Safe and preservative-free syngnathus antibacterial peptide spray as well as preparation method and application thereof

Abstract

The invention discloses a safe and preservative-free syngnathus antibacterial peptide spray as well as a preparation method and application thereof, the spray contains syngnathus antibacterial peptide SsPle as an active ingredient and an auxiliary material, and the amino acid sequence of the syngnathus antibacterial peptide SsPle is FFRSLWKGVKEGFRAGRSAYKA. The antibacterial peptide spray has the characteristics of safety, no toxicity, no traditional preservative, good stability, convenience in use and the like. The antibacterial peptide has no acute oral toxicity, no mutagenicity and no chromosome injury risk, and is high in biological safety. The spray can be stably stored for 120 days at the temperature of 30 DEG C, and still has a remarkable antibacterial effect on pseudomonas aeruginosa, methicillin-resistant staphylococcus aureus (MRSA) and the like. The preparation is simple in preparation process, can be filled by adopting a quantitative sprayer, and is convenient for clinical treatment, wound nursing and daily disinfection application. The antibacterial peptide spray solves the problem of irritation of preservatives of traditional antibacterial peptide sprays, is suitable for sensitive people, and has a wide market prospect.

Description

Technical Field

[0001] The invention belongs to the field of antibacterials, and particularly relates to a safe, preservative-free Hailong antibacterial peptide spray, a preparation method and an application thereof. Background Art

[0002] Antimicrobial peptides (AMPs) are a class of small proteins found widely in prokaryotes and eukaryotes, exhibiting a wide range of inhibitory effects against bacteria, fungi, and viruses. They exert their effects through a variety of mechanisms, including disruption of bacterial cell membranes, modulation of immune responses, and inflammation. Compared to traditional antibiotics, AMPs offer unique advantages, particularly their resistance to inducing bacterial resistance, primarily due to their targeting of microbial membranes rather than specific molecular targets. Therefore, AMPs are considered potential candidates to replace traditional antibiotics and combat multidrug-resistant bacterial infections.

[0003] The antimicrobial peptide SsPle from Syngnathus schlegeli is a novel antimicrobial peptide identified by our research team in previous studies. It consists of 22 amino acid residues. Synthesized via solid-phase chemistry, SsPle exhibits excellent water solubility and bactericidal activity under high temperature, high salt, acidic, and alkaline conditions. Previous experiments have shown that SsPle exhibits significant antimicrobial activity against common pathogens, including methicillin-resistant Staphylococcus aureus, multidrug-resistant Escherichia coli, and Pseudomonas aeruginosa.

[0004] Commercially available antimicrobial peptide sprays contain traditional chemical preservatives, such as phenoxyethanol and parabens (parabens), which can cause skin irritation, redness, itching, contact dermatitis, and other allergic reactions, making them particularly unsuitable for sensitive individuals. Furthermore, the use of traditional preservatives can affect the activity of antimicrobial peptides, reducing their antimicrobial efficacy and limiting their clinical application. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a new antimicrobial peptide spray, which uses the antimicrobial peptide SsPle from Sea Dragon as the core active ingredient and has the advantages of being safe and non-toxic, preservative-free, stable and easy to use.

[0006] The purpose of the present invention is to provide a sea dragon antimicrobial peptide spray, which contains sea dragon antimicrobial peptide SsPle as an active ingredient. The amino acid sequence of the sea dragon antimicrobial peptide SsPle is: FFRSLWKGVKEGFRAGRSAYKA

[0007] Preferably, the antibacterial spray contains antimicrobial peptides as active ingredients and other excipients, 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 Hailong antimicrobial peptide spray is a Hailong antimicrobial peptide spray against Escherichia coli (Methicilin-resistant Escherichia coli), Pseudomonas aeruginosa (Pseudomonas aeruginosa), and Staphylococcus aureus (staphylococcus aureus); preferably, the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA).

[0013] Preferably, the formula of the antibacterial spray is calculated by mass fraction, including formula 1: 0.1% sea dragon antimicrobial 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 antimicrobial peptide SsPle, 1% mannitol, 20mM citrate-citric acid, 1.0% compound antioxidant preservative system, and the balance is water.

[0014] The present invention provides a safe, preservative-free antimicrobial peptide spray of Sea Dragon SsPle. Sea Dragon SsPle antimicrobial peptide has no acute oral toxicity, will not cause bacterial gene mutations or mammalian red blood cell chromosome damage, will not cause mammalian cell chromosome aberrations, and has good biological integrity. The present invention provides two formulas. Formula 1 does not contain preservatives, completely avoiding the skin irritation and allergic reactions that may be caused by traditional preservatives. Formula 2 adopts a compound antioxidant preservative system (octanoylhydroxamic acid, hexylene glycol, propylene glycol), does not contain traditional preservatives, is mild in nature, does not irritate the skin, is easy to operate, and is very suitable for formulas claiming to be "preservative-free" or "paraben-free".

[0015] The present invention provides an antibacterial spray with good stability and convenient use and filling. After being placed at 30°C for 120 days, the spray still has excellent inhibitory effects on Pseudomonas aeruginosa, clinical multidrug-resistant Staphylococcus aureus (MRSA), etc., with good stability and reliable preparation quality. The spray is easy to use and can be sprayed directly on the affected area. In addition, its preparation process is simple, and the liquid medicine is canned using a quantitative sprayer. The convenience and stability of the spray give it broad application prospects in clinical treatment, wound care and daily disinfection.

[0016] The present invention solves the problem of preservative irritation in traditional antimicrobial peptide sprays through innovative formula design, providing a safer and more stable antimicrobial peptide spray with broad application prospects, especially suitable for sensitive people. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The Hailong antimicrobial peptide spray of formula 1 still has a good antibacterial effect when placed at 30℃ for 120 days. The bacterial solution of the control group grew normally, but the bacterial solution became turbid, and its OD 600 The value increased over time. When the bacterial solution was treated with antimicrobial peptide spray solution and SsPle peptide, bacterial growth was inhibited and the bacterial solution was always clear. 600 The value remains at a very low level.

[0018] Figure 2 The Hailong antimicrobial peptide spray of formula 2 still has a good antibacterial effect when placed at 30℃ for 120 days. The bacterial solution of the control group grew normally, but the bacterial solution became turbid, and its OD 600 The value increased over time. When the bacterial solution was treated with antimicrobial peptide spray solution and SsPle peptide, bacterial growth was inhibited and the bacterial solution was always clear. 600 The value remains at a very low level. DETAILED DESCRIPTION

[0019] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0020] Example 1. Preparation of Hailong antimicrobial peptide spray

[0021] (1) Raw materials and excipients information

[0022] Information and physicochemical properties of the antimicrobial peptide SsPle from H. schoenleinensis.

[0023] Table 1 Physicochemical properties of the antimicrobial peptide SsPle from Pseudomonas schoenostoma

[0024]

[0025] Table 2 Excipient information

[0026]

[0027] (2) Xue's sea dragon antimicrobial peptide spray formula and its preparation process

[0028] Formula ingredients. Formula percentages are expressed as (w / w, %). Formula 1: 0.1% Sea Dragon Antimicrobial Peptide SsPle, 1% glycerol, 0.31% histidine, 0.05% methionine, and finally, 1M hydrochloric acid is added to adjust the pH to approximately pH 6. The balance is pure water. Formula 2: 0.1% Sea Dragon Antimicrobial Peptide SsPle, 1% mannitol, 20mM citrate-citric acid, 1.0% compounded antioxidant preservative system, and the balance is water.

[0029] Table 3: Ingredients of Formula 1

[0030]

[0031] Table 4 Ingredients of Formula 2

[0032]

[0033] The method for preparing the Hailong antimicrobial peptide spray as described above comprises the following steps:

[0034] Preparation process for Formulation 1: Glycerol and methionine are highly water-soluble and dissolve rapidly in purified water (90% by weight) after stirring. After visual inspection, no undissolved solids are observed. Stirring is continued for 5 minutes. The excipient solution rapidly passes through a 0.45μm filter membrane, and no visible impurities remain on the filter membrane after filtration, indicating complete dissolution of the excipients. Before adding the antimicrobial peptide, the pH of the excipient solution is adjusted to approximately 6.0 by adding a buffer system of histidine-hydrochloric acid. At this point, the total weight of the excipient solution is significantly lower than the final weight. The antimicrobial peptide dissolves rapidly after addition. After adding purified water to volume, the solution remains clear and transparent, and the pH remains around 6.0 with no significant fluctuation. Finally, the solution is filtered through a 0.22μm filter membrane and dispensed into sterile metered-dose sprayers to produce the antimicrobial spray. The antimicrobial spray is then chilled at -20°C overnight to freeze the solution. Finally, the spray is stored at 30°C for 120 days.

[0035] Preparation process of formula 2: Take 90% of the total weight of purified water, add mannitol and the compound antioxidant and preservative system while stirring, and continue stirring for 5 minutes after visually no solid undissolved matter is found to prepare the auxiliary material solution. 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 fixed volume total weight. After adding the antimicrobial peptide, it can dissolve quickly. After adding purified water to the fixed volume, the solution is clear and transparent, and the pH value of the solution is measured to be around 4.5. After the solution is sterilized by filtration with a 0.22μm filter membrane, it is divided into sterile quantitative sprayers to obtain an antimicrobial spray. The entire production is carried out under non-sterile conditions. Finally, the antimicrobial spray is placed at 30°C for 120 days.

[0036] Example 2. Antibacterial activity of Hailong antimicrobial peptide spray

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

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

[0039] After the antimicrobial peptide spray was placed at 30°C for 120 days, its antimicrobial activity was tested. Figure 1 and Figure 2 ), while the bacterial solution of the control group (treated with DPBS) grew normally. The antimicrobial peptide spray solutions of Formula 1 and Formula 2, 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 antimicrobial peptide SsPle from Pseudomonas schoenostoma

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

[0042] (1) Acute oral toxicity test

[0043] 1. Materials and Animals

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

[0045] 2 Experimental animals and breeding environment:

[0046] 2.1 Experimental Animals: Ten SPF-grade ICR mice, half male and half female, weighing 18.4 g to 21.2 g for females and 19.4 g to 21.2 g for males, were provided by Nanjing Medical University, production license number SCXK(Su)2021-0001, and certificate number A202405220023. All animals were acclimated for 5 days prior to the experiment and had free access to water and food.

[0047] 2.2 Experimental environment: The experimental animals were raised in a barrier system of our center with a temperature of 20-26°C and a relative humidity of 30%-70%. The experimental animal use license number is: SYXK(Su)2020-0006.

[0048] 2.3 Feed: Irradiated sterilized experimental mouse maintenance feed, provided by Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd., production license: Su Feed Certificate (2019) 01008.

[0049] 3. Test methods:

[0050] 3.1 Method: According to the single maximum limit dose design requirements in the test standard, the dose was set at 5000 mg / kg·bw. Animals were fasted overnight (16 hours) before the experiment and had free access to water. Take 5.00 g of sample, add purified water to 10 mL, and prepare a concentration of 500 mg / mL. Orally gavage once at 10 mL / kg·bw, with an actual exposure dose of 5000 mg / kg·bw. Five males and five females were observed for signs of poisoning and the number of deaths within 14 days. Surviving animals were weighed weekly. At the end of the 14-day observation period, gross anatomical examinations were performed on the animals, and abnormalities observed with the naked eye were recorded. Source of purified water: homemade by Millipore pure water instrument, batch number: 20240527.

[0051] 3.2 Toxicity evaluation: LD 50 Those greater than 5000 mg / kg body weight are practically non-toxic; LD 50 501~5000mg / kg body weight is low toxicity; LD 50 51-500 mg / kg body weight is moderately toxic; LD 50 Less than or equal to 50 mg / kg body weight is considered highly toxic.

[0052] 2. Test results

[0053] No abnormal signs or deaths were observed in either male or female mice until the end of the observation period. Gross autopsies of surviving animals at the end of the observation period revealed no gross 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

[0055]

[0056] 3. Test Conclusion

[0057] Acute oral toxicity test LD50 of 1 mg / mL solution of SsPle in female and male mice 50 Greater than 5000mg / kg·bw. According to the toxicity evaluation regulations of the "Technical Specifications for Safety of Cosmetics" (2015 edition), the sample is actually non-toxic.

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

[0059] 1. Materials and Methods

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

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

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

[0063] Table 6 Positive substances used in this test

[0064]

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

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

[0067] The test substance was sterilized by filtration and prepared with purified water to a concentration of 50,000 μg / mL. It was diluted to other concentrations in a stepwise manner. 100 μL was added to each dish when used, as follows:

[0068] Table 7 Dilution volume and concentration of the dose group in the first trial

[0069]

[0070] 4.2 Confirmation Test: Based on the results of the initial test, a confirmation test was conducted using the pre-culture plate incorporation method, with dose groups of 5000 μg / dish, 1000 μg / dish, 200 μg / dish, 40 μg / dish, and 8 μg / dish. The samples were completely dissolved in the solvent, and no interfering precipitation or bacterial toxicity was observed in any of the groups.

[0071] The test substance was sterilized by filtration and prepared with purified water to a concentration of 50,000 μg / mL. It was diluted to other concentrations in a stepwise manner. 100 μL was added to each dish when used, as follows:

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

[0073]

[0074]

[0075] 5. Test method:

[0076] 5.1 Experimental Grouping: In addition to the test substance dosage groups, blank control, solvent control, positive substance control, and sterile control groups shall be set up. Each dosage shall be repeated three times.

[0077] 5.2 Test method:

[0078] The first test used the plate incorporation method: 0.1 mL of the test strain enrichment solution, 0.1 mL of the test sample solution, and 0.5 mL of S9 mixture (if activation was required) were added to 2 mL of top agar. Mix thoroughly and pour onto the bottom agar plate. Three plates of each strain at each dose were incubated at 37°C for 48 hours, and the number of revertant colonies on each plate was counted.

[0079] The confirmation test uses the pre-culture plate incorporation method: before adding the top culture medium, first mix 0.1mL of the test strain enrichment solution, 0.1mL of the test sample solution and 0.5mL of S9 mixed solution (added when activation is required), and incubate at 37℃ for 20min. Then add 2mL of top agar, mix well and pour it onto the bottom culture medium plate. For each dose and each strain, 3 plates are plated and incubated at 37℃ for 48h. The number of reverted colonies on each plate is counted.

[0080] 5.3 Data processing and positive result judgment criteria: Record the number of reverted colonies on each dish of each dose group of the test substance, blank control (spontaneous reversion), solvent control and positive mutagen control, and calculate the average value and standard deviation.

[0081] If the number of revertant colonies of the test substance is twice or more than that of the solvent control, and one of the following situations occurs, the test substance is judged to be mutagenic.

[0082] (1) Dose-response relationship

[0083] (2) A positive response occurs and is reproducible under any dose condition

[0084] If a test substance is tested with the four test strains mentioned above and one of the test strains is positive, regardless of whether S9 is added or not, the test substance can be reported as mutagenic-positive in the bacterial reverse mutation test. If a test substance is tested with the four test strains and all of them are negative, regardless of whether S9 is added or not, the test substance can be reported as mutagenic-negative.

[0085] 2. Test results: See Table 9 and Table 10.

[0086] 3. Test Conclusion

[0087] Results of the Salmonella typhimurium / reverse mutation test with SsPle 1 mg / mL solution: negative.

[0088] Table 9 Number of revertant colonies of Ames test strains (mean ± standard deviation) (first test)

[0089]

[0090]

[0091] * The number of revertant colonies is twice or more than that of the solvent control.

[0092] Table 10 Number of revertant colonies of Ames test strains (mean ± standard deviation) (confirmation test)

[0093]

[0094]

[0095] * The number of revertant colonies is twice or more than that of the solvent control.

[0096] (3) Chromosome aberration in mammalian cells in vitro

[0097] 1. Materials and Methods

[0098] 1. Cell line: Chinese hamster lung (CHL) cell line was selected, which was obtained from the cell bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0099] 2. Metabolite Activation System: The activation system is rat liver S9 induced by β-naphthoflavone and phenobarbital sodium. The S9 protein content is 30 mg / mL, and its activity has good metabolic activation ability for cyclophosphamide. S9, manufactured by Jiangsu Qishi Biotechnology Co., Ltd., batch number: 24FS002C.

[0100] 3. Positive Substance: Under metabolic activation conditions, use the indirect mutagen cyclophosphamide as a positive control. Cyclophosphamide is manufactured by Baxter Oncology GmbH, lot number 3C595A. It is prepared in culture medium at a concentration of 3 mg / mL. Add 25 μL to each 5 mL dish for a final concentration of 15 μg / mL. Under non-metabolic activation conditions, use the direct mutagen mitomycin as a positive control. Mitomycin is manufactured by San Chemical Technology Co., Ltd., lot number 93SEROFX. It is prepared in culture medium at a concentration of 0.05 mg / mL. Add 25 μL to each 5 mL dish for a final concentration of 0.25 μg / mL.

[0101] 4. Test Substance: SsPle, a white powder, was produced by Jiangsu GenScript Biotechnology Co., Ltd. and provided by the South China Sea Institute of Oceanology, Chinese Academy of Sciences. SsPle was dissolved in sterile, endotoxin-free Hanks' balanced salt solution to prepare a 1 mg / mL solution as the test substance. Based on cytotoxicity and test sample dissolution, three dose groups (5 μL / mL, 2.5 μL / mL, and 1.25 μL / mL) were selected for chromosomal aberration testing.

[0102] 5. Test method:

[0103] 5.1 Cytotoxicity assay

[0104] 5.1.1 Preparation: 1 day before the experiment, inoculate 5×10 cells 5 Place the cells in a culture dish and culture in a 37°C, 5% CO2 incubator for 24 hours.

[0105] 5.1.2 Exposure: During the test, the culture medium in the culture dish was aspirated, and a certain concentration of the test substance, S9-mix (if the test substance is not added to S9-mix, the amount is supplemented with culture medium) and a certain amount of serum-free culture medium were added. After the cells were placed in an incubator for 3 hours, the culture medium was aspirated, and the cells were washed three times with Hank's solution. Culture medium containing fetal bovine serum was added and cultured for a further 20 hours.

[0106] 5.1.3 Cytotoxicity Assay: The above cells were re-digested, counted, diluted, and re-seeded. 200 cells were seeded per dish, with 5 dishes per dose group. After incubation at 37°C, 5% CO2 for 7 days, colonies were formed. The culture medium was discarded, and the cells were fixed with methanol and stained with Giemsa. The number of cell colonies in each dish was counted, and the cell survival rate of each dose group was calculated (see Table 12).

[0107] 5.2 Chromosome aberration test steps

[0108] 5.2.1 Cell inoculation: One day before the experiment, inoculate 1×10 cells 6 Place the cells in a culture dish and culture in a 37°C, 5% CO2 incubator for 24 hours.

[0109] 5.2.2 Exposure Method: The test must be conducted with and without the addition of S9 mixture. During the test, 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, the amount must be supplemented with culture medium), and a certain amount of serum-free culture medium are added. The cells are placed in an incubator for 3 hours. After completion, 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 cells are returned to the incubator and harvested within 24 hours. Before harvesting, the metaphase arrestor colchicine is added for 4 hours at a final concentration of 1 μg / mL.

[0110] 5.2.3 Specimen Preparation: When harvesting cells, digest them with 0.25% trypsin solution. After the cells have detached, add culture medium containing 10% fetal bovine or calf serum to terminate the trypsin. Mix thoroughly, centrifuge at 1000 rpm for approximately 7 minutes, discard the supernatant, and treat with 0.075 mol / L KCl solution for hypotonicity. Fix the specimen with freshly prepared methanol and glacial acetic acid (3:1 by volume). Prepare conventional slides by air-drying or flame-drying, and stain with Giemsa stain. Prepare two slides for each dose group.

[0111] 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 analyzed for chromosome number and structural aberrations. The percentage of polyploid and endoreduplication cells was determined by counting 200 cells. Chromosome structural aberrations should include gaps (not used for analysis), breaks, chromatid and chromosome fragments (such as acentric chromosomes or chromatid fragments), and should be classified as breaks, deletions, exchanges, rings, and microbodies. Results are expressed as percentages. Cells with comminuted and severely damaged chromosomes (≥10 aberrations) should also be recorded.

[0112] 5.3 Data processing and result determination

[0113] The chromosomal aberration rate was calculated using the χ 2 Test 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 circumstances:

[0114] a) The rate of chromosome structural aberration caused by the test substance is statistically significant and dose-related.

[0115] b) The test substance causes a statistically significant and reproducible increase in the aberration rate at any dose.

[0116] 2. Test results

[0117] 1. Determination of the maximum dose of the test substance and test results

[0118] 1.1 Cytotoxicity results after 3-hour exposure to the test substance without metabolic activation

[0119] Cytotoxicity testing was performed using dose groups ranging from 0.0390 μL / mL to 5 μL / mL. The test substances were completely dissolved upon addition to the culture medium, as shown in Table 11. Cytotoxicity results were expressed as relative cell viability, calculated by comparing each dose group with the negative control. The results are shown in Table 12. Based on the results of the aforementioned cytotoxicity tests, the doses for the chromosome aberration assay in the absence of metabolic activation were set as follows: 5 μL / mL, 2.5 μL / mL, and 1.25 μL / mL.

[0120] 1.2 Cytotoxicity results of the test substance exposed to the test substance for 3 hours under metabolic activation conditions

[0121] Cytotoxicity testing was performed using dose groups ranging from 0.0098 μL / mL to 5 μL / mL. The test substances were completely dissolved after addition to the culture medium, as shown in Table 11. Cytotoxicity results were expressed as relative cell viability, calculated by comparing each dose group with the negative control. The results are shown in Table 12. Based on the results of the aforementioned cytotoxicity testing, the doses for the chromosome aberration assay in the absence of metabolic activation were set as follows: 5 μL / mL, 2.5 μL / mL, and 1.25 μL / mL.

[0122] Table 11 Solubility of the test substances in the selected solvents

[0123]

[0124]

[0125] a Rating standard: ++ = After the test substance solution is added to the culture medium solution, the test substance precipitates to form a large amount of precipitation; + = After the test substance solution is added to the culture medium solution, the test substance precipitates to form a suspension; - = Completely dissolved.

[0126] Table 12 Toxicity of the test substances to cells (clone formation rate)

[0127]

[0128] 2 Chromosome aberration rates and statistical results of each treatment group and control group

[0129] In the chromosome aberration test, the test results of each dosage group are shown in Table 13.

[0130] 2.1 No metabolic activation treatment

[0131] Without metabolic activation for 3 hours, the chromosome aberration rate in each test group was less than 5%. Compared with the solvent control group, no significant increase in chromosome aberration rate was observed in any of the test groups (p>0.05, χ2 test). The chromosome structural aberration rate in the positive control group (mitomycin) was 27.0%, a statistically significant increase compared with the solvent control group (p<0.05, χ2 test).

[0132] Without metabolic activation for 24 hours, the chromosome aberration rate in each test group was less than 5%. Compared with the solvent control group, no significant increase in chromosome aberration rate was observed in any of the test groups (p>0.05, χ2 test). The chromosome structural aberration rate in the positive control group (mitomycin) was 30.0%, a statistically significant increase compared with the solvent control group (p<0.05, χ2 test).

[0133] 2.2 Metabolic activation treatment

[0134] After 3 hours of metabolic activation, the chromosome aberration rate in cells in each dose group was less than 5%. Compared with the solvent control group, no significant increase in chromosome aberration rate was observed in any dose group (p>0.05, χ2 test). The chromosome 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).

[0135] Table 13 In vitro mammalian cell chromosome aberration test results

[0136]

[0137]

[0138] * = P < 0.05

[0139] 3. Test Conclusion

[0140] Under the experimental conditions described in this report, SsPle 1 mg / mL solution did not induce chromosomal aberrations in the presence or absence of an in vitro assay activation system. SsPle 1 mg / mL solution was negative for chromosomal aberrations in CHL cells.

[0141] (4) In vitro mammalian cell gene mutation test

[0142] 1. Materials and Methods

[0143] 1. Cell line: V-79 cell line, provided by the Cell Bank of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Cell lines were stored in liquid nitrogen. MEM (Eagle's Methionine) medium (Gibco) was used, supplemented with antibiotics (penicillin and streptomycin at 100 IU / mL) and 10% fetal bovine serum. Cells were cultured in a 37°C, 5% CO2 incubator.

[0144] 2. Metabolite Activation System: The activation system uses rat liver S9 induced by β-naphthoflavone and phenobarbital sodium. The S9 protein content is 30 mg / mL, and its activity has excellent metabolic activation ability for cyclophosphamide. S9, manufactured by Jiangsu Qishi Biotechnology Co., Ltd., batch number: 24FS002C. Store in a -80°C freezer.

[0145] 3. Positive Substances: The negative control group consisted of a solvent control group (serum-free MEM medium). In the absence of metabolic activation, a direct mutagen, ethyl methanesulfonate (EMS), was used as a positive control. The manufacturer was SIGMA, lot number BCBF07364, at a final concentration of 0.5 μL / mL. In the presence of metabolic activation, an indirect mutagen, benzopyrene (B(a)P), was used as a positive control. The manufacturer was Yuanye Bio, lot number M12GS141406, at a final concentration of 5 μg / mL.

[0146] 4. Test Substance: SsPle, a white powder, was produced by Jiangsu GenScript Biotechnology Co., Ltd. and provided by the South China Sea Institute of Oceanology, Chinese Academy of Sciences. SsPle was dissolved in sterile, endotoxin-free Hanks' balanced salt solution to prepare a 1 mg / mL solution as the test substance. Based on cytotoxicity and sample dissolution, five dose groups were selected for testing: 5 μL / mL, 2.5 μL / mL, 1.25 μL / mL, 0.625 μL / mL, and 0.312 μL / mL.

[0147] 5. Test method:

[0148] 5.1 Cytotoxicity test (preliminary test)

[0149] 5.1.1 Preparation: 1 day before the experiment, inoculate 5×10 cells 5 Place the cells in a culture dish and incubate at 37°C for 24 hours.

[0150] 5.1.2 Exposure: During the test, the culture medium in the culture flask was aspirated, and a certain concentration of the test substance, S9-mix (if the test substance is not added to S9-mix, the amount is supplemented with culture medium) and a certain amount of serum-free culture medium were added. After the cells were placed in an incubator for 3 hours, the culture medium was aspirated, and the cells were washed three times with Hank's solution. Culture medium containing fetal bovine serum was added and the cells were cultured for a further 20 hours.

[0151] 5.1.3 Cytotoxicity Assay: The above cells were re-digested, the cells were counted and diluted, and re-seeded. 200 cells were seeded per culture dish, with 5 dishes per dose group. After incubation at 37°C, 5% CO2 for 7 days, colonies were formed. The culture medium was discarded, and the cells were fixed with methanol and stained with Giemsa. The number of cell colonies in each dish was counted, and the cell survival rate of each dose group was calculated (see Table 14).

[0152] 5.2 HPRT site mutation analysis

[0153] 5.2.1 Based on the results of the cytotoxicity test, the relative colony formation rate of each dose group was calculated, with the colony formation rate of the solvent control group as 100%. Based on the cytotoxicity and solubility of the test substance, the highest concentration was set at 5 μL / mL. Four dose groups were established as the formal test doses. A negative control group (untreated) and a positive control group (EMS for -S9 and B(a)P for +S9) were also established. Each group included both the addition and removal of the S9 mixture. In this experiment, the high dose was 5 μL / mL, with or without metabolic activation. The concentrations were diluted in multiples to form four dose groups (i.e., 2.5 μL / mL, 1.25 μL / mL, 0.625 μL / mL, and 0.312 μL / mL).

[0154] 5.2.2 Cell inoculation: Digest the well-growing cells with trypsin-EDTA solution and inoculate 5.0×10 cells per dish. 5 The cells were placed in a 37°C, 5% CO2 incubator for 24 h before adding samples.

[0155] 5.2.3 Exposure: After 24 hours of incubation, add various concentrations of test substance solution or S9 mixture to each plate. For the negative control (solvent control), only culture medium is added. Three hours after addition, discard the culture medium, rinse three times with calcium- and magnesium-free PBS, and incubate in culture medium containing 10% serum for 20 hours.

[0156] 5.2.4 Expression of mutant cells: After the test substance interacts with the cells, continue to culture for 20 hours, then digest the cells, count them, and perform expression and cytotoxicity assays. During the expression period, cells in each dose group were seeded at a low density every 3 days, and the cell concentration was controlled at 1×10 6 After 7 days of culture, mutants were selected and colony formation rates were determined.

[0157] 5.2.5 Cytotoxicity Assay: During expression, 200 cells were seeded per dish, with 5 dishes per dose group. The cells were cultured at 37°C, 5% CO2 for 7 days, then fixed with methanol and stained with Giemsa. The number of cell colonies in each dish was counted, and the cell survival rate of each dose group was calculated (see Table 15).

[0158] 5.2.6 Selection of mutants and determination of colony formation rate: After the expression was completed, the cells were digested and inoculated separately, with 5 plates per group and 2×10 5 After the cells adhered, 6-TG was added to the plate at a final concentration of 5 μg / mL. Simultaneously, 200 cells were inoculated per dish after the digestion described above (without 6-TG). Each group of 5 dishes was placed in a 37°C, 5% CO2 incubator along with the above dishes and cultured for 7 days. After fixation with methanol and Giemsa staining, the number of colonies in each dish was counted, and the total colony number (see Table 16), colony formation rate (see Table 17), and mutation frequency (MF) (see Table 18) were calculated.

[0159] 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 the positive control substance instead of the test substance, and the untreated control group uses culture medium.

[0160] 6. Data processing and result determination

[0161] 6.1 Calculation of indicators: Calculate the relevant indicators according to the following formula:

[0162]

[0163] 6.2 Result determination

[0164] Use χ 2 The test is statistically processed. When the MF of each dose group is significantly increased compared with the negative control group and shows a dose-response relationship, or when only one dose group has a statistically significant increase and is confirmed by repeated tests, it can be judged as a positive result, that is, the test substance is mutagenic to the HGPRT system of V79 cells.

[0165] 2. Test results

[0166] 1. Cytotoxicity test results

[0167] Cytotoxicity testing was performed using dose groups ranging from 0.0098 μL / mL to 5 μL / mL. The test substance in each dose group completely dissolved after addition to the culture medium, with or without metabolic activation. 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 (see Table 14). Based on the results of the above cytotoxicity tests and the solubility of the test substance, the doses for the gene mutation test were set as follows: 5 dose groups (5 μL / mL, 2.5 μL / mL, 1.25 μL / mL, 0.625 μL / mL, and 0.312 μL / mL) were tested, with or without metabolic activation.

[0168] 2. Mutation frequency and statistical results of the experimental and control groups

[0169] No significant increase in mutation frequency (see Table 18) was observed in any dose group of the test substance compared with the negative control group, regardless of whether metabolic activation was present or not (p>0.05, χ 2 Under conditions without metabolic activation, the mutation frequency of the positive control group (EMS) was 256.62×10 -6 , which was statistically significantly increased compared with the solvent control group (p < 0.05, χ 2 Under metabolic activation conditions, the mutation frequency of the positive control group (B(a)P) was 148.46×10 -6 , which was statistically significantly increased compared with the solvent control group (p < 0.05, χ 2 test).

[0170] Table 14 Colony formation rate (cytotoxicity) at different concentrations

[0171]

[0172]

[0173] a Rating standard: ++ = After the test substance solution is added to the culture medium solution, the test substance precipitates to form a large amount of precipitation; + = After the test substance solution is added to the culture medium solution, the test substance precipitates to form a suspension; - = Completely dissolved.

[0174] Table 15 Cell colony formation rate at each concentration during the expression period

[0175]

[0176] a Rating standard: ++ = After the test substance solution is added to the culture medium solution, the test substance precipitates to form a large amount of precipitation; + = After the test substance solution is added to the culture medium solution, the test substance precipitates to form a suspension; - = Completely dissolved.

[0177] Table 16 The number of mutant cell colonies at different concentrations

[0178]

[0179]

[0180] Table 17 Cell colony formation rate for mutation selection

[0181]

[0182]

[0183] Table 18 Results of in vitro mammalian cell gene mutation test

[0184]

[0185] “*” indicates P < 0.05, and “—” indicates not applicable.

[0186] 3. Test Conclusion

[0187] In vitro mammalian cell gene mutation test results: Regardless of whether metabolic activation is present or absent, the test substance at any dose group did not cause a significant increase in the mutation frequency of the test cell lines (see Table 18). However, the positive controls EMS and B(a)P significantly increased the mutation frequency of the test cell lines, with significant differences compared to the solvent control. Therefore, it can be concluded that under these experimental conditions, the test results are negative and the test substance (SsPle 1 mg / mL solution) has no significant mammalian cell gene mutagenic effect.

Claims

1. A Hailong antimicrobial peptide spray, characterized in that: The invention contains sea dragon antimicrobial peptide SsPle as an active ingredient and excipients. The amino acid sequence of the sea dragon antimicrobial peptide SsPle is: FFRSLWKGVKEGFRAGRSAYKA. The excipients include stabilizers, antioxidants, buffers, pH regulators and antiseptic systems.

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

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

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

5. The Hailong antimicrobial peptide spray according to claim 1, characterized in that The antiseptic system is caprylhydroxamic acid, hexylene glycol and propylene glycol.

6. The Hailong antimicrobial peptide spray according to claim 1, characterized in that The Hailong antimicrobial peptide spray is a Hailong antimicrobial peptide spray that is resistant to Escherichia coli (Methicilin-resistant Escherichia coli), Pseudomonas aeruginosa (Pseudomonas aeruginosa), and Staphylococcus aureus (Staphylococcus aureus); preferably, the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus.

7. The Hailong antimicrobial peptide spray according to claim 1, characterized in that The formula of the antibacterial spray is calculated by mass fraction, including 0.1% sea dragon antibacterial peptide SsPle, 1% glycerol, 0.31% histidine, 0.05% methionine, and finally hydrochloric acid is added to adjust the pH value to pH 6, and the balance is pure water.

8. The Hailong antimicrobial peptide spray according to claim 1, characterized in that The formula of the antibacterial spray is calculated by mass fraction and includes 0.1% sea dragon antibacterial peptide SsPle, 1% mannitol, 20mM citrate-citric acid, 1.0% of the compound antioxidant and preservative system, and the balance is water.

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