Composition for inhibiting hemolysis of melittin and application thereof

Abstract

The present invention belongs to the field of biopharmaceuticals, and relates to a composition for inhibiting hemolysis of melittin and its application, wherein the composition comprises an equal volume mixture of 2-64 μmol / L melittin solution and 12.5-50 mg / L tannic acid solution, and the solvent is a phosphate buffer. The addition of tannic acid in the composition of the present invention does not change the original structure of melittin, and does not require a synthetic reaction, and the operation is simple. The combination of the two can basically eliminate the hemolytic side effect of melittin, and the feasibility is high. The composition of the present invention is compounded with tannic acid and melittin in different proportions, which has no effect on the antibacterial effect, so that it still has the characteristics of rapid sterilization, and is safe and has no side effects.

Description

Technical Field

[0001] The invention belongs to the field of biopharmaceuticals and relates to a composition for inhibiting melittin hemolysis and application thereof. Background Art

[0002] Melittin can form an amphipathic helical structure, which destroys the bacterial cell membrane by binding to the membrane to achieve the purpose of sterilization, so it has a strong broad-spectrum antibacterial activity. Melittin (Mel) has also been reported to have a synergistic anti-tumor effect when used in combination with certain chemotherapeutic drugs, and can effectively treat cancers that have already developed multidrug resistance. However, Mel has the disadvantages of strong hemolytic toxicity, poor distribution specificity, and easy metabolic degradation, and studies have found that Mel's antibacterial ability against Gram-positive bacteria is not ideal, which greatly limits its application in clinical practice. Most of the currently known methods for eliminating hemolytic activity use mutations or modifications to the melittin sequence, which are complex in the early experimental stage, have a long experimental cycle, and are costly. In addition, in the study of Mel sequence modification, the effects on hemolysis and antimicrobial effects are relatively independent.

[0003] Mel is a strong alkaline peptide with a half-hemolytic concentration of about 0.8μM for mouse erythrocytes and about 0.51μM for human cord blood erythrocytes. It acts directly on the cell phospholipid membrane to dissolve it mainly through carpet and perforation models. It can inhibit the growth of tumor cells and microbial cells, but its clinical application is also limited by its hemolytic property. As a membrane active peptide, Mel has a typical amphipathic α-helical structure, hydrophobic structure and cationicity, which are the basis for the antimicrobial peptide to interact with biological membranes. Among them, the leucine zipper region plays a vital role in the hemolytic activity of Mel. Scanning electron microscopy technology found that Mel can form cracks and holes on the surface of the erythrocyte membrane, lyse the erythrocyte membrane, and cause hemolysis.

[0004] Tannic acid is a secondary metabolite produced during plant growth. The antibacterial activity of tannic acid has long been recognized. The toxicity of tannic acid to bacteria, fungi and yeast has also been reported, among which the antibacterial activity against Staphylococcus bacteria is good. Tannic acid exerts its antibacterial effect by inhibiting extracellular microbial enzymes, depriving microorganisms of the substrate required for growth, inhibiting microbial metabolism through oxidative phosphorylation, depriving metal ions or forming cell membrane complexes with bacteria that cause changes in cell wall morphology and increased membrane permeability. However, due to the wide range of sources of tannic acid and its great diversity of antibacterial activity, screening and identifying the target microorganisms of tannic acid is a complex research work. On the other hand, when used alone, the bactericidal effect is not as good as Mel, and the antibacterial effect is often long-lasting. It is easy to produce precipitation with culture media such as TSB, which is not conducive to the operation of antibacterial tests.

[0005] Therefore, it is necessary to develop a simple antibacterial composition that improves the safety of melittin and simultaneously improves the antibacterial timeliness of tannic acid. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a composition for inhibiting hemolysis of melittin in view of the deficiencies in the prior art. The composition, without changing the sequence of melittin, retains the antibacterial and bactericidal abilities of melittin and eliminates its hemolytic side effects, thereby achieving a safe and rapid antibacterial effect.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0008] The invention discloses a composition for inhibiting melittin hemolysis. The composition comprises 2-64 micromol / L melittin solution and 12.5-50 mg / L tannic acid solution mixed in equal volumes, and the solvent is a phosphate buffer solution.

[0009] Specifically, the composition comprises a 2-8 μmol / L bee venom peptide solution and a 25-50 mg / L tannic acid solution mixed in equal volumes, and the solvent is a phosphate buffer solution.

[0010] Specifically, the phosphate buffer (PBS), 10 mM, pH = 7.4.

[0011] Wherein, the preparation method of the above-mentioned composition is to mix bee venom peptide, tannic acid and phosphate buffer according to the ratio of the formula to prepare the composition.

[0012] Furthermore, the use of the above-mentioned composition for inhibiting melittin hemolysis in the preparation of a drug for inhibiting melittin hemolysis is also within the protection scope of the present invention.

[0013] Furthermore, the use of the above-mentioned composition for inhibiting melittin hemolysis in the preparation of a drug for inhibiting melittin hemolysis and killing bacteria is also within the protection scope of the present invention.

[0014] Specifically, the bacteria are Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Enterococcus faecalis or Escherichia coli.

[0015] Beneficial effects:

[0016] (1) The addition of tannic acid to the composition of the present invention does not change the original structure of melittin, and does not require a synthetic reaction, so the operation is simple. The combined use of the two can substantially eliminate the hemolytic side effect of melittin, and the feasibility is high.

[0017] (2) The composition of the present invention is compounded with tannic acid and melittin in different proportions, which has no effect on the antibacterial effect, so that it still has the characteristics of rapid bactericidal effect and is safe and has no side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0019] Figure 1 The figure shows the effect of compositions with different ratios on the hemolysis rate.

[0020] Figure 2 The figure shows the effect of the premixing time of the composition on the hemolysis rate.

[0021] Figure 3 It is the CD spectrum diagram of Example 6; wherein, mel is a 4 μM bee venom peptide solution group, mel+TA100 is a composition group of equal volume mixture of 4 μM bee venom peptide solution + 100 μg / mL tannic acid solution, and mel+TA1000 is a composition group of equal volume mixture of 4 μM bee venom peptide solution + 1000 μg / mL tannic acid solution.

[0022] Figure 4 The antibacterial test diagram of the composition in Examples 6 to 7; Figure 4 The bacteria in (a) are Enterococcus faecalis, a0 is a blank group without adding the composition, a1 is a 50 μg / mL tannic acid + 4 μM melittin group, a2 is a 50 μg / mL tannic acid + 2 μM melittin group, a3 is a 100 μg / mL tannic acid + 2 μM melittin group, and a4 is a 200 μg / mL tannic acid + 2 μM melittin group; Figure 4 The bacteria in (b) are Escherichia coli, b0 is a blank group without adding the composition, b1 is a 50 μg / mL tannic acid + 8 μM melittin group, b2 is a 50 μg / mL tannic acid + 2 μM melittin group, b3 is a 100 μg / mL tannic acid + 2 μM melittin group, and b4 is a 300 μg / mL tannic acid + 2 μM melittin group; Figure 4 The bacteria in (c) are methicillin-resistant Staphylococcus aureus, c0 is a blank group without adding the composition, c1 is a 25 μg / mL tannic acid + 2 μM melittin group, c2 is a 25 μg / mL tannic acid + 1 μM melittin group, c3 is a 10 μg / mL tannic acid + 0.5 μM melittin group, and c4 is a 100 μg / mL tannic acid + 1 μM melittin group; Figure 4 The bacteria in (c) are Staphylococcus aureus, d0 is a blank group without adding the composition, d1 is a 50 μg / mL tannic acid + 2 μM melittin group, d2 is a 50 μg / mL tannic acid + 1 μM melittin group, d3 is a 100 μg / mL tannic acid + 0.5 μM melittin group, and d4 is a 200 μg / mL tannic acid + 1 μM melittin group.

[0023] Figure 5 This is a graph showing flocculent precipitation when the tannic acid solution used in the composition exceeds 100 μg / mL. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below with reference to the embodiments.

[0025] In the following examples and comparative examples, tannic acid was purchased from Sigma and has a molecular formula of C 76 H 52 O 46 , purity 99%; bee venom peptide is synthesized in the laboratory, and the sequence of bee venom peptide is: GLY-ILE-GLY-ALA-VAL-LEU-LYS-VAL-LEU-THR-THR-GLY-LEU-PRO-ALA-LEU-ILE-SER-TRP-ILE-LYS-ARG-LYS-ARG-GLN-GLN, purity 99%.

[0026] The phosphate buffer solution (PBS) used in the examples of the present invention is 10 mM, pH=7.4.

[0027] Example 1

[0028] (1) Preparation of a composition for reducing hemolysis of melittin: prepare a 64 μM melittin solution with PBS solution; prepare 0 μg / mL, 3.125 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL and 100 μg / mL tannic acid solutions with PBS solution; mix the 64 μM melittin solution with tannic acid solutions of different concentrations in equal volumes to prepare a composition, and use the prepared composition to conduct a hemolytic experiment. Figure 1 For the hemolysis rate data, a portion of the composition with the optimal concentration ratio was re-prepared and pre-mixed for 2 hours, and the hemolysis test was performed again after pre-mixing for 2 hours.

[0029] (2) Hemolytic test:

[0030] Take 2 mL of horse blood, wash the cells with PBS three times until the supernatant is clear, remove the supernatant, dilute the red blood cells with PBS at a concentration of 4% (v / v), add to a 96-well plate, 100 μL per well, use PBS as a negative control, Triton X-100 (polyethylene glycol octylphenyl ether) as a positive control, and add 100 μL of a composition of different concentrations of equal volume of bee venom peptide solution and tannic acid solution to each well, incubate at 37°C for 5 minutes to 2 hours, then centrifuge at 1000g for 5 minutes, and take 100 μL of the supernatant to measure A 570 , Among them, A sample is the sample absorbance, A NS is the absorbance value of the negative group, A Triton x It is the absorbance value of the positive group.

[0031] Example 2

[0032] Preparation of a composition for reducing hemolysis of melittin: prepare an 8μM melittin solution (maximum bactericidal working concentration) with PBS solution; prepare 0μg / mL, 3.125μg / mL, 6.25μg / mL, 12.5μg / mL, 25μg / mL, 50μg / mL and 100μg / mL tannic acid solutions with PBS solution; mix the 8μM melittin solution with tannic acid solutions of different concentrations in equal volumes to prepare a composition, and use the prepared composition to measure hemolysis according to the hemolysis experimental steps in Example 1. Figure 1 For the hemolysis rate data, a portion of the composition with the optimal concentration ratio was re-prepared and pre-mixed for 2 hours, and the hemolysis test was performed again after pre-mixing for 2 hours.

[0033] Example 3

[0034] Preparation of a composition for reducing hemolysis of melittin: prepare a 4 μM melittin solution with PBS solution; prepare 0 μg / mL, 3.125 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL and 100 μg / mL tannic acid solutions with PBS solution; mix the 4 μM melittin solution with tannic acid solutions of different concentrations in equal volumes to prepare a composition, and use the prepared composition to measure hemolysis according to the hemolysis experimental steps in Example 1. Figure 1 For the hemolysis rate data, a portion of the composition with the optimal concentration ratio was re-prepared and pre-mixed for 2 hours, and the hemolysis test was performed again after pre-mixing for 2 hours.

[0035] Example 4

[0036] Preparation of a composition for reducing hemolysis of melittin: prepare a 2μM melittin solution with PBS solution; prepare 0μg / mL, 3.125μg / mL, 6.25μg / mL, 12.5μg / mL, 25μg / mL, 50μg / mL and 100μg / mL tannic acid solutions with PBS solution; mix the 2μM melittin solution with tannic acid solutions of different concentrations in equal volumes to prepare a composition, and use the prepared composition to measure hemolysis according to the hemolysis experimental steps in Example 1. Figure 1 For the hemolysis rate data, a portion of the composition with the optimal concentration ratio was re-prepared and pre-mixed for 2 hours, and the hemolysis test was performed again after pre-mixing for 2 hours.

[0037] The effects of the compositions of different proportions prepared in Examples 1 to 4 on the hemolysis rate are shown in Figure 1In the limited range of the embodiment, the 64 μM melittin solution alone exceeds the bactericidal working concentration, and the hemolysis rate of blood cells at this concentration is 100%. Figure 1 It can be seen that the use of compositions with different concentration ratios reduces the hemolysis rate. When the concentration of the tannic acid solution used in the composition is 12.5-50 μg / mL, the effect of reducing the hemolysis rate is better. Even the composition prepared by mixing equal volumes of 50 μg / mL tannic acid solution and 64 μM bee venom peptide solution can reduce the hemolysis rate to 0.89%. The composition prepared by the present invention effectively controls the problem of cell hemolysis. When using a high concentration tannic acid solution (100 μg / mL), the hemolysis reaction is not well controlled, which may be due to the flocculation effect of tannic acid.

[0038] The optimal concentration ratio compositions in Examples 1 to 4 were premixed for 2 hours and then subjected to hemolytic test again. The specific hemolytic rate data are shown in Figure 2 .from Figure 2 It can be seen that short-term premixing of the composition has little effect on the hemolysis rate.

[0039] Example 5

[0040] CD spectroscopy was used to determine the effect of tannic acid on the secondary structure of Mel:

[0041] (1) using TFE solution to dilute tannic acid and melittin, respectively, to prepare a 4 μM melittin solution, a composition of a 4 μM melittin solution + a 100 μg / mL tannic acid solution in equal volumes, and a composition of a 4 μM melittin solution + a 1000 μg / mL tannic acid solution in equal volumes;

[0042] (2) placing the three solutions of different concentrations prepared in step (1) in quartz cuvettes with a light length of 0.1 cm respectively;

[0043] (3) Set the spectral parameters: scanning time is 1 min, scanning wavelength is 190-250 nm, and step is 0.5 nm;

[0044] (4) After the scan is completed, the baseline is adjusted using 0% data.

[0045] The CD spectra of the above three solutions with different concentration ratios are shown in Figure 3 ,from Figure 3It can be seen from the results that when the antimicrobial peptide (melittin) is bound to or close to the cell membrane, the melittin is mainly in an α-helical structure when it contacts the hydrophobic environment in the membrane. When a 4 μM melittin solution is used in combination with a 100 μg / mL tannic acid solution, the conformation of melittin will not change, which further proves that the composition of the present invention can still maintain the biological activity of melittin while reducing the hemolytic effect of melittin. However, when the concentration of the tannic acid solution used is increased to 1000 μg / mL, the conformation of melittin changes significantly, and at this time, not only can the hemolytic effect of melittin not be reduced, but the biological activity of melittin is also basically lost.

[0046] Example 6

[0047] The test strain was cultured to logarithmic phase and diluted to 5 × 10 6 CFU / mL, 198 μL of bacterial solution was added to a 96-well culture plate, and then 1 μL of bee venom peptide solution and tannic acid solution with different concentration ratios (Table 1) were added, and the positive control well was 200 μL of bacterial solution. The plates were cultured at 37°C for 2 min, 10 min and 30 min respectively, and 10 μL of culture solution was spotted on NB medium for counting, and the ratio concentration for complete sterilization was recorded.

[0048] Table 1 Compositions of equal volumes mixed with different concentration ratios

[0049]

[0050] Table 2 Minimum bactericidal concentration MBC of the test solution

[0051]

[0052] As can be seen from Table 2, the antibacterial effect of the composition on Escherichia coli is not as good as that on Gram-positive bacteria. The composition of different concentration ratios in Example 7 uses bee venom peptide as the main bactericidal component, and low concentration of tannic acid has no effect on the antibacterial effect of Mel. The composition prepared by equal volume ratio according to the solution concentration in Table 2 starts to have a bactericidal effect in 5 to 10 minutes, shortening the bactericidal time. However, when the concentration of the tannic acid solution used is too high, especially when it exceeds the minimum inhibitory concentration (100 μg / mL) of the tannic acid solution when used alone, the mixture of the two will produce flocculent precipitation (see Figure 5 ) and affect the antibacterial effect.

[0053] The minimum bactericidal concentrations of the test solutions in Table 2 of this embodiment are shown in Table 2. Figure 4 (d) in group d1, Figure 4 (c) in group c1, Figure 4 The a1 group and Figure 4 Group b1 in (b).

[0054] Example 7

[0055] The antibacterial test was carried out according to the antibacterial test method of Example 6, except that: the concentration of the melittin solution was 0.5-2 μmol / L, which was lower than the MIC of melittin alone (the minimum inhibitory concentration of melittin solution for positive bacteria was generally 2 μmol / L, and for negative bacteria was generally 4 μmol / L). At this time, the concentration of the tannic acid solution needed to be increased, and the concentration of the tannic acid solution was adjusted to 100-300 μg / mL. At this time, due to the low concentration of antimicrobial peptides, flocculation would not occur. The culture was carried out at 37°C for 2h, 4h and 16h, and 10 μL of the culture solution was taken for counting on the NB medium, and the ratio concentration when complete sterilization was recorded.

[0056] Table 3 Compositions of different ratios and concentrations

[0057]

[0058] Table 4 Minimum bactericidal concentration MBC of the test solution

[0059]

[0060] In this embodiment, since the concentration of the melittin solution used is relatively low, flocculation and precipitation will not occur; at the same time, since the concentration of the melittin solution is lower than the MIC concentration, tannic acid plays a leading role in sterilization. The concentration of the melittin solution in this embodiment is relatively low, so there is no need to consider the problem of hemolysis.

[0061] It can be seen from Table 4 that when the concentration of Mel (2 μM) is less than the minimum inhibitory concentration, its antibacterial effect is weakened. When used in combination with tannic acid, the high concentration of tannic acid solution plays a leading role in the antibacterial effect. The antibacterial effect of tannic acid alone is generally manifested after 5 to 6 hours, while the bactericidal effect of the composition in Example 7 begins to take effect after 4 hours, and the antibacterial effect is better as the concentration of the tannic acid solution increases, while the bactericidal time is shortened.

[0062] The minimum bactericidal concentrations of the test solutions in Table 4 of this embodiment are shown in Table 4. Figure 4 (d) in group d4, Figure 4 (c) group c4, Figure 4 The a4 group in (a) and Figure 4 Group b4 in (b).

[0063] Comparative Examples 1 to 7 show that when the concentrations of the tannic acid solution and the Mel solution exceed the specified range of the present invention, the hemolysis reduction and antibacterial effects are both reduced, indicating that the formula of the present invention can effectively eliminate the hemolytic side effect of melittin without affecting the antibacterial effect of melittin.

[0064] The present invention provides a composition for inhibiting hemolysis of melittin and its application ideas and methods. There are many methods and ways to implement the technical solution. 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 protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A composition for inhibiting hemolysis of melittin, characterized in that: The composition is composed of 2-64 μmol / L bee venom peptide solution and 12.5-50 mg / L tannic acid solution mixed in equal volumes, and the solvent is phosphate buffer.

2. The composition according to claim 1, characterized in that The composition is composed of 2-8 μmol / L bee venom peptide solution and 25-50 mg / L tannic acid solution mixed in equal volumes, and the solvent is phosphate buffer.

3. The composition according to claim 1, characterized in that The phosphate buffer, 10 mM, pH=7.

4.

4. Use of the composition for inhibiting melittin hemolysis according to any one of claims 1 to 3 in the preparation of a drug for inhibiting melittin hemolysis.

5. Use of the composition for inhibiting melittin hemolysis according to any one of claims 1 to 3 in the preparation of a drug for inhibiting melittin hemolysis and killing bacteria.

6. The use according to claim 5, characterized in that: The bacteria are Staphylococcus aureus, Enterococcus faecalis or Escherichia coli.

7. The use according to claim 5, characterized in that: The bacteria is methicillin-resistant Staphylococcus aureus.

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