Hexapeptide compound, preparation method and application thereof

Abstract

The present application provides a hexapeptide compound shown in formula (I), a preparation method thereof and its application. The amino acid sequence of the hexapeptide compound shown in formula (I) is shown in SEQ ID No. 1, specifically: H‑Gly‑Pro‑Hyp‑Gly‑Pro‑Hyp‑OH, which can significantly promote the secretion of type VII collagen, and thus exhibits good effects in firming, repairing and other aspects. At the same time, the hexapeptide compound shown in formula (I) can inhibit the secretion of inflammatory factors NO and IL‑6, and has anti-inflammatory and soothing effects. Based on this, the hexapeptide compound shown in formula (I) provided in the present application can be used in cosmetics to achieve firming, repairing, anti-inflammatory and soothing effects. Formula (I).

Description

Technical Field

[0001] The present invention relates to the technical field of daily chemical industry, and in particular to a hexapeptide compound, a preparation method thereof and application thereof. Background Art

[0002] Collagen is one of the most important structural proteins in the skin. It is mainly distributed in the dermis of the skin and accounts for 25% to 30% of the total skin protein. The mesh structure of collagen fibers provides elasticity and support for the skin and is an important biochemical reaction site for the skin. As we age, the collagen content in the skin will gradually decrease, causing the skin to lose elasticity and become loose.

[0003] Type VII collagen (Col7) is a special member of the collagen family. It is mainly found in the basement membrane area of ​​the skin and is the main component of anchor fibers. Anchor fibers are complexes that adhere to the basement membrane of the skin and the dermis below it, and they connect the surface layer of the skin with the dermis. Type VII collagen plays an important role in maintaining the integrity and structural stability of the skin. Type VII collagen produced by keratinocytes or fibroblasts alone is sufficient to form mechanically strong skin. The abundance and distribution of type VII collagen are crucial to the firming and repair functions of the skin. However, products that specifically promote the secretion of type VII collagen do not 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, a preparation method thereof and an application thereof. The hexapeptide compound provided in the present application can significantly promote the secretion of type VII collagen, inhibit inflammatory response, and has a firming and soothing effect, which can better improve the skin condition.

[0005] The present application provides a hexapeptide compound represented by formula (I):

[0006] Formula (I).

[0007] The amino acid sequence of the hexapeptide compound shown in formula (I) is shown in SEQ ID No. 1, specifically: H-Gly-Pro-Hyp-Gly-Pro-Hyp-OH, which can significantly promote the secretion of type VII collagen, and thus exhibits good effects in firming, repairing, etc. At the same time, the hexapeptide compound shown in formula (I) can inhibit the secretion of inflammatory factors NO and IL-6, and has anti-inflammatory and soothing effects. Based on this, the hexapeptide compound shown in formula (I) provided in the present application can be used in cosmetics to achieve firming, repairing, anti-inflammatory and soothing effects.

[0008] The present application also provides a method for preparing the hexapeptide compound represented by formula (I), comprising:

[0009] 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;

[0010] 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).

[0011] The present application adopts a solid phase synthesis method to sequentially connect hydroxyproline (Hyp(protecting group)), proline (Pro), glycine (Gly), hydroxyproline (Hyp(protecting group)), proline (Pro) and glycine (Gly) protected by a protecting group on a solid phase synthesis resin to obtain a resin containing a peptide chain protected by a protecting group.

[0012] The present invention uses a solid phase synthetic resin as a synthesis starting point, first reacts with a hydroxyproline raw material, and connects the hydroxyproline protected by a protective group to the resin. Specifically, the present invention mixes a solid phase synthetic resin, a hydroxyproline raw material, a reaction solvent, and a base for a first reaction, and then mixes with an alcohol solvent for a second reaction, and after deprotection, obtains a Hyp (protecting group)-resin.

[0013] In some specific implementations, the solid phase synthetic 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 synthetic 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.

[0014] After the first reaction is completed, an alcohol solvent is directly added thereto to carry out a second reaction. In some specific implementations, the time of the second reaction 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, etc., preferably methanol. After the second reaction is completed, the reaction product is filtered, and the obtained resin is washed with a reaction solvent such as DMF, preferably washed twice, and then deprotected. The present application has no special restrictions on the deprotection method, and 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 a 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°C to 30°C, preferably 15°C to 25°C, and the reaction time is preferably 0.5h to 2h, more preferably 1h to 1.5h. After the reaction is completed, the obtained product is washed with a reaction solvent such as DMF, preferably 6 times, and the resin is drained to obtain Hyp(OtBu)-resin.

[0015] After obtaining the Hyp(OtBu)-resin, proline (Pro) is connected thereto. In the present application, a proline raw material, a condensing agent and a solvent are first mixed and reacted, and then the obtained reaction solution is mixed with the Hyp(OtBu)-resin for reaction, and 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, the present application first mixes and dissolves Fmoc-Pro-OH and HOBt with DMF, and then mixes with DIC for reaction. In some specific implementations, the molar ratio of the Fmoc-Pro-OH, HOBt and DIC is preferably 20~40:20~40:20~40, and more preferably 25~35:25~35:25~35. In some specific implementations, the reaction is carried out under stirring, the temperature of the reaction is preferably 2°C~5°C, and the time is preferably 10min~15min. After the reaction is completed, the obtained reaction product is mixed with Hyp(OtBu)-resin for reaction, the temperature of the reaction is preferably 10°C~30°C, preferably 15°C~25°C, the time is preferably 1h~3h, and more preferably 1.5h~2.5h. After the reaction is completed, the reaction product is filtered, and the obtained resin is washed with a reaction solvent such as DMF, preferably washed three times, and then deprotected. The present application has no special restrictions on the deprotection method, and 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%~30%, more preferably 15%~25%. The reaction temperature is preferably 10°C to 30°C, preferably 15°C to 25°C, and the reaction time is preferably 0.5h to 2h, more preferably 1h to 1.5h. After the reaction is completed, the obtained product is washed with a reaction solvent such as DMF, preferably 6 times, and the resin is drained to obtain Pro-Hyp(OtBu)-resin.

[0016] After obtaining the Pro-Hyp(OtBu)-resin, glycine (Gly) is connected thereto. In the present application, a glycine (Gly) raw material, a condensing agent and a solvent are first mixed and reacted, and then the obtained reaction solution is mixed with the Pro-Hyp(OtBu)-resin for reaction, and after deprotection, a 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, the present application first mixes and dissolves Fmoc-GLy-OH and HOBt with DMF, and then mixes with DIC for reaction. In some specific implementations, the molar ratio of the Fmoc-Gly-OH, HOBt and DIC is preferably 20~40:20~40:20~40, and more preferably 25~35:25~35:25~35. In some specific implementations, the reaction is carried out under stirring, the temperature of the reaction is preferably 2°C to 5°C, and the time is preferably 10min to 15min. After the reaction is completed, the obtained reaction product is mixed with Pro-Hyp (OtBu)-resin for reaction, the temperature of the reaction is preferably 10°C to 30°C, preferably 15°C to 25°C, and the time is preferably 1h to 3h, more preferably 1.5h to 2.5h. After the reaction is completed, the reaction product is filtered, and the obtained resin is washed with a reaction solvent such as DMF, preferably washed three times, and then deprotected. The present application has no special restrictions on the deprotection method, and 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% to 30%, more preferably 15% to 25%. The reaction temperature is preferably 10°C to 30°C, preferably 15°C to 25°C, and the reaction time is preferably 0.5h to 2h, more preferably 1h to 1.5h. After the reaction is completed, the obtained product is washed with a reaction solvent such as DMF, preferably washed 6 times, and the resin is drained to obtain Gly-Pro-Hyp(OtBu)-resin.

[0017] Gly-Pro-Hyp(OtBu)-resin is obtained, and hydroxyproline (Hyp(protecting group)), proline (Pro) and glycine (Gly) protected by protecting groups are sequentially connected according to the same method as above to obtain H-Gly-Pro-Hyp(OtBu)- Gly-Pro-Hyp(OtBu)-CTC resin, which will not be described in detail in this application.

[0018] After obtaining H-Gly-Pro-Hyp(OtBu)- Gly-Pro-Hyp(OtBu)-CTC resin, it is deprotected. In the present application, trifluoroacetic acid is preferably used for deprotection. The specific steps are as follows:

[0019] H-Gly-Pro-Hyp(OtBu)-Gly-Pro-Hyp(OtBu)-CTC resin is mixed with a mixture of trifluoroacetic acid / triisopropylsilane / water, reacted, and filtered. The obtained filtrate is crystallized in a solvent to obtain a crude peptide.

[0020] 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°C, 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), ether, isopropyl ether, etc., preferably MTBE. The crystallized solid is centrifuged and vacuum dried to obtain a crude peptide.

[0021] After obtaining the crude peptide, it is preferably purified by high performance liquid chromatography (HPLC), and after freeze-drying, the hexapeptide represented by formula (I) can be obtained, and its 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 reverse phase C18 column.

[0022] The present application also provides a cosmetic comprising a hexapeptide compound represented by formula (I) and auxiliary materials.

[0023] In this application, according to the cosmetics classification principle, cosmetics can be divided into: cleaning cosmetics, care cosmetics and beauty / decorative cosmetics.

[0024] Among them, the cleaning cosmetics refer to cosmetics that are applied to the human body surface (such as epidermis, hair, nails, lips, etc.) by smearing, spraying or other similar methods to play a role in cleaning and hygiene or eliminating bad odors. The nursing cosmetics refer to cosmetics that are applied to the human body surface (such as epidermis, hair, nails, lips, etc.) by smearing, spraying or other similar methods to play a role in maintenance. The beauty / modification cosmetics refer to cosmetics that are applied to the human body surface (such as epidermis, hair, nails, lips, etc.) by smearing, spraying or other similar methods to play a role in beautification, modification, and increasing the charm of the human body.

[0025] Skin-applicable cleansing cosmetics include but are not limited to cleansers, makeup removers (lotions), cleansing creams (honeys), facial masks, floral water, prickly heat powder, talcum powder or bath gel; skin-care cosmetics include but are not limited to skin care creams, lotions or toners; skin-beautification / modification cosmetics include but are not limited to pressed powder, rouge, eye shadow, eyeliner (liquid), eyebrow pencil, perfume or cologne; hair-applicable cleansing cosmetics include but are not limited to shampoos, shampoo or shaving creams; hair-care cosmetics include but are not limited to conditioners, hair creams, hair oils / waxes, etc.; hair-beautification / modification cosmetics include but are not limited to powder, rouge, eye shadow, eyeliner (liquid), eyebrow pencils, perfume or cologne; hair-applicable cleansing cosmetics include but are not limited to shampoos, shampoo or shaving creams; hair-care cosmetics include but are not limited to conditioners, hair creams, hair oils / waxes, etc.; hair-beautification / modification cosmetics include but are not limited to Makeup / decoration cosmetics include but are not limited to styling mousse / hair spray, hair dye, perm, mascara (cream), hair growth agent or depilatory agent; cleaning cosmetics for nails include but are not limited to nail polish remover; care cosmetics for nails include but are not limited to nail water (cream), nail hardener; beauty / decoration cosmetics for nails include but are not limited to nail polish; cleaning cosmetics for lips include but are not limited to lip makeup remover; care cosmetics for lips include but are not limited to lip balm; beauty / decoration cosmetics for lips include but are not limited to lipstick, lip gloss or lip liner.

[0026] The amino acid sequence of the hexapeptide compound of formula (I) provided in the present application is shown in SEQ ID No. 1, specifically: H-Gly-Pro-Hyp-Gly-Pro-Hyp-OH, which can significantly promote the secretion of type VII collagen, and thus exhibits good effects in firming, repairing, etc. At the same time, the hexapeptide compound of formula (I) can inhibit the secretion of inflammatory factors NO and IL-6, and has anti-inflammatory and soothing effects. Based on this, the hexapeptide compound of formula (I) provided in the present application can be used in cosmetics to achieve firming, repairing, anti-inflammatory and soothing effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the NMR spectrum of the hexapeptide compound prepared in Example 1 of the present application;

[0028] Figure 2 This is the result of the effect of hexapeptide on human keratinocytes;

[0029] Figure 3 is the collagen expression level in the hexapeptide experimental group;

[0030] Figure 4 is the expression level of inflammatory factor NO in the hexapeptide experimental group;

[0031] Figure 5 is the expression level of inflammatory factor IL-6 in the hexapeptide experimental group. DETAILED DESCRIPTION

[0032] The present invention provides a hexapeptide compound, a preparation method thereof and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0033] The present invention will be further described below in conjunction with embodiments:

[0034] In the following embodiments, the abbreviations and their English meanings are as follows:

[0035]

[0036] Example 1

[0037] Place CTC resin (10.0 g, 10.08 mmol) in a solid phase synthesis reactor, add 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), react at 20°C for 3 h, add 10 ml of methanol (MeOH), react for 5 min, filter, and wash the resin twice with 100 ml of DMF. Then add 100 mL of 20% piperidine (Pip) / DMF solution to the solid phase synthesis reactor, react for 1 h, filter, discard the deprotection solution, wash 6 times with 100 ml of DMF, drain the resin, and proceed to the next step.

[0038] Take Fmoc-Pro-OH (10.20g, 30.24mmol) and HOBt (4.09g, 30.24mmol) in a 100mL beaker, add 100mL of DMF and stir until completely dissolved, then cool to 2~5℃, add N,N-diisopropylcarbodiimide (DIC, 3.82g, 30.24mmol), stir and react for 10~15min. Add the solution in the 100mL beaker to the solid phase synthesis reactor and react at 20℃ for 2h. After the reaction is completed, filter and wash three times with 100mL of DMF. After washing, add 100mL of 20% Pip / DMF solution, react for 1h, filter, discard the deprotection solution, wash 6 times with 100ml of DMF, drain the resin, and proceed to the next step.

[0039] Take Fmoc-Gly-OH (8.99g, 30.24mmol) and HOBt (4.09g, 30.24mmol) in a 100mL beaker, add 100mL of DMF and stir until completely dissolved, cool to 2~5℃, add DIC (3.82g, 30.24mmol), stir and react for 10~15min. Add the solution in the 100mL beaker to the solid phase synthesis reactor and react at 20℃ for 2h. After the reaction is completed, filter and wash three times with 100mL of DMF. After washing, add 100mL of 20% Pip / DMF solution, react for 1h, filter, discard the deprotection solution, wash 6 times with 100mL of DMF, drain the resin, and proceed to the next step.

[0040] Take Fmoc-Hyp(OtBu)-OH (12.38g, 30.24mmol) and HOBt (4.09g, 30.24mmol) in a 100mL beaker, add 100mL of DMF and stir until completely dissolved, cool to 2~5℃, add DIC (3.82g, 30.24mmol), stir and react for 10~15min. Add the solution in the 100mL beaker to the solid phase synthesis reactor and react at 20℃ for 2h. After the reaction is completed, filter and wash three times with 100mL of DMF. After washing, add 100mL of 20% Pip / DMF solution, react for 1h, filter, discard the deprotection solution, wash 6 times with 100mL of DMF, drain the resin, and proceed to the next step.

[0041] Take Fmoc-Pro-OH (10.20g, 30.24mmol) and HOBt (4.09g, 30.24mmol) in a 100mL beaker, add 100ml of DMF and stir until completely dissolved, cool to 2~5℃, add DIC (3.82g, 30.24mmol), stir and react for 10~15min. Add the solution in the 100mL beaker to the solid phase synthesis reactor and react at 20℃ for 2h. After the reaction is completed, filter and wash three times with 100mL of DMF. After washing, add 100mL of 20% Pip / DMF solution, react for 1h, filter, discard the deprotection solution, wash 6 times with 100mL of DMF, drain the resin, and proceed to the next step.

[0042] Take Fmoc-Gly-OH (8.99g, 30.24mmol) and HOBt (4.09g, 30.24mmol) in a 100mL beaker, add 100mL of DMF and stir until completely dissolved, cool to 2~5℃, add DIC (3.82g, 30.24mmol), stir and react for 10~15min. Add the solution in the 100mL beaker to the solid phase synthesis reactor and react at 20℃ for 2h. After the reaction is completed, filter and wash three times with 100mL of DMF. After washing, add 100mL of 20% Pip / DMF solution, react for 1h, filter, discard the deprotection solution, wash 6 times with 100mL of DMF, wash 3 times with 100mL of MeOH, and wash 3 times with 100mL of dichloromethane (DCM). The residue was dried in vacuo to obtain H-Gly-Pro-Hyp(OtBu)-Gly-Pro-Hyp(OtBu)-CTC resin.

[0043] At 0-5°C, H-Gly-Pro-Hyp(OtBu)-Gly-Pro-Hyp(OtBu)-CTC resin was added to a solution of trifluoroacetic acid (TFA) / triisopropylsilane (TIS) / water = 90 / 5 / 5 (100 mL) and reacted for 1 hour. The mixture was filtered, the filtrate was collected, and the resin was discarded. At 0-5°C, the filtrate was added to 1.0 L of methyl tert-butyl ether (MTBE), a white solid was precipitated, and the mixture was centrifuged to obtain a white solid crude peptide, which was dried under vacuum to obtain a crude peptide powder H-Gly-Pro-Hyp-Gly-Pro-Hyp-OH.

[0044] The crude peptide powder H-Gly-Pro-Hyp-Gly-Pro-Hyp-OH was purified by reverse phase C18 preparative chromatography and lyophilized to obtain the hexapeptide compound.

[0045] The hexapeptide compound was subjected to NMR analysis, and the results are shown in Figure 1 , Figure 1 This is the NMR spectrum of the hexapeptide compound prepared in Example 1 of the present application, and its NMR data are as follows:

[0046] 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).

[0047] 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 a structure of formula (I):

[0048] Formula (I).

[0049] Test Example 1 Cytotoxicity Detection

[0050] Human keratinocytes (HaCaT) were cultured at 2 × 10 4 The cells were cultured in a 96-well cell culture plate at a density of 1 / 96well and placed in a 37°C, 5% CO2 incubator. After 48 hours, the culture medium was aspirated and discarded, and the cells were washed once with 1×PBS. The cells were treated with bioactive substances and different concentrations of the linear collagen hexapeptide prepared in Example 1 were added. After 24 hours of treatment, the culture medium was removed, 100 μL of culture medium containing 10% cck-8 solution was added, and the cells were placed in an incubator for 1 hour. The absorbance at 450 nm was detected by an ELISA reader. At the same time, the solvent without the sample was used as the control group, and the cell survival rate was calculated according to the following formula:

[0051] Cell survival rate %= (absorbance of experimental group / absorbance of control group) × 100%.

[0052] Results Figure 2 , Figure 2 The results of the effect of hexapeptide on human keratinocytes are shown in Table 1. Figure 2It can be seen that the linear collagen hexapeptide in the experimental groups (1, 10, 100 and 1000 μg / mL) had no cytotoxicity.

[0053] Test Example 2 Firming Effect

[0054] Detection reagent: Human type VII collagen (COL7) enzyme-linked immunosorbent assay kit (Cat. No.: E-EL-H0781, Elabscience, China).

[0055] Test cells: fibroblasts (HFF-1), obtained from the Cell Bank of Type Culture Collection Committee of the Chinese Academy of Sciences (CAS Cell Bank).

[0056] Detection method: Fibroblasts (HFF-1) were cultured at 5×10 4 / 6wel was cultured in a 6-well cell culture plate and placed in a 37°C, 5% CO2 incubator. After 48 hours, the culture medium was discarded, washed once with 1×PBS, and 500 μg / mL of the linear collagen hexapeptide prepared in Example 1 was added. The volume of each well was 2 mL. A normal control group and an experimental group were set, with three biological replicates in each group, and the cells were placed in a cell culture incubator for further incubation for 48 hours. The cell supernatant was then aspirated, centrifuged at 1000 g, 4°C for 20 min, the supernatant was collected, and the precipitate was discarded. Subsequent operations were performed according to the instructions of the collagen ELISA kit.

[0057] Results Figure 3 , Figure 3 is the collagen expression in the hexapeptide experimental group, Figure 3 It can be seen that the expression 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.

[0058] Test Example 3 Soothing Effect

[0059] Detection reagents: mouse interleukin 6 (IL-6) enzyme-linked immunosorbent assay kit (catalog number: E-EL-M0044, Elabscience, China); nitric oxide detection kit (catalog number: S0021S, beyotime, China).

[0060] Detection method: Mouse macrophages (RAW264.7) were cultured at 1×10 6 / 6wel was cultured in a 6-well cell plate at a density of 100 μg / mL and placed in a 37°C, 5% CO2 incubator. After 24 hours, the culture medium was discarded, and dexamethasone (DEX) and 100 μg / mL of the linear collagen hexapeptide prepared in Example 1 were added for pre-incubation for 1.5 hours, followed by the addition of lipopolysaccharide (LPS) at a final concentration of 1 μg / mL. A normal control group and an experimental group were set up, with three biological replicates in each group. The cells were placed in a cell culture incubator and continued to be incubated for 24 hours. The cell supernatant was then aspirated, centrifuged at 1000 g, 4°C for 20 min, the supernatant was collected, and the precipitate was discarded. Subsequent operations were performed according to the instructions of the ELISA kit and the nitric oxide kit.

[0061] Results Figure 4 and Figure 5 , Figure 4 is the expression level of inflammatory factor NO in the hexapeptide experimental group, Figure 5 is the expression of inflammatory factor IL-6 in the hexapeptide experimental group, 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, which indicates that linear collagen hexapeptide can inhibit the secretion of inflammatory factors and has anti-inflammatory and soothing effects.

[0062] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

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

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

3. Application of the hexapeptide compound represented by formula (I) in the preparation of cosmetics for promoting the secretion of type VII collagen: Formula (I).

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

Images