Modified peptide derived from periplaneta americana and application thereof

Abstract

This invention discloses a modified peptide derived from the traditional Chinese medicine *Periplaneta americana* and its applications, belonging to the field of biomedicine. The invention provides a modified peptide derived from the traditional Chinese medicine *Periplaneta americana*, comprising a zinc ion chelate tripeptide and a truncated nonapeptide from natural *Periplaneta americana*; the amino acid residues of the zinc ion chelate tripeptide chelate Zn. 2+ The invention provides ion modification and the application of modified peptides from the Chinese medicinal herb *Periplaneta americana* in the preparation of drugs for treating and / or alleviating gout. The modified peptides from *Periplaneta americana* provided by this invention exhibit significantly better therapeutic effects on gout compared to natural peptides. At high concentrations, they do not significantly affect cell viability or cause significant toxicity to erythrocytes. Furthermore, the preparation process is convenient, reproducible, has stable and reliable purity, is simple, and has low preparation costs, thus meeting the needs of product development and application.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a modified peptide derived from the Chinese medicinal herb *Periplaneta americana* and its applications. Background Technology

[0002] Gout is a common inflammatory arthritis characterized by the sudden onset of severe pain and swelling in a single joint, primarily caused by the deposition of sodium urate (MSU) crystals in the joint and surrounding tissues. The formation of MSU crystals is due to serum uric acid (SU) levels exceeding 6.8 mg / dL (hyperuricemia). Only about 9% of individuals with serum uric acid levels between 7.0 and 8.9 mg / dL will experience a gout attack within 15 years, while only about 50% of individuals with serum uric acid levels above 10 mg / dL will develop noticeable clinical symptoms of gout. This is because other risk factors increase the risk of developing gout, particularly those related to innate immune responses.

[0003] Uric acid is a byproduct of purine metabolism, produced through diet or the body, catalyzed by xanthine oxidase, an enzyme primarily found in the liver, intestines, kidneys, and endothelial cells. It is a weak dicarboxylic acid, existing as a monovalent urate anion (approximately 98%) in plasma, extracellular fluid, and synovial fluid under physiological pH conditions. Uric acid has various physiological functions, including acting as an oxidant, antioxidant, pro-inflammatory agent, and nitric oxide regulator. It promotes an increase in NADPH oxidase, which translocates to mitochondria to produce reactive oxygen species (ROS), thus exacerbating oxidative stress. Uric acid also reacts with peroxynitric oxide compounds to generate free radicals, including aminocarbonyl and triurylcarbonyl radicals. These mechanisms of free radical and ROS generation activate the NLRP3 inflammasome and further initiate pro-inflammatory signaling and subsequent inflammatory responses (Lancet. 2008, 372(9654):1953-1961).

[0004] Like other autoinflammatory diseases, gout is caused by aseptic inflammation. Sodium urate crystals act as damage-associated molecular patterns (DAMPs), activating the innate immune response by binding to TLR2 / 4. This binding activates the NLRP3 inflammasome, thereby promoting cytokine expression. The NLRP3 inflammasome plays a crucial role in the development of gout. When macrophages encounter sodium urate crystals, the NLRP3 inflammasome is activated. This activation leads to the release of pro-inflammatory cytokines, including IL-1β, IL-6, IL-8, IL-18, and TNF-α. This response illustrates the autoinflammatory mechanism associated with gout. Genetic polymorphisms in genes regulating the formation and activity of the NLRP3 inflammasome are associated with gout, supporting its classification as an autoinflammatory disease. Clinically, gout treatment mainly includes anti-inflammatory analgesia during acute attacks, long-term uric acid-lowering therapy, and lifestyle interventions. While a relatively mature drug system has been developed for clinical treatment, issues remain regarding efficacy, safety, long-term adherence, and individualized treatment. The main disadvantages of gout treatment techniques are: 1. Acute phase medications have certain adverse reactions and usage restrictions; 2. Uric acid-lowering treatments mainly target uric acid and cannot completely resolve the inflammation problem; 3. Long-term treatment has compliance issues.

[0005] With the development of proteomics, peptidomics, and mass spectrometry, the technology for discovering and identifying bioactive peptides from the traditional Chinese medicine *Periplaneta americana* has matured. Studies have shown that peptides / low molecular weight peptides derived from *Periplaneta americana* possess certain anti-inflammatory, antioxidant, antibacterial, and tissue-repairing activities. For example, recent studies have identified a large number of peptide sequences from *Periplaneta americana* using mass spectrometry and observed that related peptide components exhibit antioxidant and anti-inflammatory activities. *Periplaneta americana* is an animal-derived medicinal material, and its protein and peptide composition is highly complex. Traditional extraction methods typically yield a mixture of multiple peptide fragments, making it difficult to directly identify the "key bioactive peptide" that truly exerts the pharmacological effect. Furthermore, natural bioactive proteins and peptides also face challenges such as preparation difficulties, potential immunogenicity, and poor stability.

[0006] Therefore, developing a modified peptide derived from the American cockroach has become an urgent need in this field. Summary of the Invention

[0007] The purpose of this invention is to provide a modified peptide derived from the animal-derived traditional Chinese medicine American cockroach, its preparation technology, and its application in the treatment of gout.

[0008] In a first aspect, the present invention provides a modified peptide derived from the Chinese medicinal herb *Periplaneta americana*, wherein the modified peptide derived from *Periplaneta americana* includes an artificial tripeptide and a natural truncated nonapeptide from *Periplaneta americana*. The amino acid sequence of the modified peptide derived from the Chinese medicinal plant *Periplaneta americana* is shown in SEQ ID NO:1; the amino acid sequence of the artificial tripeptide is EHG; and the amino acid sequence of the natural *Periplaneta americana* truncated nonapeptide is shown in SEQ ID NO:2.

[0009] Preferably, the amino acid residues of the artificial tripeptide chelate Zn. 2+ Ions are used for modification.

[0010] Preferably, Zn is added to the modified peptide derived from the Chinese medicinal herb *Periplaneta americana*. 2+ Ionic solutions chelate Zn to the amino acid residues of the artificial tripeptide. 2+ ion.

[0011] Secondly, the present invention provides the use of the modified peptide derived from the Chinese herb *Periplaneta americana* in the preparation of drugs for treating and / or relieving gout.

[0012] Preferably, the modified peptide derived from the Chinese herb *Periplaneta americana* treats and / or alleviates gout by inhibiting the expression of pro-inflammatory cytokines interleukin-1β and tumor necrosis factor-α, and promoting the expression of the anti-inflammatory cytokine interleukin-10.

[0013] Thirdly, the present invention provides a drug comprising the above-mentioned modified peptide derived from the Chinese medicinal plant American cockroach.

[0014] Preferably, the modified peptide derived from the Chinese medicinal plant *Periplaneta americana* is synthesized using natural amino acids as raw materials through polypeptide solid-phase synthesis technology.

[0015] Preferably, the drug further includes biopharmaceutical acceptable salts, hydrates, solvates and / or acceptable excipients; the excipients include at least one of fillers, flavoring agents, pH adjusters, stabilizers, water for injection, antioxidants, and osmotic pressure regulators.

[0016] Preferably, the dosage form of the drug includes at least one of the following: injection, tablet, powder, granule, capsule, oral liquid, pill, ointment, and cream.

[0017] Preferably, the method of administration of the drug includes at least one of injection, oral administration, nasal drops, and eye drops, and the site of drug delivery includes any one of muscle, endothelium, subcutaneous tissue, vein, and mucosal tissue.

[0018] Compared with the prior art, the present invention has the following advantages: This invention provides a modified peptide derived from the Chinese medicinal herb *Periplaneta americana*. The natural polypeptide from *Periplaneta americana* is truncated to obtain a short peptide sequence consisting of 9 amino acid residues. This peptide is then used as a template to modify its amino terminus with an amide bond, resulting in a tripeptide that chelates with zinc ions. The modified peptide provided by this invention exhibits significantly superior therapeutic effects on gout compared to the natural polypeptide. Furthermore, at high concentrations, the modified peptide does not significantly affect cell viability and shows no obvious toxicity to erythrocytes.

[0019] The modified peptides of this invention are prepared using inexpensive natural amino acids as raw materials through polypeptide solid-phase synthesis technology and then purified by chromatography. The process is convenient, reproducible, has stable and reliable purity, is simple, and has low preparation cost, which can meet the needs of product development and application. Attached Figure Description

[0020] Figure 1 The molecular structure of the modified peptide from the American cockroach (Periplaneta americana); Figure 2 The HPLC chromatogram of the modified peptide from American cockroaches in Example 1 is shown below. Figure 3 This is the mass spectrum of the American cockroach-modified peptide in Example 1; Figure 4 This is a graph showing the hemolytic activity of the American cockroach-modified peptide in Example 2; Figure 5 The figure shows the effect of the American cockroach-modified peptide in Example 3 on the viability of HepG2, THP-1, and SW982 cells. Figure 6 This is a graph showing the changes in joint swelling in rats before and after treatment with the American cockroach-modified peptide in Example 4, indicating the effects of this treatment on a gout model rat. Figure 7 The graph shows the changes in joint swelling in rats before and after treatment with zinc ion chelate tripeptide, natural American cockroach truncated peptide, and American cockroach modified peptide in Example 5. Figure 8 The graph shows the changes in serum IL-1β levels in gout model rats before and after treatment with the American cockroach-modified peptide in Example 6. Figure 9 This is a graph showing the changes in serum TNF-α levels in rats with gout before and after treatment with the American cockroach-modified peptide in Example 6. Figure 10 The graph shows the changes in serum IL-10 levels in rats with gout before and after treatment with the American cockroach-modified peptide in Example 6. Detailed Implementation

[0021] To better understand the present invention, the following embodiments are further illustrations of the invention, but the scope of the invention is not limited to these embodiments. Unless otherwise specified, the experimental methods used in the embodiments of the present invention are conventional methods. The reagents and materials used in the embodiments are all commercially available, and the quantitative experiments involved in the embodiments are all performed in at least three replicates.

[0022] The present invention adopts the following technical solution: This invention provides a modified peptide derived from the Chinese medicinal herb *Periplaneta americana*. The natural polypeptide of *Periplaneta americana* (NCBIID: XP_069694223.1) is truncated, and the middle segment is taken to obtain a short peptide sequence PLDSLDLKY composed of 9 amino acid residues. Using this peptide segment as a template, the tripeptide EHG is modified on its amino terminus through an amide bond.

[0023] Preferably, a modified peptide derived from the Chinese medicinal herb *Periplaneta americana* is added to a solution containing zinc ions, allowing the tripeptide EHG to chelate metallic zinc ions.

[0024] The present invention does not limit the preparation method of the modified peptide derived from the Chinese herbal medicine American cockroach, as long as the amino acid sequence shown in SEQ ID NO:1 can be synthesized.

[0025] Preferably, modified peptide molecules are prepared using solid-phase peptide synthesis technology, and then purified by semi-preparative reversed-phase high-performance liquid chromatography (HPLC) to remove impurities and obtain a pure product that meets the requirements of subsequent experiments. The purity of the modified peptide is determined by analytical HPLC, and its precise molecular weight is determined by electrospray ionization tandem mass spectrometry (ESI-MS).

[0026] Based on the above design, a truncated modified peptide derived from the Chinese medicinal herb *Periplaneta americana* was successfully prepared. It consists of 12 amino acid residues and has a theoretical molecular weight of 1386.52 Da. Its molecular structure is shown below. Figure 1 As shown.

[0027] The effects of the present invention will be described in detail below with reference to the embodiments.

[0028] In this invention, GraphPad Prism 9.0 software was used for data processing, and Student's t-test was used for statistical analysis. Data are expressed as mean ± standard deviation (SD). The significance level was defined as follows: p <0.05, p <0.01, p <0.001. Example 1: Preparation of American cockroach-modified peptides This modified peptide was prepared using solid-phase peptide synthesis technology. Natural L-amino acids were used as raw materials, with 9-fluorenemethoxycarbonyl (FMOC) as the protecting group at the amino terminus. 4-(2',4'-dimethoxyphenyl-fluorenemethoxycarbonyl-aminomethyl)-phenoxyacetamido-methyldiphenylamine resin (Rink amide MBHA resin) was used as the solid-phase support for peptide synthesis, and 1-hydroxybenzotriazole / dicyclohexylcarbodiimide (HOBt / DCC) was used as the condensing agent. Starting with tyrosine (Tyr), the peptide chain was extended from the carboxyl terminus to the amino terminus by sequentially bonding lysine-leucine-aspartic acid-leucine-serine-aspartic acid-leucine-proline-glycine-histidine-glutamic acid. The synthesized linear artificial peptide was cleaved from a solid-phase MBHA resin using a mixture of 96% trifluoroacetic acid (TFA), 1.5% water, and 2.5% triisopropylsilane (TIA) by mass percentage (m / m). After repeated precipitation with diethyl ether, the peptide was purified by preparative RP-HPLC, lyophilized, and stored at -80°C for later use.

[0029] A Bondapak C18 (30 mm × 300 mm, 15 μm) semi-preparative column was used, with a mobile phase of 5‰ trifluoroacetic acid and 0–75% acetonitrile (v / v) at a flow rate of 5 mL / min for gradient elution. The results were then analyzed by RP-HPLC. Figure 2 As shown, the retention time of the prepared American cockroach-modified peptide molecule was 11.41 min. The purity of the prepared linear molecule was 97.82% when the main peak was calculated using the area normalization method.

[0030] Measured using electrospray ionization mass spectrometry (ESI / MS), such as Figure 3 As shown, its molecular weight is m / z 1387.8 [M+H]. + m / z 694.6 [M+2H] 2+ m / z 463.7 [M+3H] 3+ The molecular weight is consistent with the theoretical molecular weight of the proposed American cockroach-modified peptide (1386.52 Da), indicating successful preparation.

[0031] Example 2: Determination of hemolytic activity of modified peptide molecules from American cockroaches Fresh blood was collected from healthy volunteers, human red blood cells were separated and prepared into a cell suspension, sterile physiological saline was used as a negative control and 1% Triton X-100 was used as a positive control, and the hemolytic activity of the American cockroach modified peptide obtained in Example 1 was determined.

[0032] The specific steps are as follows: Take fresh blood into an EDTA anticoagulant tube, invert it to ensure the blood fully contacts the anticoagulant at the bottom and on the inner wall of the tube. Centrifuge at 1000 rpm for 5 minutes at room temperature, discard the upper plasma layer, and retain the red blood cells in the lower layer. Add 3-5 times the volume of medical saline injection, invert the tube to suspend the red blood cells at the bottom, and repeat the above steps until the upper supernatant becomes colorless. Prepare a 2% (v / v) cell suspension using medical saline injection. The *Periplaneta americana* modified peptide obtained in Example 1 was dissolved in medical saline injection to prepare solutions of 32 μM, 16 μM and 8 μM, and 4 μM and 2 μM. 100 μL of 2% (v / v) erythrocyte suspension was mixed with 100 μL of each of the different concentrations of *Periplaneta americana* modified peptide solutions to achieve final concentrations of 16 μM, 8 μM, 4 μM, 2 μM, and 1 μM, respectively. The positive control consisted of erythrocytes suspended in sterile water for injection, and the negative control consisted of erythrocytes suspended in medical saline injection. The samples were incubated at 37°C for 1 hour at 50 rpm. After the samples cooled to room temperature, centrifuged the 96-well plate at 2000 rpm for 8 min. Transfer 100 μL of supernatant from each well to another newly opened 96-well plate. Measure the absorbance of the samples at 490 nm using a microplate reader. Calculate the hemolysis percentage of the modified peptide using the following formula: Hemolysis rate (%) = ( H sample - H negative ) / ( H positive - H negative )×100% in, H The absorbance value is at a wavelength of 490 nm. "sample" refers to the sample, "negative" to the negative control, and "positive" to the positive control.

[0033] The measured hemolysis rate is as follows Figure 4 As shown, the modified peptide of American cockroaches did not exhibit significant hemolytic activity at a concentration of 16 μM, with a hemolysis rate of less than 3%, indicating that it has extremely low hemolytic activity.

[0034] Example 3: Effects of American cockroach-modified peptides on the viability of HepG2, THP-1, and SW982 cells. In this embodiment, the human liver cancer cell line HepG2 (ATCC HB-8065) ​​and the human synovial sarcoma cell line SW982 (ATCC HTB-93) were adherent cells, and the human monocytic leukemia THP-11 (ATCC TIB-202) was a suspension culture cell.

[0035] 1. HepG2 cell viability assay HepG2 cells were cultured in a 37°C air incubator containing 5% CO2 using DMEM medium containing 10% fetal bovine serum. The doubling time was approximately 48-72 hours. A 1% penicillin-streptomycin mixed antibiotic was added to prevent bacterial contamination.

[0036] HepG2 cells were digested with 0.25% trypsin at 37°C for 20-40 seconds, and the cells were gently detached from the bottom of the culture dish by tapping. The cells were washed 2-3 times with sterile PBS buffer after incubation at 37°C, and then gently pipetted into a single-cell suspension in DMEM medium containing 10% fetal bovine serum. The cells were then diluted to a concentration of approximately (3-5) × 10⁻⁶. 5 Cells / mL were seeded into 6-well plates, 2 mL per well, and cultured for 24 h.

[0037] The American cockroach-modified peptides obtained in Example 1 were diluted in culture medium to concentrations of 16 μM, 8 μM, 4 μM, 2 μM, and 1 μM. The original culture medium was discarded, and 2 mL of culture medium containing different concentrations of American cockroach-modified peptides was added to each well, with fresh culture medium used as a control.

[0038] After incubation at 37°C for 24 hours, cell proliferation was measured using a CCK-8 assay kit, and the absorbance of the culture medium at 450 nm in each well was measured using a Multiskan FC (Thermo Fisher Scientific Inc.) microplate reader. Cell viability was calculated as the ratio of the absorbance values ​​of the experimental group to those of the control group, using the following formula: Cell viability (%) = [(Experimental group OD - Blank group OD) / (Control group OD - Blank group OD)] × 100%.

[0039] 2. THP-1 cell viability assay Human monocytic leukemia cells (THP-1) were cultured in a 37°C, 5% CO2 incubator using RPMI-1640 medium containing 10% fetal bovine serum, supplemented with 2 mM L-glutamine to maintain cell metabolic stability and a suitable pH environment for suspension growth. The cell density was controlled at 2 × 10⁻⁶ cells / mL. 5 up to l×l0 6cells / mL, add 1% penicillin-streptomycin mixed antibiotic to prevent bacterial contamination.

[0040] Take THP-1 cell culture medium, centrifuge and discard the culture medium. Wash 2-3 times with sterile PBS buffer incubated at 37°C. Add RPMI-1640 medium containing 10% fetal bovine serum and gently pipette to prepare a single-cell suspension. Then, use the method in step 1 to detect and calculate cell viability.

[0041] 3. SW982 cell viability assay Human synovial sarcoma cells SW982 were cultured at 37°C in a CO2-free incubator using Leibovitz's L-15 medium containing 10% fetal bovine serum and high glucose (final glucose concentration 10 mmol / L), with 1% penicillin-streptomycin mixed antibiotics added to prevent bacterial contamination.

[0042] SW982 cells were digested with 0.25% trypsin at 37°C for 20-40 seconds, and the cells were gently detached from the bottom of the culture dish by tapping. The cells were washed 2-3 times with sterile PBS buffer after incubation at 37°C, and then gently pipetted into a single-cell suspension using L-15 medium containing 10% fetal bovine serum and high glucose. Cell viability was then assessed and calculated using the method described in step 1.

[0043] HepG2, THP-1, and SW982 cell line viability assays, such as Figure 5 As shown, the modified peptides from American cockroaches at concentrations of 1 μM to 16 μM did not have significant toxic effects on any of the three cell lines mentioned above.

[0044] Example 4: Construction of a rat model of gout and evaluation of the therapeutic effect of American cockroach-modified peptides The rats used in this embodiment were all male, 8 weeks old, and weighing 200-220 grams, Wistar rats from Wuhan Shulaibao Biotechnology Co., Ltd., and were cultured in cages (3 rats per cage) in a specific pathogen-free environment. The temperature in the facility was maintained at (24±1)℃, the relative humidity at (60±5)%, and a 12-hour light / dark cycle was followed. All rats had free access to standard laboratory feed and purified water.

[0045] 1. Construction of a rat model of gout Rats were injected intra-articularly with 0.25 mL of 30 mg / mL monosodium urate (MSU) crystal suspension once a day for three consecutive days. The degree of swelling was judged by measuring the ankle joint circumference with calipers. An increase of ≥10% indicated that the model was successful.

[0046] 2. Evaluation of the therapeutic effects of American cockroach-modified peptides Rats were randomly divided into four groups (n=6 in each group): control group, model group, colchicine group, and American cockroach modified peptide treatment group. Gout was modeled in three of the groups, and the following methods were used for continuous intervention for 8 days.

[0047] Among them, the American cockroach-modified peptide treatment group consisted of gout model rats, who were given 1 μmol / kg body weight of American cockroach-modified peptide daily via intra-articular injection, with ZnCl2 added, the molar ratio of American cockroach-modified peptide to ZnCl2 being 1:1.5, and dissolved in 10 μL of physiological saline; the colchicine group consisted of gout model rats, who were given 1 μmol / kg body weight of colchicine daily via gavage, dissolved in 10 μL of physiological saline; the model group consisted of gout model rats, who were given 10 μL of physiological saline daily via gavage; and the control group consisted of healthy rats, who were given 10 μL of physiological saline daily via gavage.

[0048] The degree of joint swelling at 12 hours, 24 hours, and 48 hours was as follows for the control group, model group, colchicine group, and American cockroach modified peptide treatment group. Figure 6 As shown, the joint swelling in the model group rats was significantly higher than that in the control group at 12, 24, and 48 hours, proving the successful establishment of the model. After treatment with colchicine or American cockroach-modified peptides, the joint swelling in the gout model rats at all three time points was significantly improved. Although both the colchicine and American cockroach-modified peptide groups showed significant symptom improvement at different time points, differences still existed at different time points. At 12 hours, there was no significant difference between the two groups. At 24 and 48 hours, the American cockroach-modified peptide group had a more significant effect on relieving joint swelling than the colchicine group. Furthermore, after 48 hours of treatment with American cockroach-modified peptides, there was no statistically significant difference between the model group and the control group, indicating that after 48 hours of treatment, the joint morphology basically returned to normal.

[0049] Example 5: Evaluation of the therapeutic effects of American cockroach-modified peptides, zinc ion chelated tripeptides, and natural American cockroach truncated nonapeptides. Using the method described in Example 3, the differences in the effects of zinc ion chelate tripeptide EHG, natural American cockroach truncated nonapeptide PLDSLDLKY, and American cockroach modified peptide EHGPLDSLDLKY on gout model rats before and after treatment were compared; rats were further divided into a zinc ion chelate tripeptide group and a natural American cockroach truncated peptide group.

[0050] The zinc ion chelate tripeptide group consisted of rats with gout. The tripeptide EHG was administered intra-articularly at a dose of 1 μmol / kg body weight per day, with ZnCl2 added. The molar ratio of tripeptide EHG to ZnCl2 was 1:1.5, and the solution was dissolved in 10 μL of physiological saline. The natural American cockroach truncated peptide group consisted of rats with gout. The natural American cockroach truncated peptide was administered intra-articularly at a dose of 1 μmol / kg body weight per day, and the solution was dissolved in 10 μL of physiological saline.

[0051] The degree of joint swelling after 48 hours is as follows Figure 7 As shown, zinc ion chelate tripeptide, natural American cockroach truncated peptide, and American cockroach modified peptide can all improve the degree of joint swelling in rats with gout. However, compared with zinc ion chelate tripeptide and natural American cockroach truncated peptide, American cockroach modified peptide showed a more significant therapeutic effect, and after 48 hours of treatment with American cockroach modified peptide, there was no statistically significant difference between it and the control group.

[0052] Example 6: Changes in serum inflammation-related cytokines before and after treatment with American cockroach-modified peptides On day 3 of treatment, blood was collected from the tail vein of rats in the control group, model group, colchicine group, and American cockroach-modified peptide treatment group (as described in Example 3). Serum was separated after centrifugation at 5000 rpm at 4°C and stored at 4°C for subsequent analysis. After blood collection, rats were euthanized by intraperitoneal injection of sodium pentobarbital. The levels of inflammatory-related cytokines in the serum of rats in the normal group, model group, colchicine group, and American cockroach-modified peptide group were measured using an ELISA kit via enzyme-linked immunosorbent assay (ELISA).

[0053] Inflammation-related cytokines include: pro-inflammatory cytokines interleukin-1β (IL-1β) (using Elabscience kit, catalog number: E-HSEL-R0002), pro-inflammatory cytokines tumor necrosis factor-alpha (NF-α) (using Elabscience kit, catalog number: E-EL-R2856), and anti-inflammatory cytokines interleukin-10 (IL-10) (using Elabscience kit, catalog number: E-EL-R0016).

[0054] Results of the detection of the pro-inflammatory cytokine IL-1β are as follows Figure 8As shown, compared with the control group, the serum IL-1β content of rats in the model group was significantly increased. After treatment with colchicine or American cockroach modified peptide, its content was significantly reduced, and the American cockroach modified peptide group was significantly better than the colchicine group.

[0055] Results of the detection of pro-inflammatory cytokine TNF-α are as follows Figure 9 As shown, the serum TNF-α level in the model group rats was significantly higher than that in the normal group. After treatment, the serum levels of this cytokine in the colchicine group and the American cockroach modified peptide group were significantly reduced, but there was no significant difference between the American cockroach modified peptide group and the colchicine group.

[0056] Results of IL-10 assay for anti-inflammatory cytokine are as follows Figure 10 As shown, the serum levels of the anti-inflammatory cytokine IL-10 in the model group rats were significantly lower than those in the normal group. After treatment, the serum levels of this cytokine in the colchicine group and the American cockroach modified peptide group were significantly increased, and the American cockroach modified peptide group was significantly better than the colchicine group.

[0057] Example 7: American cockroach-modified peptide tablets Take 0.5 g of the American cockroach-modified peptide obtained in Example 1, 3.0 g of starch, and 6.5 g of dextrin, mix them thoroughly, add 50% pharmaceutical grade polyvinylpyrrolidone (PVP) as a binder, granulate, sizing, and compress to obtain American cockroach-modified peptide tablets.

[0058] Example 8: American cockroach-modified peptide gel Take 20 mL of distilled water and evenly sprinkle pharmaceutical-grade carbomer 940 onto the surface, stirring immediately to allow it to swell fully. Add propylene glycol and mix thoroughly. While stirring, add triethylamine dropwise to prepare the gel matrix. Dissolve 0.5 g of the American cockroach-modified peptide obtained in Example 1 in 20 mL of PBS solution (0.15 M, pH=7.3), and slowly add it to the gel matrix while stirring. Finally, add sterile distilled water to obtain the American cockroach-modified peptide gel.

[0059] Example 9: Small-volume injection of American cockroach-modified peptides Take 1 g of the American cockroach-modified peptide obtained in Example 1, dissolve it in 40 mL of water for injection, adjust the pH to 6.9-7.1, and add an isotonic adjuster (such as sodium chloride or glucose) if necessary to make the osmotic pressure close to the physiological level. After stirring until completely dissolved, pre-filter through a 0.45 μm filter to remove insoluble particles, and then sterilize by filtering through a 0.22 μm sterile filter membrane. Dispense the filtrate into 2 mL portions in sterile small-volume glass ampoules or vials under Class A laminar flow protection, controlling the filling volume error within ±2%. Process using an aseptic filling process to obtain the small-volume injection of the American cockroach-modified peptide.

[0060] Example 10: American cockroach-modified peptide lyophilized powder for injection Take 5 g of the American cockroach-modified peptide obtained in Example 1 and 50 g of mannitol, dissolve them in an appropriate amount of PBS buffer (0.15 M, pH 7.4), add sterile water for injection to 750 mL, mix thoroughly, add 20-30 g of activated charcoal for injection, stir at room temperature for 1 hour, coarsely filter, and immediately filter through a 0.22 μm sterile filter membrane for sterilization, dispense into 2.5 mL vials. Cool down by rapid freezing at a rate of 10-20°C per minute until the temperature reaches -55 to -60°C. Maintain this low temperature for 3 hours, then apply vacuum. Under vacuum, slowly raise the temperature at a rate of (3-5)°C / hour. Stop heating when the temperature reaches (24±2)°C. Once the temperature is close to room temperature, remove the sample from the freeze dryer, seal the container, and obtain the American cockroach-modified peptide lyophilized powder for injection.

[0061] When using the drugs obtained in Examples 7-10 above, zinc ion solution is added to chelate zinc ions onto the tripeptide.

[0062] SEQ ID NO:1 EHGPLDSLDLKY SEQ ID NO:2 PLDSLDLKY The above description is merely a preferred embodiment of the present invention, and should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A modified peptide derived from the Chinese medicinal herb *Periplaneta americana*, characterized in that, The modified peptides derived from the Chinese medicinal plant *Periplaneta americana* include artificial tripeptides and natural truncated nonapeptides from *Periplaneta americana*. The amino acid sequence of the modified peptide derived from the Chinese medicinal plant *Periplaneta americana* is shown in SEQ ID NO:1; the amino acid sequence of the artificial tripeptide is EHG; and the amino acid sequence of the natural *Periplaneta americana* truncated nonapeptide is shown in SEQ ID NO:

2.

2. The modified peptide derived from the Chinese medicinal herb *Periplaneta americana* according to claim 1, characterized in that, The amino acid residues of the artificial tripeptide chelate Zn 2+ ions are modified.

3. The modified peptide derived from the Chinese medicinal herb *Periplaneta americana* according to claim 2, characterized in that, By adding Zn to the modified peptide derived from the Chinese medicinal herb *Periplaneta americana*. 2+ Ionic solutions chelate Zn to the amino acid residues of the artificial tripeptide. 2+ ion.

4. The use of the modified peptide derived from the Chinese medicinal herb *Periplaneta americana* as described in any one of claims 1 to 3 in the preparation of drugs for treating and / or relieving gout.

5. The application according to claim 4, characterized in that, The modified peptides derived from the Chinese herb *Periplaneta americana* treat and / or alleviate gout by inhibiting the expression of pro-inflammatory cytokines interleukin-1β and tumor necrosis factor-α, and promoting the expression of the anti-inflammatory cytokine interleukin-10.

6. A drug, characterized in that, It includes the modified peptide derived from the Chinese medicinal plant *Periplaneta americana* as described in any one of claims 1 to 3.

7. The drug according to claim 6, characterized in that, The modified peptide derived from the Chinese medicinal plant *Periplaneta americana* was synthesized using natural amino acids as raw materials through polypeptide solid-phase synthesis technology.

8. The drug according to claim 6, characterized in that, The drug also includes biopharmaceutical acceptable salts, hydrates, solvates and / or acceptable excipients; The excipients include at least one of the following: fillers, flavoring agents, pH adjusters, stabilizers, water for injection, antioxidants, and osmotic pressure regulators.

9. The drug according to claim 6, characterized in that, The dosage form of the drug includes at least one of the following: injection, tablet, powder, granule, capsule, oral liquid, pill, ointment, and cream.

10. The medicament according to claim 6, characterized in that, The drug can be administered via at least one of injection, oral administration, nasal drops, or eye drops, and the drug can be introduced into any one of muscle, endothelium, subcutaneous tissue, vein, or mucosal tissue.

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