Use of a cyclic peptide compound for the preparation of an anti-inflammatory product

Abstract

The application discloses application of a cyclic peptide compound in preparation of anti-inflammatory products and belongs to the technical field of polypeptide compounds, and particularly relates to a solid-phase synthesis method for preparing H-Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-CTC resin, i.e., a fully-protected peptide resin; the fully-protected peptide resin is subjected to cutting treatment and cyclization treatment to obtain a cyclic peptide compound, and the structure of the cyclic peptide compound is Cyclo(Phe-Lys-Tyr-Pro-Phe-); the cyclization treatment further comprises deprotection cutting, and the cutting solution in the deprotection cutting is formed by mixing TFA, Tis, EDT, PhOH and H2O. The application is a cyclic peptide compound with anti-inflammatory function, which has small cytotoxicity, good safety, can inhibit inflammatory factors and can inhibit gene expression of inflammatory mediators and application thereof.

Description

[0001] This application is a divisional application of Chinese invention patent application filed on January 15, 2026, with application number 202610049852.2 and invention title "A Cyclic Peptide Compound with Anti-inflammatory Function and Its Application". Technical Field

[0002] This invention belongs to the field of polypeptide compound technology, specifically relating to the application of a cyclic peptide compound in the preparation of anti-inflammatory products. Background Technology

[0003] In recent years, peptide compounds have been widely used in the pharmaceutical and cosmetic fields due to their bioactivity and targeting properties. However, traditional peptide compounds typically suffer from the following problems: existing peptide molecules usually target only a single target or a single biological pathway. Due to the limitations of their single function, the effects of traditional peptide compounds used alone as cosmetic raw materials are often not ideal, requiring combination with other active ingredients, which increases the complexity and cost of formulations. Summary of the Invention

[0004] The purpose of this invention is to provide a cyclic peptide compound with anti-inflammatory function that has low cytotoxicity, good safety, can inhibit inflammatory factors and the gene expression of inflammatory mediators, and its application.

[0005] The technical solution adopted by the present invention to achieve the above objectives is as follows: A fully protected peptide resin with the structure: H-Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-CTC resin.

[0006] This invention discloses a fully protected polypeptide with the structure: H-Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-OH.

[0007] This invention discloses a fully protected cyclic peptide with the structure: Cyclo(Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-).

[0008] This invention discloses a cyclic peptide compound with the structure: Cyclo(Phe-Lys-Tyr-Pro-Phe-).

[0009] This invention discloses a method for preparing a cyclic peptide compound, comprising: preparing H-Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-CTC resin, i.e., a fully protected peptide resin, by solid-phase synthesis. The fully protected peptide resin was cleaved and cyclized to obtain a cyclic peptide compound with the structure: Cyclo(Phe-Lys-Tyr-Pro-Phe-).

[0010] Preferably, in the preparation of the fully protected peptide resin, the first amino acid coupling resin is synthesized first, and Fmoc-Phe-OH is coupled to the CTC resin.

[0011] Preferably, the preparation of the fully protected peptide resin uses an activated amino acid reagent, which includes an amino acid reagent, HOBT and DIC. The amino acid reagent includes Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH or Fmoc-Phe-OH.

[0012] This invention discloses a method for preparing a fully protected peptide resin, comprising: the synthesis of a first amino acid coupling resin, the activation of an amino acid reagent, and the synthesis of the fully protected peptide resin.

[0013] Preferably, in the synthesis of the first amino acid coupling resin, Fmoc-Phe-OH is coupled to CTC resin. The coupling is performed using a solid-phase synthesis method. After swelling with DCM, the CTC resin is coupled with Fmoc-Phe-OH in a DIEA-DCM solution.

[0014] Preferably, in the synthesis of the first amino acid coupling resin, Fmoc-Phe-OH is coupled to the CTC resin in a DCM solution containing DIEA.

[0015] Preferably, in the synthesis of the first amino acid coupling resin, under a nitrogen atmosphere, CTC resin is added to DCM and swollen at 20-40℃ for 5-30 min. DCM is removed by filtration, Fmoc-Phe-OH is added, and then DIEA-DCM solution is added at 10-20℃. The reaction is carried out at 20-30℃ for 2-5 h. After the reaction is completed, methanol is added for end-capping for 10-60 min, followed by filtration and washing to obtain Fmoc-Phe-CTC resin, which is the first amino acid coupling resin.

[0016] More preferably, in the synthesis of the first amino acid coupling resin, the molar amount of Fmoc-Phe-OH used is 100-300% of the reaction sites on the CTC resin.

[0017] More preferably, in the synthesis of the first amino acid coupling resin, the DIEA-DCM solution is a mixture of DIEA and DCM, and the molar volume ratio of DIEA to DCM in the DIEA-DCM solution is 0.1-1 mol: 0.1-1 L. The amount of DIEA-DCM solution used is measured by the amount of DIEA used, and the molar amount of DIEA used is 200-300% of the molar amount of Fmoc-Phe-OH used.

[0018] More preferably, in the synthesis of the first amino acid coupling resin, methanol is used for end-capping, and only an appropriate amount is needed. Washing is performed using DMF.

[0019] Preferably, in the activation of the amino acid reagent, the amino acid reagent and HOBT are added to DMF, DIC is added at 0-10℃, and activation is carried out for 3-20 minutes to obtain the activated amino acid reagent.

[0020] More preferably, in the activation of the amino acid reagent, the molar volume ratio of the amino acid reagent to DMF is 0.1-0.5 mol: 0.1-0.5 L.

[0021] More preferably, in the activation of the amino acid reagent, the molar amount of HOBT used is 50-200% of the molar amount of the amino acid reagent used.

[0022] More preferably, in the activation of the amino acid reagent, the molar amount of DIC used is 50-200% of the molar amount of the amino acid reagent used. In the activation of the amino acid reagent, the amino acid reagent includes Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, or Fmoc-Phe-OH.

[0023] Preferably, in the synthesis of the fully protected peptide resin, the first amino acid coupling resin is mixed with the deprotection solution for deprotection treatment, the liquid is removed by filtration, an activating amino acid reagent is added, and the reaction is carried out under a nitrogen atmosphere for 10-60 min. After the reaction is completed, the mixture is filtered and washed. Then, the coupling with the activating amino acid reagent is repeated. After the coupling is completed, the mixture is washed and dried to finally obtain H-Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-CTC resin, which is the fully protected peptide resin.

[0024] More preferably, in the synthesis of the fully protected peptide resin, the coupling sequence of the activating amino acid reagent is: Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH.

[0025] More preferably, in the synthesis of fully protected peptide resin, during the deprotection treatment, the resin coupled with the amino acid reagent is first immersed in a 20% Pip / DMF solution, and then deprotected at 20-30°C for 10-60 min.

[0026] More preferably, in the synthesis of the fully protected peptide resin, each time the activating amino acid reagent is coupled repeatedly, it undergoes deprotection treatment. After coupling, each reagent is filtered and washed with DMF. The washing of the last amino acid reagent is carried out sequentially with methyl ether, tetrahydrofuran, and methyl ether.

[0027] More preferably, in the synthesis of fully protected peptide resin, the molar amount of amino acid reagent used in the activating amino acid reagent is 100-300% of the reaction sites on the CTC resin.

[0028] Preferably, a cutting fluid is used in the cutting process, which is a mixture of TFA and DCM; or, the cutting fluid is a mixture of HFIP and DCM.

[0029] This invention discloses a method for preparing a fully protected peptide, comprising: subjecting a fully protected peptide resin to a fully protected cleavage process to obtain a fully protected peptide.

[0030] Preferably, the fully protective cutting process uses a fully protective cutting fluid, which includes a DCM solution containing TFA or a DCM solution containing HFIP.

[0031] Preferably, in the preparation of the fully protected peptide, the fully protected peptide resin is mixed with the cleavage solution and treated at 20-40℃ for 10-60 min. The resin is removed by filtration, petroleum ether is added to the filtrate for sedimentation, the supernatant is removed by centrifugation, the peptide is washed with petroleum ether and centrifuged, and then vacuum dried to obtain the fully protected peptide H-Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-OH.

[0032] More preferably, in the preparation of the fully protected peptide, the cleavage fluid is a mixture of TFA and DCM, and the content of TFA in the cleavage fluid is 0.1-5 vol%.

[0033] More preferably, in the preparation of the fully protected peptide, the cutting solution is used to immerse the fully protected peptide resin, and petroleum ether is used for sedimentation and washing in appropriate amounts.

[0034] Preferably, in the preparation of the fully protected peptide, the fully protected peptide resin is mixed with the cleavage solution and treated at 20-40℃ for 10-60 min. The resin is removed by filtration, and the filtrate is concentrated and evaporated to dryness to obtain the fully protected peptide H-Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-OH.

[0035] More preferably, in the preparation of the fully protected peptide, the cleavage fluid is a mixture of HFIP and DCM, and the content of HFIP in the cleavage fluid is 20-40 vol%.

[0036] More preferably, in the preparation of the fully protected peptide, the cleavage fluid is immersed in the fully protected peptide resin.

[0037] Preferably, the cyclization treatment includes a fully protected cyclization treatment, in which HATU and DIEA are used for cyclization; or, in which HOOBT, NMM and DIC are used for cyclization.

[0038] Preferably, the cyclization treatment further includes deprotection cutting, wherein the cutting fluid in the deprotection cutting is a mixture of TFA, Tis, EDT, PhOH and H2O.

[0039] This invention discloses a method for preparing a fully protected cyclic peptide, comprising: subjecting a fully protected polypeptide to a fully protected cyclic peptide by a fully protected cyclization process to obtain a fully protected cyclic peptide.

[0040] Preferably, the fully protected cyclization treatment uses HATU-DIEA solution to perform fully protected cyclization of the peptide; or, the fully protected cyclization treatment uses DMF solution containing HOBT, DIC, and NMM to perform fully protected cyclization.

[0041] Preferably, in the preparation of the fully protected cyclic peptide, the fully protected peptide is mixed with DMF to obtain a fully protected peptide DMF solution; HATU, DIEA and DMF are mixed to obtain a HATU-DIEA solution, and the temperature of the HATU-DIEA solution is controlled at 20-30℃. Then, the fully protected peptide DMF solution is added dropwise to the HATU-DIEA solution, and the reaction is carried out for 20-120 min. The reaction is monitored by HPLC. After the reaction is completed, water and ethyl acetate are added for extraction. The aqueous phase is extracted again with ethyl acetate. The organic phases are combined and then washed successively with saturated sodium bicarbonate solution, water and saturated sodium chloride solution. The mixture is dried with anhydrous sodium sulfate, filtered, and rotary evaporated to obtain the fully protected cyclic peptide Cyclo(Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-).

[0042] More preferably, in the preparation of the fully protected cyclic peptide, the fully protected peptide and DMF in the fully protected peptide DMF solution are mixed at a mass-volume ratio of 1-10g:50-200mL.

[0043] More preferably, in the preparation of the fully protected cyclic peptide, HATU, DIEA and DMF in the HATU-DIEA solution are mixed in a mass-volume ratio of 5-20g: 5-20mL: 20-40mL.

[0044] More preferably, in the preparation of the fully protected cyclic peptide, the amount of the fully protected peptide DMF solution is based on the fully protected peptide, and the amount of HATU in the HATU-DIEA solution is based on HATU, with the amount of HATU being 100-200 wt% of the fully protected peptide.

[0045] Preferably, in the preparation of the fully protected cyclic peptide, the fully protected peptide is mixed with DMF to obtain a fully protected peptide DMF solution; then HOOBT is added, and NMM and DIC are added while controlling the reaction temperature at 0-10℃. The reaction is then stirred at 20-40℃ for 8-24 hours, and the reaction is monitored by HPLC. After the reaction is completed, water is added to precipitate the solid, which is dissolved in ethyl acetate. The solid is then washed successively with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and rotary evaporated to obtain the fully protected cyclic peptide Cyclo(Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-).

[0046] More preferably, in the preparation of the fully protected cyclic peptide, the fully protected peptide and DMF in the fully protected peptide DMF solution are mixed at a mass-volume ratio of 1-10g:1-10L.

[0047] More preferably, in the preparation of the fully protected cyclic peptide, the amount of HOOBT used is 10-30 wt% of the fully protected peptide.

[0048] More preferably, in the preparation of the fully protected cyclic peptide, the amount of NMM used is 5-20 wt% of the fully protected peptide.

[0049] More preferably, in the preparation of the fully protected cyclic peptide, the amount of DIC used is 5-20 wt% of the fully protected peptide.

[0050] More preferably, in the preparation of the fully protected cyclic peptide, an appropriate amount of water is used when precipitating the solid, an appropriate amount of ethyl acetate is used to dissolve the solid, and an appropriate amount of saturated sodium bicarbonate solution, water, and saturated sodium chloride solution are used in the washing process.

[0051] Preferably, in the preparation of the cyclic peptide compound, the fully protected cyclic peptide is mixed with the cleavage solution for 1-4 hours, concentrated, precipitated with ice-cold ether, washed, centrifuged, and evaporated to dryness to obtain the cyclic peptide compound Cyclo (Phe-Lys-Tyr-Pro-Phe-).

[0052] More preferably, in the preparation of the cyclic peptide compound, the cleavage solution is a mixture of TFA, Tis, EDT, PhOH and H2O, wherein TFA, Tis, EDT, PhOH and H2O are mixed in a volume ratio of 80-90:2-10:1-5:1-5:1-5.

[0053] More preferably, in the preparation of the cyclic peptide compound, the ratio of the amount of fully protected cyclic peptide to the amount of cleavage solution used is 1-10g: 20-100mL.

[0054] More preferably, in the preparation of cyclic peptide compounds, an appropriate amount of icy diethyl ether is used during precipitation.

[0055] This invention discloses the use of the above-mentioned cyclic peptide compounds in the preparation of anti-inflammatory products and / or cosmetics and / or pharmaceuticals.

[0056] This invention utilizes a solid-phase synthesis method to prepare H-Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-CTC resin, i.e., a fully protected peptide resin. The fully protected peptide resin is then cleaved and cyclized to obtain a cyclic peptide compound with the structure Cyclo(Phe-Lys-Tyr-Pro-Phe-). The cyclization process also includes deprotection cleavage, where the cleavage solution is a mixture of TFA, Tis, EDT, PhOH, and H2O. Therefore, this invention offers the following advantages: Cyclo(Phe-Lys-Tyr-Pro-Phe-) exhibits low cytotoxicity and good safety. Cyclo(Phe-Lys-Tyr-Pro-Phe-) can inhibit inflammatory factors and the gene expression of inflammatory mediators. Thus, this invention provides a cyclic peptide compound with low cytotoxicity, good safety, and the ability to inhibit inflammatory factors and the gene expression of inflammatory mediators, along with its applications. Attached Figure Description

[0057] Figure 1 This is an HPLC chromatogram.

[0058] Figure 2 This is a mass spectrum.

[0059] Figure 3 This is a graph showing the results of the cytotoxicity test.

[0060] Figure 4 This is a graph showing TNF-α levels under LPS-induced inflammatory conditions.

[0061] Figure 5 This is a graph showing IL-6 levels under LPS-induced inflammatory conditions.

[0062] Figure 6 This is a graph showing IL-1β levels under LPS-induced inflammatory conditions.

[0063] Figure 7 This is a graph showing the expression levels of COX-2 and iNOD genes under LPS-induced inflammatory conditions. Detailed Implementation

[0064] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0065] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0066] Example 1: A method for preparing a fully protected peptide resin Synthesis of the first amino acid coupling resin: Under a nitrogen atmosphere, CTC resin was added to DCM and swollen at 25°C for 10 min. DCM was removed by filtration. Fmoc-Phe-OH was added, followed by DIEA-DCM solution at 10°C. The reaction was carried out at 25°C for 2.5 h. After the reaction was complete, methanol was added for end-capping for 30 min. The mixture was then filtered and washed to obtain Fmoc-Phe-CTC resin, the first amino acid coupling resin. The molar amount of Fmoc-Phe-OH used was 200% of the reaction sites on the CTC resin. The DIEA-DCM solution was a mixture of DIEA and DCM, with a molar volume ratio of DIEA to DCM of 0.5 mol:0.5 L. The amount of DIEA-DCM solution used was measured based on the amount of DIEA, and the molar amount of DIEA used was 250% of the molar amount of Fmoc-Phe-OH used. Methanol end-capping was used in appropriate amounts. Washing was performed using DMF.

[0067] Activation of the amino acid reagent: The amino acid reagent and HOBT were added to DMF, and DIC was added at 2℃. Activation was carried out for 3 minutes to obtain the activated amino acid reagent. The molar volume ratio of the amino acid reagent to DMF was 0.24 mol: 0.25 L. The molar amount of HOBT used was 100% of the molar amount of the amino acid reagent. The molar amount of DIC used was 100% of the molar amount of the amino acid reagent. The amino acid reagent used in the activation process included Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Phe-OH.

[0068] Synthesis of fully protected peptide resin: The first amino acid coupling resin was mixed with a deprotection solution for deprotection treatment. The liquid was removed by filtration, and an activating amino acid reagent was added. The reaction was carried out under a nitrogen atmosphere for 40 min. After the reaction was completed, the mixture was filtered and washed. The coupling process with the activating amino acid reagent was then repeated. After coupling, the resin was washed and dried to obtain H-Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-CTC resin, i.e., the fully protected peptide resin. The coupling sequence of the activating amino acid reagent was: Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH. In the deprotection treatment, the resin coupled with the amino acid reagent was first immersed in a 20% Pip / DMF solution and deprotected at 25°C for 30 min. Each subsequent coupling with the activating amino acid reagent was followed by deprotection treatment, and after coupling, the resin was filtered and washed with DMF. The washing of the last amino acid reagent after coupling was performed sequentially with methyl ether, tetrahydrofuran, and methyl ether. The molar amount of the amino acid reagent used in the activating amino acid reagent was 240% of the reaction sites on the CTC resin.

[0069] Example 2: A method for preparing a fully protected polypeptide Preparation of the fully protected peptide: The fully protected peptide resin was mixed with the cleavage fluid and treated at 30°C for 30 min. The resin was removed by filtration, and petroleum ether was added to the filtrate for precipitation. The supernatant was removed by centrifugation, followed by washing with petroleum ether and centrifugation. The peptide was then vacuum dried to obtain the fully protected peptide H-Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-OH. The cleavage fluid was a mixture of TFA and DCM, with a TFA content of 1 vol%. The fully protected peptide resin was immersed in the cleavage fluid, and the amount of petroleum ether used for precipitation and washing was appropriate. The fully protected peptide resin was from Example 1. The yield of the fully protected peptide H-Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-OH prepared in this example was 80.4%, and the purity was 83.8%.

[0070] Example 3: A method for preparing a fully protected polypeptide Preparation of the fully protected peptide: The fully protected peptide resin was mixed with the cleavage fluid and treated at 30°C for 30 min. The resin was removed by filtration, and the filtrate was concentrated and evaporated to dryness to obtain the fully protected peptide H-Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-OH. The cleavage fluid was a mixture of HFIP and DCM, with HFIP content of 33 vol%. The cleavage fluid was used to immerse the fully protected peptide resin. The fully protected peptide resin was from Example 1. The yield of the fully protected peptide H-Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-OH prepared in this example was 72.5%, and the purity was 85.3%.

[0071] Example 4: A method for preparing a fully protected cyclic peptide Preparation of the fully protected cyclic peptide: The fully protected peptide was mixed with DMF to obtain a fully protected peptide DMF solution; HATU, DIEA, and DMF were mixed to obtain a HATU-DIEA solution, and the temperature of the HATU-DIEA solution was controlled at 25℃. Then, the fully protected peptide DMF solution was added dropwise to the HATU-DIEA solution, and the reaction was carried out for 60 min. The reaction was monitored by HPLC. After the reaction was completed, water and ethyl acetate were added for extraction. The aqueous phase was extracted again with ethyl acetate. The organic phases were combined and then washed successively with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution. The mixture was dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain the fully protected cyclic peptide Cyclo(Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-). The fully protected peptide DMF solution was mixed with DMF at a mass-to-volume ratio of 10 g: 100 mL. The HATU-DIEA solution was mixed with HATU, DIEA, and DMF at a mass-to-volume ratio of 11.8 g: 10.2 mL: 30 mL. The amount of the fully protected peptide DMF solution was based on the fully protected peptide itself, and the amount of HATU in the HATU-DIEA solution was based on HATU, with HATU used at 118 wt% of the fully protected peptide. The fully protected peptide was derived from Example 2. The fully protected cyclic peptide Cyclo (Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-) prepared in this example had a yield of 69.4% and a purity of 73.6%.

[0072] Example 5: A method for preparing a fully protected cyclic peptide Preparation of the fully protected cyclic peptide: The fully protected peptide was mixed with DMF to obtain a fully protected peptide DMF solution; then HOOBT was added, and NMM and DIC were added while controlling the reaction temperature at 0℃. The reaction was then stirred at 25℃ for 16 h, and the reaction was monitored by HPLC. After the reaction was completed, water was added to precipitate the solid, which was dissolved in ethyl acetate and then washed successively with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution. The solid was dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain the fully protected cyclic peptide Cyclo(Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-). The fully protected peptide and DMF in the fully protected peptide DMF solution were mixed at a mass-to-volume ratio of 10 g:10 L. The amount of HOOBT used was 17 wt% of the fully protected peptide. The amount of NMM used was 11 wt% of the fully protected peptide. The amount of DIC used was 13 wt% of the fully protected peptide. Appropriate amounts of water, ethyl acetate for dissolving the solid, and saturated sodium bicarbonate solution, water, and saturated sodium chloride solution used in the washing were all appropriate amounts. The fully protected peptide was obtained from Example 2. The fully protected cyclic peptide Cyclo(Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-) prepared in this example had a yield of 87.9% and a purity of 84.8%.

[0073] Example 6: A method for preparing a cyclic peptide compound Preparation of the cyclic peptide compound: The fully protected cyclic peptide was mixed with the cleavage buffer for 1-4 hours, concentrated, precipitated with ice-cold diethyl ether, washed, centrifuged, and evaporated to dryness to obtain the cyclic peptide compound Cyclo (Phe-Lys-Tyr-Pro-Phe-). The cleavage buffer was a mixture of TFA, Tis, EDT, PhOH, and H2O, with a volume ratio of 87.5:5:2.5:2.5:2.5. The ratio of the fully protected cyclic peptide to the cleavage buffer was 5 g:40 mL. An appropriate amount of ice-cold diethyl ether was used during precipitation. The fully protected cyclic peptide was from Example 5. The yield of the cyclic peptide compound Cyclo (Phe-Lys-Tyr-Pro-Phe-) prepared in this example was 78.5%, and the purity was 83.8%.

[0074] The cyclic peptide compound Cyclo (Phe-Lys-Tyr-Pro-Phe-) in Example 6 of this invention was further purified by HPLC using a reverse-phase C18 column to obtain purified cyclic peptide compound Cyclo (Phe-Lys-Tyr-Pro-Phe-) with a purity of 99.%, as shown in the HPLC chromatogram. Figure 1 As shown, the mass spectrum is as follows Figure 2As shown. In this invention, the cyclic peptide compound Cyclo (Phe-Lys-Tyr-Pro-Phe-) can also be named cyclic peptide PR2928. Cyclo, Cyclo (Phe-Lys-Tyr-Pro-Phe-), and cyclic peptide PR2928 all refer to the same product in this application.

[0075] The structure of Cyclo(Phe-Lys-Tyr-Pro-Phe-) is shown below: .

[0076] Experimental example: 1. Cytotoxicity testing of cyclic peptide compounds This invention tested the potential toxicity of the prepared cyclic peptide PR2928 to cells in order to determine the safe concentration range of cyclic peptide PR2928.

[0077] In this invention, HFF-1 fibroblasts were seeded into 96-well plates with 200 μL of culture medium per well, approximately 10,000 cells / well, ensuring a uniform cell density. The plates were cultured overnight at 37°C with 5% CO2 to allow for cell adhesion and growth. After 24 hours, the cell culture medium was aspirated, and 200 μL of DMEM medium containing different concentrations of the cyclic peptide PR2928 was added. The 96-well plates were then returned to the incubator for further culture. After 24 hours, the absorbance was measured at 490 nm using the MTT assay with a microplate reader. A control group (containing only culture medium and no cells) was also designed. Cell viability was calculated as (experimental group absorbance - control group absorbance) / (control group absorbance - control group absorbance) × 100% to determine the cytotoxicity of the cyclic peptide PR2928 to fibroblasts and its safe concentration range. The concentrations of the cyclic peptide PR2928 were 0 μM, 1 μM, 5 μM, 10 μM, 50 μM, 100 μM, and 500 μM.

[0078] In this invention, the macrophage cell line RAW264.7 was also used for testing. The testing steps were the same as those for the HFF-1 test, except that the HFF-1 was replaced with RAW264.7, to determine the toxicity of the cyclic peptide PR2928 to macrophages and its safe concentration range.

[0079] The cytotoxicity test results in this invention are as follows: Figure 3 As shown, HFF-1 and RAW264.7 cells treated with different concentrations of cyclic peptide PR2928 did not show a significant decrease in cell viability. At a concentration of 500 μM, the cell viability remained at 95% relative to the control group, confirming that cyclic peptide PR2928 has extremely low cytotoxicity.

[0080] 2. Detection of the effect of cyclic peptide PR2928 on the secretion of inflammatory factors In inflammatory responses, cytokines (such as TNF-α, IL-6, and IL-1β) are important regulatory molecules. Through complex network interactions, they jointly participate in the regulation of immune responses, tissue repair, and pathological damage. TNF-α is primarily responsible for activating immune cells, IL-6 promotes the persistence of the immune response, while IL-1β, produced by inflammasome activation, is closely related to tissue damage. Overexpression of these cytokines under external stimuli can lead to tissue damage and organ dysfunction. Macrophages generate inflammatory stress under lipopolysaccharide (LPS) stimulation. In this context, this invention tested the ability of the cyclic peptide PR2928 to inhibit the production of inflammatory factors in LPS-stimulated macrophages.

[0081] In this invention, RAW264.7 macrophage cell line was seeded at 50,000 cells / well in 6-well plates and cultured for 24 hours. Treatment groups, negative control groups, and blank control groups were established. The treatment groups received DMEM medium containing 10 μM cyclic peptide PR2928, while the negative control group received no cyclic peptide PR2928, and the blank control group received no cyclic peptide PR2928. Two hours after drug administration, 200 μL of LPS working solution was added to each well of all groups except the blank control group, and the cells were incubated for another 22 hours. The cell culture supernatant was collected, and TNF-α, IL-6, and IL-1β were detected and analyzed according to the ELISA kit instructions.

[0082] The t-test was used for statistical analysis to compare the groups. P < 0.05 was considered statistically significant (marked as * in the figure), and P < 0.01 was considered highly significant (marked as ** in the figure).

[0083] The inhibition rate is calculated using the formula: Inhibition rate % = 100% - (Negative control group - Treatment group) / (Control group - Blank control) × 100%, and then normalized.

[0084] The detection results of TNF-α are as follows Figure 4 As shown, compared with the blank control group, the TNF-α level in the LPS-stimulated negative control group was significantly increased, indicating that the stimulation conditions can increase the TNF-α level. The IL-6 detection results are as follows... Figure 5 As shown, compared with the blank control group, the IL-6 level in the LPS-stimulated negative control group was significantly increased, indicating that the stimulation conditions can increase the IL-6 level. The IL-1β detection results are as follows... Figure 6As shown, compared with the blank control group, the IL-1β level in the LPS-stimulated negative control group was significantly increased, indicating that the stimulation conditions can increase the IL-1β level. Compared with the negative control group, the inhibition rates of TNF-α, IL-6, and IL-1β after LPS stimulation and treatment with 10 μM cyclic peptide PR2495 were 58.2%, 37.9%, and 31.9%, respectively. This demonstrates that treatment with cyclic peptide PR2928 exhibits a significant ability to inhibit the production of inflammatory factors.

[0085] 3. Effects of cyclic peptide PR2928 on the expression of inflammatory mediator genes. COX-2 (cyclooxygenase-2) and iNOS (inducible nitric oxide synthase) are two extremely important and representative "enzymatic" inflammatory mediators. Both are inducible enzymes, meaning they are not expressed by normal cells in a resting state, but can be strongly induced by LPS. They are not directly cytokines, but are responsible for catalyzing the production of inflammatory factors. COX-2 catalyzes the synthesis of prostaglandins, which lead to increased local vascular permeability, resulting in tissue edema and a lowered pain threshold, causing hyperalgesia. The large amount of NO produced by iNOS catalysis combines with superoxide anions to form the more toxic peroxynitrite, leading to protein nitration, DNA damage, and cell death, thereby exacerbating tissue destruction.

[0086] This invention uses qPCR to test the mRNA expression levels of COX-2 and iNOS.

[0087] In this invention, RAW264.7 macrophage cell line was seeded at 50,000 cells / well in 6-well plates and cultured for 24 hours. Treatment and control groups were established, and the treatment groups were treated with DMEM medium containing the cyclic peptide PR2928 at a concentration of 10 μM. The control group was treated with DMEM without the peptide. Simultaneously with drug administration, 200 μL of LPS working solution was added to each well, and the cells were incubated for another 6 hours. Cells were washed twice with PBS, and RNA was extracted from each well using the TRIZOL method. GAPDH was used as an internal control for gene expression to assess the mRNA expression levels of COX-2 and iNOS. Calculation formula: ; ; .

[0088] The results were statistically analyzed using the t-test method. Compared with the control group, significance was indicated by *, where P value < 0.05 was * indicating a significant difference, and P value < 0.01 was ** indicating a highly significant difference.

[0089] The gene expression inhibition rate is calculated by normalizing the formula: Inhibition rate % = (Control group - Treatment group) / Control group × 100%.

[0090] Test results are as follows Figure 7 As shown, compared with the control group macrophages treated with LPS alone, the addition of cyclic peptide PR2928 did not significantly change the expression level of COX-2 gene, but significantly reduced the expression level of iNOS gene, with an inhibition rate of 57.84%.

[0091] In summary, the cyclic peptide PR2928 in this method exhibits low cytotoxicity and good safety. It effectively inhibits key pro-inflammatory factors such as TNF-α, IL-6, and IL-1β in LPS-induced macrophages, and also effectively inhibits the gene expression of inflammatory mediators such as iNOS in LPS-induced macrophages. This demonstrates that the cyclic peptide PR2928 of this invention not only significantly inhibits the expression of multiple pro-inflammatory factors but also inhibits the expression of the inflammatory mediator iNOS, exhibiting good anti-inflammatory capabilities.

[0092] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.

[0093] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.

Claims

1. The application of a cyclic peptide compound in the preparation of anti-inflammatory products, characterized in that, The structure of the cyclic peptide compound is Cyclo(Phe-Lys-Tyr-Pro-Phe-), and the anti-inflammatory product is used to inhibit inflammatory factors and / or inhibit the expression of inflammatory mediator genes and / or reduce inflammatory responses.

2. The application according to claim 1, characterized in that, The anti-inflammatory product is an anti-inflammatory cosmetic.

3. The application according to claim 2, characterized in that, The anti-inflammatory cosmetic is used to inhibit the expression of one or more pro-inflammatory factors among TNF-α, IL-6 and IL-1β in cells.

4. The application according to claim 2, characterized in that, The anti-inflammatory cosmetic is used to inhibit the expression of the iNOS gene in cells.

5. The application according to claim 1, characterized in that, The anti-inflammatory product is an anti-inflammatory drug.

6. The application according to claim 5, characterized in that, The anti-inflammatory drug is used to inhibit the production of one or more pro-inflammatory factors in macrophages, including TNF-α, IL-6, and IL-1β.

7. The application according to claim 5, characterized in that, The anti-inflammatory drug is used to inhibit the expression of the iNOS gene in macrophages.

8. The application according to claim 5, characterized in that, The anti-inflammatory drug is used to prevent or treat inflammatory diseases associated with the overexpression of at least one of the pro-inflammatory factors TNF-α, IL-6, and IL-1β, wherein the inflammatory disease is selected from at least one of skin inflammation, tissue damage, or organ dysfunction.

9. The application according to claim 5, characterized in that, The anti-inflammatory drug is used to prevent or treat inflammatory diseases associated with iNOS gene overexpression, wherein the inflammatory disease is selected from at least one of protein nitration damage, DNA damage, or cell death-related tissue destruction diseases.

10. The application according to any one of claims 1-9, characterized in that, The concentration of the cyclic peptide compound used in the anti-inflammatory product is 10-500 μM.

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