A group of anti-aging cyclic peptide compounds and their preparation method and application

Abstract

The present invention provides a group of anti-aging cyclic peptide structures, preparation methods and application methods. The sequences of the anti-aging cyclic peptides are c(DWYGFGDW), c(YGGFLKTTKS) and c(YGGFLAKYP). The three cyclic peptides have a zebrafish embryo ROS clearance rate of 32%, 28% and 30% at a concentration of 0.001%; ​​the promotion rates of zebrafish Elna gene expression are 54%, 53% and 60%; the promotion rates of zebrafish collagen col1a1a gene expression are 62%, 99% and 71%; the promotion rates of zebrafish collagen col1a1b gene expression are 36%, 80% and 108%; the promotion rates of zebrafish collagen col1a2 gene expression are 49%, 52% and 51%.

Description

Technical Field

[0001] The invention relates to the preparation and application of a group of cyclic peptides, belonging to the technical field of daily chemical products, and in particular to a cyclic peptide with anti-aging function, a preparation method thereof, and application in daily chemical products. Background Art

[0002] The skin is the largest organ in the human body and plays a role in protecting the internal organs of the body from external damage. Ultraviolet radiation, temperature, air pollution and other factors will accelerate the aging of the skin. As the core ingredient that has won the Nobel Prize many times, peptides have the characteristics of definite structure, clear mechanism of efficacy, strong efficacy, and low effective dosage (generally only a few to several hundred ppm are needed to have a significant effect). Beauty peptides with different functions such as anti-wrinkle, whitening, moisturizing, anti-oxidation, and repair are widely developed and applied.

[0003] However, most of the skin care products on the market that use peptides as active ingredients directly add peptides or peptide concentrates into conventional skin care product matrices. Active peptides will degrade over time in conventional matrix formulas, and after being applied to the skin, they are easily broken down by enzymes in the human body, thus affecting their efficacy. Summary of the invention

[0004] The purpose of the present invention is to provide a group of anti-skin aging cyclic peptide compounds and their preparation method and application. The prepared cyclic peptides have significant anti-aging effects and good stability and can be better applied in the field of daily chemicals.

[0005] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0006] In the first aspect, the present invention provides three anti-aging cyclic peptides with excellent performance, the anti-aging cyclic peptides comprising:

[0007] Anti-aging cyclic peptide-1:

[0008] The sequence of anti-aging cyclic peptide-1 is: c(DWYGFGDW);

[0009] The structural formula of anti-aging cyclic peptide-1 is as shown in Formula Ⅰ:

[0010]

[0011] Anti-aging cyclic peptide-2:

[0012] The sequence of anti-aging cyclic peptide-2 is: c(YGGFLKTTKS);

[0013] The structural formula of anti-aging cyclic peptide-2 is as shown in Formula II:

[0014]

[0015] Anti-aging cyclic peptide-3:

[0016] The sequence of anti-aging cyclic peptide-3 is: c(YGGFLAKYP);

[0017] The structural formula of anti-aging cyclic peptide-3 is as follows:

[0018]

[0019] In a second aspect, the present invention provides a method for preparing an anti-aging cyclic peptide, wherein the preparation steps include: Step 1, linear peptide synthesis:

[0020] 2-Chlorotriphenylmethyl chloride (2-CTC resin, 0.98mmol / g loading) was weighed in a reaction tube, and dichloromethane (DCM) was added to swell at room temperature for 1 hour. After the solvent was drained, the first amino acid monomer (1.5equiv) at the C-terminus and N,N-diisopropylethylamine (DIEA, 5equiv) in dichloromethane solution were added. After shaking at room temperature for 1 hour, the resin was washed 3 times with N,N-dimethylformamide (DMF) and DCM in turn, and then drained. Subsequently, Fmoc protection was removed, and 20% piperidine / DMF solution was added for deprotection twice, each time for 10 minutes, and then the resin was washed 3 times with DCM and DMF in turn. After drying, the second amino acid was coupled, and the activated second amino acid monomer DMF solution (including 5equiv of amino acid monomer, 4.75equiv of condensing agent HBTU and 5equiv of DIEA) was added. After shaking at room temperature for 1 hour, the resin was washed 3 times with DMF and DCM in turn, and then drained. Repeat the above steps of removing Fmoc protection, washing, coupling, and washing until the last amino acid monomer. Add the cleavage reagent hexafluoroisopropanol (HFIP) / DCM (20%) to the dried resin, shake and react at room temperature for 0.5 h, repeat once, collect the combined filtrate into a centrifuge tube, and remove the solvent by rotary evaporation to obtain a crude product.

[0021] Step 2: Cyclization:

[0022] The crude peptide was dissolved in DMF (0.005 M), and HATU (2 equiv) and DIEA (3-6 equiv) were added in sequence after complete dissolution, and the reaction was carried out at room temperature for 2 h. The cyclization process was monitored by LCMS. After the reaction was complete, the solvent was removed by vacuum distillation to obtain a crude cyclopeptide product, which was dissolved in acetonitrile, and the insoluble solid was removed by centrifugation. The filtrate was collected and dried to carry out the next step of deprotection.

[0023] Step 3: Deprotection:

[0024] Prepare a deprotection solution of TFA / triisopropylsilane (TIPS) / DCM = 50:5:45 or TFA / TIPS / 1,2-ethanedithiol (EDT) / DCM = 50:2.5:2.5:45, add it to the crude cyclic peptide product, shake at room temperature for 0.5-2h, and monitor the deprotection process by LCMS. After the deprotection is complete, the solvent is blown dry by an air pump, and the product is purified by a column machine.

[0025] The beneficial effects of the present invention are:

[0026] 1) The present invention designs and develops a group of anti-aging cyclic peptides with excellent performance, including:

[0027] Anti-aging cyclic peptide-1: c(DWYGFGDW);

[0028] Anti-aging cyclic peptide-2: c(YGGFLKTTKS);

[0029] Anti-aging cyclic peptide-3: c(YGGFLAKYP);

[0030] 2) The present invention provides a method for preparing a group of anti-aging cyclic peptides, wherein the purity of the prepared anti-aging cyclic peptides is greater than 95%;

[0031] 3) The anti-aging cyclic peptides designed and developed by the present invention have significant anti-aging effects. The three cyclic peptides have a scavenging rate of 32%, 28% and 30% for zebrafish embryo ROS at a concentration of 0.001%; ​​the promotion rate of zebrafish Elna gene expression is 54%, 53% and 60%; the promotion rate of zebrafish collagen col1a1a gene expression is 62%, 99% and 71%; the promotion rate of zebrafish collagen col1a1b gene expression is 36%, 80% and 108%; the promotion rate of zebrafish collagen col1a2 gene expression is 49%, 52% and 51%. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the LCMS spectrum of anti-aging cyclic peptide-1;

[0033] Figure 2 This is the LCMS spectrum of anti-aging cyclic peptide-2;

[0034] Figure 3 This is the LCMS spectrum of anti-aging cyclic peptide-3;

[0035] Figure 4 This is an example diagram of the zebrafish reactive oxygen species (ROS) clearance rate of anti-aging cyclic peptides-1 to 3;

[0036] Figure 5 This is a bar graph of zebrafish elastin gene expression of anti-aging cyclopeptide-1;

[0037] Figure 6This is a bar graph of zebrafish elastin gene expression of anti-aging cyclopeptide-2;

[0038] Figure 7 This is a bar graph of zebrafish elastin gene expression of anti-aging cyclopeptide-3;

[0039] Figure 8 This is a bar graph of the zebrafish collagen gene expression of anti-aging cyclic peptide-1;

[0040] Fig. 9 This is a bar graph of the zebrafish collagen gene expression of anti-aging cyclic peptide-2;

[0041] Fig.10 This is a bar graph of the zebrafish collagen gene expression levels of the anti-aging cyclic peptide-3. DETAILED DESCRIPTION

[0042] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below.

[0043] As used herein, the term "prepared from" is synonymous with "comprising." As used herein, the terms "comprising," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0044] When amount, concentration or other value or parameter is expressed as range, preferred range or a series of upper preferred value and lower preferred value limit range, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value and any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when disclosing range "1 to 5", described range should be interpreted as including range "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5" etc. When numerical range is described in this article, unless otherwise stated, the range is intended to include its end value and all integers and fractions within the range.

[0045] Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited to the specific embodiments disclosed below.

[0046] The present invention provides three anti-aging cyclic peptides with excellent performance, and the anti-aging peptides include:

[0047] Anti-aging cyclic peptide-1:

[0048] The sequence of anti-aging cyclic peptide-1 is: c(DWYGFGDW); the structural formula of anti-aging cyclic peptide-1 is as shown in Formula I:

[0049]

[0050] Anti-aging cyclic peptide-2:

[0051] The sequence of anti-aging cyclic peptide-2 is: c(YGGFLKTTKS); the structural formula of anti-aging cyclic peptide-2 is as shown in Formula II:

[0052]

[0053] Anti-aging cyclic peptide-3:

[0054] The sequence of anti-aging cyclic peptide-3 is: c(YGGFLAKYP); the structural formula of anti-aging cyclic peptide-3 is as shown in Formula III:

[0055]

[0056] Examples 1 to 3

[0057] The preparation steps of anti-aging cyclic peptides-1 to 3 include:

[0058] Step 1: Synthesis of linear peptides:

[0059] 2-Chlorotriphenylmethyl chloride (2-CTC resin, 0.98mmol / g loading) was weighed in a reaction tube, and dichloromethane (DCM) was added to swell at room temperature for 1 hour. After the solvent was drained, the first amino acid monomer (1.5equiv) at the C-terminus and N,N-diisopropylethylamine (DIEA, 5equiv) in dichloromethane solution were added. After shaking at room temperature for 1 hour, the resin was washed 3 times with N,N-dimethylformamide (DMF) and DCM in turn, and then drained. Subsequently, Fmoc protection was removed, and 20% piperidine / DMF solution was added for deprotection twice, each time for 10 minutes, and then the resin was washed 3 times with DCM and DMF in turn. After drying, the second amino acid was coupled, and the activated second amino acid monomer DMF solution (including 5equiv of amino acid monomer, 4.75equiv of condensing agent HBTU and 5equiv of DIEA) was added. After shaking at room temperature for 1 hour, the resin was washed 3 times with DMF and DCM in turn, and then drained. Repeat the above steps of removing Fmoc protection, washing, coupling and washing until the last amino acid monomer.

[0060] Add the cleavage reagent hexafluoroisopropanol (HFIP) / DCM (20%) to the dried resin, and shake the reaction at room temperature for 0.5 h. Repeat once, and collect the combined filtrate into a centrifuge tube. Remove the solvent by rotary evaporation to obtain a crude product.

[0061] Step 2: Cyclization:

[0062] The crude peptide was dissolved in DMF (0.005 M), and HATU (2 equiv) and DIEA (3-6 equiv) were added in sequence after complete dissolution, and the reaction was carried out at room temperature for 2 h. The cyclization process was monitored by LCMS. After the reaction was complete, the solvent was removed by vacuum distillation to obtain a crude cyclopeptide product, which was dissolved in acetonitrile, and the insoluble solid was removed by centrifugation. The filtrate was collected and dried to carry out the next step of deprotection.

[0063] Step 3: Deprotection:

[0064] Prepare a deprotection solution of TFA / triisopropylsilane (TIPS) / DCM = 50:5:45 or TFA / TIPS / 1,2-ethanedithiol (EDT) / DCM = 50:2.5:2.5:45, add it to the crude cyclic peptide product, shake at room temperature for 0.5-2h, and monitor the deprotection process by LCMS. After the deprotection is complete, the solvent is blown dry by an air pump, and the product is purified by a column machine.

[0065] The LCMS spectra of the prepared anti-aging cyclic peptides-1 to 3 are as follows: Figures 1 to 3 shown.

[0066] The following are the relevant contents of the efficacy test examples of the present invention.

[0067] Efficacy test example

[0068] 1. LCMS Identification Method of Anti-Aging Cyclic Peptides

[0069] LCMS test conditions: Shimadzu LCMS2050; chromatographic column Shimazu shim-pack GISTC18 (2 μm, 100 mm x 2.1 mm); flow rate 0.3 mL / min; mobile phase A (H2O); mobile phase B (acetonitrile, containing 0.01% HCOOH);

[0070] Test method: Wavelength 204nm. Elution gradient: 0-0.5min, maintain 5% B; 0.5-5min, the proportion of B increases to 95%; 5-8min, the proportion of B remains at 95%; then, decrease to 5% within 0.5min and maintain for 1.5min to equilibrate the column.

[0071] 2. Zebrafish embryo ROS clearance rate test method

[0072] The test method for the active oxygen scavenging rate of zebrafish embryos is tested using the group standard T / HPCIA 001-2023 "Cosmetic Anti-wrinkle and Firming Effect-Zebrafish Embryo Active Oxygen Species (ROS) Scavenging Test Method".

[0073] 3. Test method for the promotion rate of zebrafish collagen and elastin genes

[0074] The test method for the promotion rate of zebrafish collagen and elastin genes is tested using the group standard T / QLMZ 2-2022 "Evaluation Method for the Firming Efficacy of Cosmetics Based on the Zebrafish Model".

[0075] Test results:

[0076] Table 1 shows the test results of the 0.001% peptide zebrafish embryo reactive oxygen species (ROS) clearance rate and the zebrafish collagen and elastin gene promotion rate.

[0077] Table 1 Free radical scavenging rate of anti-aging cyclic peptides and promotion rate of collagen and elastin gene expression

[0078]

[0079] The clearance rate of reactive oxygen species (ROS) in zebrafish of anti-aging cyclic peptides-1-3 is as follows Figure 4 As shown, the ROS clearance rate of anti-aging cyclic peptide-1 was 32%, the ROS clearance rate of anti-aging cyclic peptide-2 was 28%, and the ROS clearance rate of anti-aging cyclic peptide-3 was 30%, which were significantly higher than the ROS clearance rate of glutathione in the positive control group of 15%.

[0080] Depend on Figures 5 to 7 The histogram of zebrafish elastin gene expression of anti-aging cyclic peptides-1 to 3 shows that the promotion rates of anti-aging cyclic peptides-1 to 3 on zebrafish elastin gene expression were 54%, 53% and 60% respectively.

[0081] Depend on Figures 8 to 10 The bar graph of zebrafish collagen gene expression of anti-aging cyclic peptides-1 to 3 shows that anti-aging cyclic peptides-1 to 3 have a significant promoting effect on the expression of zebrafish collagen gene.

[0082] 4. Population testing of anti-wrinkle essence

[0083] The anti-aging cyclic peptide of the present invention can be applied to various daily chemical products, including but not limited to essences, lotions, creams, essential oils, shampoos and the like.

[0084] In order to demonstrate the anti-aging effect of the cyclic peptide of the present invention in daily chemical products, the present invention uses an anti-wrinkle essence to test the product, and the formula of the anti-wrinkle essence is shown in Table 2 below:

[0085] Table 2 Anti-wrinkle essence formula of application examples 1-3 and application comparative example 1

[0086]

[0087] The preparation method of the anti-wrinkle essence of application examples 1-3 and application comparative examples 1-3 of the present invention comprises the following steps:

[0088] S1. Add the water and sclerotium gum of phase A into the emulsifier, homogenize until completely dispersed, start stirring, heat to 75-85°C, and keep warm for 20-30 minutes;

[0089] S2. Add p-hydroxyacetophenone and phenoxyethanol in phase B into the emulsifying pot and stir at 75°C for 5-10 minutes;

[0090] S3, cooling the material prepared in step S2 to below 40°C, adding the premixed phase C, i.e., the premix of the essence and PEG-40 hydrogenated castor oil, and stirring and dispersing for 5-10 minutes;

[0091] S4. Add the premixed phase D to the material prepared in step S3, and stir for 5-20 minutes until the material is evenly dispersed to obtain the anti-wrinkle essence.

[0092] Anti-wrinkle effect test:

[0093] Test subjects: aged 20 to 50 years old, healthy subjects (healthy men and women are defined as those without chronic diseases, major diseases or allergies, and not currently taking any medications). The 30 subjects were randomly divided into three groups, with 10 people in each group.

[0094] Testing period: 14 days.

[0095] Method of use: Application Examples 1-3 and Application Comparative Examples 1-3 were blindly tested and numbered DWR, KSR, YPR, DWL, KSL, and YPL respectively. The first group of subjects used DWR (Application Example 1) on the right face and DWL (Application Comparative Example 1) on the left face. The second group of subjects used KSR (Application Example 2) on the right face and KSL (Application Comparative Example 2) on the left face. The third group of subjects used YPR (Application Example 3) on the right face and YPL (Application Comparative Example 3) on the left face. The test area is the corner of the eye. No products (cosmetics or topical medicines) can be used on the test area 3 days before the test. Before the test, the subjects need to clean their faces uniformly and wipe them clean with a dandruff-free absorbent dry paper towel. The subjects used it once a day for 14 consecutive days, and the amount used each time was 0.2 ml.

[0096] Evaluation method: Use the American Visia skin tester to test the wrinkle improvement of the subjects before and after using the product on the 14th day, and calculate the average improvement of 10 people in each group. At the same time, on the 14th day, the subjects self-evaluated the wrinkle removal effect according to the following scoring scale, and calculate the average score of 10 people in each group.

[0097] Rating rating:

[0098] Very good: 5 points;

[0099] Good: 4 points;

[0100] Slightly better: 3 points;

[0101] Normal: 2 points;

[0102] Slightly worse: 1 point;

[0103] Bad: 0 points.

[0104] Table 3 shows the results of the use evaluation of application examples 1-3 and application comparative examples 1-3.

[0105]

[0106]

[0107] It can be seen from the application examples and application comparison examples in Table 3 that the wrinkle improvement effects of the application examples containing the cyclic peptide of the present invention are all greater than 20%, which is much better than the wrinkle improvement effect of the application comparison examples.

[0108] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Therefore, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. An anti-aging cyclic peptide compound, characterized in that: The anti-aging cyclic peptide compound is anti-aging cyclic peptide-1, the sequence is: c(DWYGFGDW); or anti-aging cyclic peptide-2, the sequence is: c(YGGFLKTTKS); or anti-aging cyclic peptide-3, the sequence is: c(YGGFLAKYP); Among them, the structural formula of anti-aging cyclic peptide-1 is as shown in Formula I: The structural formula of anti-aging cyclic peptide-2 is as shown in Formula II: The structural formula of anti-aging cyclic peptide-3 is as follows:

2. The method for preparing the anti-aging cyclic peptide compound according to claim 1, characterized in that: The preparation steps include: Step 1: Synthesis of linear peptides: 2-Chlorotriphenylmethyl chloride was weighed in a reaction tube, and DCM was added to swell at room temperature for 1 hour; after the solvent was drained, the DCM solution of the first amino acid monomer at the C-terminus and DIEA was added, and after shaking reaction at room temperature for 1 hour, the resin was washed with DMF and DCM three times in sequence, and then drained; Then, the Fmoc protection was removed by adding 20% ​​piperidine / DMF solution for 2 times, each time for 10 min, and then the resin was washed with DCM and DMF for 3 times; After drying, the second amino acid was coupled, and the activated second amino acid monomer DMF solution was added. After oscillating at room temperature for 1 hour, the resin was washed 3 times with DMF and DCM in sequence, and then dried. The above steps of removing Fmoc protection, washing, coupling, and washing were repeated until the last amino acid monomer; Add the cleavage reagent hexafluoroisopropanol (HFIP) / DCM (20%) to the dried resin, shake and react at room temperature for 0.5 h, repeat once, collect the combined filtrate into a centrifuge tube, and remove the solvent by rotary evaporation to obtain a crude product; Step 2: Cyclization: The crude peptide product was dissolved in DMF, and HATU and DIEA were added in sequence after complete dissolution, and the reaction was carried out at room temperature for 2 hours; LCMS monitored the cyclization process, and after the reaction was complete, the solvent was removed by vacuum distillation to obtain a crude cyclopeptide product, which was dissolved in acetonitrile, and the insoluble solid was removed by centrifugation, and the filtrate was collected and spin-dried to carry out the next step of deprotection; Step 3: Deprotection: Prepare a deprotection solution of TFA / TIPS / DCM or TFA / TIPS / EDT / DCM, add it to the crude cyclic peptide product, shake at room temperature for 0.5-2h, and monitor the deprotection progress by LCMS; after the deprotection is complete, blow dry the solvent with an air pump, and purify the product using a column machine.

3. The method for preparing the anti-aging cyclic peptide compound according to claim 2, characterized in that: In step 1, the 2-chlorotriphenylmethyl chloride is 0.98 mmol / g loading; In the DCM solution of the first amino acid monomer at the C-terminus and DIEA, the first amino acid monomer at the C-terminus is 1.5 equiv and DIEA is 5 equiv; The second amino acid monomer DMF solution includes 5 equiv of amino acid monomer, 4.75 equiv of condensing agent HBTU and 5 equiv of DIEA.

4. The method for preparing the anti-aging cyclic peptide compound according to claim 2, characterized in that: In step 2, the crude peptide product is dissolved in 0.005 M DMF; the HATU is 2 equiv, and the DIEA is 3-6 equiv.

5. The method for preparing the anti-aging cyclic peptide compound according to claim 2, characterized in that: In step three, The deprotection solution is TFA / TIPS / DCM=50:5:45, or TFA / TIPS / EDT / DCM=50:2.5:2.5:

45.

6. The method for preparing the anti-aging cyclic peptide compound according to claim 2, characterized in that: The purity of the prepared anti-aging cyclic peptide is greater than 95%.

7. Use of the anti-aging cyclic peptide compound according to any one of claims 1 to 6 in daily chemicals, characterized in that: The application forms include but are not limited to oil control essence, oil control cream, oil control mask, oil control scalp essence, oil control shampoo, and acne essence.

8. The use of the anti-aging cyclic peptide compound as claimed in claim 7 in daily chemicals, characterized in that: The application method in daily chemical products is to disperse the anti-aging cyclic peptide into glycerol, propylene glycol and butylene glycol solvents and then add the solvents into daily chemical products.

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