Application of isofulvic alcohol in preparation of medicine for preventing and treating clostridium perfringens infection
By using isoflurane alcohol to inhibit the IV pili function of C. perfringens, the problem of degradation of antibiotic treatment effect in the prior art was solved, and a drug that effectively prevents and treats C. perfringens infection was developed, which significantly inhibits the inhibition and controls of C. perfringens infection. Bacterial sliding movement and biological film formation improve the prevention and treatment effect of gas gangrene.
Patent Information
- Application Number
- CN202411347893.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-26
- Publication Date
- 2024-12-13
AI Technical Summary
The prior art is difficult to effectively inhibit the function of C. perfringens IV type pili, resulting in reduced antibiotic treatment effect and drug residues problems, and lacks new drug solutions for C. perfringens infection.
Isofluorin is used to inhibit the function of C. perfringens IV type pili, block the formation and adhesion of bacteria, and develop drugs to prevent and treat C. perfringens infection, including tablets, capsules and Granules.
Isofluorin significantly inhibits the sliding movement of C. perfringens and the formation of biological membranes, reduces the colonization of infected receptors, has good prevention and treatment effects, and has ideal prevention and treatment effects on gas gangrene in mice.
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Figure CN119112871A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of biomedicine, and in particular to application of isoxanthohumol in preparing medicines for preventing and treating Clostridium perfringens infection. Background Art
[0002] Clostridium perfringens is a Gram-positive anaerobic bacillus, one of the most common pathogens in nature. It can be isolated from the gastrointestinal tract of humans and animals and is widely distributed in soil, contaminated water and decaying organic matter. The bacterium is also one of the main pathogens that cause necrotic enteritis in poultry, gas gangrene in humans and livestock, enterotoxemia and food poisoning in humans. The pathogenicity of the bacterium is related to the various extracellular toxins it produces. So far, 13 different toxins have been reported, among which α, β, ε and ι are the most important, and the bacterium can be divided into five toxin types: A, B, C, D and E. Each serotype of Clostridium perfringens can produce α-toxin, which is also the main virulence factor. It has phospholipase C and sphingomyelinase activity, and can simultaneously hydrolyze phosphatidylcholine and sphingomyelin on the cell membrane, leading to cell lysis. Therefore, it has the characteristics of cytotoxicity, hemolytic activity, lethality and platelet aggregation, which seriously threatens the life and health of livestock and poultry and hinders the development of animal husbandry. Poultry are mainly infected with type A and type C, especially type A. After poultry are infected, it mainly causes necrotizing enteritis. The lesions are mainly concentrated in the small intestine, manifested as thinning of the intestinal wall accompanied by congestion and bleeding, and focal or patchy necrosis and shedding of the intestinal mucosa.
[0003] Type IV pili (TFP) is a kind of motility organ existing on the bacterial cell membrane. TFP provides bacteria with the ability to move on the surface of natural environment and other media, which is called gliding motility. At the same time, TFP is also an important virulence factor in the process of bacterial infection, which can mediate many biological functions, such as bacterial movement, adhesion to host cells, biofilm formation, DNA uptake and protein secretion. At present, necrotic enteritis in poultry is mainly prevented and treated by adding antibiotics to feed. However, the long-term and large-scale use of antibiotics and their irregular use have continuously induced the production of drug-resistant bacteria, and the effect of antibiotic treatment has been continuously reduced or even lost. At the same time, it is accompanied by the problem of drug residues, which endangers food safety. Therefore, by inhibiting the function of TFP of Clostridium perfringens, interfering with the adhesion and colonization of bacteria in the intestine, and blocking the pathogenic process of Clostridium perfringens infection, it has become a new drug development strategy for anti-Clostridium perfringens infection.
[0004] Isoxanthohumol, also known as "isoxanthohumol". It is abundant in the root of Sophora flavescens and can also be extracted from hops. Studies have shown that it has multiple pharmacological effects such as anti-tumor, antioxidant and promotion of estrogen activity. Although there are also reports that isoxanthohumol has certain antibacterial effects, it is mainly resistant to Staphylococcus aureus and Staphylococcus aureus. At present, there are no reports at home and abroad that isoxanthohumol can resist Clostridium perfringens infection by inhibiting the function of type IV pili of Clostridium perfringens. Summary of the invention
[0005] The purpose of the present invention is to provide an application of isoxanthohumol in the preparation of a drug for preventing and treating Clostridium perfringens infection, so as to solve the problems existing in the above-mentioned prior art. The present invention verifies through experiments that isoxanthohumol significantly inhibits the function of type IV pili (TFP) of Clostridium perfringens, has no bactericidal / antibacterial activity against Clostridium perfringens, and has an ideal preventive and therapeutic effect on gas gangrene caused by Clostridium perfringens infection in mice.
[0006] To achieve the above object, the present invention provides the following solutions:
[0007] The invention provides application of isoxanthohumol in preparing medicine for preventing and treating diseases caused by bacterial infection, wherein the bacteria is Clostridium perfringens.
[0008] Furthermore, the diseases include necrotizing enterocolitis, enterotoxemia and gas gangrene.
[0009] Furthermore, isoxanthohumol prevents and treats infection of Clostridium perfringens by inhibiting the function of type IV pili of Clostridium perfringens, inhibiting the biofilm formation of Clostridium perfringens and reducing the colonization number of Clostridium perfringens in the infection receptor.
[0010] The present invention also provides a medicine for preventing and treating diseases caused by bacterial infection, comprising an active ingredient and pharmaceutically acceptable adjuvants; the active ingredient comprises isoxanthohumol.
[0011] Furthermore, the dosage form of the drug includes tablets, capsules and granules.
[0012] The present invention discloses the following technical effects:
[0013] The invention proves that isoxanthohumol can significantly inhibit the function of type IV pili (TFP) of Clostridium perfringens through a slidable movement inhibition test, a biofilm formation inhibition test, a growth curve test, a cell adhesion test and a mouse gas gangrene model treatment test, and has no bactericidal or antibacterial activity against Clostridium perfringens. An in vivo infection model treatment test shows that isoxanthohumol has a good preventive and therapeutic effect on Clostridium perfringens gas gangrene in mice.
[0014] The invention provides a theoretical basis for using isoxanthohumol for preventing and treating Clostridium perfringens infection, increases the selectivity of drugs for preventing and treating Clostridium perfringens infection, has broad application prospects, and is of great significance to the development of new drugs. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. 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 creative work.
[0016] Figure 1 This is a diagram showing the inhibitory effect of isoxanthohumol on the sliding movement of Clostridium perfringens;
[0017] Figure 2 This is a graph showing the inhibitory effect of isoxanthohumol on the biofilm formation of Clostridium perfringens;
[0018] Figure 3 The effect of isoxanthohumol on the growth of Clostridium perfringens within the effective concentration range;
[0019] Figure 4 This is a diagram showing the inhibitory effect of isoxanthohumol on the adhesion of Clostridium perfringens cells;
[0020] Figure 5 This is a graph showing the effect of isoxanthohumol on the survival rate of mice infected with Clostridium perfringens;
[0021] Figure 6 This graph shows the effect of isoxanthohumol on the number of colonies colonizing the thigh muscles of mice infected with Clostridium perfringens. DETAILED DESCRIPTION
[0022] 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0023] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.
[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content of this specification shall prevail.
[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.
[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0027] The isoxanthohumol used in the present invention has a CAS number of 70872-29-6 and a molecular formula of C 21 H 22 O 5 , molecular weight is 354.4, chemical structure is as follows:
[0028]
[0029] Clostridium perfringens was the standard strain ATCC13124 purchased from American Type Culture Collection (ATCC).
[0030] The present invention uses a mouse model of gas gangrene caused by Clostridium perfringens infection as an example to verify the effect of isoxanthohumol in preventing and treating diseases caused by Clostridium perfringens infection. Isoxanthohumol can also be used to prevent and treat necrotic enteritis, enterotoxemia, gas gangrene in humans and animals, and food poisoning caused by Clostridium perfringens infection.
[0031] Example
[0032] 1. Clostridium perfringens sliding movement inhibition test
[0033] The standard strain of Clostridium perfringens ATCC13124 was cultured anaerobically in liquid BHI medium at 37°C for 12h. 300μL of the culture was taken and continued to be cultured in 2mL of liquid BHI, and the culture was continued anaerobically at 37°C for 5h. After the culture was completed, 1mL of the culture was taken, centrifuged at 12000rpm for 5min, and the supernatant was discarded. The bacteria were resuspended in 500μL of BHI liquid medium to prepare a bacterial suspension (WT). 5μL of the above bacterial solution was added dropwise to 0.7% BHI semi-solid agar plates containing different concentrations (8μg / mL and 16μg / mL) of isoxanthohumol, recorded as experimental groups (WT+8μg / mL and WT+16μg / mL), and a positive control group of DMSO (without isoxanthohumol) and a negative control group of 400μL of liquid TSB medium were set up. The culture was anaerobically cultured at 37°C for 72h, and the results of bacterial sliding movement were counted.
[0034] The results showed that at a concentration of 8 μg / mL, isoxanthohumol significantly inhibited the sliding movement of Clostridium perfringens ATCC13124, and the inhibitory effect was more obvious at a concentration of 16 μg / mL (see Figure 1 ).
[0035] 2. Clostridium perfringens biofilm formation inhibition test
[0036] The standard strain of Clostridium perfringens ATCC13124 was cultured anaerobically in 2 mL of liquid BHI medium at 37 °C for 12 h, centrifuged at 12000 rpm for 5 min, discarded the supernatant, washed once with sterile PBS, and then resuspended in TSB liquid medium and adjusted to OD 600nm =0.1, take 400μL of the adjusted bacterial culture and add it to a 24-well plate, then add isoxanthohumol respectively, so that the final concentration of isoxanthohumol in the 24-well plate is 2μg / mL, 4μg / mL, 8μg / mL and 16μg / mL, respectively, set up a positive control group of DMSO (without isoxanthohumol) and a negative control group of 400μL liquid TSB medium, three replicates for each group, and place the 24-well plate at 30℃ for anaerobic culture for 120h. After the culture is completed, discard the supernatant, wash twice with sterile PBS, dry at 37℃ for 1h, add 400μL of 0.1% crystal violet sterilized by filtration, incubate at room temperature for 45min, discard the crystal violet, and wash three times with sterile PBS. 33% acetic acid was added to each well to dissolve crystal violet. After mixing by pipetting, 100 μL was added to each well of the 96-well plate. Three replicate wells were set up in each group. The absorbance was measured at 570 nm on an ELISA reader. The group without isoxanthohumol was set as 100%. The biofilm formation rate was calculated based on the absorbance of crystal violet.
[0037] The results showed that at a concentration of 8 μg / ml, isoxanthohumol significantly inhibited the formation of biofilm of Clostridium perfringens, and the inhibitory effect was more obvious when the concentration was 16 μg / mL (see Figure 2 ).
[0038] 3. Growth curve of Clostridium perfringens
[0039] C. perfringens was cultured anaerobically in 2 mL of liquid BHI medium at 37°C for 12 h. The next day, it was inoculated into 200 mL of fresh BHI medium and continued to be cultured until OD 600nm =0.3, dispensed into 6 conical bottles in equal amounts, 20 mL per bottle. Add isoxanthohumol to each bottle to make the final concentrations 4, 8, 16, 32 and 64 μg / mL, respectively. Set up a DMSO control group, place the culture at 37°C for anaerobic culture, and measure the OD of the bacterial culture every 1 hour. 600nm The values were recorded until the bacteria grew to the plateau phase and a growth curve was drawn.
[0040] The results showed that within the effective concentration range (4-64 μg / mL), isoxanthohumol did not affect the growth of Clostridium perfringens (see Figure 3 ).
[0041] 4. Clostridium perfringens cell adhesion test
[0042] The Caco-2 cells were cultured in DMEM medium without serum and antibiotics at a concentration of 3 × 10 5 1 mL of cell suspension was added to each well of a 24-well plate and placed at 37°C and 5% CO 2 Incubate overnight.
[0043] A single colony of the standard strain of Clostridium perfringens ATCC13124 was picked and placed in 2 mL of liquid BHI medium and cultured anaerobically at 37°C overnight. The next day, the culture was expanded to 2 mL of fresh liquid BHI medium at a ratio of 1:50, and different final concentrations of isoxanthohumol (2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL and 32 μg / mL) were added, and a negative control without isoxanthohumol was set up. Then, the culture was placed at 37°C for 4 h of anaerobically. After the culture was completed, the supernatant was discarded after centrifugation at 12000 rpm for 2 min, and the culture was washed twice with sterile PBS. Then, the concentration of the bacterial suspension was adjusted to 6×10 7 CFUs / mL, infect cells for 1h at MOI=200. Wash away bacteria that do not adhere to cells with sterile PBS, wash each well 3 times (positive control wells are not washed), add 1ml of 0.2% TritonX-100, blow and beat 50 times repeatedly, and place on a horizontal shaker for 10min, spread on BHI plates by doubling dilution method, and culture anaerobically at 37℃ overnight, count the colonies in each group, and calculate the cell adhesion rate of Clostridium perfringens in each group, taking the negative control group as 100%.
[0044] The results showed that in the concentration range of 2 μg / mL to 32 μg / mL, isoxanthohumol significantly inhibited the adhesion of Clostridium perfringens to Caco-2 cells (see Figure 4 ).
[0045] 5. Study on the treatment of Clostridium perfringens gas gangrene model in mice
[0046] 5.1 Establishment of Clostridium perfringens gas gangrene model in mice
[0047] Female Balb / c mice aged 6 to 8 weeks were injected intramuscularly with 50 μL of a suspension of Clostridium perfringens ATCC13124 prepared in sterile PBS (concentration of 1×10 10 CFUs / mL) were used to establish a mouse model of Clostridium perfringens gas gangrene, and an equal volume of sterile PBS was set as a healthy control group.
[0048] 5.2 Protective effect of isoxanthohumol on the mouse model of Clostridium perfringens gas gangrene
[0049] According to the above method, a mouse model of Clostridium perfringens gas gangrene was established. The treatment group was given 50 mg / kg body weight of isoxanthohumol by subcutaneous injection in the neck, and the drug was administered once every 12 hours for three consecutive days, which was recorded as the isoxanthohumol treatment group. At the same time, a healthy control group (no treatment) and an infection control group (infection control was not given). The number of samples in each group of the healthy control group, infection control group and isoxanthohumol treatment group was 10. After infection, the survival of each group of mice was recorded within 96 hours, and the survival rate was statistically calculated.
[0050] The results showed that 48 hours after infection, all mice in the model group died (survival rate 0%), while the survival rate of the isoxanthohumol-treated group was 60%, indicating that isoxanthohumol effectively improved the survival rate of mice infected with Clostridium perfringens (see Figure 5 ).
[0051] 5.3 Analysis of colonization of thigh muscle
[0052] At the end of the experiment, thigh muscles of mice in each group were ground under sterile conditions to prepare 1% PBS tissue homogenate, which was diluted in multiples and spread on BHI blood plates. The plates were cultured anaerobically at 37°C overnight, and the number of colonies in thigh muscles of each group was counted.
[0053] The results showed that compared with the infected control group, isoxanthohumol treatment significantly reduced the number of colonies in the thigh muscles of mice infected with Clostridium perfringens (see Figure 6 ). In summary, isoxanthohumol has a good alleviating effect on gas gangrene caused by Clostridium perfringens infection.
[0054] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.
Claims
1. Use of isoxanthohumol in the preparation of a medicament for preventing and treating diseases caused by bacterial infection, characterized in that: The bacteria is Clostridium perfringens.
2. The use according to claim 1, characterized in that: Such diseases include necrotizing enterocolitis, enterotoxemia, and gas gangrene.
3. The use according to claim 1, characterized in that: Isoxanthohumol prevents and treats Clostridium perfringens infection by inhibiting the function of type IV pili of Clostridium perfringens, inhibiting the biofilm formation of Clostridium perfringens and reducing the number of colonization of Clostridium perfringens in the infection receptor.
4. A drug for preventing and treating diseases caused by bacterial infection, characterized in that: The invention comprises active ingredients and pharmaceutically acceptable auxiliary materials; the active ingredients comprises isoxanthohumol.
5. The drug according to claim 4, characterized in that The dosage forms of the drug include tablets, capsules and granules.