Preparation method of polypeptide with anti-inflammatory effect

Abstract

The invention discloses a preparation method of polypeptide with an anti-inflammatory effect. The preparation method comprises the following steps: S1, swelling resin; s2, removing Fmoc protecting groups of the resin; s3, washing the resin, and draining the cleaned resin to ensure that the resin reaches the cleanliness required by the next-step reaction; s4, amino acid coupling; s5, removing an Fmoc protecting group in the amino acid; s6, polypeptide cutting: preparing a cutting reagent, after the last amino acid is synthesized and the Fmoc group is removed, cleaning the resin, adding the cutting reagent, putting a synthesis tube into a shaking table, oscillating, cutting, collecting a cutting liquid into a centrifugal tube, and drying by blowing; s7, polypeptide purification; and S8, polypeptide freeze-drying: performing freeze-drying treatment on the purified polypeptide solution through a low-temperature freeze-drying machine to finally obtain solid powder of the target polypeptide. The anti-inflammatory effect can be improved, adverse reactions are reduced, toxic and side effects on the body in the anti-inflammatory process are reduced or eliminated, and the varieties of anti-inflammatory polypeptides are enriched.

Description

Technical Field

[0001] The present invention belongs to the technical field of compound preparation, and in particular relates to a method for preparing a polypeptide with anti-inflammatory effect. Background Art

[0002] Inflammation is a basic pathological response of living tissue to the stimulation of various damaging factors, which is mainly manifested as an active response of the vascular system. Inflammation can be divided into infectious inflammation and non-infectious inflammation. Infectious inflammation is usually caused by pathogens such as bacteria, viruses, and parasites, while non-infectious inflammation is caused by tissue necrosis, allergic reactions, foreign bodies in the body, or autoimmune diseases. When the body is damaged, infected, or otherwise stimulated, the immune system will initiate an inflammatory response to prevent further damage and promote recovery.

[0003] Although inflammation is usually used as a defense mechanism by the human body, excessive or persistent inflammatory responses can be harmful, especially to the skin. Many common skin problems, such as acne, are closely related to inflammation of the sebaceous glands of the hair follicles. Excessive oil secretion and clogged pores can cause redness, pain, pustules and nodules, which not only affect the appearance, but may also leave scars and increase the psychological burden of patients. In addition, chronic inflammation can accelerate skin aging as we age. Inflammatory reactions destroy collagen and elastic fibers in the skin, causing skin sagging, wrinkle proliferation, loss of luster and firmness, and making the skin dull and rough. Therefore, more and more people are looking for cosmetics to improve aging problems caused by inflammation.

[0004] Anti-inflammatory is the process of inhibiting or reducing inflammatory responses by regulating the body's physiological mechanisms or external intervention methods. Physiologically, macrophages release specific cytokines after clearing pathogens, inhibiting overactive immune cells, preventing excessive expansion of inflammation, and maintaining the body's internal environment balance. In terms of external intervention, key molecules in the inflammatory signaling pathway are often targeted, such as inhibiting cyclooxygenase activity and reducing the synthesis of inflammatory mediators such as prostaglandins, to relieve inflammatory symptoms and promote recovery.

[0005] At present, inhibiting the synthesis or activity of inflammatory mediators has become the mainstream way to treat inflammation. However, existing anti-inflammatory products still face many challenges in the cosmetics field. On the one hand, some anti-inflammatory ingredients, such as plant extracts, have poor stability and are easily affected by environmental factors such as light, temperature and oxygen, thereby reducing their activity and effect; on the other hand, the transdermal absorption efficiency of some anti-inflammatory ingredients is low, and it is difficult to effectively penetrate into the deep layer of the skin to exert its effect, which limits the actual efficacy of the product. In contrast, peptides have shown unique advantages in the anti-inflammatory field. Peptides have excellent biocompatibility, high affinity with skin cells, and can be more efficiently absorbed by the skin, thereby better exerting their anti-inflammatory effects. Therefore, the development of new anti-inflammatory peptides that are safe, efficient and have low side effects has important research and application value. Summary of the invention

[0006] The present invention aims to at least solve the technical problems existing in the above-mentioned prior art, and provides a method for preparing a polypeptide with anti-inflammatory effect, which can improve the anti-inflammatory effect, reduce adverse reactions, reduce or eliminate the toxic and side effects on the body during the anti-inflammatory process, and enrich the types of anti-inflammatory polypeptides.

[0007] The technical solution of the present invention is a method for preparing a polypeptide with anti-inflammatory effect, comprising the following steps:

[0008] S1, resin swelling;

[0009] S2, removal of resin Fmoc protecting group;

[0010] S3, resin washing, the washed resin needs to be drained to ensure that it reaches the cleanliness required for the next step of reaction;

[0011] S4, amino acid coupling, adding the prepared amino acid solution to the resin washed in step S3, and shaking the reaction to ensure that the amino acid and the resin are completely coupled;

[0012] S5, removal of Fmoc protecting groups in amino acids;

[0013] S6. Peptide cleavage: prepare the cleavage reagent. After the last amino acid is synthesized and its Fmoc group is removed, wash the resin and add the cleavage reagent. Place the synthesis tube in a shaker and perform at least one cleavage. Collect the cleavage solution in a centrifuge tube and blow dry.

[0014] S7, peptide purification;

[0015] S8. Polypeptide freeze-drying: The purified polypeptide solution is freeze-dried by a low-temperature freeze dryer to finally obtain a solid powder of the target polypeptide.

[0016] Preferably, the numerical swelling in step S1 is specifically as follows: accurately weigh an appropriate amount of resin, place it in a T-shaped polypeptide solid phase synthesis tube, add dried dichloromethane, and ensure that the resin is completely covered by dichloromethane; use a ziplock bag to tightly seal the lid of the synthesis tube and tighten it, then place the synthesis tube in a shaker and shake for 15 minutes to ensure that the resin is fully swollen.

[0017] Preferably, the removal of the Fmoc protecting group of the resin in step S2 is specifically as follows: the dichloromethane in the swollen resin is drained, and an appropriate amount of a pre-prepared 50% morpholine / DMF solution is added to remove the Fmoc protecting group; the synthesis tube is placed in a shaker and shaken for 10 minutes, then the deprotection solution is drained, and fresh deprotection solution is added and shaken for 20 minutes.

[0018] Preferably, the numerical washing in step S3 is specifically as follows: take out the polypeptide solid phase synthesis tube, drain the deprotection solution, wash twice with DMF, wash twice with methanol, wash twice with dichloromethane, and finally wash twice with DMF.

[0019] Preferably, the preparation of the amino acid solution in step S4 is as follows: according to the amino acid sequence of the target polypeptide, weigh the first amino acid, take 4 times the single amount as the standard, put it into a bottle, weigh 4 times the single amount of HBTU, HOBT and 8 times the single amount of diisopropylethylamine respectively, add an appropriate amount of dry DMF to dissolve, ultrasonically treat for 2 minutes, ensure complete dissolution and seal for later use; the shaking reaction time in step S4 is 2 hours.

[0020] Preferably, the removal of the Fmoc protecting group in the amino acid in step S5 is specifically as follows: after the first amino acid condensation is completed, the resin is washed according to the previous washing steps, and a 50% morpholine / DMF solution is added to remove the Fmoc protecting group on the amino acid. Then, the resin is washed and the second amino acid is added, and the cyclic synthesis is carried out according to the polypeptide sequence.

[0021] Preferably, the cleavage reagent prepared in step S6 is composed of 95% TFA, 2.5% TiS and 2.5% water; the synthesis tube is placed in a shaker for 1 hour, the number of cuts is two, and nitrogen is used to blow dry.

[0022] Preferably, the polypeptide purification in step S7 is as follows: when the sample is blown dry until only a small amount of solvent remains, ether is added and placed in a -20°C refrigerator for freezing precipitation for 15 minutes, followed by centrifugation, and the supernatant is discarded; a small amount of sample is taken, the molecular weight of the polypeptide is determined by mass spectrometry, and separation and purification is performed by reversed-phase high performance liquid chromatography, and the effluent is collected.

[0023] Preferably, in step S8, the purity of the polypeptide is analyzed using high performance liquid chromatography to ensure that the purity of the target polypeptide reaches the expected standard.

[0024] The beneficial effects of the present invention are: it can improve the anti-inflammatory effect, reduce adverse reactions, reduce or eliminate the toxic and side effects on the body during the anti-inflammatory process, and enrich the types of anti-inflammatory polypeptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The structural formula of the polypeptide with anti-inflammatory effect prepared by the present invention;

[0026] Figure 2 This is a high performance liquid phase diagram of the polypeptide with anti-inflammatory effect prepared by the present invention;

[0027] Figure 3 The hydrogen spectrum and carbon spectrum of the polypeptide structure prepared by the present invention (solvent DMSO-d6);

[0028] Figure 4 This is a graph showing the activity and cytotoxicity test results of different concentrations of the polypeptide on RAW264.7 cells in Example 2 of the present invention;

[0029] Figure 5 This is a graph showing the activity and cytotoxicity test results of different concentrations of polypeptides on HaCaT cells in Example 2 of the present invention;

[0030] Figure 6 This is a graph showing the effects of different concentrations of the polypeptide in Example 2 of the present invention on the levels of inflammatory factors in the RAW264.7 cell inflammation model. DETAILED DESCRIPTION

[0031] In order to make the invention purpose, technical solution and technical effect of the present invention clearer, the present invention is further described in detail below in conjunction with specific implementation methods. It should be understood that the specific implementation methods described in this specification are only for explaining the present invention, not for limiting the present invention.

[0032] In the following examples, sweet orange essential oil was purchased from Shanghai Xingye Biotechnology Co., Ltd., and other experimental materials and reagents were conventional consumables and reagents available from commercial channels.

[0033] Example 1

[0034] The invention is applied in the synthesis process and in the field of daily chemicals, and the method for synthesizing 0.1 mmol of a tetrapeptide of an SLVH amino acid sequence comprises the following steps:

[0035] a) Resin swelling: 143 mg of Fmoc-His(Trt)-Wang resin with a substitution value of 0.7 mmol / g was accurately weighed and placed in a T-type peptide solid phase synthesis tube, and 5 mL of dry DMF was added to ensure that the resin was completely immersed. The synthesis tube was sealed with a ziplock bag and the lid was tightened, placed in a shaker (speed of 280 rpm), and shaken for 15 minutes to ensure that the resin was fully swollen.

[0036] b) Removal of Fmoc protecting group from resin: After draining the dichloromethane from the fully swollen resin, add 5 mL of pre-prepared 50% morpholine / DMF deprotection solution, put it into a shaker, set the speed to 220 rpm and shake for 10 minutes. After the shaking, drain the deprotection solution, add 5 mL of deprotection solution again, and continue shaking for 20 minutes to ensure that the Fmoc protecting group is completely removed.

[0037] c) Resin washing: Take out from the shaker, drain the deprotection solution, wash twice with DMF, twice with methanol, twice with dichloromethane, and finally wash twice with DMF. The washed resin needs to be drained to ensure that it reaches the cleanliness required for the next step of the reaction.

[0038] d) Amino acid coupling: According to the amino acid sequence of the target peptide, weigh 0.4mmol of Fmoc-Val-OH (135mg) and put it into a bottle. Weigh 0.4mmol of HBTU (151mg), HOBT (54mg) and 0.8mmol of DIEA (103mg) respectively, add 5mL DMF to dissolve, ultrasonicate for 2 minutes, and seal the solution after ensuring that it is completely dissolved. After washing the resin according to the above washing steps, add the prepared amino acid solution and shake for 2 hours to ensure that the amino acid is completely coupled with the resin.

[0039] e) Removal of Fmoc protecting groups in amino acids: After the condensation reaction of the first amino acid is completed, the resin is washed according to the aforementioned washing steps, 5 mL of 50% morpholine / DMF deprotection solution is added, and the mixture is shaken on a shaker for 10 minutes. After the shaking is completed, the deprotection solution is drained, and 5 mL of deprotection solution is added again, and the mixture is shaken for 20 minutes. After washing the resin, amino acids Fmoc-Leu-OH (141 mg) and Fmoc-Ser-OH (141 mg) are added in sequence according to the peptide sequence, and the amounts of HOBT, HBTU and DIEA are the same as before. Finally, Ser-Leu-Val-His-Wang resin is obtained.

[0040] f) Peptide cleavage: Prepare the cleavage reagent in advance, including 95% TFA, 2.5% Tis and 2.5% water. After completing the last amino acid synthesis and removing the Fmoc group, wash the resin and add 8 mL of cleavage reagent, put it in a shaker for 1 hour, perform two cleavages, collect the two cleavage solutions in a centrifuge tube, and blow dry with nitrogen.

[0041] g) Peptide purification: When only 1 mL of solvent is left after drying, add 40 mL of ether and place in a -20°C refrigerator to freeze the precipitate for 15 minutes. Then centrifuge, pour off the supernatant, and take a small amount of sample for mass spectrometry analysis to determine the molecular weight of the polypeptide. Separate and purify by reversed-phase HPLC, and collect the effluent. Reverse-phase HPLC purification conditions: mobile phase A: 0.1% TFA / water; mobile phase B: acetonitrile; gradient elution, collect the effluent of the main absorption peak.

[0042] h) Peptide freeze drying: The purified peptide solution is freeze-dried using a low-temperature freeze dryer to obtain a peptide solid powder, i.e., the target peptide chain. At the same time, the purity of the peptide is analyzed by high-performance liquid chromatography to ensure that its purity meets the requirements.

[0043] Example 2

[0044] 1. Material characterization test results of the polypeptide with anti-inflammatory effect prepared by the present invention (such as Figure 1-3 shown),

[0045] The polypeptide synthesized by the present invention exhibits excellent anti-inflammatory effects. In the quality control link, the present invention uses high-performance liquid chromatography analysis and nuclear magnetic resonance technology to conduct in-depth analysis: the results of high-performance liquid chromatography showed that the purity of the polypeptide was as high as 99.04%, and the impurity content was negligible; at the same time, the molecular structure was finely analyzed with the help of nuclear magnetic resonance spectra, and the polypeptide structure was accurately verified to be correct. 1 H NMR(500MHz, DMSO-d6)δ9.03(s,1H),7.96(d,J=9.2Hz,1H),7.87–7.77(m,2H),7.52(dd,J=5.9,1.7Hz,1H),7.13–7.07(m,2H),6.79(ddt,J= 4.9,1.7,0.8Hz,1H),4.54(dt,J=10.1,6.4Hz,1H),4.36(dt,J=7.8,6.4Hz,1H),4.11(dddt,J=9.1,6.0,2.9,1.5Hz,1H),3.89–3.81(m,1H), 3.66–3.57(m,3H),3.14(ddd,J=14.7,6.4,0.7Hz,1H),2.93(ddd,J=14.7,6.4,0.7Hz,1H),2.67(dd,J=5.9,4.8Hz,1H),2.23–2.10(m,J=6.5 Hz,1H),1.76–1.68(m,1H),1.60(dtt,J=13.1,7.9,6.5Hz,1H),1.47(ddd,J=14.4,8.0,6.4Hz,1H),0.97–0.88(m,8H),0.84(d,J=6.5Hz,3H). 13C NMR(150MHz,DMSO-d6)δ172.60,172.31,171.88,171.40,169.91,168.49,159.65,159 .30,158.95,158.60,134.30,134.22,129.94,129.75,120.78,117.88,117.43,117.34 ,114.97,112.06,59.24,58.06,51.96,51.59,51.43,49.10,46.14,31.02,30.04,27. 20,26.76,26.54,24.59,23.79,23.45,22.72,21.78,19.44,18.44.ESI-MS:calcd.for C20H34N6O6(M+H + )454.53found 454.53.

[0046] 2. Efficacy Test Results

[0047] Test subjects: The synthetic tetrapeptide (SLVH) was evaluated and verified for efficacy.

[0048] Test method:

[0049] 1. RAW264.7 cytotoxicity assay

[0050] 1.1 Preparation of test samples: Weigh 40 mg of the test sample accurately, add 1 mL of dimethyl sulfoxide (DMSO) to dissolve, and prepare a sample solution with a concentration of 40 mg / mL. During the experiment, use high-glucose DMEM medium containing 10% fetal bovine serum for appropriate dilution.

[0051] 1.2 Cell culture: RAW264.7 cell line was cultured in high-glucose DMEM medium containing 10% fetal bovine serum in a 37°C, 5% CO2 incubator. After the cell growth density reached 80%, it was used for subsequent experiments.

[0052] 1.3 Cytotoxicity assay: The test samples were diluted to concentrations of 6.25, 12.5, 25, 50, 100 and 200 μg / mL. RAW264.7 cells in the logarithmic growth phase were selected and the cell concentration was adjusted to 1×10 5 / mL, inoculated in a 96-well plate, 100μL per well. Cultured in a 37℃, 5% CO2 incubator for 24 hours. The experiment set up a control group, a drug group and a blank group, with 6 replicates in each group. Discard the culture medium in the 96-well plate, add 100μL of the sample solution to the drug group, and add 100μL of high-glucose DMEM culture medium containing 10% fetal bovine serum to the control group and the blank group, and continue to culture for 24 hours. Subsequently, discard the culture medium, add 100μL of culture medium containing 10% CCK-8 reagent to each well, and continue incubation for 1-2 hours. Finally, use an enzyme reader to measure the absorbance (A) value at 450nm, and calculate the cell activity. Cell activity = (A drug group-A blank group) / (A control group-A blank group)×100%. Six parallel replicates were set for each sample, and the results are as follows Figure 4 shown.

[0053] 1.4 From Figure 4 It can be seen that within the experimental range, the novel tetrapeptide did not show obvious cytotoxicity to RAW264.7 cells.

[0054] 2. HaCaT cytotoxicity test

[0055] 2.1 Preparation of test samples: 40 mg of the test sample was accurately weighed and dissolved in 1 mL of dimethyl sulfoxide (DMSO) to prepare a sample solution with a concentration of 40 mg / mL. During the experiment, high-glucose DMEM medium containing 10% fetal bovine serum was used for appropriate dilution.

[0056] 2.2 Cell culture: HaCaT cell lines were cultured in high-glucose DMEM medium containing 10% fetal bovine serum in a 37°C, 5% CO2 incubator. After the cell growth density reached 80%, they were used in subsequent experiments.

[0057] 2.3 Cytotoxicity assay: The test samples were diluted to concentrations of 6.25, 12.5, 25, 50, 100 and 200 μg / mL. HaCaT cells in the logarithmic growth phase were selected and the cell concentration was adjusted to 1×10 5 / mL, inoculated in a 96-well plate, 100μL per well. Cultured in a 37℃, 5% CO2 incubator for 24 hours. The experiment set up a control group, a drug group and a blank group, with 6 replicates in each group. Discard the culture medium in the 96-well plate, add 100μL of the sample solution to the drug group, and add 100μL of high-glucose DMEM culture medium containing 10% fetal bovine serum to the control group and the blank group, and continue to culture for 24 hours. Subsequently, discard the culture medium, add 100μL of culture medium containing 10% CCK-8 reagent to each well, and continue incubation for 1-2 hours. Finally, use an enzyme reader to measure the absorbance (A) value at 450nm, and calculate the cell activity. Cell activity = (A drug group-A blank group) / (A control group-A blank group)×100%. Six parallel replicates were set for each sample, and the results are as follows Figure 5 shown.

[0058] 2.4 From Figure 5 It can be seen that within the experimental range, the novel tetrapeptide did not show obvious cytotoxicity to HaCaT cells.

[0059] 3. Anti-inflammatory activity detection experiment

[0060] In order to evaluate the anti-inflammatory activity of the compounds, this experiment established a model for the inhibition of LPS-induced nitric oxide (NO) production in RAW264.7 cells. RAW264.7 cells in the logarithmic growth phase were taken and the cell concentration was adjusted to 4×10 5 / mL, inoculated in a 96-well plate, 100μL of cell suspension was added to each well, and cultured in an incubator at 37℃ and 5% CO2 for 24 hours. The experiment set up a normal group, a model group and a drug-treated group. The normal group only added cell culture medium, the model group added 100ng / mL of LPS, and the drug-treated group added different concentrations of sample solutions (25μg / mL, 50μg / mL, 100μg / mL) on the basis of 100ng / mL of LPS, and each group set up 6 replicates. After discarding the culture medium, 100μL of the above solution was added to each well and cultured for 24 hours. 50μL of cell supernatant was collected, mixed with 50μL of Griess I reagent, and then 50μL of Griess II reagent was added, and it was kept away from light for 10 minutes. The absorbance (A) value was measured at a wavelength of 540nm using an enzyme marker. The NO content in the cell culture medium was further detected according to the method in the NO kit manual.

[0061] from Figure 6 It can be seen that the new tetrapeptide can effectively inhibit the LPS-induced inflammatory response of RAW 264.7 cells at concentrations of 100 μg / mL, 50 μg / mL and 25 μg / mL, showing certain anti-inflammatory activity.

[0062] 4. In vitro antibacterial test

[0063] Minimum inhibitory concentration (MIC) determination: The minimum inhibitory concentration (MIC) of the sample against the experimental strain was determined by the micro-dilution method to evaluate its antibacterial activity. The selected experimental strains include: Escherichia coli ATCC8739, Pseudomonas aeruginosa ATCC27853 and Staphylococcus aureus ATCC6538. The specific experimental steps are as follows: First, the bacteria were inoculated in LB medium and cultured to the logarithmic growth phase (OD 620 =0.4-0.6), and then diluted to OD 620 =0.02. The sample was dissolved in DMSO and diluted twice to prepare a series of sample solutions with different concentrations. Then, 10 μL of the diluted sample was mixed with 90 μL of bacterial suspension, added to a 96-well plate, and incubated in a shaker at 37°C for 24 hours. The minimum inhibitory concentration (MIC) of the sample was calculated by measuring the turbidity of the microplate reader at a wavelength of 620 nm. In the experiment, DMSO was used as a negative control. Three parallel experiments were set for each sample, and the results are shown in Table 1.

[0064] Table 1 Minimum inhibitory concentration (μM) of the present invention against standard strains

[0065]

[0066] The results in Table 1 show that the polypeptides of the present invention exhibit strong antibacterial activity against common experimental strains.

Claims

1. A method for preparing a polypeptide having an anti-inflammatory effect, characterized in that: Here are the steps: S1, resin swelling; S2, removal of resin Fmoc protecting group; S3, resin washing, the washed resin needs to be drained to ensure that it reaches the cleanliness required for the next step reaction; S4, amino acid coupling, adding the prepared amino acid solution to the resin washed in step S3, and shaking the reaction to ensure that the amino acid and the resin are completely coupled; S5, removal of Fmoc protecting groups in amino acids; S6. Peptide cleavage: prepare the cleavage reagent. After the last amino acid is synthesized and its Fmoc group is removed, wash the resin and add the cleavage reagent. Place the synthesis tube in a shaker and perform at least one cleavage. Collect the cleavage solution in a centrifuge tube and blow dry. S7, peptide purification; S8. Polypeptide freeze-drying: The purified polypeptide solution is freeze-dried by a low-temperature freeze dryer to finally obtain a solid powder of the target polypeptide.

2. The method for preparing a polypeptide with anti-inflammatory effect according to claim 1, characterized in that: The numerical swelling in step S1 is specifically as follows: accurately weigh an appropriate amount of resin, place it in a T-shaped polypeptide solid phase synthesis tube, add dried dichloromethane, and ensure that the resin is completely covered by dichloromethane; use a ziplock bag to tightly seal the lid of the synthesis tube and tighten it, then put the synthesis tube into a shaker and shake it for 15 minutes to ensure that the resin is fully swollen.

3. The method for preparing a polypeptide with anti-inflammatory effect according to claim 1, characterized in that: The removal of the Fmoc protecting group of the resin in step S2 is specifically as follows: the dichloromethane in the swollen resin is drained, and an appropriate amount of a pre-prepared 50% morpholine / DMF solution is added to remove the Fmoc protecting group; the synthesis tube is placed in a shaker and shaken for 10 minutes, then the deprotection solution is drained, and fresh deprotection solution is added and shaken for 20 minutes.

4. The method for preparing a polypeptide with anti-inflammatory effect according to claim 1, characterized in that: The numerical washing in step S3 is specifically as follows: take out the polypeptide solid phase synthesis tube, drain the deprotection solution, wash twice with DMF, wash twice with methanol, wash twice with dichloromethane, and finally wash twice with DMF.

5. The method for preparing a polypeptide with anti-inflammatory effect according to claim 1, characterized in that: The preparation of the amino acid solution in step S4 is as follows: according to the amino acid sequence of the target polypeptide, the first amino acid is weighed, and 4 times the single amount is used as the standard, and it is put into a bottle, and 4 times the single amount of HBTU, HOBT and 8 times the single amount of diisopropylethylamine are weighed respectively, and an appropriate amount of dry DMF is added to dissolve, and ultrasonic treatment is performed for 2 minutes. After ensuring complete dissolution, it is sealed for use; the shaking reaction time in step S4 is 2 hours.

6. The method for preparing a polypeptide with anti-inflammatory effect according to claim 1, characterized in that: The removal of the Fmoc protecting group in the amino acid in step S5 is specifically as follows: after the first amino acid condensation is completed, the resin is washed according to the previous washing steps, and a 50% morpholine / DMF solution is added to remove the Fmoc protecting group on the amino acid. Then, the resin is washed and the second amino acid is added, and the cyclic synthesis is carried out according to the polypeptide sequence.

7. The method for preparing a polypeptide with anti-inflammatory effect according to claim 1, characterized in that: The cleavage reagent prepared in step S6 is composed of 95% TFA, 2.5% Tis and 2.5% water; the synthesis tube is placed in a shaker for 1 hour, cut twice, and blown dry with nitrogen.

8. The method for preparing a polypeptide with anti-inflammatory effect according to claim 1, characterized in that: The polypeptide purification in step S7 is specifically as follows: when the sample is blown dry until only a small amount of solvent remains, ether is added and placed in a -20°C refrigerator for freezing precipitation for 15 minutes, then centrifuged using a centrifuge and the supernatant is poured out; a small amount of sample is taken, the molecular weight of the polypeptide is determined by mass spectrometry, and it is separated and purified by reversed-phase high-performance liquid chromatography, and the effluent is collected.

9. The method for preparing a polypeptide with anti-inflammatory effect according to claim 1, characterized in that: In step S8, the purity of the polypeptide is analyzed using high performance liquid chromatography to ensure that the purity of the target polypeptide meets the expected standard.

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