Application of schisandra chinensis extract in preparation of preparation for treating alopecia or promoting hair regeneration

Abstract

The invention discloses application of a schisandra chinensis extract in a preparation for preventing and treating alopecia or promoting hair regeneration, and belongs to the field of alopecia treatment. The schizandrin provided by the invention comprises one or a composition of a plurality of components selected from schizandrin A and schizandrin B. According to the invention, the deoxyschizandrin A and the deoxyschizandrin B are found to be capable of remarkably promoting proliferation of hair follicle cells; the schisandrin B can remarkably promote hair regeneration after androgenetic alopecia, and meanwhile, the hair regeneration promoting effect is related to the concentration of the schisandrin B. The invention relates to a medicine for treating alopecia, which comprises schizandrin A, schizandrin B, a pharmaceutical composition and an optional pharmaceutically acceptable carrier. The medicine can be prepared into different dosage forms such as an external solution, a lotion, a liniment, an ointment, a plaster, a paste, a patch and the like, and is convenient to use.

Description

Technical Field

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

[0002] With the development of the times, hair loss has gradually become a health problem that people are widely concerned about. Hair loss types can be divided into scarring and non-scarring types according to whether scars are produced. Scarring alopecia is caused by chemical and physical damage, skin tumors, and disease infections, which cause the hair follicle epithelium to be replaced by scar tissue, and the hair follicles disappear permanently. Non-scarring alopecia can be divided into androgenic alopecia (AGA), alopecia areata (AA), telogen effluvium, etc. according to the cause of hair loss. Non-scarring alopecia usually retains hair follicles and sebaceous gland components, providing a basis for hair regeneration. Among the types of hair loss, androgenic alopecia is the most common type. About 58% of men over 50 years old will suffer from androgenic alopecia, and about 73% of men and 57% of women over 80 years old will be affected. Although hair loss is not a fatal disease, it troubles people's daily lives and gradually develops towards younger people, resulting in a decline in the quality of life of patients and even inducing the occurrence of psychological diseases in patients. The demand for hair regeneration is increasing year by year, and it has broad market prospects.

[0003] Currently, the drugs approved by the U.S. Food and Drug Administration (FDA) for the treatment of hair loss include minoxidil, finasteride, and baricitinib. In 1986, minoxidil was first marketed for the treatment of androgenic alopecia in men and women at a 2% topical concentration, and a 5% concentration was introduced in 1993; in 1998, the U.S. FDA approved an oral dose of 1 mg of finasteride for the treatment of androgenic alopecia in men. The JAK inhibitor baricitinib has shown good results in clinical trials for the treatment of severe alopecia areata and has been approved for severe alopecia areata. Existing hair loss treatments and effects are limited. Minoxidil, as a topical drug, is prone to cause side effects such as dermatitis, eczema, and allergies; finasteride is taken orally and has a systemic effect, with a certain probability of causing side effects such as impaired male reproductive function, sexual dysfunction, and breast development. In addition to drug treatment, the U.S. FDA has also approved low-level laser therapy (LLLT) for hair regeneration, but the effectiveness of LLLT is relatively limited and is usually used in combination with minoxidil or finasteride. Drug administration and laser treatment are both aimed at activating the function of hair follicles. If the hair follicles have atrophied and become necrotic and conventional methods are ineffective, hair transplantation can be used for treatment. Hair transplantation is the process of transplanting hair from other parts of the body to the part of the hair loss area, usually hair 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 hair loss again.

[0004] Natural compounds are easy to extract from low-cost natural ingredients and often have low toxicity. Recently, some natural compounds have been widely studied in the field of promoting hair growth, such as quercetin and curcumin. Schisandrin A and Schisandrin B are the main active ingredients in Schisandrin, of which Schisandrin B has been identified to have anti-inflammatory and autophagy-promoting effects, but there has been no research on hair regeneration. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an application of a Schisandra chinensis extract in the preparation of a preparation for treating hair loss or promoting hair follicle regeneration. The Schisandra chinensis 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 extract in preparing a preparation for treating hair loss or promoting hair follicle regeneration. The schisandra extract is one or more of schisandrin A or schisandrin B, preferably schisandrin B.

[0007] Among them, 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 alopecia indications selected from: androgenic alopecia, alopecia areata, telogen effluvium, cicatricial alopecia, endocrine alopecia, nutritional alopecia or chemical alopecia, preferably the alopecia 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 an external medicine or an oral medicine, and the dosage form can be an external solution, a lotion, a liniment, an ointment, a plaster, a paste or a 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 also 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, 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 to improve 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 the preparation of 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 in cells and improve the antioxidant capacity of cells. Schisandrin B treatment 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 user. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The primary cell line of the hair papilla cells in Example 1 of the present invention is constructed. A shows the process of hair papilla cell cluster extraction and the cell free state of the hair papilla cell cluster on days 1, 2, and 3, with a scale bar of 100 μm; B shows the expression of α-SMA and ALP proteins in the extracted hair papilla cells, with a scale bar of 200 μm; C shows the cell viability after adding 10 ng / ml of FGF2 cytokine. ***P<0.001, compared with the control group, the cell viability increased significantly after adding 10 ng / ml of FGF2 cytokine.

[0018] Figure 2Cell viability of dermal papilla cells treated with the main components of Schisandra chinensis. A is the cell viability of dermal papilla cells treated with different concentrations of Schisandrae Chinensis A; B is 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 increased cell viability with significant differences.

[0019] Figure 3 Schisandrin B inhibits apoptosis of hair papilla cells. A is a flow cytometric analysis of different concentrations of Schisandrin B inhibiting apoptosis of hair papilla cells; B is a statistical graph of the apoptosis ratio of hair papilla cells. *P<0.05, **P<0.01, compared with the control group, the addition of Schisandrin B reduced the apoptosis ratio of cells with significant difference.

[0020] Figure 4 Schisandrin B removes reactive oxygen species in hair papilla cells. A is a fluorescence image of different concentrations of Schisandrin B removing reactive oxygen species in hair papilla cells, with a scale bar of 200 μm; B is a statistical graph of the fluorescence intensity of reactive oxygen species in hair papilla cells. **P < 0.01, compared with the control group, the addition of Schisandrin B reduced the reactive oxygen species in cells with significant difference.

[0021] Figure 5 Gene expression of dermal papilla cells treated with different Schisandrin B for 24 hours. A is the expression of autophagy gene Atg7. B is the expression of autophagy gene Atg5. C is the expression of inflammatory gene TGF-α. D is the expression of inflammatory gene IL-1β. ***P<0.001, compared with the control group, the gene expression of Schisandrin B treatment group was significantly different, which significantly promoted 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 in the figure is 500 μm.

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

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

[0025] Fig. 9 The effect of Schisandrin B on promoting hair regeneration in mice.

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

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

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

[0029] Fig.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 implementation modes.

[0031] Example 1

[0032] C57BL / 7 mice aged 4-6 weeks were killed by cervical dislocation and soaked in 75% ethanol solution for disinfection. The whiskers of the mice were cut off and trimmed, and washed four times with PBS containing 2% double antibody. The hair follicles at the whiskers were plucked with tweezers, and the hair follicles were collected and washed five times with PBS. The collected hair follicles were digested with 0.2% type I collagenase for half an hour, and the hair follicles were blown and observed under a microscope to separate the hair papilla cell clusters in the hair follicles, 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 800rpm for 3 minutes. The precipitate was resuspended with fresh culture medium and dispersed in DMEM complete culture medium. After 24 hours, it was observed that the hair papilla cell clusters adhered 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 in the figure. After culturing the hair papilla cell cluster for 4 days, subculture was performed and cells of passages 2-5 were used. The hair papilla cells were plated, fixed and stained as shown in 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 C, the cell viability of hair papilla cells increased significantly after supplementation with FGF2. In subsequent experiments, 10 ng / ml of FGF2 cytokine was added to the culture system of hair papilla cells.

[0033] Example 2

[0034] Dermal papilla cells were digested, counted and plated, with 1×104 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 for 24, 48, 72, and 96 hours, and 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] Digest and count the dermal papilla cells. 4 Cells were plated into six-well plates, and after the cells adhered, they were treated with different concentrations of Schisandrin B for 24 hours. The dermal papilla cells were first washed with PBS, then digested with trypsin, and digested with complete medium. The cells were collected and centrifuged at 1200 rpm for 5 minutes, washed with PBS, treated with an apoptosis detection kit, and then tested with a flow cytometer. The test results are shown in Figure 2. Figure 3 As shown, there is a certain apoptotic ratio in primary cells, and the addition of 20μM and 40μM schisandrin B can significantly inhibit the apoptosis of hair papilla cells.

[0036] The dermal papilla cells were digested and counted, with 20 × 10 4 Cells were plated, and different concentrations of Schisandrin B were added to treat the cells for 4 hours after the cells adhered 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 the DCFH-DA probe was added and incubated for half an hour. The cells were washed twice with serum-free DMEM, and the intensity of reactive oxygen species was detected using a laser confocal instrument, and pictures were taken as shown in the figure. Figure 4 As shown in A, Rosup incubation can significantly increase the level of reactive oxygen species in hair papilla cells, and the addition of schisandrin B can protect hair 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 generation of reactive oxygen species and protect dermal papilla cells from damage by reactive oxygen species.

[0037] The dermal papilla cells were digested and counted, with 1×10 4 The cells were plated into 96-well plates. After the cells adhered to the wall, different concentrations of Schisandrin B were added for 24 hours, washed twice with PBS, and added Reagent was used to extract RNA, primers were designed, and the expression of autophagy genes Atg7 and Atg5 and inflammatory genes TGF-α and IL-1β was detected by Q-PCR. Figure 5As shown in A and B in Figure 3, the expression level of autophagy genes in the hair papilla cells of the group treated with Schisandrin B was significantly increased. Figure 5 As shown in C and D, the expression level of inflammatory genes in hair papilla cells of the schisandrin B-treated group was significantly reduced.

[0038] Example 3

[0039] Six-week-old C57BL / 6 mice were selected and housed in an SPF-grade mouse house. After one week of adaptation, the back was depilated and medication was started on the second day, which was recorded as DAY0. Photos were taken every 4 days. The androgenic alopecia model was constructed by applying 0.5% testosterone (50% ethanol aqueous solution) every day, and the dosage was 50 μl / cm 2 Apply testosterone solution half an hour later every day, and the dosage is 50μl / cm 2 5% minoxidil was used as a positive control, and the concentrations of schisandrin B were selected to be 80 μg / ml, 160 μg / ml, and 320 μg / ml, and were prepared using 50% propylene glycol and 50% anhydrous ethanol. The experiment was carried out for a total of 28 days.

[0040] On the 14th day, one rat was taken from each group and killed by dislocation of the cervical vertebra. The skin of the treated area on the back was taken and fixed, dehydrated, embedded, sliced, and stained with hematoxylin-eosin (HE staining). The staining results are shown in Figure 6 As shown in the figure, the hair follicles of the mice in the AGA model group and the group treated with 80μg / ml Schisandrae Chinensis B were short and small in number; the hair follicles of the group treated with 320μg / ml Schisandrae Chinensis B were short and round in length; the hair follicles of the 5% minoxidil group and the group treated with 160μg / ml Schisandrae Chinensis B were complete in shape and large in number, and the hair regeneration effect was the best. The thickness of the skin after 14 days of drug administration was calculated based on the HE-stained sections, as shown in the figure. Figure 7 Compared with the model group, the skin of mice in the 5% minoxidil group, 160μg / ml, and 320μg / ml schisandrin B groups was significantly thickened, which is consistent with the development of hair follicles. The removed skin was stained with Ki67 protein fluorescence, and the removed tissue was fixed, 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 80μg / ml schisandrin B-treated group had less green fluorescence in the hair follicles, indicating that there were fewer proliferating cells; 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 dislocating the cervical vertebrae. Fig. 9 and Fig.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; 80μg / ml, 160μg / ml, and 320μg / ml of schisandrin B were effective for hair regeneration, among which 160μg / ml of schisandrin B had the best effect on hair regeneration. After hair removal, the skin of the mouse depilatory area turned pink and 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 back of the mouse. 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 a significant difference over time. The hair of each group was plucked and SEM pictures of the hair were taken, as shown Fig.11 and 12 As shown in the figure, the hair area of ​​the AGA model group was the smallest and the hair diameter was the smallest, and the hair diameter of the drug-treated group was significantly increased compared with the AGA model group. Fig.13 There was no significant difference in body weight among the groups of mice on day 28, and topical drug treatment had no significant effect on the body weight and health of the mice.

[0042] Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention, and these changes and improvements fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached 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 a 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 also 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.

9. Use of schisandrin B in the preparation of an autophagy gene expression promoter or an inflammatory gene expression inhibitor for hair papilla cells; the autophagy genes are Atg7 and Atg5, and the inflammatory genes are TGF-α and IL-1β.

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