Cyclic polypeptide and application thereof

Abstract

The invention provides a cyclic polypeptide and application thereof, the cyclic polypeptide is derived from Blastomyces dermatidis, the amino acid sequence of the cyclic polypeptide is as shown in SEQ ID NO.1, and a cyclic structure is formed through three pairs of disulfide bonds (Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6) in a molecule. The cyclic peptide has multiple functions of resisting gram-positive bacteria (including MRSA), inhibiting inflammatory response and promoting wound healing. The polypeptide has a wide application prospect in the fields of medicines, cosmetics and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a cyclic polypeptide derived from the fungus Blastomyces dermatitidis and its applications. Background Art

[0002] The skin is the largest organ of the human body and undertakes many important physiological functions. It is not only the first barrier between us and the external environment, protecting the body from harmful substances such as bacteria, viruses, and ultraviolet rays, but also regulates body temperature and excretes waste through sweat glands and sebaceous glands. The nerve endings in the skin enable us to perceive external stimuli such as touch, temperature, and pain, and the color and texture of the skin also reflect the health status of the human body. Skin injuries are usually caused by daily traumas. Once they occur, they break the body's main antibacterial barrier, making it vulnerable to various microbial infections, and the occurrence of infections will delay wound healing.

[0003] In addition, wounds that cannot heal in a timely manner may be prone to further infections and persistent inflammatory reactions. Given the dosage limitations and potential systemic toxicity of antibiotics, the healing of infectious wounds has received increasing attention and emphasis in recent years.

[0004] Among various wound infections, Staphylococcus aureus accounts for approximately 80% of all skin and soft tissue infections. More than 30% of patients with Staphylococcus aureus infections develop chronic or recurrent infections within 3 months, even after receiving antibacterial drug treatment. Among the various factors contributing to this problem, the ability of Staphylococcus aureus to survive within host cells is widely regarded as a key determinant of the persistence and recurrence of these infections. Increasing evidence indicates that Staphylococcus aureus can resist the fusion of phagosomes and lysosomes, thereby multiplying within the phagolysosomes of macrophages and persisting in human keratinocytes. This property enables Staphylococcus aureus to evade the immune system and antibiotics and has the potential to regenerate when the environment permits. According to the data published in the international authoritative journal The Lancet Microbe in 2023, there are approximately 5 million cases of infectious wound patients globally per year, and the proportion of delayed healing due to drug-resistant bacteria colonization exceeds 40%. Methicillin-resistant Staphylococcus aureus (MRSA), as a type of Staphylococcus aureus resistant to multiple drugs, poses a major threat to human health in diseases such as skin infections and sepsis.

[0005] Therefore, it is necessary to develop an antibacterial product with high efficiency, low toxicity, and scalability. Summary of the Invention

[0006] The object of the present invention is to provide a cyclic polypeptide derived from the fungus Blastomyces dermatitidis, which has multiple functions of anti-Gram-positive bacteria (including MRSA), inhibiting inflammatory responses, and promoting wound healing. The preparation method includes solid-phase synthesis of a linear peptide followed by oxidation to form disulfide bonds and purification by high-performance liquid chromatography. The polypeptide has broad application prospects in the fields of medicine, cosmetics, etc.

[0007] To solve the above technical problems, the present invention adopts the following technical solutions:

[0008] In the first aspect of the present invention, a cyclic polypeptide is provided. The cyclic polypeptide is derived from the fungus Blastomyces dermatitidis, and its nucleotide sequence is as shown in SEQ ID NO.1. The linking mode of disulfide bonds in the cyclic polypeptide is: Cys1-Cys4, Cys2-Cys5, Cys3-Cys6.

[0009] In the second aspect of the present invention, the application of the cyclic polypeptide in the preparation of an antimicrobial agent is provided, and the microorganisms include at least one of Staphylococcus aureus, Staphylococcus epidermidis, and Micrococcus luteus.

[0010] In the third aspect of the present invention, the application of the cyclic polypeptide in the preparation of an anti-inflammatory product is provided.

[0011] The anti-inflammatory product is a product that inhibits inflammatory factors, and the inflammatory factors include at least one of IL-1β, IL-6, and TNF-α.

[0012] In the fourth aspect of the present invention, the application of the cyclic polypeptide in the preparation of a drug for promoting cell proliferation is provided.

[0013] As a specific embodiment, the drug can promote the proliferation of fibroblasts NIH-3T3 and vascular endothelial cells HUVEC.

[0014] In the fifth aspect of the present invention, the application of the cyclic polypeptide in the preparation of a product for promoting the healing of infectious wounds is provided.

[0015] Furthermore, the product includes at least one of drugs, cosmetics, or daily necessities.

[0016] In the sixth aspect of the present invention, a product for antioxidant or promoting wound repair and healing is provided, including the cyclic polypeptide.

[0017] Furthermore, the product further includes a pharmaceutically or cosmetically acceptable salt and / or acceptable excipients.

[0018] In the seventh aspect of the present invention, there is provided a pharmaceutical composition comprising the cyclic polypeptide, and the composition is an ointment, a gel, an injection or a freeze-dried powder injection.

[0019] Furthermore, the product can be introduced into muscle, endothelial, subcutaneous or mucosal tissues by injection, smearing, dressing, acupuncture, and other physically or chemically mediated methods, or can be introduced into the body after being mixed or / and wrapped with other substances.

[0020] In the eighth aspect of the present invention, there is provided a method for preparing the cyclic polypeptide, and the method includes:

[0021] Purifying the linear polypeptide shown in SEQ ID NO.1 by solid-phase synthesis to obtain a purified polypeptide;

[0022] Oxidizing the purified polypeptide in Tris-HCl buffer to form three pairs of intramolecular disulfide bonds;

[0023] Purifying by semi-preparative HPLC and lyophilizing to obtain a cyclic polypeptide derived from the fungus Blastomyces dermatitidis.

[0024] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0025] The present invention provides a cyclic polypeptide derived from the fungus Blastomyces dermatitidis and its application. The cyclic peptide of the present invention forms a stable cyclic structure through three pairs of intramolecular disulfide bonds and has the following multiple biological activities:

[0026] (1) Antibacterial activity: The MIC value against the clinical drug-resistant bacterium MRSA is 4 μM, and bactericidal effect is achieved by destroying the integrity of the cell membrane (Example 3 and Figure 6 );

[0027] (2) Anti-inflammatory activity: Significantly inhibits the secretion of inflammatory factors (IL-1β, IL-6, TNF-α) induced by LPS, and the inhibition rate is ≥ 65% (Example 4 and Figure 7 );

[0028] (3) Promoting repair ability: Accelerates the healing of infectious wounds in an animal model (the healing rate at 14 days is 90%), and has no hemolytic toxicity (Example 6 and Figure 9 );

[0029] (4) Structural stability: Intramolecular disulfide bonds endow it with the property of being resistant to enzymatic hydrolysis, which is suitable for the development of topical preparations (Examples 7-10).

[0030] The above effects indicate that the cyclic peptide combines high efficiency and safety and has the potential to be developed into a new anti-infective drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0032] Figure 1 It is a schematic diagram of the pairing mode of the cyclic polypeptide of the present invention.

[0033] Figure 2 It is a high performance liquid chromatography chart of the cyclic peptide formed after the prepared linear peptide is paired with the intramolecular disulfide bond;

[0034] Figure 3 It is a mass spectrometry chart of the cyclic peptide prepared from fungi;

[0035] Figure 4 It is a three-dimensional structure diagram of the cyclic peptide prepared from fungi constructed by a computer;

[0036] Figure 5 It is the hemolytic activity result of the cyclic peptide prepared from fungi;

[0037] Figure 6 It is the change in the state of MRSA bacteria before and after the action of the cyclic peptide prepared from fungi; among them, Figure A is the control group MRSA ATCC43300 bacteria without the action of the cyclic peptide; Figure B is the morphology of the bacteria after the action of the cyclic peptide;

[0038] Figure 7 It is the cyclic peptide prepared from fungi inhibiting the secretion and expression of inflammation-related cytokines IL-1β ( Figure 7 A), IL-6 ( Figure 7 B), and TNF-α ( Figure 7 C);

[0039] Figure 8 It is the cyclic peptide prepared from fungi promoting the proliferation of fibroblasts NIH-3T3 and vascular endothelial cells HUVEC; among them, Figure A is the result of promoting the proliferation of mouse fibroblasts NIH-3T3, and Figure B is the result of promoting the proliferation of human umbilical vein endothelial cells HUVEC;

[0040] Figure 9 It is the cyclic peptide prepared from fungi promoting the healing of infectious wounds in mice; among them, Figure A shows that the cyclic peptide prepared from fungi can significantly accelerate the wound healing, and Figure B shows significant differences on the 4th, 7th, and 11th days (P<0.05);

[0041] Figure 10The in vivo antibacterial effect of a cyclic peptide derived from fungi on infected wounds in mice. Detailed implementation manners

[0042] The present invention will be specifically described below in combination with the detailed implementation manners and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these detailed implementation manners and examples are used to illustrate the present invention rather than to limit the present invention.

[0043] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present invention belongs. In case of conflict, this specification shall prevail.

[0044] Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment, etc. used in the present invention can be obtained through market purchases or can be obtained by existing methods.

[0045] A cyclic polypeptide derived from the fungus Blastomyces dermatitidis and its application provided by the embodiment of the present invention have the following general idea:

[0046] Active polypeptides derived from fungi can be classified into antibacterial peptides, antiviral peptides, antitumor peptides, immunomodulatory peptides, enzyme inhibitory peptides, etc. according to their structures and biological functions. These polypeptides play important roles in biological defense, inflammation regulation and signal transduction. For example, antibacterial peptides can destroy the cell membranes of pathogenic bacteria, antitumor peptides can induce apoptosis of cancer cells, and immunomodulatory peptides can enhance the body's immune response. With the progress of technology, active polypeptides derived from fungi can be prepared by chemical synthesis (polypeptide solid-phase synthesis technology) to obtain high-purity chemical entities, overcoming the difficulties of low secretion and expression levels of natural polypeptides and difficult extraction and purification. This promotes their application in the clinical and industrial fields. In order to develop and effectively utilize natural polypeptides derived from fungi and solve scientific problems such as difficult healing of clinical infectious wounds, guided by the treatment of diseases and the needs of patients, to break through the existing technical and industrial bottlenecks:

[0047] First, the present invention discloses a cyclic polypeptide derived from fungi, whose linear sequence is: GWGCNIFGGNDYRCHRHCKSISGYKGGYCKLGGICKCY (SEQ ID NO.1), and forms a cyclic spatial structure through the pairing of three pairs of intramolecular disulfide bonds (Cys1-Cys4, Cys2-Cys5, Cys3-Cys6).

[0048] The natural polypeptide involved in the present invention is derived from Blastomyces dermatitidis. Through previous transcriptome and proteome studies, the amino acid sequence was mined and obtained. Further bioinformatics analysis showed that it consists of three parts, namely signal peptide, propeptide and mature peptide: MRFSVFAIISALSMTALA (signal peptide) IPVPAPEDLDIAEATADLAARDAPVEAIPDDFAGDVSGLDDDDDDDDEDENSAGALQKR (propeptide) GWGCNIFGGNDYRCHRHCKSISGYKGGYCKLGGICKCY (mature peptide).

[0049] By using the solid-phase peptide synthesis technology, the mature peptide sequence derived from fungi was prepared. It consists of 38 amino acid residues and forms a cyclic peptide through three pairs of disulfide bond pairing (the pairing mode is as Figure 1 shown), with a theoretical molecular weight of 4199.85 Da and a theoretical isoelectric point pI of 9.06.

[0050] Second, the present invention also provides a preparation method for the cyclic peptide derived from fungi: First, a linear polypeptide is prepared by using the solid-phase peptide synthesis technology; then, oxidation is carried out in a buffer solution with appropriate concentration and pH. For every 2 hydrogen atoms removed, a pair of intramolecular disulfide bonds can be formed, and a total of 6 hydrogen atoms are removed to form three pairs of intramolecular disulfide bonds; next, separation and purification are carried out by semi-preparative high-performance liquid chromatography technology based on the difference in retention time between the linear peptide and the cyclic peptide; finally, after freezing in a -80 °C refrigerator for 6 hours, it is placed in a freeze dryer and freeze-dried for 12 - 18 hours.

[0051] Third, the present invention provides the application of the cyclic peptide derived from fungi described above in anti-microbial, anti-inflammatory, promoting the proliferation of fibroblasts and vascular endothelial cells. At the same time, the constructed animal model of infectious skin injury was treated with this cyclic peptide. The results showed that compared with the blank control group, in the 14-day external treatment process of the gel treatment group containing the cyclic peptide of the present invention, the wound healing speed of the skin lesion treated with the gel containing the cyclic peptide was significantly faster than that of the control group.

[0052] From the above three points, it can be seen that compared with the prior art, the cyclic peptide derived from fungi described in the present invention is an active substance with multiple biological functions and has unique advantages. It not only has the functions of antibacterial, anti-inflammatory, promoting cell proliferation and accelerating wound healing, but also can be prepared by the solid-phase peptide synthesis technology, with low cost and energy consumption, easy quality control, and stable and uniform quality among batches. Therefore, it has good technological advancement.

[0053] The following will combine examples and experimental data to elaborate in detail on a cyclic polypeptide derived from the fungus Blastomyces dermatitidis of the present application and its applications.

[0054] Example 1: Cyclic polypeptide derived from the fungus Blastomyces dermatitidis

[0055] First, 9-fluorenylmethyloxycarbonyl (FMOC) was used as the protecting group at the amino terminus. 4-methyl-benzhydrylamine resin·HCl (MBHA resin) was selected as the solid-phase carrier for polypeptide synthesis. HOBt / DCC was used as the condensing agent for the reaction, and the peptide chain GWGCNIFGGNDYRCHRHCKSISGYKGGYCKLGGICKCY was extended from the carboxyl terminus to the amino terminus (C-terminal → N-terminal).

[0056] The polypeptide sequence containing 38 amino acid residues was cleaved from the MBHA resin with a mixture of 95.0% trifluoroacetic acid, 2.0% water, and 3.0% triisopropylsilane (TIA) (by mass percentage). After repeated precipitation with ether several times, it was initially purified by preparative reverse-phase high-performance liquid chromatography RP-HPLC. Next, three pairs of intermolecular disulfide linkages were formed through the oxidative dehydrogenation of sulfhydryl groups to fold into a cyclic peptide molecule. The specific operation was as follows:

[0057] 1 mg of the above-prepared linear polypeptide was diluted in an ionic buffer, and the pH was adjusted to deviate appropriately from the isoelectric point to avoid precipitation. Subsequently, the reaction solution was incubated overnight at room temperature. The cyclic peptide formed with three pairs of intramolecular disulfide bonds was centrifuged at 10,000 rpm for 10 minutes at 4 °C, and the supernatant was further purified by RP-HPLC (Shimadzu, Japan). Purification process: Using a C18 reversed-phase preparative column (250 mm × 20 mm, 5 μm); mobile phase: 1‰ trifluoroacetic acid, 0%-70% (by volume percentage) acetonitrile as the mobile phase, and gradient elution was carried out at a flow rate of 1.0 mL / min. The main peak eluate was collected, frozen at -80 °C for 4 hours, then placed in a freeze dryer for freeze-drying. After 12 - 16 hours of freeze-drying to form a loose solid powder, it was stored in a -20 °C refrigerator for standby. Before and after the formation of three pairs of intramolecular disulfide bonds, their retention times were 8.67 min and 10.05 min respectively, indicating that after the formation of three pairs of intramolecular disulfide bonds to form a cyclic peptide, the molecular polarity became smaller and the retention time was extended ( Figure 2 ). Identified by time-of-flight mass spectrometry TOF-MS, its molecular weight was 4199.9 Da, which was consistent with the theoretical molecular weight of this cyclic peptide ( Figure 3) According to the simulated spatial structure of this cyclic peptide, the pairing mode of its intramolecular disulfide bonds is Cys1-Cys4, Cys2-Cys5, Cys3-Cys6( Figure 4 )

[0058] Example 2. Hemolytic activity of the cyclic peptide derived from fungi of the present invention

[0059] Fresh blood was obtained from healthy male volunteers. After separating human red blood cells, a suspension was prepared. Using sterile PBS buffer as the negative control and sterilized Milli-Q ultrapure water as the positive control, the hemolytic activity of this cyclic peptide was measured. The specific steps are as follows:

[0060] Collect whole blood using a heparin anticoagulant tube. Gently invert the anticoagulant tube 3-5 times to allow the blood to fully contact the anticoagulant to achieve the anticoagulation effect. Centrifuge at 800 rpm for 15 min at room temperature, aspirate all the upper plasma and retain the lower red blood cells; Add 3-5 times the volume of sterile PBS buffer, gently pipette and blow to suspend the red blood cells precipitated at the bottom of the centrifuge tube, centrifuge at 800 rpm for 10 min at room temperature, discard the supernatant and retain the precipitated red blood cells, repeat the above operation 3-5 times until the supernatant becomes colorless; Use sterile PBS buffer to make a 2%-3% (v / v) concentration cell suspension of red blood cells; Use sterilized normal saline to prepare solutions of this cyclic peptide of the present invention at concentrations of 128 μmol / L, 64 μmol / L, 32 μmol / L, 16 μmol / L, 8 μmol / L, 4 μmol / L, 2 μmol / L and 1 μmol / L; Mix 100 μL of this zinc ion chelating modified peptide solution and 100 μL of red blood cell suspension and add them to a 96-well plate so that the final concentrations of the cyclic peptide are 64 μmol / L, 32 μmol / L, 16 μmol / L, 8 μmol / L, 4 μmol / L, 2 μmol / L, 1 μmol / L and 0.5 μmol / L respectively; The negative control group is red blood cells suspended with sterile PBS buffer, and the positive control uses sterilized Milli-Q ultrapure water to suspend red blood cells; Place the samples in a thermostatic shaker and incubate at 37 °C at 80 rpm - 120 rpm for 40 min; Centrifuge the samples at 5000 rpm for 5 min, transfer 150 μL of the supernatant from each well to another clean 96-well plate; Use a full-wavelength microplate reader to measure the absorbance at a wavelength of 490 nm, and calculate the hemolysis percentage through the following formula. In the formula, H is the absorbance at a wavelength of 490 nm: Hemolysis rate (%) = (H sample -H negative ) / (H positive -H negative )×100%,

[0061] As Figure 5As shown, the results indicate that the fungal-derived cyclic peptides of the present invention did not show obvious hemolytic effects (hemolysis rate ≤ 5%) when the final concentration ranged from 0.5 to 64 μmol / L.

[0062] Example 3. Antimicrobial Activity of the Fungal-Derived Cyclic Peptides of the Present Invention

[0063] The test strains were inoculated onto sterilized Luria-Bertani (LB) solid medium plates by the three-zone streaking method and incubated upside down in a constant temperature incubator at 37 °C for 14 hours. A single colony was picked with an inoculation loop and transferred to sterilized liquid LB medium, and cultured with shaking at 37 °C and 160 rpm until the logarithmic growth phase. The absorbance value (OD600) of the bacterial solution at a wavelength of 600 nm was measured using an ultraviolet spectrophotometer.

[0064] According to the conversion relationship of 1 OD ≈ 1×10 9 CFU / mL, the bacterial solution was diluted with the medium to (1 - 2)×10 5 CFU / mL. First, 100 μL of sterilized 2×LB medium was added to a sterile 96-well plate; next, 100 μL of a cyclic peptide solution with a concentration of 256 μmol / L dissolved in sterilized LB medium was added to the first well. After mixing evenly, 100 μL was taken and added to the second well, and 100 μL was aspirated and discarded from the eighth well, and serially diluted two-fold; finally, 100 μL of the diluted bacterial solution with a concentration of 1×105 CFU / mL was added to each well and mixed evenly. The wells without the addition of cyclic peptides served as negative controls. The above groups were cultured with shaking at 37 °C for 18 - 24 hours, and the absorbance value at a wavelength of 600 nm was measured. The minimum inhibitory concentration (MIC) was taken as the lowest value of the final concentration of the polypeptide in the wells where bacterial growth could not be detected (OD 600 ≤ 0.05). The bacterial solutions in the negative control wells were all turbid, and the absorbance OD 600 > 3 at 600 nm. The results are shown in Table 1, indicating that the cyclic peptide has good inhibitory effects on Gram-positive bacteria but no antibacterial activity against Gram-negative bacteria.

[0065] Table 1. In Vitro Antibacterial Activity of Fungal-Derived Cyclic Peptides

[0066]

[0067] To observe the changes in the bacterial cell state before and after treatment with the cyclic peptide, the cell morphology was observed by scanning electron microscopy (SEM). The results are shown in the following figure. The control group of MRSA ATCC43300 without the action of the cyclic peptide had plump and round cells ( Figure 6A); After the action of the cyclic peptide, obvious wrinkles and depressions appeared on the surface of the bacterial cells, and some bacterial cells burst, and the outflow of the contents was visible. Figure 6 B). The above changes observed by SEM indicate that the cyclic peptide caused substantial damage to the bacterial cells, resulting in the death of the bacteria.

[0068] Example 4. Anti-inflammatory activity of the cyclic peptide derived from fungi of the present invention

[0069] RAW264.7 cells cultured to 70%-80% were taken, the culture medium was aspirated, and the cells were washed twice with D-Hanks buffer; an appropriate amount of trypsin was added and digested at 37°C for 20-30 seconds, and the cells were gently pipetted with DMEM medium containing 10% fetal bovine serum (FBS) to make a single-cell suspension; the cell concentration was adjusted to 2.5×10 5 Cells / mL, inoculated into a 48-well plate, 0.5 mL per well. The culture plate was incubated at 37°C, 5% CO2 for 8-12 hours, and the supernatant was aspirated and discarded. In the blank group, 0.5 mL of DMEM medium containing 10% FBS was added; in the LPS model group and the experimental group, 0.5 mL of DMEM medium containing 10% FBS with LPS (the final concentration of LPS was 10 μg / mL) was added. The culture plate was incubated at 37°C, 5% CO2 for 1 hour. In the blank group and the LPS model group, 0.5 mL of DMEM medium containing 10% FBS was added; in the experimental group, 0.5 mL of DMEM medium containing 10% FBS with the polypeptide of the present invention (the final concentration was 64 μmol / L) was added. The culture plate was incubated at 37°C, 5% CO2 for about 16 hours, and centrifuged at 1500 rmp for 10 min. The supernatant was diluted by an appropriate multiple, and the contents of inflammatory-related cytokines IL-1β, IL-6, and TNF-α in the supernatant were measured by Enzyme-Linked Immuno Sorbent Assay (ELISA).

[0070] The results show that the cyclic peptide of the present invention can significantly inhibit the expression of inflammatory-related cytokines IL-1β Figure 7 A), IL-6 Figure 7 B), and TNF-α Figure 7 C) in murine macrophages RAW 264.7 induced by LPS, indicating that the cyclic peptide has good anti-inflammatory effects.

[0071] Example 5. Proliferation-promoting activity of the cyclic peptide derived from fungi of the present invention

[0072] Take mouse fibroblast cell line NIH-3T3 cells or human umbilical vein endothelial cells HUVEC cultured to a confluence rate of 70%-80%, aspirate and discard the culture medium, and wash 2-3 times with sterile PBS buffer incubated at 37°C; add an appropriate amount of 0.25% trypsin and digest at 37°C for 20 - 40 seconds, gently tap the culture dish or flask to detach the cells from the bottom of the culture container, add an appropriate amount of DMEM or M200PRF / LSGS medium containing 10% fetal bovine serum (FBS) and gently pipette the cells to make a single-cell suspension; dilute the cells with the medium and adjust the cell concentration to approximately (3-5)×10 5 cells / mL, and inoculate into a 6-well plate, 2 mL per well. Inoculate NIH / 3T3 or HUVECs cells into a 96-well plate at a density of 6000-8000 cells per well and culture in complete culture medium for 24 h to reach monolayer culture. Dilute the cyclic peptide described in the present invention to concentrations of 64 μM, 32 μM, 16 μM, and 8 μM in complete medium. Replace the original medium with medium containing different concentrations of defensin. Use fresh medium as a control. After incubating at 37°C and 5% CO2 for 24 hours, use a CCK-8 kit to determine cell proliferation. Use a Multiskan FC (Thermo Fisher Scientific Inc.) microplate reader to measure the absorbance of the medium at 450 nm in each well. Calculate the cell viability as the ratio of the absorbance value of the experimental group to the absorbance value of the control group.

[0073] The results are as Figure 8 shown. When the concentration of the cyclic peptide is in the range of 16 μM - 64 μM, it can promote the proliferation of mouse fibroblast NIH-3T3, and the difference is statistically significant (P<0.05)( Figure 8 A); when the concentration of the cyclic peptide is 64 μM, it can promote the proliferation of human umbilical vein endothelial cells HUVEC, and the difference is statistically significant (P<0.05)( Figure 8 B).

[0074] Example 6. The cyclic peptide derived from fungi described in the present invention promotes the healing of infectious wounds in mice

[0075] To evaluate the therapeutic effect of the cyclic peptides of the present invention, a mouse infectious full-thickness skin lesion model was constructed. Mice aged between 7 and 8 weeks were housed in a specific pathogen-free environment with standard diet and water. All procedures were approved by the Experimental Animal Ethics Committee of Renmin Hospital of Wuhan University (approval number: WDRM 20240807 A). After anesthesia with isoflurane, the backs of the mice were shaved and disinfected with 75% ethanol. Under sterile conditions, two full-thickness wounds were symmetrically excised from the left and right sides of the dorsal skin of the mice using a circular skin sampler, with each wound having a diameter of 6 mm. Then, the wounds were infected with 50 μL of MRSA ATCC3400 suspension (1×10 8 CFU / mL). Two days later, 50 μL of the solution containing the cyclic peptide was dropped onto the left wound, and the same volume of sterile PBS was dropped onto the right wound using a sterile syringe. After natural absorption for 20 minutes, the wounds were covered with sterile gauze. The drug was administered once every 48 hours. The appearance of the wounds was recorded on days 0, 2, 4, 7, 11, and 14, and the wound healing rate was quantified using Image J software. The in vivo antibacterial effect of the cyclic peptide was evaluated by plating the number of MRSA in the wound rinsate. On days 0, 2, 4, and 7, the wounds were rinsed with PBS and then cultured in solid medium, and the in vivo antibacterial effect was observed by the change in the number of colony-forming units on the culture plates.

[0076] The results showed that the cyclic peptide derived from fungi of the present invention could significantly accelerate wound healing ( Figure 9 A), and there were significant differences on days 4, 7, and 11 (P<0.05) ( Figure 9 B). The results of the in vivo antibacterial effect study showed that starting from day 4, the number of colonies cultured on the culture plates in the cyclic peptide treatment group of the present invention was significantly less than that in the control group; by day 7, MRSA was completely cleared, indicating that the cyclic peptide also had good in vivo antibacterial activity when applied topically ( Figure 9 )

[0077] Example 7. Tablets

[0078] 1 g of the cyclic peptide derived from fungi of the present invention, 22 g of starch, and 3 g of dextrin were mixed, and a pharmaceutical-grade polyvinylpyrrolidone (PVP) with a mass concentration of 65% was used as a binder to granulate, size the granules, and press into tablets to obtain the tablets.

[0079] Example 8. Cream

[0080] 0.5 g of the cyclic peptide derived from fungi of the present invention, 1.0 g of glyceryl monostearate, 2.5 g of stearic acid, 0.2 g of white petrolatum, 1.2 g of liquid paraffin, 1.0 g of lanolin, 0.1 g of triethanolamine, and 15 mL of distilled water were mixed evenly to obtain the cream.

[0081] Example 9. Gel

[0082] Take 5 mL of distilled water, sprinkle an appropriate amount of carbomer (about 0.2 g) on the liquid surface, place it in a water bath at 45 °C and stir to make it fully swell. Add propylene glycol and mix evenly, then add triethanolamine dropwise with stirring to form a gel matrix; dissolve 0.2 g of the fungal-derived cyclic peptide of the present invention in PBS buffer solution with a concentration of 0.15 M (pH = 7.2), and slowly add it to the gel matrix with continuous stirring. Finally, add the remaining distilled water to prepare 10.0 g of the cyclic peptide gel.

[0083] Example 10, freeze-dried powder for injection

[0084] Take 3 g of the fungal-derived cyclic peptide of the present invention and 18 g of mannitol, place them in a container, dissolve them with an appropriate amount of PBS buffer solution (0.15 M, pH 7.4), add injection water to 250 mL, shake well, add 10 - 15 g of activated carbon for injection, stir gently at room temperature for about 60 minutes, filter roughly, filter and sterilize with a 0.22 μm sterilizing filter membrane, dispense into vials, 2.0 mL per vial, use the quick-freezing method, cool down 10 - 15 °C per minute, cool down to -45 - 50 °C, maintain for 1 hour, and evacuate. After freeze-drying for 12 - 18 hours, slowly warm up under vacuum state, with a warming rate of 4 - 5 °C per hour, stop warming up when the temperature rises to room temperature (25 °C), take it out after the temperature approaches room temperature, cover and seal to obtain the freeze-dried powder injection.

[0085] Finally, it should also be noted that the terms "comprise", "include" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device.

[0086] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0087] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A cyclic polypeptide, characterized in that, The cyclic polypeptide is derived from the fungus Blastomyces dermatitidis, and its nucleotide sequence is as shown in SEQ ID NO.

1. The linking mode of disulfide bonds in the cyclic polypeptide is: Cys1-Cys4, Cys2-Cys5, Cys3-Cys6.

2. Use of the cyclic polypeptide according to claim 1 in the preparation of an antimicrobial agent, characterized in that, The microorganism includes at least one of Staphylococcus aureus, Staphylococcus epidermidis, and Micrococcus luteus.

3. Use of the cyclic polypeptide according to claim 1 in the preparation of an anti-inflammatory product.

4. Use of the cyclic polypeptide according to claim 1 in the preparation of a product for promoting cell proliferation.

5. Use of the cyclic polypeptide according to claim 1 in the preparation of a product for promoting the healing of infectious wounds.

6. The application according to claims 2-5, characterized in that, The product includes at least one of drugs, cosmetics, or daily necessities.

7. A product for antioxidant or promoting wound repair or healing, characterized in that, It includes the cyclic polypeptide according to claim 1.

8. The product according to claim 7, characterized in that, The product further includes pharmaceutically or cosmetically acceptable salts and / or acceptable excipients.

9. A pharmaceutical composition comprising the cyclic polypeptide according to claim 1, characterized in that, The composition is an ointment, a gel, an injection, or a lyophilized powder injection.

10. A method for preparing the cyclic polypeptide according to claim 1, characterized in that, The method includes: Purifying the linear polypeptide shown in SEQ ID NO.1 by solid-phase synthesis to obtain the purified polypeptide; Oxidizing the purified polypeptide in Tris-HCl buffer to form three pairs of intramolecular disulfide bonds; Purifying by semi-preparative HPLC and freeze-drying to obtain the cyclic polypeptide derived from the fungus Blastomyces dermatitidis.

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