Application of Schisandra chinensis extract in preparing preparations for treating hair loss or promoting hair regeneration

Abstract

The present invention discloses the use of schisandra extract in a preparation for preventing and treating hair loss or promoting hair regeneration, belonging to the field of hair loss treatment. The schisandra of the present invention includes a combination of one or more of schisandra A and schisandra B. The present invention found that both schisandra A and schisandra B can significantly promote the proliferation of hair follicle cells; schisandra B can significantly promote hair regeneration after androgenic alopecia, and at the same time, the effect of promoting hair regeneration is related to the concentration of schisandra B. The present invention relates to a drug for treating hair loss, which includes schisandra A and schisandra B and a drug combination, as well as an optional pharmaceutically acceptable carrier. The drug can be prepared into different dosage forms such as external use solution, lotion, liniment, ointment, plaster, paste, patch, etc. for convenient use.

Description

Technical Field

[0001] The invention belongs to the field of hair loss treatment, and in particular relates to the application of a Schisandra chinensis extract in preparing a preparation for treating hair loss or promoting hair regeneration. Background Art

[0002] With the passage of time, hair loss has become a widespread health concern. Hair loss can be categorized into scarring and non-scarring types based on whether scarring occurs. Scarring forms of hair loss are caused by chemical or physical damage, skin tumors, and infections, which lead to the permanent loss of hair follicles due to the replacement of the hair follicle epithelium with scar tissue. Non-scarring forms of hair loss can be categorized by the cause, including androgenic alopecia (AGA), alopecia areata (AA), and telogen effluvium. Non-scarring forms of hair loss typically retain hair follicles and sebaceous glands, providing a foundation for hair regeneration. Androgenic alopecia is the most common type of hair loss. While not a life-threatening condition, hair loss is a daily nuisance and is increasingly affecting younger people. This can lead to a decline in quality of life and even lead to psychological problems. The demand for hair regeneration is growing annually, presenting a promising market opportunity.

[0003] Currently, the US Food and Drug Administration (FDA)-approved medications for hair loss include minoxidil, finasteride, and baricitinib. Minoxidil was first marketed in 1986 for the treatment of androgenetic alopecia in both men and women at a 2% topical concentration, with a 5% concentration introduced in 1993. In 1998, the FDA approved a 1 mg oral dose of finasteride for the treatment of androgenetic alopecia in men. The JAK inhibitor baricitinib has shown promising results in clinical trials for the treatment of severe alopecia areata and is currently approved for this condition. Existing hair loss treatments are limited in effectiveness. Minoxidil, as a topical medication, is prone to side effects such as dermatitis, eczema, and allergies. Finasteride, taken orally, has systemic effects and a certain chance of causing side effects such as impaired male reproductive function, sexual dysfunction, and breast development. In addition to medication, the US FDA has also approved low-level laser therapy (LLLT) for hair regrowth, but its effectiveness is limited and it is usually used in combination with minoxidil or finasteride. Both medication and laser therapy are aimed at activating hair follicle function. If the hair follicles have already atrophied and become necrotic, conventional treatments are ineffective. Hair transplantation is the process of transplanting hair from other parts of the body to the area of ​​hair loss, typically from the back of the head. However, hair transplantation is expensive, the number of autologous hair follicle donors is limited, and there is a risk of further hair loss.

[0004] Natural compounds are easily extracted from low-cost natural ingredients and often have low toxicity. Recently, some natural compounds, such as quercetin and curcumin, have been widely studied in the field of promoting hair growth. Schisandrin A and Schisandrin B are the main active ingredients in Schisandrin. Schisandrin B has been identified as having anti-inflammatory and autophagy-promoting effects, but its application in hair regeneration has not yet been studied. Summary of the Invention

[0005] The present invention aims to overcome the deficiencies of the prior art and provide a method for preparing a preparation for treating hair loss or promoting hair follicle regeneration. The schisandra extract is schisandrin A or schisandrin B. The present invention provides a new approach or a wider range of options for treating hair loss.

[0006] The present invention provides an application of a schisandra chinensis extract in preparing a preparation for treating hair loss or promoting hair follicle regeneration. The schisandra chinensis extract is one or more of schisandrin A and schisandrin B, preferably schisandrin B.

[0007] The chemical structure of schisandrin A is shown in I, and the chemical structure of schisandrin B is shown in II:

[0008]

[0009] The Schisandra chinensis extract of the present invention can treat hair loss indications selected from: androgenic alopecia, alopecia areata, telogen effluvium, cicatricial alopecia, endocrine alopecia, nutritional alopecia or chemical alopecia, preferably the hair loss is androgenic alopecia.

[0010] The Schisandra chinensis extract of the present invention can be used to prepare a preparation for promoting hair follicle regeneration, wherein the hair follicle regeneration is hair papilla cell regeneration.

[0011] Preferably, the preparation is a medicine or a daily chemical product. The medicine can be a topical medicine or an oral medicine, and the dosage form can be a topical solution, lotion, liniment, ointment, plaster, paste, or patch, etc. The medicine comprises a Schisandra chinensis extract and a pharmaceutically acceptable carrier or excipient. Daily chemical products include, but are not limited to, cosmetics, care products, or beauty products, such as shampoo, conditioner, hair essence, hair oil, hair lotion, scalp cream, hair gel, hair spray, hair mask, or eyebrow care solution, etc.

[0012] Preferably, the total concentration of the Schisandra chinensis extract in the preparation is 50 μg / mL-5000 μg / mL. Further preferably, the Schisandra chinensis extract in the preparation is schisandrin B, and the concentration of schisandrin B is 50 μg / mL-5000 μg / mL.

[0013] Preferably, the preparation further includes a solubilizer to obtain a better dissolution effect of the Schisandra chinensis extract, and the solubilizer is a combination of one or more of propylene glycol, ethanol, and glycerol, preferably propylene glycol and ethanol, more preferably, 50% propylene glycol and 50% anhydrous ethanol are prepared in a 1:1 ratio.

[0014] According to an embodiment of the present invention, the present invention also provides schisandrin B in protecting hair papilla cells from damage by reactive oxygen species. The schisandrin B can be used to prepare a reactive oxygen species scavenger for hair papilla cells, thereby improving the antioxidant capacity of hair papilla cells under oxidative stress.

[0015] According to an embodiment of the present invention, the present invention also provides the use of schisandrin B in preparing a hair papilla cell autophagy gene expression promoter or a hair papilla cell inflammatory gene expression inhibitor; the autophagy gene is Atg7 or Atg5, and the inflammatory gene is TGF-α or IL-1β.

[0016] The present invention has discovered the application of schisandrin A and schisandrin B in treating hair loss and promoting cell regeneration. The present invention provides a new approach and option for treating hair loss, especially androgenic alopecia. Schisandrin A and schisandrin B can significantly promote the proliferation of hair papilla cells in hair follicles and inhibit the apoptosis of hair papilla cells. When cells are in a state of oxidative stress, they significantly remove reactive oxygen species in cells and improve the antioxidant capacity of cells. Treatment with schisandrin B can significantly increase the expression of autophagy genes and inflammatory factors in hair papilla cells. In an animal model of androgenic alopecia, the effect of treating hair loss is obvious. In addition, schisandrin A or schisandrin B is derived from the natural Chinese medicine Schisandra chinensis, which is easy to obtain, low in cost, and safe for the subjects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the primary cell line established for dermal papilla cells described in Example 1 of the present invention. Figure A shows the extraction process of dermal papilla cell clusters and the cell isolation of dermal papilla cell clusters on days 1, 2, and 3 (scale bar, 100 μm); Figure B shows the expression of α-SMA and ALP proteins in extracted dermal papilla cells (scale bar, 200 μm); Figure C shows cell viability after addition of 10 ng / ml FGF2. ***P < 0.001. Compared with the control group, the addition of 10 ng / ml FGF2 significantly increased cell viability.

[0018] Figure 2Cell viability of dermal papilla cells treated with the main components of Schisandra chinensis. A shows the cell viability of dermal papilla cells treated with different concentrations of Schisandrae Chinensis A; B shows the cell viability of dermal papilla cells treated with different concentrations of Schisandrae Chinensis B. *P<0.05, **P<0.01, ***P<0.001. Compared with the control group, the addition of Schisandrae Chinensis A and Schisandrae Chinensis B significantly increased cell viability.

[0019] Figure 3 Schisandrin B inhibits apoptosis in dermal papilla cells. A shows flow cytometric analysis of the inhibition of dermal papilla cell apoptosis by schisandrin B at different concentrations; B shows a statistical graph of the apoptotic percentage in dermal papilla cells. *P < 0.05, **P < 0.01. Compared with the control group, the apoptotic percentage was significantly reduced in the schisandrin B group.

[0020] Figure 4 Schisandrin B eliminates reactive oxygen species in dermal papilla cells. A is a fluorescence image showing the elimination of reactive oxygen species in dermal papilla cells by different concentrations of Schisandrin B. Scale bar, 200 μm. B is a statistical graph showing the fluorescence intensity of reactive oxygen species in dermal papilla cells. **P < 0.01. Compared with the control group, the reduction in reactive oxygen species after the addition of Schisandrin B was significant.

[0021] Figure 5 Gene expression in dermal papilla cells after 24 hours of treatment with different schisandrin B compounds. A shows the expression of the autophagy gene Atg7. B shows the expression of the autophagy gene Atg5. C shows the expression of the inflammatory gene TGF-α. D shows the expression of the inflammatory gene IL-1β. ***P < 0.001. Compared with the control group, the gene expression in the schisandrin B-treated group was significantly different, significantly promoting the biological activity and gene expression of dermal papilla cells.

[0022] Figure 6 HE staining of mouse skin 14 days after administration of schisandrin B. The scale bar is 500 μm.

[0023] Figure 7 The skin thickness of mice was measured after 14 days of schisandrin B administration. ***P<0.001, the skin thickness of the drug-treated group increased significantly compared with the model group.

[0024] Figure 8 Ki67 staining of mouse skin 14 days after administration of schisandrin B. Scale bar: 100 μm.

[0025] Figure 9 The effect of schisandra chinensis B on promoting hair regeneration in mice.

[0026] Figure 10The grayscale changes of the back of mice over time. *P<0.05, **P<0.01, ***P<0.001. Compared with the model group, the grayscale of the skin in the drug-treated group increased significantly.

[0027] Figure 11 SEM image of mouse hair after 28 days of schisandrin administration. The scale bar is 20 μm.

[0028] Figure 12 Statistical graph of hair diameter of mice after 28 days of schisandrin B administration. ***P<0.001, hair diameter of the administration group increased significantly compared with the model group.

[0029] Figure 13 The body weight of mice was counted 28 days after administration of schisandrin B. DETAILED DESCRIPTION

[0030] The present invention will be further described and illustrated below in conjunction with specific embodiments.

[0031] Example 1

[0032] C57BL / 7 mice aged 4-6 weeks were killed by cervical dislocation and immersed in 75% ethanol solution for disinfection. The whiskers of the mice were cut off, trimmed, washed 4 times with PBS containing 2% double antibody, and the hair follicles in the whiskers were plucked with tweezers. After the hair follicles were collected, they were washed 5 times with PBS. The collected hair follicles were digested with 0.2% type I collagenase for half an hour, the hair follicles were blown, and the hair papilla cell clusters in the hair follicles were observed under a microscope to separate, and the digestion was stopped with culture medium containing fetal bovine serum. Impurities in the hair shaft were removed with tweezers, and the supernatant was centrifuged twice at 800 rpm for 3 minutes. The precipitate was resuspended with fresh culture medium and dispersed in DMEM complete culture medium. After 24 hours, the hair papilla cell clusters were observed to adhere to the wall, and the cells in the hair papilla cell clusters began to free themselves. Fresh culture medium was replaced as Figure 1 As shown in A. After culturing the hair papilla cell cluster for 4 days, subculture was performed and cells of passages 2-5 were used. Figure 1 As shown in Figure B, the extracted dermal papilla cells expressed α-SMA and ALP proteins. Because dermal papilla cells gradually lost their proliferation ability in ordinary DMEM complete medium, 10 ng / ml of FGF2 cytokine was added to the culture system. Figure 1 As can be seen from Figure C, the cell viability of dermal papilla cells increased significantly after supplementation with FGF2. In subsequent experiments, 10 ng / ml of FGF2 cytokine was added to the culture system of dermal papilla cells.

[0033] Example 2

[0034] Dermal papilla cells were digested, counted, and plated, with 1×10 4 The system was 200 μl, and the drug was added after 24 hours of adhesion. Then the cell culture plate was placed in a cell culture incubator and cultured for 24, 48, 72, and 96 hours. The cell viability was tested by MTT method. Figure 2 Schisandrin A in the formula can significantly promote the proliferation of hair papilla cells; Figure 2 B in it, schisandrin B can also significantly promote the proliferation of hair papilla cells. The cell viability of hair papilla cells treated with schisandrin B is higher than that treated with schisandrin A. Therefore, schisandrin B was selected for subsequent experiments.

[0035] The dermal papilla cells were digested and counted. 10×10 4 Cells were plated into six-well plates and treated with different concentrations of schisandrin B for 24 hours after cell attachment. Dermal papilla cells were first washed with PBS, then digested with trypsin. Complete culture medium was added to terminate digestion, cells were collected, centrifuged at 1200 rpm for 5 minutes, washed with PBS, treated with an apoptosis detection kit, and analyzed by flow cytometry. The test results are shown in Figure 2. Figure 3 As shown in the figure, there is a certain apoptosis ratio in primary cells, and the addition of 20 μM and 40 μM schisandrin B can significantly inhibit the apoptosis of dermal papilla cells.

[0036] The dermal papilla cells were digested and counted, with 20 × 10 4 Cells were plated and treated with different concentrations of schisandrin B for 4 hours after the cells attached to the wall. The cells were washed twice with serum-free DMEM. Except for the control cells, the remaining cells were incubated with Rosup in the reactive oxygen species detection kit for half an hour, washed twice with serum-free DMEM, and DCFH-DA probe was added. The cells were incubated for half an hour and washed twice with serum-free DMEM. The reactive oxygen species intensity was detected using a laser confocal instrument and pictures were taken as shown below. Figure 4 As shown in A, Rosup incubation can significantly increase the level of reactive oxygen species in dermal papilla cells, and the addition of schisandrin B can protect dermal papilla cells. Figure 4 As shown in B, the process was repeated three times and the fluorescence intensity of reactive oxygen species was statistically analyzed. Schisandrin B can significantly reduce the production of reactive oxygen species and protect dermal papilla cells from the damage of reactive oxygen species.

[0037] The dermal papilla cells were digested and counted, with 1×10 4 Cells were plated into 96-well plates. After the cells adhered, different concentrations of schisandrin B were added for 24 hours, washed twice with PBS, and TRIzol® Reagent was added to extract RNA. Primers were designed and Q-PCR was performed to detect the expression of autophagy genes Atg7 and Atg5 and inflammatory genes TGF-α and IL-1β. Figure 5 As shown in Figures A and B, the expression levels of autophagy genes in the dermal papilla cells of the Schisandrin B-treated group were significantly increased. Figure 5 As shown in Figures C and D, the expression levels of inflammatory genes in the dermal papilla cells of the schisandrin B-treated group were significantly reduced.

[0038] Example 3

[0039] Six-week-old C57BL / 6 mice were selected and housed in an SPF-grade mouse facility. After a week of acclimation, hair was removed from the back. Dosing began on the second day, designated Day 0, and photographs were taken every four days. An androgenic alopecia model was established by applying 0.5% testosterone (50% ethanol in water) daily at a dose of 50 μl / cm. 2 Apply testosterone solution half an hour later every day, and the dosage is 50 μl / cm 2 5% minoxidil was used as a positive control, and the concentrations of schisandrin B were selected as 80 μg / ml, 160 μg / ml, and 320 μg / ml, using 50% propylene glycol and 50% anhydrous ethanol for preparation. The experiment was carried out for a total of 28 days.

[0040] On the 14th day, one animal was taken from each group and sacrificed by cervical dislocation. The skin of the treated area on the back was taken and fixed, dehydrated, embedded, sliced, and stained with hematoxylin-eosin (HE). The staining results were as follows: Figure 6 As shown in the figure, the hair follicles in the AGA model group and the 80 μg / ml Schisandrae Chinensis B treatment group were short and small in number. In the 320 μg / ml Schisandrae Chinensis B treatment group, the hair follicles were short and round. In the 5% minoxidil group and the 160 μg / ml Schisandrae Chinensis B treatment group, the hair follicles were intact and numerous, indicating the best hair regeneration effect. The thickness of the skin after 14 days of drug administration was calculated based on HE-stained sections. Figure 7 Compared with the model group, the skin of mice in the 5% minoxidil group, 160 μg / ml, and 320 μg / ml schisandrin B-treated groups showed significant thickening, which is consistent with the development of hair follicles. The removed skin was stained with Ki67 protein fluorescence. The removed tissue was fixed, membrane-permeated, incubated with primary antibody, incubated with secondary antibody, and the image was scanned with a fluorescence microscope. Figure 8 It can be seen that on the 14th day, the AGA model group and the 80 μg / ml schisandrin B-treated group had less green fluorescence in the hair follicles, indicating that there were fewer proliferating cells in them; in comparison, the other groups had more green fluorescence, and the fluorescence was at the bottom of the hair follicles, indicating that there were more proliferating cells at the bottom of the hair follicles. The experimental drug significantly promoted hair regeneration.

[0041] After 28 days of the experiment, photos were taken and all experimental mice were killed by cervical dislocation. Figure 9 and Figure 10As shown, most of the back skin in the AGA model group did not grow hair, indicating that the androgenic alopecia model was successfully constructed; 80 μg / ml, 160 μg / ml, and 320 μg / ml of schisandrin B were effective in hair regeneration, among which 160 μg / ml of schisandrin B had the best hair growth effect. After hair removal, the skin of the mouse depilatory area was pink, first turned gray, then black, and finally hair grew out of the skin. The changes in the hair on the back of the mouse can be seen by counting the grayscale values ​​of the mouse's back. There was no obvious change in the grayscale value of the back of the mice in the AGA model group within 28 days, while the grayscale of the back of the drug-treated group gradually increased over time. Compared with the AGA model group, the increase in skin grayscale in the drug-treated group showed significant differences over time. The hair of each group was plucked out and SEM pictures of the hair were taken, as shown below. Figure 11 and 12 As shown in the figure, the hair area and hair diameter of the AGA model group were the smallest, and the hair diameter of the drug-treated group was significantly increased compared with the AGA model group. Figure 13 There was no significant difference in the body weight of mice in each group on day 28, and topical drug treatment had no significant effect on the body weight and health of mice.

[0042] Those skilled in the art will appreciate that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Use of a Schisandra chinensis extract in preparing a preparation for treating hair loss or promoting hair regeneration, wherein the Schisandra chinensis extract is one or more of schisandrin A or schisandrin B.

2. The use according to claim 1, characterized in that The Schisandra chinensis extract is Schisandrin B.

3. The use according to claim 1, characterized in that The preparation for promoting hair regeneration is a preparation for promoting the regeneration of hair papilla cells.

4. The use according to claim 1, characterized in that The preparation is a medicine or a daily chemical product.

5. The use according to claim 4, characterized in that The medicine comprises schisandra chinensis extract and a pharmaceutically acceptable carrier.

6. The use according to claim 1, characterized in that The total concentration of the Schisandra chinensis extract in the preparation is 50 μg / mL to 5000 μg / mL.

7. The use according to any one of claims 1 to 6, characterized in that The preparation further includes a dissolving agent to obtain a better dissolving effect of the Schisandra chinensis extract, and the dissolving agent is a combination of one or more of propylene glycol, ethanol, and glycerol.

8. The use according to claim 1, characterized in that The dosage form of the preparation is an external solution, lotion, liniment, ointment, plaster, paste or patch.

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