Application of baicalin in preparation of anti-vibrio parahaemolyticus preparation
By using baicalin to inhibit the formation of Vibrio parahaemolyticus, the high transmission and drug resistance of the bacteria are solved, and a green and natural antibacterial strategy is provided, which provides a new prevention and treatment plan for the marine aquaculture industry and reduces the disease the occurrence and spread of
Patent Information
- Application Number
- CN202411637027.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-15
- Publication Date
- 2024-12-27
AI Technical Summary
The high transmission, pathogenicity and drug resistance of Vibrio parahaemolyticus have led to huge challenges in the marine aquaculture industry. The abuse of existing antibiotics has led to the emergence of drug-resistant strains, which are difficult to effectively prevent and control, and the biological membrane is difficult to remove. Looking for new antibacterials Infection strategies and drugs are of great significance.
Baicalin is used as an anti-Vibiral parahemolytic preparation. By inhibiting the formation of biological membranes, the bacterial pathogenic ability is weakened. Baicalin and NaOH are arranged in a solution at a concentration of 0.44 mg/mL, which is used to inhibit the formation of biological membranes of Vibrio parahemolytic.
Baicalin significantly inhibits the formation of Vibrio parahemolytic biofilm, reduces its pathogenic ability, and provides a green and natural anti-Vbrio parahemolytic inhibitor, which is suitable for the healthy and green development of the marine aquaculture industry, avoiding the problem of antibiotic resistance. .
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microbial control preparations, and particularly relates to use of baicalin in preparing an anti-Vibrio parahaemolyticus preparation. Background Art
[0002] Vibrio parahaemolyticus is a pathogen that poses a serious threat to the aquaculture industry. It can infect a variety of marine organisms and is highly transmissible, pathogenic, and drug-resistant. It has caused huge economic losses to the marine aquaculture industry, and its pathogenic mechanism needs to be studied urgently.
[0003] Currently, antibiotics are the main means of preventing and controlling Vibrio parahaemolyticus. However, the abuse of antibiotics can lead to the emergence of drug-resistant strains, making the prevention and control of Vibrio parahaemolyticus difficult. Biofilm is a key problem leading to bacterial infection. Vibrio parahaemolyticus has a strong ability to form biofilms, and biofilms are difficult to remove. Therefore, it is of great significance to find new anti-bacterial infection strategies and develop corresponding anti-biofilm drugs.
[0004] The antibacterial effect of traditional Chinese medicine is characterized by multi-target, and bacteria are not easy to develop resistance to it. Therefore, searching for effective ingredients with anti-biofilm effects from traditional Chinese medicine has become a new way to develop biofilm inhibitors. Summary of the invention
[0005] The purpose of the present invention is to overcome at least one disadvantage of the above-mentioned prior art and to provide a use of baicalin in the preparation of an anti-Vibrio parahaemolyticus preparation.
[0006] In order to solve the above technical problems, the present invention provides the use of baicalin in preparing an anti-Vibrio parahaemolyticus preparation.
[0007] Preferably, baicalin is used to inhibit the formation of biofilm of Vibrio parahaemolyticus. Biofilm is an important virulence factor of Vibrio parahaemolyticus. Baicalin can inhibit the formation of biofilm of Vibrio parahaemolyticus to achieve the effect of weakening the toxicity of Vibrio parahaemolyticus.
[0008] Preferably, baicalin and NaOH are configured into a solution.
[0009] Preferably, the concentration of baicalin in the solution is 0.44 mg / mL. Baicalin and NaOH can be prepared into a 28 mg / mL stock solution and stored at room temperature.
[0010] The present invention provides the use of baicalin in the preparation of an anti-Vibrio parahaemolyticus preparation. Experiments show that baicalin has a good inhibitory effect on the formation of biofilm of Vibrio parahaemolyticus without inhibiting the growth of Vibrio parahaemolyticus. Therefore, baicalin can be used as a biofilm inhibitor of Vibrio parahaemolyticus and an anti-Vibrio parahaemolyticus preparation. The present invention opens up a new use for baicalin, uses it for Vibrio parahaemolyticus biofilm inhibitor, and provides a new candidate preparation for the prevention and treatment of Vibrio parahaemolyticus; in the case of increasingly common and increasingly serious drug resistance of clinical Vibrio parahaemolyticus, a green and natural anti-Vibrio parahaemolyticus inhibitor is developed, thereby providing a reference for the healthy and green development of marine aquaculture. BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Figure 1 A to Figure 1 E is a graph showing the results of the determination of MIC in Example 1 of the present invention, wherein: Figure 1 A is a 96-well plate culture diagram. Figure 1 B is the crystal violet staining picture. Figure 1 C is the bacterial growth OD 600 , Figure 1 D is the result of crystal violet staining for biofilm formation. Figure 1 E is the result graph showing the effect of solvent NaOH on the growth of Vibrio parahaemolyticus.
[0012] Figure 2 A to Figure 2 B is a graph showing the biofilm formation results at different baicalin concentrations in Example 2 of the present invention, wherein: Figure 2 A is the result of observing the biofilm formation in the test tube. Figure 2 B is the result of crystal violet staining of biofilm formation.
[0013] Figure 3 This is a diagram showing the morphological results of Vibrio parahaemolyticus colonies on Congo red agar plates at different baicalin concentrations in Example 3 of the present invention.
[0014] Figure 4 This is a graph showing the effect of baicalin on the outer membrane permeability of Vibrio parahaemolyticus in Example 4 of the present invention.
[0015] Figure 5 This is a graph showing the effect of baicalin on the inner membrane integrity of Vibrio parahaemolyticus in Example 5 of the present invention. DETAILED DESCRIPTION
[0016] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments.
[0017] The present invention starts with the natural medicinal plant resource library and the "Aquatic Drug Information Sheet" to search for natural antibacterial substances, and finds that baicalin has a definite effect on inhibiting the biofilm of Vibrio parahaemolyticus. At the same time, baicalin, as an effective ingredient derived from traditional Chinese medicine, is not easy to produce drug resistance.
[0018] The present invention detects the effect of baicalin on the inhibition of biofilm formation of Vibrio parahaemolyticus through an experiment, indicating that baicalin has a strong inhibitory effect on biofilm of Vibrio parahaemolyticus. When baicalin is added, the formation of biofilm of Vibrio parahaemolyticus can be significantly inhibited, thereby reducing the pathogenicity of Vibrio parahaemolyticus. Therefore, baicalin can be used as an anti-Vibrio parahaemolyticus preparation.
[0019] The discovery of the present invention also provides a new research direction and application prospect for marine aquaculture. Through in-depth research on the mechanism of action and effect evaluation of baicalin, its application value in marine aquaculture can be further explored, providing a strong guarantee for the sustainable development of marine aquaculture.
[0020] raw material
[0021] Baicalin (HPLC≥98%, CAS: 21967-41-9), 1-N-phenyl-naphtylamine (NPN), and propidium iodide (PI) were purchased from Shanghai Yuanye Technology Co., Ltd.; Congo red was purchased from Shenggong Biotechnology (Shanghai) Co., Ltd.;
[0022] Sterile water: obtained by high temperature sterilization at 121℃ for 20min;
[0023] Each test drug was stored at 4°C. Take it out before the experiment, mix it thoroughly and then test it.
[0024] strain
[0025] The strain used in this study was Vibrio parahaemolyticus, which was stored at -80°C for a long time.
[0026] Culture medium
[0027] LB liquid culture medium: 1% (w / v) NaCl, 1% (w / v) Tryptone, and 0.5% (w / v) Yeast Extract were mixed in an aqueous solution, sterilized by high pressure (121° C., 20 min), and stored at 4° C. for future use.
[0028] Congo red solid medium: 1% (w / v) NaCl, 1% (w / v) Tryptone, 0.5% (w / v) Yeast Extract, 0.04% (w / v) Congo red, and 1.5% (w / v) agar were mixed in aqueous solution and sterilized by high pressure (121°C, 20min).
[0029] Instruments and Equipment
[0030] 96-well plate (Hunan Beekman Biotechnology Co., Ltd.); electronic balance (Shanghai Jinke Tianmei Co., Ltd.); high-speed desktop centrifuge (Hangzhou Haosheng Instrument Co., Ltd.); electric constant temperature incubator (Shanghai Minquan Instrument Co., Ltd.); SPX-150B-Z biochemical incubator (Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory); SW-CJ-IFD single-person single-sided clean bench (Suzhou Purification Equipment Co., Ltd.)
[0031] Configuration of main reagents
[0032] 0.1% crystal violet solution: weigh 0.1 g of crystal violet, add 100 mL of deionized water and mix well.
[0033] 33% glacial acetic acid solution: Measure 33 mL of glacial acetic acid, add 67 mL of deionized water, and mix well.
[0034] Baicalin stock solution: Accurately weigh baicalin, dissolve it in NaOH solution, filter it through a 0.22 mm organic phase filter membrane, prepare a stock solution with a concentration of 28 mg / mL, and store it at 4°C for later use.
[0035] 1mmol NPN: First prepare 10mL of 50mmol NPN, take an appropriate amount of NPN and dissolve it in 10mL of acetone to prepare 50mmol / L NPN, take 100uL and dissolve it in 4900μl of acetone and mix it 50 times to dilute it 1mmol / LNPN.
[0036] Preparation of 5mmol / L HEPES + 5mmol / L glucose: prepare 10mL of 50mmol HEPES, take 0.11915g of HEPES and 0.09008g of glucose and dissolve them in 10mL of deionized water, then dilute 10 times, take 1mL and dissolve it in 9mL of ddH2O to prepare 5mmol / L HEPES + 5mmol / L glucose.
[0037] Preparation of Congo red agar plates with different baicalin concentrations
[0038] Sterile Congo red solid culture medium was heated in a microwave oven to melt, and the corresponding volume of baicalin stock solution was accurately calculated and added when pouring the plates to make the final concentrations of 0, 0.44, 0.88, 1.75, 3.5, and 7 mg / mL.
[0039] Bacterial liquid preparation
[0040] During the experiment, 1% of the inoculum of the strain was inoculated into LB medium, 0.1% (v / v) carbenicillin was added, and activation and recovery culture was performed at 200 r / min and 37° C. for 12 h. Before all the test operations in the present invention, Vibrio parahaemolyticus was subjected to the above activation culture.
[0041] Example 1
[0042] Determination of Minimum Inhibitory Concentration (MIC) of Baicalin against Vibrio parahaemolyticus
[0043] The dilution method determines the antibacterial efficacy of a drug by measuring the growth of equal amounts of bacterial liquid in liquid culture medium containing different concentrations of drugs, as well as the turbidity of the culture medium after growth. The method is simple and quick. The lowest concentration of the drug that inhibits the growth of the test bacteria measured by the dilution method is called the "minimum inhibitory concentration", which can accurately and quantitatively reflect the antibacterial activity of the drug. This experiment specifically uses the 96-well plate two-fold dilution method to determine the MIC.
[0044] Adjust the OD of the bacterial culture 600 is 0.5 for subsequent use; first add 100μl LB culture medium to each well of the experimental group in the well plate. Add 200μl LB to the first well, add 100μl drug stock solution to the second well, mix well and add 100μl to the second well, mix well and add 100μl to the third well, and so on, mix well in the 11th well and then take 100μl and discard. Add 100μl bacterial solution to wells 2 to 12, and add 100μl LB to well 12. Culture in a 37℃ incubator for 24h. Measure the absorbance value (OD) of each well at 600nm 600 ).
[0045] After OD test 600 Then, stain with crystal violet, discard all the liquid in the well, and rinse gently with sterile PBS 3 times (to rinse the floating bacteria and reduce error interference). Then put it in an oven for drying, and then stain it with 200μl 0.1% crystal violet solution for 30min. After discarding the crystal violet solution, rinse it with deionized water and dry it. Finally, add 200μl 33% glacial acetic acid solution to dissolve it. Use an enzyme marker to measure the absorbance at 595nm. The size of the OD value at different drug concentrations can reflect the ability of baicalin to intervene in biofilm.
[0046] The results are as follows Figure 1 A to Figure 1 As shown in E, Figure 1 A is a 96-well plate culture diagram. Figure 1 B is the crystal violet staining picture. Figure 1 C is the bacterial growth OD 600 , Figure 1 D is the result of crystal violet staining for biofilm formation. Figure 1E is the effect of the solvent NaOH on the growth of Vibrio parahaemolyticus. When the concentration of baicalin is ≤28 mg / mL, no clear wells are observed, indicating that the MIC of baicalin against Vibrio parahaemolyticus is greater than 28 mg / mL, and the solvent NaOH used has no effect on the growth of the bacteria. In addition, the OD 600 The large difference is due to the formation of biofilm in the well plate.
[0047] Example 2
[0048] Effect of baicalin on biofilm formation of Vibrio parahaemolyticus
[0049] Take 250μl of overnight culture of Vibrio parahaemolyticus, inoculate it in 5mL LB medium, add a certain amount of baicalin solution with mother solution concentration to make the final concentration 0, 0.1, 0.44, 0.88, 1.75, 3.5, 7mg / mL, and culture at 30℃ for 24, 48, 72h. After the culture, take out the test tube, select 0, 0.44, 1.75, 7mg / mL groups, discard all the liquid in the tube (biofilm generally exists on the surface of the liquid and the tube wall), and gently rinse 3 times with sterile PBS (rinse the floating bacteria to reduce error interference). Then put it in an oven for drying, then stain it with 200μl 0.1% crystal violet solution for 30min, discard the crystal violet solution, rinse it with deionized water, and dry it. Finally, add 33% glacial acetic acid solution to dissolve it. Use an enzyme marker to measure the absorbance at 595nm. The size of the OD value at different drug concentrations can reflect the ability of baicalin to intervene in biofilm.
[0050] The results of biofilm formation under different baicalin concentrations are shown in Figure 2 A and Figure 2 As shown in B, Figure 2 A is the result of observing the biofilm formation in the test tube. From right to left, the concentrations are 0, 0.1, 0.44, 0.88, 1.75, 3.5, and 7 mg / mL. Figure 2 B is the result of crystal violet staining biofilm formation. In the experimental group treated with baicalin, the amount of biofilm formation showed a significant downward trend. In the three treatment groups of 24h, 48h and 72h, the amount of biofilm formation of baicalin at a concentration of 0.44mg / mL was significantly reduced. In the 24h treatment group, the amount of biofilm formation of baicalin at a concentration of 7mg / mL was the lowest due to the inhibition of bacterial growth. This result shows that baicalin significantly inhibits the formation of biofilm of Vibrio parahaemolyticus.
[0051] Example 3
[0052] Congo red plate method to test biofilm formation ability
[0053] Congo red is an organic compound (C 32 H22 N6Na2O6S2), which can form a red complex with the bacterial extracellular matrix. The extracellular matrix is the main component of the biofilm. When it combines with Congo red in the culture medium, it forms a rough and wrinkled structure, so it can be used to identify biofilm-positive strains.
[0054] Take the overnight culture of Vibrio parahaemolyticus and measure its OD in a spectrophotometer. 600 , adjust its OD 600 0.5, dilute to 10 with PBS 7 10 μl was plated on Congo red agar plates with different concentrations of baicalin. After 2 days of incubation, all plates were visually inspected and the morphological types were divided into: red, dry and rough indicated the expression of extracellular matrix, representing the ability of bacteria to produce biofilm, while smooth and white indicated the absence of biofilm.
[0055] Results Figure 3 The control group (final concentration 0 mg / mL) showed dry and rough colonies, indicating that the bacteria had a strong ability to form biofilms. After adding baicalin, they obviously became smooth and moist colonies, indicating that baicalin inhibited the formation of biofilms of Vibrio parahaemolyticus.
[0056] Example 4
[0057] Effects of baicalin on outer membrane permeability of Vibrio parahaemolyticus
[0058] 1-N-phenyl-naphtylamine (NPN) is a hydrophobic fluorescent probe that produces strong blue fluorescence in a phospholipid environment, but not in an aqueous environment. The intact bacterial cell outer membrane repels 1-N-phenyl-naphtylamine. Once the membrane is damaged, 1-N-phenyl-naphtylamine can enter the membrane phospholipid layer and show fluorescence enhancement. The absorption peak of the blue fluorescent group it produces is 350nm, and the scattering peak is 420nm. It has become a commonly used fluorescence detection signal to evaluate the degree of bacterial membrane damage.
[0059] Transfer the activated bacterial solution to 5 mL LB liquid culture medium at 5%, add baicalin to the bacterial solution to make the final concentration of 0, 0.44, 0.88, 1.75, 3.5 mg / mL, and shake and culture at 200 r / min for 3 hours. Centrifuge at 8000 r / min for 2 minutes to collect the bacterial precipitate. Wash the precipitate with 5 mmol / L HEPES buffer, wash repeatedly until there is no color, collect the precipitate by centrifugation, and then add a certain amount of 5 mmol / L HEPES buffer to resuspend the precipitate, and prepare the OD 600The bacterial suspension with a value of about 0.5 is reserved. The bacterial suspension is divided into 5 2mL centrifuge tubes, and 1mL of bacterial suspension is added to each. 10μl of 1mmol / L NPN is added to the bacterial suspension, mixed thoroughly to make the final concentration of NPN 10μmol / L, and incubated at 37℃ for 90min. Add 200μl of each sample to a 96-well white reaction plate and set up 3 parallels. The fluorescence intensity is measured on a multifunctional microplate reader with an excitation wavelength of 350nm and an emission wavelength of 420nm.
[0060] Results Figure 4 ,Compared with the control group (concentration 0 mg / mL), after the addition of baicalin, the fluorescence intensity increased, indicating that baicalin destroyed the outer membrane of Vibrio parahaemolyticus, and showed an upward trend with the increase of baicalin concentration.
[0061] Example 5
[0062] Effects of baicalin on the inner membrane integrity of Vibrio parahaemolyticus
[0063] Propidium iodide (PI) is a nucleic acid dye that cannot pass through intact cell membranes, but can enter dead cells and bind to nucleic acids in the cells, causing dead cells to emit red fluorescence. Therefore, the fluorescent staining of PI can be used to determine the activity of bacteria and the integrity of cell membranes.
[0064] Transfer the activated bacterial solution to 5 mL LB liquid culture medium at 5%, add baicalin to the bacterial solution to make the final concentration of 0, 0.44, 0.88, 1.75, 3.5 mg / mL, and culture at 37°C, 200 r / min for 3 hours. Centrifuge at 8000 r / min for 2 minutes to collect the bacterial precipitate. Wash the precipitate with 5 mmol / L PBS, wash repeatedly until there is no color, collect the precipitate by centrifugation, and then add a certain amount of PBS to resuspend the precipitate, and prepare the OD 600 The bacterial suspension with a value of about 0.5 is reserved. The bacterial suspension is divided into 5 2mL centrifuge tubes, and 1mL of bacterial suspension is added to each. 5μl of 1.5mmol / L PI is added to the bacterial suspension, mixed thoroughly to make the final concentration of PI 15μmol / L, and incubated at 37℃ for 90min. Add 200μl of each sample to a 96-well white reaction plate and set up 3 parallels. The fluorescence intensity is measured on a multifunctional microplate reader with an excitation wavelength of 535nm and an emission wavelength of 615nm.
[0065] Results Figure 5 Compared with the control group (concentration 0 mg / mL), there was no significant change in fluorescence intensity after adding 0.44 mg / mL baicalin; 0.88, 1.75, and 3.5 mg / mL baicalin had a destructive effect on the inner membrane of Vibrio parahaemolyticus, and showed an upward trend with the increase of baicalin concentration.
[0066] The present invention shows through experiments that baicalin has a good inhibitory effect on the formation of Vibrio parahaemolyticus biofilm. This discovery provides a new idea and method for preventing and treating Vibrio parahaemolyticus diseases. The baicalin of the present invention, as a natural inhibitor, has the advantages of being non-toxic, harmless, environmentally friendly and safe, and can provide a new candidate antibiotic substitute for prevention and treatment for marine aquaculture. By using baicalin as an inhibitor, the growth and reproduction of Vibrio alginolyticus can be effectively inhibited, the occurrence and spread of diseases can be reduced, and the stability and sustainable development of the aquaculture industry can be guaranteed.
[0067] In this specification, the present invention has been described with reference to specific embodiments thereof. However, it is apparent that various modifications and variations may be made without departing from the spirit and scope of the present invention. Therefore, the description should be regarded as illustrative rather than restrictive.
Claims
1. Use of baicalin in the preparation of anti-Vibrio parahaemolyticus preparations.
2. The use according to claim 1, characterized in that Baicalin is used to inhibit biofilm formation of Vibrio parahaemolyticus.
3. The use according to claim 1, characterized in that Baicalin and NaOH are prepared into a solution.
4. The use according to claim 3, characterized in that The concentration of baicalin in the solution is 0.44 mg / mL.