Preparation method and application of toad skin polypeptide, toad skin antibacterial peptide A or toad skin antibacterial peptide B

Abstract

The invention provides a preparation method and application of toad skin polypeptide, toad skin antibacterial peptide A or toad skin antibacterial peptide B, and belongs to the technical field of biological medicine. The preparation method of the toad skin polypeptide comprises the following steps: crushing dried toad skin to obtain toad skin powder, mixing the toad skin powder with neutral protease, adding water, performing enzymolysis, filtering, and collecting filtrate to obtain enzymatic hydrolysate; and adding the enzymatic hydrolysate into an ultrafiltration centrifuge tube filled with an ultrafiltration membrane with the molecular weight cutoff of 1kDa, centrifuging, and collecting component permeate with the molecular weight less than 1kDa, thereby obtaining the product. The toad skin polypeptide disclosed by the invention has a bacteriostatic effect, and the bacteriostatic activity of the toad skin polypeptide is further improved by the toad skin antibacterial peptide A. The toad skin antibacterial peptide B obtained by further separation has a remarkable antibacterial effect and also has anti-tumor activity. Therefore, the invention provides more thoughts and choices for preparing antibacterial drugs and antitumor drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a preparation method and application of toad skin polypeptide, toad skin antibacterial peptide A or toad skin antibacterial peptide B. Background Art

[0002] Toad skin is the dried epidermis of toads obtained by squeezing out the toad venom and removing the internal organs from Bufo bufo gargarizans Cantor or Bufo melanostictus Schneider of the genus Bufo in the order Anura. It was first recorded in the section of insects in "Supplement to Ben Jing Feng Yuan" and is included in the list of ethnic medicines as a traditional ethnic medicine. In Zhuang language, it is called "Neng Gong Chou". Amphibians live in humid and dark environments. Under the long-term natural environment selection, their skin has evolved into a complex tissue with multiple biological functions such as defense, respiration, and excretion, which makes their skin and skin secretions contain extremely rich multifunctional active peptides. As the "head of the five poisons", toad-based medicinal materials can "use poison to fight poison" and have been widely used by ethnic groups in the treatment of heat-toxic syndromes such as tumors and carbuncles. The dried skin of the toad peeled off and dried is the medicinal material dried toad skin, which is one of the few amphibian skin-based traditional Chinese medicines that are still widely circulated in the market today. Currently, the application of toad skin medicinal materials is mainly as the raw material for clinical anti-tumor and anti-inflammatory drugs represented by cinobufacini preparations. Most studies believe that the clinical anti-tumor and anti-inflammatory activities exhibited by such drugs are caused by bufadienolide compounds and indole alkaloid compounds contained in toad skin. Therefore, the research on the components and pharmacological activities of these two types of compounds in toad skin and traditional Chinese medicine preparations using toad skin as the raw material is relatively extensive, but less attention has been paid to the toad skin polypeptide components with great potential for drug development. Currently, the research on the multifunctional active peptides in the skin of frogs at home and abroad is only increasing, while there are few reports on the components and pharmacological activities of the polypeptide components in toad skin. There is no relevant content on the separation, purification, and identification of antibacterial peptide components in dried toad skin, which is not conducive to the comprehensive utilization of toad skin medicinal materials and the in-depth study of the clinical efficacy mechanism of traditional Chinese medicine preparations using toad skin as the raw material. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a preparation method and application of toad skin polypeptide, toad skin antibacterial peptide A or toad skin antibacterial peptide B.

[0004] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0005] The present invention provides a preparation method of toad skin polypeptide, comprising the following steps:

[0006] After crushing dried toad skin, the obtained toad skin powder is mixed with neutral protease, enzymolyzed after adding water, filtered, and the filtrate is collected to obtain an enzymolysis solution;

[0007] The enzymatic hydrolysate is added into an ultrafiltration centrifuge tube equipped with an ultrafiltration membrane with a molecular weight cutoff of 1 kDa, centrifuged, and the permeate of the components with a molecular weight less than 1 kDa is collected to obtain the product.

[0008] Preferably, the dried toad skin is the dried whole skin of the Chinese giant toad Bufobufogargarizans Cantor.

[0009] Preferably, the enzyme material ratio of the neutral protease to the toad skin powder is 7500-8500:1U / g; the mass volume ratio of the toad skin powder to water is 1:15-25g / mL; the enzymolysis temperature is 45-55°C, and the enzymolysis time is 3-5h.

[0010] The present invention also provides a toad skin polypeptide obtained by the above preparation method.

[0011] The present invention also provides a method for preparing toad skin antimicrobial peptide A, comprising the following steps:

[0012] The toad skin polypeptide solution was subjected to Sephadex G-25 column chromatography and eluted with water at a rate of 0.4 to 0.6 mL·min -1 After 175 minutes, the eluate was collected, and 12 to 13 mL of the eluate was collected to obtain the peak A eluate, thereby obtaining toad skin antimicrobial peptide A.

[0013] The present invention also provides a method for preparing toad skin antimicrobial peptide B, comprising the following steps:

[0014] The peak A eluate is purified and eluted by reverse phase high performance liquid chromatography, and the eluate after 10min to 12min is collected to obtain toad skin antimicrobial peptide B.

[0015] Preferably, the chromatographic column of the reversed-phase high-performance liquid chromatography is a HPLCONE C18 reversed-phase column; the mobile phase A is a 0.1% trifluoroacetic acid solution, and the mobile phase B is acetonitrile; the elution procedure is isocratic elution of 10% volume fraction A phase and 90% volume fraction B phase; the elution rate is 1 mL·min -1 .

[0016] The present invention also provides a use of the toad skin polypeptide, the toad skin antimicrobial peptide A prepared by the preparation method, or the toad skin antimicrobial peptide B prepared by the preparation method in the preparation of antibacterial products.

[0017] Preferably, the bacteria include one or more of Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa.

[0018] The present invention also provides an application of toad skin antimicrobial peptide B prepared by the above preparation method in the preparation of anti-tumor drugs.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention provides a preparation method and application of toad skin polypeptide, toad skin antimicrobial peptide A or toad skin antimicrobial peptide B. The present invention uses neutral protease enzymatic hydrolysis to collect components with a molecular weight less than 1 kDa in the enzymatic hydrolysis solution to obtain a toad skin polypeptide, which has an antibacterial effect. The toad skin antimicrobial peptide A obtained by the present invention through purification by dextran gel G-25 column chromatography further improves its antibacterial activity. The toad skin antimicrobial peptide B obtained by the present invention through further purification and separation by reversed-phase high-performance liquid chromatography has a significant antibacterial effect and also has anti-tumor activity. Therefore, the present invention provides a new idea and option for the preparation of antibacterial drugs and anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The standard curve for determining peptide concentration by Bradford method;

[0022] Figure 2 It is the G-25 gel chromatography spectrum of the polypeptide solution with molecular weight less than 1 kDa;

[0023] Figure 3 is the RP-HPLC spectrum of the eluent of peak A;

[0024] Figure 4 The results of the inhibition of toad skin antimicrobial peptide B on different bacteria; the upper left corner is the test group, the upper right corner is the positive control group, and the bottom is the negative control group;

[0025] Figure 5 The results are the detection results of the proliferation inhibition rate of MDA-MB-231 cells by toad skin antimicrobial peptide B at different concentrations and action time;

[0026] Figure 6 This is the effect of toad skin antibiotic peptide B at different concentrations on the migration and invasion ability of MDA-MB-231 cells. DETAILED DESCRIPTION

[0027] The present invention provides a method for preparing a toad skin polypeptide, comprising the following steps:

[0028] The dried toad skin is crushed, the obtained toad skin powder is mixed with neutral protease, water is added to perform enzymolysis, the filtrate is filtered, and the filtrate is collected to obtain an enzymolysis solution;

[0029] The enzymatic hydrolysate is added into an ultrafiltration centrifuge tube equipped with an ultrafiltration membrane with a molecular weight cutoff of 1 kDa, centrifuged, and the permeate of the components with a molecular weight less than 1 kDa is collected to obtain the product.

[0030] After the dry toad skin is crushed, the obtained toad skin powder is mixed with a neutral protease, and water is added for enzymolysis, and the filtrate is filtered and collected to obtain an enzymolysis solution. The dry toad skin is the dried whole skin of the Chinese giant toad Bufobufogargarizans Cantor. The crushing method refers to crushing the dry toad skin and passing it through a 50-70 mesh sieve. The enzyme material ratio of the neutral protease to the toad skin powder is 7500-8500:1U / g, further 7600-8300:1U / g, and more preferably 8000:1U / g; the mass volume ratio of the toad skin powder to water is 1:15-25g / mL, further 1:17-23g / mL, and more preferably 1:20g / mL; the enzymolysis temperature is 45-55°C, further 46-53°C, and more preferably 50°C, and the enzymolysis time is 3-5h, further 3.5-4.5h, and more preferably 4h. The present invention has no special limitation on the source of neutral protease, and any preparation method known in the art or commercially available products can be used. The polypeptide prepared by neutral protease in the present invention has antibacterial effect.

[0031] The present invention has found that not all polypeptide components with any molecular weight have antibacterial effects. In order to further enhance the antibacterial effect of toad skin polypeptide, the present invention adds the enzymatic hydrolysate to an ultrafiltration centrifuge tube equipped with an ultrafiltration membrane with a molecular weight cutoff of 1 kDa, centrifuges, and collects the permeate of the component with a molecular weight less than 1 kDa to obtain the antibacterial polypeptide. The present invention has found that the antibacterial activity of the collected component with a molecular weight less than 1 kDa is higher than that of polypeptides with other molecular weights.

[0032] The present invention also provides a toad skin polypeptide obtained by the above preparation method.

[0033] The present invention also provides a method for preparing toad skin antimicrobial peptide A, comprising the following steps:

[0034] The toad skin polypeptide solution was subjected to Sephadex G-25 column chromatography and eluted with water at a rate of 0.4 to 0.6 mL·min -1 After 175 minutes, the eluate was collected, and 12 to 13 mL of the eluate was collected to obtain the peak A eluate, thereby obtaining toad skin antimicrobial peptide A.

[0035] When preparing the toad skin antimicrobial peptide A, the concentration of the toad skin peptide solution is 45-55 mg·mL -1 The sample loading amount of the chromatography is 90 to 110 mg. The elution rate is preferably 0.5 mL·min -1 The amount of the collected eluate is preferably 12.5 mL. In the present invention, different eluate peaks are obtained by Sephadex G-25 column chromatography, and only the eluate of peak A has an antibacterial effect.

[0036] The present invention also provides a method for preparing toad skin antimicrobial peptide B, comprising the following steps:

[0037] The peak A eluate is purified and eluted by reverse phase high performance liquid chromatography, and the eluate after 10min to 12min is collected to obtain toad skin antimicrobial peptide B.

[0038] When preparing toad skin antimicrobial peptide B, the chromatographic column of the reversed phase high performance liquid chromatography is a HPLCONE C18 reversed phase column; the mobile phase A is a 0.1% trifluoroacetic acid solution, and the mobile phase B is acetonitrile; the elution procedure is isocratic elution of 10% volume fraction A phase and 90% volume fraction B phase; the elution rate is 1 mL·min -1 The present invention can obtain a variety of different components through reverse phase high performance liquid chromatography. The present invention has found that only toad skin antimicrobial peptide B has an antibacterial effect, while other components do not have antibacterial function.

[0039] The present invention also provides a use of the toad skin polypeptide, the toad skin antimicrobial peptide A prepared by the preparation method, or the toad skin antimicrobial peptide B prepared by the preparation method in the preparation of antibacterial products.

[0040] In the present invention, the bacteria include one or more of Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa, and further include one or more of Escherichia coli ATCC25922, Staphylococcus aureus ATCC25923, Pseudomonas aeruginosa ATCC27853 and Bacillus subtilis ATCC6633.

[0041] The present invention also provides an application of toad skin antimicrobial peptide B prepared by the above preparation method in the preparation of anti-tumor drugs.

[0042] The present invention unexpectedly found that the toad skin antimicrobial peptide B prepared by the present invention also has an anti-tumor effect, especially for breast cancer. In the present invention, the drug also includes a pharmaceutically acceptable excipient. The drug can be used for antibacterial purposes using toad skin polypeptide, toad skin antimicrobial peptide A or toad skin antimicrobial peptide B as the only active ingredient, or can be used in combination with other antimicrobial active ingredients for antibacterial purposes. The present invention can also use toad skin antimicrobial peptide B as the only active ingredient for antitumor, such as breast cancer, and can also be used in combination with other antitumor active ingredients for antitumor purposes.

[0043] The present invention separates and purifies antimicrobial peptides from dried toad skin, and obtains not only polypeptide components with significant antibacterial activity, but also polypeptide components with anti-tumor activity, thus filling the gap in the study of the composition and activity of toad skin polypeptide components, providing a theoretical basis for their clinical application and product development, and providing new ideas for the current research on the mechanism of action of toad skin traditional Chinese medicine preparations.

[0044] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0045] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0046] In this example, the neutral protease was purchased from Beijing Solebow Technology Co., Ltd., item number Z8031. The Escherichia coli ATCC25922, Staphylococcus aureus ATCC25923, Pseudomonas aeruginosa ATCC27853 and Bacillus subtilis ATCC6633 were all purchased from Shandong Top Bioengineering Co., Ltd.

[0047] Example 1

[0048] A method for preparing a toad skin polypeptide, which is prepared by the following method:

[0049] (1) The dried whole skin of the Chinese giant toad Bufo bufogargarizans Cantor, an animal of the Toad family with good properties and quality, was used. The dried toad skin was cut into pieces with scissors, dried in an oven at 40° C. to constant weight, ground into powder with a pulverizer, and passed through a 60-mesh sieve to obtain toad skin powder for later use.

[0050] (2) Accurately weigh 5.0 g of toad skin powder into a conical flask and add 8000 Ug -1 40000U neutral protease was added to the enzyme-to-material ratio, and 100mL of pure water was injected to perform enzymolysis in a constant temperature water bath at 50°C to extract the dried toad skin polypeptide for 4 hours. After enzymolysis, the extract was filtered and the filtrate was collected to obtain the enzymolysis solution.

[0051] (3) Ultrafiltration technology was used for preliminary fractionation and separation. 15 mL of the enzymatic hydrolysate was added to an ultrafiltration centrifuge tube equipped with an ultrafiltration membrane with a molecular weight cutoff of 1 kDa. The ultrafiltration was carried out at 4 °C and 3000 r / min. -1 The first retentate and the first permeate (i.e., the component with a molecular weight less than 1 kDa) were collected; the first retentate was added to an ultrafiltration centrifuge tube equipped with an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and the mixture was centrifuged at 4°C and 3000 r / min. -1 The mixture was centrifuged at 4 °C and 3000 r / min for 30 min to collect the second retentate and the second permeate (i.e., the component with a molecular weight ≥1 kDa and <3 kDa); the second retentate was added to an ultrafiltration centrifuge tube equipped with an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and the mixture was centrifuged at 4 °C and 3000 r / min -1 The mixture was centrifuged at 4 °C and 3000 r / min for 30 min to collect the third retentate and the third permeate (i.e., the component with a molecular weight ≥3 kDa and <10 kDa); the third retentate was added to an ultrafiltration centrifuge tube equipped with an ultrafiltration membrane with a molecular weight cutoff of 50 kDa, and the mixture was centrifuged at 4 °C and 3000 r / min -1The mixture was centrifuged at 370 °C for 30 min to collect the fourth retentate (i.e., the component with a molecular weight of ≥50 kDa) and the fourth permeate (i.e., the component with a molecular weight of ≥10 kDa and <50 kDa).

[0052] After freeze-drying each component, the minimum inhibitory concentration MIC value and the peptide yield were measured respectively, and the difference in antibacterial activity of each component was compared. The screening and comparison results are shown in Table 1. Among them, the peptide yield was determined by Bradford method. Plot a standard curve, measure the absorbance value, and substitute it into the standard curve to calculate the sample peptide concentration. The standard curve is as follows Figure 1 shown.

[0053] x=(y-0.5119)÷1.1934

[0054] Where: y is the absorbance at 595nm; x is the peptide concentration of the solution at this absorbance, mg·mL -1 .

[0055] Peptide yield (%) = x × v × f ÷ m × 100%

[0056] Wherein: x represents the sample polypeptide concentration, mg / mL; v represents the extraction solution volume, mL; f represents the dilution factor; m represents the dry toad skin mass, mg.

[0057] Among them, the minimum inhibitory concentration of different components of toad skin polypeptide was determined by TTC microdilution method:

[0058] (1) Strain activation

[0059] Take out four test bacterial strains, including Escherichia coli ATCC25922, Staphylococcus aureus ATCC25923, Pseudomonas aeruginosa ATCC27853 and Bacillus subtilis ATCC6633, from the refrigerator, spread 100 μL of bacterial solution on LB solid culture medium in a sterile operating table, culture at 37°C for 2 h, then pick a single colony with good growth condition and streak it into LB slant culture medium, and culture it again for 24 h.

[0060] (2) Preparation of bacterial suspension

[0061] The activated strains were inoculated into LB liquid medium and cultured at 37°C overnight, then diluted 100 times to adjust the bacterial solution concentration to 10 6 ~10 8 cfu / mL, for future use.

[0062] (3) Determination of minimum inhibitory concentration (MIC) value

[0063] The TTC microdilution method was used to detect the minimum inhibitory concentration (MIC) values ​​of components with a molecular weight of less than 1 kDa, components with a molecular weight of ≥1 kDa and <3 kDa, components with a molecular weight of ≥3 kDa and <10 kDa, components with a molecular weight of ≥10 kDa and <50 kDa, and components with a molecular weight of ≥50 kDa. Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa were used as test bacteria to investigate the antibacterial activity of each polypeptide component. An appropriate amount of polypeptide sample was dissolved in sterile water, and LB medium was used as the strain culture medium to dilute the sample at the same time, so that the final concentration of the polypeptide components above was 250 mg mL -1 , 125mg·mL -1 、62.5mg·mL -1 、31.25g·mL -1 , 15.63mg·mL -1 、7.81mg·mL -1 、3.91mg·mL -1 , add 100 μL to a 96-well plate. Dilute the bacterial suspension after secondary activation to a concentration of 10 6 ~10 8 cfu·mL -1 Then add 5 μL of bacterial solution to each well, incubate in a 37°C incubator for 16 to 18 hours, then add 5 μL of 0.5% TTC solution to each well, incubate for 1 hour and observe the color change in the wells with the naked eye. If there is bacterial growth, the wells will turn red. The minimum sample concentration of the colorless wells is the MIC value of the sample.

[0064] The MIC of each polypeptide component against four strains of Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa was used as an indicator of the antibacterial strength. The comprehensive MIC was calculated using the following formula:

[0065] SMIC=(MIC1+MIC2+MIC3+MIC4)÷4

[0066] Wherein: SMIC represents comprehensive MIC; MIC1, MIC2, MIC3, MIC4 represent the MIC values ​​of each component against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, respectively.

[0067] The results in Table 1 show that the components with a molecular weight less than 1 kDa have the strongest comprehensive antibacterial activity and exhibit certain antibacterial activity against the four tested bacterial species. Therefore, the components with a molecular weight less than 1 kDa were collected for further separation and purification.

[0068] The present invention names the component with a molecular weight less than 1 kDa as toad skin polypeptide.

[0069] Table 1 Extraction rate of different components extracted from toad skin and minimum inhibitory concentration (MIC) determination results

[0070]

[0071]

[0072] Note: “+” means bacterial growth; “-” means no bacterial growth, and “ / ” means no SMIC value.

[0073] Example 2

[0074] A method for preparing toad skin antimicrobial peptide A, the steps are as follows:

[0075] The molecular weight <1 kDa component (toad skin polypeptide) solution obtained in Example 1 was concentrated and freeze-dried into powder, and then prepared into 50 mg mL -1 The toad skin polypeptide solution was set aside for use, and the sample amount was 100 mg of toad skin polypeptide solution each time on a dextran gel G-25 gel column (chromatographic column specification: 1.6 cm × 60 cm), and the constant flow pump was turned on to control the flow rate to 0.5 mL min -1 After the sample solution completely enters the gel layer, add 3-5 cm of deionized water to the upper end of the gel, seal the chromatography column, and reconnect the deionized water to start elution. 5min / tube, 2.5mL of eluent can be collected in each tube, and a single chromatography lasts a total of 5.8h. The ultraviolet absorption of each tube of eluent is detected at 280nm and the absorption peak shape is recorded. The A peak eluent is the 36th to 40th tube (the A peak eluent is collected after 175min and collected until 200min), and 12.5mL of A peak eluent can be collected in a single chromatography; the B peak eluent is the 42nd to 44th tube (the B peak eluent is collected after 205min and collected until 220min), and 7.5mL of B peak eluent can be collected in a single chromatography; the C peak eluent is the 50th to 56th tube (the C peak eluent is collected after 245min and collected until 280min), and 17.5mL of C peak eluent can be collected in a single chromatography The eluent of peak C and peak D are from tubes 60 to 66 (the eluent of peak D is collected starting from 295 min and lasting until 330 min). 17.5 mL of the eluent of peak D can be collected at a time.

[0076] Among them, the G-25 gel chromatography spectrum of the polypeptide solution with a molecular weight less than 1 kDa is as follows Figure 2 As shown, four elution peaks were obtained by G-25 gel chromatography of a polypeptide solution with a molecular weight less than 1 kDa.

[0077] The eluted peaks were collected and concentrated by rotary evaporation under reduced pressure. The antibacterial activity of each absorption peak component was determined by the inhibition zone method. The peak position of the toad skin active peptide was tracked. The results are shown in Table 2.

[0078] Determination method of the inhibition zone:

[0079] (1) Strain activation

[0080] Take out four test bacterial strains, including Escherichia coli ATCC25922, Staphylococcus aureus ATCC25923, Pseudomonas aeruginosa ATCC27853 and Bacillus subtilis ATCC6633, from the refrigerator respectively, spread 100 μL of bacterial solution on LB solid culture medium in a sterile operating table, culture at 37°C for 24 h, then pick a single colony with good growth condition and streak it into LB slant culture medium, and culture it again for 24 h.

[0081] (2) Preparation of bacterial suspension

[0082] The activated strains were inoculated into LB liquid medium, cultured overnight at 37°C, and diluted 100 times to adjust the bacterial solution concentration to 10 6 ~10 8 cfu / mL, for future use.

[0083] (3) Plate culture

[0084] Pipette 100 μL of the above four test bacterial suspensions onto the solid culture medium and spread evenly. Place the sterile drug-susceptibility paper strips soaked with peak A, peak B, peak C and peak D eluates on the plate, with 3 parallels in each group. After culturing in a 37°C incubator overnight, observe whether there is a transparent antibacterial zone around the drug-susceptibility paper strips.

[0085] The results in Table 2 show that the fraction less than 1 kDa after dextran G-25 gel chromatography showed a certain antibacterial activity against the four tested bacterial species.

[0086] The present invention names the A peak eluate as toad skin antibiotic peptide A.

[0087] Table 2Sephadex G-25 chromatography separation of each component inhibition zone experiment

[0088] Eluent Bacillus subtilis Staphylococcus aureus Escherichia coli Pseudomonas aeruginosa Peak A eluent + + + + Peak B eluent － － － － Peak C eluent － － － － Peak D eluent － － － －

[0089] Note: “+” represents the presence of inhibition zone; “-” represents the absence of inhibition zone.

[0090] Example 3

[0091] A method for preparing toad skin antimicrobial peptide B, comprising the following steps:

[0092] The eluate from peak A prepared in Example 2 was collected and lyophilized to powder and then redissolved in deionized water to prepare 1 mg mL -1The solution was centrifuged and the collected supernatant was filtered through a 0.22 μm water filter membrane. The filtrate was collected for reversed-phase high-performance liquid chromatography purification. The reversed-phase high-performance liquid chromatography column was HPLCONEC18 (250×10 mm) (Merchant: Suzhou Suyan Pharmaceutical Technology Co., Ltd., item number: 14001-019) reversed-phase column. The mobile phase of the system was 0.1% trifluoroacetic acid solution (A) and acetonitrile (B); the volume fraction of 10% A phase and 90% B phase was used for isocratic elution; the volume flow rate was 1 mL·min -1 , single injection volume 100μL, total process 30min, manually collect each elution peak component, collect 10min~12min eluate as A1 component, a total of 2mL A1 component eluate can be collected at a time; collect 15min~18min eluate as A2 component, a total of 3mL A2 component eluate can be collected at a time; collect 24min~27min eluate as A3 component, a total of 3mL A3 component eluate can be collected at a time.

[0093] The mobile phase is prepared by measuring 1.0 mL of trifluoroacetic acid solution for phase A, diluting it to 1000 mL with pure water, mixing it, filtering it with suction, and ultrasonicating it to prepare a 0.1% trifluoroacetic acid solution; and measuring 1000 mL of acetonitrile for phase B, mixing it, filtering it with suction, and ultrasonicating it to obtain phase B.

[0094] Among them, the RP-HPLC spectrum of the A peak eluate is shown in Figure 3 ,Depend on Figure 3 It can be seen that three components were separated from the eluent of peak A.

[0095] After collecting the eluate of each component, it was concentrated under reduced pressure and the inhibition zone experiment was repeated. The method for determining the inhibition zone is shown in Example 2. The results are shown in Table 3.

[0096] The results in Table 3 show that the final antimicrobial active peptide tracked and locked was the eluate of component A1.

[0097] Table 3 RP-HPLC purification and separation of each component inhibition zone experiment

[0098] Components Bacillus subtilis Staphylococcus aureus Escherichia coli Pseudomonas aeruginosa A1 fraction eluent + + + + A2 fraction eluent － － － － A3 fraction eluent － － － －

[0099] Note: “+” represents the presence of inhibition zone; “-” represents the absence of inhibition zone.

[0100] (3) Collect the eluate of the A1 component, concentrate under reduced pressure, and freeze-dry to obtain polypeptide powder, thereby obtaining toad skin antimicrobial peptide B for later use.

[0101] Example 4

[0102] Antibacterial experimental study

[0103] 1. Experimental Materials

[0104] Escherichia coli ATCC25922, Staphylococcus aureus ATCC25923, Pseudomonas aeruginosa ATCC27853 and Bacillus subtilis ATCC6633 were purchased from Shandong Top Bioengineering Co., Ltd.

[0105] 2. Test methods

[0106] 2.1 Activation of strains and preparation of bacterial suspension

[0107] Take out four test bacterial strains, including Escherichia coli ATCC25922, Staphylococcus aureus ATCC25923, Pseudomonas aeruginosa ATCC27853 and Bacillus subtilis ATCC6633, from the refrigerator, spread 100 μL of bacterial solution on LB solid culture medium in a sterile operating table, culture at 37°C for 24 hours, then pick a single colony with good growth condition and streak it into LB slant culture medium, and culture it again for 24 hours.

[0108] 2.2 Determination of antibacterial activity by inhibition zone method

[0109] Sterile drug-sensitive paper discs were soaked in 2 mg mL -1 of kanamycin (positive control group), 5 mg·mL -1 The toad skin antimicrobial peptide B (test group) and sterile purified water (negative control group) prepared in Example 3 were placed on the plate coated with the bacterial solution, respectively, and cultured in a 37° C. incubator overnight, and then the diameter of each inhibition zone was measured.

[0110] Results Figure 4 And as shown in Table 4.

[0111] Figure 4 The results in Table 4 show that the toad skin antimicrobial peptide B prepared by the present invention can inhibit Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa.

[0112] Table 4 Results of the diameter of the inhibition zone of toad skin antimicrobial peptide B against different bacteria

[0113] project Bacillus subtilis Staphylococcus aureus Escherichia coli Pseudomonas aeruginosa Diameter of inhibition zone (mm) 21.17±0.58 17.43±0.61 11.69±0.35 2.27±0.22

[0114] 2.3 Determination of minimum inhibitory concentration (MIC) value

[0115] Pipette 100 μL of LB liquid medium into wells 1-9 of each row of a 96-well plate, and pipette 100 μL of the 5 mg mL -1 The toad skin antimicrobial peptide B prepared in Example 3 was added to the first well of each row and pipetted 3 to 5 times to ensure sufficient mixing. At this time, the concentration of toad skin antimicrobial peptide B prepared in Example 3 in the first well of each row was 2.5 mg·mL -1, then draw 100 μL of liquid from the first well and add it to the second well, and mix thoroughly. At this time, the concentration of toad skin antimicrobial peptide B prepared in Example 3 in the second well is 1.25 mg·mL -1 Repeat this doubling dilution step to the 9th well, and finally discard the 100 μL liquid aspirated from the 9th well. The concentration of toad skin antimicrobial peptide B prepared in Example 3 in each row of 9 wells is 2.50 mg·mL -1 , 1.25mg·mL -1 , 0.625mg·mL -1 , 0.312mg·mL -1 , 0.156mg·mL -1 , 0.078mg·mL -1 , 0.039mg·mL -1 , 0.020mg·mL -1 , 0.010mg·mL -1 Finally, add 5 μL of Escherichia coli ATCC25922, Staphylococcus aureus ATCC25923, Pseudomonas aeruginosa ATCC27853 and Bacillus subtilis ATCC6633 bacterial solutions into each of the 9 wells, with 3 parallels per well. After culturing in a 37°C incubator for 16 to 18 hours, add 5 μL of 0.5% TTC solution and cultivate in a constant temperature incubator for 1 hour before observing the results.

[0116] Table 5 Antibacterial activity test results of toad skin antimicrobial peptide B against 4 tested bacteria (n=3)

[0117] project Bacillus subtilis Staphylococcus aureus Escherichia coli Pseudomonas aeruginosa <![CDATA[MIC(mg·mL -1 )]]> 0.039 0.078 0.156 2.50

[0118] The results in Table 5 show that toad skin antimicrobial peptide B has an inhibitory effect on Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa.

[0119] Example 5

[0120] Study on the antitumor activity of toad skin antimicrobial peptide B prepared in Example 3

[0121] Determination method of antitumor activity of toad skin antimicrobial peptide B:

[0122] 8 × 10 cells were seeded per well in a 96-well plate. 3 After 24 hours, 0.25 mg mL -1 , 0.5mg·mL -1 and 1.0 mg mL -110 μL of CCK8 solution was added to the MDA-MB-231 cells as a control group after adding different concentrations of toad skin antimicrobial peptide B for 24h and 48h, and the absorbance value was detected at 450nm by a microplate reader after culturing for 3-4h in a cell culture incubator. Cell proliferation inhibition rate (%) = (1-average OD value of the experimental group) / average OD value of the control group.

[0123] Figure 5 The results showed that the proliferation ability of MDA-MB-231 cells treated with toad skin antimicrobial peptide B was significantly decreased. At 24 h, the cell inhibition rate decreased with the increase of toad skin antimicrobial peptide B concentration and action time, showing a time-dependent relationship. At 48 h, the toad skin antimicrobial peptide B concentration was 0.5 mg·mL -1 The cell inhibition rate was as high as 57.99%. At the same concentration, the inhibitory effect on cell proliferation was more obvious after 48 hours than after 24 hours.

[0124] Transwell cell migration and invasion assay:

[0125] (1) Migration test

[0126] Refer to the cell passage steps of human breast cancer cell MDA-MB-231 to obtain cell suspension and adjust the concentration to 2×10 5 200 μL of cell suspension was inoculated into the uncoated chamber, and a control group and groups treated with different concentrations of toad antimicrobial peptide B (0.25, 0.5, 1.0 mg mL -1 ), add 10% fetal bovine serum culture medium to the lower chamber, transfer to a cell culture incubator and culture for 24 hours. Take out the transwell chamber, discard the culture medium and wash with PBS, add methanol to fix for 30 minutes, air dry and stain with 0.1% crystal violet, wash the excess dye with PBS, select 5 fields of view under a 10x microscope to count and record.

[0127] (2) Invasion assay

[0128] The upper chamber surface needs to be coated with diluted matrix gel to simulate the extracellular matrix layer. After air drying at room temperature, culture medium is added to hydrate the matrix membrane and step (1) is repeated.

[0129] Figure 6 The results showed that after treatment with toad skin antimicrobial peptide B, the migration and invasion abilities of MDA-MB-231 cells were significantly decreased, and the higher the drug concentration, the more obvious the decrease in the migration and invasion abilities of cells.

[0130] It can be seen that the toad skin antibacterial active peptide B has certain potential for the development of clinical anti-tumor drugs.

[0131] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a toad skin polypeptide, characterized in that: The following steps are involved: The dried toad skin is crushed, the obtained toad skin powder is mixed with neutral protease, water is added to perform enzymolysis, the filtrate is filtered, and the filtrate is collected to obtain an enzymolysis solution; The enzymatic hydrolysate is added into an ultrafiltration centrifuge tube equipped with an ultrafiltration membrane with a molecular weight cutoff of 1 kDa, centrifuged, and the permeate of the components with a molecular weight less than 1 kDa is collected to obtain the product.

2. The preparation method according to claim 1, characterized in that: The dried toad skin is the dried whole skin of the Chinese giant toad Bufobufogargarizans Cantor.

3. The preparation method according to claim 1, characterized in that: The enzyme material ratio of the neutral protease to the toad skin powder is 7500-8500:1U / g; the mass volume ratio of the toad skin powder to water is 1:15-25g / mL; the enzymolysis temperature is 45-55°C, and the enzymolysis time is 3-5h.

4. The toad skin polypeptide obtained by the preparation method according to any one of claims 1 to 3.

5. A method for preparing toad skin antimicrobial peptide A, characterized in that: The following steps are involved: The toad skin polypeptide solution according to claim 4 is subjected to Sephadex G-25 column chromatography and eluted with water at an elution rate of 0.4 to 0.6 mL·min -1 After 175 minutes, the eluate was collected, and 12 to 13 mL of the eluate was collected to obtain the peak A eluate, thereby obtaining toad skin antimicrobial peptide A.

6. A method for preparing toad skin antimicrobial peptide B, characterized in that: The following steps are involved: The peak A eluate of claim 5 is subjected to reverse phase high performance liquid chromatography purification and elution, and the eluate after 10 to 12 minutes is collected to obtain toad skin antimicrobial peptide B.

7. The preparation method according to claim 6, characterized in that: The chromatographic column of the reversed-phase high-performance liquid chromatography is a HPLCONE C18 reversed-phase column; the mobile phase A is a 0.1% trifluoroacetic acid solution, and the mobile phase B is acetonitrile; the elution procedure is isocratic elution of 10% volume fraction A phase and 90% volume fraction B phase; the elution rate is 1 mL·min -1 .

8. Use of the toad skin polypeptide according to claim 4, the toad skin antimicrobial peptide A prepared by the preparation method according to claim 5, or the toad skin antimicrobial peptide B prepared by the preparation method according to claim 6 or 7 in the preparation of antibacterial products.

9. The use according to claim 8, characterized in that: The bacteria include one or more of Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa.

10. Use of toad skin antimicrobial peptide B prepared by the preparation method according to claim 6 or 7 in the preparation of anti-tumor drugs.

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