A polyarginine modified peptide derived from the traditional Chinese medicine American cockroach and its application

Abstract

The present invention provides a polyarginine modified peptide derived from the traditional Chinese medicine American cockroach and its application. The nucleotide sequence of the polyarginine modified peptide derived from the traditional Chinese medicine American cockroach is shown in SEQ ID NO.1. The modified peptide can be prepared by using a polypeptide solid phase synthesis technology, and has the characteristics and advantages of mature preparation technology, high yield, good purity, strong stability, etc. At the same time, it has a wide antibacterial spectrum and has good antibacterial (including Gram-positive cocci and Gram-negative bacilli) and antifungal (Candida fungi) activities. MTT experiments and hemolytic activity studies show that it also has low cytotoxicity and has good application prospects and development value.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a polyarginine modified peptide derived from the traditional Chinese medicine American cockroach and application thereof. Background Art

[0002] Infections caused by pathogenic microorganisms, especially infections caused by drug-resistant bacteria, fungi and viruses, are currently a serious threat to people's health and a thorny problem that clinicians in medical institutions will encounter in the process of treating diseases. In September 2004, Professor Wang Fu from the Institute of Antibiotics of Huashan Hospital Affiliated to Fudan University and eight domestic hospitals that have been conducting bacterial resistance monitoring for many years jointly established CHINET. Currently, the monitoring network has 71 member units. Data from the monitoring network show that in 2022, member units isolated a total of 339,513 strains of clinical pathogenic bacteria, and the top five isolated strains were: Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii.

[0003] Regarding fungal surveillance, the National China Hospital Invasive Fungal Surveillance Net (CHIF-NET) was established in 2009. It was the first domestic fungal surveillance network organized by Peking Union Medical College Hospital and 12 well-known tertiary-level A hospitals in China. Currently, more than 100 medical institutions have been included in the network, covering 31 provinces across the country. Based on CHIF-NET, the National Health Commission announced the establishment of the China Fungal Disease Surveillance System (CFDSS) in 2019 and formally commissioned the Peking Union Medical College Hospital of the Chinese Academy of Medical Sciences as the national center of the surveillance network. In actual clinical work, fungal infections mainly occur in immunocompromised populations, such as cancer patients and post-transplant patients. Fungal infections bring serious economic burdens to the healthcare system. It is estimated that the medical expenses for fungal diseases in the United States in 2017 reached 7.2 billion US dollars. Among the infections caused by fungi, invasive fungal diseases (IFD) pose the greatest direct threat to human health, especially immunocompromised populations. Candida, Cryptococcus, and Aspergillus are the main pathogens that cause IFD in clinical practice. In recent years, emerging opportunistic fungi can also cause life-threatening infections. For example, Candida auris has become a public threat worldwide due to its multidrug resistance and small-scale outbreaks reported in medical institutions.

[0004] At present, the antimicrobial drugs used in clinical practice include antibacterial and antifungal drugs, which are mainly divided into antibiotics, semi-synthetic or synthetic antimicrobial agents. With the long-term use of traditional anti-hygiene drugs, especially the unreasonable abuse, the isolation rate of clinical drug-resistant strains has become increasingly high. In order to delay the emergence of bacterial resistance and avoid the lack of drugs in the future, while actively responding to the challenges brought by microbial resistance, implement the "Biosafety Law of the People's Republic of China", and better protect people's health, on October 25, 2022, the National Health Commission and 13 other departments jointly formulated the "National Action Plan for Containing Microbial Resistance (2022-2025)", which pointed out that "promote the research and development and transformation and application of new antimicrobial drugs, diagnostic tools, vaccines, and antimicrobial drug substitutes. Establish a joint research mechanism for multidisciplinary collaborative innovation, promote the output and transformation of key technologies and major products for the prevention and control of microbial resistance", "support the diagnosis, treatment and prevention and control of drug-resistant bacterial infections, including new treatment options, prevention and control strategies for drug-resistant bacterial infections, and post-marketing evaluation of antimicrobial drugs".

[0005] Therefore, researching and developing new safe, effective and cheap antimicrobial drugs is a feasible way to solve the above problems, and biological drugs (such as antimicrobial peptides) are a practical strategy. Antimicrobial peptides can act on the membrane structure of bacteria. Therefore, compared with traditional antibiotics and synthetic antibacterial agents, they are not easy to develop drug resistance. Most of the antimicrobial peptides previously developed have stopped at the basic research stage due to their low stability, high hemolytic activity, and certain immunogenicity, and rarely enter the subsequent new drug development and clinical research.

[0006] Therefore, it is necessary to develop a new type of safe, effective and inexpensive antimicrobial drug. Summary of the invention

[0007] The present invention aims to provide a polyarginine modified peptide derived from the traditional Chinese medicine American cockroach and its application, which has a wide antibacterial spectrum and good antibacterial (including Gram-positive cocci and Gram-negative bacilli) and antifungal (Candida fungi) activities. MTT experiments and hemolytic activity studies show that it also has low cytotoxicity and has good application prospects and development value.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] In a first aspect of the present invention, a poly-arginine modified peptide derived from the traditional Chinese medicine American cockroach is provided. The nucleotide sequence of the poly-arginine modified peptide derived from the traditional Chinese medicine American cockroach is shown in SEQ ID NO.1.

[0010] As a specific embodiment, the preparation method of the polyarginine modified peptide derived from the traditional Chinese medicine American cockroach is:

[0011] Through amide bond linkage, three arginines were introduced in front of the N-terminus (amino terminus) of the natural polypeptide isolated from the American cockroach, i.e., tri-arginine modification. The sequence of the complete modified peptide is RRRLRHKVYGYCVLGP (SEQ ID NO. 1). The molecular design idea is as follows:

[0012]

[0013] The tri-arginine modified peptide was prepared by peptide solid phase synthesis technology. It consists of 16 amino acid residues, has a theoretical molecular weight of 1973.38 Da, and an isoelectric point pI of 10.94.

[0014] The natural polypeptide template involved in the present invention is derived from the natural polypeptide separated from the body of the traditional Chinese medicine American cockroach Periplaneta americana. The natural polypeptide consists of 13 amino terminal residues, and its sequence is: LRHKVYGYCVLGP.

[0015] (Reference: Kim IW, Lee JH, Subramaniyam S, Yun EY, Kim I, Park J, HwangJS. De Novo Transcriptome Analysis and Detection of Antimicrobial Peptides of the American Cockroach Periplaneta americana (Linnaeus).PLoS One.2016,11(5):e0155304.doi:10.1371 / journal.pone.0155304.)

[0016] The preparation method has reasonable raw material selection, low price and easy availability, simple preparation process, and easy large-scale preparation and industrialization.

[0017] In other embodiments, the poly-arginine modified peptide derived from the traditional Chinese medicine American cockroach can also be prepared by other methods.

[0018] In a second aspect of the present invention, there is provided a use of the polyarginine modified peptide derived from the traditional Chinese medicine American cockroach in the preparation of a product for inhibiting fungi and / or bacteria.

[0019] Furthermore, the bacteria include Gram-positive bacteria and Gram-negative bacteria, the Gram-positive bacteria include at least one of Staphylococcus aureus, Staphylococcus epidermidis and clinically isolated methicillin-resistant Staphylococcus aureus; the Gram-negative bacteria include at least one of Escherichia coli, Pseudomonas aeruginosa and clinically isolated Acinetobacter baumannii.

[0020] Further, the fungus includes at least one of Candida albicans, Candida parapsilosis and clinically isolated Candida parapsilosis.

[0021] Furthermore, the product for inhibiting fungi and / or bacteria includes antimicrobial drugs or daily chemicals.

[0022] In the third aspect of the present invention, a product for inhibiting fungi and / or bacteria is provided, comprising the polyarginine modified peptide derived from the traditional Chinese medicine American cockroach.

[0023] Furthermore, the product for inhibiting fungi and / or bacteria includes antimicrobial drugs or daily chemicals, and the product also includes salts and / or excipients acceptable in medicine or daily chemicals.

[0024] The product for inhibiting fungi and / or bacteria can be prepared into various pharmaceutical dosage forms or / and daily chemical products such as injections, sterile powders for injection, powders, ointments, creams, gels, microneedles, etc. according to conventional preparation methods.

[0025] Furthermore, the product for inhibiting fungi and / or bacteria can be introduced into muscle, endothelial, subcutaneous or mucosal tissues by injection, smearing, application, acupuncture, and other physical or chemical mediated methods, or can be mixed with and / or wrapped with other substances and then introduced into the body.

[0026] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0027] The present invention provides a polyarginine modified peptide derived from the traditional Chinese medicine American cockroach and its application. The present invention introduces three arginines in front of the N-terminus (amino terminus) of the natural polypeptide (LRHKVYGYCVLGP) separated from the American cockroach through an amide bond link, i.e., a tri-arginine modified peptide (RRRLRHKVYGYCVLGP). The polyarginine modified peptide derived from the traditional Chinese medicine American cockroach of the present invention has stronger antibacterial activity and a wider antibacterial spectrum than the natural polypeptide. The modified peptide can be prepared by using a polypeptide solid phase synthesis technology, and has the characteristics and advantages of mature preparation technology, high yield, good purity, strong stability, etc. At the same time, it has a wide antibacterial spectrum and has good antibacterial (including Gram-positive cocci and Gram-negative bacilli) and antifungal (Candida fungi) activities. MTT experiments and hemolytic activity studies show that it also has lower cytotoxicity and has good application prospects and development value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0029] Figure 1 HPLC chart of the polyarginine modified peptide derived from the traditional Chinese medicine American cockroach prepared by the present invention;

[0030] Figure 2 The mass spectrum of the polyarginine modified peptide derived from the traditional Chinese medicine American cockroach prepared by the present invention;

[0031] Figure 3 The polyarginine modified peptide derived from the traditional Chinese medicine American cockroach is used to resist Gram-positive bacteria;

[0032] Figure 4 The polyarginine modified peptide derived from the traditional Chinese medicine American cockroach is used to resist Gram-negative bacteria;

[0033] Figure 5 The antifungal effect of the polyarginine modified peptide derived from the traditional Chinese medicine American cockroach of the present invention;

[0034] Figure 6 To evaluate the toxicity of the polyarginine modified peptide derived from the traditional Chinese medicine American cockroach on fibroblasts;

[0035] Figure 7 To evaluate the toxicity of the polyarginine modified peptide derived from the traditional Chinese medicine American cockroach on endothelial cells;

[0036] Figure 8This is the hemolytic activity result of the polyarginine modified peptide derived from the traditional Chinese medicine American cockroach of the present invention. DETAILED DESCRIPTION

[0037] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0038] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0039] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0040] The following will describe in detail a polyarginine modified peptide derived from the traditional Chinese medicine American cockroach and its application in combination with examples and experimental data.

[0041] Example 1. Preparation of the polyarginine modified peptide RRRLRHKVYGYCVLGP from the Chinese medicine American cockroach according to the present invention

[0042] In the synthesis of this linear peptide segment, 9-fluorenylmethyloxycarbonyl (FMOC) was used as the protecting group of the amino terminus, 4-methyl-benzhydrylamine resin·HCl (MBHAresin) was selected as the solid phase carrier for peptide synthesis, and HOBt / DCC was used as the condensing agent for the reaction to extend the peptide chain from the carboxyl terminus to the amino terminus (C terminus → N terminus). The polyarginine modified peptide containing 16 amino acid residues was cleaved from the MBHA resin using a mixture of 92.0% trifluoroacetic acid, 3.5% water and 6.5% triisopropylsilane (TIA) (mass percentage). After repeated precipitation with ether, it was purified by preparative reverse phase high performance liquid chromatography RP-HPLC. Purification process: C18 reverse phase preparative column (250mm×20mm, 5μm) was used; mobile phase: 2.5‰ trifluoroacetic acid, 0%~55% (volume percentage) acetonitrile as the mobile phase, and gradient elution was performed at a flow rate of 2.5mL / min. The retention time (RT) was 9.42 min after RP-HPLC detection, and the purity was >95% ( Figure 1 The main peak eluate was collected, frozen at -80°C for 12 hours, and then placed in a freeze dryer for freeze drying overnight.

[0043] The molecular weight of the product was identified by ESI-Q-TOF-MS, which was consistent with the theoretical molecular weight of the peptide intermediate to be prepared, m / z 987.06 [M+2H] 2+ ,m / z 658.34[M+3H] 3+ ,m / z 494.83[M+4H] 4+ ( Figure 2 ).

[0044] Example 2: Determination of the anti-Gram-positive bacterial activity of the polyarginine modified peptide derived from the traditional Chinese medicine American cockroach according to the present invention

[0045] The Gram-positive bacteria to be tested include standard strains (Staphylococcus aureus S.aureus GDMCC1.178 and Staphylococcus epidermidis S.epidermidis AB 208187) and clinically isolated methicillin-resistant Staphylococcus aureus (MRSA). The standard strains were purchased from Guangdong Microbial Culture Collection Center (GDMCC), and the clinically isolated drug-resistant pathogens were provided by the Laboratory Medicine Center of Renmin Hospital of Wuhan University. The minimum inhibitory concentration (MIC) was determined by the multiple dilution method, and the antibacterial activity of the polyarginine modified peptide was characterized by the MIC value. The specific method is as follows:

[0046] (1) The strain to be tested was inoculated into sterile Luria-Bertani (LB) solid medium plates using the three-zone streak method and incubated in an incubator at 37°C for 12 hours. Single colonies were picked up with an inoculation loop and transferred to newly prepared sterile liquid LB medium and cultured at 37°C and 180 rpm with shaking until the logarithmic growth phase. The absorbance of the bacterial solution at a wavelength of 600 nm (OD) was measured using an ELISA reader. 600 ), according to 1OD≈1×10 9 The conversion relationship of CFU / mL was calculated by diluting the bacterial suspensions of S. aureus, S. epidermidis standard strains and clinical drug-resistant strain MRSA with sterile liquid culture medium to a concentration of 2×10 6 CFU / mL.

[0047] (2) First, add 75 μL of sterilized LB medium to a sterile 96-well plate; then, add 75 μL of a 128 μmol / L peptide solution dissolved in sterilized LB medium to the first well, mix well, and add 75 μL to the second well, and repeat the steps one by one. Finally, aspirate 75 μL from the sixth well and discard it, and then dilute it twice in sequence; finally, add a diluted solution with a concentration of 2×10 6 75 μL of CFU / mL bacterial solution was mixed evenly. The wells without peptide were used as negative controls.

[0048] (3) The above groups were cultured at 37°C and 150 rpm for 12 to 18 hours, and the absorbance value OD at a wavelength of 600 nm was measured. 600 The minimum inhibitory concentration (MIC) is taken as the minimum inhibitory concentration at which no bacterial growth can be detected (OD 600 The lowest final concentration of polyarginine modified peptides from the Chinese medicine American cockroach in the wells with a concentration of ≤0.8. The bacterial solution in the negative control wells was turbid, and the absorbance OD at 600nm was 600 >3.00. The absorbance of each well of modified peptide with different concentrations is as follows Figure 3 As shown, the MIC value results of the poly-arginine modified peptide derived from the traditional Chinese medicine American cockroach against Gram-positive bacteria are shown in Table 1.

[0049] Table 1 - Anti-G peptides derived from the American cockroach polyarginine modified peptides of the present invention and natural peptides before modification + Bacterial activity

[0050]

[0051] From Table 1, we can see that:

[0052] The polyarginine modified peptide derived from the traditional Chinese medicine American cockroach of the present invention is resistant to the above three G + The MIC values ​​of the strains (Staphylococcus aureus, Staphylococcus epidermidis, and methicillin-resistant Staphylococcus aureus) were 4 μM, 8 μM, and 16 μM, respectively.

[0053] The unmodified American cockroach natural peptides are resistant to the three G + The MIC values ​​of the strains were 16μM, 32μM, and 32μM, respectively.

[0054] It can be seen from the above that the polyarginine modified peptide derived from the traditional Chinese medicine American cockroach described in the present invention has stronger antibacterial activity than natural polypeptides.

[0055] Example 3: Determination of the anti-Gram-negative bacterial activity of the polyarginine modified peptide derived from the traditional Chinese medicine American cockroach according to the present invention

[0056] The Gram-negative bacteria to be tested include standard strains (Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC27853) and clinically isolated Acinetobacter baumannii. The standard strains were purchased from Guangdong Microbial Culture Collection Center (GDMCC), and the clinically isolated drug-resistant pathogens were provided by the Laboratory Medicine Center of Renmin Hospital of Wuhan University. The minimum inhibitory concentration (MIC) was determined by the multiple dilution method, and the antibacterial activity of the polyarginine modified peptide was characterized by the MIC value. The specific method is the same as Example 2.

[0057] The absorbance of each well of poly-arginine modified peptide with different concentrations is as follows Figure 4 As shown, the MIC value results of the polyarginine modified peptide derived from the traditional Chinese medicine American cockroach against Gram-positive bacteria are shown in Table 2.

[0058] Table 2 - Anti-G - Bacterial activity

[0059]

[0060] From Table 2, we can see that:

[0061] The polyarginine modified peptide derived from the traditional Chinese medicine American cockroach of the present invention is resistant to the above three G - The MIC values ​​of the strains (Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii) were 32 μM, 16 μM, and 32 μM, respectively.

[0062] The unmodified American cockroach natural peptide had no significant effect on the three G - The strains had no antibacterial effect.

[0063] It can be seen from the above that the polyarginine modified peptides of the present invention have a wider antibacterial activity than natural polypeptides.

[0064] Example 4: Determination of antifungal activity of the polyarginine modified peptide derived from the traditional Chinese medicine American cockroach according to the present invention

[0065] The fungal strains to be tested include standard strains of Candida (Candida albicans ATCC10231, Candida parapsilosis ATCC 22019) and clinically isolated Candida parapsilosis C. parapsilosis. The standard strains were purchased from Guangdong Microbial Culture Collection Center (GDMCC), and the clinically isolated drug-resistant pathogens were provided by the Laboratory Medicine Center of Renmin Hospital of Wuhan University. The minimum inhibitory concentration (MIC) was determined by the doubling dilution method, and the antibacterial activity of the polyarginine modified peptide derived from the traditional Chinese medicine American cockroach was characterized by the MIC value.

[0066] The Candida fungi were cultured in Sabouraud Dextrose Broth (SDB) at a temperature of 28° C. The specific method was the same as in Example 2.

[0067] The absorbance of each well of modified peptide with different concentrations is as follows Figure 5 As shown, the antifungal MIC values ​​of the polyarginine modified peptides derived from the traditional Chinese medicine American cockroach described in the present invention are shown in Table 3.

[0068] Table 3 - Polyarginine modified peptides anti-G - Bacterial activity

[0069]

[0070] From Table 3, we can see that:

[0071] The MIC values ​​of the poly-arginine modified peptide of the present invention against the above three strains are 32 μM, 16 μM and 32 μM respectively.

[0072] The unmodified natural peptide from American cockroach had no antibacterial effect on the other two fungi except Candida parapsilosis ATCC 22019, with an MIC value of 32 μM.

[0073] It can be seen from the above that the polyarginine modified peptides of the present invention have a wider antibacterial activity than natural polypeptides.

[0074] Example 5: Evaluation of the toxicity of the polyarginine modified peptide derived from the traditional Chinese medicine American cockroach to fibroblasts

[0075] The MTT method was used to detect the toxicity of the polyarginine modified peptide from the traditional Chinese medicine American cockroach on mouse fibroblast NIH-3T3 after intervention. The specific operation was as follows:

[0076] (1) Digest the cells in the logarithmic phase with trypsin for 50 seconds, add culture medium to stop digestion, and gently disperse and suspend the cells with a pipette. Centrifuge the cell suspension at 1000 rpm for 3 minutes, discard the supernatant, add an appropriate amount of culture medium to resuspend the precipitate to make a cell suspension, and adjust the cell count to an appropriate concentration.

[0077] (2) After the cell suspension is prepared, mix it gently, take a 96-well plate, add 100 μL of cell suspension (5000 cells / well) to each well, place the culture plate in a 37°C, 5% CO2 incubator, and culture for 4-6 hours to allow the cells to adhere to the wall. Carefully aspirate and discard the culture medium. Control group (Group A): only add low-serum culture medium (~3% FBS) without poly-arginine modified peptide; experimental group (Group BH): add 100 μL of low-serum culture medium containing poly-arginine modified peptide (the final concentrations of poly-arginine modified peptide in Group BH are 1 μM, 2 μM, 4 μM, 8 μM, 16 μM and 32 μM, respectively). Each group is a column, and 6 duplicate wells are set. Use sterile PBS to seal the edges of the experimental wells to prevent the evaporation of the culture system at the edge of the experimental wells due to the long experimental time.

[0078] (3) After 12 hours of incubation, add 10 μL of MTT solution (5 mg / ml, i.e., 0.5% MTT) to each well and continue incubation for 4-5 hours. Aspirate and discard the supernatant, add 150 μL of DMSO to each well, and incubate at 37°C and 100 rpm in a shaker for 10-15 minutes to fully dissolve the crystals. Use an ELISA reader to measure the ultraviolet absorption at a wavelength of 490 nm.

[0079] Since the succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystalline formazan and deposit it in the cells, while dead cells do not have this function. Therefore, compared with the absorbance ratio of the control group A (cell viability is 100%), it can reflect the effect of different concentrations of the polyarginine modified peptide derived from the traditional Chinese medicine American cockroach on the viability of mouse fibroblast NIH-3T3 cells.

[0080] The results are as follows Figure 6 As shown, different concentrations of poly-arginine modified peptides derived from the traditional Chinese medicine American cockroach had no effect on the viability of the cells, and even at a high concentration of 32 μmol / L, there was no obvious toxicity to the NIH-3T3 cell line.

[0081] Example 6: Evaluation of the toxicity of the polyarginine-modified peptide derived from the traditional Chinese medicine American cockroach on endothelial cells

[0082] The MTT method was used to detect the effect of the polyarginine modified peptide derived from the traditional Chinese medicine American cockroach on the activity of human umbilical vein endothelial cells (HUVEC) after intervention.

[0083] For specific operation steps, refer to the steps and methods of the MTT experiment in Example 5:

[0084] The results are as follows Figure 7 As shown, different concentrations of polyarginine-modified peptides derived from the traditional Chinese medicine American cockroach had no effect on the viability of the cells. Even at a high concentration of 32 μmol / L, there was no obvious toxicity to the immune cell line, and at this concentration, there was a certain effect of promoting the proliferation of HUVEC (P<0.05).

[0085] Example 7. Hemolytic activity results of the polyarginine modified peptide derived from the traditional Chinese medicine American cockroach according to the present invention

[0086] Fresh blood was obtained from a healthy male (the inventor of this patent), and human red blood cells were separated and prepared into a suspension. Sterile saline was used as a negative control and sterile water for injection was used as a positive control to determine the hemolytic activity of the polyarginine-modified peptide derived from the traditional Chinese medicine American cockroach.

[0087] The specific steps are as follows: collect whole blood with a heparin anticoagulation tube, gently invert the blood to fully anticoagulate, centrifuge at room temperature at 800rpm for 5min, absorb the upper plasma as much as possible and retain the lower red blood cells; add 3 times the volume of sterile saline, gently blow and suck with a pipette to suspend the red blood cells at the bottom, centrifuge at room temperature at 800rpm for 3min, discard the supernatant and retain the precipitated red blood cells, repeat the operation 3 times until the supernatant is colorless; prepare the red blood cells into a 2% (v / v) cell suspension with sterile saline; configure the polyarginine modified peptide derived from the traditional Chinese medicine American cockroach of the present invention into solutions with concentrations of 64μmol / L, 32μmol / L, 16μmol / L, 8μmol / L, 4μmol / L and 2μmol / L with sterile saline; add 75μL of the polyarginine modified peptide The amino acid modified peptide solution and 75 μL of red blood cell suspension were mixed and added to a 96-well plate, and the final concentrations of the peptides were 32 μmol / L, 16 μmol / L, 8 μmol / L, 4 μmol / L, 2 μmol / L and 1 μmol / L, respectively; the negative control group was red blood cells suspended in sterile saline, and the positive control was red blood cells suspended in sterile water for injection; the samples were placed in a constant temperature shaker and incubated at 100 rpm for 45 min at (36.5±0.5)°C; the samples were centrifuged at 4000 rpm for 3 min at room temperature, and 150 μL of supernatant was taken from each well and transferred to another 96-well plate; the absorbance at a wavelength of 490 nm was measured with a full-wavelength microplate reader, and the hemolysis percentage was calculated by the following formula, where H is the absorbance at a wavelength of 490 nm: Hemolysis rate (%) = (H sample -H negative ) / (H positive -H negative )×100%, such as Figure 8 shown.

[0088] The results showed that the polyarginine modified peptide derived from the traditional Chinese medicine American cockroach of the present invention did not show obvious hemolysis (hemolysis rate ≤ 8%) when the final concentration was as high as 32 μmol / L.

[0089] Example 8. Preparation of different dosage forms

[0090] 1. Tablets

[0091] 0.5 g of the polyarginine modified peptide derived from the traditional Chinese medicine American cockroach of the present invention, 8 g of starch and 3.5 g of dextrin are mixed, 40% of pharmaceutical grade polyvinyl pyrrolidone (PVP) is used as a binder, granulated, granulated and tableted to obtain tablets.

[0092] 2. Cream

[0093] Take 0.5 g of the polyarginine modified peptide derived from the traditional Chinese medicine American cockroach, 1.0 g of glyceryl monostearate, 2.5 g of stearic acid, 0.2 g of white vaseline, 1.2 g of liquid paraffin, 1.0 g of lanolin, 0.1 g of triethanolamine, and 15 mL of distilled water, and mix well to obtain the product.

[0094] 3. Gel

[0095] Take 5 mL of distilled water, sprinkle carbomer on the liquid surface, warm water bath at 40°C and stir to fully swell. Add propylene glycol and mix evenly, then add triethanolamine while stirring to form a gel matrix; dissolve 0.1 g of the polyarginine modified peptide derived from the traditional Chinese medicine American cockroach in PBS buffer (pH=7.4) with a concentration of 0.15 M, and slowly add it to the gel matrix under continuous stirring. Finally, add the remaining distilled water to obtain 10.0 g of the modified peptide gel.

[0096] 4. Lyophilized powder for injection

[0097] 3 g of the polyarginine modified peptide derived from the traditional Chinese medicine American cockroach and 15 g of mannitol are taken, placed in a container, an appropriate amount of PBS buffer (0.15 M, pH 7.4) is added to dissolve, water for injection is added to 250 mL, shaken well, 8-10 g of activated carbon for injection is added, the mixture is gently stirred at room temperature for about 60 minutes, coarsely filtered, sterilized by filtering with a 0.22 μm sterilizing filter membrane, and packaged in 2.0 mL bottles. The mixture is quickly frozen by cooling by 10-15° C. per minute to -45--50° C., maintained for 3 hours, vacuumed, and slowly heated under vacuum at a heating rate of 2-6° C. per hour. When the temperature reaches room temperature (-25° C.), the mixture is stopped, taken out after the temperature approaches room temperature, and sealed with a cover to obtain a lyophilized powder injection.

[0098] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0099] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0100] 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 equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A polyarginine modified peptide derived from the traditional Chinese medicine American cockroach, characterized in that: The amino acid sequence of the polyarginine modified peptide derived from the traditional Chinese medicine American cockroach is shown in SEQ ID NO.

1.

2. Use of the polyarginine modified peptide derived from the traditional Chinese medicine American cockroach according to claim 1 in the preparation of a product for inhibiting fungi and / or bacteria, wherein the bacteria include Gram-positive bacteria and Gram-negative bacteria, the Gram-positive bacteria include at least one of Staphylococcus aureus, Staphylococcus epidermidis and clinically isolated methicillin-resistant Staphylococcus aureus; the Gram-negative bacteria include at least one of Escherichia coli, Pseudomonas aeruginosa and clinically isolated Acinetobacter baumannii; the fungi include at least one of Candida albicans, Candida parapsilosis and clinically isolated Candida parapsilosis.

3. The use according to claim 2, characterized in that: The products for inhibiting fungi and / or bacteria include antimicrobial drugs or daily chemicals.

4. A product for inhibiting fungi and / or bacteria, characterized in that The invention comprises the poly-arginine modified peptide derived from the traditional Chinese medicine American cockroach as described in claim 1.

5. The product according to claim 4, characterized in that The product for inhibiting fungi and / or bacteria includes antimicrobial drugs or daily chemicals, and the product also includes salts and / or excipients acceptable in medicine or daily chemicals.

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