Preparation for cloning hair follicle cells and promoting hair growth as well as preparation method and application of preparation
By constructing a preparation containing hair follicle cell proliferation factor and plant extract, the problem that existing hair loss treatment methods fail to effectively promote hair follicle cell proliferation and hair growth has been solved, and excellent hair growth effect has been achieved.
Patent Information
- Application Number
- CN202411977572.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2025-04-11
AI Technical Summary
Existing hair loss treatment methods have not fundamentally solved the problem of hair loss, and there is a lack of effective methods to promote hair follicle cell proliferation and hair growth.
By constructing a preparation that includes hair follicle cell proliferation factor, glycine, mannitol, sodium hyaluronate, Ligustrum extract and Polygonum multiflorum extract for promoting hair follicle cell cloning and hair growth.
This preparation can effectively promote the proliferation and hair growth of hair follicle stem cells, and the preparation method is simple and inexpensive.
Smart Images
Figure CN119792489A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of hair growth, and in particular to a preparation for hair follicle cell cloning and hair growth promotion, and a preparation method and application thereof. Background Art
[0002] Hair loss refers to the phenomenon of hair loss. Normally lost hair is in the catagen and telogen phases. Since the hair entering the catagen and the hair entering the growth phase are constantly in a dynamic balance, the normal amount of hair can be maintained. Pathological hair loss refers to abnormal or excessive hair loss, which has many causes. Method for preparing hair follicle stem cells.
[0003] At present, the treatment methods for hair loss include drug treatment, such as minoxidil and finasteride, laser treatment, such as using low-energy laser to irradiate the scalp to stimulate the growth activity of hair follicles and promote healthy hair growth, surgical treatment, such as hair transplantation, and traditional Chinese medicine treatment, such as astragalus, Polygonum multiflorum, Cuscuta australis, Rehmannia glutinosa, etc. However, the current hair loss treatment methods have not fundamentally solved the problem of hair loss. In view of this, the present invention aims to provide an effective hair loss solution. Summary of the invention
[0004] In view of the technical problems existing in the prior art, the present invention provides a preparation for hair follicle cell cloning and hair growth promotion, and its preparation method and application. The present invention uses hair follicle cell proliferation promoting factor and cooperates with Ligustrum lucidum extract and Polygonum multiflorum extract to construct a preparation for hair follicle cell cloning and hair growth promotion. After verification and analysis, the preparation has excellent effects of promoting hair follicle stem cell proliferation and hair growth.
[0005] Specifically, the present invention first provides a preparation for hair follicle cell cloning and promoting hair growth, which includes the following components: 1-5 parts of hair follicle cell proliferation promoting factor, 20-50 parts of glycine, 10-30 parts of mannitol, 30-60 parts of sodium hyaluronate, 50-100 parts of Ligustrum lucidum extract, 50-100 parts of Polygonum multiflorum extract and 500-1000 parts of purified water.
[0006] Preferably, the preparation comprises the following components: 1 part of hair follicle cell proliferation promoting factor, 20 parts of glycine, 10 parts of mannitol, 30 parts of sodium hyaluronate, 50 parts of Ligustrum lucidum extract, 50 parts of Polygonum multiflorum extract and 500 parts of purified water.
[0007] Preferably, the preparation comprises the following components: 2 parts of hair follicle cell proliferation promoting factor, 30 parts of glycine, 20 parts of mannitol, 40 parts of sodium hyaluronate, 70 parts of Ligustrum lucidum extract, 80 parts of Polygonum multiflorum extract and 800 parts of purified water.
[0008] Preferably, the preparation comprises the following components: 5 parts of hair follicle cell proliferation promoting factor, 50 parts of glycine, 30 parts of mannitol, 60 parts of sodium hyaluronate, 100 parts of Ligustrum lucidum extract, 100 parts of Polygonum multiflorum extract and 1000 parts of purified water.
[0009] Preferably, the hair follicle cell proliferation promoting factor provided by the present invention is an active polypeptide shown in SEQ ID NO.1, which can increase the expression level of SETDB1 in hair follicle stem cells and promote hair regeneration.
[0010] Preferably, the method for preparing the extract of Ligustrum lucidum or Polygonum multiflorum comprises the following steps: 1) Weigh 100-200g of Ligustrum lucidum or Polygonum multiflorum, grind mechanically, pass through a 100-mesh sieve, and set aside; 2) Add deionized water to the powder of step 1) at a mass / volume ratio of 1:(5-10) (g:mL), heat to boil, keep warm at 75-90°C for 3-4h, cool, centrifuge and collect the filtrate; 3) adding cellulase at a final concentration of 100-200 IU / mL to the filtrate of step 2), maintaining the temperature at 55-65°C for enzymatic hydrolysis for 4-6 hours, and then rapidly heating to 95°C for 10-20 minutes after enzymatic hydrolysis; 4) The enzymatic hydrolyzate of step 3) is filtered through an ultrafiltration membrane, and the filtrate with a molecular weight cutoff of less than or equal to 3000 Da is the extract of Ligustrum lucidum or Polygonum multiflorum.
[0011] Preferably, the method for preparing the extract of Ligustrum lucidum or Polygonum multiflorum comprises the following steps: 1) Weigh 100g of Ligustrum lucidum or Polygonum multiflorum, grind it mechanically, pass it through a 100-mesh sieve, and set aside; 2) Add deionized water to the powder of step 1) at a mass / volume ratio of 1:5 (g:mL), heat to boil, keep warm at 85°C for 3.5h, cool, centrifuge and collect the filtrate; 3) adding cellulase at a final concentration of 100 IU / mL to the filtrate of step 2), maintaining the temperature at 55°C for 4 hours of enzymolysis, and then rapidly heating to 95°C for 10 minutes after enzymolysis; 4) The enzymatic hydrolyzate of step 3) is filtered by an ultrafiltration membrane, and the filtrate with a molecular weight cutoff of less than or equal to 3000 Da is the extract of Ligustrum lucidum and Polygonum multiflorum.
[0012] Preferably, the method for preparing the extract of Ligustrum lucidum or Polygonum multiflorum comprises the following steps: 1) Weigh 150g of Ligustrum lucidum or Polygonum multiflorum, grind it mechanically, pass it through a 100-mesh sieve, and set aside; 2) Add deionized water to the powder of step 1) at a mass / volume ratio of 1:8 (g:mL), heat to boil, keep at 80°C for 4 hours, cool, centrifuge and collect the filtrate; 3) adding cellulase at a final concentration of 150 IU / mL to the filtrate of step 2), maintaining the temperature at 60°C for enzymatic hydrolysis for 5 hours, and then rapidly heating to 95°C for 15 minutes after enzymatic hydrolysis; 4) The enzymatic hydrolyzate of step 3) is filtered through an ultrafiltration membrane, and the filtrate with a molecular weight cutoff of less than or equal to 3000 Da is the extract of Ligustrum lucidum or Polygonum multiflorum.
[0013] Preferably, the method for preparing the extract of Ligustrum lucidum and Polygonum multiflorum comprises the following steps: 1) Weigh 200g of Ligustrum lucidum or Polygonum multiflorum, grind mechanically, pass through a 100-mesh sieve, and set aside; 2) Add deionized water to the powder of step 1) at a mass / volume ratio of 1:10 (g:mL), heat to boil, keep at 90°C for 3h, cool, centrifuge and collect the filtrate; 3) adding cellulase at a final concentration of 200 IU / mL to the filtrate of step 2), maintaining the temperature at 65°C for enzymatic hydrolysis for 6 hours, and then rapidly heating to 95°C for 20 minutes after enzymatic hydrolysis; 4) The enzymatic hydrolyzate of step 3) is filtered through an ultrafiltration membrane, and the filtrate with a molecular weight cutoff of less than or equal to 3000 Da is the extract of Ligustrum lucidum or Polygonum multiflorum.
[0014] Preferably, the extract of Ligustrum lucidum and Polygonum multiflorum is prepared by decoction and enzymatic hydrolysis to effectively extract active ingredients from Ligustrum lucidum and Polygonum multiflorum, which can help hair follicle cell proliferation factor to promote hair growth.
[0015] Another aspect of the present invention also provides a method for preparing the preparation, comprising the following steps: 1) Weigh the above number of portions for later use; 2) Add glycine, mannitol and sodium hyaluronate to purified water in sequence and mix well; 3) adding the extracts of Ligustrum lucidum and Polygonum multiflorum successively into step 2) and stirring to dissolve; 4) Add the hair follicle cell proliferation promoting factor into step 3) and stir to dissolve. After stirring and dissolving, perform microfiltration. After filtration, perform packaging to obtain a finished preparation.
[0016] On the other hand, the present invention also provides use of the preparation obtained by the above method in preparing a preparation for preventing or treating hair loss.
[0017] Preferably, the preparation is a liquid preparation.
[0018] Preferably, the preparation is applied by spraying evenly on the hair loss area, once in the morning, noon and evening.
[0019] Another object of the present invention is to provide a hair follicle stem cell, characterized in that the hair follicle stem cell specifically expresses a hair follicle cell proliferation-promoting factor.
[0020] Preferably, the present invention further provides a hair follicle stem cell exosome, characterized in that the hair follicle stem cells specifically express hair follicle cell proliferation promoting factor.
[0021] The advantages of the present invention are as follows: the present invention extracts and separates a hair follicle cell proliferation factor for the first time, which can effectively promote the expression of the SETDB1 gene in hair follicle stem cells and promote the proliferation of hair follicle stem cells. Thus, the present invention constructs the hair growth preparation, which is a preparation for hair follicle cell cloning and hair growth promotion using the hair follicle cell proliferation factor and cooperating with Ligustrum lucidum extract and Polygonum multiflorum extract. After verification and analysis, the preparation has excellent effects on promoting the proliferation of hair follicle stem cells and hair growth, and the preparation method of the preparation is simple and low in cost. At the same time, the present invention also provides hair follicle stem cells or hair follicle stem cell exosomes that specifically express hair follicle cell proliferation factor, and the use of the stem cells or their exosomes can also achieve the purpose of hair follicle cell cloning and promoting hair growth. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 . Microscopic observation results of hair follicle stem cell proliferation promotion; Figure 2 . Analysis of relative expression of SETDB1 gene in hair follicle stem cells. DETAILED DESCRIPTION
[0023] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.
[0024] The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention.
[0025] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.
[0026] Example 1 A preparation for cloning hair follicle cells and promoting hair growth, comprising the following components: 1 part of hair follicle cell proliferation promoting factor, 20 parts of glycine, 10 parts of mannitol, 30 parts of sodium hyaluronate, 50 parts of Ligustrum lucidum extract, 50 parts of Polygonum multiflorum extract and 500 parts of purified water.
[0027] The hair follicle cell proliferation promoting factor provided by the present invention is an active polypeptide shown in SEQ ID NO.1, which can increase the expression level of SETDB1 in hair follicle stem cells and promote hair regeneration.
[0028] The method for obtaining the hair follicle cell proliferation promoting factor is to extract the active polypeptide from ginger by enzymatic hydrolysis, and the specific steps are as follows: 1) Wash the ginger, cut it into pieces, and physically crush it to prepare a homogenate; 2) adding a composite protease with an enzyme dosage of 220 IU / mL of ginger homogenate to the ginger homogenate, maintaining the temperature at 45°C and stirring continuously for 3 hours, and then performing enzyme inactivation treatment; wherein the composite protease is a combination of alkaline protease and fibrinase, and the enzyme dosage ratio of the two is 2:3; 4) The enzymatic hydrolysate is centrifuged and filtered to retain polypeptides with a molecular weight between 40,000 Da and 60,000 Da, and then analyzed and identified by HPLC-MS.
[0029] Wherein, the preparation method of the extract of Ligustrum lucidum or Polygonum multiflorum comprises the following steps: 1) Weigh 100g of Ligustrum lucidum or Polygonum multiflorum, grind it mechanically, pass it through a 100-mesh sieve, and set aside; 2) Add deionized water to the powder of step 1) at a mass / volume ratio of 1:5 (g:mL), heat to boil, keep at 85°C for 3.5h, cool to 25°C, centrifuge at 2500rpm, and collect the filtrate; 3) adding cellulase at a final concentration of 100 IU / mL to the filtrate of step 2), maintaining the temperature at 55°C for 4 hours of enzymolysis, and then rapidly heating to 95°C for 10 minutes after enzymolysis; 4) The enzymatic hydrolyzate of step 3) is filtered by an ultrafiltration membrane, and the filtrate with a molecular weight cutoff of less than or equal to 3000 Da is the extract of Ligustrum lucidum and Polygonum multiflorum.
[0030] Among them, the extract of Ligustrum lucidum and Polygonum multiflorum described in the present invention utilizes water decoction and enzymatic hydrolysis methods to effectively extract active ingredients in Ligustrum lucidum and Polygonum multiflorum, and the active ingredients help the hair follicle cell proliferation promoting factor to exert the effect of promoting hair growth.
[0031] The preparation method of the preparation comprises the following steps: 1) Weigh the above number of portions for later use; 2) Add glycine, mannitol and sodium hyaluronate to purified water in sequence and mix well; 3) adding the extracts of Ligustrum lucidum and Polygonum multiflorum successively into step 2) and stirring to dissolve; 4) Add the hair follicle cell proliferation promoting factor into step 3) and stir to dissolve. After stirring and dissolving, perform microfiltration. After filtration, perform packaging to obtain a finished preparation.
[0032] Example 2 A preparation for hair follicle cell cloning and hair growth promotion, comprising the following components: 2 parts of hair follicle cell proliferation promoting factor, 30 parts of glycine, 20 parts of mannitol, 40 parts of sodium hyaluronate, 70 parts of Ligustrum lucidum extract, 80 parts of Polygonum multiflorum extract and 800 parts of purified water.
[0033] The preparation methods are the same as in Example 1.
[0034] Example 3 A preparation for cloning hair follicle cells and promoting hair growth, comprising the following components: 5 parts of hair follicle cell proliferation promoting factor, 50 parts of glycine, 30 parts of mannitol, 60 parts of sodium hyaluronate, 100 parts of Ligustrum lucidum extract, 100 parts of Polygonum multiflorum extract and 1000 parts of purified water.
[0035] The preparation methods are the same as in Example 1.
[0036] Comparative Example 1 A hair growth preparation comprises the following components: 55 parts of glycine, 30 parts of mannitol, 60 parts of sodium hyaluronate, 100 parts of Ligustrum lucidum fruit extract, 100 parts of Polygonum multiflorum extract and 1000 parts of purified water.
[0037] The preparation method is similar to that of Example 1.
[0038] Comparative Example 2 A hair growth preparation comprises the following components: 5 parts of hair follicle cell proliferation promoting factor, 50 parts of glycine, 30 parts of mannitol, 60 parts of sodium hyaluronate and 1200 parts of purified water.
[0039] The preparation method is similar to that of Example 1.
[0040] Verification Example 1 Analysis of the proliferation-promoting test of hair follicle cell proliferation-promoting factor: Hair follicle stem cells were collected and separated, and pretreated with DMEM complete medium. When cell proliferation was stable, hair follicle cell proliferation-promoting factor with a final concentration of 2 ng / mL was added to the DMEM complete medium, and culture was continued for 12 hours. The blank group was added with a volume of DMEM complete medium. After the culture was completed, the number of hair follicle stem cells in each field of view was recorded by statistical microscopy, and the expression of the SETDB1 gene in the hair follicle stem cells was quantitatively analyzed by qPCR.
[0041] The results are as follows Figure 1-2 As shown, compared with the blank group, the hair follicle cell proliferation factor effectively increased the number of cells in each field of view, and the expression of the SETDB1 gene also showed an upward trend, proving that the hair follicle cell proliferation factor of the present invention can effectively regulate the proliferation potential of hair follicle stem cells by promoting the expression of the SETDB1 gene.
[0042] Verification Example 2
[0043] Hair growth promotion test: 70 Balb / c male mice were divided into 6 groups, 10 mice in each group, and Na 2 S Hair Remover for Back 4x4cm 2 Regional hair. The treatment groups were smeared with the preparations described in Examples 1-3 and Comparative Examples 1-2, 0.2 mL / time per mouse, three times a day, the positive control group was smeared with an equal amount of minoxidil tincture, and the negative control group was smeared with an equal amount of distilled water. After smearing for 7 consecutive days, the hair growth on the back of the mice was recorded and counted.
[0044] The results are shown in Table 1. The hair on the back of the mice in Examples 1-3 grew rapidly and densely. Compared with Comparative Examples 1-2, the hair growth preparation prepared by the present invention has good hair growth promoting performance.
[0045] Table 1. Analysis of hair growth promotion test Group Hair length (mm) <![CDATA[Hair density (roots / cm 2 ).]]> Example 1 8.12±0.21262 115.2454±5.3665 Example 2 9.26±0.32151 138.5455±4.8985 Example 3 11.35±0.33214 157.8254±8.5412 Comparative Example 1 3.26±0.41554 86.5468±9.1254 Comparative Example 2 4.65±0.32126 90.1364±5.2545 Negative control group 1.23±0.02364 85.3225±2.3326 Positive control group 9.15±0.45985 135.6672±5.2654
[0046] It is necessary to point out that the above embodiments are limited to further elaboration and explanation of the technical solution of the present invention, and are not further limitations of the technical solution of the present invention. The method of the present invention is only a preferred implementation scheme, and is not used to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A preparation for hair follicle cell cloning and hair growth promotion, characterized in that: The preparation comprises the following components: 1-5 parts of hair follicle cell proliferation promoting factor, 20-50 parts of glycine, 10-30 parts of mannitol, 30-60 parts of sodium hyaluronate, 50-100 parts of Ligustrum lucidum fruit extract, 50-100 parts of Polygonum multiflorum extract and 500-1000 parts of purified water.
2. The preparation according to claim 1, characterized in that The preparation comprises the following components: 1 part of hair follicle cell proliferation promoting factor, 20 parts of glycine, 10 parts of mannitol, 30 parts of sodium hyaluronate, 50 parts of Ligustrum lucidum extract, 50 parts of Polygonum multiflorum extract and 500 parts of purified water.
3. The preparation according to claim 1, characterized in that The preparation comprises the following components: 2 parts of hair follicle cell proliferation promoting factor, 30 parts of glycine, 20 parts of mannitol, 40 parts of sodium hyaluronate, 70 parts of Ligustrum lucidum extract, 80 parts of Polygonum multiflorum extract and 800 parts of purified water.
4. The preparation according to claim 1, characterized in that The preparation comprises the following components: 5 parts of hair follicle cell proliferation promoting factor, 50 parts of glycine, 30 parts of mannitol, 60 parts of sodium hyaluronate, 100 parts of Ligustrum lucidum extract, 100 parts of Polygonum multiflorum extract and 1000 parts of purified water.
5. The preparation according to claim 1, characterized in that The hair follicle cell proliferation promoting factor is an active polypeptide shown in SEQ ID NO.1, which can increase the expression level of SETDB1 in hair follicle stem cells and promote hair regeneration.
6. The preparation according to claim 1, characterized in that The preparation method of the extract of Ligustrum lucidum or Polygonum multiflorum comprises the following steps: 1) Weigh 100-200g of Ligustrum lucidum or Polygonum multiflorum, grind mechanically, pass through a 100-mesh sieve, and set aside; 2) Add deionized water to the powder of step 1) at a mass / volume ratio of 1:(5-10), heat to boil, keep warm at 75-90°C for 3-4h, cool, centrifuge and collect the filtrate; 3) adding cellulase at a final concentration of 100-200 IU / mL to the filtrate of step 2), maintaining the temperature at 55-65°C for enzymatic hydrolysis for 4-6 hours, and then rapidly heating to 95°C for 10-20 minutes after enzymatic hydrolysis; 4) The enzymatic hydrolyzate of step 3) is filtered through an ultrafiltration membrane, and the filtrate with a molecular weight cutoff of less than or equal to 3000 Da is the extract of Ligustrum lucidum or Polygonum multiflorum.
7. A method for preparing the preparation according to any one of claims 1 to 6, characterized in that: The steps include: 1) Weigh the above number of portions for later use; 2) Add glycine, mannitol and sodium hyaluronate to purified water in sequence and mix well; 3) adding the extracts of Ligustrum lucidum and Polygonum multiflorum successively into step 2) and stirring to dissolve; 4) Add the hair follicle cell proliferation promoting factor into step 3) and stir to dissolve. After stirring and dissolving, perform microfiltration. After filtration, perform packaging to obtain a finished preparation.
8. Use of the preparation obtained by the method of claim 7 in preparing a preparation for preventing or treating hair loss.
9. The use according to claim 8, characterized in that The preparation is a liquid preparation.
10. A hair follicle stem cell or hair follicle stem cell exosome, characterized in that: The hair follicle stem cells or hair follicle stem cell exosomes specifically express hair follicle cell proliferation promoting factor, and the hair follicle cell proliferation promoting factor is an active polypeptide shown in SEQ ID NO.1, and the active polypeptide can increase the expression level of SETDB1 in hair follicle stem cells.