Use of melissa oil in the preparation of clostridium perfringens TFP inhibitors

Abstract

The application discloses application of a Melissa oil in preparation of a Clostridium perfringens type IV pilus system inhibitor product. Type IV pilus (TFP) is an important cell surface functional structure of Clostridium perfringens, and through dynamic assembly, stretching and retraction of the pilus, mediates non-flagellum-dependent sliding movement of the bacteria on a solid surface, promotes diffusion of the bacteria and cooperative movement between different cells. Meanwhile, the type IV pilus participates in formation of a biological membrane of the bacteria, thereby enhancing tolerance of Clostridium perfringens to adverse environments such as oxidative stress. Research shows that the Melissa oil can inhibit sliding movement and biological membrane formation of Clostridium perfringens mediated by the type IV pilus, destroy a pilus morphological structure and down-regulate expression of genes related to the type IV pilus structure and assembly under conditions without significantly affecting growth of Clostridium perfringens, thereby providing a new anti-toxicity strategy for green and safe prevention and control of Clostridium perfringens infection.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of lemon balm oil in the preparation of Clostridium perfringens TFP inhibitors. Background Technology

[0002] Clostridium perfringens ( Clostridium perfringens It is a Gram-positive anaerobic bacterium widely found in soil, feed, and animal intestines, and is also an important animal-derived opportunistic pathogen. Excessive proliferation of this bacterium can cause a variety of intestinal infectious diseases, including necrotizing enteritis in poultry farming, which can cause damage to the intestinal mucosa, decreased digestive and absorptive functions, and reduced production performance. In severe cases, it can lead to death, causing significant economic losses to the poultry farming industry.

[0003] Type IV pili (TFPs) are filamentous structures attached to the surface of *Clostridium perfringens*. They participate in various infection processes, including gliding movement, adhesion, colonization of host cells, DNA uptake, protein secretion, and biofilm formation. *Clostridium perfringens* encodes TFPs through multiple gene clusters, such as... pilA , pilB , pilD and pilT These gene clusters are involved in the assembly, function, and contraction of pili. Furthermore, the type IV pili system plays a crucial regulatory role in the formation of Clostridium perfringens biofilms. pilT The significantly reduced biofilm-forming ability of the gene-deleted mutant further confirms the crucial role of TFP in bacterial adhesion, aggregation, and biofilm maturation. Therefore, TFP can be considered a potential therapeutic target for intervention in Clostridium perfringens infection. By inhibiting its assembly or biological function, it may effectively weaken bacterial colonization, biofilm formation, and persistent infection capacity, providing a new strategy for the prevention and treatment of Clostridium perfringens infection.

[0004] Plant essential oils are highly volatile and exhibit broad-spectrum antibacterial properties, along with physiological functions such as inhibiting inflammatory responses, providing antioxidant effects, and regulating gut microbiota balance. They hold great potential in improving animal production performance and reducing disease incidence. Studies have shown that lemon balm oil possesses various biological activities, including antibacterial, antioxidant, anti-inflammatory, and sedative effects, demonstrating broad potential for clinical applications. Regarding antibacterial activity, it exhibits significant inhibitory effects against a variety of pathogenic microorganisms, including certain Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis, Listeria monocytogenes), Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Salmonella enteritidis), and some fungi. Therefore, this study aims to investigate the role of lemon balm oil in inhibiting the sliding motility of Clostridium perfringens and in the preparation of Clostridium perfringens fimbriae inhibitors. Summary of the Invention

[0005] This invention explores for the first time the pharmaceutical application potential of lemon balm oil in the preparation of Clostridium perfringens fimbriae inhibitors, providing a new research approach for the prevention of Clostridium perfringens infection and discovering a potential natural compound.

[0006] This invention verifies that lemon balm oil can inhibit the sliding movement and adhesion colonization process mediated by bacterial pili, thereby reducing the infectivity of Clostridium perfringens, through experiments such as Clostridium perfringens sliding movement inhibition test, minimum inhibitory concentration determination, growth curve determination test, transmission electron microscopy (TEM) observation, biofilm formation inhibition test, real-time quantitative PCR, hemolytic activity test, cell viability and cytotoxicity detection, and Clostridium perfringens adhesion to Caco-2 cells. The Clostridium perfringens ATCC 13124 used in the experiment is a publicly preserved strain, purchased from the American Type Culture Collection (ATCC), strain number ATCC® 13124™.

[0007] The positive effects of this invention are as follows: This invention is the first to use lemon balm oil in the preparation of Clostridium perfringens fimbriae inhibitors, discovering that it can effectively inhibit the sliding ability mediated by Clostridium perfringens fimbriae, thereby weakening its adhesion, colonization, and dispersal-related abilities, and reducing the infection risk and pathogenic potential of Clostridium perfringens. This application expands the scope of application of lemon balm oil in the pharmaceutical field, providing a new direction for drug development for the prevention and treatment of Clostridium perfringens infection, and has good application prospects and development value. Attached Figure Description

[0008] Figure 1 A: Melissa oil inhibits the sliding motion of Clostridium perfringens ATCC13124 on a semi-solid BHI plate; B: Melissa oil measurement results on the sliding diameter of Clostridium perfringens ATCC13124.

[0009] Figure 2 Lemon balm oil does not inhibit the growth of Clostridium perfringens ATCC13124 within the effective concentration range.

[0010] Figure 3 The MIC value of lemon balm oil against Clostridium perfringens ATCC13124 was greater than 512 μg / mL.

[0011] Figure 4 TFP was absent on the surface of Clostridium perfringens ATCC13124 cells after treatment with lemon balm oil.

[0012] Figure 5A represents the inhibition of Clostridium perfringens ATCC13124 biofilm formation by lemon balm oil within its effective concentration range, and B represents the quantification of the inhibition of Clostridium perfringens ATCC13124 biofilm formation by lemon balm oil.

[0013] Figure 6 Lemon balm oil inhibits the transcriptional level of the fimbrial regulatory gene of Clostridium perfringens ATCC13124. Detailed Implementation

[0014] The present invention will be further described with reference to specific embodiments, but these embodiments are only used to illustrate the technical solutions of the present invention and do not constitute a limitation on the scope of protection of the present invention. Without departing from the essence of the technical solutions of the present invention, all equivalent substitutions, improvements, modifications, or other easily achievable changes made by those skilled in the art based on the disclosure of the present invention should be covered within the scope of protection of the claims of the present invention.

[0015] Example 1 Lemon balm oil is used to prepare Clostridium perfringens fimbriae inhibitors for application in any pharmaceutically acceptable form of drug carrier.

[0016] Example 2 Lemon balm oil can be used as a component in the preparation of medicines for treating infectious diseases, and is an option for inhibiting Clostridium perfringens fimbriae.

[0017] Example 3 Lemon balm oil can be used as a novel drug for treating infectious diseases caused by Clostridium perfringens.

[0018] Experiment 1 Clostridium perfringens sliding motion inhibition test The sliding motility of *Clostridium perfringens* was determined using a semi-solid method. *Clostridium perfringens* ATCC 13124 was anaerobically cultured at 37 °C, and the culture was collected and resuspended in sterile FT medium. 5 × 10⁻⁶ samples were taken. 6 CFU bacterial suspension was vertically dropped into the center of BHI plates containing 0.5% agar and final concentrations of 4, 8, 16, and 32 μg / mL lemon balm oil, respectively, and anaerobically cultured at 37 ℃ for 72 h. After incubation, photographs were taken and the diameter of the sliding region was measured to evaluate the inhibitory effect of lemon balm oil on the TFP-mediated sliding movement of Clostridium perfringens.

[0019] Conclusion: Compared with the untreated control group, lemon balm oil significantly inhibited the sliding function of Clostridium perfringens (see Appendix). Figure 1 AB).

[0020] Experiment 2 Growth curve of Clostridium perfringens ATCC13124 Overnight-cultured Clostridium perfringens ATCC 13124 was inoculated into BHI medium at a ratio of 1:100 and anaerobically cultured at 37 °C until OD500. 600 =0.1. Subsequently, lemon balm oil was added to the groups to achieve final concentrations of 0, 4, 8, 16, 32, 64, and 128 μg / mL, respectively, and anaerobic culture continued. OD was measured every 1 h. 600 Until the bacteria reach a plateau phase.

[0021] Conclusion: Melissa oil within the effective concentration range had no effect on the growth of Clostridium perfringens ATCC13124 (see Appendix). Figure 2 ).

[0022] Experiment 3 Determination of the minimum inhibitory concentration (MIC) of lemon balm oil against Clostridium perfringens ATCC13124 Following the guidelines of the Clinical and Laboratory Standards Institute (CLSI), the minimum inhibitory concentration (MIC) of anaerobic bacteria was determined using the standard agar dilution method to identify the minimum effective concentration of lemon balm oil in inhibiting the growth of Clostridium perfringens.

[0023] Brunner's blood agar plates containing different final concentrations of lemon balm oil were prepared and supplemented with heme chloride (5 μg / mL), vitamin K1 (1 μg / mL), and lysed sheep blood (5% v / v). The overnight-cultured Clostridium perfringens ATCC 13124 precipitate was collected, the supernatant was discarded, and the plate was resuspended in sterile FT medium, with OD adjusted. 600 To 0.1. (The value is 1×10⁻⁶.) 5 CFU bacterial suspension was vertically dropped into the center of a plate and incubated anaerobically at 37 °C for 48 h.

[0024] Conclusion: At the highest tested concentration of 512 μg / mL, lemon balm oil did not significantly inhibit the growth of Clostridium perfringens ATCC 13124 on blood agar plates, indicating that its minimum inhibitory concentration (MIC) for this strain is higher than 512 μg / mL (see Appendix). Figure 3 ).

[0025] Experiment 4 Transmission electron microscopy (TEM) was used to observe the inhibitory effect of lemon balm oil on the pili assembly of Clostridium perfringens ATCC13124. Clostridium perfringens ATCC 13124 bacterial suspensions incubated with different concentrations of lemon balm oil for 6 h were centrifuged, and the precipitate was collected, washed twice with autoclaved phosphate-buffered saline (PBS), and resuspended in PBS. 10 μL of the culture was placed on a carbon-coated nickel grid plate for 1 minute, followed by staining with 0.2% phosphotungstic acid solution for 30 seconds. The samples were visualized using a Tecnai Spirit (120 kV) transmission electron microscope.

[0026] Conclusion: With increasing lemon balm oil concentration, the colony edges of Clostridium perfringens ATCC 13124 gradually became smoother, showing a clear dose-dependent trend (see Appendix). Figure 4 ).

[0027] Experiment 5 Lemon balm oil inhibits biofilm formation in Clostridium perfringens ATCC13124 Clostridium perfringens ATCC 13124 in logarithmic growth phase was washed three times with sterile PBS, resuspended in TSB, and OD adjusted. 600 To a final concentration of 8, 16, and 32 μg / mL, add 400 μL of bacterial suspension to each well of a 24-well plate. Use an equal volume of DMSO as a 0 μg / mL control and sterile TSB with DMSO as a negative control. Incubate anaerobically at 30 °C for 120 h. After incubation, gently wash the biofilm with PBS, dry, and stain with 400 μL of 0.1% crystal violet for 1 h. Wash and dry again, then dissolve the crystal violet in 400 μL of 33% acetic acid. Mix well and transfer 100 μL to a 96-well plate. Measure the absorbance at 570 nm to evaluate the biofilm formation capacity.

[0028] Conclusion: Lemon balm oil (16-32 μg / mL) can significantly inhibit the formation of Clostridium perfringens biofilm (see Appendix). Figure 5 AB).

[0029] Experiment 6 Effects of lemon balm oil on fimbrial regulatory genes of Clostridium perfringens ATCC13124 Gene expression analysis was performed using real-time quantitative polymerase chain reaction (qPCR). Clostridium perfringens ATCC 13124 in logarithmic growth phase was anaerobically incubated with a specified concentration of lemon balm oil at 37°C for 10 hours. The pili regulatory genes in Clostridium perfringens ATCC 13124 were analyzed. PilA1 , PilA2-1 , PilA2-2 , PilA3 , PilB2-2 , PilC , PilD , PilT The expression levels of messenger ribonucleic acid (mRNA) of the gene were determined. Total RNA was extracted from bacteria using the Trizol method, and its complementary deoxyribonucleic acid (cDNA) was synthesized by reverse transcription using a cDNA reverse transcription kit. Quantitative polymerase chain reaction (RT-qPCR) was performed using a real-time quantitative polymerase chain reaction kit. Gene expression levels were determined by 2... -ΔΔCt The method is used for quantification.

[0030] Conclusion: Lemon balm oil has a significant inhibitory effect on the gene regulation of pili genes in Clostridium perfringens (see appendix). Figure 6 ).

[0031] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The medicinal use of lemon balm oil in the preparation of Clostridium perfringens fimbriae inhibitors.

2. The use as described in claim 1, characterized in that, The Clostridium perfringens pili are type IV pili, and the lemon balm oil exerts its pili-inhibiting effect by inhibiting the sliding movement mediated by type IV pili of Clostridium perfringens.

3. The use as described in claim 1 or 2, characterized in that, The Clostridium perfringens fimbriae inhibitor can inhibit Clostridium perfringens fimbriae-mediated bacterial motility.

4. The use as described in any one of claims 1 to 3, characterized in that, The Clostridium perfringens fimbriae inhibitor is used to prepare drugs for the prevention and / or treatment of diseases related to Clostridium perfringens infection.

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