Baicalin and boric acid antibacterial peptide assembly, and preparation method and application thereof

Abstract

This invention discloses a baicalin and boric acid antimicrobial peptide assembly, its preparation method, and its application, belonging to the field of biomedical technology. The baicalin and boric acid antimicrobial peptide assembly comprises baicalin and a boric acid antimicrobial peptide; the amino acid sequence of the boric acid antimicrobial peptide is one of the sequences shown in 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. This invention provides a baicalin and boric acid antimicrobial peptide assembly in which the phenylboronic acid molecule introduced at the C-terminus of the boric acid antimicrobial peptide can form a borate ester bond with baicalin, improving the water solubility and membrane permeability of baicalin, making it easier for it to enter bacterial cells and exert its effect. It can form a multi-target synergistic antimicrobial effect with baicalin, thereby significantly improving the antimicrobial efficacy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and particularly relates to an assembly of baicalin and boric acid antimicrobial peptides, its preparation method and application. Background Technology

[0002] Single-component antibacterial agents, due to their single target, are prone to inducing drug resistance mutations in bacteria during long-term use. Bacteria can rapidly adapt to the mechanism of action of single antibacterial agents through gene mutation, horizontal gene transfer, and other means, thereby developing drug resistance.

[0003] Baicalin, as a natural plant extract, possesses certain antibacterial, anti-inflammatory, and antioxidant activities; however, its use alone has significant drawbacks. Baicalin has poor water solubility, limiting its application and bioavailability in aquatic environments. Furthermore, its insufficient membrane permeability makes it difficult to effectively penetrate bacterial cell membranes, thus affecting its antibacterial efficacy. These limitations significantly restrict the antibacterial properties of baicalin in practical applications, making it difficult to meet the growing demand for antibacterial products.

[0004] While existing antimicrobial peptides possess broad-spectrum antimicrobial activity, some exhibit poor stability and are prone to degradation or inactivation under physiological conditions or during storage, thus affecting their antimicrobial efficacy. Furthermore, some antimicrobial peptides possess high cytotoxicity, potentially damaging host cells and limiting their application in medicine and other fields. These issues make the development of novel and highly effective antimicrobial systems an urgent 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 a composite microsphere with enhanced antibacterial properties of baicalin, its preparation method, and its 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, it cannot simultaneously inhibit the growth of multiple pathogens, resulting in a single antibacterial mechanism. Chinese patent application CN116375745A discloses an invention that provides a carrier-free supramolecular hydrogel structure, preparation, and application formed by the self-assembly of baicalin and zinc ions. However, when baicalin reacts with many 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, 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 antibacterial properties.

[0006] Therefore, the development of a composite material of baicalin and antimicrobial peptides with synergistic antibacterial properties as a novel antibacterial agent to replace antibiotics is a question worthy of serious consideration. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention proposes an antimicrobial peptide assembly of baicalin and boric acid, its preparation method, and its application. 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 (MIC) and broad-spectrum antimicrobial activity.

[0008] To achieve the above objectives, 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 boric acid antimicrobial peptide is one of the sequences shown in 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 to 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 was mixed with deionized water, ultrasonically treated, and the pH was adjusted to 6-7 to obtain baicalin mother liquor;

[0013] (2) The boric acid antimicrobial peptide was mixed with deionized water, ultrasonically treated, and the pH was adjusted to 6-7 to obtain the antimicrobial peptide mother liquor.

[0014] (3) Mix the baicalin mother liquor obtained in step (1) with the antimicrobial peptide mother liquor obtained in step (2) to obtain a mixture. Vortex the mixture for 3-7 minutes to obtain the baicalin and boric acid antimicrobial peptide assembly.

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

[0016] Preferably, the ultrasonic treatment in step (1) has a power of 50-100W, a frequency of 20-40kHz, and a duration of 50-70min.

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

[0018] Preferably, the ultrasonic treatment in step (2) has a power of 50-100W, a frequency of 20-40kHz, and a duration of 50-70min.

[0019] Preferably, the mixing ratio of the baicalin mother liquor obtained in step (1) and the antimicrobial peptide mother liquor obtained in step (2) is calculated according to the molar ratio of baicalin to boric acid antimicrobial peptide of 1:0.1 to 10.

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

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

[0022] Preferably, the antibacterial product is an anti-drug-resistant bacteria product, and the drug-resistant bacteria include Escherichia coli, Staphylococcus aureus, Salmonella, and Bacillus subtilis.

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

[0024] This invention provides an assembly of baicalin and boric acid antimicrobial peptides, which self-assembles into core-shell nanoparticles with a particle size of 50-300 nm. The boric acid antimicrobial peptides of this invention possess antimicrobial activity and can specifically recognize the cis-diol structure of bacterial surface polysaccharides through the phenylboronic acid group, achieving targeted anchoring. Simultaneously, the cationic sequence disrupts the bacterial membrane potential through electrostatic adsorption, achieving highly efficient antimicrobial activity. Furthermore, baicalin further blocks the biosynthesis of extracellular polysaccharides (EPS) by inhibiting bacterial β-glucuronidase activity, thereby increasing the degradation rate of the biofilm matrix. The boric acid antimicrobial peptides possess unique structural and functional properties. The phenylboronic acid molecule introduced at the C-terminus of the peptide can form a borate ester bond with baicalin, thus forming an antimicrobial assembly. By compounding with boric acid antimicrobial peptides, the water solubility and membrane permeability of baicalin can be improved, making it easier for baicalin to enter bacterial cells and exert its effects. This allows for multi-target synergistic antimicrobial action with baicalin, increasing the difficulty for bacteria to develop drug resistance mutations, thereby significantly improving the antimicrobial effect. In acidic environments (such as infection sites), borate ester bonds easily break, allowing the antibacterial system to intelligently adjust to changes in environmental pH. This compound system not only overcomes the limitations of single antibacterial agents but also provides a new approach to solving bacterial resistance problems, showing broad application prospects. It not only improves antibacterial performance but also reduces the risk of resistance, providing a safer and more effective option for antibacterial therapy.

[0025] This invention employs a micro-broth dilution method to study the in vitro antibacterial activity of the baicalin-boric acid antimicrobial peptide assembly. It exhibits significant antibacterial activity against both Gram-negative and Gram-positive bacteria, and also demonstrates a significant synergistic effect against drug-resistant Escherichia coli and Staphylococcus aureus. With a MIC as low as 1.88 μM, it has significant research implications and holds promise as a novel antibacterial agent to replace antibiotics.

[0026] The baicalin and boric acid antimicrobial peptide assembly of this invention exhibits high stability at physiological pH and can intelligently release the drug in the slightly acidic environment of infection. Through its advantages such as multi-target synergistic effect, reduced risk of drug resistance, enhanced antimicrobial efficiency, and intelligent pH-responsive regulation, it provides a more efficient and safer option for antimicrobial therapy.

[0027] The preparation process of this invention is simple, requires no high temperature or organic solvents, and is applicable to fields such as antibacterial drugs and medical device coatings, providing an efficient and safe solution to the problem of bacterial resistance. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 The structure of the boric acid antimicrobial peptide with the amino acid sequence shown in SEQ ID NO.3 is as follows;

[0030] Figure 2 The structure of the boric acid antimicrobial peptide with the amino acid sequence shown in SEQ ID NO.1 is as follows;

[0031] Figure 3 The structure of the boric acid antimicrobial peptide with the amino acid sequence shown in SEQ ID NO.5 is as follows;

[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 results 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 (MIC) determination of the baicalin and boric acid antimicrobial peptide BFFR4 assembly against Escherichia coli and Staphylococcus aureus.

[0036] Figure 8 The structure of the boric acid antimicrobial peptide with the amino acid sequence shown in SEQ ID NO.2 is as follows;

[0037] Figure 9 The structure of the boric acid antimicrobial peptide with the amino acid sequence shown in SEQ ID NO.4 is as follows;

[0038] Figure 10 The structure of the boric acid antimicrobial peptide with the amino acid sequence shown in SEQ ID NO.6 is as follows;

[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 (MIC) determination of the baicalin and boric acid antimicrobial peptide BFFK4 assembly against Escherichia coli and Staphylococcus aureus.

[0041] Figure 13 This is a schematic diagram illustrating the binding of baicalin and boric acid antimicrobial peptides according to the present invention. Detailed Implementation

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of 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 terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0044] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0045] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0047] The materials used in this invention were sourced from: Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) used in this invention were purchased from Beijing Bio-Bio Biotechnology Co., Ltd., with E. coli being bio-67405 and S. aureus being bio-52471; baicalin was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0048] The liquid broth culture medium formula used in this invention is as follows: 10g of LB broth powder is added to 400mL of distilled water, stirred and dissolved, the pH is adjusted to 7, and the mixture is autoclaved at 121℃ for 20min. After sterilization, it is stored at room temperature for later use.

[0049] The formula for the solid broth culture medium used in this invention is as follows: 10g of LB broth powder, 7g of agar powder, 400mL of distilled water, stirred until completely dissolved, pH adjusted to 7, autoclaved at 121℃ for 20min, and stored at room temperature for later use.

[0050] Example 1

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

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

[0053] Example 2

[0054] (1) Mix 0.2g of baicalin with 2mL of deionized water, use 50W power and 40KHz frequency, sonicate for 60min, adjust pH to 7 with 0.1M NaOH to obtain baicalin mother liquor;

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

[0056] (3) Mix the baicalin mother liquor and the antimicrobial peptide mother liquor according to the molar ratio of baicalin to boric acid antimicrobial peptide of 1:0.5 to obtain a mixture. Vortex the mixture at 1000 rpm for 5 min to obtain the baicalin and boric acid antimicrobial peptide BFFR4 assembly.

[0057] Example 3

[0058] (1) Mix 0.2g of baicalin with 2mL of deionized water, sonicate at 50W and 40KHz for 60min, and adjust the pH to 7 with 0.1M NaOH to obtain baicalin mother liquor;

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

[0060] (3) Mix the baicalin mother liquor and the antimicrobial peptide mother liquor according to the molar ratio of baicalin to boric acid antimicrobial peptide of 1:0.5 to obtain a mixture. Vortex the mixture at 1000 rpm for 5 min to obtain the baicalin and boric acid antimicrobial peptide BFFK4 assembly.

[0061] Example 4

[0062] (1) Mix 0.2g of baicalin with 2mL of deionized water, sonicate at 50W and 40KHz for 60min, and adjust the pH to 7 with 0.1M NaOH to obtain baicalin mother liquor;

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

[0064] (3) Mix the baicalin mother liquor and the antimicrobial peptide mother liquor according to the molar ratio of baicalin to boric acid antimicrobial peptide of 1:0.5 to obtain a mixture. Vortex the mixture at 1000 rpm for 5 min to obtain the baicalin and boric acid antimicrobial peptide BFFRKRK assembly.

[0065] Example 5

[0066] (1) Mix 0.1g of baicalin with 0.5mL of deionized water, sonicate at 50W power and 20kHz frequency for 50min, and adjust the pH to 7 with 0.1M NaOH to obtain baicalin mother liquor;

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

[0068] (3) Mix the baicalin mother liquor and the antimicrobial peptide mother liquor according to the molar ratio of baicalin to boric acid antimicrobial peptide of 1:0.1 to obtain a mixture. Vortex the mixture at 800 rpm for 3 min to obtain the baicalin and boric acid antimicrobial peptide BK4FF assembly.

[0069] Example 6

[0070] (1) Mix 0.1g of baicalin with 1.5mL of deionized water, sonicate at 100W and 40kHz for 70min, and adjust the pH to 7 with 0.1M NaOH to obtain baicalin mother liquor;

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

[0072] (3) Mix the baicalin mother liquor and the antimicrobial peptide mother liquor according to the molar ratio of baicalin to boric acid antimicrobial peptide of 1:10 to obtain a mixture. Vortex the mixture at 1500 rpm for 7 min to obtain the baicalin and boric acid antimicrobial peptide BR4FF assembly.

[0073] Example 7

[0074] (1) Mix 0.2g of baicalin with 2mL of deionized water, sonicate at 75W power and 30kHz frequency for 60min, and adjust the pH to 7 with 0.1M NaOH to obtain baicalin mother liquor;

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

[0076] (3) Mix the baicalin mother liquor and the antimicrobial peptide mother liquor according to the molar ratio of baicalin to boric acid antimicrobial peptide of 1:5 to obtain a mixture. Vortex the mixture at 1200 rpm for 7 min to obtain the baicalin and boric acid antimicrobial peptide BRKRKFF assembly.

[0077] Experimental Example 1

[0078] Characterization analysis of baicalin-antimicrobial peptide assemblies:

[0079] The baicalin and BFFR4 antimicrobial peptide assembly solutions obtained in Example 2 and the baicalin and BFFK4 antimicrobial peptide assemblies obtained in Example 3 were subjected to particle size analysis, zeta potential analysis, and transmission electron microscopy morphology observation. Dynamic light scattering (DLS) determined the particle sizes of baicalin, the antimicrobial peptide BFFR4, and the baicalin and BFFR4 antimicrobial peptide assembly to be 67.5 nm, 98.2 nm, and 120.6 nm, respectively, indicating that baicalin and the antimicrobial peptide can obtain larger assemblies through borate ester bonds. Figure 4 As shown, the morphology of the baicalin and BFFR4 antimicrobial peptide assembly, observed by transmission electron microscopy, is spherical. This indicates that the hydrophobic water region (FF sequence) of the baicalin and BFFR4 antimicrobial peptide assembly together constitutes a hydrophobic core, promoting the micellization of the assembly. Figure 11 As shown, the baicalin and BFFK4 antimicrobial peptide assembly presents as a chain-like structure, indicating that the antimicrobial target is not fully exposed. Figure 5 The figure shows the results of the Zeta potential analysis. The Zeta potentials of BFFR4, baicalin, and the baicalin-BFFR4 antimicrobial peptide assembly are 13.0 mV, -16.4 mV, and 18.6 mV, respectively. This indicates that baicalin is coated in the "core", while the antimicrobial peptide has more positive charge exposed on the surface of the assembly, thereby enhancing the antimicrobial properties of the assembly.

[0080] Experiment Example 2

[0081] In vitro antibacterial assay of baicalin-antimicrobial peptide assemblies:

[0082] 1) Selection and resuscitation of bacterial strains

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

[0084] 2) Take the baicalin mother liquor, BFFR4 mother liquor, baicalin and BFFR4 assembly, and baicalin and BFFK4 assembly from Example 2, and respectively mix them with 10 -6 Equal volumes of Escherichia coli and Staphylococcus aureus at a concentration of CFU / mL were mixed and incubated together at 37°C and 170 rpm for 2 hours.

[0085] 3) The co-incubated bacterial suspension was diluted with sterile water and plated onto nutrient agar plates. It was then incubated at 37°C for 20 hours, and the colony count was performed. The measurements were repeated three times.

[0086] like Figure 6 The image shows the viable bacterial counts of *E. coli* and *Staphylococcus aureus* on nutrient agar plates after treatment with sterile water (blank), baicalin, BFFR4, and the baicalin / BFFR4 assembly, respectively. It can be seen that the antibacterial effect of the baicalin / BFFR4 assembly is significantly higher than that of baicalin and BFFR4 alone. The minimum inhibitory concentration (MIC) of the baicalin / BFFR4 assembly against *E. coli* and *Staphylococcus aureus* can reach 1.88 μM (e.g., 1.88 μM). Figure 7 As shown in the figure, its antibacterial activity is far higher than that of most existing antimicrobial peptides. Additionally, as... Figure 12 As shown, the minimum inhibitory concentration of baicalin and the BFFK4 assembly against Escherichia coli and Staphylococcus aureus is 7.5 μM, which is lower than that of the BFFR4 assembly.

[0087] This invention aims to reduce antibiotic use by preparing a series of synergistic antibacterial assemblies of baicalin and boric acid antimicrobial peptides through the formation of boric acid ester bonds. Baicalin exerts its antibacterial effect primarily by disrupting the integrity of bacterial cell membranes and inhibiting bacterial DNA and protein synthesis, while the boric acid antimicrobial peptides interfere with bacterial metabolic processes by binding to specific receptors on the bacterial surface. This invention achieves self-assembly through simple pH adjustment and solution blending, without requiring high temperatures, high pressures, or organic solvents, aligning with green chemistry principles and making it suitable for large-scale production.

[0088] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope 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 includes baicalin and boric acid antimicrobial peptide; The structural formula of the boric acid antimicrobial peptide is as follows: 、 、 、 、 、 One of them; The phenylboronic acid molecules in the boric acid antimicrobial peptide form borate ester bonds with baicalin.

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 as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Baicalin was mixed with deionized water, sonicated, and the pH was adjusted to 6-7 to obtain baicalin mother liquor; (2) The boric acid antimicrobial peptide was mixed with deionized water, ultrasonically treated, and the pH was adjusted to 6-7 to obtain the antimicrobial peptide mother liquor; (3) Mix the baicalin mother liquor obtained in step (1) with the antimicrobial peptide mother liquor obtained in step (2) to obtain a mixture. Vortex the mixture for 3-7 minutes to obtain the baicalin and boric acid antimicrobial peptide assembly.

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

5. The preparation method according to claim 3, characterized in that, The ultrasonic treatment in step (1) has a power of 50~100W, a frequency of 20~40kHz, and a duration of 50~70min.

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

7. The preparation method according to claim 3, characterized in that, The ultrasonic treatment in step (2) has a power of 50~100W, a frequency of 20~40kHz, and a duration of 50~70min.

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

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

10. The use of the baicalin and boric acid antimicrobial peptide assembly as described in claim 1 or 2 in the preparation of antimicrobial drugs.

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