Safe preservative-free sea dragon antibacterial peptide spray and preparation method and application thereof

Abstract

The application discloses a safe preservative-free sea dragon antibacterial peptide spray and a preparation method and application thereof. The spray contains sea dragon antibacterial peptide SsPle as an active ingredient and excipients. The amino acid sequence of the sea dragon antibacterial peptide SsPle is FFRSLWKGVKEGFRAGRSAYKA. The antibacterial peptide spray has the characteristics of safety, non-toxicity, non-inclusion of traditional preservatives, good stability, convenient use and the like. The antibacterial peptide has no acute oral toxicity, no mutagenicity and no chromosome damage risk, and has high biological safety. The spray is stable at 30 DEG C for 120 days, and still has a significant bacteriostatic effect on pseudomonas aeruginosa, methicillin-resistant staphylococcus aureus (MRSA) and the like. The preparation process is simple, the spray can be filled by using a quantitative sprayer, and the spray is convenient for clinical treatment, wound care and daily disinfection application. The application solves the problem of irritation of traditional antibacterial peptide sprays, is suitable for sensitive people, and has a broad market prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of antibacterial, and particularly relates to a safe preservative-free sea dragon antimicrobial peptide spray and a preparation method and application thereof. BACKGROUND

[0002] Antimicrobial peptides (AMP) are a class of small molecule proteins widely existing in prokaryotes and eukaryotes, which have a wide range of inhibitory effects on bacteria, fungi and viruses. They play a role by destroying bacterial cell membranes, regulating immune responses and regulating inflammation, etc. Compared with traditional antibiotics, antimicrobial peptides have unique advantages, especially their resistance to bacterial resistance, mainly due to their target being microbial membranes rather than specific molecular targets. Therefore, antimicrobial peptides are considered as potential candidate drugs to replace traditional antibiotics and cope with multi-drug resistant bacterial infections.

[0003] Syngnathus schlegeli antimicrobial peptide SsPle is an antimicrobial peptide identified by the research team in the previous study, which is composed of 22 amino acid residues. The SsPle antimicrobial peptide synthesized by solid phase chemistry has good water solubility, and has excellent bactericidal activity under high temperature, high salt, acidic and alkaline conditions. Previous experiments have found that the SsPle antimicrobial peptide has significant antibacterial activity against common pathogenic bacteria such as methicillin-resistant Staphylococcus aureus, multi-drug resistant Escherichia coli and Pseudomonas aeruginosa.

[0004] The antimicrobial peptide sprays on the market contain traditional chemical preservatives such as phenoxyethanol, p-hydroxybenzoic acid esters (nipagin) and the like, which may cause skin irritation, swelling, itching, contact dermatitis and other allergic reactions, especially not friendly to sensitive groups. In addition, the use of traditional preservatives may also affect the activity of antimicrobial peptides, reduce their antibacterial effect, and limit their application in clinical practice. SUMMARY

[0005] In order to solve the above technical problems, the present application provides an antimicrobial peptide spray, which takes Syngnathus schlegeli antimicrobial peptide SsPle as the core active ingredient, and has the advantages of safety, non-toxicity, preservative-free, good stability and convenient use, etc.

[0006] The object of the present application is to provide a sea dragon antimicrobial peptide spray containing sea dragon antimicrobial peptide SsPle as an active ingredient, wherein the sea dragon antimicrobial peptide SsPle has an amino acid sequence of: FFRSLWKGVKEGFRAGRSAYKA

[0007] Preferably, the antimicrobial peptide spray takes antimicrobial peptides as active ingredients, and also contains other auxiliary materials such as stabilizers, antioxidants, buffers, pH regulators and preservative systems.

[0008] Preferably, the stabilizer is glycerol or mannitol.

[0009] Preferably, the buffer system is histidine or citrate-citric acid.

[0010] Preferably, the antioxidant is methionine.

[0011] Preferably, the preservative system is caprylhydroxamic acid & hexylene glycol & propylene glycol.

[0012] Preferably, the sea dragon antibacterial peptide spray is a sea dragon antibacterial peptide spray against Methicilin-resistant Escherichia coli, Pseudomonas aeruginosa, staphylocccus aureus; preferably, the staphylocccus aureus is Methicilin-resistant staphylocccus aureus (MRSA).

[0013] Preferably, the formula of the antibacterial peptide spray includes Formula 1: 0.1% sea dragon antibacterial peptide SsPle, 1% glycerol, 0.31% histidine, 0.05% methionine, and finally adding hydrochloric acid to adjust the pH value to pH 6, and the balance is pure water, or Formula 2: 0.1% sea dragon antibacterial peptide SsPle, 1% mannitol, 20 mM citrate-citric acid, 1.0% of the compounded antioxidant and preservative system, and the balance is water.

[0014] The present application provides a safe and preservative-free sea dragon antibacterial peptide spray. The sea dragon antibacterial peptide SsPle has no acute oral toxicity, does not cause bacterial gene mutation or mammalian red blood cell chromosome damage, and does not cause mammalian cell chromosome aberration, and has good biological integrity. The present application provides two formulas, Formula One does not contain preservatives, completely avoiding the skin irritation and allergic reactions that may be caused by traditional preservatives. Formula Two uses a compounded antioxidant and preservative system (caprylhydroxamic acid, hexylene glycol, propylene glycol), does not contain traditional preservatives, is mild to the skin, is easy to operate, and is very suitable for use in formulations that claim to be "preservative-free" or "do not contain nepotin esters".

[0015] The application provides an antibacterial peptide spray which is good in stability, convenient to use and fill, and has excellent inhibitory effect on Pseudomonas aeruginosa, multiple drug-resistant Staphylococcus aureus (MRSA) and the like after being placed at 30 DEG C for 120 days, is good in stability, and is reliable in preparation quality. The spray is convenient to use and can be directly sprayed on a lesion. In addition, the preparation process is simple, and the liquid medicine is filled in a quantitative spray tank. The convenience and stability of the spray make it have a wide application prospect in clinical treatment, wound care and daily disinfection.

[0016] The application solves the problem of irritability of a preservative in a traditional antibacterial peptide spray, provides a safer and more stable antibacterial peptide spray, and has a wide application prospect, and is especially suitable for sensitive people. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is the antibacterial peptide spray of formula one of Seahorse which still has good inhibitory effect after being placed at 30 DEG C for 120 days. The bacterial liquid of the control group Control grows normally, the bacterial liquid becomes turbid, and the OD value thereof increases with the lapse of time. The bacterial liquid is inhibited in growth after being treated with the antibacterial peptide spray solution and SsPle peptide, the bacterial liquid is always clear, and the OD value is kept at a very low level. 600 600

[0018] Figure 2 is the antibacterial peptide spray of formula two of Seahorse which still has good inhibitory effect after being placed at 30 DEG C for 120 days. The bacterial liquid of the control group Control grows normally, the bacterial liquid becomes turbid, and the OD value thereof increases with the lapse of time. The bacterial liquid is inhibited in growth after being treated with the antibacterial peptide spray solution and SsPle peptide, the bacterial liquid is always clear, and the OD value is kept at a very low level. 600 600 DETAILED DESCRIPTION

[0019] The following examples are further illustrations of the application, but not limitations of the application.

[0020] Example 1. Preparation of the antibacterial peptide spray of Seahorse

[0021] (1) Information of raw materials and auxiliary materials

[0022] Information and physicochemical properties of SsPle antibacterial peptide of Seahorse.

[0023] Table 1 Physicochemical properties of SsPle antibacterial peptide of Seahorse

[0024]

[0025] Table 2 Information of auxiliary materials ​​​​

[0026]

[0027] (2) Spray formulation of SsPle and its preparation process

[0028] Formulation ingredients. The formulation ratio is expressed as (w / w, %). Formulation one: 0.1% SsPle, 1% glycerol, 0.31% histidine, 0.05% methionine, and 1M hydrochloric acid was added to adjust the pH to about 6, and the rest was pure water. Formulation two: 0.1% SsPle, 1% mannitol, 20 mM citrate-citric acid, 1.0% complex antioxidant preservative system, and the rest was water.

[0029] Table 3. Ingredient table of formulation one

[0030]

[0031] Table 4. Ingredient table of formulation two

[0032]

[0033] The preparation method of the SsPle spray as described above includes the following steps:

[0034] Preparation process of formulation one: Glycerol and methionine are well soluble in water, and can be quickly dissolved after stirring in 90% of the total weight of purified water. After visual inspection of no solid undissolved material, continue stirring for 5 min. The auxiliary solution can quickly pass through a 0.45 μm filter membrane, and there is no obvious impurity on the filter membrane after filtration, indicating that the auxiliary materials have been completely dissolved. Before adding the antibacterial peptide, add the buffer system histidine-hydrochloric acid to adjust the pH of the auxiliary solution to about 6.0, at which time the total weight of the auxiliary solution is much lower than the final total weight. The antibacterial peptide can be quickly dissolved after being added. After adding purified water to constant volume, the solution is clear and transparent, and the pH value of the solution is still about 6.0 without obvious fluctuation. Finally, filter with a 0.22 μm filter membrane, and then divide and pack in a sterile quantitative sprayer to obtain the antibacterial peptide spray. Then place the antibacterial peptide spray at -20℃ for overnight low temperature treatment, so that the solution freezes, and finally place the spray at 30℃ for 120 days.

[0035] Preparation process of Formula Two: Take 90% of the total weight of purified water, add mannitol and the complex antioxidant and preservative system while stirring, and continue stirring for 5 minutes after the solid undissolved substances are visually removed. Before adding the antimicrobial peptide, add the buffer system citrate-citric acid to adjust the pH of the auxiliary material solution to the target value. At this time, the total weight of the auxiliary material solution is much lower than the final total weight. After adding the antimicrobial peptide, it can be quickly dissolved. After adding purified water to the constant volume, the solution is clear and transparent, and the pH value of the solution is about 4.5. After filtering and sterilizing the solution with a 0.22 μm filter membrane, it is packaged in a sterile quantitative nebulizer to obtain an antimicrobial peptide spray. The entire production process is carried out under non-sterile conditions. Finally, the antimicrobial peptide spray is placed at 30°C for 120 days.

[0036] Example 2. Antimicrobial activity of the sea dragon antimicrobial peptide spray

[0037] Strains: Escherichia coli, Pseudomonas aeruginosa, Methicilin-resistant staphylocccus aureus (MRSA).

[0038] Method: Bacteria were cultured in nutrient broth (NB), and when the bacteria reached the logarithmic growth phase, they were centrifuged at 4000 × g for 5 minutes to collect the bacterial cells. The bacteria were diluted to 10 4 CFU / mL with a mixture of Dulbecco's phosphate buffered saline (DPBS) and hydrolyzed casein medium (MH) (volume ratio 3:2). The diluted bacterial solution was added to a 96-well microbial culture plate, and an equal volume of antimicrobial agent was added. The "Control" group was treated with an equal volume of DPBS; the "SsPle" group was treated with an equal volume of SsPle (48 μM); and the "antimicrobial peptide spray" group was treated with an equal volume of antimicrobial peptide spray solution. The OD 600 values were measured at 0h, 12h, 24h using a microplate reader, and all experiments were repeated three times.

[0039] After the antimicrobial peptide spray was placed at 30°C for 120 days, its antimicrobial activity was detected. The results showed that Figure 1 and Figure 2 The control group (treated with DPBS) grew normally. The antimicrobial peptide spray solutions of Formula One and Formula Two, as well as the SsPle peptide, could significantly inhibit the growth of Escherichia coli, Pseudomonas aeruginosa, and MRSA.

[0040] Example 3. Toxicological study of the sea dragon antimicrobial peptide SsPle

[0041] Entrusted Jiangsu Nanjing Medical University Health Testing Center to conduct acute oral toxicity analysis, bacterial reverse mutation analysis, in vitro mammalian cell chromosome aberration analysis, and mammalian cell gene mutation test on the hailong antibacterial peptide SsPle. The sample of Xue's hailong antibacterial peptide was provided by Jiangsu GenScript Biotechnology Co., Ltd., with the production batch number C3868DQHGO-5 / PE4489. The inspection acceptance number was 24NYHZ-WT070, and the sample name during toxicology testing was SaMoronecidin.

[0042] (1)Acute oral toxicity test

[0043] I. Materials and animals

[0044] 1 Test substance: SsPle peptide white powder, produced by Jiangsu GenScript Biotechnology Co., Ltd., provided by the South China Sea Institute of Oceanology, Chinese Academy of Sciences. The SsPle was dissolved in sterile and endotoxin-free Hanks balanced salt solution to prepare a 1mg / mL solution as the test substance.

[0045] 2 Experimental animals and rearing environment:

[0046] 2.1 Experimental animals: 10 SPF-grade ICR mice, 5 males and 5 females. The body weight of female animals was 18.4g - 21.2g, and the body weight of male animals was 19.4g - 21.2g. They were provided by Nanjing Medical University, with the production license number SCXK (Su) 2021-0001 and the certificate number A202405220023. All animals were acclimated to the environment for 5 days before the experiment, with free access to water and food.

[0047] 2.2 Test environment: The experimental animals were reared in the central barrier system at a temperature of 20 - 26°C and a relative humidity of 30% - 70%. The experimental animal use license number: SYXK (Su) 2020-0006.

[0048] 2.3 Feed: Irradiated and sterilized maintenance feed for experimental mice, provided by Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd., production license: Su饲证 (2019) 01008.

[0049] 3 Test method:

[0050] 3.1 Methods: Based on the single maximum dose design requirements in the testing standard, the dose was set at 5000 mg / kg·bw. Animals were fasted overnight (16 hours) but had free access to water. 5.00 g of sample was taken and diluted with purified water to 10 mL to prepare a concentration of 500 mg / mL. The sample was administered orally once at a dose of 10 mL / kg·bw, resulting in an actual exposure dose of 5000 mg / kg·bw. Five males and five females were used. The animals were observed for 14 days for signs of poisoning and the number of deaths. Animals that did not die were weighed weekly. At the end of the 14-day observation period, a gross necropsy was performed on the animals, and any abnormalities observed were recorded. Purified water source: Millipore pure water system (self-made), batch number: 20240527.

[0051] 3.2 Toxicity evaluation: LD50 50 LD50+ is practically non-toxic; 50 Those with a concentration of 501-5000 mg / kg body weight are considered to have low toxicity; LD50 50 A dose of 51-500 mg / kg body weight is considered moderately toxic; LD50 50 A dose of 50 mg / kg body weight or less is considered highly toxic.

[0052] II. Test Results

[0053] No abnormal signs or deaths were observed in either male or female mice by the end of the observation period. Gross dissections of the surviving animals at the end of the observation period revealed no obvious abnormalities. The effects of the test substance on mouse body weight and mortality are shown in Table 5.

[0054] Table 5. Effects of the test substances on acute oral toxicity in mice ( ±s)

[0055]

[0056] III. Experimental Conclusions

[0057] Acute oral toxicity test of 1 mg / mL SsPle solution in male and female mice: LD50 50 With a concentration greater than 5000 mg / kg·bw, according to the toxicity evaluation provisions of the "Cosmetic Safety Technical Specifications" (2015 edition), this sample is practically non-toxic.

[0058] (2) Salmonella Typhimurium / reverse mutation test

[0059] I. Materials and Methods

[0060] 1. Test strains: Four standard mutant strains of Salmonella Typhimurium TA97a, TA98, TA100 and TA102 were provided by Molecular Toxicology, Inc., with batch numbers 5083D, 5052D, 5008D and 5007D respectively.

[0061] 2. Metabolite activation system: S9 induced by β-naphthylflavonoid and phenobarbital sodium was provided by Jiangsu Qishi Biotechnology Co., Ltd., with a protein content of 30 mg / mL. Batch number: 24FS002C.

[0062] 3. Positive reagents: The positive reagents used in this test are as follows:

[0063] Table 6 Positive samples used in this experiment

[0064]

[0065] 4. Test substance: SsPle, white powder, produced by Jiangsu Genscript Biotech Co., Ltd., and provided by the South China Sea Institute of Oceanology, Chinese Academy of Sciences. SsPle was dissolved in sterile, endotoxin-free Hanks salt equilibrium solution to prepare a 1 mg / mL solution as the test substance.

[0066] 4.1 Initial test: The plate incorporation method was used, with dosage groups of 5000 µg / plate, 1581 µg / plate, 500 µg / plate, 158.1 µg / plate, and 50 µg / plate. The samples were completely dissolved in the solvent, and no interfering precipitation or bacterial toxicity was observed in any group.

[0067] The test substance was filtered and sterilized, and prepared with purified water to a concentration of 50,000 µg / mL. It was then serially diluted to other concentrations. 100 µL was added to each dish before use, as detailed below:

[0068] Table 7. Dilution volumes and concentrations for the first trial

[0069]

[0070] 4.2 Confirmatory Test: Based on the initial test results, the confirmatory test adopted the pre-culture plate incorporation method, with dosage groups of 5000 µg / plate, 1000 µg / plate, 200 µg / plate, 40 µg / plate, and 8 µg / plate. The samples were completely dissolved in the solvent, and no interfering precipitation or bacterial toxicity was observed in any group.

[0071] The test substance was filtered and sterilized, and prepared with purified water to a concentration of 50,000 µg / mL. It was then serially diluted to other concentrations. 100 µL was added to each dish before use, as detailed below:

[0072] Table 8. Dilution volumes and concentrations of the dose groups in the confirmation experiment.

[0073]

[0074] 5. Test methods:

[0075] 5.1 Experimental Grouping: In addition to the test substance dose groups, blank control, solvent control, positive control, and sterile control groups were also set up in the experiment. Each dose was tested in triplicate.

[0076] 5.2 Test Methods:

[0077] The initial experiment used the plate incorporation method. 0.1 mL of enrichment broth for the test strain, 0.1 mL of the test sample solution, and 0.5 mL of S9 mixture (added if activation is required) were added to 2 mL of top agar. After mixing, the mixture was poured onto bottom agar plates, with three plates per dose and strain. The plates were incubated at 37 °C for 48 h, and the number of revertant colonies per plate was counted.

[0078] The experiment confirmed the use of the pre-culture plate incorporation method: before adding the top layer of culture medium, 0.1 mL of the test strain enrichment broth, 0.1 mL of the test sample solution and 0.5 mL of S9 mixture (added when activation is required) were mixed and incubated at 37°C for 20 min. Then, 2 mL of top layer agar was added, mixed, and poured onto the bottom layer culture medium plate. Three plates were used for each dose and each strain, and the plates were incubated at 37°C for 48 h. The number of revertant colonies in each plate was counted.

[0079] 5.3 Data processing and criteria for judging positive results: Record the number of reverted colonies per plate for each dose group of the test substance, blank control (spontaneous reversion), solvent control and positive mutagen control, and calculate the mean and standard deviation.

[0080] If the number of revertant colonies of the test substance is twice or more than twice the number of revertant colonies of the solvent control, and one of the following conditions is met, the test substance is determined to be mutagenic positive.

[0081] (1) A dose-response relationship exists.

[0082] (2) A positive reaction occurred and was repeatable under any dosage condition.

[0083] If any one of the four test strains is positive after testing the test substance, regardless of whether S9 is added or not, the bacterial reverse mutation test of the test substance can be reported as mutagenic positive. If all four test strains are negative after testing the test substance, regardless of whether S9 is added or not, the test substance can be reported as mutagenic negative.

[0084] II. Experimental results: see Tables 9 and 10.

[0085] III. Experimental Conclusions

[0086] Salmonella Typhimurium / reverse mutation test result in SsPle 1 mg / mL solution: negative.

[0087] Table 9. Reversion colony count of Ames test strains (mean ± standard deviation) (first test)

[0088]

[0089] * The number of revertant colonies was twice or more than twice that of the solvent control.

[0090] Table 10. Reversion colony counts of Ames test strains (mean ± standard deviation) (confirmatory test)

[0091]

[0092] * The number of revertant colonies was twice or more than twice that of the solvent control.

[0093] (3) Chromosomal aberrations in in vitro mammalian cells

[0094] I. Materials and Methods

[0095] 1. Cell line: The Chinese hamster lung (CHL) cell line was selected and obtained from the cell bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0096] 2. Metabolite activation system: The activation system is rat liver S9 induced by β-naphthylflavonoid and phenobarbital sodium. The S9 protein content is 30 mg / mL, and its activity has a good metabolic activation ability for cyclophosphamide. S9 is manufactured by Jiangsu Qishi Biotechnology Co., Ltd., batch number: 24FS002C.

[0097] 3. Positive control: Under conditions of metabolic activation, cyclophosphamide, an indirect mutagen, was used as a positive control. The manufacturer of cyclophosphamide was Baxter Oncology Gubh, batch number: 3C595A. The culture medium was prepared at a concentration of 3 mg / mL, with 25 μL added to each 5 mL dish, resulting in a final concentration of 15 μg / mL. Under conditions of no metabolic activation, mitomycin, a direct mutagen, was used as a positive control. The manufacturer of mitomycin was San Chemical Technology Co., Ltd., batch number: 93SEROFX. The culture medium was prepared at a concentration of 0.05 mg / mL, with 25 μL added to each 5 mL dish, resulting in a final concentration of 0.25 μg / mL.

[0098] 4. Test Substance: SsPle, a white powder, produced by Jiangsu Genscript Biotech Co., Ltd., and provided by the South China Sea Institute of Oceanology, Chinese Academy of Sciences. SsPle was dissolved in sterile, endotoxin-free Hanks salt-balanced solution to prepare a 1 mg / mL solution as the test substance. Based on cytotoxicity and sample solubility, three dosage groups (5 μL / mL, 2.5 μL / mL, and 1.25 μL / mL) were selected for chromosomal aberration assays.

[0099] 5. Test methods:

[0100] 5.1 Cytotoxicity test

[0101] 5.1.1 Preparation: One day before the experiment, seed cells at a rate of 5 × 10⁶ cells / day. 5 Place each in a petri dish and incubate at 37°C in a 5% CO2 incubator for 24 hours.

[0102] 5.1.2 Exposure: During the experiment, the culture medium in the culture dish was aspirated, and a certain concentration of the test substance, S9-mix (for test substances without S9-mix, the concentration was made up with culture medium) and a certain amount of serum-free culture medium were added. After being placed in an incubator for 3 h, the culture medium was aspirated, the cells were washed three times with Hank's solution, and culture medium containing fetal bovine serum was added and cultured for another 20 h.

[0103] 5.1.3 Cytotoxicity assay: The cells were re-digested, counted, diluted, and re-seeded. 200 cells were seeded per culture dish, 5 dishes per dose group. After culturing at 37 ℃ and 5% CO2 for 7 days, colonies were formed. The culture medium was discarded, the cells were fixed with methanol and stained with Giemsa. The number of cell colonies in each dish was counted, and the cell viability of each dose group was calculated (see Table 12).

[0104] 5.2 Procedure for Chromosomal Aberration Testing

[0105] 5.2.1 Cell seeding: One day before the experiment, seed cells at a rate of 1×10⁶ cells / day. 6 Place each in a petri dish and incubate at 37°C in a 5% CO2 incubator for 24 hours.

[0106] 5.2.2 Exposure Method: The experiment must be conducted with and without the addition of S9 mixture. During the experiment, the culture medium in the culture dish is aspirated, and a certain concentration of the test substance, S9 mixture (if S9 mixture is not added, it needs to be replenished with culture medium), and a certain amount of serum-free culture medium are added. The dish is then incubated for 3 hours. After the experiment, the culture medium containing the test substance is aspirated, the cells are washed three times with Hank's solution, and culture medium containing 10% fetal bovine serum is added. The dish is then returned to the incubator, and the cells are harvested within 24 hours. Before harvesting, colchicine, a cell division metaphase inhibitor, is added for 4 hours at a final concentration of 1 μg / mL.

[0107] 5.2.3 Specimen Preparation: Upon cell harvesting, cells were digested with 0.25% trypsin solution. After cell detachment, culture medium containing 10% fetal bovine or calf serum was added to terminate the trypsin effect. The mixture was then centrifuged at 1000 r / min for approximately 7 min. The supernatant was discarded, and the cells were hypotonic with 0.075 mol / L KCl solution. Fixation was then performed using freshly prepared methanol and glacial acetic acid solution (volume ratio 3:1). Slides were prepared using standard air-drying or flame-drying methods and stained with Giemsa stain. Two slides were prepared for each dose group.

[0108] 5.2.4 Analysis of Metaphase Cells: 200 metaphase cells were observed in each experimental group (100 metaphase cells were observed in the positive control group), and chromosome number and structural aberrations were analyzed. The percentage of polyploidy and intranuclear replicating cells was obtained by counting 200 cells. Chromosomal structural aberrations should include clefts (not used for analysis), breaks, chromatids, and chromosome fragments (acentric chromosomes or chromatid fragments, etc.), classified as breaks, deletions, exchanges, rings, and microbodies. Results are expressed as percentages. Cells with fragmented chromosomes and severe damage (aberration count ≥ 10) should also be recorded.

[0109] 5.3 Data Processing and Result Determination

[0110] The percentage of cells with chromosomal aberrations was calculated using the χ² method. 2 Testing is conducted to evaluate the mutagenicity of the test substance. The test substance can be determined to be mutagenic in this test system under the following two conditions:

[0111] a) The test substance induced a statistically significant rate of chromosomal structural aberrations, which was dose-dependent.

[0112] b) The test substance causes a statistically significant increase in distortion rate at any dosage condition, and this increase is reproducible.

[0113] II. Test Results

[0114] 1. Determination of the maximum dose of the test substance and test results

[0115] 1.1 Cytotoxicity results after 3 hours of exposure to the test substance under conditions without metabolic activation

[0116] Cytotoxicity assays were performed using dose groups ranging from 0.0390 µL / mL to 5 µL / mL. All test substances dissolved completely upon addition to the culture medium, as detailed in Table 11. Cytotoxicity results were expressed as relative cell viability, calculated by comparing each dose group with the negative control, as shown in Table 12. Based on the results of the cytotoxicity assays, the dose settings for the chromosomal aberration assay under non-metabolic activation conditions were as follows: 5 μL / mL, 2.5 μL / mL, and 1.25 μL / mL.

[0117] 1.2 Cytotoxicity results after 3 hours of exposure to the test substance under metabolic activation conditions

[0118] Cytotoxicity assays were performed using dose groups ranging from 0.0098 µL / mL to 5 µL / mL. All test substances dissolved completely upon addition to the culture medium, as detailed in Table 11. Cytotoxicity results were expressed as relative cell viability, calculated by comparing each dose group with the negative control, as shown in Table 12. Based on the results of the cytotoxicity assays, the dose settings for the chromosomal aberration assay under non-metabolic activation conditions were as follows: 5 μL / mL, 2.5 μL / mL, and 1.25 μL / mL.

[0119] Table 11 Solubility of the test substance in the selected solvent

[0120]

[0121] Rating criteria: ++= The test substance precipitates out in the culture medium solution after the test substance solution is added to the culture medium solution, forming a large amount of precipitate; += The test substance precipitates out in the culture medium solution after the test substance solution is added to the culture medium solution, forming a suspension; -= Completely dissolved.

[0122] Table 12. Cell toxicity of test substances (clonal formation rate)

[0123]

[0124] 2. Chromosomal aberration rates and statistical results in each treatment group and control group

[0125] The results of the chromosomal aberration test for each dose group are shown in Table 13.

[0126] 2.1 No metabolic activation treatment

[0127] Under conditions of no metabolic activation treatment for 3 hours, the chromosomal aberration rate in each dose group of the test substance was less than 5%. Compared with the solvent control group, the rate of chromosomal aberration in each dose group was significantly increased (p>0.05, χ2 test). The chromosomal structural aberration rate in the positive control group (mitomycin C) was 27.0%, which was statistically significantly increased compared with the solvent control group (p<0.05, χ2 test).

[0128] Under conditions of no metabolic activation treatment for 24 hours, the chromosomal aberration rate in each dose group of the test substance was less than 5%. Compared with the solvent control group, the rate of chromosomal aberration in each dose group was significantly increased (p>0.05, χ2 test). The chromosomal structural aberration rate in the positive control group (mitomycin C) was 30.0%, which was statistically significantly increased compared with the solvent control group (p<0.05, χ2 test).

[0129] 2.2 Metabolic activation treatment

[0130] Under 3 hours of metabolic activation treatment, the chromosomal aberration rate in each dose group of the test substance was less than 5%. Compared with the solvent control group, the rate of chromosomal aberration in each dose group was significantly increased (p>0.05, χ² test). The chromosomal structural aberration rate in the positive control group (cyclophosphamide) was 25.0%, which was statistically significantly increased compared with the solvent control group (p<0.05, χ² test).

[0131] Table 13 Results of in vitro mammalian cell chromosome aberration assay

[0132]

[0133] * = P < 0.05

[0134] III. Experimental Conclusions

[0135] Under the experimental conditions described in this report, with or without the presence of an in vitro assay activation system, SsPle 1 mg / mL solution could not induce chromosomal aberrations, and the results of the CHL cell chromosomal aberration assay with SsPle 1 mg / mL solution were negative.

[0136] (4) In vitro mammalian cell gene mutation assay

[0137] I. Materials and Methods

[0138] 1. Cell line: V-79 cell line, provided by the Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The cell line was stored in liquid nitrogen. MEM (Eagle) medium solution, provided by Gibco, was used, with antibiotics (penicillin and streptomycin, at 100 IU / mL) and fetal bovine serum added at 10%. Cells were cultured in a cell culture incubator at 37°C and 5% CO2.

[0139] 2. Metabolite Activation System: The activation system consisted of rat liver S9 induced by β-naphthylflavonoid and phenobarbital sodium. The S9 protein content was 30 mg / mL, and its activity showed good metabolic activation ability for cyclophosphamide. S9 was manufactured by Jiangsu Qishi Biotechnology Co., Ltd., batch number: 24FS002C. It was stored at -80℃.

[0140] 3. Positive control: The negative control group was a solvent control group, using serum-free MEM culture medium as the solvent. Under conditions without metabolic activation, the direct mutagen ethyl methanesulfonate (EMS) was used as a positive control. The manufacturer was SIGMA, batch number: BCBF07364, with a final concentration of 0.5 μL / mL. Under conditions with metabolic activation, the indirect mutagen benzo[a]pyrene (B(a)P) was used as a positive control. The manufacturer was Yuanye Biotechnology, batch number: M12GS141406, with a final concentration of 5 μg / mL.

[0141] 4. Test Substance: SsPle, a white powder, produced by Jiangsu Genscript Biotech Co., Ltd., and provided by the South China Sea Institute of Oceanology, Chinese Academy of Sciences. SsPle was dissolved in sterile, endotoxin-free Hanks salt equilibrium solution to prepare a 1 mg / mL solution as the test substance. Based on cytotoxicity and sample solubility, five dosage groups (5 μL / mL, 2.5 μL / mL, 1.25 μL / mL, 0.625 μL / mL, and 0.312 μL / mL) were selected for testing.

[0142] 5. Test methods:

[0143] 5.1 Cytotoxicity assay (preliminary assay)

[0144] 5.1.1 Preparation: One day before the experiment, seed cells at a rate of 5 × 10⁶ cells / day. 5 Place each in a petri dish and incubate at 37°C for 24 hours.

[0145] 5.1.2 Exposure: During the experiment, the culture medium in the culture flask was aspirated, and a certain concentration of the test substance, S9-mix (for test substances without S9-mix, the concentration was made up with culture medium) and a certain amount of serum-free culture medium were added. After being placed in an incubator for 3 h, the culture medium was aspirated, the cells were washed three times with Hank's solution, and culture medium containing fetal bovine serum was added and cultured for another 20 h.

[0146] 5.1.3 Cytotoxicity assay: The cells were re-digested, counted, diluted, and re-seeded. 200 cells were seeded per culture dish, 5 dishes per dose group. After culturing at 37℃ and 5% CO2 for 7 days, colonies were formed. The culture medium was discarded, the cells were fixed with methanol and stained with Giemsa. The number of cell colonies in each dish was counted, and the cell viability of each dose group was calculated (see Table 14).

[0147] 5.2 HPRT site mutation analysis

[0148] 5.2.1 Based on the cytotoxicity test results, the relative colony formation rate of each dose group was calculated with the colony formation rate of the solvent control group being 100%. According to the cytotoxicity and solubility of the test substance, the highest concentration was set at 5 μL / mL. Four additional dose groups were set as the formal test doses. A negative control group (untreated) and a positive control group (-S9 using EMS, +S9 using B(a)P) were also set. Each group included both the addition and non-addition of the S9 mixture. In this experiment, the high dose was selected as 5 μL / mL under both metabolic activation and non-metabolically activated conditions, and was diluted to four dose groups (i.e., 2.5 μL / mL, 1.25 μL / mL, 0.625 μL / mL, and 0.312 μL / mL).

[0149] 5.2.2 Cell seeding: After digesting well-grown cells with trypsin-EDTA solution, seed 5.0 × 10⁶ cells per dish. 5 Cells were incubated at 37°C in a 5% CO2 incubator for 24 hours, ready for sample addition.

[0150] 5.2.3 Exposure: After 24 hours of incubation, different concentrations of test substance solution or S9 mixture were added to each of the above-mentioned petri dishes. The negative control group (solvent control group) was only given culture medium. Three hours after sample addition, the culture medium was discarded, and the dishes were rinsed three times with calcium- and magnesium-free PBS solution, and then incubated for 20 hours with culture medium containing 10% serum.

[0151] 5.2.4 Expression of mutant cells: After the test substance was applied to the cells, they were cultured for another 20 hours, then digested, counted, and their expression and cytotoxicity were determined. In each dosage group, cells were seeded at low density every 3 days during the expression period, controlling the cell concentration at 1 × 10⁻⁶. 6 Less than one per dish. After 7 days of culture, mutants were selected and colony formation rate was determined.

[0152] 5.2.5 Cytotoxicity assay: During expression, 200 cells were seeded in each culture dish, 5 dishes per dose group, and cultured at 37°C and 5% CO2 for 7 days. After 7 days, the cells were fixed with methanol and stained with Giemsa. The number of cell colonies in each dish was counted, and the cell viability of each dose group was calculated (see Table 15).

[0153] 5.2.6 Selection of mutants and determination of colony formation rate: After expression, cells were digested and seeded separately, with 5 plates per group and 2 × 10⁶ cells per plate. 5 Cells were cultured and 6-TG was added to each plate after cell adhesion, with a final concentration of 5 μg / mL. Simultaneously, 200 cells were seeded per plate (without 6-TG), with 5 plates per group. These plates were incubated at 37°C in a 5% CO2 incubator for 7 days. After fixation with methanol and Giemsa staining, the number of colonies in each plate was counted, and the total number of colonies (see Table 16), colony formation rate (see Table 17), and mutation frequency (MF) (see Table 18) were calculated.

[0154] 5.2.7 The operating procedures for the positive and negative control groups are the same as those for the experimental group, except that the positive control group uses a positive control instead of the test substance, and the untreated control group uses culture medium.

[0155] 6. Data processing and result determination

[0156] 6.1 Indicator Calculation: Calculate the relevant indicators using the following formulas:

[0157]

[0158] 6.2 Result Determination

[0159] Using χ 2 The test was statistically analyzed. When the MF of each dose group increased significantly compared with the negative control group and showed a dose-response relationship, or when only one dose group showed a statistically significant increase that was confirmed by repeated tests, it was considered a positive result, that is, the test substance was mutagenic to the HGPRT system of V79 cells.

[0160] II. Test Results

[0161] 1. Cytotoxicity test results

[0162] Cytotoxicity assays were performed using dose groups ranging from 0.0098 µL / mL to 5 µL / mL. With or without metabolic activation, the test substance in each dose group dissolved completely upon addition to the culture medium. The pH values ​​for each dose group are shown in Table 14. Cytotoxicity results were expressed as relative colony formation rates, calculated by comparing each dose group of the test substance with the negative control, as shown in Table 14. Based on the results of the cytotoxicity assays and the solubility of the test substance, the dosage settings for the gene mutation assay were as follows: five dose groups—5 µL / mL, 2.5 µL / mL, 1.25 µL / mL, 0.625 µL / mL, and 0.312 µL / mL—were selected for the assays with or without metabolic activation.

[0163] 2. Mutation frequency and statistical results in the experimental and control groups.

[0164] Compared with the negative control group, no mutation frequency was significantly increased in any of the test substance dose groups (see Table 18) regardless of whether metabolic activation was present or absent (p>0.05, χ²). 2 (Test). Under conditions without metabolic activation, the mutation frequency in the positive control group (EMS) was 256.62 × 10⁻⁶. -6 Compared with the solvent control group, it showed a statistically significant increase (p<0.05, χ²). 2 (Test). Under metabolic activation conditions, the mutation frequency in the positive control group (B(a)P) was 148.46 × 10⁻⁶. -6 Compared with the solvent control group, it showed a statistically significant increase (p<0.05, χ²). 2 test).

[0165] Table 14 Colony formation rate (cytotoxicity) at different concentrations

[0166]

[0167] Rating criteria: ++ = After the test substance solution is added to the culture medium solution, the test substance precipitates and forms a large amount of precipitate; + = After the test substance solution is added to the culture medium solution, the test substance precipitates and forms a suspension; - = Completely dissolved.

[0168] Table 15. Cell colony formation rate at different expression concentrations during the expression phase.

[0169]

[0170] Rating criteria: ++ = After the test substance solution is added to the culture medium solution, the test substance precipitates and forms a large amount of precipitate; + = After the test substance solution is added to the culture medium solution, the test substance precipitates and forms a suspension; - = Completely dissolved.

[0171] Table 16 Colony numbers of mutant cells at different concentrations

[0172]

[0173] Table 17. Colony formation rate of cells undergoing mutation selection

[0174]

[0175] Table 18 Results of in vitro mammalian cell gene mutation assay

[0176]

[0177] "*" indicates P < 0.05, and "—" indicates not applicable.

[0178] III. Experimental Conclusions

[0179] Results of in vitro mammalian cell gene mutation assay: Regardless of whether metabolic activation was present or absent, none of the test substance dosage groups significantly increased the mutation frequency in the test cell lines (see Table 18). However, the mutation frequency of the positive control EMS and B(a)P test cell lines significantly increased, showing a significant difference compared to the solvent control. Therefore, it can be concluded that under the conditions of this experiment, the results were negative, and the test substance (SsPle 1 mg / mL solution) did not have a significant effect on inducing gene mutations in mammalian cells.

Claims

1. A type of antimicrobial peptide spray, characterized in that, The product contains seahorse antimicrobial peptide SsPle as an active ingredient and excipient. The seahorse antimicrobial peptide SsPle has the following amino acid sequence: FFRSLWKGVKEGFRAGRSAYKA. The excipients include stabilizers, antioxidants, buffers, pH adjusters, and a preservative system. The preservative system is capryloyl hydroxamic acid, hexanediol, and propylene glycol.

2. The antimicrobial peptide spray according to claim 1, characterized in that, The stabilizer is glycerol or mannitol.

3. The antimicrobial peptide spray according to claim 1, characterized in that, The buffer is histidine or citrate-citric acid.

4. The antimicrobial peptide spray according to claim 1, characterized in that, The antioxidant mentioned is methionine.

5. The antimicrobial peptide spray according to claim 1, characterized in that, The Hailong antimicrobial peptide spray is effective against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus.

6. The antimicrobial peptide spray according to claim 5, characterized in that, The Staphylococcus aureus mentioned is methicillin-resistant Staphylococcus aureus.

7. The antimicrobial peptide spray according to claim 5, characterized in that, The Escherichia coli mentioned is methicillin-resistant Escherichia coli.

8. The antimicrobial peptide spray according to claim 1, characterized in that, The antimicrobial peptide spray comprises, by mass fraction, 0.1% Hailong antimicrobial peptide SsPle, 1% mannitol, 20 mM citrate-citric acid, 1.0% capryloyl hydroxamic acid & hexanediol & propylene glycol, with the balance being water.

9. A type of antimicrobial peptide spray, characterized in that, The antimicrobial peptide spray comprises, by mass fraction, 0.1% antimicrobial peptide SsPle, 1% glycerol, 0.31% histidine, and 0.05% methionine. Hydrochloric acid is added to adjust the pH to 6, and the remainder is pure water. The amino acid sequence of the antimicrobial peptide SsPle is: FFRSLWKGVKEGFRAGRSAYKA.

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