Composition for promoting hair growth and combined hair restorer prepared from composition

Abstract

The invention provides a composition for promoting hair growth and a combined hair growth agent prepared from the composition, and belongs to the technical field of hair growth. The composition is mainly prepared from the saarcodon imbricatum polysaccharide and gamma-hydroxy-N-methyl-L-leucine 4-cyclosporin A (CsA-4-OH), and is prepared from the saarcodon imbricatum polysaccharide and the gamma-hydroxy-N-methyl-L- The sarcodon aspratus polysaccharide is polysaccharide with the molecular weight range of 10,000-30,000 Daltons and is obtained through specific extraction and purification processes, so that the special biological activity of the sarcodon aspratus polysaccharide is ensured. The composition disclosed by the invention shows a remarkable effect in the aspect of promoting proliferation of hair follicle stem cells, so that the composition can be used for increasing the hair growth speed and reducing the potential side effects of the modified cyclosporin A at the same time.

Description

Technical Field

[0001] The invention belongs to the technical field of hair growth, and in particular relates to a composition for promoting hair growth and a combined hair growth agent prepared therefrom. Background Art

[0002] Hair loss is becoming a health challenge that cannot be ignored in China. According to statistics, about one in six people suffer from hair loss. Hair loss not only affects appearance, but can also lead to patients' damaged self-esteem, increased social anxiety, and in some cases, mental health issues such as depression, which seriously affect their overall quality of life. With the increasing social pressure, changes in lifestyle, and the prevalence of malnutrition, physiological hair loss, that is, hair loss that occurs naturally with aging, has begun to occur frequently in younger groups, which has attracted widespread attention.

[0003] Cyclosporine A (CsA) is an immunosuppressant widely used to prevent rejection after organ transplantation and to treat certain autoimmune diseases. In recent years, researchers have begun to explore the potential of CsA in the treatment of hair loss, especially for some immune-related hair loss types, such as alopecia areata. Although CsA shows the potential to promote hair growth, as an immunosuppressant, its side effects cannot be ignored, including immunosuppression, nephrotoxicity and hypertension.

[0004] In order to overcome these shortcomings of CsA, researchers modified its structure by adding a hydroxyl group to the γ carbon atom of the 4th position of N-methyl-L-leucine (N-methyl-L-Leu4) in CsA to obtain γ-hydroxy-N-methyl-L-leucine 4-cyclosporin A (CsA-4-OH). Studies have shown that this modification reduces the immunosuppressive activity of CsA while still maintaining its function of promoting hair growth. Therefore, CsA-4-OH is regarded as a hair loss treatment drug with potential clinical application value.

[0005] However, the synthesis process of CsA-4-OH is complex and inefficient, resulting in a high production cost, which limits the extensiveness of its clinical application. Moreover, although the immunosuppressive activity is reduced compared to that of CsA, CsA still has a high immunosuppressive activity, which limits its application. In order to solve this problem, the present invention attempts to combine it with other bioactive ingredients to achieve a synergistic effect, thereby reducing the amount of CsA-4-OH used and reducing the possibility of side effects. This strategy not only enables CsA-4-OH to achieve better hair growth effects at low concentrations, but also reduces the possibility of side effects of CsA-4-OH. Summary of the invention

[0006] The object of the present invention is to provide a new composition for promoting hair growth and a combined hair growth agent prepared therefrom, so as to achieve a better hair growth effect to treat hair loss.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] First, the present invention provides a composition for promoting hair growth, the composition comprising black tiger palm polysaccharide and γ-hydroxy-N-methyl-L-leucine 4-cyclosporin A;

[0009] The black tiger palm fungus polysaccharide is a polysaccharide extracted from the black tiger palm fungus and has a molecular weight range of 10,000 to 30,000 Daltons.

[0010] Preferably, the concentration of the black tiger palm polysaccharide in the composition is 50-200 μg / ml, and the concentration of the γ-hydroxy-N-methyl-L-leucine 4-cyclosporin A is 0.5-1 μg / ml.

[0011] Preferably, the black tiger palm mushroom polysaccharide is prepared according to the following preparation method:

[0012] (1) crushing a dried product of black tiger palm fungus, removing fat, filtering and drying to obtain a black tiger palm fungus filter residue;

[0013] (2) adding distilled water at a solid-liquid ratio of 10:1, extracting in a 50°C water bath constant temperature oscillator for 3 times, each time for 2.5 hours, filtering and collecting the filtrate after each extraction, and combining the filtrates of the 3 times to obtain a black tiger palm mushroom extract;

[0014] (3) Concentrating the black tiger palm mushroom extract to 25% of the original volume using a rotary evaporator;

[0015] (4) adding 4 times the volume of 95% ethanol, placing at 4°C for 24 hours, centrifuging and discarding the supernatant, and drying the precipitate in an oven to obtain crude polysaccharide of black tiger palm mushroom;

[0016] (5) dissolving the crude polysaccharide of black tiger palm mushroom into a solution using distilled water, and filtering using a 0.45 μm microporous filter membrane to obtain a crude polysaccharide solution of black tiger palm mushroom;

[0017] (6) treating the crude polysaccharide of black tiger palm mushroom using an ultrafiltration membrane with a molecular weight cut-off of 3w Daltons;

[0018] (7) treating the permeate with an ultrafiltration membrane with a molecular weight cutoff of 1w Dalton to obtain a polysaccharide solution of black tiger palm mushroom;

[0019] (8) The black tiger palm mushroom polysaccharide solution is placed in a freeze dryer, and after freeze drying, black tiger palm mushroom polysaccharide is obtained.

[0020] Secondly, the present invention provides an application of a composition in the preparation of a combined hair growth promoting agent, wherein the composition is composed of black tiger palm polysaccharide and γ-hydroxy-N-methyl-L-leucine 4-cyclosporin A;

[0021] The black tiger palm fungus polysaccharide is a polysaccharide extracted from the black tiger palm fungus and has a molecular weight range of 10,000 to 30,000 Daltons.

[0022] Preferably, the concentration of the black tiger palm polysaccharide in the composition is 50-200 μg / ml, and the concentration of the γ-hydroxy-N-methyl-L-leucine 4-cyclosporin A is 0.5-1 μg / ml;

[0023] The black tiger palm mushroom polysaccharide is prepared by the above-mentioned black tiger palm mushroom polysaccharide preparation method.

[0024] Secondly, the present invention provides an application of a composition in the preparation of a hair growth agent for achieving a hair growth effect by promoting the proliferation of hair follicle stem cells, wherein the composition is composed of black tiger palm polysaccharide and γ-hydroxy-N-methyl-L-leucine 4-cyclosporin A;

[0025] The concentration of the black tiger palm polysaccharide in the composition is 50-200 μg / ml, and the concentration of the γ-hydroxy-N-methyl-L-leucine 4-cyclosporin A is 0.5-1 μg / ml;

[0026] The black tiger palm mushroom polysaccharide is prepared by the black tiger palm mushroom polysaccharide preparation method described in claim 3.

[0027] Secondly, the present invention provides a combined hair growth promoting agent, the combined hair growth agent is composed of an effective component and a liquid auxiliary component, the effective component is composed of 50-200 μg / ml black tiger palm polysaccharide and 0.5-1 μg / ml γ-hydroxy-N-methyl-L-leucine 4-cyclosporin A;

[0028] The black tiger palm mushroom polysaccharide is prepared by the black tiger palm mushroom polysaccharide preparation method described in claim 3.

[0029] Preferably, the liquid auxiliary component consists of 5% cocamidopropyl betaine, 2% glycerin, 0.5% tea tree essential oil, 87.5% distilled water, and 5% propylene glycol.

[0030] Preferably, each 100 ml of the combined hair growth agent is prepared by the following preparation method:

[0031] (1) Add 87.5 ml of distilled water into a clean container, slowly add 5 ml of cocamidopropyl betaine, and stir evenly to obtain a mixed solution A;

[0032] (2) Slowly add 2 ml of glycerol to the mixed solution A and stir to evenly disperse the glycerol to obtain a mixed solution B;

[0033] (3) adding 0.5 ml of tea tree essential oil and stirring evenly to obtain a mixed solution C;

[0034] (4) adding CsA-4-OH to 5 ml of propylene glycol to a final concentration of 10-20 μg / ml to obtain a CsA-4-OH solution;

[0035] (5) Add the CsA-4-OH solution to the mixed solution C and stir evenly to obtain a mixed solution D;

[0036] (6) Add the obtained black tiger palm polysaccharide to the mixed solution D to make the final concentration of 50-200 μg / ml to obtain a combined hair growth promoting agent.

[0037] Secondly, the present invention provides a method for preparing a combined hair growth promoting agent, wherein each 100 ml of the combined hair growth promoting agent comprises the following steps:

[0038] (1) Add 87.5 ml of distilled water into a clean container, slowly add 5 ml of cocamidopropyl betaine, and stir evenly to obtain a mixed solution A;

[0039] (2) Slowly add 2 ml of glycerol to the mixed solution A and stir to evenly disperse the glycerol to obtain a mixed solution B;

[0040] (3) adding 0.5 ml of tea tree essential oil and stirring evenly to obtain a mixed solution C;

[0041] (4) adding CsA-4-OH to 5 ml of propylene glycol to a final concentration of 10-20 μg / ml to obtain a CsA-4-OH solution;

[0042] (5) Add the CsA-4-OH solution to the mixed solution C and stir evenly to obtain a mixed solution D;

[0043] (6) Add the obtained black tiger palm polysaccharide b to the mixed solution D to make the final concentration of 50-200 μg / ml to obtain a combined hair growth promoting agent.

[0044] Secondly, the present invention provides an application of black tiger palm mushroom polysaccharide in the preparation of a drug for promoting hair regeneration by increasing the content of growth factors VEGF and IGF-1 in the skin, characterized in that the black tiger palm mushroom polysaccharide is prepared by the above-mentioned preparation method of black tiger palm mushroom polysaccharide.

[0045] The beneficial effects of the present invention are:

[0046] The present invention provides a novel composition for promoting hair growth and a combined hair growth agent prepared therefrom, wherein the composition comprises black tiger palm fungus polysaccharide and γ-hydroxy-N-methyl-L-leucine 4-cyclosporin A (CsA-4-OH). By utilizing the biological activity of black tiger palm fungus polysaccharide and its synergistic effect with modified cyclosporin A, the present invention achieves the effect of promoting hair growth at a lower concentration while reducing potential side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The effect of different concentrations of black tiger palm polysaccharide a on the proliferation of hair follicle stem cells;

[0048] Figure 2 The effect of different concentrations of black tiger palm polysaccharide b on the proliferation of hair follicle stem cells;

[0049] Figure 3 The effect of different concentrations of black tiger palm polysaccharide c on the proliferation of hair follicle stem cells;

[0050] Figure 4 The effect of different concentrations of black tiger palm polysaccharide d on the proliferation of hair follicle stem cells;

[0051] Figure 5 The effect of different concentrations of black tiger palm polysaccharide b on the migration of hair follicle stem cells;

[0052] Figure 6 The effect of black tiger palm polysaccharide b on the hair growth of mice;

[0053] Among them, a is the photographic result of mouse hair growth, b is the hair coverage rate of mice at 14 days of treatment, and c is the hair coverage rate of mice at 21 days of treatment;

[0054] Figure 7 The effect of Polysaccharide b of Black Tiger Palm Fungus on the content of VEGF and IGF-1 in mouse skin;

[0055] Figure 8 The effect of different concentrations of CsA-4-OH on the proliferation of hair follicle stem cells;

[0056] Fig. 9 This is the effect of CsA-4-OH combined with black tiger palm mushroom polysaccharide b on the proliferation of hair follicle stem cells. DETAILED DESCRIPTION

[0057] In order to clearly explain the technical details of this solution, we will illustrate it through several specific examples. These examples are only used to show the implementation method of the solution, and are not intended to limit the solution. The patent protection of the present invention not only covers the contents described in this article, but also includes all existing or future possible derivative forms related to the present invention, which also belong to the protection scope of the present invention.

[0058] Example 1

[0059] The main purpose of this example is to obtain human hair follicle stem cells

[0060] (1) Use sterilized tweezers to carefully pluck several hairs containing intact hair follicles from the donor site, place the hair in sterile phosphate-buffered saline (PBS) containing double antibiotics (penicillin and streptomycin), and gently stir and rinse the hair three times to ensure that surface impurities and bacteria are completely removed;

[0061] (2) Use sterile scissors to cut off the rest of the hair, leaving only intact hair follicles, and place each hair follicle in a well of a 24-well plate, with 2 hair follicles placed in each well;

[0062] (3) Add 50 μl of DMEM / F-12 medium to each well to ensure that the hair follicles are well attached to the wall. Place the 24-well plate at 37°C and 5% CO. 2 Incubate in a cell culture incubator;

[0063] (4) After 24 h of culture, 200 μL of DMEM / F-12 medium was slowly added to avoid disturbing the hair follicles;

[0064] (5) Replace the culture medium with fresh one every 3 days and observe the crawling of cells around the hair follicles;

[0065] (6) After 10-14 days of culture, the hair follicles were observed. When the long spindle cells began to crawl out of the hair follicles, the culture medium was replaced with DMEM / F-12 medium containing 10% fetal bovine serum (FBS) and the culture was continued;

[0066] (7) When the cell confluence reaches 80-90%, remove the culture medium, gently rinse the cells twice with sterile PBS, add 0.25% trypsin-EDTA solution, incubate in a cell culture incubator for 1-2 minutes, and gently tap the cell culture plate to make about 80% of the cells fall off;

[0067] (8) Immediately add a culture medium containing FBS to terminate the digestion reaction, collect the cell suspension, centrifuge, discard the supernatant, resuspend the cells in fresh culture medium, and plate the cells for conventional culture to obtain human hair follicle stem cells.

[0068] Example 2

[0069] The purpose of this example is to prepare black tiger palm polysaccharides with different molecular weights

[0070] (1) 200 g of dried black tiger palm mushroom was crushed into small pieces, and fat was removed by Soxhlet extraction. After filtering and drying, a black tiger palm mushroom residue was obtained;

[0071] (2) adding distilled water at a solid-liquid ratio of 10:1, extracting in a 50°C water bath constant temperature oscillator for 3 times, each time for 2.5 hours, filtering and collecting the filtrate after each extraction, and combining the filtrates of the 3 times to obtain a black tiger palm mushroom extract;

[0072] (3) Concentrating the black tiger palm mushroom extract to 25% of the original volume using a rotary evaporator;

[0073] (4) adding 4 times the volume of 95% ethanol, placing in a refrigerator at 4°C for 24 h, centrifuging at 4000 rpm, discarding the supernatant, and drying the precipitate in an oven to obtain crude polysaccharide of black tiger palm mushroom;

[0074] (5) dissolving the crude polysaccharide of black tiger palm mushroom in distilled water to a solution of 0.5 g / ml, and filtering with a 0.45 μm microporous filter membrane to obtain a crude polysaccharide solution of black tiger palm mushroom;

[0075] (6) treating the crude polysaccharide of black tiger palm fungus with an ultrafiltration membrane with a molecular weight cutoff of 10w Daltons, treating the permeate with an ultrafiltration membrane with a molecular weight cutoff of 3w Daltons, and collecting the retentate to obtain a black tiger palm fungus polysaccharide a solution (3w-10w);

[0076] (7) treating the permeate with an ultrafiltration membrane with a molecular weight cutoff of 1w Dalton, collecting the retentate to obtain a solution of black tiger palm polysaccharide b (1-3w);

[0077] (8) treating the permeate with an ultrafiltration membrane with a molecular weight cutoff of 5000 Daltons, collecting the retentate to obtain a black tiger palm mushroom polysaccharide c solution (5000-1w), and collecting the permeate to obtain a black tiger palm mushroom polysaccharide solution d (less than 5000);

[0078] (9) The obtained black tiger palm fungus polysaccharide a solution, black tiger palm fungus polysaccharide b solution, black tiger palm fungus polysaccharide c solution and black tiger palm fungus polysaccharide d solution were separately packed into glass dishes, placed in a freeze dryer and freeze-dried into powder to obtain black tiger palm fungus polysaccharide a, black tiger palm fungus polysaccharide b, black tiger palm fungus polysaccharide c and black tiger palm fungus polysaccharide d.

[0079] Example 3

[0080] The purpose of this example is to detect the effects of different molecular weights of black tiger palm polysaccharides on the proliferation of hair follicle stem cells

[0081] (1) The black tiger palm fungus polysaccharide a, black tiger palm fungus polysaccharide b, black tiger palm fungus polysaccharide c and black tiger palm fungus polysaccharide d prepared in Example 2 were prepared into 10 μg / ml, 50 μg / ml and 200 μg / ml solutions using DMEM medium containing 10% FBS, and the control group was only added with DMEM medium containing 10% FBS;

[0082] (2) The hair follicle stem cells were digested with 0.25% trypsin-EDTA solution, prepared into a cell suspension by centrifugation and resuspension, and inoculated into a 96-well cell culture plate;

[0083] (3) After the culture plate inoculated with cells was cultured in an incubator for 24 h, the original culture medium was removed, and different concentrations of black tiger palm polysaccharides a, b, c, and d prepared in step 1 were added, with 3 replicate wells set for each treatment;

[0084] (4) The cell culture plate was placed in the incubator for further culture. After 48 h, 10 μL of CCK-8 solution was added to each well and the plate was placed in the cell culture incubator for 2.5 h.

[0085] (5) After the incubation, the absorbance at 450 nm was measured using an enzyme reader to analyze the effects of different concentrations of black tiger palm polysaccharides a, b, c, and d on cell proliferation.

[0086] from Figure 1 It can be seen that compared with the control group (average absorbance 0.556), 10 μg / mL black tiger palm mushroom polysaccharide a (average absorbance 0.583) did not show a significant effect on the proliferation of hair follicle stem cells; 50 μg / mL black tiger palm mushroom polysaccharide a (average absorbance 0.702) and 200 μg / mL black tiger palm mushroom polysaccharide a (average absorbance 0.734) both showed a promoting effect on the proliferation of hair follicle stem cells, and the P values ​​of both were less than 0.05, indicating that the difference was statistically significant.

[0087] from Figure 2 It can be seen that compared with the control group (average absorbance 0.569), 10 μg / mL black tiger palm mushroom polysaccharide b (average absorbance 0.711) showed a certain promoting effect on the proliferation of hair follicle stem cells; 50 μg / mL black tiger palm mushroom polysaccharide b (average absorbance 0.837) and 200 μg / mL black tiger palm mushroom polysaccharide b (average absorbance 0.929) both showed more significant proliferation promoting effects, with P values ​​less than 0.01 and 0.001, respectively, and the differences were statistically significant.

[0088] from Figure 3 It can be seen that compared with the control group (average absorbance 0.578), 10μg / mL and 50μg / mL black tiger palm polysaccharide c (average absorbances of 0.609 and 0.635, respectively) slightly improved the proliferation of hair follicle stem cells, but because the P value was greater than 0.05, this difference was not statistically significant; however, 200μg / mL black tiger palm polysaccharide c (average absorbance 0.697) showed a significant promoting effect on the proliferation of hair follicle stem cells, with a P value less than 0.05, and the difference was statistically significant.

[0089] from Figure 4It can be seen that compared with the control group (average absorbance 0.582), whether at a concentration of 10 μg / mL (average absorbance 0.592), 50 μg / mL (average absorbance 0.594) or 200 μg / mL (average absorbance 0.591), Black Tiger Palm Mushroom polysaccharide d had no significant effect on the proliferation of hair follicle stem cells.

[0090] In summary, among the four different black tiger palm fungus polysaccharides, black tiger palm fungus polysaccharide d has no significant promoting effect on the proliferation of hair follicle stem cells. Black tiger palm fungus polysaccharide c only shows a certain promoting effect at a higher concentration (200μg / mL). Black tiger palm fungus polysaccharide a has a certain promoting effect on the proliferation of hair follicle stem cells at concentrations of 50μg / mL and 200μg / mL, but the overall effect is relatively general. Black tiger palm fungus polysaccharide b can not only promote the proliferation of hair follicle stem cells at a lower concentration, but also its promoting effect is significantly enhanced with the increase of concentration. The effect is much higher than other types of polysaccharides, and it has unexpected technical effects. Therefore, this study selected black tiger palm fungus polysaccharide b for subsequent experiments.

[0091] Example 4

[0092] The purpose of this example is to detect the effect of black tiger palm polysaccharide b on the migration of hair follicle stem cells

[0093] (1) digesting the hair follicle stem cells with a 0.25% trypsin-EDTA solution, preparing a cell suspension through centrifugation and resuspension steps, seeding the cells into a 6-well cell culture plate, and then placing the cell culture plate in a cell culture incubator for culturing until the cell density exceeds 85%;

[0094] (2) Use a sterile 200 μl pipette tip to gently scratch a straight line on the cell monolayer. The scratch should be uniform and consistent to avoid repeated scratching that may cause cell damage. At the same time, use a microscope to record the initial width of the scratch in each treatment group.

[0095] (3) Gently wash the cells with sterile PBS to remove floating cell debris and other unattached cells;

[0096] (4) 10 μg / ml, 50 μg / ml and 200 μg / ml of black tiger palm polysaccharide b solution were added, and the control group was added with DMEM medium. FBS was not added to the black tiger palm polysaccharide b solution and DMEM medium, so as to eliminate the influence of cell proliferation on the results;

[0097] (5) Place the treated 6-well cell culture plate back into the cell culture incubator and continue culturing for 24 hours;

[0098] (6) After 24 hours of culture, the scratch width of each treatment group was observed again under a microscope and recorded. The cell migration rate was calculated by comparing the initial and final scratch widths.

[0099] from Figure 5 It can be clearly observed from the analysis that, compared with the control group, the polysaccharide b of the black tiger palm mushroom has a significant promoting effect on the migration ability of hair follicle stem cells in the concentration range of 10μg / mL to 200μg / mL. Specifically:

[0100] At a lower concentration (10 μg / mL), the polysaccharide b of the black tiger palm mushroom can also be observed to have a preliminary promoting effect on the migration ability of hair follicle stem cells, which indicates that even a trace amount of polysaccharide addition is sufficient to affect the behavior of cells;

[0101] When the concentration of black tiger palm mushroom polysaccharide b was increased to 50 μg / mL, the migration ability of hair follicle stem cells was further significantly improved, which shows that polysaccharide b has a strong promoting effect on cell migration within this concentration range;

[0102] At a high concentration of 200 μg / mL, black tiger palm polysaccharide b showed the strongest promoting effect on the migration ability of hair follicle stem cells. This shows that the effect of black tiger palm polysaccharide b is concentration-dependent and can achieve optimal biological activity at high concentrations.

[0103] Overall, Black Tiger Palm Mushroom Polysaccharide B not only has a significant effect in promoting the migration of hair follicle stem cells, but also shows a clear positive correlation with the increase in concentration.

[0104] Example 5

[0105] The purpose of this example is to detect the effect of black tiger palm polysaccharide on promoting hair regeneration in mice

[0106] (1) Twelve 6-week-old male C57BL / 6J mice were subjected to adaptive feeding for 1 week;

[0107] (2) Using an animal-specific hair remover, remove the hair on the back of all mice, and then apply a depilatory cream to completely remove the remaining hair, so that the hair of the mice is in the resting phase, thereby establishing a hair loss model;

[0108] (3) The mice were randomly divided into two groups: a control group and a black tiger palm polysaccharide group. The control group was smeared with distilled water every day, while the black tiger palm polysaccharide group was smeared with distilled water containing 200 μg / mL black tiger palm polysaccharide b every day. The other conditions of the two groups were kept the same;

[0109] (4) The mice were photographed on days 1, 4, 7, 14, and 21, and the results on days 14 and 21 were used as representative data. The hair coverage was calculated using ImageJ software.

[0110] according to Figure 6 The data presented show that the group of mice treated with black tiger palm polysaccharide b showed a significant increase in hair growth rate compared with the control group. Specifically, from the hair coverage rate, it can be seen that on the 14th day, the hair coverage rate of the control group was 30.17±2.95%, while the hair coverage rate of the black tiger palm polysaccharide b group was 76.75±3.48%. This difference is statistically significant, indicating that black tiger palm polysaccharide b has a positive effect on promoting hair growth.

[0111] As the experiment continued, on day 21, the hair coverage rate of the control group was 59.59±3.42%, while the hair coverage rate of the black tiger palm polysaccharide B group was 95.92±2.83%. This result further confirmed the significant effect of black tiger palm polysaccharide B in promoting hair growth, and this effect became more obvious over time.

[0112] Based on the above data, we can conclude that black tiger palm polysaccharide b can effectively accelerate the hair growth rate of mice, and this effect is statistically significant.

[0113] Example 6

[0114] The purpose of this example is to detect the effect of black tiger palm polysaccharide on the growth factors VEGF and IGF-1 in the skin

[0115] Both vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) are closely related to hair growth. Among them, VEGF participates in the hair growth cycle by promoting the formation of blood vessels around hair follicles. IGF-1 affects hair follicle proliferation, tissue remodeling, hair growth cycle and hair follicle differentiation. Therefore, the present invention detects VEGF and IGF-1 in the skin of mice treated with black tiger palm polysaccharide b to observe whether it can affect the content of VEGF and IGF-1 in the skin.

[0116] (1) On the 21st day, the skin of the depilated area of ​​the mice in the control group and the mice in the black tiger palm polysaccharide b group was collected, and the skin samples were washed with normal saline to remove surface impurities and blood;

[0117] (2) After cutting the skin sample into small pieces, the skin tissue is prepared into a homogenate;

[0118] (3) Detect VEGF and IGF-1 by referring to the ELISA kits of VEGF and IGF-1.

[0119] according to Figure 7From the data results, we can clearly find that the content of two key growth factors, VEGF and IGF-1, in the hair removal area of ​​mice treated with black tiger palm polysaccharide B increased significantly. Compared with the untreated control group, this increase is statistically significant, indicating that black tiger palm polysaccharide B has a potential role in promoting hair growth.

[0120] Specifically, the increase in VEGF content reached 13.61%, while the increase in IGF-1 content was more significant, reaching 44.34%. This difference suggests that black tiger palm polysaccharide b may mainly promote the proliferation and migration of hair follicle cells by promoting IGF-1, thereby achieving the effect of promoting hair growth.

[0121] In summary, the present invention found in cell experiments that the black tiger palm mushroom polysaccharide b prepared by the present invention has a significant promoting effect on the proliferation and migration of hair follicle stem cells. In animal experiments, it was found that the black tiger palm mushroom polysaccharide b prepared by the present invention can promote hair growth in mice and increase the contents of VEGF and IGF-1 in the skin of the hair loss area of ​​mice, and the promoting effect on IGF-1 is very significant. Therefore, it can be developed into a new hair growth water to promote hair growth and treat hair loss problems.

[0122] Example 7

[0123] A hair growth water containing natural fungus extract

[0124] The hair growth water consists of a liquid auxiliary component and an effective component, wherein the liquid auxiliary component comprises 5% cocamidopropyl betaine, 2% glycerol, 0.5% tea tree essential oil and 92.5% distilled water; the effective component is black tiger palm mushroom polysaccharide b, and its concentration is greater than 10 μg / ml, preferably 10-200 μg / ml, and the optimal concentration is 200 μg / ml.

[0125] Example 8

[0126] A method for preparing hair growth water containing natural fungus extract

[0127] (1) Add 92.5 ml of distilled water into a clean container, slowly add 5 ml of cocamidopropyl betaine, and stir evenly to obtain a mixed solution A;

[0128] (2) Slowly add 2 ml of glycerol to the mixed solution A and stir to evenly disperse the glycerol to obtain a mixed solution B;

[0129] (3) adding 0.5 ml of tea tree essential oil and stirring evenly to obtain a mixed solution C;

[0130] (4) adding black tiger palm polysaccharide b to the mixed solution C to make the final concentration of 10-200 μg / ml to obtain a hair growth water product;

[0131] (5) Pack the combined hair growth agent into clean, sterilized brown glass bottles, seal them, and store them away from light.

[0132] Example 9

[0133] γ-Hydroxy-N-methyl-L-leucine 4-cyclosporine A (CsA-4-OH) is a modification of cyclosporine A (CsA for short), and its structural formula is as follows:

[0134]

[0135] CsA-4-OH significantly reduces immunosuppressive activity while still maintaining its function of promoting hair growth, and is therefore considered a hair loss treatment drug with potential clinical application value. However, the synthesis process of CsA-4-OH is complex and inefficient, resulting in its high production cost, which limits the breadth of its clinical application. In addition, despite the reduction of its immunosuppressive activity, the IC50 of immunosuppression is still only about 5μg / mL. If attempts are made to promote hair growth by increasing the concentration used, other side effects may occur. Therefore, the present invention attempts to combine black tiger palm polysaccharide b with CsA-4-OH to achieve better hair growth effects while maintaining a low usage of CsA-4-OH.

[0136] First, this example detects the effect of CsA-4-OH on the proliferation of hair follicle stem cells

[0137] (1) Dissolve CsA-4-OH in DMSO and dilute it to a concentration of 0.1 μg / ml, 0.5 μg / ml, 1 μg / ml, 5 μg / ml, and 25 μg / ml in DMEM medium;

[0138] (2) digesting the hair follicle stem cells with a 0.25% trypsin-EDTA solution, preparing a cell suspension by centrifugation and resuspension, and seeding the cells into a 96-well cell culture plate;

[0139] (3) placing the cell culture plate in an incubator for culture, removing the original culture medium after 24 hours, adding different concentrations of CsA-4-OH solution prepared in step (1), and each treatment has 3 replicate wells;

[0140] (4) The cell culture plate was placed in the incubator for further culture. After 48 h, 10 μL of CCK-8 solution was added to each well and the plate was placed in the cell culture incubator for 2.5 h.

[0141] (5) After incubation, use an ELISA reader to measure the absorbance at 450 nm.

[0142] from Figure 8 The results show that when 0.1μg / ml CsA-4-OH is used to treat hair follicle stem cells, it has no obvious effect on cell proliferation. When 0.5, 1 or 5μg / ml CsA-4-OH is used to treat hair follicle stem cells, the proliferation ability of hair follicle stem cells can be effectively promoted, among which 5μg / ml concentration has the best effect. Therefore, at a concentration of 0.5-5μg / ml, CsA-4-OH can promote proliferation. However, when the concentration of CsA-4-OH is increased to 25μg / ml, it will have an inhibitory effect on the proliferation ability of hair follicle stem cells.

[0143] Example 10

[0144] Combined with the results of Example 9 and the immunosuppressive properties of CsA-4-OH, the present invention selected 0.5 μg / ml and 1 μg / ml in combination with 50 μg / ml of Pseudomonas aeruginosa polysaccharide b to explore the effect of the combination of the two on the proliferation of hair follicle stem cells.

[0145] (1) hair follicle stem cells were digested with 0.25% trypsin-EDTA solution, and after digestion, a cell suspension was prepared by centrifugation and resuspension, and the cells were seeded into a 96-well cell culture plate;

[0146] (2) The cell culture plate was placed in an incubator for culture. After 24 hours, the original culture medium was removed and 0.5 μg / ml CsA-4-OH solution (experimental group a), 1 μg / ml CsA-4-OH solution (experimental group b), 50 μg / ml black tiger palm fungus polysaccharide b solution (experimental group c), 0.5 μg / ml CsA-4-OH + 50 μg / ml black tiger palm fungus polysaccharide b solution (experimental group d), 1 μg / ml CsA-4-OH + 50 μg / ml black tiger palm fungus polysaccharide b solution (experimental group e) were added respectively. Each treatment had 3 replicate wells, and DMEM culture medium was added to the control group.

[0147] (3) The cell culture plate was placed in the incubator for further culture. After 48 h, 10 μL of CCK-8 solution was added to each well and the plate was placed in the cell culture incubator for 2.5 h.

[0148] (4) After the incubation, the absorbance at 450 nm was measured using an ELISA reader.

[0149] The experimental results are as follows Fig. 9As shown, the average absorbance of the control group was 0.561±0.026, the average absorbance of the experimental group a was 0.671±0.038, the average absorbance of the experimental group b was 0.755±0.031, the average absorbance of the experimental group c was 0.829±0.030, the average absorbance of the experimental group d was 0.972±0.032, and the average absorbance of the experimental group e was 1.090±0.036.

[0150] By comparing the absorbance of the experimental group and the control group, we can calculate the proliferation promotion rate of each experimental group relative to the control group. Among them, the proliferation promotion rate of experimental group a is 19.61%, the proliferation promotion rate of experimental group b is 34.58%, the proliferation promotion rate of experimental group c is 47.77%, the proliferation promotion rate of experimental group d is 73.26%, and the proliferation promotion rate of experimental group e is 94.30%.

[0151] In order to compare whether the effect of combined use is better than that of single use, the present invention uses the following Jin formula for calculation: q=E(A+B) / (EA+EB-EA·EB), wherein E(A+B) is the effect of combined use of CsA-4-OH and black tiger palm mushroom polysaccharide b, EA is the effect of CsA-4-OH used alone, and EB is the effect of black tiger palm mushroom polysaccharide b used alone.

[0152] Taking the combination of 0.5 μg / ml CsA-4-OH solution and 50 μg / ml black tiger palm mushroom polysaccharide b as an example, q=0.7326 / (0.1961+0.4777-0.1961×0.4777)≈1.2624, which is higher than 1.15, indicating that the combination of 0.5 μg / ml CsA-4-OH solution and 50 μg / ml black tiger palm mushroom polysaccharide b can produce a synergistic effect of promoting the proliferation of hair follicle stem cells.

[0153] For 1 μg / ml CsA-4-OH solution, q=0.9430 / (0.3458+0.4777-0.3458×0.4777)≈1.4319, which is higher than 1.15, indicating that the combination of 1 μg / ml CsA-4-OH solution and 50 μg / ml black tiger palm polysaccharide b can produce a synergistic effect in promoting the proliferation of hair follicle stem cells, and the synergistic effect is more significant.

[0154] From the above results, it can be seen that when 0.5μg / ml or 1μg / ml CsA-4-OH solution is used in combination with black tiger palm mushroom polysaccharide b, a significant synergistic effect can be produced. Therefore, the two can be used in combination to promote the proliferation of hair follicle stem cells, thereby effectively promoting hair growth, and effectively reducing the amount of CsA-4-OH to achieve cost reduction, and effectively preventing the possible side effects of excessively high concentrations of CsA-4-OH.

[0155] Embodiment 11

[0156] A combined hair growth agent for promoting hair growth

[0157] The hair growth agent consists of a liquid auxiliary component and an effective component, wherein the liquid auxiliary component comprises 5% cocamidopropyl betaine, 2% glycerol, 0.5% tea tree essential oil, 87.5% distilled water and 5% propylene glycol; the effective component is black tiger palm fungus polysaccharide b and CsA-4-OH; wherein the concentration range of the black tiger palm fungus polysaccharide b is 50-200 μg / ml, and the concentration range of the CsA-4-OH is 0.5-1 μg / ml.

[0158] Example 12

[0159] A preparation method of a combined hair growth agent for promoting hair growth

[0160] (1) Add 87.5 ml of distilled water into a clean container, slowly add 5 ml of cocamidopropyl betaine, and stir evenly to obtain a mixed solution A;

[0161] (2) Slowly add 2 ml of glycerol to the mixed solution A and stir to evenly disperse the glycerol to obtain a mixed solution B;

[0162] (3) adding 0.5 ml of tea tree essential oil and stirring evenly to obtain a mixed solution C;

[0163] (4) adding CsA-4-OH to 5 ml of propylene glycol to a final concentration of 10-20 μg / ml to obtain a CsA-4-OH solution;

[0164] (5) Add the CsA-4-OH solution to the mixed solution C and stir evenly to obtain a mixed solution D;

[0165] (6) adding the obtained black tiger palm polysaccharide b to the mixed solution D to make the final concentration of 50-200 μg / ml to obtain a combined hair growth promoting agent;

[0166] (7) Pack the combined hair growth agent into clean, sterilized brown glass bottles, seal them, and store them away from light.

Claims

1. A composition for promoting hair growth, characterized in that: The composition consists of black tiger palm polysaccharide and γ-hydroxy-N-methyl-L-leucine 4-cyclosporin A; The black tiger palm fungus polysaccharide is a polysaccharide extracted from the black tiger palm fungus and has a molecular weight range of 10,000 to 30,000 Daltons.

2. A composition for promoting hair growth according to claim 1, characterized in that: The concentration of the black tiger palm polysaccharide in the composition is 50-200 μg / ml, and the concentration of the gamma-hydroxy-N-methyl-L-leucine 4-cyclosporin A is 0.5-1 μg / ml.

3. A composition for promoting hair growth according to claim 1, characterized in that: The black tiger palm mushroom polysaccharide is prepared according to the following preparation method: (1) crushing a dried product of black tiger palm fungus, removing fat, filtering and drying to obtain a black tiger palm fungus filter residue; (2) adding distilled water at a solid-liquid ratio of 10:1, extracting in a 50°C water bath constant temperature oscillator for 3 times, each time for 2.5 hours, filtering and collecting the filtrate after each extraction, and combining the filtrates of the 3 times to obtain a black tiger palm mushroom extract; (3) Concentrating the black tiger palm mushroom extract to 25% of the original volume using a rotary evaporator; (4) adding 4 times the volume of 95% ethanol, placing at 4°C for 24 hours, centrifuging and discarding the supernatant, and drying the precipitate in an oven to obtain crude polysaccharide of black tiger palm mushroom; (5) dissolving the crude polysaccharide of black tiger palm mushroom into a solution using distilled water, and filtering using a 0.45 μm microporous filter membrane to obtain a crude polysaccharide solution of black tiger palm mushroom; (6) treating the crude polysaccharide of black tiger palm mushroom using an ultrafiltration membrane with a molecular weight cut-off of 3w Daltons; (7) treating the permeate with an ultrafiltration membrane with a molecular weight cutoff of 1w Dalton to obtain a polysaccharide solution of black tiger palm mushroom; (8) The black tiger palm mushroom polysaccharide solution is placed in a freeze dryer, and after freeze drying, black tiger palm mushroom polysaccharide is obtained.

4. Use of a composition in the preparation of a combined hair growth promoting agent, characterized in that: The composition consists of black tiger palm polysaccharide and γ-hydroxy-N-methyl-L-leucine 4-cyclosporin A; The black tiger palm fungus polysaccharide is a polysaccharide extracted from the black tiger palm fungus and has a molecular weight range of 10,000 to 30,000 Daltons.

5. Use of a composition according to claim 4 in the preparation of a combined hair growth promoting agent, characterized in that: The concentration of the black tiger palm polysaccharide in the composition is 50-200 μg / ml, and the concentration of the γ-hydroxy-N-methyl-L-leucine 4-cyclosporin A is 0.5-1 μg / ml; The black tiger palm mushroom polysaccharide is prepared by the black tiger palm mushroom polysaccharide preparation method described in claim 3.

6. Use of a composition in the preparation of a hair growth agent for achieving hair growth effect by promoting the proliferation of hair follicle stem cells, characterized in that: The composition consists of black tiger palm polysaccharide and γ-hydroxy-N-methyl-L-leucine 4-cyclosporin A; The concentration of the black tiger palm polysaccharide in the composition is 50-200 μg / ml, and the concentration of the γ-hydroxy-N-methyl-L-leucine 4-cyclosporin A is 0.5-1 μg / ml; The black tiger palm mushroom polysaccharide is prepared by the black tiger palm mushroom polysaccharide preparation method described in claim 3.

7. A combined hair growth promoting agent, characterized in that: The combined hair growth agent is composed of an effective component and a liquid auxiliary component, wherein the effective component is composed of 50-200 μg / ml black tiger palm polysaccharide and 0.5-1 μg / ml γ-hydroxy-N-methyl-L-leucine 4-cyclosporin A; The black tiger palm mushroom polysaccharide is prepared by the black tiger palm mushroom polysaccharide preparation method described in claim 3.

8. A combined hair growth promoting agent according to claim 7, characterized in that: The liquid auxiliary component consists of 5% cocamidopropyl betaine, 2% glycerin, 0.5% tea tree essential oil, 87.5% distilled water and 5% propylene glycol.

9. A combined hair growth promoting agent according to claim 8, characterized in that: Each 100 ml of the combined hair growth agent is prepared by the following preparation method: (1) Add 87.5 ml of distilled water into a clean container, slowly add 5 ml of cocamidopropyl betaine, and stir evenly to obtain a mixed solution A; (2) Slowly add 2 ml of glycerol to the mixed solution A and stir to evenly disperse the glycerol to obtain a mixed solution B; (3) adding 0.5 ml of tea tree essential oil and stirring evenly to obtain a mixed solution C; (4) adding CsA-4-OH to 5 ml of propylene glycol to a final concentration of 10-20 μg / ml to obtain a CsA-4-OH solution; (5) Add the CsA-4-OH solution to the mixed solution C and stir evenly to obtain a mixed solution D; (6) Add the obtained black tiger palm polysaccharide to the mixed solution D to make the final concentration of 50-200 μg / ml to obtain a combined hair growth promoting agent.

10. A method for preparing a combined hair growth promoting agent, characterized in that: The preparation method of each 100 ml of the combined hair growth agent comprises the following steps: (1) Add 87.5 ml of distilled water into a clean container, slowly add 5 ml of cocamidopropyl betaine, and stir evenly to obtain a mixed solution A; (2) Slowly add 2 ml of glycerol to the mixed solution A and stir to evenly disperse the glycerol to obtain a mixed solution B; (3) adding 0.5 ml of tea tree essential oil and stirring evenly to obtain a mixed solution C; (4) adding CsA-4-OH to 5 ml of propylene glycol to a final concentration of 10-20 μg / ml to obtain a CsA-4-OH solution; (5) Add the CsA-4-OH solution to the mixed solution C and stir evenly to obtain a mixed solution D; (6) Add the obtained black tiger palm polysaccharide to the mixed solution D to make the final concentration of 50-200 μg / ml to obtain a combined hair growth promoting agent.

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