Application of vitamin K2 in preparation of anti-hair loss composition

Abstract

The invention discloses application of vitamin K2 in preparation of an anti-hair loss composition. Research finds that the vitamin K2 has the effects of promoting VEGF gene expression and inhibiting IL-6 and DKK-1 gene expression in an androgen type alopecia model, and has the effects of promoting Gas6, VEGF and TGF gene expression in a pressure type alopecia model, so that a good alopecia prevention and treatment effect is achieved, and toxic and side effects are few.

Description

Technical Field

[0001] The present invention relates to a new application of vitamin K2, in particular to the application of vitamin K2 in the preparation of an anti - hair - loss composition. Background Art

[0002] Hair loss is a common physiological phenomenon. Among hair - loss people, androgenetic alopecia accounts for 90%. The Wnt signaling pathway is one of the known pathogenesis mechanisms of androgenetic alopecia and is involved in basic biological processes such as cell proliferation and differentiation. In the skin hair follicle tissue, this signal promotes the restart of the hair follicle from the resting phase to the growth phase by regulating the growth cycle of hair matrix cells, thus promoting the generation of new hair. Among them, Dickkopf - related protein 1 (DKK - 1) is a Wnt signaling inhibitor protein, which further blocks the Wnt signaling pathway by directly or indirectly competitively binding to the Wnt protein receptor LRP5 / 6, resulting in the premature entry of hair follicles into the regression phase and causing hair loss.

[0003] Vascular endothelial growth factor (VEGF) is a key angiogenesis factor, which can promote the formation of blood vessels around hair follicles, increase the blood supply to hair follicles, thereby supporting hair growth. And due to the periodic changes of VEGF, it regulates the growth cycle of hair follicles; at the same time, VEGF can protect hair follicle stem cells (HFSC) from androgen - induced apoptosis through specific signaling pathways (such as the PI3K / AKT pathway), which plays an important role in maintaining the normal function of hair follicles and hair growth.

[0004] Stress - induced hair loss is a hair - loss problem caused by long - term mental stress or psychological burden, which leads to disorders of the hair follicle growth cycle, hormone imbalance, etc. Stress can prompt hair follicles to prematurely enter the resting phase from the growth phase.

[0005] The Gas6 gene refers to the Growth Arrest - Specific 6 gene. Gas6 secreted by dermal papilla cells (DP) binds to its receptor Axl in hair follicle stem cells (HFSC), which can promote the proliferation of HFSC, thereby promoting hair growth. Research shows that in mice under stress conditions, corticosterone, a stress hormone from the adrenal gland, binds to the GR receptor on DP cells, which will inhibit the expression of the Gas6 gene in DP cells, resulting in a decrease in the activity of hair follicle stem cells, hair growth arrest and hair loss; while restoring the expression of the Gas6 gene can overcome stress - induced HFSC quiescence and hair - growth inhibition.

[0006] TGF is transforming growth factor, a class of pleiotropic cytokines that play important roles in hair follicle morphogenesis, development, and hair follicle cycle regulation.

[0007] Currently, the means of treating hair loss such as drug treatments with minoxidil, dexamethasone, etc. However, drug treatment is difficult to cure, there is a risk of recurrence, and long-term drug use will have certain toxic reactions.

[0008] Although there are also research reports that plant active ingredients such as polygonum multiflorum, scallion extract, mulberry root extract, xuefuzhuyu decoction, artemisinin, saponins, and pueraria root extract have certain therapeutic effects on hair loss, the anti-hair loss effect is not ideal enough.

[0009] Vitamin K2 is composed of a 2-methyl-1,4-naphthoquinone nucleus and an isoprenoid side chain at the C3 position, and is a fat-soluble vitamin. Currently, studies have found that vitamin K2 has the effects of inhibiting vascular calcification, anti-tumor, regulating immunity and blood sugar, etc. However, there is no research report on the role of vitamin K2 in preventing hair loss and promoting hair growth. Summary of the Invention

[0010] The purpose of the present invention is to provide an application of vitamin K2 in the preparation of an anti-hair loss composition. Through research, the present invention has found that vitamin K2 has the effect of promoting the expression of the VEGF gene and inhibiting the expression of the IL-6 and DKK-1 genes in the androgenetic alopecia model, and has the effect of promoting the expression of the Gas6, VEGF, and TGF genes in the stress-induced alopecia model, thereby achieving good effects in preventing and treating hair loss, stimulating hair growth, and having few toxic and side effects.

[0011] Technical solution of the present invention: The application of vitamin K2 in the preparation of an anti-hair loss composition.

[0012] In the aforementioned application, the hair loss is androgenetic alopecia or / and stress-induced alopecia.

[0013] In the aforementioned application, the anti-hair loss includes at least one of preventing hair loss, treating hair loss, and stimulating hair growth.

[0014] In the aforementioned application, the application of vitamin K2 in the preparation of an anti-androgenetic alopecia composition for promoting VEGF expression and inhibiting IL-6 and DKK-1 gene expression.

[0015] In the aforementioned application, the application of vitamin K2 in the preparation of an anti-stress-induced alopecia composition for promoting VEGF, Gas6, and TGF gene expression.

[0016] In the aforementioned application, the cell dosage of vitamin K2 in the preparation of an anti-androgenetic alopecia composition ≤ 0.001563 wt%.

[0017] In the aforementioned application, the cell dosage of vitamin K2 in the preparation of the anti-stress alopecia composition is ≤ 5 μg / mL.

[0018] In the aforementioned application, the composition includes a pharmaceutical composition or a cosmetic composition.

[0019] In the aforementioned application, the pharmaceutical composition includes any one of creams, patches, ointments, cream preparations, capsules, injections, gels, injections, sprays, lotions, liniments.

[0020] In the aforementioned application, the cosmetic composition includes any one of shampoos, hair conditioners, hair waxes, hairsprays, hair conditioners, sprays, anti-hair loss serums, essence sprays, pre-wash gels.

[0021] The present invention also provides an anti-hair loss cosmetic, comprising vitamin K2 and a cosmetically acceptable carrier.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] The present invention provides the application of vitamin K2 in the preparation of an anti-hair loss composition. By establishing an androgen model for anti-hair loss testing, the present invention discovers that vitamin K2 has the characteristics of promoting the expression of vascular endothelial growth factor (VEGF), inhibiting the expression of IL-6 and DKK-1 genes, thereby achieving the effects of preventing androgenetic alopecia and stimulating hair growth.

[0024] In addition, the present invention also conducts anti-hair loss testing by establishing a corticosterone model, and discovers that vitamin K2 has the characteristics of promoting the expression of Gas6, VEGF and TGF genes, thereby achieving the effects of preventing stress-induced alopecia and stimulating hair growth, and having few toxic and side effects. Specific embodiments

[0025] The following examples are used to further illustrate the present invention, but shall not be used as a basis for limiting the present invention.

[0026] Verification of the application effect of vitamin K2:

[0027] The cells used in this test were dermal papilla cells, batch number: 210727, from the Shaanxi branch of Guangdong Boxi. The main reagents used in this test were MSCM culture medium (Gibco), PBS (Solarbio), MTT (Sigma), DMSO (Sigma), DHT (Sigma), minoxidil (Sigma), dexamethasone (National Institutes for Food and Drug Control), VEGF ELISA kit (Abcam), IL-6 ELISA kit (Abcam), AG RNAex Pro Reagent (Accurate Biology), reverse transcription kit (Accurate Biology), and fluorescent dye (Accurate Biology). The main equipment used in this test was a CO2 incubator (Thermo, 150I), a laminar flow hood (Suzhou Jing'an Antai, SW-CJ-1F), an inverted microscope (Olympus, CKX53), an enzyme-linked immunosorbent assay (ELISA) reader (BioTek, Epoch), an ordinary polymerase chain reaction (PCR) instrument (Bori, TC-XP-G), and a fluorescence quantitative PCR instrument (Roche, lightcycler480 II).

[0028] Experimental Example 1: Androgen model - anti-hair loss test.

[0029] Cytotoxicity test:

[0030] 1) Cell seeding: Resuscitate the dermal papilla cells. After resuscitation, when the plating rate reaches about 60%, seed the cells into a 96-well plate and incubate overnight in a CO2 incubator (37°C, 5% CO2).

[0031] 2) Test grouping: Set up a zero control group, a solvent control group (Control), a positive control group (PC), and a sample group. In the sample group, prepare sample working solutions at 8 concentrations for each sample, and set 3 replicate wells for each concentration.

[0032] 3) Solution preparation: Dilute vitamin K2 with absolute ethanol according to the test concentration setting table (Table 1) to prepare sample working solutions at different concentrations.

[0033] Table 1 Test concentration setting table

[0034]

[0035] 4) Drug administration: Administer the drugs when the plating rate of the cells in the 96-well plate reaches 50% - 60%. Add 200 μL of culture medium to each well in the solvent control group; add 200 μL of culture medium containing 10% DMSO to each well in the positive control group; add 200 μL of culture medium containing the corresponding concentration of the sample to each well in the sample group; no cells are seeded in the zero control group, and only 200 μL of cell culture medium is added. After drug administration, place the 96-well plate in a CO2 incubator (37°C, 5% CO2) and culture for 24 h.

[0036] 5) Detection: After the cells were incubated for 24 h, the supernatant was discarded, and MTT working solution (0.5 mg / mL) was added. The cells were incubated at 37 °C in the dark for 4 h. After incubation, the supernatant was discarded, 150 μL DMSO was added to each well, and the OD value was read at 490 nm.

[0037] The MTT detection results of the cytotoxicity test are shown in the following table (Table 2).

[0038] Table 2 MTT detection results of vitamin K2

[0039]

[0040] According to the MTT results, it was shown that vitamin K2 did not exhibit obvious cytotoxicity based on dermal papilla cells within the concentration range of 0.001563%.

[0041] Anti - hair loss effect test:

[0042] 1) Cell seeding: The dermal papilla cells were resuscitated. After resuscitation, when the plating rate reached about 60%, the cells were seeded into 6 - well plates and incubated overnight in a CO2 incubator (37 °C, 5% CO2).

[0043] 2) Solution preparation: Prepare the sample working solution according to the test grouping (Table 3).

[0044] Table 3 Test grouping

[0045]

[0046]

[0047] Note: Minoxidil (PC1) is the positive control for VEGF and DKK - 1 detection; Dexamethasone (PC2) is the positive control for IL - 6 detection.

[0048] 3) Stimulation and drug administration: According to the test grouping, when the plating rate of the cells in the 6 - well plates reached 80% - 90%, grouped drug administration was carried out, with 3 replicate wells in each group. The blank control (BC) group added 2 mL of culture medium to each well, the negative control (NC) group added 2 mL of culture medium containing 800 nM DHT to each well, the positive control (PC) group added 2 mL of culture medium containing 800 nM DHT and 500 μM minoxidil to each well, and the sample group added 2 mL of culture medium containing 800 nM DHT and the corresponding concentration of the test substance to each well. After drug administration, the 6 - well plates were placed in an incubator (37 °C, 5% CO2) and cultured for 24 h.

[0049] 4) ELISA test: After the incubation, collect the culture medium in a centrifuge tube. After collection, place the samples for ELISA detection in a freezer at -80 °C for storage. Analyze the VEGF index according to the operation manual of the ELISA kit.

[0050] 5) Gene expression detection: After the incubation, aspirate the old liquid, wash twice with 1 mL / well of PBS, add 1 mL of AGRNAex Pro Reagent to each well, pipette to lyse the cells, and then collect the samples. Extract RNA, reverse transcribe it into cDNA, and then perform fluorescence quantitative PCR detection. Use the method to calculate the results of the DKK-1 gene expression level.

[0051] 6) Result statistical analysis: Use GraphPad Prism for plotting, and the results are expressed as Mean ± SD. The t-test statistical analysis is used for comparison between groups. All statistical analyses are two-tailed. A significant difference is considered when P < 0.05, and a highly significant difference is considered when P < 0.01.

[0052] 7) Detection results:

[0053] 7.1) The test results of vascular endothelial growth factor (VEGF) are shown in the following table (Table 4).

[0054] Table 4 Summary table of VEGF content results

[0055]

[0056] Note: When using the t-test method for statistical analysis, compared with the BC group, the significance is indicated by #, P-value < 0.05 is indicated by #, and P-value < 0.01 is indicated by ##; compared with the NC group, the significance is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **.

[0057] According to Table 4, compared with the BC group, the VEGF content in the NC group decreased significantly, indicating that the test stimulator strip is effective. Compared with the NC group, the VEGF content in the PC1 group increased significantly, indicating that the positive control of this test is effective. Compared with the NC group, the VEGF content in the sample group increased significantly, with a promotion rate of 43.73%, indicating that vitamin K2 can promote the secretion of vascular endothelial growth factor (VEGF), increase the content of VEGF, thereby promoting the formation of blood vessels around the hair follicles, increasing the blood and nutrient supply of the hair follicles, stimulating hair growth, and improving the health of the hair follicles.

[0058] 7.2) The test results of inflammatory factor (IL-6) are shown in the following table (Table 5).

[0059] Table 5 Summary table of IL-6 secretion results

[0060]

[0061] Note: When performing statistical analysis using the t-test method, compared with the BC group, significance is indicated by #, P-value < 0.05 is indicated by #, and P-value < 0.01 is indicated by ##; compared with the NC group, significance is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **.

[0062] According to Table 5, compared with the BC group, the secretion of IL-6 in the NC group decreased significantly, indicating that the test stimulus strip was effective. Compared with the NC group, the secretion of IL-6 in the PC2 group decreased significantly, indicating that the test positive control was effective. Compared with the NC group, the secretion of IL-6 in the sample group decreased significantly, with an inhibition rate of 30.93%, indicating that vitamin K2 can inhibit the secretion of IL-6 and reduce the inflammatory response.

[0063] 7.3) The test results of the DKK-1 gene expression level are shown in the following table (Table 6).

[0064] Table 6 Summary Table of the Detection Results of the Relative Expression Level of the DKK-1 Gene

[0065]

[0066]

[0067] Note: The method is used for result calculation. When performing statistical analysis using the t-test method, the mRNA amplification multiple of the BC group is normalized. Compared with the BC group, significance is indicated by #, P-value < 0.05 is indicated by #, and P-value < 0.01 is indicated by ##; compared with the NC group, significance is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **.

[0068] According to Table 6, compared with the BC group, the relative expression level of the DKK-1 gene in the NC group increased significantly, indicating that the test stimulus conditions were effective. Compared with the NC group, the relative expression level of the DKK-1 gene in the PC group decreased significantly, indicating that the detection of the positive control in this test was effective. Compared with the NC group, the relative expression level of the DKK-1 gene in the sample group decreased significantly, with an inhibition rate of 22.44%. It shows that vitamin K2 can inhibit the expression of the DKK-1 gene, thereby avoiding hair loss caused by the premature entry of hair follicles into the regression period.

[0069] The above experiments demonstrated that, for dermal papilla cells stimulated by DHT, vitamin K2 could promote the expression of vascular endothelial growth factor (VEGF), inhibit the secretion of IL-6 and the expression of DKK-1 gene, thereby achieving the effects of preventing androgenetic alopecia and stimulating hair growth.

[0070] Experimental Example 2: Corticosterone model - anti-hair loss test.

[0071] 1) Cell seeding: Resuscitate dermal papilla cells (DP cells), observe the cell growth condition, count the cells after 2 days, and seed the cells into 96-well plates and 12-well plates. Among them, the 96-well plates are used to detect cell viability, and the 12-well plates are used to detect the expression levels of genes such as Gas6. Incubate the plates overnight in a CO2 incubator (37 °C, 5% CO2).

[0072] 2) Solution preparation: Prepare the sample working solution, which consists of corticosterone and vitamin K2.

[0073] 3) Experimental grouping: When the confluence rate of dermal papilla cells reaches about 60%, administer drugs in groups. Each group has 3 replicate wells.

[0074] 4) Select corticosterone at concentrations of 25 μM to 800 μM for cytotoxicity experiments to detect cell viability. The experimental conditions are shown in Table 7.

[0075] Table 7 Grouping of experimental conditions for different corticosterone concentrations

[0076]

[0077]

[0078] The cytotoxicity test results of corticosterone are shown in the following table (Table 8).

[0079] Table 8 Experimental results

[0080]

[0081] Select vitamin K2 at different concentrations diluted with absolute ethanol for cytotoxicity experiments to detect cell viability. The experimental conditions are shown in Table 9.

[0082] Table 9 Grouping of experimental conditions for different vitamin K2 concentrations

[0083]

[0084] The cytotoxicity test results of vitamin K2 are shown in the following table (Table 10).

[0085] Table 10 Cell viability

[0086] Serial number 1 2 3 4 5 Treatment Ctrl 0.05 μg / mL 0.5 μg / mL 5 μg / mL 50 μg / mL Average survival rate (%) 100 107 100 102 0.75

[0087] 5) According to the results of the above cytotoxicity test, the concentration of corticosterone for modeling was selected as 100 μM, and the concentration of vitamin K2 was 0.5 μg / mL. Quantitative qPCR detection of Gas6, VEGF, and TGF was performed, and the experimental conditions are shown in Table 11.

[0088] Table 11 Experimental conditions for modeling

[0089]

[0090]

[0091] After the administration was completed, the 96-well plate and 12-well plate were placed in an incubator (37 °C, 5% CO2) and incubated for 72 hours.

[0092] 6) Data processing:

[0093] 6.1) Cell viability test: Add 10 μL of CCK-8 reagent to each well in the 96-well plate and incubate in an incubator (37 °C, 5% CO2) for 3 hours. Then, use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at 450 nm.

[0094] 6.2) Calculation of cell survival rate: Cell survival rate (%) = [(As - Ab) / (Ac - Ab)] × 100%; where As: absorbance of the experimental group wells (containing cells, medium, CCK-8 solution, and drug solution); Ac: absorbance of the negative control group wells (containing cells, medium, CCK-8 solution, without drug); Ab: absorbance of the blank control group wells (containing medium, CCK-8 solution, without cells and drug).

[0095] 6.3) Sample collection: After the incubation of the 12-well plate was completed, the cells were collected for subsequent tests.

[0096] 6.4) Gene expression detection: Extract RNA, reverse transcribe it into cDNA, and perform fluorescence quantitative PCR detection. The 2 -△△Ct method was used for result calculation.

[0097] 6.5) Statistical analysis of results: GraphPad Prism was used for plotting, and the results were expressed as Mean + SD. One-way ANOVA statistical analysis was used for comparison between groups. P < 0.05 was considered to have a significant difference, and P < 0.01 was considered to have a highly significant difference.

[0098] 7) Detection results:

[0099] The results of the cytotoxicity test of vitamin K2 + corticosterone (Cort) are shown in the following table (Table 12).

[0100] Table 12 Cell survival rate

[0101]

[0102]

[0103] The test results of Gas6, VEGF, and TGF of vitamin K2 are shown in Table 13.

[0104] Table 13 Summary Table of Test Results of Gas6, VEGF, and TGF

[0105]

[0106] According to Table 12, based on dermal papilla cells, vitamin K2 + corticosterone at 0.5 μg / mL did not show obvious cytotoxicity.

[0107] According to Table 13, vitamin K2 at a certain concentration can promote the gene expression of Gas6, VEGF, and TGF, thereby playing a preventive role in stress-induced hair loss, promoting hair growth, and having few toxic and side effects.

[0108] According to the above tests, vitamin K2 can be applied in anti-androgenic and stress-induced hair loss compositions, and the anti-hair loss compositions include pharmaceutical compositions or cosmetic compositions.

[0109] The pharmaceutical composition includes any one of creams, patches, ointments, cream preparations, gels, capsules, injections, creams, gels, patches, sprays, ointments, plasters, lotions, liniments, and sprays.

[0110] The cosmetic composition includes any one of shampoos, hair conditioners, hair waxes, hairsprays, hair conditioners, sprays, anti-hair loss serums, essence sprays, and pre-wash gels.

[0111] The present invention also provides an anti-hair loss cosmetic, which contains vitamin K2 and a cosmetically acceptable carrier, and the dosage of vitamin K2 is generally 0.05 wt%.

[0112] It should be understood that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. For those skilled in the art, the technical solutions recorded in the above embodiments can be modified, or some of the technical features can be equivalently replaced; and all such modifications and replacements should fall within the protection scope of the appended claims of the present invention.

Claims

1. Use of vitamin K2 in the preparation of an anti - hair loss composition.

2. The application according to claim 1, characterized in that: The hair loss is androgenetic alopecia or / and stress - induced alopecia.

3. The application according to claim 1, characterized in that: The anti - hair loss includes at least one of preventing hair loss, treating hair loss and stimulating hair growth.

4. The application according to claim 2, wherein: Use of vitamin K2 in the preparation of an anti - androgenetic alopecia composition that promotes the expression of VEGF, inhibits the expression of IL - 6 and DKK - 1 genes.

5. The application according to claim 2, wherein: Use of vitamin K2 in the preparation of an anti - stress - induced alopecia composition that promotes the expression of VEGF, Gas6 and TGF genes.

6. The application according to claim 2, characterized in that: The cell dosage of vitamin K2 in the preparation of an anti - androgenetic alopecia composition is ≤0.001563 wt%.

7. The application according to claim 2, characterized in that: The cell dosage of vitamin K2 in the preparation of an anti - stress - induced alopecia composition is ≤5 μg / mL.

8. The application according to claim 1, characterized in that: The composition includes a pharmaceutical composition or a cosmetic composition.

9. The application according to claim 8, wherein: The cosmetic composition includes any one of shampoo, hair conditioner, hair wax, hairspray, hair conditioner, spray, anti - hair loss essence, essence spray, pre - wash gel.

10. An anti - hair - loss cosmetic, characterized in that: Contains vitamin K2 and a cosmetically acceptable carrier.

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