A composition for promoting hair growth, a preparation method thereof, and application thereof in preparing essence

Abstract

The present invention discloses a composition for promoting hair growth, a preparation method thereof, and an application thereof in preparing an essence, and relates to the technical field of compositions. The present invention provides a preparation method for a composition for promoting hair growth, comprising the following steps: (1) inoculating porcine umbilical cord mesenchymal stem cells into a culture medium for subculture, and collecting the cell culture supernatant; (2) inoculating Lactobacillus gasseri seed liquid into the cell culture supernatant for fermentation culture to obtain a crude fermentation liquid; (3) sterilizing and filtering the crude fermentation liquid to obtain a composition for promoting hair growth. The present invention achieves the effects of promoting hair growth and skin care by co-fermenting the supernatant of Lactobacillus gasseri and porcine umbilical cord mesenchymal stem cell culture.

Description

Technical Field

[0001] The invention relates to the technical field of compositions, in particular to a composition for promoting hair growth, a preparation method thereof and an application of the composition in preparing essence. Background Art

[0002] Due to the increasingly serious aging of the population and the increasing pressure of life, more and more people are facing the crisis of hair loss or even baldness. The number of people with hair loss in my country is huge. Hair loss types can be divided into androgenic alopecia (also known as seborrheic alopecia), neurotic alopecia, chemical alopecia, senile alopecia, pathological alopecia, etc. Among them, androgenic alopecia is currently the most common cause of hair loss in clinical practice. The principle of androgenic alopecia is that under the influence of male hormones, the sebaceous glands near the pores of the scalp enlarge, the sebum secretion is vigorous, the pores are blocked, and anaerobic bacteria such as Malassezia multiply, leading to inflammation of the sebaceous glands. Sebaceous gland inflammation further promotes the escape and migration of hair follicle stem cells, resulting in a decrease in stem cells in the hair follicle area and cessation of hair growth.

[0003] Currently, in the treatment of androgen-induced hair loss, oral administration of drugs such as finasteride, dutasteride and minoxidil is mainly used clinically. However, long-term use of these drugs can easily lead to adverse reactions, such as edema, tachycardia, dyspnea, fatigue and nausea, and may lead to the formation of cancer.

[0004] Therefore, providing a composition for promoting hair growth that can promote hair growth with less side effects has become an urgent problem to be solved. Summary of the invention

[0005] Based on this, the purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a composition for promoting hair growth and a preparation method and application thereof.

[0006] To achieve the above object, the technical solution adopted by the present invention is: a method for preparing a composition for promoting hair growth, comprising the following steps:

[0007] (1) inoculating porcine umbilical cord mesenchymal stem cells into a culture medium for subculture, and collecting the cell culture supernatant;

[0008] (2) inoculating Lactobacillus gasseri seed liquid into the cell culture supernatant for fermentation to obtain a crude fermentation liquid;

[0009] (3) sterilizing and filtering the crude fermentation broth to obtain a composition for promoting hair growth.

[0010] The present invention co-fermented the supernatant of Lactobacillus gasseri and porcine umbilical cord mesenchymal stem cells, and used the supernatant removed from the stem cell culture as a culture medium. The removed supernatant contained multiple active ingredients and exosomes of the stem cells, and served as a fermentation medium for Lactobacillus gasseri, providing rich nutrients for the growth of Lactobacillus gasseri. The stress metabolites of Lactobacillus gasseri can protect the structure and activity of the protein and RNA of the exosomes in the supernatant, and minimize the loss of active substances, thereby achieving a good effect of promoting hair growth and skin care, and having a good effect of promoting hair growth and repairing hair.

[0011] In one embodiment, in step (1), the culture medium is a serum-free culture medium, and the amount of the porcine umbilical cord mesenchymal stem cells is 0.8-1.5% of the mass of the culture medium; the conditions for the subculture are: temperature 35-39°C, 3-7% CO2, time 22-48h; the number of subcultures is 2-10 times.

[0012] In one embodiment, in step (2), the bacterial concentration of the Lactobacillus gasseri seed solution is 10 7 -10 9 cfu / mL, the amount of Lactobacillus gasseri seed solution added is 1-3% of the volume of the cell culture supernatant, the fermentation temperature is 35-40°C, the fermentation time is 24-48h, and the rotation speed is 60-120rpm; in the step (3), the sterilization temperature is 80-95°C, the sterilization time is 15-30min, and the filtration method is diatom filter filtration.

[0013] The inventor found in the actual experimental process that the number of cell subcultures has a great influence on the hair growth effect of the final product, and the cell culture supernatant with a subculture number of 2-10 times has the best hair growth effect. In the actual experimental process, the inventor took into account that after the first subculture, the supernatant of the cells still contained a low amount of impurities during separation, so the supernatant of the first subculture was not used. The cell supernatant after the second subculture has high cell viability, and the content of growth factors and exosomes produced by the cells is also high. With the increase of the number of subcultures, the viability of the cells will gradually decrease, and the content of growth factors and exosomes in the cell supernatant is less at this time. In addition, with the increase of the number of subcultures, the risk of cell canceration will also greatly increase, and the cell supernatant with a large number of subcultures will also contain more substances secreted by cancer cells, which is not conducive to subsequent fermentation. And with the increase of the number of subcultures, the content of growth factors and exosomes in the cell supernatant is less, and the hair growth effect of the final product is reduced.

[0014] In one embodiment, in step (2), the preparation method of the Lactobacillus gasseri seed solution is:

[0015] S1, inoculating Lactobacillus gasseri colonies into a seed culture medium and culturing once to obtain a Lactobacillus gasseri suspension;

[0016] S2, inoculating the Lactobacillus gasseri suspension into the seed culture medium for secondary culture to obtain the Lactobacillus gasseri seed solution.

[0017] In one embodiment, in the step S1, the temperature of the primary culture is 35-40°C, the time of the primary culture is 18-30h, and the rotation speed is 60-120rpm; in the step S2, the inoculation volume ratio of the Lactobacillus gasseri suspension is 1-3%, the temperature of the secondary culture is 35-40°C, the time of the secondary culture is 18-30h, and the rotation speed is 60-120rpm; the seed culture medium in the steps S1 and S2 is MRS liquid culture medium or modified MRS liquid culture medium.

[0018] In addition, the present invention provides a hair growth promoting composition prepared by the preparation method of the hair growth promoting composition.

[0019] Furthermore, the present invention provides the use of the composition for promoting hair growth in the preparation of cosmetics, daily chemicals, health products, and medicines.

[0020] Furthermore, the present invention provides a cosmetic comprising the following components in percentage by mass: 0.1-50% of a composition for promoting hair growth, 2-40% of a cosmetic base, and the balance being water.

[0021] In one embodiment, the cosmetic base includes at least one of a thickener, a functional active ingredient, an emulsifier, a chelating agent, an antioxidant, a preservative, a fragrance, and a pH adjuster.

[0022] The present invention also provides a hair follicle revitalizing essence, comprising the following components in percentage by weight: 1-5% of a composition for promoting hair growth, 0.02-0.08% of acetylated sodium hyaluronate, 0.05-0.2% of ultra-low molecular weight hyaluronic acid, 0.1-0.5% of p-hydroxyacetophenone, 0.8-1.3% of erythritol, 1-3% of fibronectin, 12-18% of ginsenosides, 0.1-0.3% of oleifera leaf enzyme, 0.5-2% of trehalose, 0.3-0.8% of transdermal polypeptide, 0.3-0.8% of yeast polypeptide, 0.5-2% of colloidal silver and water supplemented to 100% of the total weight.

[0023] In one embodiment, the molecular weight of the ultra low molecular weight hyaluronic acid is 400-2000 Da.

[0024] In one embodiment, the ultra-low molecular weight hyaluronic acid is purchased from Dongguan Juwei New Material Technology Co., Ltd.

[0025] In one embodiment, the method for preparing the hair follicle activating essence comprises the following steps:

[0026] (a) mixing water, p-hydroxyacetophenone and erythritol to obtain a mixture 1;

[0027] (b), adding acetylated sodium hyaluronate and ultra-low molecular weight hyaluronic acid to the mixture 1, and mixing them evenly to obtain a mixture 2;

[0028] (c) Adding the hair growth promoting composition, fibronectin, ginsenoside, olive leaf enzyme, trehalose, transdermal polypeptide, yeast polypeptide and colloidal silver to the mixture 2, mixing them evenly, and obtaining a hair follicle activating essence.

[0029] Compared with the prior art, the beneficial effects of the present invention are:

[0030] (1) The present invention screens out a lactobacillus-Lactobacillus gasseri through multiple strains, and co-fermentes Lactobacillus gasseri with the supernatant of umbilical cord mesenchymal stem cell culture to prepare a composition rich in cell growth factors and exosomes, which has a good effect of promoting hair growth and repairing. (2) The present invention uses the supernatant removed from the stem cell culture as a culture medium, and the removed supernatant contains multiple active ingredients and exosomes of stem cells. As a fermentation medium for Lactobacillus gasseri, it provides rich nutrients for the growth of Lactobacillus gasseri. The stress metabolites of Lactobacillus gasseri can protect the structure and activity of the protein and RNA of the exosomes in the supernatant, thereby achieving a good effect of promoting hair growth and skin care. (3) The composition of the present invention can promote the proliferation of regulatory T cells, and the proliferation of regulatory T cells can prevent T cells from attacking hair follicle stem cells, reduce the migration of hair follicle stem cells, and increase the number of hair follicle stem cells; the composition of the present invention can also reduce scalp inflammation, promote angiogenesis in the hair follicle area, and increase hair follicle nutrition, which ultimately shows that the composition of the present invention has a good effect of promoting hair growth. (4) The composition of the present invention uses the supernatant removed from pig umbilical cord mesenchymal stem cells. The pig gene has a high homology rate with human genes and is highly compatible with the human body. The supernatant does not contain animal placental serum, and the final product is not easy to cause irritation. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The figures are comparison pictures before and after using the hair growth essence of the composition for promoting hair growth of Example 1; wherein (a) is a picture of the scalp of a volunteer before using the hair growth essence of Example 1, and (b) is a picture of the scalp of a volunteer after using the hair growth essence of Example 1 for 2 months. DETAILED DESCRIPTION

[0032] In order to better illustrate the purpose, technical scheme and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. Its purpose is to understand the content of the present invention in detail, rather than to limit the present invention. All other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Unless otherwise specified, the experimental reagents and instruments designed for the implementation and comparative examples of the present invention are all commonly used ordinary reagents and instruments, which can be obtained from commercial channels. In the implementation and comparative examples, the experimental methods used are conventional methods unless otherwise specified; and unless otherwise specified, the raw materials used in parallel experiments are the same batch of raw materials.

[0033] The raw materials used in the embodiments and comparative examples are described below, but are not limited to these materials:

[0034] MRS liquid culture medium is a commonly used culture medium, and its formula is as follows: 10g / L peptone, 5g / L beef powder, 4g / L yeast powder, 2g / L glucose, 1mL / L Tween 80, 2g / L dipotassium hydrogen phosphate, 5g / L sodium acetate, 2g / L triammonium citrate, 0.2g / L magnesium sulfate, 0.05g / L manganese sulfate, and the balance water.

[0035] Modified MRS liquid medium is a commonly used culture medium, and its formula is as follows: 20 g / L glucose, 5 g / L casein hydrolysate, 5 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 2 g / L diammonium citrate, 2 g / L dipotassium hydrogen phosphate, 0.58 g / L magnesium sulfate, 0.25 g / L manganese sulfate, 1 mL / L Tween 80, and the remainder water.

[0036] The formula of the serum-free culture medium of the present invention is 50 mg / L vitamin C, 20 μg / L stem cell growth factor, 2 mol / L L-glutamine, 100 μg / L human transforming growth factor TGF-β1, 20 μg / L epidermal growth factor and 20 μg / L human fibroblast growth factor, 0.3 mg / L baicalin; other commercially available serum-free culture media such as SYL-SF serum-free culture medium of Beijing Sanyouli Technology Co., Ltd. and serum-free culture medium (article number ME000-N023) produced by Yikesai Biotechnology (Taicang) Co., Ltd. are also suitable for the present invention.

[0037] The method for isolating umbilical cord mesenchymal stem cells of the present invention is as follows: cut a fresh pig umbilical cord into small sections of 3-5 cm, wash it with physiological saline, peel off the blood vessels and umbilical cord Wharton's jelly, and digest it with digestive fluid to obtain a suspension. The suspension is filtered through a cell filter with a pore size of 150 μm, and the filtrate is collected. The filtrate is centrifuged (1500 rpm, 5 min), the supernatant is discarded, the precipitate is rinsed 3 times with PBS buffer solution, and the umbilical cord mesenchymal stem cells are separated by density gradient centrifugation. The specific isolation method of umbilical cord mesenchymal stem cells of the present invention refers to patent application number: CN201610529860.3, and the name of the invention is: human umbilical cord mesenchymal stem cell extract freeze-dried powder and preparation method thereof.

[0038] The separation method of the rabbit umbilical cord mesenchymal stem cells and the bovine umbilical cord mesenchymal stem cells of the present invention is the same as the separation method of the pig umbilical cord mesenchymal stem cells.

[0039] The porcine adipose-derived mesenchymal stem cells used in the present invention were purchased from Shanghai Lianzu Biotechnology Co., Ltd.

[0040] The acetylated sodium hyaluronate used in the present invention was purchased from Runhui Biotechnology (Weihai) Co., Ltd., the ultra-low molecular weight hyaluronic acid used in the present invention was purchased from Dongguan Juwei New Material Technology Co., Ltd., the fibronectin and ginsenoside used in the present invention were both purchased from Guangzhou Lanxi Pharmaceutical Co., Ltd., the model of the fibronectin is fibronectin OTL-0992, the model of the ginsenoside is ginsenoside OTL-1099, the olive leaf enzyme and yeast polypeptide used in the present invention were purchased from Long Sidan (Shanghai) Biotechnology Co., Ltd., the model of the olive leaf enzyme is olive leaf enzyme NT, the model of the yeast polypeptide is yeast polypeptide EW2000, the transdermal polypeptide used in the present invention was purchased from Guangzhou Hesheng Chuangyan Biotechnology Co., Ltd., the model is TDP-500 transdermal polypeptide, the colloidal silver used in the present invention was purchased from Guangzhou Hengan Fine Chemical Co., Ltd., the model is colloidal silver AC1000-D.

[0041] The Lactobacillus gasseri used in the present invention is numbered CGMCC 1.3396 and was purchased from Shanghai Xuanke Biotechnology Co., Ltd.

[0042] The plant lactobacillus used in the present invention is numbered BNCC 194165 and was purchased from Beina Chuanglian Biotechnology Co., Ltd. in the mall.

[0043] The fermented lactobacillus used in the present invention is numbered BNCC 194390 and was purchased from Beina Chuanglian Biotechnology Co., Ltd. in the mall.

[0044] The Lactobacillus rhamnosus used in the present invention is numbered ATCC 7469 and was purchased from Ruichu Biotechnology (Jiangsu) Co., Ltd.

[0045] Examples 1-7

[0046] A method for preparing a composition for promoting hair growth comprises the following steps:

[0047] (1) inoculating porcine umbilical cord mesenchymal stem cells into a serum-free culture medium at a temperature of 35-39° C. and 3-7% CO2 for 22-48 hours, subculturing for 2-10 times, and collecting the cell culture supernatant; the amount of the porcine umbilical cord mesenchymal stem cells used is 0.8-1.5% of the mass of the culture medium;

[0048] (2) inoculating the Lactobacillus gasseri seed solution into the cell culture supernatant, and fermenting and culturing at 35-40° C. and a rotation speed of 60-120 rpm for 24-48 hours to obtain a crude fermentation solution; the bacterial concentration of the Lactobacillus gasseri seed solution is 10 7 -10 9 cfu / mL, the amount of Lactobacillus gasseri seed solution added is 1-3% of the volume of the cell culture supernatant;

[0049] (3) The crude fermentation liquid is sterilized by keeping it at 80-95° C. for 15-30 min, and then filtered through a diatom filter to obtain a composition for promoting hair growth.

[0050] The preparation method of the lactobacillus gasseri seed liquid is:

[0051] S1. Inoculate the Lactobacillus gasseri colony into the seed culture medium, and culture it at 35-40° C. and 60-120 rpm for 18-30 hours to obtain a Lactobacillus gasseri suspension;

[0052] S2. Inoculate the Lactobacillus gasseri suspension into the seed culture medium, and culture once at 35-40° C. and 60-120 rpm for 18-30 hours to obtain the Lactobacillus gasseri seed solution; the amount of the Lactobacillus gasseri seed solution added is 1-3% of the volume of the cell culture supernatant.

[0053] The seed culture medium in S1 and S2 is MRS liquid culture medium or improved MRS liquid culture medium.

[0054] Example 1 A composition for promoting hair growth

[0055] The preparation method of a composition for promoting hair growth in this embodiment is as follows:

[0056] (1) Porcine umbilical cord mesenchymal stem cells were inoculated into serum-free culture medium at a mass fraction of 1%, and subcultured at 37° C. and 5% CO 2 , and the cell culture supernatant of the second generation during the subculture process was collected.

[0057] (2) Inoculating the Lactobacillus gasseri seed solution into the cell culture supernatant in step (1) at an inoculation volume ratio of 2%, and fermenting and culturing at 37° C. and 80 rpm for 36 h to obtain a crude fermentation solution.

[0058] (3) The crude fermentation liquid was sterilized by keeping it at 85° C. for 20 min, and then filtered through a diatom filter to obtain a composition for promoting hair growth.

[0059] Wherein, the preparation method of Lactobacillus gasseri seed liquid is:

[0060] S1. Inoculate the Lactobacillus gasseri colony into MRS liquid culture medium through an inoculation loop, and culture at 37° C. and 80 rpm for 24 h to obtain a Lactobacillus gasseri suspension;

[0061] S2, inoculate the Lactobacillus gasseri suspension in step S1 into MRS liquid culture medium at an inoculation volume ratio of 2%, and culture for a second time at 37° C. and 80 rpm for 24 h to obtain a Lactobacillus gasseri seed solution, wherein the bacterial concentration of the Lactobacillus gasseri seed solution is about 10 8 cfu / mL.

[0062] Example 2 A composition for promoting hair growth

[0063] The preparation method of a composition for promoting hair growth in this embodiment is as follows:

[0064] (1) Porcine umbilical cord mesenchymal stem cells were inoculated into serum-free culture medium at a mass fraction of 0.8%, and subcultured at 37° C. and 3% CO 2 , and the cell culture supernatant of the second generation during the subculture process was collected.

[0065] (2) Inoculating the Lactobacillus gasseri seed solution into the cell culture supernatant in step (1) at an inoculation volume ratio of 3%, and fermenting and culturing at 35° C. and 60 rpm for 24 h to obtain a crude fermentation solution.

[0066] (3) The crude fermentation liquid was sterilized by keeping it at 95° C. for 15 minutes, and then filtered through a diatom filter to obtain a composition for promoting hair growth.

[0067] Wherein, the preparation method of Lactobacillus gasseri seed liquid is:

[0068] S1. Inoculate the Lactobacillus gasseri colonies into a modified MRS liquid culture medium through an inoculation loop, and culture at 40° C. and 60 rpm for 18 h to obtain a Lactobacillus gasseri suspension.

[0069] S2, inoculating the Lactobacillus gasseri suspension in step S1 into the modified MRS liquid medium at an inoculation volume ratio of 1%, and culturing for 30 hours at 40° C. and 60 rpm to obtain a Lactobacillus gasseri seed solution, wherein the bacterial concentration of the Lactobacillus gasseri seed solution is about 107 cfu / mL.

[0070] Example 3 A composition for promoting hair growth

[0071] The preparation method of a composition for promoting hair growth in this embodiment is as follows:

[0072] (1) Porcine umbilical cord mesenchymal stem cells were inoculated into serum-free culture medium at a mass fraction of 1.5%, and subcultured at 37° C. and 7% CO 2 , and the cell culture supernatant of the second generation during the subculture process was collected.

[0073] (2) Inoculating the Lactobacillus gasseri seed solution into the cell culture supernatant in step (1) at an inoculation volume ratio of 1%, and fermenting and culturing at 35° C. and 120 rpm for 48 h to obtain a crude fermentation solution.

[0074] (3) The crude fermentation liquid was sterilized by keeping it at 80° C. for 30 min, and then filtered through a diatom filter to obtain a composition for promoting hair growth.

[0075] Wherein, the preparation method of Lactobacillus gasseri seed liquid is:

[0076] S1. Inoculate the Lactobacillus gasseri colonies into MRS liquid culture medium through an inoculation loop, and culture at 35° C. and 120 rpm for 30 h to obtain a Lactobacillus gasseri suspension.

[0077] S2, inoculate the Lactobacillus gasseri suspension in step S1 into MRS liquid culture medium at an inoculation volume ratio of 3%, and culture for a second time at 35° C. and 120 rpm for 18 h to obtain a Lactobacillus gasseri seed solution, wherein the bacterial concentration of the Lactobacillus gasseri seed solution is about 10 9 cfu / mL.

[0078] Example 4 A composition for promoting hair growth

[0079] The preparation method of a composition for promoting hair growth in this embodiment is as follows:

[0080] (1) Porcine umbilical cord mesenchymal stem cells were inoculated into serum-free culture medium at a mass fraction of 1%, and subcultured at 37° C. and 5% CO 2 , and the cell culture supernatant of the sixth generation during the subculture process was collected.

[0081] (2) Inoculating the Lactobacillus gasseri seed solution into the cell culture supernatant in step (1) at an inoculation volume ratio of 2%, and fermenting and culturing at 37° C. and 80 rpm for 36 h to obtain a crude fermentation solution.

[0082] (3) The crude fermentation liquid was kept at 85° C. for 20 minutes and then filtered through a diatom filter to obtain a composition for promoting hair growth.

[0083] Wherein, the preparation method of Lactobacillus gasseri seed liquid in this embodiment is the same as that in Example 1.

[0084] The only difference between this embodiment and embodiment 1 is that in embodiment 4, the cell culture supernatant of the 6th generation is collected as the culture medium of Lactobacillus gasseri.

[0085] Example 5 A composition for promoting hair growth

[0086] The preparation method of a composition for promoting hair growth in this embodiment is as follows:

[0087] (1) Porcine umbilical cord mesenchymal stem cells were inoculated into serum-free culture medium at a mass fraction of 1%, and subcultured at 37° C. and 5% CO 2 , and the cell culture supernatant of the 10th generation during the subculture process was collected.

[0088] (2) Inoculating the Lactobacillus gasseri seed solution into the cell culture supernatant in step (1) at an inoculation volume ratio of 2%, and fermenting and culturing at 37° C. and 80 rpm for 36 h to obtain a crude fermentation solution.

[0089] (3) The crude fermentation liquid was kept at 85° C. for 20 minutes and then filtered through a diatom filter to obtain a composition for promoting hair growth.

[0090] Wherein, the preparation method of Lactobacillus gasseri seed liquid in this embodiment is the same as that in Example 1.

[0091] The only difference between this embodiment and embodiment 1 is that in embodiment 5, the cell culture supernatant of the 10th generation is collected as the culture medium of Lactobacillus gasseri.

[0092] Example 6 A composition for promoting hair growth

[0093] The preparation method of a composition for promoting hair growth in this embodiment is as follows:

[0094] (1) Porcine umbilical cord mesenchymal stem cells were inoculated into serum-free culture medium at a mass fraction of 1%, and subcultured at 35° C. and 5% CO 2 , and the cell culture supernatant of the second generation during the subculture process was collected.

[0095] (2) Inoculating the Lactobacillus gasseri seed solution into the cell culture supernatant in step (1) at an inoculation volume ratio of 2%, and fermenting and culturing at 37° C. and 80 rpm for 36 h to obtain a crude fermentation solution.

[0096] (3) The crude fermentation liquid was kept at 85° C. for 20 minutes and then filtered through a diatom filter to obtain a composition for promoting hair growth.

[0097] Wherein, the preparation method of Lactobacillus gasseri seed liquid in this embodiment is the same as that in Example 1.

[0098] The difference between this embodiment and embodiment 1 is that the culture temperature of the porcine umbilical cord mesenchymal stem cells in embodiment 5 is 35°C.

[0099] Example 7 A composition for promoting hair growth

[0100] The preparation method of a composition for promoting hair growth in this embodiment is as follows:

[0101] (1) Porcine umbilical cord mesenchymal stem cells were inoculated into serum-free culture medium at a mass fraction of 1%, and subcultured at 39° C. and 5% CO 2 , and the cell culture supernatant of the second generation during the subculture process was collected.

[0102] (2) Inoculating the Lactobacillus gasseri seed solution into the cell culture supernatant in step (1) at an inoculation volume ratio of 2%, and fermenting and culturing at 37° C. and 80 rpm for 36 h to obtain a crude fermentation solution.

[0103] (3) The crude fermentation liquid was kept at 85° C. for 20 minutes and then filtered through a diatom filter to obtain a composition for promoting hair growth.

[0104] Wherein, the preparation method of Lactobacillus gasseri seed liquid in this embodiment is the same as that in Example 1.

[0105] The difference between this embodiment and embodiment 1 is that the culture temperature of the porcine umbilical cord mesenchymal stem cells in embodiment 5 is 39°C.

[0106] Comparative Example 1 A composition for promoting hair growth

[0107] The preparation method of a composition for promoting hair growth in this comparative example is as follows:

[0108] (1) Rabbit umbilical cord mesenchymal stem cells were inoculated into serum-free culture medium at a mass fraction of 1%, and subcultured at 37° C. and 5% CO 2 , and the cell culture supernatant of the second generation during the subculture process was collected.

[0109] (2) Inoculating the Lactobacillus gasseri seed solution into the cell culture supernatant in step (1) at an inoculation volume ratio of 2%, and fermenting and culturing at 37° C. and 80 rpm for 36 h to obtain a crude fermentation solution.

[0110] (3) The crude fermentation liquid was sterilized by keeping it at 85° C. for 20 min, and then filtered through a diatom filter to obtain a composition for promoting hair growth.

[0111] Wherein, the preparation method of Lactobacillus gasseri seed liquid in this embodiment is the same as that in Example 1.

[0112] The only difference between this comparative example and Example 1 is that the umbilical cord mesenchymal stem cells in Comparative Example 1 are rabbit-derived umbilical cord mesenchymal stem cells.

[0113] Comparative Example 2 A composition for promoting hair growth

[0114] The preparation method of a composition for promoting hair growth in this comparative example is as follows:

[0115] (1) Bovine umbilical cord mesenchymal stem cells were inoculated into serum-free culture medium at a mass fraction of 1%, and subcultured at 37° C. and 5% CO 2 , and the cell culture supernatant of the second generation during the subculture process was collected.

[0116] (2) Inoculating the Lactobacillus gasseri seed solution into the cell culture supernatant in step (1) at an inoculation volume ratio of 2%, and fermenting and culturing at 37° C. and 80 rpm for 36 h to obtain a crude fermentation solution.

[0117] (3) The crude fermentation liquid was sterilized by keeping it at 85° C. for 20 min, and then filtered through a diatom filter to obtain a composition for promoting hair growth.

[0118] Wherein, the preparation method of Lactobacillus gasseri seed liquid in this embodiment is the same as that in Example 1.

[0119] The only difference between this comparative example and Example 1 is that the umbilical cord mesenchymal stem cells in Comparative Example 2 are bovine umbilical cord mesenchymal stem cells.

[0120] Comparative Example 3 A composition for promoting hair growth

[0121] The preparation method of a composition for promoting hair growth in this comparative example is as follows:

[0122] (1) Porcine umbilical cord mesenchymal stem cells were inoculated into serum-free culture medium at a mass fraction of 1%, and subcultured at 37° C. and 5% CO 2 , and the cell culture supernatant of the 13th generation during the subculture process was collected.

[0123] (2) Inoculating the Lactobacillus gasseri seed solution into the cell culture supernatant in step (1) at an inoculation volume ratio of 2%, and fermenting and culturing at 37° C. and 80 rpm for 36 h to obtain a crude fermentation solution.

[0124] (3) The crude fermentation liquid was kept at 85° C. for 20 minutes and then filtered through a diatom filter to obtain a composition for promoting hair growth.

[0125] Wherein, the preparation method of Lactobacillus gasseri seed liquid in this embodiment is the same as that in Example 1.

[0126] The only difference between Comparative Example 3 and Example 1 is that the culture generations of cells in the obtained cell culture supernatant are different.

[0127] Comparative Example 4: a composition for promoting hair growth.

[0128] The preparation method of a composition for promoting hair growth in this comparative example is as follows:

[0129] (1) Porcine umbilical cord mesenchymal stem cells were inoculated into serum-free culture medium at a mass fraction of 1%, and subcultured at 37° C. and 5% CO 2 , and the cell culture supernatant of the second generation during the subculture process was collected.

[0130] (2) Inoculating Lactobacillus plantarum seed solution into the cell culture supernatant in step (1) at an inoculation volume ratio of 2%, and fermenting and culturing at 37° C. and 80 rpm for 36 h to obtain a crude fermentation solution.

[0131] (3) The crude fermentation liquid was kept at 85° C. for 20 minutes and then filtered through a diatom filter to obtain a composition for promoting hair growth.

[0132] Wherein, the preparation method of plant lactobacillus seed liquid is:

[0133] S1. Inoculate the Lactobacillus plantarum colony into MRS liquid culture medium through an inoculation loop, and culture at 37° C. and 80 rpm for 24 h to obtain a Lactobacillus plantarum suspension;

[0134] S2, inoculate the Lactobacillus plantarum suspension in step S1 into MRS liquid culture medium at an inoculation volume ratio of 2%, and culture for a second time at 37° C. and 80 rpm for 24 h to obtain Lactobacillus plantarum seed liquid, wherein the bacterial concentration of the Lactobacillus plantarum seed liquid is about 10 8 cfu / mL.

[0135] The difference between this comparative example and Example 1 is only that the fermented bacteria are different.

[0136] Comparative Example 5: a composition for promoting hair growth.

[0137] The preparation method of a composition for promoting hair growth in this comparative example is as follows:

[0138] (1) Porcine umbilical cord mesenchymal stem cells were inoculated into serum-free culture medium at a mass fraction of 1%, and subcultured at 37° C. and 5% CO 2 , and the cell culture supernatant of the second generation during the subculture process was collected.

[0139] (2) Inoculating the fermented Lactobacillus seed solution into the cell culture supernatant in step (1) at an inoculation volume ratio of 2%, and fermenting and culturing at 37° C. and 80 rpm for 36 h to obtain a crude fermentation solution.

[0140] (3) The crude fermentation liquid was kept at 85° C. for 20 minutes and then filtered through a diatom filter to obtain a composition for promoting hair growth.

[0141] Wherein, the preparation method of fermented lactobacillus seed liquid is:

[0142] S1. Inoculate the fermented Lactobacillus colony into MRS liquid culture medium through an inoculation loop, and culture at 37° C. and 80 rpm for 24 h to obtain a fermented Lactobacillus suspension;

[0143] S2, inoculate the fermented lactobacillus suspension in step S1 into MRS liquid culture medium at an inoculation volume ratio of 2%, and culture for a second time at 37° C. and 80 rpm for 24 h to obtain a fermented lactobacillus seed solution, wherein the bacterial concentration of the fermented lactobacillus seed solution is about 10 8 cfu / mL.

[0144] The difference between this comparative example and Example 1 is only that the fermented bacteria are different.

[0145] Comparative Example 6: A composition for promoting hair growth.

[0146] The preparation method of a composition for promoting hair growth in this comparative example is as follows:

[0147] (1) Porcine umbilical cord mesenchymal stem cells were inoculated into serum-free culture medium at a mass fraction of 1%, and subcultured at 37° C. and 5% CO 2 , and the cell culture supernatant of the second generation during the subculture process was collected.

[0148] (2) Inoculating the Lactobacillus rhamnosus seed solution into the cell culture supernatant in step (1) at an inoculation volume ratio of 2%, and fermenting and culturing at 37° C. and 80 rpm for 36 h to obtain a crude fermentation solution.

[0149] (3) The crude fermentation liquid was kept at 85° C. for 20 minutes and then filtered through a diatom filter to obtain a composition for promoting hair growth.

[0150] Wherein, the preparation method of fermented lactobacillus seed liquid is:

[0151] S1. Inoculate the Lactobacillus rhamnosus colony into MRS liquid culture medium through an inoculation loop, and culture at 37° C. and 80 rpm for 24 h to obtain a Lactobacillus rhamnosus suspension;

[0152] S2, inoculate the Lactobacillus rhamnosus suspension in step S1 into MRS liquid culture medium at an inoculation volume ratio of 2%, and culture for a second time at 37° C. and 80 rpm for 24 h to obtain Lactobacillus rhamnosus seed liquid, the bacterial concentration of the Lactobacillus rhamnosus seed liquid is about 10 8 cfu / mL.

[0153] The difference between this comparative example and Example 1 is only that the fermented bacteria are different.

[0154] Comparative Example 7 A composition for promoting hair growth

[0155] The preparation method of a composition for promoting hair growth in this comparative example is as follows:

[0156] (1) Porcine adipose-derived mesenchymal stem cells were inoculated into serum-free culture medium at a mass fraction of 1%, and subcultured at 37° C. and 5% CO 2 , and the cell culture supernatant of the second generation during the subculture process was collected.

[0157] (2) Inoculating the Lactobacillus gasseri seed solution into the cell culture supernatant in step (1) at an inoculation volume ratio of 2%, and fermenting and culturing at 37° C. and 80 rpm for 36 h to obtain a crude fermentation solution.

[0158] (3) The crude fermentation liquid was sterilized by keeping it at 85° C. for 20 min, and then filtered through a diatom filter to obtain a composition for promoting hair growth.

[0159] Wherein, the preparation method of Lactobacillus gasseri seed liquid in this embodiment is the same as that in Example 1.

[0160] The only difference between this comparative example and Example 1 is that the porcine umbilical cord mesenchymal stem cells are replaced with porcine adipose mesenchymal stem cells.

[0161] Application Example 1 A hair follicle activating essence

[0162] This application example provides a hair follicle revitalizing essence, comprising the following components in percentage by weight: 3% of the composition for promoting hair growth in Example 1, 0.05% of acetylated sodium hyaluronate, 0.1% of ultra-low molecular weight hyaluronic acid, 0.3% of p-hydroxyacetophenone, 1% of erythritol, 2% of fibronectin, 15% of ginsenosides, 0.2% of olive leaf enzyme, 1% of trehalose, 0.5% of transdermal polypeptide, 0.5% of yeast polypeptide, 1% of colloidal silver and the remainder of water.

[0163] The preparation method of the hair follicle activating essence of this application example includes:

[0164] (a) mixing water, p-hydroxyacetophenone and erythritol to obtain a mixture 1;

[0165] (b), adding acetylated sodium hyaluronate and ultra-low molecular weight hyaluronic acid to the mixture 1, and mixing them evenly to obtain a mixture 2;

[0166] (c) Adding the hair growth promoting composition, fibronectin, ginsenoside, olive leaf enzyme, trehalose, transdermal polypeptide, yeast polypeptide and colloidal silver to the mixture 2, mixing them evenly, and obtaining a hair follicle activating essence.

[0167] Safety Testing - Human Patch Test

[0168] Based on the skin irritation / corrosion test in the 2015 Technical Specification for Safety of Cosmetics, the composition was evaluated for cosmetic irritation through a human patch test.

[0169] Test sample: the hair growth promoting composition of Examples 1-7 and Comparative Examples 1-7, prepared into a solution with a mass concentration of 50% by using deionized water.

[0170] Test steps: 140 people (10 volunteers per group, 14 groups in total), aged between 16 and 65 years old, were selected. The subjects had healthy skin and no history of skin allergies, meeting the voluntary selection criteria. 0.020-0.025g of the test sample was placed in the patch tester, and the patch tester with the test sample was applied to the flexor side of the subject's forearm with a non-irritating tape, and the palm of the hand was gently pressed to evenly apply it to the skin for 24 hours. 30 minutes after removing the test patch tester, observe the skin reaction after the indentation disappears. If the result is negative, observe again 24 hours and 48 hours after the patch test. Record the results according to the 2015 "Technical Specifications for Safety of Cosmetics" as the reference standard.

[0171] The evaluation criteria are shown in Table 1:

[0172] Table 1

[0173]

[0174]

[0175] The test results are shown in Table 2:

[0176] Table 2

[0177] Group Skin reaction (24h) Skin reaction (48h) Example 1 10 cases of grade 0 10 cases of grade 0 Example 2 10 cases of grade 0 10 cases of grade 0 Example 3 10 cases of grade 0 10 cases of grade 0 Example 4 10 cases of grade 0 10 cases of grade 0 Example 5 10 cases of grade 0 10 cases of grade 0 Example 6 10 cases of grade 0 10 cases of grade 0 Example 7 10 cases of grade 0 10 cases of grade 0 Comparative Example 1 6 cases of grade 0; 4 cases of grade 1 8 cases of grade 0; 2 cases of grade 1 Comparative Example 2 8 cases of grade 0; 2 cases of grade 1 10 cases of grade 0 Comparative Example 3 10 cases of grade 0 10 cases of grade 0 Comparative Example 4 10 cases of grade 0 10 cases of grade 0 Comparative Example 5 10 cases of grade 0 10 cases of grade 0 Comparative Example 6 10 cases of grade 0 10 cases of grade 0 Comparative Example 7 10 cases of grade 0 10 cases of grade 0

[0178] The test results show that the patch test of the hair growth promoting composition prepared by Examples 1-7 was performed on each group of subjects. The skin reactions of the volunteers were observed 24h and 48h after the test. The skin reactions of all volunteers were negative, indicating that the hair growth promoting compositions of Examples 1-7 are safe to use. Comparative Examples 1 and 2 used rabbit-derived and bovine-derived umbilical cord mesenchymal stem cells to prepare hair growth promoting compositions, and some volunteers had skin damage of varying degrees 24 and 48h after uncovering the patch tester, indicating that the hair growth promoting compositions prepared using rabbit-derived and bovine-derived umbilical cord mesenchymal stem cells are not as safe as the hair growth promoting compositions prepared using porcine-derived umbilical cord mesenchymal stem cells.

[0179] Performance Test-1 Hair Growth Promotion Effect Test

[0180] Testing process: The hair growth promoting compositions prepared in Examples 1-7 and Comparative Examples 1-7 were prepared into hair growth essence; matrix essence.

[0181] The formula of the hair growth essence is as follows (mass percentage): Phase A: water balance, 10% hair growth promoting composition, 0.5% polyvinyl alcohol, 0.3% carbomer, 2.0% acrylic acid (ester) copolymer; Phase B: 0.06% maleic acid modified castor oil, 0.8% camellia seed oil, 0.08% vitamin C ethyl ether, 0.2% octyl dimethicone ethoxylated glucoside; Phase C: 1% parahydroxyacetophenone, 0.05% fragrance.

[0182] The formula of the matrix essence is as follows: Phase A: balance water, 0.5% polyvinyl alcohol, 0.3% carbomer, 2.0% acrylic acid (ester) copolymer; Phase B: 0.06% maleic acid modified castor oil, 0.8% camellia seed oil, 0.08% vitamin C ethyl ether, 0.2% octyl dimethicone ethoxy glucoside; Phase C: 1% p-hydroxyacetophenone, 0.05% fragrance.

[0183] The preparation method of the hair growth essence is as follows: the materials in phase A are mixed evenly at 70°C, kept warm for later use, and a mixed solution A is obtained; the materials in phase B are mixed evenly at 70°C, kept warm for later use, and a mixed solution B is obtained; at 70°C, the mixed solution B is added to the mixed solution A and mixed evenly; the temperature is lowered to 45°C, the materials in phase C are added, and the mixture is stirred evenly to prepare the hair growth essence.

[0184] The preparation method of matrix essence is the same as that of hair growth essence.

[0185] Test steps: 150 volunteers with hair loss problems were selected and randomly divided into 15 groups, 10 people in each group, and each group used a test sample. Usage: Introduce the hair growth essence into the scalp through microcrystal vibration, once every 5 days, for 2 months, a total of 12 times.

[0186] Evaluation method: After two months of use, volunteers scored the hair growth effect of the hair growth essence, and the scoring results are shown in Table 3. The score ranges from 0 to 10, with 0 being no hair growth effect and 10 being an excellent hair growth effect with a significant increase in hair volume in the area of ​​use. The scoring results were averaged.

[0187] Table 3

[0188]

[0189] The scoring effects of the matrix essences of Examples 1-7 and Comparative Examples 1-7 are shown in Table 4 below.

[0190] Table 4

[0191] Group Hair growth effect score Group Hair growth effect score Matrix Essence 0.6 / / Example 1 9.7 Comparative Example 1 3.1 Example 2 9.5 Comparative Example 2 1.6 Example 3 9.4 Comparative Example 3 5.7 Example 4 9.2 Comparative Example 4 3.6 Example 5 8.5 Comparative Example 5 2.8 Example 6 8.7 Comparative Example 6 5.1 Example 7 9.0 Comparative Example 7 5.8

[0192] The test results show that the hair growth essence prepared by the composition of the embodiment of the present invention has a good hair growth effect. The matrix essence without adding the composition of the embodiment or the comparative example has no hair growth effect, and the score is 0.6 points. The hair growth effect of Examples 1-7 is better than that of Comparative Examples 1 and 2, indicating that the source of umbilical cord mesenchymal stem cells has an important influence on the hair growth effect of the final product. The present invention preferably prepares the composition using porcine mesenchymal stem cells. Figure 1 The figures are comparison pictures before and after using the hair growth essence of the composition for promoting hair growth of Example 1; wherein (a) is a picture of the scalp of a volunteer before using the hair growth essence of Example 1, and (b) is a picture of the scalp of a volunteer after using the hair growth essence of Example 1 for 2 months.

[0193] The hair growth effects of Examples 1, 4, and 5 are better than those of Comparative Example 3, and the hair growth effect of Example 1 is better than that of Example 4. This indicates that the cell culture supernatants obtained by different subcultures have a great influence on the hair growth effect of the final product. The more subculture times the cells have, the less the content of growth factors and exosomes in the cell supernatant, resulting in a reduced hair growth effect of the final product. The composition of the present invention is preferably a cell culture supernatant that has been subcultured 2-10 times.

[0194] The hair growth effect of Examples 1-7 is better than that of Comparative Examples 4-6, indicating that the fermented bacteria species have an impact on the hair growth effect of the final product. The composition obtained by fermenting with Lactobacillus gasseri has a better hair growth effect, while when other lactobacilli are used for fermentation, such as Lactobacillus plantarum, Lactobacillus fermentum or Lactobacillus rhamnosus, the hair growth effect of the final product is poor.

[0195] The hair growth effect of Example 1 is better than that of Comparative Example 7, indicating that the type of stem cells has an impact on the hair growth effect of the final product. Comparative Example 7 uses the supernatant of adipose mesenchymal stem cells as a component of the fermentation medium. During the cell culture process, the active substances produced in the supernatant of porcine adipose mesenchymal stem cells are less than those in the supernatant of porcine umbilical cord mesenchymal stem cells, resulting in a poor hair growth effect of the final product. The present invention preferably uses the supernatant of porcine umbilical cord mesenchymal stem cells to prepare a composition for promoting hair growth.

[0196] Performance test-2 Repair performance test-zebrafish fry after sun repair experiment

[0197] Test principle: Zebrafish fins are equivalent to human arms or legs, and their ability to repair and regenerate is stronger than that of humans. The tail fin of zebrafish exposed to ultraviolet rays will become rough and wrinkled, but the tail fin of zebrafish can be repaired in a short period of time. A zebrafish fry sunburn damage model is constructed to observe the recovery of the tail fin area by the test object, so as to evaluate the sunburn repair effect of the test sample.

[0198] Test sample: The hair growth promoting composition of Examples 1-7 and Comparative Examples 1-7 was prepared into a solution with a mass concentration of 0.1% using a culture solution. The preparation method of the culture solution is as follows: 2940 mg of anhydrous calcium chloride, 1233 mg of magnesium sulfate heptahydrate, 630 mg of sodium bicarbonate, and 55 mg of potassium chloride were weighed and dissolved in 10 L of water.

[0199] Test steps: The tested zebrafish fry were divided into several groups according to the modeling group, test sample group and blank control group, with 6 zebrafish fry in each group. Before the test, the tail fins of the zebrafish fry were photographed and the initial tail fin area of ​​the zebrafish fry was recorded. The zebrafish fry in the modeling group and the test sample group were irradiated with ultraviolet light for 3 times, and the blank control group was not irradiated with ultraviolet light and cultured normally. Then, the test sample was added to the test sample group, and the same volume of culture medium was added to the modeling group and the blank control group. After incubation for 48 hours, the tail fins of the zebrafish fry in the modeling group, the test sample group and the blank control group were photographed, and the tail fin area of ​​the zebrafish fry after the experiment was recorded and compared with the initial tail fin area of ​​the zebrafish fry to obtain the fin area change rate, which was used to characterize the recovery of the tail fin area.

[0200] The test results are shown in Table 5 below.

[0201] Table 5

[0202]

[0203]

[0204] The zebrafish fry in the blank control group were not exposed to ultraviolet light, and the change rate of the fin area was 121.8%, which is the change rate of the fin area under normal growth conditions. The fin area of ​​the modeling group was atrophied by ultraviolet light, decreasing to 47.7%. In the test sample group, after ultraviolet irradiation, the composition of the embodiment or comparative example was added to the culture medium, and the fins were repaired, and the fin area was improved to varying degrees compared with the modeling group.

[0205] The test results show that the composition of the embodiment of the present invention has good repair performance and can promote the repair of sunburn damage to fish fins. The repair effect of the embodiment is better than that of comparative examples 1 and 2, indicating that the source of stem cells has a great influence on the repair performance of the final product. Comparative example 1 uses rabbit stem cells and comparative example 2 uses bovine stem cells. The repair effect of the composition prepared under the same conditions is poor, and the composition of comparative example 1 has almost no effect.

[0206] The repair effects of Examples 1, 4, and 5 are better than those of Comparative Example 3, indicating that the cell culture supernatants obtained from different subcultures have a greater influence on the repair effect of the final product. When the cell culture supernatants with fewer subcultures are used, the cell viability is greater, the active substances contained in the cell culture supernatants are also more, and the repair effect of the final product is also better. The repair effects of Examples 1-7 are better than those of Comparative Examples 4-6, indicating that the fermented bacteria have a great influence on the repair effect of the final composition, and the use of Lactobacillus gasseri to ferment porcine umbilical cord mesenchymal stem cells has the best effect. The repair effects of Examples 1-7 are better than those of Comparative Example 7, indicating that the type of stem cells has an influence on the repair effect of the final product, and the present invention preferably uses porcine umbilical cord mesenchymal stem cells to prepare the composition.

[0207] Performance test-3 soothing performance test

[0208] Test sample: The hair growth promoting composition of Examples 1-7 and Comparative Examples 1-7 was prepared into a solution with a mass concentration of 0.1% using a culture solution. The preparation method of the culture solution is as follows: 2940 mg of anhydrous calcium chloride, 1233 mg of magnesium sulfate heptahydrate, 630 mg of sodium bicarbonate, and 55 mg of potassium chloride were weighed and dissolved in 10 L of water.

[0209] Test steps: The zebrafish were divided into several groups according to the modeling group, test sample group, blank control group and positive control group, with 24 zebrafish in each group. The modeling group was added with culture medium containing C48 / 80 compounds, the test sample group was added with culture medium containing C48 / 80 compounds and test samples, the blank control group was not added with C48 / 80 compounds, but with culture medium instead, and the positive control group was added with culture medium containing 0.1% sodium cromoglycate. After adding the corresponding reagents to each group, after 24 hours of treatment, the absorbance value (OD value) of each group was detected by an enzyme marker, and the relative expression level of trypsin (Trpytase relative expression level) was used to evaluate the soothing effect of the test sample.

[0210] The test results are shown in Table 6 below.

[0211] Table 6

[0212]

[0213] As shown in the results of the above table, under the stimulation of Compound 48 / 80, the relative expression level of Tryptase secreted by the test substance in zebrafish was about 100.0%, and the relative expression level of Tryptase in the blank control group was about 5.9%. Compared with the blank control, Compound 48 / 80 stimulation can successfully induce allergic reactions in zebrafish larvae. After the positive control sodium cromoglycate was used to treat zebrafish larvae that induced allergic reactions with Compound 48 / 80, the relative expression level of Tryptase was 23.3%. Although the relative expression level of Tryptase was higher than that of the blank control group, it was significantly lower than that of the Compound 48 / 80 group, indicating that sodium cromoglycate has a soothing effect. After using the composition of the embodiment of the present invention, the relative expression level of Tryptase was significantly lower than that of the Compound 48 / 80 group, indicating that the composition of the present invention has a soothing effect.

[0214] Among them, the repair effect of the embodiment is better than that of comparative examples 1 and 2, indicating that the source of stem cells has a great influence on the soothing performance of the final product. Comparative example 1 uses rabbit stem cells and comparative example 2 uses bovine stem cells. The soothing effect of the composition prepared under the same conditions is poor. The soothing effect of Example 1 is better than that of Comparative Example 3, indicating that the generation from which the cell culture supernatant is derived has a great influence on the final product. The cell culture supernatant with a fewer number of passages is used, the cell viability is greater, the cell culture supernatant contains more active substances and exosomes, and the soothing effect of the final product is also better. The soothing effect of the embodiment is better than that of comparative examples 4-6, indicating that the fermented strain has a great influence on the soothing effect of the final composition, and the effect of fermenting porcine umbilical cord mesenchymal stem cells using Lactobacillus gasseri is the best. The soothing effect of the embodiment is better than that of comparative example 7, indicating that the type of stem cells selected in the cell supernatant has an effect on the soothing effect of the final product. The present invention preferably uses porcine umbilical cord mesenchymal stem cells to prepare cell culture supernatant.

[0215] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing a composition for promoting hair growth, characterized in that: The following steps are involved: (1) Inoculating porcine umbilical cord mesenchymal stem cells into a culture medium for subculture, and collecting the cell culture supernatant; the conditions for the subculture are: temperature 35-39°C, 3-7% CO2, time 22-48h; the number of subcultures is 2-10 times; (2) inoculating Lactobacillus gasseri seed liquid into the cell culture supernatant for fermentation to obtain a crude fermentation liquid; the number of the Lactobacillus gasseri is CGMCC 1.3396; (3) sterilizing and filtering the crude fermentation broth to obtain a composition for promoting hair growth.

2. The method for preparing the composition for promoting hair growth according to claim 1, characterized in that: In the step (1), the culture medium is a serum-free culture medium, and the amount of the porcine umbilical cord mesenchymal stem cells used is 0.8-1.5% of the mass of the culture medium.

3. The method for preparing the composition for promoting hair growth according to claim 1, characterized in that: In the step (2), the bacterial concentration of the Lactobacillus gasseri seed solution is 10 7 -10 9 cfu / mL, the amount of Lactobacillus gasseri seed solution added is 1-3% of the volume of the cell culture supernatant, the fermentation temperature is 35-40°C, the fermentation time is 24-48h, and the rotation speed is 60-120rpm; in the step (3), the sterilization temperature is 80-95°C, the sterilization time is 15-30min, and the filtration method is diatom filter filtration.

4. The method for preparing the composition for promoting hair growth according to claim 1, characterized in that: In the step (2), the preparation method of the Lactobacillus gasseri seed solution is: S1, inoculating Lactobacillus gasseri colonies into a seed culture medium and culturing once to obtain a Lactobacillus gasseri suspension; S2, inoculating the Lactobacillus gasseri suspension into the seed culture medium for secondary culture to obtain the Lactobacillus gasseri seed solution.

5. The method for preparing the composition for promoting hair growth according to claim 4, characterized in that: In the step S1, the temperature of the primary culture is 35-40°C, the time of the primary culture is 18-30h, and the rotation speed is 60-120rpm; in the step S2, the inoculation volume ratio of the Lactobacillus gasseri suspension is 1-3%, the temperature of the secondary culture is 35-40°C, the time of the secondary culture is 18-30h, and the rotation speed is 60-120rpm; the seed culture medium in the steps S1 and S2 is MRS liquid culture medium or improved MRS liquid culture medium.

6. A composition for promoting hair growth prepared by the method for preparing a composition for promoting hair growth according to any one of claims 1 to 5.

7. Use of the composition for promoting hair growth as claimed in claim 6 in the preparation of daily chemicals for promoting hair growth or medicines for promoting hair growth.

8. Use of the composition for promoting hair growth according to claim 6 in the preparation of cosmetics for promoting hair growth.

9. A cosmetic, characterized in that: The cosmetic comprises the following components in percentage by mass: 0.1-50% of the hair growth promoting composition according to claim 6, 2-40% of the cosmetic base, and the balance of water.

10. The cosmetic according to claim 9, characterized in that The cosmetic matrix includes at least one of a thickener, a functional active substance, an emulsifier, a chelating agent, an antioxidant, a preservative, a flavor, and a pH adjuster.

11. A hair follicle activating essence, characterized in that: The invention comprises the following components in percentage by weight: 1-5% of the composition for promoting hair growth according to claim 6, 0.02-0.08% of acetylated sodium hyaluronate, 0.05-0.2% of ultra-low molecular weight hyaluronic acid, 0.1-0.5% of p-hydroxyacetophenone, 0.8-1.3% of erythritol, 1-3% of fibronectin, 12-18% of ginsenoside, 0.1-0.3% of olive leaf enzyme, 0.5-2% of trehalose, 0.3-0.8% of transdermal polypeptide, 0.3-0.8% of yeast polypeptide, 0.5-2% of colloidal silver and water supplemented to 100% of the total weight.

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