 mins, the cells are separated, washed, and collected. 【Chemistry 1】 Step S-2 involves dispersing the collected cells in one or a combination of physiological saline, 5% glucose solution, phosphate buffer, and Tris buffer to a density of 5.0 × 10⁶ cells / mL to 5.0 × 10⁷ cells / mL, and then sonicating the mixture at 2°C to 8°C to prepare a cell lysate. Step S-3 involves centrifuging the cell lysate prepared in step S-2, and then sequentially filtering the resulting separation through 0.45 μm and 0.22 μm filtration membranes. The aforementioned mesenchymal passaged cells are derived from the umbilical cord. Hair follicle repair protein composition.

2. The ultrasonic conditions for step S-2 are 2°C to 8°C, 25kHz, and 360W, and the ultrasonic treatment is performed for 1 to 5 minutes, with a 3-second interval between operations. The hair follicle repair protein composition according to claim 1.

3. The separation in step S-3 is centrifugation at 2000 to 8000 rpm for 10 to 30 minutes. The hair follicle repair protein composition according to claim 1.

4. The separation in step S-3 is multi-stage centrifugal separation, wherein the multi-stage centrifugal separation is performed in order at 3000-4000 rpm * 3-5 min, 5000-6000 rpm * 3-5 min, and 7000 rpm * 5-8 min. The hair follicle repair protein composition according to claim 1.

5. The molecular weight of the hair follicle repair protein composition is 20 kDa to 300 kDa. The hair follicle repair protein composition according to claim 1.

6. A method for producing a hair follicle repair protein composition, specifically, Step (1) involves adding 20 U / mL to 35 U / mL of nuclease to a cell protein extract and performing enzymatic decomposition at 37°C ± 1°C for 15 min to 40 min to prepare an enzymatic decomposition solution. Step (2) includes preparing the enzyme hydrolysate prepared in step (1) at 2°C to 8°C, mixing it to a concentration of 5 to 15 mg / ml with an elution solvent consisting of 300 mmol / L sodium chloride in 50 mmol / L phosphate buffer at pH 6.8, passing it through a chromatography column at an elution flow rate of 0.1 to 1 ml / min, and monitoring and collecting the elution site at an ultraviolet wavelength of 280 nm. The preparation of the aforementioned cell protein extract is as follows: Step S-1 involves placing mesenchymal passaged cells with a density of 5.0 × 10⁶ cells / mL to 1.0 × 10⁷ cells / mL into a culture medium containing 40–50% DMEM / F12, 40–50% RPMI1640, 0.1–2% bovine serum protein (BSA), 1–15 ug / mL epidermal growth factor (EGF), 1–15 ug / mL fibroblast growth factor (FGF), 1–15 ug / mL insulin transferrin, 0.01–0.1% amino acid compound 18AA, and 2–10 μmol / L of a stress substance represented by the following formula. After culturing at 37.0°C ± 0.5°C and 5% ± 1.0% CO₂ for 10–60 mins, the cells are separated, washed, and collected. 【Chemistry 2】 Step S-2 involves dispersing the collected cells in one or a combination of physiological saline, 5% glucose solution, phosphate buffer, and Tris buffer to a density of 5.0 × 10⁶ cells / mL to 5.0 × 10⁷ cells / mL, and then sonicating the mixture at 2°C to 8°C to prepare a cell lysate. Step S-3 involves centrifuging the cell lysate prepared in step S-2, and then sequentially filtering the resulting separation through 0.45 μm and 0.22 μm filtration membranes. The aforementioned mesenchymal passaged cells are derived from the umbilical cord. A method for producing a hair follicle repair protein composition.

7. A lyophilized hair follicle repair protein composition preparation prepared by adding a lyophilized protective agent, which is one or a combination selected from mannitol, sorbitol, dextran, glycerol, sucrose, trehalose, glucose, lactose, maltose, glucan, polyethylene glycol, phosphate, acetate, citrate, and starch, to the hair follicle repair protein composition described in Claim 1, and then lyophilizing the mixture.

8. A cell protein extract having a hair follicle repair effect, wherein the method for producing the extract is: Step S-1 involves placing mesenchymal passaged cells with a density of 5.0 × 10⁶ cells / mL to 1.0 × 10⁷ cells / mL into a culture medium containing 40–50% DMEM / F12, 40–50% RPMI1640, 0.1–2% bovine serum protein (BSA), 1–15 ug / mL epidermal growth factor (EGF), 1–15 ug / mL fibroblast growth factor (FGF), 1–15 ug / mL insulin transferrin, 0.01–0.1% amino acid compounds, and a stress substance represented by the following formula at a concentration of 2–10 μmol / L. This is then cultured for 10–60 mins at 37.0°C ± 0.5°C and 5% ± 1.0% CO₂, followed by separation, washing, and cell collection. 【Transformation 3】 Step S-2 involves dispersing the collected cells in one or a combination of physiological saline, 5% glucose solution, phosphate buffer (PBS), and Tris buffer to a density of 5.0 × 10⁶ cells / mL to 5.0 × 10⁷ cells / mL, and then sonicating the mixture at 2°C to 8°C to prepare a cell lysate. Step S-3 involves centrifuging the cell lysate prepared in step S-2, and then sequentially filtering the resulting separation through 0.45 μm and 0.22 μm filtration membranes. The aforementioned mesenchymal passaged cells are derived from the umbilical cord. A cell protein extract that has hair follicle repair effects.

9. A method for producing a cell protein extract having a hair follicle repair effect, more specifically, Step S-1 involves placing mesenchymal passaged cells with a density of 5.0 × 10⁶ cells / mL to 1.0 × 10⁷ cells / mL into a culture medium containing 40–50% DMEM / F12, 40–50% RPMI1640, 0.1–2% bovine serum protein (BSA), 1–15 ug / mL epidermal growth factor (EGF), 1–15 ug / mL fibroblast growth factor (FGF), 1–15 ug / mL insulin transferrin, 0.01–0.1% amino acid compounds, and a stress substance represented by the following formula at a concentration of 2–10 μmol / L. This is then cultured for 20–40 minutes at 37.0°C ± 0.5°C and 5% ± 1.0% CO₂, followed by separation, washing, and cell collection. 【Chemistry 4】 Step S-2 involves dispersing the collected cells in one or a combination of physiological saline, 5% glucose solution, phosphate buffer (PBS), and Tris buffer to a density of 5.0 × 10⁶ cells / mL to 5.0 × 10⁷ cells / mL, and then sonicating the mixture at 2°C to 8°C to prepare a cell lysate. Step S-3 involves centrifuging the cell lysate prepared in step S-2, and then sequentially filtering the resulting separation through 0.45 μm and 0.22 μm filtration membranes. The aforementioned mesenchymal passaged cells are derived from the umbilical cord. A method for producing a cell protein extract that has a hair follicle repair effect.

10. A lyophilized preparation of a cell protein extract having a hair follicle repair effect, wherein a lyophilized protective agent, which is one or a combination thereof selected from mannitol, sorbitol, dextran, glycerol, sucrose, trehalose, glucose, lactose, maltose, glucan, polyethylene glycol, phosphate, acetate, citrate, and starch, is added to the cell protein extract according to claim 8, and the preparation is lyophilized to produce a lyophilized preparation. A freeze-dried preparation containing cell protein extracts that have hair follicle repair effects.

11. A hair follicle repair composition comprising the hair follicle repair protein composition described in claim 1 and a pharmaceutically acceptable carrier.

12. A hair follicle repair composition comprising a cell protein extract having the hair follicle repair effect described in Claim 8 and a pharmaceutically acceptable carrier.

13. The composition is one selected from a freeze-drying agent, a gelling agent, a nasal spray, an ointment, a cream, an emulsion, a liquid dressing, an injection, and a suppository. The composition according to claim 11.

14. The method of administering the composition is one or a combination selected from smearing, rolling needle, microneedling, and massage. The composition according to claim 11.

15. Use of the cell protein extract having a hair follicle repair effect according to Claim 8 in the preparation of a product for repairing oxidatively damaged skin.

16. Use of the hair follicle repair protein composition according to claim 1 in the preparation of a hair follicle repair product.

17. The product is one selected from a hair growth product, a product for preventing and treating hair diseases, a product for delaying physiological hair loss, and a product for improving the scalp environment. The use described in claim 16.

18. The hair disorder is one or a combination selected from thinning hair, abnormal hair distribution, and abnormal hair density. The use described in claim 17.

19. The physiological hair loss is one selected from natural hair loss, seasonal hair loss, infant hair loss, senile hair loss, and postpartum hair loss. The use described in claim 17.

20. The product is a product for preventing and treating pathological alopecia, wherein the pathological alopecia is any one selected from androgenous alopecia, alopecia areata, syphilitic alopecia, trichotillomania, physical alopecia, alopecia folliculitis, perifolliculitis capitis abscedens et suffodiens, congenital hypotrichosis, scalp lichen planus, hair lichen planus, scalp lupus discoid erythematous, radioactive alopecia, female fibrous alopecia, and scalp burns. The use described in claim 16.

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