XVII type collagen truncated peptide and application thereof in anti-hair loss products

Abstract

The invention provides an XVII type collagen truncated peptide and application thereof in an anti-hair loss product, and belongs to the technical field of peptide products. The XVII type collagen truncated peptide provided by the invention can remarkably improve the oxidation resistance of hair follicles, effectively enhance the structural stability of the hair follicles, strengthen the toughness of hair roots and obviously delay the senescence process of scalp cells, so that the problem of alopecia is synergistically improved from multiple dimensions. According to the product provided by the invention, large protein is replaced by small molecule peptide, so that the technical obstacles that traditional collagen is difficult to absorb transdermally and easy to degrade are solved, and a brand new solution is provided for anti-hair-loss active ingredients; a core component which is high in activity, easy to absorb and high in safety is provided for the anti-hair-loss field, and the urgent demand of consumers for efficient anti-hair-loss products is met; the peptide technology capable of being industrialized is used for achieving integration of the three effects of hair loss prevention, hair consolidation and aging resistance, and hair loss prevention products are promoted to be upgraded to be efficient, convenient and universal.

Description

Technical Field

[0001] The present invention relates to the technical field of peptide products, and particularly to a truncated peptide of type XVII collagen and its application in anti - hair loss products. Background Art

[0002] Hair follicle stem cells are mainly located in the bulge area of the hair follicle and around the dermal papilla, maintaining periodic hair regeneration. Hair follicle stem cells are often in a dormant state in the hair follicle, but will rapidly activate and divide during the hair growth cycle to generate hair or repair damaged parts of the hair follicle. Therefore, the vitality of hair follicle stem cells is an important manifestation of the hair - growing ability of the hair follicle. At present, the core mechanism of hair loss is the dysfunction of hair follicle stem cells. For example, the activity of hair follicle stem cells is inhibited and they remain in a dormant state for a long time, or the stem cells directly differentiate into epidermal keratinocytes instead of hair follicle cells, etc.

[0003] Research shows that with age, the expression of COL17A1 decreases, leading to the destruction of the hair follicle stem cell microenvironment and the loss of stem cells. This is one of the key mechanisms of hair thinning and gray hair. Type XVII collagen (COL17A1) is a key component of hemidesmosomes, which helps to anchor stem cells in the basal layer of the epidermis to the basement membrane and maintain the microenvironment stability of hair follicle stem cells (located in the bulge area of the hair follicle). COL17A1 maintains the self - renewal of stem cells and inhibits differentiation by stabilizing the Wnt signaling pathway (such as Fzd4, Nfatc2, Nfatc4, and Tcf7, etc.). Therefore, supplementing type XVII collagen is a method to maintain the differentiation activity of hair follicle stem cells and is a feasible anti - hair loss means.

[0004] However, type XVII collagen is a large protein of 295.2 kDa, which is limited in application in absorbable daily - used smear products. Truncated functional polypeptides are an important form of its application in daily - used products. Polypeptides have a small molecular weight, good transdermal ability and absorbability, and at the same time have high stability and formulation compatibility. Therefore, functional type XVII collagen peptides have great potential in daily - used anti - hair loss products. Summary of the Invention

[0005] The purpose of the present invention is to provide a truncated peptide of type XVII collagen and its application in anti - hair loss products, providing an effective means for the development of daily - used anti - hair loss products.

[0006] In order to achieve the above - mentioned invention purpose, the present invention provides the following technical solutions: The present invention provides an application of a truncated peptide of type XVII collagen in the preparation of an anti - hair loss product, and the amino acid sequence of the truncated peptide of type XVII collagen is as shown in SEQ ID NO.1.

[0007] The present invention also provides an application of a type XVII collagen truncated peptide in the preparation of a hair follicle stem cell activator, and the amino acid sequence of the type XVII collagen truncated peptide is as shown in SEQ ID NO.1.

[0008] The present invention also provides an application of a type XVII collagen truncated peptide in the preparation of an antioxidant scalp care product, and the amino acid sequence of the type XVII collagen truncated peptide is as shown in SEQ ID NO.1.

[0009] The present invention also provides an application of a type XVII collagen truncated peptide in the preparation of a product for inhibiting scalp cell aging, and the amino acid sequence of the type XVII collagen truncated peptide is as shown in SEQ ID NO.1.

[0010] The present invention also provides an application of a type XVII collagen truncated peptide in the preparation of a gene expression regulator, wherein the gene is selected from at least one of COL17A1, ITGβ1, ITGA6, NES, and SOX10, and the amino acid sequence of the type XVII collagen truncated peptide is as shown in SEQ ID NO.1.

[0011] The present invention also provides an application of a type XVII collagen truncated peptide in the preparation of a product for promoting the expression of TGF-β, ALPL, NOG or CD44 genes, and the amino acid sequence of the type XVII collagen truncated peptide is as shown in SEQ ID NO.1.

[0012] The present invention also provides an anti-hair loss topical product, using the type XVII collagen truncated peptide as an active ingredient, and the amino acid sequence of the type XVII collagen truncated peptide is as shown in SEQ ID NO.1; The dosage form of the anti-hair loss topical product includes shampoo, essence, lotion or spray.

[0013] The present invention also provides an anti-hair loss composition containing the type XVII collagen truncated peptide, and the amino acid sequence of the type XVII collagen truncated peptide is as shown in SEQ ID NO.1.

[0014] Preferably, the anti-hair loss composition further contains minoxidil and / or finasteride.

[0015] Advantages of the present invention: The truncated peptide of type XVII collagen provided by the present invention can significantly enhance the antioxidant capacity of hair follicles, effectively strengthen the stability of hair follicle structure, strongly enhance the toughness of hair roots, and significantly delay the aging process of scalp cells, thereby synergistically improving hair loss problems from multiple dimensions. The product provided by the present invention uses small molecule peptides to replace large proteins, solving the technical obstacles of traditional collagen being difficult to penetrate the skin and being easily degraded, and providing a new solution for anti-hair loss active ingredients; the active ingredients provided by the present invention can be flexibly developed into daily care products such as shampoos, serums, sprays, etc., or integrated into medical devices such as microneedle patches and hair growth instruments, providing core ingredients with high activity, easy absorption, and strong safety in the anti-hair loss field, meeting consumers' urgent needs for highly effective anti-hair loss products; with an industrializable peptide technology, it realizes the integration of the triple effects of "anti-hair loss - hair strengthening - anti-aging", and promotes the upgrading of anti-hair loss products to be more efficient, convenient, and universal. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a diagram showing the results of the influence of Hair-Col17 on the expression of antioxidant-related genes; Figure 2 It is a diagram showing the results of the influence of Hair-Col17 on the expression of genes related to stabilizing hair follicles; Figure 3 It is a diagram showing the results of the influence of Hair-Col17 on the expression of genes related to strengthening hair roots; Figure 4 It is a diagram showing the results of the influence of Hair-Col17 on senescent cells in hair follicles. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention is based on a truncated peptide of type XVII collagen, and the amino acid sequence of the truncated peptide of type XVII collagen is GRGDNP, as shown in SEQ ID NO.1. In the present invention, the truncated peptide refers to a functional short peptide obtained by selectively designing and cleaving type XVII collagen, and has excellent characteristics of enhanced transdermal absorption, improved stability, and retention of hair follicle biological activity.

[0018] The present invention provides an anti-hair loss composition containing the truncated peptide of type XVII collagen provided by the present invention. In the present invention, preferably, the anti-hair loss composition further contains minoxidil and / or finasteride. In the present invention, the anti-hair loss composition may further contain plant anti-hair loss active ingredients (such as Platycladus orientalis extract, ginsenoside), vitamin hair growth factors (such as biotin, niacinamide), trace elements (such as zinc compounds), penetration enhancers (such as azone, lecithin), and scalp conditioners (such as panthenol, ceramide).

[0019] The present invention also provides the use of the above-mentioned type XVII collagen truncated peptide in the preparation of anti-hair loss products. In the present invention, the anti-hair loss products can cover daily chemical care products (such as anti-hair loss shampoos, hair growth essences), medical devices (such as preparations for hair growth devices, microneedle patches), professional line products (such as scalp perfusion solutions, anti-hair loss treatment sets), and household instrument consumables (such as hair growth comb introduction solutions).

[0020] The present invention also provides the use of the above-mentioned type XVII collagen truncated peptide in the preparation of follicular stem cell activators. The activator may further comprise stem cell proliferation assisting factors (such as EGF, FGF), follicular microenvironment regulators (such as Wnt pathway activators), cell energy metabolism promoters (such as ATP precursors), and stem cell protection components (such as mitochondrial antioxidants).

[0021] The present invention also provides the use of the above-mentioned type XVII collagen truncated peptide in the preparation of antioxidant scalp care products. The product may further incorporate free radical scavengers (such as vitamin C derivatives, glutathione), photo-damage repair components (such as ectoine, melatonin), oxidative stress relievers (such as ergothioneine, astaxanthin), and barrier repair lipids (such as squalane, cholesterol).

[0022] The present invention also provides the use of the above-mentioned type XVII collagen truncated peptide in the preparation of products for inhibiting scalp cell aging. The product may be synergistically added with cell aging inhibitors (such as Senolytics components, NAD+ precursors), telomere protection factors (such as telomerase activators), DNA repair agents (such as photolyase), and inflammation relieving components (such as bisabolol, dipotassium glycyrrhizinate).

[0023] The present invention also provides the use of the above-mentioned type XVII collagen truncated peptide in the preparation of gene expression regulators, and the genes are selected from at least one of COL17A1, ITGβ1, ITGA6, NES, and SOX10. The regulator may further comprise gene transcription enhancing cofactors (such as histone deacetylase inhibitors), signal pathway co-activators (such as Notch pathway regulators), epigenetic modification components (such as methyl donors), and gene delivery vectors (such as lipid nanoparticles), etc.

[0024] The present invention also provides the use of the above-mentioned type XVII collagen truncated peptide in the preparation of products for promoting the expression of TGF-β, ALPL, NOG, or CD44 genes. The product may be combined with follicular growth cycle regulators (such as prostaglandin analogs), extracellular matrix synthesis promoters (such as silicon compounds), dermal papilla cell activators (such as adenosine), and follicular blood supply improving components (such as minoxidil derivatives).

[0025] The present invention also provides an anti - hair - loss topical product, which uses the type XVII collagen truncation peptide provided by the present invention as an active ingredient; the dosage forms of the anti - hair - loss topical product include shampoo, essence, lotion or spray. The dosage form can also be extended to gel, foam, roll - on liquid, ampoule concentrate, ointment, scalp scrub, conditioner, hair mask, hair - growth pen and dissolvable microneedle array; product excipients may include thickeners (such as carbomer), emulsifiers (such as PEG - 40 hydrogenated castor oil), preservatives (such as phenoxyethanol), pH regulators (such as lactic acid) and sensory improvers (such as menthol).

[0026] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0027] Embodiment The sequence of the type XVII collagen anti - hair - loss peptide provided by the present invention is GRGDNP, as shown in SEQ ID NO.1.

[0028] This short peptide was synthesized by a conventional method and used as a test sample for the following experiments: Test on the expression of antioxidant - related genes of type XVII collagen peptide (Hair - Col17) Dermal papilla cells in the logarithmic growth phase were seeded in 6 - well plates at a density of 1×10 6 cells / well and cultured in an incubator at 37 °C and 5% CO2 for 24 h; the culture medium was discarded, and the cells were treated with UVB (50 mJ / cm 2 )(the blank control group did not need to be treated with UVB); in the experimental groups, samples of each concentration were added, and the blank control group and the model group were added with serum - free medium, and cultured in an incubator at 37 °C and 5% CO2 for 24 h; the supernatant was discarded, and the cells were collected after washing twice with PBS; RNA was extracted, reverse - transcribed into cDNA, and then RT - qPCR was performed. The data were expressed as mean ± standard error. The inter - group differences between the control group and the negative control group were analyzed using the T - test; the differences between the sample groups and the negative control group were analyzed using one - way ANOVA. P < 0.05 (* and #) was considered to have a significant difference, P < 0.01 (** and ##) was considered to have a highly significant difference, and P < 0.001 (*** and ) was considered to have an extremely significant difference.

[0029] Blank control (BC) group: cultured with serum - free medium; Model (NC) group: cultured with serum - free medium after UVB irradiation at 50 mJ / cm 2 ; Experimental groups: cultured with the addition of Hair - Col17 solution at the corresponding concentration after UVB irradiation at 50 mJ / cm 2 ; Positive control group (PC): 50 mJ / cm 2 Cultured with 50 μg / mL finasteride solution after UVB irradiation As Figure 1 shown, compared with the model group, Hair-Col17 could significantly promote the expression of antioxidant genes such as SOD2 and GPX3. Hair-Col17 at 5 ppm, 10 ppm, and 50 ppm increased the expression level of the SOD2 gene by 60.36% (P<0.001), 80.07% (P<0.001), and 99.70% (P<0.001), respectively; Hair-Col17 at 10 ppm and 50 ppm significantly increased the expression level of the GPX3 gene by 143.75% (P<0.001) and 208.41% (P<0.001), respectively.

[0030] The results showed that Hair-Col17 at 5, 10, and 50 ppm had the effect of scavenging free radicals.

[0031] Expression test of type XVII collagen peptide (Hair-Col17) on stabilizing hair follicle-related genes The experimental process and experimental groups were the same as above.

[0032] As Figure 2 shown, compared with the blank control group, the relative expression levels of COL17A1, ITGβ1, ITGA6, NES, and SOX10 genes in the model group after UVB irradiation were significantly down-regulated, indicating that the animal model was successfully established in this experiment and the model group was effective.

[0033] Compared with the model group, adding Hair-Col17 after UVB stimulation could promote the expression of genes related to hair follicle stability, such as COL17A1, ITGβ1, ITGA6, NES, and SOX10. 5 ppm and 50 ppm of Hair-Col17 increased the expression levels of the COL17A1 gene by 19.23% (P<0.01) and 42.04% (P<0.001) respectively, while 50 μg / mL of finasteride inhibited the expression of the COL17A1 gene, with the expression level decreasing by 30.99% (P<0.001). 5 ppm and 50 ppm of Hair-Col17 increased the expression levels of the ITGβ1 gene by 42.89% (P<0.01) and 63.52% (P<0.001) respectively, while finasteride had no significant effect on the expression of the ITGβ1 gene. Both 50 ppm of Hair-Col17 and 50 μg / mL of finasteride could promote the expression of the ITGA6 gene, with the expression levels increasing by 60.17% (P<0.01) and 66.71% (P<0.01) respectively. 5 ppm and 50 ppm of Hair-Col17 increased the expression levels of the NES gene by 68.99% (P<0.05) and 253.55% (P<0.001) respectively, while finasteride had no significant effect on the expression of the NES gene. 50 ppm of Hair-Col17 increased the expression level of the SOX10 gene by 65.69% (P<0.05), while finasteride had no significant effect on the expression of the SOX10 gene.

[0034] Expression test of genes related to strengthening hair roots by type XVII collagen peptide (Hair-Col17) The experimental procedure and experimental groups were the same as above.

[0035] As Figure 3 shown, compared with the blank control group, the relative expression levels of TGF-β, ALPL, NOG, and CD44 genes in the model group after UVB irradiation were significantly downregulated, indicating that the model was successfully established in this experiment and the model group was effective.

[0036] Compared with the model group, adding Hair-Col17 after UVB stimulation could promote the gene expression of strong hair roots such as TGF-β, ALPL, NOG, and CD44. 10 ppm, 50 ppm Hair-Col17, and 50 μg / mL finasteride increased the TGF-β gene expression levels by 333.30% (P<0.001), 394.44% (P<0.001), and 149.58% (P<0.05), respectively. 10 ppm and 50 ppm Hair-Col17 increased the ALPL gene expression levels by 52.66% (P<0.05) and 93.66% (P<0.001), respectively, while finasteride had no significant effect on ALPL gene expression. 10 ppm, 50 ppm Hair-Col17, and 50 μg / mL finasteride increased the NOG gene expression levels by 312.92% (P<0.001), 378.79% (P<0.001), and 130.32% (P<0.001), respectively. 10 ppm, 50 ppm Hair-Col17, and 50 μg / mL finasteride increased the CD44 gene expression levels by 94.29% (P<0.001), 145.22% (P<0.001), and 44.66% (P<0.01), respectively.

[0037] Senescence cell staining test Dermal papilla cells in the logarithmic growth phase were seeded in 6-well plates at a density of 1×10 6 cells / well and cultured in an incubator at 37°C with 5% CO2 for 24 h; the culture medium was discarded, and the cells were treated with UVB (50 mJ / cm 2 2) (the blank control group did not need to be treated with UVB); in the experimental groups, samples at various concentrations were added, and the blank control group and the model group were added with serum-free medium, and cultured in an incubator at 37°C with 5% CO2 for 24 h; the supernatant was discarded, and the cells were washed twice with PBS, and 1 mL of β-galactosidase staining fixative was added and fixed at room temperature for 15 min; the cell fixative was aspirated, and the cells were washed three times with PBS, 3 min each time; the PBS was aspirated, and 1 mL of staining working solution was added to each well; pictures were taken under a microscope, and the proportion of β-galactosidase-positive cells in the pictures was analyzed using software; β-galactosidase-positive cells represent senescent cells, and the formula for calculating their relative content is as follows:

[0038] As Figure 4As shown, compared with the blank control group, the proportion of senescent cells in the model group increased after UVB irradiation, indicating that the model was effective. Compared with the model group, adding 5, 10, 50 ppm Hair-Col17 and 50 μg / mL finasteride after UVB stimulation could reduce the proportion of senescent cells, and the cell senescence inhibition rates were 58.39% (P<0.05), 63.59% (P<0.01), 74.35% (P<0.01), and 61.71% (P<0.01), respectively.

[0039] As can be seen from the above examples, dermal papilla cells were used to analyze the expression of SOD2 and GPX3 genes by RT-qPCR to evaluate the effect of Hair-Col17 on scavenging free radicals. Compared with the blank control group, the relative expression levels of SOD2 and GPX3 genes in the model group were significantly down-regulated, indicating that the modeling of this experiment was successful and the model group was effective. Compared with the model group, 5, 10, 50 ppm Hair-Col17 and 50 μg / mL finasteride significantly increased the expression levels of the SOD2 gene by 60.36% (P<0.001), 80.07% (P<0.001), 99.70% (P<0.001), and 41.44% (P<0.01), respectively; 10, 50 ppm Hair-Col17 and 50 μg / mL finasteride significantly increased the expression levels of the GPX3 gene by 143.75% (P<0.001), 208.41% (P<0.001), and 71.12% (P<0.05), respectively. The above experimental results show that 5, 10, 50 ppm of Hair-Col17 has the effect of scavenging free radicals.

[0040] Dermal papilla cells were used to analyze the expression of follicle-related genes COL17A1, ITGβ1, ITGA6, NES, and SOX10 by RT-qPCR to evaluate the effect of Hair-Col17 on follicle stabilization. Compared with the blank control group, the relative expression levels of COL17A1, ITGβ1, ITGA6, NES, and SOX10 genes in the model group were significantly downregulated, indicating successful modeling in this experiment and the effectiveness of the model group. Compared with the model group, 5 and 50 ppm of Hair-Col17 increased the expression levels of the COL17A1 gene by 19.23% (P<0.01) and 42.04% (P<0.001), respectively, while 50 μg / mL of finasteride decreased the expression level of the COL17A1 gene by 30.99% (P<0.001); 5 and 50 ppm of Hair-Col17 increased the expression levels of the ITGβ1 gene by 42.89% (P<0.001) and 63.52% (P<0.001), respectively, while finasteride had no significant effect on the expression of the ITGβ1 gene; 50 ppm of Hair-Col17 and 50 μg / mL of finasteride increased the expression levels of the ITGA6 gene by 60.17% (P<0.01) and 66.71% (P<0.01), respectively; 5 and 50 ppm of Hair-Col17 increased the expression levels of the NES gene by 68.99% (P<0.05) and 253.55% (P<0.001), respectively, while finasteride had no significant effect on the expression of the NES gene; 50 ppm of Hair-Col17 increased the expression level of the SOX10 gene by 65.69% (P<0.05), while finasteride had no significant effect on the expression of the SOX10 gene. The above experimental results indicate that Hair-Col17 has the effect of stabilizing hair follicles.

[0041] Dermal papilla cells were used to measure and analyze the expression levels of genes TGF-β, ALPL, CD44, and NOG related to strong and tough hair roots by RT-qPCR method, so as to evaluate the effect of Hair-Col17 on strengthening hair roots. Compared with the blank control group, the relative expression levels of TGF-β, ALPL, NOG, and CD44 genes in the model group were significantly down-regulated, indicating that the modeling of this experiment was successful and the model group was effective. Compared with the model group, 10, 50 ppm Hair-Col17 and 50 μg / mL finasteride increased the expression level of TGF-β gene by 333.30% (P<0.001), 394.44% (P<0.001), 149.58% (P<0.05) respectively; 10, 50 ppm Hair-Col17 increased the expression level of ALPL gene by 52.66% (P<0.05), 93.66% (P<0.001) respectively, while finasteride had no significant effect on the expression of ALPL gene; 10, 50 ppm Hair-Col17 and 50 μg / mL finasteride increased the expression level of NOG gene by 312.92% (P<0.001), 378.79% (P<0.001), 130.32% (P<0.001) respectively; 10, 50 ppm Hair-Col17 and 50 μg / mL finasteride increased the expression level of CD44 gene by 94.29% (P<0.001), 145.22% (P<0.001), 44.66% (P<0.01) respectively. The above experimental results show that Hair-Col17 has the effect of strengthening hair roots.

[0042] Dermal papilla cells were used to measure and analyze the proportion of senescent cells by β-galactosidase staining method to evaluate the anti-aging effect of Hair-Col17. Compared with the blank control group, after UVB irradiation, the proportion of senescent cells in the model group increased, indicating that the model was effective. Compared with the model group, the cell senescence inhibition rates of 5, 10, 50 ppm Hair-Col17 and 50 μg / mL finasteride were 58.39% (P<0.05), 63.59% (P<0.01), 74.35% (P<0.01), 61.71% (P<0.01) respectively. The above experimental results show that Hair-Col17 has anti-aging efficacy.

[0043] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of a type XVII collagen truncated peptide in the preparation of an anti - hair loss product, characterized in that, The amino acid sequence of the type XVII collagen truncated peptide is as shown in SEQ ID NO.

1.

2. Use of a type XVII collagen truncated peptide in the preparation of a hair follicle stem cell activator, characterized in that, The amino acid sequence of the type XVII collagen truncated peptide is as shown in SEQ ID NO.

1.

3. Use of a type XVII collagen truncated peptide in the preparation of an antioxidant scalp care product, characterized in that, The amino acid sequence of the type XVII collagen truncated peptide is as shown in SEQ ID NO.

1.

4. Use of a type XVII collagen truncated peptide in the preparation of a product for inhibiting scalp cell aging, characterized in that, The amino acid sequence of the type XVII collagen truncated peptide is as shown in SEQ ID NO.

1.

5. Use of a type XVII collagen truncated peptide in the preparation of a gene expression regulator, characterized in that, The gene is selected from at least one of COL17A1, ITGβ1, ITGA6, NES, and SOX10, and the amino acid sequence of the type XVII collagen truncated peptide is as shown in SEQ ID NO.

1.

6. Use of a type XVII collagen truncated peptide in the preparation of a product for promoting the expression of TGF-β, ALPL, NOG, or CD44 genes, wherein the amino acid sequence of the type XVII collagen truncated peptide is as shown in SEQ ID NO.

1.

7. An anti - hair - loss topical product, characterized in that, Using the type XVII collagen truncated peptide as an active ingredient, the amino acid sequence of the type XVII collagen truncated peptide is as shown in SEQ ID NO.1; The dosage form of the anti-hair loss topical product includes shampoo, essence, lotion, or spray.

8. An anti - hair loss composition containing a truncated peptide of type XVII collagen, characterized in that, The amino acid sequence of the type XVII collagen truncated peptide is as shown in SEQ ID NO.

1.

9. The anti-hair loss composition according to claim 8, characterized in that, The anti-hair loss composition further comprises minoxidil and / or finasteride.

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