Application of cardamom seed ketone in preparation of anti-hair loss composition

Abstract

The invention discloses an application of cardamom seed ketone in preparation of an anti-hair loss composition. Researches find that the cardamom seed ketone has the effects of promoting VEGF gene expression and inhibiting DKK-1 gene expression in an androgenetic alopecia model, and has the effects of promoting Gas6, VEGF and TGF gene expression in a pressure-type alopecia model, so that the cardamom seed ketone plays a good role in preventing and treating alopecia, and has few toxic and side effects.

Description

Technical Field

[0001] The present invention relates to a new application of cardamom ketone, in particular to the application of cardamom ketone 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 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 inhibitory protein, which further blocks the Wnt signaling pathway by directly or indirectly competitively binding to the Wnt protein receptors 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 angiogenic factor, which can promote the formation of blood vessels around hair follicles, increase the blood supply to hair follicles, thus supporting hair growth. And due to the cyclic 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 in 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. The Gas6 secreted by dermal papilla cells (DP) binds to its receptor Axl on hair follicle stem cells (HFSC), which can promote the proliferation of HFSC, thus promoting hair growth. Research shows that in mice under stress, the stress hormone corticosterone 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, which plays an important role 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 use of drugs 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 kudzu root extract have a certain therapeutic effect on hair loss, the anti-hair loss effect is not ideal enough.

[0009] Cardamonin is a natural plant active ingredient, also known as hydroxymethoxyphenyldecanone or paradol (6-Paradol), with a CAS number of 27113-22-0 and a chemical formula of C 17 H 26 O3.

[0010] Currently, it has been found that cardamonin has a variety of biological activities, such as scavenging free radicals, antioxidant, anti-inflammatory, soothing the skin, etc. However, there is no report on the role of cardamonin in preventing hair loss. Summary of the Invention

[0011] The purpose of the present invention is to provide an application of cardamonin in the preparation of an anti-hair loss composition. Through research, the present invention has found that cardamonin has the effect of promoting the expression of the VEGF gene and inhibiting the expression of the DKK-1 gene 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 a good effect of preventing and treating hair loss, stimulating hair growth, and having few toxic and side effects.

[0012] The technical solution of the present invention: the application of cardamonin in the preparation of an anti-hair loss composition.

[0013] In the aforementioned application, the hair loss is androgenetic alopecia or / and stress-induced alopecia.

[0014] In the aforementioned application, the anti-hair loss includes at least one of preventing hair loss, treating hair loss, and stimulating hair growth.

[0015] In the aforementioned application, the application of cardamonin in the preparation of an anti-androgenetic alopecia composition that promotes the expression of the VEGF gene and inhibits the expression of the DKK-1 gene.

[0016] In the aforementioned application, the application of cardamonin in the preparation of an anti-stress-induced alopecia composition that promotes the VEGF, Gas6, and TGF genes.

[0017] In the aforementioned application, the cell dosage of cardamom ketone in the preparation of the anti-androgenic alopecia composition is ≤ 0.0625 wt%.

[0018] In the aforementioned application, the cell dosage of cardamom ketone in the preparation of the anti-stress alopecia composition is ≤ 0.002 wt%.

[0019] In the aforementioned application, the composition includes a pharmaceutical composition or a cosmetic composition.

[0020] In the aforementioned application, 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.

[0021] The present invention also provides an anti-hair loss cosmetic, which contains cardamom ketone or a plant extract containing cardamom ketone.

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

[0023] The present invention provides the application of cardamom ketone in the preparation of an anti-hair loss composition. By establishing an androgen model for anti-hair loss testing, the present invention finds that cardamom ketone has the characteristics of promoting the expression of vascular endothelial growth factor (VEGF) and inhibiting the expression of DKK-1 gene, thereby playing a role in preventing androgenic alopecia and stimulating hair growth.

[0024] In addition, the present invention also conducts anti-hair loss testing by establishing a corticosterone model, and finds that cardamom ketone has the characteristics of promoting the expression of Gas6, VEGF and TGF genes, thereby playing a role in preventing stress alopecia and promoting hair growth, and having few toxic and side effects. Detailed implementation manners

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

[0026] Verification of the application effect of cardamom ketone:

[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), VEGF ELISA kit (Abcam), AG RNAex Pro Reagent (Acorray Biotech), reverse transcription kit (Acorray Biotech), and fluorescent dye (Acorray Biotech). 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 PCR instrument (Bori, TC-XP-G), and a fluorescence quantitative PCR instrument (Roche, lightcycler480II).

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

[0029] Cytotoxicity test:

[0030] 1) Cell seeding: The dermal papilla cells were resuscitated. After resuscitation, when the plating rate reached about 60%, the cells were seeded into 96-well plates and incubated overnight in a CO2 incubator (37°C, 5% CO2).

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

[0032] 3) Solution preparation: Different concentrations of sample working solutions were prepared according to the test concentration setting table (Table 1). The stock solution of cardamom ketone was: 1% cardamom ketone + 99% dipropylene glycol, and then diluted with water to the required concentration of the sample working solution.

[0033] Table 1 Test concentration setting table

[0034]

[0035]

[0036] 4) Administration: Administration is carried out when the cell seeding rate in the 96-well plate reaches 50% - 60%. In the solvent control group, 200 μL of culture medium is added to each well; in the positive control group, 200 μL of culture medium containing 10% DMSO is added to each well; in the sample group, 200 μL of culture medium containing the sample at the corresponding concentration is added to each well; in the zero-adjustment group, no cells are inoculated, and only 200 μL of cell culture medium is added. After the administration is completed, the 96-well plate is placed in a CO2 incubator (37 °C, 5% CO2) and cultured for 24 h.

[0037] 5) Detection: After the cells are incubated for 24 h, the supernatant is discarded, and MTT working solution (0.5 mg / mL) is added. Incubate at 37 °C in the dark for 4 h. After the incubation is completed, the supernatant is discarded, and 150 μL of DMSO is added to each well. The OD value is read at 490 nm.

[0038] The MTT detection results of the cytotoxicity test are shown in the following table (Table 2).

[0039] Table 2 MTT detection results of cardamom ketone

[0040]

[0041] According to the MTT results, it is shown that cardamom ketone does not show obvious cytotoxicity based on dermal papilla cells within the concentration range of 0.0625%.

[0042] Anti - hair loss effect test:

[0043] 1) Cell seeding: Resuscitate the dermal papilla cells. After resuscitating the cells, when the seeding rate reaches about 60%, inoculate the cells into a 6-well plate and incubate overnight in a CO2 incubator (37 °C, 5% CO2).

[0044] 2) Solution preparation: Prepare the sample working solution according to the test grouping (Table 3).

[0045] Table 3 Test grouping

[0046]

[0047] 3) Stimulation and administration: According to the test grouping, when the cell seeding rate in the 6-well plate reaches 80% - 90%, carry out grouped administration, with 3 replicates in each group. In the blank control (BC) group, 2 mL of culture medium is added to each well; in the negative control (NC) group, 2 mL of culture medium containing 800 nM DHT is added to each well; in the positive control (PC) group, 2 mL of culture medium containing 800 nM DHT and 500 μM minoxidil is added to each well; in the sample group, 2 mL of culture medium containing 800 nM DHT and the test substance at the corresponding concentration is added to each well. After the administration is completed, the 6-well plate is placed in an incubator (37 °C, 5% CO2) and cultured for 24 h.

[0048] 4) ELISA test: After the incubation, collect the culture medium in a centrifuge tube. After collection, place the samples for ELISA detection in a -80 °C freezer for storage, and perform the detection and analysis of the VEGF index according to the operation manual of the ELISA kit.

[0049] 5) Gene expression detection: After the incubation, aspirate and discard 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. After extracting RNA and reverse transcribing it into cDNA, perform fluorescence quantitative PCR detection, and use the method to calculate the results of the DKK-1 gene expression level.

[0050] 6) Result statistical analysis: Use GraphPad Prism to plot the graph, 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.

[0051] 7) Detection results:

[0052] 7.1) The test results of vascular endothelial growth factor (VEGF) are shown in the following table (Table 4).

[0053] Table 4 Summary table of VEGF content results

[0054]

[0055] 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 **.

[0056] According to Table 4, compared with the BC group, the VEGF content in the NC group decreased significantly, indicating that the test stimulator strip was effective. Compared with the NC group, the VEGF content in the PC group increased significantly, indicating that the test positive control was effective. Compared with the NC group, the VEGF content in the sample group increased significantly, with a promotion rate of 11.96%, indicating that cardamonin 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 to the hair follicles, stimulating hair growth, and improving the health of the hair follicles.

[0057] 7.2) The test results of the DKK-1 gene expression level are shown in the following table (Table 5).

[0058] Table 5 Summary table of the detection results of the relative expression level of DKK-1 gene

[0059]

[0060] Note: When using the method for result calculation and performing statistical analysis using the t-test method, the mRNA amplification multiples of the BC group were normalized. When compared with the BC group, significance is indicated by #, P-value < 0.05 is indicated by #, and P-value < 0.01 is indicated by ##; when compared with the NC group, significance is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **.

[0061] According to Table 5, compared with the BC group, the relative expression level of the DKK-1 gene in the NC group was significantly upregulated, indicating that the stimulation conditions of this test were effective. Compared with the NC group, the relative expression level of the DKK-1 gene in the PC group was significantly downregulated, indicating that the positive control detection of this test was effective. Compared with the NC group, the relative expression level of the DKK-1 gene in the sample group was significantly downregulated, and the inhibition rate was 39.74%, indicating that cardamonin can inhibit the expression of the DKK-1 gene, thereby preventing hair loss caused by the premature entry of hair follicles into the regression period.

[0062] The above experiments demonstrated that based on DHT-stimulated dermal papilla cells, cardamonin can promote the expression of vascular endothelial growth factor (VEGF) and inhibit the expression of the DKK-1 gene, thereby achieving the effects of preventing androgenetic alopecia and stimulating hair growth.

[0063] Experimental Example 2: Corticosterone model - anti-hair loss test.

[0064] 1) Cell seeding: Resuscitate dermal papilla cells (DP cells), observe the cell growth situation, count the cells after 2 days, and seed the cells into 96-well plates and 12-well plates. Among them, 96-well plates are used to detect cell viability, and 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).

[0065] 2) Solution preparation: Prepare the sample working solution.

[0066] 3) Experimental grouping: When the plating rate of dermal papilla cells reaches about 60%, group and administer drugs. Each group has 3 replicate wells.

[0067] 4) Select corticosterone with a concentration of 25 μM to 800 μM for cytotoxicity experiments to detect cell viability. The experimental conditions are shown in Table 6.

[0068] Table 6 Grouping of experimental conditions with different corticosterone concentrations

[0069]

[0070] The results of the cytotoxicity test of cortisone are shown in the following table (Table 7).

[0071] Table 7 Experimental results

[0072]

[0073] Cytotoxicity experiments were conducted on different concentrations of cardamom ketone diluted with water to detect cell viability. The experimental conditions are shown in Table 8.

[0074] Table 8 Grouping of experimental conditions for different cardamom ketone concentrations

[0075]

[0076]

[0077] The results of the cytotoxicity test of cardamom ketone are shown in the following table (Table 9).

[0078] Table 9 Cell viability

[0079] Serial number 1 2 3 4 5 Treatment Ctrl 0.002％ 0.005％ 0.01％ 0.02％ Average survival rate (%) 100 99 100 79 64

[0080] 5) According to the above cytotoxicity test results, the concentration of cortisone for modeling was selected as 100 μM, and the concentration of cardamom ketone was 0.002%. Quantitative qPCR detection of Gas6, VEGF, and TGF was carried out, and the experimental conditions are shown in Table 10.

[0081] Table 10 Experimental conditions for modeling

[0082]

[0083] After drug administration, the 96-well plate and 12-well plate were placed in an incubator (37 °C, 5% CO2) and incubated for 72 hours.

[0084] 6) Data processing:

[0085] 6.1) Cell viability test: Add 10 μL of CCK-8 reagent to each well of the 96-well plate and incubate in an incubator (37 °C, 5% CO2) for 3 hours. Then, use a microplate reader to measure the absorbance at 450 nm.

[0086] 6.2) Calculation of cell viability: Cell viability (%) = [(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).

[0087] 6.3) Sample collection: After the incubation of the 12-well plate is completed, collect the cells for subsequent tests.

[0088] 6.4) Gene expression detection: Extract RNA, reverse transcribe it into cDNA, and perform fluorescence quantitative PCR detection. Use the 2 -△△Ct method for result calculation.

[0089] 6.5) Result statistical analysis: Use GraphPad Prism to plot graphs, and the results are expressed as Mean + SD. One-way ANOVA statistical analysis is used for comparison between groups. A significant difference is considered when P < 0.05, and a highly significant difference is considered when P < 0.01.

[0090] 7) Test results:

[0091] The results of the cytotoxicity test of cardamom ketone + corticosterone (Cort) are shown in the following table (Table 11).

[0092] Table 11 Cell survival rate

[0093] Serial number 1 2 3 Treatment Ctrl 0.002%-Cort 0.002％ Average survival rate (%) 100 90 99

[0094] The test results of Gas6, VEGF, and TGF of cardamom ketone are shown in Table 12.

[0095] Table 12 Summary table of test results of Gas6, VEGF, and TGF

[0096]

[0097] According to Table 11, based on dermal papilla cells, 0.002% cardamom ketone + corticosterone did not show obvious cytotoxicity.

[0098] According to Table 12, cardamom ketone at a certain concentration can promote the gene expression of Gas6, VEGF, and TGF, thus playing a preventive role in stress-induced hair loss, promoting hair growth, and having few toxic and side effects.

[0099] According to the above tests, cardamom ketone can be applied in anti-androgenetic and stress-induced hair loss compositions, and the anti-hair loss compositions include pharmaceutical compositions or cosmetic compositions.

[0100] 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.

[0101] The cosmetic composition includes any one of shampoos, hair conditioners, hair waxes, hairsprays, hair conditioners, sprays, anti-hair loss essence liquids, essence sprays, and pre-wash gels.

[0102] The present invention also provides an anti - hair - loss cosmetic, which contains cardamonin or a plant extract containing cardamonin, and the content of cardamonin is 1 - 2 wt%.

[0103] It should be understood that the above - mentioned embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. For those skilled in the art, the technical solutions recorded in the above - mentioned 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. Application of cardamom seed ketone in preparing an anti-hair loss composition.

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

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

4. The use according to claim 2, characterized in that: The cardamom seed ketone is used in preparing an anti-androgenic alopecia composition which promotes VEGF gene expression and inhibits DKK-1 gene expression.

5. The use according to claim 2, characterized in that: The cardamom seed ketone is used in preparing an anti-stress type hair loss composition for promoting VEGF, Gas6 and TGF genes.

6. The use according to claim 2, characterized in that: The amount of cardamom seed ketone used in the preparation of the anti-androgenic alopecia composition is ≤0.0625wt%.

7. The use according to claim 2, characterized in that: The amount of cardamom seed ketone used in the preparation of the anti-stress type hair loss composition is ≤0.002wt%.

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

9. The use according to claim 8, characterized in that: The cosmetic composition comprises any one of shampoo, conditioner, hair wax, hair spray, hair conditioner, spray, anti-hair loss essence, refinement spray and pre-wash gel.

10. An anti-hair loss cosmetic, characterized in that: Contains cardamom seed ketone or a plant extract containing cardamom seed ketone.

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