Application of glycerol glucoside in preparation of product for treating alopecia seborrhoeica

Abstract

The invention provides application of glycerol glucoside in preparation of a product for treating alopecia seborrhoeica, and belongs to the technical field of alopecia prevention. Glycerin can promote the proliferation and repair capacity of hair follicle stem cells by activating a Wnt / beta-catenin signal channel, inhibiting the enzyme activity of 5alpha-reductase and reducing the generation of dihydrotestosterone, so that hair follicle regeneration and hair growth are promoted, and the effect of treating alopecia seborrhoeica is achieved.

Description

Technical Field

[0001] The invention relates to the technical field of anti-hair loss, in particular to application of glycerol glucoside in preparing a product for treating seborrheic alopecia. Background Art

[0002] Glyceryl Glucoside (GG) is a multifunctional natural glycoside that was first discovered in lilies in the 1970s. It has six stereostructures, as follows: Figure 1 As shown, 2-αGG and 2-βGG are natural configurations. The 2-αGG configuration has significant effects in moisturizing, anti-oxidation, and promoting cell activity because it connects a molecule of glucose and glycerol through an α-1,2-glycosidic bond. It has broad application potential in the fields of medicine, food, and cosmetics.

[0003] In the medical field, glyceryl glucoside can maintain the hydration state of the skin and enhance the skin barrier function by absorbing and retaining water. In the food field, glyceryl glucoside can not only be used as an emulsifier and stabilizer in foods such as ice cream, candy, chocolate, and dairy products, but can also effectively inhibit the growth of intestinal pathogens by promoting the growth of probiotics. In the cosmetics field, due to its small molecular weight, glyceryl glucoside can efficiently penetrate into the dermis of the skin to achieve deep moisturizing and long-lasting moisturizing. Studies have shown that glyceryl glucoside can enhance the water retention function of the skin, reduce water loss, and provide a lasting moisturizing effect by increasing the expression of aquaporin AQP3; for example, glyceryl glucoside has a good effect in the treatment of dry skin, especially when used in combination with other ingredients such as natural moisturizing factors and ceramides, it can more comprehensively improve the skin's water retention capacity and barrier function. In addition, glyceryl glucoside also has the ability to activate and promote cell regeneration, enhance cell vitality, and accelerate cell metabolism. It can effectively reduce the release of inflammatory cytokines, and improve skin inflammation, acne, and relieve skin redness and allergies. In terms of safety, glyceryl glucoside can be used as a substitute for glycerol in cosmetics with lower cytotoxicity and genotoxicity.

[0004] Seborrheic alopecia (SA) is a common androgen-dependent hair loss disease in clinical practice. After the onset of the disease, patients may experience symptoms such as gradual hair loss on the forehead and top of the head, increased scalp oil, and a large amount of dandruff, which affects their appearance and mental health. Modern medicine believes that SA is an androgen-dependent autosomal dominant polygenic hereditary alopecia. Excessive androgen or increased sensitivity to androgen is the main factor for the occurrence of the disease. The occurrence of SA is directly related to the increase in the levels of testosterone (T) and its metabolite dihydrotestosterone (DHT), and androgen receptor (AR), and 5α-reductase (5α-R) plays an important role in it. In addition, endocrine dysfunction, inflammation, genetics and other factors can all lead to the occurrence of the disease. Minoxidil is a commonly used drug for the treatment of seborrheic alopecia, but it can cause adverse reactions such as hirsutism during use, and it only treats the symptoms but not the root cause, so the overall effect is not ideal. In addition to minoxidil, traditional Chinese medicine has also conducted research on the effects of some Chinese herbal components on seborrheic alopecia, such as "Liu Na. Research on the efficacy mechanism of Xinyi on seborrheic alopecia and analysis of active ingredients", "Li Menghuan, Xue Yujie, Chen Xiaoyan. Effect of Astragalus Granules combined with Minoxidil in the treatment of seborrheic alopecia and its impact on growth factor levels" and other literatures have explored the therapeutic effects of Chinese herbal components in seborrheic alopecia.

[0005] The hair follicle is a unique organ located between the dermis and epidermis of the skin and is responsible for the production of hair. The hair follicle contains two types of stem cells: epithelial stem cells (such as hair follicle stem cells) and mesenchymal stem cells (such as dermal papilla cells). These two types of stem cells work together to maintain the structure and function of the hair follicle and are also involved in the regeneration and repair of the skin.

[0006] The patent application with the publication number "CN113908072A" discloses a hair growth liquid containing glycerol glucoside, which uses glycerol glucoside as the main active ingredient to promote hair growth. Through the synergistic effects of scalp moisturizing, anti-oxidation, reducing inflammation, regulating sebum, and repairing damaged cells, it improves the growth environment of scalp hair follicles, prevents hair loss and promotes hair growth. The patent application with the publication number "CN112716963A" discloses the use of glycerol glucoside in hair growth products, wherein glycerol glucoside can enhance the ability of hair papilla cells to secrete VEGF and glycerol glucoside has no toxic effect on hair papilla cells, thereby increasing the blood vessel density around the hair follicles and the permeability of the blood vessels, promoting the supply of oxygen and nutrients required by the hair, and then promoting scalp hair growth. However, the above patents all conduct research on the promoting effect of glycerol glucoside on hair papilla cells, and no detailed research is conducted on specific types of hair loss.

[0007] In addition to hair papilla cells, there is currently no specific research on whether glycerol glucoside has a promoting effect on hair follicle stem cells, another core cell in hair growth, and what its mechanism of action is. There is also no report on the targeted therapeutic effect of glycerol glucoside in seborrheic alopecia. Summary of the invention

[0008] In view of the deficiencies of the prior art, the present invention provides the use of glycerol glucoside in preparing a product for treating seborrheic alopecia.

[0009] The technical solution of the present invention is as follows: Application of glyceryl glucoside in preparing products for treating seborrheic alopecia.

[0010] Preferably, the concentration of the glycerol glucoside is 45-410 mg / mL.

[0011] Further preferably, the concentration of the glycerol glucoside is 410 mg / mL.

[0012] Preferably, the glyceryl glucoside treats seborrheic alopecia by activating the Wnt / β-catenin signaling pathway.

[0013] Preferably, the glyceryl glucoside treats seborrheic alopecia by inhibiting the enzymatic activity of 5α-reductase and reducing the production of dihydrotestosterone.

[0014] Preferably, the glyceryl glucoside treats seborrheic alopecia by promoting the proliferation and repair ability of hair follicle stem cells.

[0015] A product for treating seborrheic alopecia, comprising glyceryl glucoside.

[0016] Preferably, the concentration of the glycerol glucoside is 410 mg / mL.

[0017] Beneficial effects: The present invention has found through experimental studies that glyceryl glucoside can promote the proliferation and repair ability of hair follicle stem cells by activating the Wnt / β-catenin signaling pathway, inhibiting the enzymatic activity of 5α-reductase, and reducing the production of dihydrotestosterone, thereby promoting hair follicle regeneration and hair growth, thereby achieving the effect of treating seborrheic alopecia. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Different configurations of GG.

[0019] Figure 2 The effect of different concentrations of GG solution on the activity of human hair follicle stem cells.

[0020] Figure 3 The fluorescence intensity detection result.

[0021] Figure 4 This is the result of Edu staining rate.

[0022] Figure 5 The results of the scratch test.

[0023] Figure 6 The results are wound healing rate.

[0024] Figure 7 To verify the experimental results that GG activates the Wnt / β-catenin signaling pathway.

[0025] Figure 8 To verify the experimental results of the effect of GG on the Wnt / β-catenin signaling pathway.

[0026] Fig. 9 These are the experimental results of GG promoting the expression of Wnt / β-catenin signaling pathway and nuclear translocation.

[0027] Fig.10 The results show the effect of GG on hair growth in mouse skin.

[0028] Fig.11 This is the effect of GG on seborrheic alopecia in mice at the biochemical level.

[0029] Fig.12 This is the therapeutic effect of GG on seborrheic alopecia in mice. DETAILED DESCRIPTION

[0030] The following is explained in conjunction with specific embodiments: Source of experimental materials: Human hair follicle stem cells were purchased from Wuhan Pronocell Life Science Co., Ltd.

[0031] Example 1: Glycerol glucoside promotes the proliferation of human hair follicle stem cells As the primitive cells in hair follicles, the activation and division of hair follicle stem cells are the basis of hair regeneration. They can produce new cells by proliferation to promote the regeneration of hair follicles and hair growth. Hair follicle stem cells play an important role in preventing hair loss and hair regeneration.

[0032] (1) CCK-8 experiment was used to investigate the effect of different concentration gradients of glycerol glucoside on the proliferation of human hair follicle stem cells. The concentrations of glycerol glucoside used were: 1% (10 mg / mL), 2% (20 mg / mL), 3% (30 mg / mL), 4% (40 mg / mL), and 5% (50 mg / mL). The same volume of cell culture medium was added to human hair follicle stem cells as the control (CTR) group. The same volume of minoxidil solution with a concentration of 0.03 mmol / L was added to human hair follicle stem cells as the positive control group (Minoxidil).

[0033] The experimental results are as follows Figure 2 As shown (* P <0.05;** P <0.01;*** P <0.001; ns, no significant difference). Figure 2 It can be seen that compared with the control group, the 1% GG solution significantly increased the cell viability of human hair follicle stem cells, and the promoting effect on the viability of human hair follicle stem cells even exceeded that of the positive control group. Therefore, the 1% GG solution was used to explore its effect on the proliferation of human hair follicle stem cells.

[0034] (2) Edu is a new type of thymidine deoxynucleoside analog. It can replace thymidine and be incorporated into newly synthesized DNA during DNA replication. It can covalently react with fluorescent or biotin-labeled azide, so the ability of cell proliferation can be detected by fluorescence intensity.

[0035] In this study, the Edu experiment was used to further detect the effect of 1% GG solution on the proliferation ability of human hair follicle stem cells. The same volume of cell culture medium was used as the Control (CTR) group, and the cell nucleus was stained with Hoechest 33342 fluorescent dye.

[0036] The fluorescence intensity detection results are as follows Figure 3 As shown, the Edu staining rate results are as follows Figure 4 As shown (** P <0.01). Figure 3~4 It was found that glycerol glucoside significantly enhanced the proliferation ability of human hair follicle stem cells.

[0037] Example 2: Glycerol glucoside promotes the repair of human hair follicle stem cells The repair of hair follicle stem cells is of great significance for promoting hair follicle regeneration, and the cell scratch experiment can reflect the cell repair ability to a certain extent.

[0038] This study used a cell scratch test to detect the effect of 1% GG solution on the wound healing ability of human hair follicle stem cells after 20 hours of treatment. The specific steps are as follows: (1) Select human hair follicle stem cells with good growth status, digest with trypsin, prepare cell suspension and count the cells; 5 The cells were inoculated into a 24-well plate at a density of 100 / well, shaken to mix, and placed in an incubator; (2) When the cell growth density reaches 80%-90%, remove the cell culture medium and scratch each well with a 200 μL sterile pipette tip to make the scratch area and surface of each well as consistent as possible. Wash the cells three times with PBS buffer to remove floating cells. (3) Randomly select the scratch area of ​​each hole to take a photo, that is, the scratch width at 0h, and record the photo position; (4) Continue to add cell culture medium containing 1% serum (Control group, CTR) or 1% GG solution prepared with cell culture medium containing 1% serum to each well, with 3 replicate wells set up for each group; (5) After 20 hours of treatment, take a photo at the original location and calculate the mobility according to the following formula: Mobility = (0h scratch width - 20h scratch width) / 0h scratch width × 100% The scratch test results are as follows Figure 5 The wound healing rate results are shown in Figure 6 As shown (** P <0.01). Figures 5 and 6 It was found that 1% GG solution significantly promoted the wound healing of hair follicle stem cells, which indicated that glycerol glucoside could promote the repair of human hair follicle stem cells.

[0039] Example 3: Glycerol glucoside activates the Wnt / β-catenin signaling pathway The Wnt / β-catenin signaling pathway is one of the most critical signaling pathways in the regulation of hair follicle growth and cycle. This signaling pathway regulates hair growth and maintenance by regulating the proliferation, migration, intercellular adhesion and angiogenesis of hair follicle cells, and plays a potential targeted role in the prevention and treatment of hair loss.

[0040] In this study, human hair follicle stem cells were treated with 1% GG solution for 24 hours, and cell culture medium was used as the Control (CTR) group. qPCR and Western blot experiments were used to detect the relative expression of Wnt10b and β-catenin in the Wnt / β-catenin signaling pathway at the mRNA level and protein level, respectively.

[0041] The results are as follows Figure 7As shown, Figure A shows the relative expression of Wnt10b at the mRNA level, Figure B shows the relative expression of β-catenin at the mRNA level, Figure C shows the relative expression of Wnt10b at the protein level, Figure D shows the relative expression of β-catenin at the protein level, Figure E shows the quantitative analysis of the relative expression of Wnt10b at the protein level, and Figure F shows the quantitative analysis of the relative expression of β-catenin at the protein level (* P <0.05;** P <0.01;*** P <0.001). Figure 7 It can be seen that glycerol glucoside significantly promoted the transcription of the Wnt / β-catenin signaling pathway at the mRNA level and the expression at the protein level, and had an activating effect on the pathway.

[0042] Example 4: Glycerol glucoside promotes the proliferation and repair of human hair follicle stem cells by activating the Wnt / β-catenin signaling pathway In order to further verify the regulatory effect of glycerol glucoside on the Wnt / β-catenin signaling pathway, this experiment introduced the Wnt inhibitor KYA1797K (1797K) to detect the effect of glycerol glucoside on the signaling pathway. The specific steps are as follows: 1% GG solution, 25 μM 1797K solution, and 1% GG solution + 25 μM 1797K solution were prepared and used to treat human hair follicle stem cells for 24 hours, respectively. Cell culture medium was used as the Control (CTR) group. The relative expression of β-catenin, a key downstream molecule of Wnt, was then detected by Western blot.

[0043] The results are as follows Figure 8 As shown, Figure A shows the relative expression of β-catenin at the protein level in different treatment groups, and Figure B shows the quantitative analysis of the relative expression of β-catenin at the protein level in different treatment groups (* P <0.05,** P <0.01,*** P <0.001). Figure 8 It was found that glycerol glucoside significantly upregulated the expression of β-catenin, 1797K significantly downregulated the expression of β-catenin, and the addition of glycerol glucoside reversed the inhibitory effect of 1797K on β-catenin. Therefore, it is speculated that glycerol glucoside promotes the proliferation and repair of human hair follicle stem cells by activating the Wnt / β-catenin signaling pathway.

[0044] Example 5: Glycerol Glucoside Promotes Nuclear Translocation of Wnt / β-catenin Signaling Pathway The Wnt / β-catenin signaling pathway plays a vital role in the activation of hair follicle stem cells. β-catenin is a key signal transduction factor in this signaling pathway. In adult tissues, the activation of β-catenin increases its expression in the cytoplasm and transfers it to the nucleus, thereby activating downstream target genes such as c-myc and cyclin D1, which are essential for cell division and growth. In hair follicle stem cells, high levels of β-catenin activity can maintain the undifferentiated state of stem cells and promote their self-renewal ability.

[0045] In this study, the expression and localization of the Wnt / β-catenin signaling pathway were detected by cell immunofluorescence experiments. The cell culture medium was used as the Control (CTR) group, and DAPI fluorescent dye was used to stain the cell nucleus.

[0046] The results are as follows Fig. 9 As shown, Figure A shows the expression and localization of Wnt10b, and Figure B shows the expression and localization of β-catenin. Fig. 9 It was found that glycerol glucoside not only significantly activated the expression of Wnt10b and β-catenin in human hair follicle stem cells, but also promoted the translocation of Wnt10b and β-catenin from the cytoplasm to the nucleus. The above results indicate that glycerol glucoside promotes the proliferation and repair of hair follicle stem cells by activating the Wnt / β-catenin signaling pathway to cause nuclear translocation.

[0047] Example 6: Effect of glyceryl glucoside in treating seborrheic alopecia in mice Thirty male C57BL / 6L mice (23±2g) were selected and randomly divided into 6 groups, 5 mice in each group, namely blank group, modeling group (model group), positive control group, low concentration group, medium concentration group and high concentration group; each mouse was depilated at the same position with the spine as the midline, and the depilation area was about 2.5cm×2cm. The back injection method was adopted, and the blank group was injected with sterilized soybean oil, and the mice in the other groups were subcutaneously injected with a testosterone propionate solution with a concentration of 5mg / mL to construct a mouse seborrheic alopecia model. After the model was successfully constructed, the positive control group applied minoxidil tincture (4.5g / 90mL) to the mice, with an application dose of 50μL / mouse / day, and massaged for 1 minute each time; the low concentration group, medium concentration group and high concentration group applied glycerol glucoside solution with a concentration of 45mg / mL, 137mg / mL and 410mg / mL to the mice, respectively, and the application dose was 50μL / mouse / day. Before applying the sample the next day, clean the hair removal area with 37°C saline, remove the sample from the previous day and then apply a new sample, and continue to apply for 28 days.

[0048] After 28 days of continuous administration, the hair on the back of the mice was photographed and observed. Fig.10 As shown, Figure A shows the hair removal effect on the back of mice, and Figure B shows the hair growth on the back of mice in each group after 28 days of the experiment. Fig.10 It was found that after 28 days of the experiment, the back hair of the mice in the blank group had returned to normal, with almost no exposed skin; a large amount of exposed skin appeared in the hair removal area on the back of the mice in the modeling group; the back hair of the mice in the low-concentration group, medium-concentration group and high-concentration group grew to varying degrees. Specifically, from the analysis of the results of the apparent growth of hair, it was found that the effect of the high-concentration group was similar to that of the minoxidil tincture group, and the hair growth effect from high to low was: high-concentration group>medium-concentration group>low-concentration group.

[0049] 5α-reductase plays a key role in seborrheic alopecia. It can convert testosterone in the body into dihydrotestosterone (DHT), which is the main factor leading to hair follicle atrophy, shortened hair growth cycle and final hair loss. After 28 consecutive days of administration, blood was collected from each group of mice after anesthesia. After the whole blood was precipitated for 30 minutes, the supernatant was centrifuged at 4°C and the levels of type II 5α-reductase, testosterone and dihydrotestosterone in the serum of each group of mice were detected using ELISA kits.

[0050] The results are as follows Fig.11 As shown, Figure A shows the relative enzyme activity of 5α-reductase in the serum of each group of mice, Figure B shows the relative content of dihydrotestosterone in the serum of each group of mice, and Figure C shows the relative content of testosterone in the serum of each group of mice (*** P <0.001; **** P <0.0001; ns, no significant difference). Fig.11 It can be seen that compared with the blank group, the 5α-reductase activity and dihydrotestosterone content in the model group were significantly increased, indicating that the seborrheic alopecia model of mice was successfully established; compared with the model group, different concentration gradients of glycerol glucoside (low concentration group, medium concentration group and high concentration group) significantly reduced the activity of 5α-reductase and the content of dihydrotestosterone, and the effect was significantly better than that of the positive control group. Since testosterone level has no direct correlation with hair loss and hair growth, testosterone level is only used as an auxiliary indicator for the analysis of dihydrotestosterone and 5α-reductase indicators. As shown in Figure C, the testosterone level in the serum of the model group was significantly increased compared with that in the blank group. This result indicates that the modeling process may induce testosterone expression in mice, while the positive control group and the high concentration group can restore the testosterone in the serum of mice to a level close to that of the blank group. The above results show that different concentrations of glycerol glucoside can inhibit the 5α-reductase activity in the serum of the seborrheic alopecia mouse model and reduce the level of dihydrotestosterone, thereby reducing the inhibitory effect of dihydrotestosterone on hair follicle hair growth, and play a role in preventing hair loss and hair growth.

[0051] After 28 days of continuous administration, ophthalmic scissors were used to remove skin tissue of approximately 1.5 cm × 1.5 cm from the hair removal area on the back of the mice. Tissue sections were made and HE staining was performed. The changes in hair follicle morphology in each group of sections were observed and photographed. The ratio of the number of terminal hair follicles to the number of vellus hair follicles, as well as the ratio of the number of growing hair follicles to the number of resting hair follicles were counted and calculated.

[0052] The results are as follows Fig.12 Figure A shows the HE staining results of the back skin tissue sections of each group of mice, Figure B shows the ratio of the number of terminal hair follicles to the number of vellus hair follicles in the back skin hair follicles of each group of mice, and Figure C shows the ratio of the number of growing hair follicles to the number of resting hair follicles in the back skin hair follicles of each group of mice (* P <0.05;** P <0.01;*** P <0.001; **** P <0.001; ns, no significant difference). Fig.12 It was found that compared with the blank group, the model group not only had a large number of terminal hairs missing at the hair follicles, but most of the hair follicles were in the resting phase; compared with the model group, different concentration gradients of glycerol glucoside significantly promoted the number of terminal hairs, and made the hair growth gradually shift from the resting phase to the growth phase, and the effect of glycerol glucoside in the high concentration group was better than that in the positive control group.

[0053] The above results show that glyceryl glucoside has a significant effect on the treatment of seborrheic alopecia, which gives it greater application value and market prospects in the field of hair loss prevention.

Claims

1. The application of glycerol glucoside is characterized in that: The invention is applied to prepare products for treating seborrheic alopecia; the glycerol glucoside is applied by promoting the proliferation and repairing ability of hair follicle stem cells.

2. The use according to claim 1, characterized in that The concentration of the glycerol glucoside is 45-410 mg / mL.

3. The use according to claim 1, characterized in that The concentration of the glycerol glucoside is 410 mg / mL.

4. The use according to claim 1, characterized in that The glyceryl glucoside treats seborrheic alopecia by activating the Wnt / β-catenin signaling pathway.

5. The use according to claim 1, characterized in that The glyceryl glucoside treats seborrheic alopecia by inhibiting the enzymatic activity of 5α-reductase and reducing the generation of dihydrotestosterone.

6. A product for treating seborrheic alopecia, characterized in that: Contains Glyceryl Glucoside.

7. The product according to claim 6, characterized in that The concentration of the glycerol glucoside is 410 mg / mL.

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