Hexapeptide compound as well as preparation method and application thereof

Abstract

The invention provides a hexapeptide compound as shown in a formula (I), and a preparation method and application thereof. The amino acid sequence of the hexapeptide compound shown in the formula (I) is shown in SEQ ID No.1, specifically H-Gly-Pro-Hyp-Gly-Pro-Hyp-OH, and the hexapeptide compound can remarkably promote secretion of VII type collagen, so that the hexapeptide compound has good effects in the aspects of tightening, repairing and the like. Meanwhile, the hexapeptide compound shown in the formula (I) can inhibit secretion of inflammatory factors NO and IL-6, and has anti-inflammatory and soothing effects. On the basis, the hexapeptide compound as shown in the formula (I) provided by the invention can be applied to cosmetics and has the effects of tightening, repairing, resisting inflammation and relieving. # imgabs0 # formula (I): (1).

Description

Technical Field

[0001] This invention relates to the field of daily chemical technology, and in particular to a hexapeptide compound, its preparation method and its application. Background Technology

[0002] Collagen is one of the most important structural proteins in the skin, mainly distributed in the dermis, accounting for 25% to 30% of the total skin protein. The network structure of collagen fibers provides elasticity and support to the skin and is an important site for biochemical reactions in the skin. As we age, the amount of collagen in the skin gradually decreases, leading to loss of skin elasticity and sagging.

[0003] Collagen type VII (Col7) is a unique member of the collagen family, primarily found in the basement membrane region of the skin, and is a major component of anchor fibers. Anchor fibers are complexes in which the skin's basement membrane adheres to the underlying dermis, connecting the epidermis and dermis. Collagen type VII plays a crucial role in maintaining skin integrity and structural stability; the amount of collagen produced solely by keratinocytes or fibroblasts is sufficient to form mechanically strong skin. The abundance and distribution of collagen type VII are critical to skin firmness and repair. However, current products specifically targeting the promotion of collagen type VII secretion do not yet meet market demand. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a hexapeptide compound, its preparation method and its application. The hexapeptide compound provided by this application can significantly promote the secretion of type VII collagen, inhibit inflammatory response, and has firming and soothing effects, which can better improve skin condition.

[0005] This application provides a hexapeptide compound of formula (I): Formula (I).

[0006] The amino acid sequence of the hexapeptide compound represented by formula (I) is shown in SEQ ID No. 1, specifically: H-Gly-Pro-Hyp-Gly-Pro-Hyp-OH. It significantly promotes the secretion of type VII collagen, thus exhibiting good effects in firming and repair. Simultaneously, the hexapeptide compound represented by formula (I) can inhibit the secretion of inflammatory factors NO and IL-6, possessing anti-inflammatory and soothing effects. Based on this, the hexapeptide compound represented by formula (I) provided in this application can be used in cosmetics to achieve firming, repairing, anti-inflammatory, and soothing effects.

[0007] This application also provides a method for preparing the hexapeptide compound shown in formula (I), comprising: a) Hydroxyproline, proline, glycine, hydroxyproline, proline and glycine protected by protecting groups are sequentially linked onto a solid-phase synthetic resin to obtain a resin containing peptide chains protected by protecting groups. b) Remove the protecting group and resin of the resin containing the protecting group to obtain the hexapeptide compound shown in formula (I).

[0008] This application employs a solid-phase synthesis method, sequentially linking hydroxyproline (Hyp(protecting group)), proline (Pro), glycine (Gly), hydroxyproline (Hyp(protecting group)), proline (Pro), and glycine (Gly) protected by protecting groups onto a solid-phase synthesis resin to obtain a resin containing peptide chains protected by protecting groups.

[0009] This application uses a solid-phase synthetic resin as the starting point for synthesis. First, it reacts with a hydroxyproline raw material to attach a protecting group-protected hydroxyproline onto the resin. Specifically, this application mixes the solid-phase synthetic resin, the hydroxyproline raw material, the reaction solvent, and a base for a first reaction, then mixes with an alcohol solvent for a second reaction. After deprotection, Hyp (protecting group)-resin is obtained.

[0010] In some specific implementations, the solid-phase synthesis resin includes, but is not limited to, Wang-resin, CTC-resin, etc., preferably CTC-resin. The hydroxyproline raw material is preferably Fmoc-Hyp(OtBu)-OH. The reaction solvent includes, but is not limited to, N,N-dimethylformamide (DMF), dichloromethane (DCM), dimethyl sulfoxide (DMSO), etc., preferably DMF. The base includes, but is not limited to, N,N-diisopropylethylamine (DIEA), triethylamine (TEA), pyridine (Py), etc., preferably DIEA. In some specific implementations, the molar ratio of the solid-phase synthesis resin, hydroxyproline raw material, and base is 5~15:15~30:0.01~0.1, preferably 8~12:20~25:0.03~0.08. In some specific implementations, the temperature of the first reaction is preferably 10℃~30℃, preferably 15℃~25℃, and the time is preferably 2h~5h, more preferably 2.5h~4.5h.

[0011] After the first reaction is complete, an alcohol solvent is added directly to initiate a second reaction. In some specific implementations, the second reaction time is preferably 1 min to 10 min, more preferably 3 min to 8 min. In some specific implementations, the alcohol solvent includes, but is not limited to, methanol, ethanol, or isopropanol, preferably methanol. After the second reaction is complete, the reaction product is filtered, and the obtained resin is washed with a reaction solvent such as DMF, preferably twice, and then deprotected. This application does not impose any particular limitation on the deprotection method; the deprotection method can be selected according to the type of protecting group. For example, when the solid-phase synthetic resin is CTC-resin and the hydroxyproline raw material is Fmoc-Hyp(OtBu)-OH, piperidine is preferably used for deprotection, specifically including: mixing the obtained resin with a piperidine solution to obtain Hyp(OtBu)-resin. In some specific implementations, the piperidine solution is preferably a DMF solution of piperidine, wherein the mass concentration of piperidine is preferably 10% to 30%, more preferably 15% to 25%. The reaction temperature is preferably 10℃~30℃, more preferably 15℃~25℃, and the reaction time is preferably 0.5h~2h, more preferably 1h~1.5h. After the reaction is complete, the product is washed with a reaction solvent such as DMF, preferably 6 times, and the resin is dried to obtain Hyp(OtBu)-resin.

[0012] After obtaining Hyp(OtBu)-resin, proline (Pro) is attached to it. In this application, proline raw material, condensing agent, and solvent are first mixed and reacted. Then, the resulting reaction solution is mixed with Hyp(OtBu)-resin and reacted. After deprotection, Pro-Hyp(OtBu)-resin is obtained. In some specific implementations, the proline raw material is Fmoc-Pro-OH; the condensing agent is a mixture of HOBt / DIC; and the solvent is DMF. Specifically, in this application, Fmoc-Pro-OH and HOBt are first mixed and dissolved with DMF, and then reacted with DIC. In some specific implementations, the molar ratio of Fmoc-Pro-OH, HOBt, and DIC is preferably 20~40:20~40:20~40, more preferably 25~35:25~35:25~35. In some specific implementations, the reaction is carried out under stirring conditions, with the reaction temperature preferably between 2°C and 5°C, and the reaction time preferably between 10 min and 15 min. After the reaction is complete, the obtained reaction product is mixed with Hyp(OtBu)-resin for further reaction, with the reaction temperature preferably between 10°C and 30°C, more preferably between 15°C and 25°C, and the reaction time preferably between 1 h and 3 h, more preferably between 1.5 h and 2.5 h. After the reaction is complete, the reaction product is filtered, and the obtained resin is washed with a reaction solvent such as DMF, preferably three times, and then deprotected. This application does not impose any particular limitation on the deprotection method; the deprotection method can be selected according to the type of protecting group. For example, when the proline raw material is Fmoc-Pro-OH, piperidine is preferably used for deprotection, specifically including: mixing the obtained resin with a piperidine solution to obtain Pro-Hyp(OtBu)-resin. In some specific implementations, the piperidine solution is preferably a DMF solution of piperidine, wherein the mass concentration of piperidine is preferably 10% to 30%, more preferably 15% to 25%. The reaction temperature is preferably 10℃~30℃, more preferably 15℃~25℃, and the reaction time is preferably 0.5h~2h, more preferably 1h~1.5h. After the reaction is complete, the product is washed with a reaction solvent such as DMF, preferably 6 times, and the resin is dried to obtain Pro-Hyp(OtBu)-resin.

[0013] After obtaining Pro-Hyp(OtBu)-resin, glycine (Gly) is attached to it. In this application, glycine (Gly) raw material, condensing agent, and solvent are first mixed and reacted. The resulting reaction solution is then mixed with Pro-Hyp(OtBu)-resin for further reaction. After deprotection, Gly-Pro-Hyp(OtBu)-resin is obtained. In some specific implementations, the glycine raw material is Fmoc-GLy-OH; the condensing agent is a mixture of HOBt / DIC; and the solvent is DMF. Specifically, in this application, Fmoc-GLy-OH and HOBt are first mixed and dissolved with DMF, and then reacted with DIC. In some specific implementations, the molar ratio of Fmoc-Gly-OH, HOBt, and DIC is preferably 20~40:20~40:20~40, more preferably 25~35:25~35:25~35. In some specific implementations, the reaction is carried out under stirring conditions, with the preferred reaction temperature being 2℃~5℃ and the preferred reaction time being 10min~15min. After the reaction is complete, the obtained reaction product is mixed with Pro-Hyp(OtBu)-resin for further reaction, with the preferred reaction temperature being 10℃~30℃, preferably 15℃~25℃, and the preferred reaction time being 1h~3h, more preferably 1.5h~2.5h. After the reaction is complete, the reaction product is filtered, and the obtained resin is washed with a reaction solvent such as DMF, preferably three times, and then deprotected. This application does not have any particular limitation on the deprotection method; the deprotection method can be selected according to the type of protecting group. For example, when the glycine raw material is Fmoc-Gly-OH, piperidine is preferably used for deprotection, specifically including: mixing the obtained resin with a piperidine solution to obtain Gly-Pro-Hyp(OtBu)-resin. In some specific implementations, the piperidine solution is preferably a DMF solution of piperidine, wherein the mass concentration of piperidine is preferably 10%~30%, more preferably 15%~25%. The reaction temperature is preferably 10℃~30℃, more preferably 15℃~25℃, and the reaction time is preferably 0.5h~2h, more preferably 1h~1.5h. After the reaction is complete, the obtained product is washed with a reaction solvent such as DMF, preferably 6 times, and the resin is dried to obtain Gly-Pro-Hyp(OtBu)-resin.

[0014] Gly-Pro-Hyp(OtBu)-resin was obtained, and hydroxyproline (Hyp(protecting group)), proline (Pro) and glycine (Gly) protected by the protecting group were sequentially linked in the same manner as described above to obtain H-Gly-Pro-Hyp(OtBu)-Gly-Pro-Hyp(OtBu)-CTC resin, which will not be described in detail here.

[0015] After obtaining the H-Gly-Pro-Hyp(OtBu)-Gly-Pro-Hyp(OtBu)-CTC resin, it is deprotected. In this application, trifluoroacetic acid is preferably used for deprotection. The specific steps are as follows: The H-Gly-Pro-Hyp(OtBu)-Gly-Pro-Hyp(OtBu)-CTC resin was mixed with a mixture of trifluoroacetic acid / triisopropylsilane / water and reacted. The mixture was then filtered, and the resulting filtrate was crystallized in a solvent to obtain crude peptides.

[0016] In some specific implementations, the volume ratio of trifluoroacetic acid / triisopropylsilane / water is 80~90:5~10:5~10, more preferably 90:5:5. In some specific implementations, the reaction temperature is 0~5℃, and the reaction time is 0.h~1.5h. In some specific implementations, the solvent includes, but is not limited to, methyl tert-butyl ether (MTBE), diethyl ether, isopropyl ether, etc., preferably MTBE. The crystallized solid is centrifuged and vacuum dried to obtain crude peptide.

[0017] After obtaining the crude peptide, it is preferably purified by high-performance liquid chromatography (HPLC), and then lyophilized to obtain the hexapeptide shown in formula (I), whose amino acid sequence is shown in SEQ ID No. 1, specifically: H-Gly-Pro-Hyp-Gly-Pro-Hyp-OH. In some specific implementations, the HPLC column is a reversed-phase C18 column.

[0018] This application also provides a cosmetic product comprising a hexapeptide compound of formula (I) and excipients.

[0019] In this application, according to the principles of cosmetic classification, cosmetics can be divided into: cleansing cosmetics, care cosmetics, and beauty / decorative cosmetics.

[0020] The term "cleansing cosmetics" refers to cosmetics applied to the surface of the human body (such as epidermis, hair, nails, lips, etc.) by smearing, spraying, or other similar methods to cleanse and hygienically eliminate unpleasant odors. The term "care cosmetics" refers to cosmetics applied to the surface of the human body (such as epidermis, hair, nails, lips, etc.) by smearing, spraying, or other similar methods to maintain health and appearance. The term "beauty / decorative cosmetics" refers to cosmetics applied to the surface of the human body (such as epidermis, hair, nails, lips, etc.) by smearing, spraying, or other similar methods to beautify, decorate, and enhance personal attractiveness.

[0021] Skin-friendly cleansing cosmetics include, but are not limited to, facial cleansers, makeup removers (lotions), cleansing creams (moisturizers), face masks, floral water, prickly heat powder, talcum powder, or bath gel; skin-friendly care cosmetics include, but are not limited to, skin creams, lotions, or toners; skin-friendly beauty / makeup cosmetics include, but are not limited to, pressed powder, blush, eyeshadow, eyeliner (liquid), eyebrow pencil, perfume, or cologne; hair-friendly cleansing cosmetics include, but are not limited to, shampoos, hair creams, or shaving creams; hair-friendly care cosmetics include, but are not limited to, conditioners, hair lotions, hair oils / waxes, etc.; hair-friendly beauty... Makeup / decorative cosmetics include, but are not limited to, styling mousse / hairspray, hair dye, perm solution, mascara, hair growth product, or hair removal product; nail cleaning cosmetics include, but are not limited to, nail polish remover; nail care cosmetics include, but are not limited to, nail conditioner (cream) and nail hardener; nail beauty / decorative cosmetics include, but are not limited to, nail polish; lip cleaning cosmetics include, but are not limited to, lip makeup remover; lip care cosmetics include, but are not limited to, lip balm; and lip beauty / decorative cosmetics include, but are not limited to, lipstick, lip gloss, or lip liner.

[0022] The amino acid sequence of the hexapeptide compound of formula (I) provided in this application is shown in SEQ ID No. 1, specifically: H-Gly-Pro-Hyp-Gly-Pro-Hyp-OH. It can significantly promote the secretion of type VII collagen, thus exhibiting good effects in firming and repair. Simultaneously, the hexapeptide compound of formula (I) can inhibit the secretion of inflammatory factors NO and IL-6, possessing anti-inflammatory and soothing effects. Based on this, the hexapeptide compound of formula (I) provided in this application can be used in cosmetics to achieve firming, repairing, anti-inflammatory, and soothing effects. Attached Figure Description

[0023] Figure 1 The NMR spectrum of the hexapeptide compound prepared in Example 1 of this application; Figure 2 This is the result of the effect of hexapeptide on human keratinocytes; Figure 3 The collagen expression level in the hexapeptide experimental group; Figure 4 The expression level of the inflammatory factor NO in the hexapeptide experimental group; Figure 5 The expression level of the inflammatory factor IL-6 in the hexapeptide experimental group. Detailed Implementation

[0024] This invention provides a hexapeptide compound, its preparation method, and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0025] The present invention will be further illustrated below with reference to the embodiments: In the following embodiments, the abbreviations and their English meanings are as follows:

[0026] Example 1 CTC resin (10.0 g, 10.08 mmol) was placed in a solid-phase synthesis reactor, and amino acid Fmoc-Hyp(OtBu)-OH (8.25 g, 20.16 mmol), 100 mL of N,N-dimethylformamide (DMF), and 8.0 mL of N,N-diisopropylethylamine (DIEA) were added. The reaction was carried out at 20 °C for 3 h. Then, 10 mL of methanol (MeOH) was added, and the reaction was carried out for 5 min. The mixture was filtered, and the resin was washed twice with 100 mL of DMF. Then, 100 mL of 20% piperidine (Pip) / DMF solution was added to the solid-phase synthesis reactor, and the reaction was carried out for 1 h. The mixture was filtered, the deprotection solution was discarded, and the resin was washed 6 times with 100 mL of DMF. The resin was dried under vacuum and proceeded to the next step.

[0027] Take Fmoc-Pro-OH (10.20 g, 30.24 mmol) and HOBt (4.09 g, 30.24 mmol) in a 100 mL beaker, add 100 mL of DMF and stir until completely dissolved. Cool to 2–5 °C, add N,N-diisopropylcarbodiimide (DIC, 3.82 g, 30.24 mmol), and stir for 10–15 min. Add the resulting 100 mL solution to a solid-phase synthesis reactor and react at 20 °C for 2 h. After the reaction is complete, filter and wash three times with 100 mL of DMF. After washing, add 100 mL of 20% Pip / DMF solution, react for 1 h, filter, discard the deprotection solution, wash six times with 100 mL of DMF, dry the resin, and proceed to the next step.

[0028] Take Fmoc-Gly-OH (8.99 g, 30.24 mmol) and HOBt (4.09 g, 30.24 mmol) in a 100 mL beaker, add 100 mL of DMF and stir until completely dissolved. Cool to 2–5 °C, add DIC (3.82 g, 30.24 mmol), and stir for 10–15 min. Add the resulting 100 mL solution to a solid-phase synthesis reactor and react at 20 °C for 2 h. After the reaction is complete, filter and wash three times with 100 mL of DMF. After washing, add 100 mL of 20% Pip / DMF solution, react for 1 h, filter, discard the deprotection solution, wash six times with 100 mL of DMF, dry the resin, and proceed to the next step.

[0029] Take Fmoc-Hyp(OtBu)-OH (12.38 g, 30.24 mmol) and HOBt (4.09 g, 30.24 mmol) in a 100 mL beaker, add 100 mL of DMF and stir until completely dissolved. Cool to 2–5 °C, add DIC (3.82 g, 30.24 mmol), and stir for 10–15 min. Add the resulting 100 mL solution to a solid-phase synthesis reactor and react at 20 °C for 2 h. After the reaction is complete, filter and wash three times with 100 mL of DMF. After washing, add 100 mL of 20% Pip / DMF solution, react for 1 h, filter, discard the deprotection solution, wash six times with 100 mL of DMF, dry the resin, and proceed to the next step.

[0030] Take Fmoc-Pro-OH (10.20 g, 30.24 mmol) and HOBt (4.09 g, 30.24 mmol) in a 100 mL beaker, add 100 mL of DMF and stir until completely dissolved. Cool to 2–5 °C, add DIC (3.82 g, 30.24 mmol), and stir for 10–15 min. Add the resulting 100 mL solution to a solid-phase synthesis reactor and react at 20 °C for 2 h. After the reaction is complete, filter and wash three times with 100 mL of DMF. After washing, add 100 mL of 20% Pip / DMF solution, react for 1 h, filter, discard the deprotection solution, wash six times with 100 mL of DMF, dry the resin, and proceed to the next step.

[0031] Take Fmoc-Gly-OH (8.99 g, 30.24 mmol) and HOBt (4.09 g, 30.24 mmol) in a 100 mL beaker, add 100 mL of DMF and stir until completely dissolved. Cool to 2–5 °C, add DIC (3.82 g, 30.24 mmol), and stir for 10–15 min. Add the resulting 100 mL solution to a solid-phase synthesis reactor and react at 20 °C for 2 h. After the reaction is complete, filter and wash three times with 100 mL of DMF. After washing, add 100 mL of 20% Pip / DMF solution, react for 1 h, filter, discard the deprotection solution, and wash 6 times with 100 mL of DMF, 3 times with 100 mL of MeOH, and 3 times with 100 mL of dichloromethane (DCM). Vacuum drying yielded H-Gly-Pro-Hyp(OtBu)-Gly-Pro-Hyp(OtBu)-CTC resin.

[0032] H-Gly-Pro-Hyp(OtBu)-Gly-Pro-Hyp(OtBu)-CTC resin was added to a 100 mL solution of trifluoroacetic acid (TFA) / triisopropylsilane (TIS) / water = 90 / 5 / 5 and reacted for 1 h. The mixture was filtered, the filtrate was collected, and the resin was discarded. The filtrate was then added to 1.0 L of methyl tert-butyl ether (MTBE) at 0–5 °C, resulting in the precipitation of a white solid. This solid was centrifuged to obtain a white crude peptide, which was then dried under vacuum to obtain crude peptide powder H-Gly-Pro-Hyp-Gly-Pro-Hyp-OH.

[0033] The crude peptide powder H-Gly-Pro-Hyp-Gly-Pro-Hyp-OH was purified by reverse-phase C18 preparative chromatography and then lyophilized to obtain the hexapeptide compound.

[0034] The hexapeptide compound was subjected to NMR analysis, and the results are shown in [reference needed]. Figure 1 , Figure 1 The NMR spectrum of the hexapeptide compound prepared in Example 1 of this application is shown below: 1 H NMR (400 MHz, MeOD), δ ppm: 7.51 (t, J = 6.0 Hz, 1H), 4.62-4.54 (m,2H), 4.48-4.42 (m, 2H), 4.41-4.36 (m, 1H), 4.33 (ddd, J = 7.1, 5.4, 0.9 Hz,1H), 3.89 (d, J = 6.0 Hz, 2H), 3.75 (td, J = 6.8, 2.4 Hz, 2H), 3.68-3.63 (m,1H), 3.59 (dt, J = 12.2, 4.5 Hz, 3H), 3.49 (dd, J = 6.8, 2.7 Hz, 1H), 3.48-3.39 (m, 3H), 3.32 (dd, J = 28.6, 5.5 Hz, 2H), 2.77 (dq, J = 33.3, 6.8 Hz, 2H), 2.29 (dt, J = 12.0, 5.9 Hz, 1H), 2.11-1.98 (m, 3H), 1.96-1.82 (m, 7H).

[0035] The hexapeptide compound is a linear collagen hexapeptide, and its amino acid sequence is shown in SEQ ID No. 1, specifically: H-Gly-Pro-Hyp-Gly-Pro-Hyp-OH, which has the structure of formula (I): Formula (I).

[0036] Experimental Example 1: Cytotoxicity Detection Human keratinocytes (HaCaT) were used at a rate of 2 × 10⁻⁶. 4 Cells were cultured at a density of 96 wells in 96-well cells and incubated at 37°C in a 5% CO2 incubator. After 48 h, the culture medium was discarded, and the cells were washed once with 1×PBS. Then, the cells were treated with bioactive substances, with different concentrations of the linear collagen hexapeptide prepared in Example 1 added. After 24 h of treatment, the culture medium was removed, and 100 μL of medium containing 10% CCK-8 solution was added. The cells were incubated for 1 h, and the absorbance at 450 nm was measured using a microplate reader. Simultaneously, a solvent without the sample was used as a control group. Cell viability was calculated using the following formula: Cell viability % = (experimental group absorbance / control absorbance) × 100%.

[0037] See results Figure 2 , Figure 2 The effect of hexapeptide on human keratinocytes, from Figure 2 It can be seen that the linear collagen hexapeptide in the experimental groups (1, 10, 100 and 1000 μg / mL) showed no cytotoxicity.

[0038] Experimental Example 2: Firming Effect Test reagent: Human type VII collagen (COL7) enzyme-linked immunosorbent assay kit (Catalog No.: E-EL-H0781, Elabscience, China).

[0039] Cells used for testing: fibroblasts (HFF-1), from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee (CAS Cell Bank).

[0040] Detection method: Fibroblasts (HFF-1) were prepared at a concentration of 5 × 10⁻⁶. 4 Cells at a density of 6 μL were cultured in 6-well cell culture plates and incubated at 37°C in a 5% CO2 incubator for 48 h. After incubation, the culture medium was aspirated, and the cells were washed once with 1×PBS. 500 μg / mL of the linear collagen hexapeptide prepared in Example 1 was added, bringing the volume to 2 mL per well. Normal control and experimental groups were established, with three biological replicates for each group. Cells were incubated for another 48 h. The cell supernatant was then aspirated and centrifuged at 1000 g at 4°C for 20 min. The supernatant was collected, and the precipitate was discarded. Subsequent procedures were performed according to the collagen ELISA kit instructions.

[0041] See results Figure 3 , Figure 3 The collagen expression level in the hexapeptide experimental group was determined by... Figure 3 It can be seen that the expression level of type VII collagen increased significantly after treatment with linear collagen hexapeptide, indicating that linear collagen hexapeptide can specifically promote the secretion of type VII collagen and has a firming effect.

[0042] Experimental Example 3: Soothing Effect Detection reagents: Mouse interleukin-6 (IL-6) enzyme-linked immunosorbent assay kit (Catalog No.: E-EL-M0044, Elabscience, China); Nitric oxide assay kit (Catalog No.: S0021S, beyotime, China).

[0043] Detection method: Mouse macrophages (RAW264.7) were used at a concentration of 1×10⁻⁶. 6Cells at a density of 6wel were cultured in 6-well plates and incubated at 37°C in a 5% CO2 incubator. After 24 h, the culture medium was discarded, and dexamethasone (DEX) and linear collagen hexapeptide prepared in Example 1 were added to a final concentration of 100 μg / mL for pre-incubation for 1.5 h. Lipopolysaccharides (LPS) were then added to a final concentration of 1 μg / mL. Normal control and experimental groups were set up, with three biological replicates for each group. Cells were incubated for another 24 h. The cell supernatant was then aspirated, centrifuged at 1000 g at 4°C for 20 min, and the supernatant was collected and the precipitate discarded. Subsequent procedures were performed according to the ELISA kit and nitric oxide kit instructions.

[0044] See results Figure 4 and Figure 5 , Figure 4 The expression level of the inflammatory factor NO in the hexapeptide experimental group. Figure 5 The expression level of the inflammatory cytokine IL-6 in the hexapeptide experimental group was determined by... Figure 4 and Figure 5 It can be seen that the expression levels of NO and IL-6 were significantly reduced after treatment with linear collagen hexapeptide, indicating that linear collagen hexapeptide can inhibit the secretion of inflammatory factors and has anti-inflammatory and soothing effects.

[0045] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Hexapeptide compound represented by formula (I): Formula (I).

2. Application of the hexapeptide compound represented by formula (I) in the preparation of cosmetics: Formula (I).

3. Use of the hexapeptide compound represented by formula (I) in the preparation of firming and / or repairing products: Formula (I).

4. Use of the hexapeptide compound represented by formula (I) in the preparation of products for promoting the secretion of type VII collagen: Formula (I).

5. Application of the hexapeptide compound represented by formula (I) in the preparation of soothing products: Formula (I).

6. Application of the hexapeptide compound represented by formula (I) in the preparation of anti-inflammatory products: Formula (I).

7. A cosmetic comprising the hexapeptide compound according to claim 1 and auxiliary materials.

8. A method for preparing a hexapeptide compound represented by formula (I), comprising: a) sequentially connecting hydroxyproline, proline, glycine, hydroxyproline, proline and glycine protected by protecting groups to a solid phase synthetic resin to obtain a resin containing a peptide chain protected by protecting groups; b) removing the protective group of the resin containing the peptide chain protected by the protective group and the resin to obtain the hexapeptide compound represented by formula (I): Formula (I).

9. The preparation method according to claim 8, characterized in that: The protecting group is tert-butoxycarbonyl.

10. The preparation method according to claim 9, characterized in that: Also includes: c) Purifying the hexapeptide compound represented by formula (I) by high performance liquid chromatography.

Images