Baicalin and boric acid antibacterial peptide assembly as well as preparation method and application thereof

Abstract

The invention discloses a baicalin and boric acid antibacterial peptide assembly and a preparation method and application thereof, belongs to the technical field of biological medicine, and provides a baicalin and boric acid antibacterial peptide assembly which comprises baicalin and boric acid antibacterial peptide. The amino acid sequence of the boric acid antibacterial peptide is one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6. According to the baicalin and boric acid antibacterial peptide assembly, phenylboronic acid molecules introduced to the C end of boric acid antibacterial peptide polypeptide can form boric acid ester bonds with the baicalin, the water solubility and membrane permeability of the baicalin can be improved, the baicalin can enter bacterial cells more easily to play a role, and the baicalin and the boric acid antibacterial peptide can form a multi-target synergistic antibacterial effect; therefore, the antibacterial effect is obviously improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a baicalin and boric acid antimicrobial peptide assembly, and a preparation method and application thereof. Background Art

[0002] Single-ingredient antimicrobial agents, due to their single target, are prone to inducing bacterial resistance mutations during long-term use. Bacteria rapidly adapt to the mechanism of action of a single antimicrobial agent through gene mutation and horizontal gene transfer, thus developing resistance.

[0003] Baicalin, a natural plant extract, possesses certain antibacterial, anti-inflammatory, and antioxidant activities, but it has significant drawbacks when used alone. Baicalin has poor water solubility and low solubility in water, which limits its application and bioavailability in aqueous environments. Furthermore, baicalin's membrane permeability is insufficient, making it difficult to effectively penetrate bacterial cell membranes, thus affecting its antibacterial efficacy. These drawbacks significantly limit baicalin's antibacterial properties in practical applications, making it difficult to meet the growing demand for antibacterial agents.

[0004] While existing antimicrobial peptides possess broad-spectrum antimicrobial activity, some suffer from poor stability and are susceptible to degradation or inactivation in physiological environments or during storage, compromising their antimicrobial efficacy. Furthermore, some antimicrobial peptides exhibit high cytotoxicity, potentially damaging host cells and limiting their application in pharmaceuticals and other fields. These challenges make the development of novel, highly effective antimicrobial systems a pressing priority.

[0005] Currently, various technologies have been developed to prepare antimicrobial peptides or baicalin composite materials for antibacterial purposes. For example, Chinese patent application CN119454615A discloses composite microspheres that enhance the antibacterial properties of baicalin, as well as their preparation and application. In this method, baicalin is embedded in the hydrophobic interior of zein, reducing the minimum inhibitory concentration of baicalin and its cell membrane conductivity. However, these methods are unable to simultaneously inhibit the growth of multiple pathogens and exhibit a single antibacterial mechanism. Chinese patent application CN116375745A discloses an invention providing the structure, preparation, and application of a carrier-free supramolecular hydrogel formed by the self-assembly of baicalin and metallic zinc ions. However, when baicalin reacts with various metal ions, it can only self-assemble into a hydrogel with zinc ions; reactions with other metal ions result in precipitation, which greatly limits the application of baicalin antibacterial materials. Chinese patent application CN114404672A discloses a fiber membrane combining polyphenols and antimicrobial peptides, as well as its preparation method and application. The fiber membrane is prepared by modifying the surface of the fiber membrane substrate by combining polyphenol compounds and antimicrobial peptides. Although this invention reduces the cytotoxicity of the material, it does not significantly enhance the antimicrobial properties.

[0006] Therefore, it is a question worth pondering at present to prepare a composite material of baicalin and antimicrobial peptides with synergistic antibacterial properties as a new antibacterial agent to replace antibiotics. Summary of the Invention

[0007] To solve the above technical problems, the present invention proposes a baicalin and boric acid antimicrobial peptide assembly, a preparation method and application thereof, and in vitro antibacterial experiments were conducted on the baicalin and boric acid antimicrobial peptide assembly using representative Gram-negative bacteria Escherichia coli and representative Gram-positive bacteria Staphylococcus aureus. The results showed that the baicalin and boric acid antimicrobial peptide assembly exhibited excellent minimum inhibitory concentration and broad antibacterial spectrum.

[0008] To achieve the above object, the present invention provides a baicalin and boric acid antimicrobial peptide assembly, wherein the baicalin and boric acid antimicrobial peptide assembly comprises baicalin and boric acid antimicrobial peptide;

[0009] The amino acid sequence of the boronic acid antimicrobial peptide is one of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6.

[0010] Preferably, the molar ratio of baicalin to boric acid antimicrobial peptide in the baicalin and boric acid antimicrobial peptide assembly is 1:0.1-10.

[0011] The present invention also provides a method for preparing the baicalin and boric acid antimicrobial peptide assembly, comprising the following steps:

[0012] (1) Baicalin is mixed with deionized water, subjected to ultrasonic treatment, and the pH is adjusted to 6-7 to obtain a baicalin mother solution;

[0013] (2) mixing the boric acid antimicrobial peptide with deionized water, ultrasonically treating the mixture, and adjusting the pH to 6-7 to obtain an antimicrobial peptide mother solution;

[0014] (3) The baicalin mother liquor obtained in step (1) is mixed with the antimicrobial peptide mother liquor obtained in step (2) to obtain a mixed solution, and the mixed solution is vortexed for 3 to 7 minutes to obtain an assembly of baicalin and boric acid antimicrobial peptide.

[0015] Preferably, the mixing ratio of baicalin and deionized water in step (1) is 0.1 g: 0.5-1.5 mL.

[0016] Preferably, the power of the ultrasonic treatment in step (1) is 50 to 100 W, the frequency of the ultrasonic treatment is 20 to 40 kHz, and the time of the ultrasonic treatment is 50 to 70 min.

[0017] Preferably, the mixing ratio of the boric acid antimicrobial peptide and deionized water in step (2) is 0.01 g: 0.5-1.5 mL.

[0018] Preferably, the power of the ultrasonic treatment in step (2) is 50 to 100 W, the frequency of the ultrasonic treatment is 20 to 40 kHz, and the time of the ultrasonic treatment is 50 to 70 min.

[0019] Preferably, in step (3), the mixing ratio of the baicalin mother solution obtained in step (1) and the antimicrobial peptide mother solution obtained in step (2) is calculated based on a molar ratio of baicalin to boric acid antimicrobial peptide of 1:0.1-10.

[0020] Preferably, the rotation speed of the vortex in step (3) is 800 to 1500 rpm.

[0021] The present invention also provides the use of the baicalin and boric acid antimicrobial peptide assembly in the preparation of antimicrobial products.

[0022] Preferably, the antibacterial product is a product for treating drug-resistant bacteria, and the drug-resistant bacteria include Escherichia coli, Staphalococcus aureus, Salmonella and Bacillus subtilis.

[0023] Compared with the prior art, the present invention has the following advantages and technical effects:

[0024] The present invention provides an assembly of baicalin and boric acid antimicrobial peptide, which self-assembles to form core-shell nanoparticles with a particle size of 50 to 300 nm. The boric acid antimicrobial peptide of the present invention has antimicrobial activity itself and can specifically recognize the cis-diol structure of bacterial surface polysaccharides through the phenylboronic acid group, achieving targeted anchoring. At the same time, the cationic sequence destroys the bacterial membrane potential through electrostatic adsorption, achieving high antibacterial efficiency. In addition, baicalin inhibits the activity of bacterial β-glucuronidase, further blocking the biosynthesis of extracellular polysaccharides (EPS), thereby improving the degradation rate of the biofilm matrix. The boric acid antimicrobial peptide has unique structural and functional properties. The phenylboronic acid molecule introduced at the C-terminus of the polypeptide can form a boronate ester bond with baicalin, thereby forming an antimicrobial assembly. By compounding with the boric acid antimicrobial peptide, the water solubility and membrane permeability of baicalin can be improved, making it easier for it to enter the bacterial cell and exert its effect. It can form a multi-target synergistic antimicrobial effect with baicalin, increasing the difficulty of bacteria developing drug-resistant mutations, thereby significantly improving the antibacterial effect. In acidic environments (such as those at the site of infection), the borate ester bond easily breaks, allowing the antimicrobial system to intelligently adjust to changes in environmental pH. This complex system not only overcomes the limitations of a single antimicrobial agent but also offers a new approach to addressing bacterial resistance, with broad application prospects. It not only improves antimicrobial performance but also reduces the risk of resistance, providing a safer and more effective option for antimicrobial treatment.

[0025] This study used a broth microdilution method to investigate the in vitro antibacterial activity of a baicalin-boronic acid antimicrobial peptide assembly. The results demonstrated significant antibacterial activity against both Gram-negative and Gram-positive bacteria, with a significant synergistic effect against drug-resistant Escherichia coli and Staphylococcus aureus, achieving a low MIC of 1.88 μM. This study demonstrates significant research significance and suggests the potential for a novel antimicrobial agent to replace antibiotics.

[0026] The baicalin and boric acid antimicrobial peptide assembly of the present invention is highly stable at physiological pH and can intelligently release the drug in the slightly acidic environment of infection. Through multi-target synergy, reduced risk of drug resistance, enhanced antimicrobial efficacy, and intelligent pH-responsive regulation, it provides a more efficient and safer option for antimicrobial treatment.

[0027] The preparation process of the present invention is simple, does not require high temperature or organic solvents, is suitable for the fields of antibacterial drugs, medical device coatings, etc., and provides an efficient and safe solution to solve bacterial resistance. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0029] Figure 1 The amino acid sequence is the structural formula of the boronic acid antimicrobial peptide shown in SEQ ID NO.3;

[0030] Figure 2 The amino acid sequence is the structural formula of the boronic acid antimicrobial peptide shown in SEQ ID NO.1;

[0031] Figure 3 The amino acid sequence is the structural formula of the boronic acid antimicrobial peptide shown in SEQ ID NO.5;

[0032] Figure 4 Transmission electron microscopy image of the assembly of baicalin and boric acid antimicrobial peptide BFFR4, scale bar is 100 nm;

[0033] Figure 5 Zeta potential analysis of baicalin, boric acid antimicrobial peptide BFFR4, and the assembly of baicalin and boric acid antimicrobial peptide BFFR4. In the figure, BFFR4 represents boric acid antimicrobial peptide BFFR4, BA represents baicalin, and R4 / BA represents the assembly of baicalin and boric acid antimicrobial peptide BFFR4.

[0034] Figure 6The figure shows a comparison of the antibacterial effects of baicalin, boric acid antimicrobial peptide BFFR4, and the assembly of baicalin and boric acid antimicrobial peptide BFFR4. In the figure, BFFR4 represents boric acid antimicrobial peptide BFFR4, and baicalin / BFFR4 represents the assembly of baicalin and boric acid antimicrobial peptide BFFR4.

[0035] Figure 7 The results show the minimum inhibitory concentration of the assembly of baicalin and boric acid antimicrobial peptide BFFR4 against Escherichia coli and Staphylococcus aureus;

[0036] Figure 8 The amino acid sequence is the structural formula of the boronic acid antimicrobial peptide shown in SEQ ID NO.2;

[0037] Figure 9 The amino acid sequence is the structural formula of the boronic acid antimicrobial peptide shown in SEQ ID NO.4;

[0038] Figure 10 The amino acid sequence is the structural formula of the boronic acid antimicrobial peptide shown in SEQ ID NO.6;

[0039] Figure 11 Transmission electron microscopy image of the assembly of baicalin and boric acid antimicrobial peptide BFFK4, scale bar is 1000 nm;

[0040] Figure 12 The results show the minimum inhibitory concentration of the assembly of baicalin and boric acid antimicrobial peptide BFFK4 against Escherichia coli and Staphylococcus aureus;

[0041] Figure 13 Schematic diagram of the combination of baicalin and boric acid antimicrobial peptide of the present invention. DETAILED DESCRIPTION

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0044] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0045] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0046] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0047] Sources of materials used in the present invention: Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) used in the present invention were purchased from Beijing Biobo Biotechnology Co., Ltd., Escherichia coli (E. coli) was E. coli bio-67405, and Staphylococcus aureus (S. aureus) was Staphylococcus aureus bio-52471; baicalin was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0048] The liquid broth culture medium used in the present invention is formulated as follows: 10 g of LB broth powder is added to 400 mL of distilled water, stirred and dissolved, the pH value is adjusted to 7, and sterilized by high pressure at 121° C. for 20 min. After sterilization, the mixture is stored at room temperature for later use.

[0049] The solid broth medium used in the present invention is formulated as follows: 10 g of LB broth powder, 7 g of agar powder, 400 mL of distilled water, stirring until completely dissolved, adjusting the pH to 7, autoclaving at 121° C. for 20 min, and storing at room temperature for later use.

[0050] Example 1

[0051] The six antimicrobial peptides described in this paper were synthesized by Shanghai Jiepeptide Biotechnology Co., Ltd. using a solid-phase method. They all contain hydrophobic amino acids, which can insert into and disrupt bacterial cell membranes. Combined with the bactericidal effect of boron ions, they achieve a synergistic and highly effective bactericidal effect. All chemicals used were analytical grade.

[0052] The abbreviations and amino acid sequences of the six antimicrobial peptides are: BFFK4: B(OH)2-FFGPLGLAGKKKK-NH2 (SEQ ID NO.1) The specific structural formula is as follows Figure 2 As shown; BK4FF: B(OH)2-KKKKGPLGLAGFF-NH2 (SEQ ID NO.2) The specific structural formula is as follows Figure 8 As shown; BFFR4: B (OH) 2 -FFGPLGLAGRRRR-NH 2 (SEQ ID NO.3) The specific structural formula is as follows Figure 1 As shown; BR4FF: B (OH) 2 -RRRRGPLGLAGFF-NH 2 (SEQ ID NO. 4) The specific structural formula is as shown Figure 9 As shown; BFFRKRK: B(OH)2-FFGPLGLAGRKRK-NH2 (SEQ ID NO.5) The specific structural formula is as follows Figure 3 As shown; BRKRKFF: B (OH) 2 -RKRKGPLGLAGFF-NH 2 (SEQ ID NO.6) The specific structural formula is as follows Figure 10 As shown, Figure 1 、 Figure 2 、 Figure 3 、 Figure 8 、 Figure 9 and Figure 10 As shown, the red structural part is rich in high-density positively charged amino acids: arginine (R) and lysine (K).

[0053] Example 2

[0054] (1) 0.2 g of baicalin was mixed with 2 mL of deionized water, ultrasonically treated at a power of 50 W and a frequency of 40 kHz for 60 min, and the pH was adjusted to 7 with 0.1 M NaOH to obtain a baicalin mother solution;

[0055] (2) 0.02 g of the boric acid antimicrobial peptide BFFR4 (SEQ ID NO. 3) was mixed with 2 mL of deionized water, ultrasonicated at a power of 50 W and a frequency of 40 kHz for 60 min, and the pH was adjusted to 7 with 0.1 M NaOH to obtain the antimicrobial peptide mother solution;

[0056] (3) The baicalin mother solution and the antimicrobial peptide mother solution were mixed according to the molar ratio of baicalin to boric acid antimicrobial peptide of 1:0.5 to obtain a mixed solution, and the mixed solution was vortexed at a speed of 1000 rpm for 5 minutes to obtain a baicalin and boric acid antimicrobial peptide BFFR4 assembly.

[0057] Example 3

[0058] (1) 0.2 g of baicalin was mixed with 2 mL of deionized water and ultrasonically treated at a power of 50 W and a frequency of 40 kHz for 60 min. The pH was adjusted to 7 with 0.1 M NaOH to obtain a baicalin mother solution.

[0059] (2) 0.02 g of the boric acid antimicrobial peptide BFFK4 (SEQ ID NO. 1) was mixed with 2 mL of deionized water, ultrasonicated at a power of 50 W and a frequency of 40 kHz for 60 min, and the pH was adjusted to 7 with 0.1 M NaOH to obtain the antimicrobial peptide mother solution;

[0060] (3) The baicalin mother solution and the antimicrobial peptide mother solution were mixed according to the molar ratio of baicalin to boric acid antimicrobial peptide of 1:0.5 to obtain a mixed solution, and the mixed solution was vortexed at a speed of 1000 rpm for 5 minutes to obtain a baicalin and boric acid antimicrobial peptide BFFK4 assembly.

[0061] Example 4

[0062] (1) 0.2 g of baicalin was mixed with 2 mL of deionized water and ultrasonically treated at a power of 50 W and a frequency of 40 kHz for 60 min. The pH was adjusted to 7 with 0.1 M NaOH to obtain a baicalin mother solution.

[0063] (2) 0.02 g of the boric acid antimicrobial peptide BFFRKRK (SEQ ID NO. 5) was mixed with 2 mL of deionized water, ultrasonicated at a power of 50 W and a frequency of 40 kHz for 60 min, and the pH was adjusted to 7 with 0.1 M NaOH to obtain the antimicrobial peptide mother solution;

[0064] (3) The baicalin mother solution and the antimicrobial peptide mother solution were mixed according to the molar ratio of baicalin to boric acid antimicrobial peptide of 1:0.5 to obtain a mixed solution, and the mixed solution was vortexed at a speed of 1000 rpm for 5 minutes to obtain a baicalin and boric acid antimicrobial peptide BFFRKRK assembly.

[0065] Example 5

[0066] (1) 0.1 g of baicalin was mixed with 0.5 mL of deionized water, and ultrasonicated at 50 W and 20 kHz for 50 min. The pH was adjusted to 7 with 0.1 M NaOH to obtain the baicalin mother solution.

[0067] (2) 0.01 g of the boric acid antimicrobial peptide BK4FF (SEQ ID NO. 2) was mixed with 0.5 mL of deionized water, ultrasonicated at 50 W and 20 kHz for 50 min, and the pH was adjusted to 7 with 0.1 M NaOH to obtain the antimicrobial peptide mother solution;

[0068] (3) The baicalin mother solution and the antimicrobial peptide mother solution were mixed according to the molar ratio of baicalin to boric acid antimicrobial peptide of 1:0.1 to obtain a mixed solution, and the mixed solution was vortexed at a speed of 800 rpm for 3 minutes to obtain a baicalin and boric acid antimicrobial peptide BK4FF assembly.

[0069] Example 6

[0070] (1) 0.1 g of baicalin was mixed with 1.5 mL of deionized water, and ultrasonicated for 70 min at 100 W and 40 kHz. The pH was adjusted to 7 with 0.1 M NaOH to obtain the baicalin mother solution.

[0071] (2) 0.01 g of the boric acid antimicrobial peptide BR4FF (SEQ ID NO. 4) was mixed with 1.5 mL of deionized water, ultrasonicated at 100 W and 40 kHz for 70 min, and the pH was adjusted to 7 with 0.1 M NaOH to obtain the antimicrobial peptide mother solution;

[0072] (3) The baicalin mother solution and the antimicrobial peptide mother solution were mixed according to the molar ratio of baicalin to boric acid antimicrobial peptide of 1:10 to obtain a mixed solution, and the mixed solution was vortexed at a speed of 1500 rpm for 7 minutes to obtain a baicalin and boric acid antimicrobial peptide BR4FF assembly.

[0073] Example 7

[0074] (1) 0.2 g of baicalin was mixed with 2 mL of deionized water, and ultrasonicated at 75 W and 30 kHz for 60 min. The pH was adjusted to 7 with 0.1 M NaOH to obtain a baicalin mother solution.

[0075] (2) 0.02 g of the boric acid antimicrobial peptide BRKRKFF (SEQ ID NO. 6) was mixed with 2 mL of deionized water and sonicated at 75 W and 30 kHz for 60 min. The pH was adjusted to 7 with 0.1 M NaOH to obtain the antimicrobial peptide mother solution.

[0076] (3) The baicalin mother solution and the antimicrobial peptide mother solution were mixed according to the molar ratio of baicalin to boric acid antimicrobial peptide of 1:5 to obtain a mixed solution, and the mixed solution was vortexed at a speed of 1200 rpm for 7 minutes to obtain a baicalin and boric acid antimicrobial peptide BRKRKFF assembly.

[0077] Experimental Example 1

[0078] Characterization and analysis of baicalin-antimicrobial peptide assembly:

[0079] The baicalin and BFFR4 antimicrobial peptide assembly solution obtained in Example 2 and the baicalin and BFFK4 antimicrobial peptide assembly obtained in Example 3 were subjected to particle size analysis, zeta potential analysis, and transmission electron microscopy morphology observation. Dynamic light scattering (DLS) determined that the particle sizes of baicalin, antimicrobial peptide BFFR4, and baicalin and BFFR4 antimicrobial peptide assembly were 67.5 nm, 98.2 nm, and 120.6 nm, respectively. This indicates that the boronate ester bond between baicalin and antimicrobial peptide can produce an assembly with a larger particle size. Figure 4 As shown in Figure 1, the morphology of the baicalin and BFFR4 antimicrobial peptide assembly was observed by transmission electron microscopy and was spherical, which indicates that the hydrophobic domain (FF sequence) of the baicalin and BFFR4 antimicrobial peptide assembly together constitutes the hydrophobic core, promoting the micellization of the assembly. Figure 11 As shown in the figure, the assembly of baicalin and BFFK4 antimicrobial peptide is in the shape of a spherical ball, and the antimicrobial target is not fully exposed. Figure 5 The figure shows the Zeta potential analysis results, in which the Zeta potentials of BFFR4, baicalin, and the assembly of baicalin and BFFR4 antimicrobial peptide are 13.0 mV, -16.4 mV, and 18.6 mV, respectively. This indicates that baicalin is coated in the "core" and more positive charges of the antimicrobial peptide are exposed on the surface of the assembly, thereby enhancing the antimicrobial properties of the assembly.

[0080] Experimental Example 2

[0081] In vitro antibacterial experiment of baicalin-antimicrobial peptide assembly:

[0082] 1) Selection and recovery of bacterial strains

[0083] Escherichia coli and Staphalococcus aureus were streaked on agar nutrient medium and cultured overnight at 37°C. Monoclonal Escherichia coli and Staphylococcus aureus were picked and inoculated into broth culture medium and cultured at 37°C and 170 rpm for 8 hours. The cultured Escherichia coli and Staphylococcus aureus were then cultured in broth culture medium until 10 -6 The concentration of CFU / mL was used for in vitro antibacterial experiments.

[0084] 2) The baicalin mother solution, BFFR4 mother solution, baicalin and BFFR4 assembly, and baicalin and BFFK4 assembly in Example 2 were respectively mixed with 10 -6 Equal volumes of Escherichia coli and Staphylococcus aureus with a CFU / mL concentration were mixed and incubated at 37°C and 170 rpm in a shaker for 2 h.

[0085] 3) The bacterial suspension after co-incubation was diluted with sterile water by a certain multiple and plated onto nutrient agar plates. The plates were incubated in a constant temperature incubator at 37°C for 20 hours and the number of colonies was counted. This measurement was performed three times.

[0086] like Figure 6 As shown, the nutrient agar plates show the amount of live bacteria of Escherichia coli and Staphylococcus aureus after treatment with sterile water (blank), baicalin, BFFR4, and the assembly of baicalin and BFFR4. It can be seen that the antibacterial effect of the assembly of baicalin and BFFR4 is significantly higher than that of baicalin and BFFR4 antimicrobial peptide alone. The minimum inhibitory concentration of the assembly of baicalin and BFFR4 against Escherichia coli and Staphylococcus aureus can reach 1.88μM (as shown in Figure 2). Figure 7 As shown in Figure 2), the antibacterial activity of the antimicrobial peptides is much higher than that of most existing antimicrobial peptides. Figure 12 As shown, the minimum inhibitory concentration of baicalin and BFFK4 assembly against Escherichia coli and Staphylococcus aureus is 7.5 μM, and its antibacterial ability is lower than that of BFFR4 assembly.

[0087] This invention aims to reduce antibiotic use. By creating boronate bonds, a series of synergistic antimicrobial assemblies of baicalin and boronic acid antimicrobial peptides are prepared. Baicalin primarily exerts its antimicrobial effects by disrupting the integrity of bacterial cell membranes and inhibiting bacterial DNA and protein synthesis, while boronic acid antimicrobial peptides can interfere with bacterial metabolic processes by binding to specific receptors on bacterial surfaces. This invention achieves self-assembly through simple pH control and solution blending, eliminating the need for high temperature, high pressure, or organic solvents. This approach aligns with the principles of green chemistry and is suitable for large-scale production.

[0088] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A baicalin and boric acid antimicrobial peptide assembly, characterized in that: The baicalin and boric acid antimicrobial peptide assembly comprises baicalin and boric acid antimicrobial peptide; The amino acid sequence of the boronic acid antimicrobial peptide is one of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.

6.

2. The baicalin and boric acid antimicrobial peptide assembly according to claim 1, characterized in that: The molar ratio of baicalin to boric acid antimicrobial peptide in the baicalin and boric acid antimicrobial peptide assembly is 1:0.1-10.

3. The method for preparing the baicalin and boric acid antimicrobial peptide assembly according to claim 1 or 2, characterized in that: The following steps are involved: (1) Baicalin is mixed with deionized water, subjected to ultrasonic treatment, and the pH is adjusted to 6-7 to obtain a baicalin mother solution; (2) mixing the boric acid antimicrobial peptide with deionized water, ultrasonically treating the mixture, and adjusting the pH to 6-7 to obtain an antimicrobial peptide mother solution; (3) The baicalin mother liquor obtained in step (1) is mixed with the antimicrobial peptide mother liquor obtained in step (2) to obtain a mixed solution, and the mixed solution is vortexed for 3 to 7 minutes to obtain an assembly of baicalin and boric acid antimicrobial peptide.

4. The preparation method according to claim 3, characterized in that The mixing ratio of baicalin and deionized water in step (1) is 0.1 g: 0.5-1.5 mL.

5. The preparation method according to claim 3, characterized in that: The power of the ultrasonic treatment in step (1) is 50 to 100 W, the frequency of the ultrasonic treatment is 20 to 40 kHz, and the time of the ultrasonic treatment is 50 to 70 min.

6. The preparation method according to claim 3, characterized in that: The mixing ratio of the boric acid antimicrobial peptide and deionized water in step (2) is 0.01 g: 0.5-1.5 mL.

7. The preparation method according to claim 3, characterized in that: The power of the ultrasonic treatment in step (2) is 50 to 100 W, the frequency of the ultrasonic treatment is 20 to 40 kHz, and the time of the ultrasonic treatment is 50 to 70 min.

8. The preparation method according to claim 3, characterized in that: In step (3), the mixing ratio of the baicalin mother liquor obtained in step (1) and the antimicrobial peptide mother liquor obtained in step (2) is calculated based on a molar ratio of baicalin to boric acid antimicrobial peptide of 1:0.1 to 10.

9. The preparation method according to claim 3, characterized in that: The rotation speed of the vortex in step (3) is 800-1500 rpm.

10. Use of the baicalin and boric acid antimicrobial peptide assembly according to claim 1 or 2 in the preparation of antibacterial products.

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