Stem cell exosome composition for repairing skin and hair follicle injury as well as preparation method and application of stem cell exosome composition

Abstract

The invention provides a stem cell exosome composition for repairing skin and hair follicle injury as well as a preparation method and application thereof, and belongs to the technical field of cell repair. The invention provides a stem cell exosome composition for repairing skin and hair follicle injury. The stem cell exosome composition comprises the following components in parts by weight: 30-50 parts of umbilical cord mesenchymal stem cell exosome, 15-25 parts of a ginseng extract, 15-25 parts of lotus placenta, 10-20 parts of a saffron crocus extract, 10-20 parts of tea bran and 10-20 parts of a mulberry leaf extract. The traditional Chinese medicine extract and the umbilical cord blood mesenchymal stem cell exosome are mixed and then immediately applied to damaged skin or scalp, the mixture is applied 3-5 times every day, one treatment course comprises 7 days, and after 1-3 treatment courses, the damaged skin is remarkably healed, the scalp oil phenomenon is remarkably relieved, and hair of hair follicles is remarkably increased.

Description

Technical Field

[0001] The present invention relates to the field of cell repair technology, and in particular to a stem cell exosome composition for repairing skin and hair follicle damage, and a preparation method and application thereof. Background Art

[0002] Burns and scalds are common types of skin injuries in clinical medicine. The healing process of such injuries is complex and time-consuming. Improper treatment can easily lead to infection and may even induce scar formation, resulting in unsightly wounds after treatment, which are easily subject to abnormal gazes from outsiders, seriously affecting the patient's quality of life and physical and mental health. Moreover, most of the existing skin injury repair products are limited to providing short-term analgesic and anti-inflammatory effects, and have no significant long-term effects on promoting skin injury healing and preventing scar formation. Therefore, how to develop a drug that can prevent scar formation is a problem that technicians currently need to solve.

[0003] Hair loss is a common disease in dermatology. With the intensification of social competition, social pressures and psychological and physiological factors such as work pressure, mental stress, and hormone imbalance trigger or aggravate the occurrence of hair loss, significantly affecting patients' mental health and quality of life. Hair loss can be divided into reversible non-scarring alopecia and irreversible scarring alopecia. Irreversible scarring alopecia is caused by damage to hair follicles or loss of hair follicle pores, and the fallen hair cannot regenerate. However, non-scarring alopecia can promote the regeneration of fallen hair or reverse hair follicle damage and mild atrophy through specific treatments.

[0004] Stem cell therapy has shown great potential in the field of regenerative medicine, especially umbilical cord mesenchymal stem cells, which have attracted more and more attention due to their wide sources, convenient extraction, low immunogenicity and other advantages. Studies have found that exosomes, a vesicle structure with a diameter of about 100 nm, secreted by mesenchymal stem cells, have obvious medicinal effects on skin damage by carrying cytokines, growth factors, mRNA, lipids and proteins from source cells. However, the use of single umbilical cord stem cell exosomes has limited repair effects. Therefore, combining stem cell exosomes with traditional Chinese medicine compositions to repair skin and hair follicle damage provides a new idea for solving the above problems. Summary of the invention

[0005] The purpose of the present invention is to provide a stem cell exosome composition for repairing skin and hair follicle damage, which can effectively promote the repair of skin caused by burns and scalds and the growth of hair.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a stem cell exosome composition for repairing skin and hair follicle damage, comprising the following components in parts by weight: 30-50 parts of umbilical cord mesenchymal stem cell exosomes, 15-25 parts of ginseng extract, 15-25 parts of lotus placenta, 10-20 parts of saffron extract, 10-20 parts of tea bran extract, and 10-20 parts of mulberry leaf extract.

[0008] The present invention also provides a method for preparing the composition, comprising the following steps:

[0009] (1) mixing and stirring ginseng extract, lotus placenta extract, saffron extract, tea bran extract, and mulberry leaf extract, and filtering to obtain a Chinese medicine composition;

[0010] (2) When in use, the Chinese medicine composition is mixed with umbilical cord mesenchymal stem cell exosomes at 4-6°C.

[0011] Preferably, the stirring is carried out at 4-6° C. and at a speed of 200-500 r / min for 5-10 min, and the filtration is carried out using a 0.22 μm filter membrane.

[0012] The present invention also provides an application of the composition or the composition obtained by the preparation method in preparing a drug for repairing skin and hair follicle damage.

[0013] The present invention provides a stem cell exosome composition for repairing skin and hair follicle damage, wherein camellia seeds (tea bran) are rich in oil, tea polyphenols, protein, vitamins and various trace elements, which can improve hair quality, reduce the formation of dandruff or oil, promote blood circulation, improve the scalp environment, prevent hair loss, and make hair more shiny.

[0014] A large amount of ginseng raw materials are obtained by tissue culture of 50-year-old ginseng, and the effective ingredients such as saponins and polysaccharides are extracted. These ingredients are easily absorbed, can dilate scalp capillaries, promote blood circulation, regulate water and oil balance, improve scalp environment, and promote hair growth efficiency.

[0015] Lotus ovary extract is known as ovary collagen, 100% natural plant placenta. It is rich in billions of moisturizing antioxidant factors and a large number of amino acids, vitamins, trace elements and other nutrients. It can repair damaged skin, reshape skin activity, promote skin regeneration, stimulate metabolism, improve cell nutrition, and revitalize the skin.

[0016] Frosted mulberry leaves, that is, mulberry leaves harvested after frost, are leaves of the deciduous tree mulberry of the Moraceae family. They are sweet, bitter and cold in nature. They enter the lung and liver meridians. They have the functions of dispersing wind-heat, clearing lung dryness, treating eye diseases, cooling blood, quenching thirst, stopping sweating, preventing hair loss and promoting hair growth. Studies have shown that the main chemical components in mulberry leaves are flavonoids, alkaloids, phenylpropanoids, volatile oils, steroids, organic acids, etc.

[0017] Saffron, also known as saffron and crocus, is a precious Chinese medicinal material. According to the "Compendium of Materia Medica", it is mild in nature and sweet in taste. It has the effects of promoting blood circulation, nourishing blood, dispersing blood stasis and relieving swelling and relieving pain, cooling blood and detoxifying, relieving depression and calming the mind, and regulating qi and strengthening the stomach.

[0018] Tea bran, also known as tea oil cake and tea meal, is the cake produced after pressing tea seeds for oil. Tea bran contains protein, amino acids and up to 12-17% tea saponin. Tea saponin belongs to triterpenoid saponin and is a natural non-ionic surfactant. In addition, tea saponin can restore the permeability of capillaries and has anti-seepage and anti-inflammatory effects. Long-term use of tea bran has the effects of stopping dandruff, relieving itching, degreasing, sterilizing, and repairing damaged hair.

[0019] The present invention mixes the above-mentioned Chinese medicinal extracts with the exosomes of umbilical cord blood mesenchymal stem cells and then applies them to the damaged skin or scalp immediately, 3 to 5 times a day, 7 days as a course of treatment, and 1 to 3 courses of treatment can significantly heal the damaged skin, significantly alleviate the oily scalp phenomenon, and significantly increase the hair of the hair follicles. DETAILED DESCRIPTION

[0020] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0021] Example 1

[0022] A stem cell exosome composition for repairing skin and hair follicle damage is composed of the following components in parts by weight: 40 parts of umbilical cord mesenchymal stem cell exosomes, 20 parts of ginseng extract, 20 parts of lotus placenta, 15 parts of saffron extract, 15 parts of tea bran, and 15 parts of mulberry leaf extract.

[0023] Among them, the method for extracting exosomes from umbilical cord mesenchymal stem cells refers to Example 1 in the Chinese invention patent application number: CN202210808952.0, and the specific steps are:

[0024] (1) The umbilical cord of a healthy full-term newborn was washed three times with sterile PBS; the blood vessels in the umbilical cord were removed and the umbilical cord was cut into 3 mm 2 The left and right tissue blocks were placed in a cell culture bottle, MEM-α complete medium containing 10% FBS was added, and the cells were cultured in a hypoxic environment (37°C, CO2 volume fraction of 5%, N2 volume fraction of 94%, O2 volume fraction of 1%), and the medium was changed every 3 days; when fibroblasts crawled out around the tissue blocks and merged in large pieces, the supernatant and tissue blocks were removed, and 0.25% trypsin-EDTA was used for subculture. The subcultured cells at this time were recorded as P1, and the cells of the subsequent generations were P2, P3...Pn.

[0025] (2) The P3-P9 human umbilical cord mesenchymal stem cells obtained in step (1) were concentrated to 1×10 5 The density of cells / mL was inoculated on a bottom area of ​​75 cm 2 In a culture bottle, umbilical cord mesenchymal stem cells are cultured in a serum-free MEM-α medium; after 48 hours, the cell culture fluid is collected and centrifuged at 200 g for 5 minutes, and the obtained supernatant is the umbilical cord mesenchymal stem cell culture supernatant;

[0026] (3) subjecting the culture supernatant of human umbilical cord mesenchymal stem cells obtained in step (2) to gradient centrifugation, and the resulting precipitate is the exosomes secreted by the mesenchymal stem cells;

[0027] The step of gradient centrifugation is as follows: the culture supernatant of human umbilical cord mesenchymal stem cells obtained in step (2) is centrifuged at 300g for 10 minutes to obtain supernatant No. 1, supernatant No. 1 is centrifuged at 2000g for 30 minutes to obtain supernatant No. 2, supernatant No. 2 is centrifuged at 10000g for 30 minutes to obtain supernatant No. 3, supernatant No. 3 is centrifuged at 120000g for 180 minutes, and the obtained precipitate is the exosomes secreted by mesenchymal stem cells, wherein the temperature of the four centrifugations is 4°C.

[0028] Preparation method of ginseng extract: select five-year artificially cultivated ginseng obtained by tissue culture, take 250g of ginseng, wash it, remove the mud and sand, dry it and cut it into blocks of 0.5×0.5cm~0.5×1.0cm, mix it with 2500mL of production water, and distill it at a distillation temperature of 70°C and a vacuum degree of 0.07Mpa. When the volume of the effluent is equivalent to half of the amount of water added, stop distillation and collect the first distillate. Add production water to the remaining liquid, the amount of water added is the same as the first time, the distillation temperature is 80°C, the vacuum degree is 0.06Mpa, and when the volume of the effluent is equivalent to half of the amount of water added, stop distillation and collect the second distillate. Combine the first distillate and the second distillate to obtain the ginseng extract.

[0029] Lotus placenta extraction method: Pick unopened lotus seeds, clean them, let them stand for 10 minutes, use a sealed bag to pack the lotus, and store them in a 5°C refrigerator. After 24 hours, take the lotus out of the refrigerator and wash it again twice in tap water. Then wash it three times in deionized water. The lotus and the extract are crushed together at a mass ratio of 5:1 for 5 minutes to obtain lotus slurry. The extract formula is: 1wt% glycerol, 1wt% propylene glycol, and 98wt% water. The lotus slurry is put into a three-roller mill and rolled three times, and weighed after completion. The lotus slurry and the extract are poured into a stirring pot at a mass ratio of 1:1 again and stirred for 60 minutes. The obtained extraction mixture is first filtered through a 200-mesh filter to separate the large-particle lotus residue. Then it is filtered through a filter element pressure filter with a pore size of 100 microns, 10 microns and 0.2 microns in turn. After decolorization and desalination, and then sterilized by a bacterial filter, a colorless and transparent lotus extract is obtained.

[0030] The saffron extract is prepared by the following method: taking a whole saffron, soaking it in pure water for 3 days; filtering it with gauze to obtain the soaking solution, evaporating and concentrating it into a paste; dissolving and extracting it with 80% ethanol for 4 times, evaporating and removing the solvent, and spray drying to obtain the saffron extract. When used, pure water is added to dissolve it in a mass volume ratio of 1:2, and filtered through a filter membrane with a pore size of 0.22 microns for use.

[0031] Preparation method of tea bran: crush the tea bran, then mix it with deionized water at a solid-liquid mass ratio of 1:2, add 0.2g / ml enzyme solution at 35℃ for 4h to obtain enzymatic solution; the enzymatic solution is prepared by mixing cellulase, lipase and amylase at a mass ratio of 2:1:1, the enzyme activity of cellulase is 600u / g, the enzyme activity of lipase is 800u / g, and the enzyme activity of amylase is 1100u / g. Add deionized water twice as much as the enzymatic solution to the enzymatic solution and grind it into a fine slurry; add probiotic solution to the fine slurry for probiotic fermentation for 4h, heat to 80℃ for sterilization for 15min, and cool to room temperature; the amount of probiotic solution added is 0.5% of the volume of the finely ground slurry, which includes thermophilic streptococcus, aspergillus niger, yeast and bacillus subtilis in a content ratio of 1:1:1:1, and the effective live bacteria concentration of the probiotic solution is 3.2×10 4 The obtained fermented liquid is again emulsified by water grinding to obtain the liquid which is the tea bran extract. It is filtered through a filter membrane with a pore size of 0.22 microns for later use.

[0032] The preparation method of mulberry leaf extract is as follows: weigh naturally dried mulberry leaves, mix them with water at a mass volume ratio of 1:10 Kg / L, and perform ultrasonic treatment with an ultrasonic frequency of 100 kHz, a power of 300 W, and an ultrasonic treatment of 40 minutes; after completion, place the mulberry leaves in water at a temperature of 45°C for water extraction, adjust the pH value to 7.50, stir at a speed of 30 rpm, and perform water extraction for 60 minutes; filter the feed liquid with a 200-mesh sieve; repeat the previous steps for the residue and stir and extract it twice with hot water, remove the residue and leave the filtrate, combine the filtrates obtained three times, centrifuge at 3000 rpm to remove impurities for 20 minutes, and obtain a supernatant; seal and store for later use.

[0033] The ginseng extract, lotus placenta extract, soapberry extract, tea bran extract and mulberry leaf extract were mixed, stirred at 400 r / min for 5 min at 4° C., and filtered through a 0.22 μm filter membrane to obtain a Chinese medicine composition;

[0034] When in use, the above-mentioned Chinese medicine composition is mixed with umbilical cord mesenchymal stem cell exosomes at 4°C.

[0035] Experimental Example 1

[0036] 1. Healthy ICR mice, weighing 20±2g, half male and half female, were provided by the Experimental Animal Center of Sun Yat-sen University.

[0037] 2. Experimental Methods

[0038] Set up the following groups:

[0039] Example 1: The original solution of the composition was diluted 5 times with water.

[0040] Comparative Example 1: The composition formula is adjusted to: 20 parts of umbilical cord mesenchymal stem cell exosomes, 30 parts of ginseng extract, 30 parts of lotus placenta, 15 parts of saffron extract, 15 parts of tea bran, and 15 parts of mulberry leaf extract. When used, the original solution of the composition is diluted 5 times with water.

[0041] Comparative Example 2: The composition formula is adjusted to: 70 parts of umbilical cord mesenchymal stem cell exosomes, 10 parts of ginseng extract, 10 parts of lotus placenta, 10 parts of saffron extract, 10 parts of tea bran, and 10 parts of mulberry leaf extract. When used, the original solution of the composition is diluted 5 times with water.

[0042] Comparative Example 3: The composition formula is adjusted to: 40 parts of umbilical cord mesenchymal stem cell exosomes, 10 parts of ginseng extract, 10 parts of lotus placenta, 10 parts of wolfberry, 10 parts of Polygonum multiflorum, 10 parts of Ligustrum lucidum, 10 parts of black sesame, and 10 parts of fig. When used, the original solution of the composition is diluted 5 times with water.

[0043] Comparative Example 4: The composition formula is adjusted to: 40 parts of umbilical cord mesenchymal stem cell exosomes, 10 parts of Platycladus orientalis extract, 10 parts of lotus placenta, 10 parts of wolfberry, 10 parts of tea bran, and 10 parts of mulberry leaf extract

[0044] 10 parts of black sesame. When using, dilute the original solution of the composition with water 5 times.

[0045] A total of 42 healthy adult ICR mice were selected. The mice were anesthetized with sodium pentobarbital injection, and the hair removal was performed on the back, with the hair removal area of ​​1cm×1cm. After the back was scalded (second degree scald, paraffin oil 180°C), they were randomly divided into a positive control group, a model group (no drug application), and an experimental group (including Example 1 group, comparative examples 1 to 4 groups, a total of 5 groups), with 6 mice in each group. After modeling, the drug was administered 3 times a day, and the dosage of each administration was 0.2mL / cm 2 The drugs were administered for 21 consecutive days. The blank group and the model group were not given any drugs. The positive control group was given Jingwanhong ointment at a dosage of 0.2 g / cm 2 The scarring time and scarring rate were used to investigate the therapeutic effect of the composition on burns. The scarring rate was the percentage of the scab area to the skin lesion area, and the average value was taken for each group. The specific values ​​are shown in Table 1.

[0046] Table 1 Scarring rate of each treatment group at different time

[0047] Group 3-day scarring rate (%) 7-day scarring rate (%) 14-day scarring rate (%) Model Group 11.1 21.5 55.4 Positive control group 32.7 62.7 80.6 Example 1 Group 62.5 80.6 100 Comparative Example 1 49.2 60.5 80.6 Comparative Example 2 49.6 63.8 85.4 Comparative Example 3 47.3 56.3 80.9 Comparative Example 4 48.4 58.9 81.5

[0048] As can be seen from Table 1, the composition of Example 1 group has the best effect, and scabs can be formed faster in 3 days, which is more significant than other groups.

[0049] Experimental Example 2

[0050] 70 healthy mice were selected and randomly divided into 7 groups according to body weight after one week of adaptive feeding, with 10 mice in each group. The hair on the back of the mice was cut short and a depilatory cream was applied to the back of the mice for about 5 minutes. The depilatory area was about 3×4 cm. The skin was washed with warm water and pink without damage, confirming that the hair follicles of the mice were in the resting phase.

[0051] Animal modeling was performed according to the reference "Effect of Ligustrum lucidum alcohol extract on hair growth in mice with androgenic alopecia model". Except for the blank group, each group of mice was injected with testosterone propionate solution 5 mg / (kg·d) at multiple points under the skin of the depilatory area on the back, and treated with testosterone propionate for 30 minutes. The blank group and the model group were smeared with 200 μL of distilled water, and the experimental group (Example 1 group in Experimental Example 1, Comparative Examples 1 to 4 groups, a total of 5 groups) was smeared with 200 μL of the composition liquid, and massaged for 1 minute after smearing for 14 days. The skin and hair status of the mice were observed, and the hair growth of each group of mice was recorded and scored 7 and 14 days after administration. The scoring criteria are as follows:

[0052] 0 points: no growth, the skin in the hair removal area is pink;

[0053] 1 point: the skin in the hair removal area is gray (less than 20% growth);

[0054] 2 points: The skin in the hair removal area is black with a little hair growth (more than 20% and less than 40%);

[0055] 3 points: The skin in the hair removal area is black with a little hair growth (greater than 40% and less than 60%);

[0056] 4 points: The skin in the hair removal area is black and some hair grows (60% to 80% growth);

[0057] 5 points: The hair in the depilated area has basically grown completely (80% to 100% growth).

[0058] The results are shown in Table 2.

[0059] Table 2 The scoring results of hair growth of mice in each group

[0060] Group 7 days 14 days Blank Group 1 4 Model Group 0 2 Example 1 Group 2 5 Comparative Example 1 1 4 Comparative Example 2 1 4 Comparative Example 3 1 4 Comparative Example 4 1 4

[0061] Table 2 shows that the hair growth of the composition of Example 1 was significantly improved compared with other groups during the 7-14 days of treatment, and the hair follicle damage recovered fastest.

[0062] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A stem cell exosome composition for repairing skin and hair follicle damage, characterized in that: The invention comprises the following components in parts by weight: 5-10 parts of umbilical cord mesenchymal stem cell exosomes, 30-50 parts of ginseng extract, 15-30 parts of lotus placenta, 10-20 parts of saffron extract, 10-20 parts of tea bran extract and 10-20 parts of mulberry leaf extract.

2. A method for preparing the composition according to claim 1, characterized in that: The steps include: (1) mixing and stirring ginseng extract, lotus placenta extract, saffron extract, tea bran extract, and mulberry leaf extract, and filtering to obtain a Chinese medicine composition; (2) When in use, the Chinese medicine composition is mixed with umbilical cord mesenchymal stem cell exosomes at 4-6°C.

3. The preparation method according to claim 2, characterized in that: The stirring is carried out at 4-6° C. and at a rotation speed of 200-500 r / min for 5-10 min, and the filtration is carried out using a 0.22 μm filter membrane.

4. Use of the composition according to claim 1 or the composition obtained by the preparation method according to claims 2 to 3 in preparing drugs for repairing skin and hair follicle damage.