Application of matrine in preparation of anti-vibrio anguillarum preparation

Abstract

The invention relates to application of matrine in preparation of an anti-vibrio anguillarum preparation. The anti-vibrio anguillarum preparation is a vibrio anguillarum biofilm inhibitor which does not influence vibrio anguillarum growth. Compared with the prior art, the matrine is used as a natural bacteriostatic agent for inhibiting the formation of the vibrio anguillarum biofilm, the effect of reducing the vibrio anguillarum toxicating capability can be achieved by inhibiting the formation of the vibrio anguillarum biofilm, and the matrine has the characteristic advantages of being environment-friendly, safe and non-toxic.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to the application of matrine in the preparation of anti-Vibrio anguillarum preparations. Background Art

[0002] As an important pathogen in the field of aquaculture, Vibrio anguillarum has wide infectivity. Its strong transmission ability, high pathogenic characteristics and significant drug resistance pose a serious threat to the seawater aquaculture industry. The diseases caused by this bacterium not only result in huge economic losses, but also highlight the urgency of in-depth exploration of its pathogenic mechanism. Vibrio anguillarum has a strong ability to form biofilms, which are difficult to remove. Moreover, as an important virulence factor of Vibrio anguillarum, the formation of biofilms is also the core problem leading to the difficulty in preventing and controlling the bacterium in the mechanism of bacterial infection. Therefore, when exploring antibacterial strategies, it is of great significance to develop biofilm inhibitors.

[0003] Existing drugs are less applied in the infection of Vibrio anguillarum, and the treatment effects are not ideal. Antibiotics such as tetracycline, sulfamethoxazole and sulfadimidine have poor treatment effects on the infection of Vibrio anguillarum. There is literature showing that oxytetracycline can be used as a drug for treating Vibrio anguillarum infection and added to fish feed at a ratio of 10 g / 100 kg of feed. However, oxytetracycline degrades slowly in water, and some unmetabolized drugs will enter the environment through the excretion of fish, resulting in the residue of antibiotics in water and having a negative impact on the water ecological environment. As an effective component of natural Chinese herbal medicines, matrine shows its unique advantages. The literature shows that matrine is easily degraded in different natural waters, and its half-life is 6.26 - 12.81 days. Its photolysis characteristics in water indicate that matrine is easily degraded in different waters such as pond water and river water, especially in natural waters, and the photolysis half-life is within 0.5 h. Matrine can not only effectively prevent and control diseases, but also reduce the potential threats of drugs to water, aquatic organisms and human health, and is an ideal choice for realizing ecological environmental protection and sustainable aquaculture.

[0004] CN109890958A discloses a novel Vibrio anguillarum phage VIB-ANP-1 for inhibiting the proliferation of Vibrio anguillarum bacteria. CN103110887A also discloses a Chinese herbal medicine formula for inhibiting Vibrio anguillarum, including at least one of Schisandra chinensis, pomegranate peel, Artemisia argyi, Aloe vera, burnt alum, catechu, Siegesbeckia orientalis, Sanguisorba officinalis, Lobelia chinensis, Loropetalum chinense, Areca catechu, Chaenomeles sinensis, Sapium sebiferum, Myrrh, Pulsatilla chinensis, Artemisia capillaris, Arisaema heterophyllum, Scutellaria baicalensis, Andrographis paniculata, Polygonum cuspidatum, Quisqualis indica, Taraxacum mongolicum, Humulus lupulus, Portulaca oleracea, Forsythia suspensa, Gentiana scabra, Atractylodes lancea, Paeonia suffruticosa, Pharbitis nil, Eugenia caryophyllata, Viola philippica, Clematis chinensis, Solanum nigrum, Galla chinensis, Astragalus membranaceus, Hedyotis diffusa, Bupleurum chinense, Evodia rutaecarpa, Prunus mume, Isatis indigotica, Plantago asiatica, Atractylodes lancea, etc. Among them, the bacteriostatic agent formed by mixing pomegranate peel, Artemisia argyi, Schisandra chinensis and Aloe vera in a ratio of 1:4:1:4 has the best bacteriostatic effect, and its minimum inhibitory concentration MIC is 3.906 mg / mL. However, neither of them records whether it can inhibit the formation of Vibrio anguillarum biofilm, and currently there is no publicly available effective preparation that can inhibit the formation of Vibrio anguillarum biofilm. Summary of the Invention

[0005] The purpose of the present invention is to develop an effective preparation for inhibiting the formation of Vibrio anguillarum biofilm, thereby providing the application of matrine in the preparation of anti-Vibrio anguillarum preparations.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] One of the technical solutions of the present invention is to provide the application of matrine in the preparation of anti-Vibrio anguillarum preparations.

[0008] In some specific embodiments, the anti-Vibrio anguillarum preparation is a Vibrio anguillarum biofilm inhibitor.

[0009] In some specific embodiments, the anti-Vibrio anguillarum preparation is a Vibrio anguillarum biofilm inhibitor that does not affect the growth of Vibrio anguillarum.

[0010] Another technical solution of the present invention is to provide an anti-Vibrio anguillarum preparation for inhibiting the formation of Vibrio anguillarum biofilm. The anti-Vibrio anguillarum inhibitor is a Vibrio anguillarum biofilm inhibitor that does not affect the growth of Vibrio anguillarum, and it is composed of matrine and pharmaceutically acceptable adjuvants.

[0011] In some specific embodiments, the final concentration of matrine is ≥0.5 mg / mL and <10 mg / mL.

[0012] In some specific embodiments, the final concentration of matrine is 1 mg / mL.

[0013] In some specific embodiments, the anti-Vibrio anguillarum preparation is composed of matrine, DMSO, and TSB culture medium.

[0014] TSB liquid medium: 0.5% (w / v) NaCl, 1.7% (w / v) tryptone, 0.3% (w / v) soy peptone, 0.25% (w / v) glucose, 0.25% (w / v) KH2PO4 are mixed in an aqueous solution, autoclaved (121 °C, 15 min), and stored at 4 °C for later use.

[0015] In some specific embodiments, the final concentration of matrine is ≥0.5 mg / mL and <10 mg / mL, and the volume percentage of DMSO is (0.1 - 2)%.

[0016] In some specific embodiments, the final concentration of matrine is 1 mg / mL.

[0017] The third technical solution of the present invention is to provide a method for inhibiting the formation of Vibrio anguillarum biofilm by using matrine, including the following steps:

[0018] Inoculate the Vibrio anguillarum liquid into the TSB liquid medium containing matrine, and statically culture at (20 - 30) °C for at least 24 h, wherein the final concentration of matrine is ≥0.5 mg / mL and <10 mg / mL, and the inoculation amount of Vibrio anguillarum is 5% of the TSB liquid medium.

[0019] In some specific embodiments, the method for inhibiting the formation of Vibrio anguillarum biofilm by using matrine includes the following steps:

[0020] Inoculate the Vibrio anguillarum liquid into the TSB liquid medium containing matrine, and statically culture at 20 °C for at least 48 h, wherein the final concentration of matrine is 1 mg / mL, and the inoculation amount of Vibrio anguillarum is 5% of the TSB liquid medium.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) Starting from the natural medicinal plant resource library and the "Aquaculture Medicine Instruction Sheet", the present invention searches for natural antibacterial substances. Through detection, it is found that matrine can have a good inhibitory effect on the formation of Vibrio anguillarum biofilm without affecting the growth of Vibrio anguillarum. The biofilm of Vibrio anguillarum is an important virulence factor of Vibrio anguillarum. Matrine can reduce the virulence of Vibrio anguillarum by inhibiting the formation of Vibrio anguillarum biofilm. Therefore, matrine can be used as an anti-Vibrio anguillarum preparation to reduce the pathogenic ability of Vibrio anguillarum, opening up an innovative idea for disease prevention and control.

[0023] (2) In the realistic background of the continuous deterioration of the Vibrio anguillarum drug resistance problem, as an effective component of traditional Chinese medicine, matrine has multi-target antibacterial characteristics, and it is not easy for Vibrio anguillarum to develop drug resistance, which opens up a new technical direction for the prevention and treatment of aquatic diseases.

[0024] (3) The present invention uses matrine as a natural antibacterial agent to inhibit the formation of Vibrio anguillarum biofilms, which has the characteristics of environmental protection, safety, and non-toxicity. It not only provides a scientific basis for improving the prevention and control system of Vibrio anguillarum infection, but also injects innovative impetus into ensuring the ecological safety of the aquaculture industry and the sustainable development of the industry. It provides a new research direction and practical path for the marine aquaculture industry, demonstrating significant industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a graph showing the determination results of MIC in Example 1 of the present invention.

[0026] Figure 2 It is a graph showing the formation results of Vibrio anguillarum biofilms under the influence of different matrine concentrations in Example 2 of the present invention.

[0027] Figure 3 It is a graph showing the morphological results of Vibrio anguillarum colonies on Congo red agar plates under the influence of different matrine concentrations in Example 3 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented on the premise of the technical solution of the present invention, and gives the detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0029] The raw materials involved in the following embodiments:

[0030] (1) Matrine (HPLC≥98%, CAS: 519-02-8) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.;

[0031] (2) Congo red was purchased from Sangon Biotech (Shanghai) Co., Ltd.;

[0032] Each test drug was stored at 4°C, taken out before the experiment, and thoroughly mixed before testing.

[0033] The strains involved in the following embodiments:

[0034] Vibrio anguillarum is Vibrio anguillarum MVM425, and Vibrio anguillarum is stored at -80°C for a long time.

[0035] The culture media involved in the following embodiments:

[0036] (1) TSB liquid medium: 0.5% (w / v) NaCl, 1.7% (w / v) tryptone, 0.3% (w / v) soy peptone, 0.25% (w / v) glucose, 0.25% (w / v) KH2PO4 were mixed in an aqueous solution, autoclaved (121°C, 15 min), and stored at 4°C for later use.

[0037] (2) Congo red solid medium: 1% (w / v) NaCl, 1% (w / v) tryptone, 0.5% (w / v) yeast extract, 0.04% (w / v) Congo red, 1.5% (w / v) agar are mixed in an aqueous solution and autoclaved (121 °C, 20 min).

[0038] The main preparation configurations involved in the following examples:

[0039] (1) Matrine stock solution: Weigh matrine precisely, dissolve it in TSB liquid medium with 2% DMSO content, filter through a 0.22 mm organic phase filter membrane, and prepare a stock solution with a concentration of 20 mg / mL, which is stored at 4 °C for later use.

[0040] (2) Preparation of Congo red agar plates with different matrine concentrations

[0041] Take the sterilized Congo red solid medium, heat it to melt on a microwave oven, and accurately calculate and add the corresponding volume of matrine stock solution when pouring the plate to make the final concentrations 0, 0.25, 0.5, and 1 mg / mL respectively.

[0042] (3) Bacterial suspension preparation

[0043] During the experiment, Vibrio anguillarum is inoculated into TSB liquid medium, and the inoculation amount of the strain is 1% of the volume of the medium. It is activated and resuscitated for 12 h at 200 r / min and 30 °C. Before all the test operations in the present invention, Vibrio anguillarum is cultured in this way for activation.

[0044] In addition, if there is no special indication of raw materials or treatment techniques, it means that they are all conventional commercially available raw material products or conventional treatment techniques in this field.

[0045] Example 1 Determination of the minimum inhibitory concentration (MIC) of matrine against Vibrio anguillarum

[0046] In this example, the dilution method is used, that is, to determine the growth of an equal amount of Vibrio anguillarum bacterial suspension in liquid media containing different concentrations of matrine drugs and the turbidity of the medium after growth to determine the antibacterial efficacy of the drugs.

[0047] The minimum inhibitory concentration is the lowest concentration at which the drug inhibits the growth of the test bacteria measured by the above dilution method, which can quantitatively and accurately reflect the antibacterial characteristics of the drug.

[0048] In this experiment, the 96-well plate two-fold dilution method is specifically used to determine the MIC. First, adjust the OD of the bacterial suspension 600 to 0.5 for subsequent use. The experimental steps are as follows:

[0049] Add 100 μL of TSB liquid medium to wells 2 to 12 first. Then, add 200 μL of matrine stock solution to well 1. After that, take 100 μL and add it to well 2. Mix well and then take 100 μL and add it to well 3, and so on. After mixing well in well 11, aspirate 100 μL and discard it. Finally, add 100 μL of bacterial solution to wells 1 to 12. Incubate in an incubator at 30 °C for 24 h. Measure the absorbance (OD 600 ) at 600 nm for each well.

[0050] The results are as Figure 1 shown. Figure 1 For the OD 600 of bacterial growth, when the matrine concentration is 10 mg / mL, the corresponding well is clear, indicating that the MIC of matrine against Vibrio anguillarum = 10 mg / mL.

[0051] Example 2 Effect of matrine on biofilm formation by Vibrio anguillarum

[0052] Take 250 μL of overnight culture of Vibrio anguillarum, inoculate it into 5 mL of TSB liquid medium, and add a certain amount of matrine stock solution to make the final concentrations 0, 0.125, 0.25, 0.5, 1 mg / mL respectively. Incubate statically at 20 °C for 24, 48, 72, 96 h.

[0053] The results of biofilm formation by Vibrio anguillarum under the influence of different matrine concentrations are as Figure 2 shown. Among them, Figure 2 are the results of observing the amount of biofilm formation by Vibrio anguillarum in test tubes. From left to right, the matrine concentrations are 0, 0.125, 0.25, 0.5, 1 mg / mL. In the experimental groups treated with matrine, the amount of biofilm formation by Vibrio anguillarum shows an obvious decreasing trend. In the 48 h treatment group, under the influence of matrine at a concentration of 1 mg / mL, the amount of biofilm formation by Vibrio anguillarum decreases significantly.

[0054] This result indicates that matrine significantly inhibits the biofilm formation of Vibrio anguillarum.

[0055] Example 3 Congo red plate method for testing biofilm formation ability

[0056] Congo red is an organic compound (C 32 H 22 N6Na2O6S2), which can form a red complex with the extracellular matrix of bacteria. The extracellular matrix is the main component of the biofilm. After it binds to Congo red in the culture medium, a rough and wrinkled structure will be formed. Therefore, it can be used to identify biofilm-positive strains.

[0057] Take the overnight culture of Vibrio anguillarum bacterial solution, measure its OD 600 using a spectrophotometer, and adjust its OD 600was 0.5 and diluted to 10-fold with PBS. 10 μL was taken and spread on Congo red agar plates containing different concentrations of matrine. After incubation at 30 °C for 7 days, all plates were visually inspected. The morphological types were classified as follows: ① Red, dry and rough indicated the expression of extracellular matrix, representing that the bacteria were able to produce biofilms; ② Smooth and white indicated no biofilm production. 6 The control group (the final concentration of matrine in the medium was 0 mg / mL) showed dry and rough colonies, indicating that V. anguillarum had a strong ability to form biofilms. After adding different concentrations of matrine to the medium, it significantly changed to smooth and moist colonies, indicating that matrine could effectively inhibit the formation of V. anguillarum biofilms.

[0058] The results are shown in Figure 3 , the control group (the final concentration of matrine in the medium was 0 mg / mL) showed dry and rough colonies, indicating that V. anguillarum had a strong ability to form biofilms. After adding different concentrations of matrine to the medium, it significantly changed to smooth and moist colonies, indicating that matrine could effectively inhibit the formation of V. anguillarum biofilms.

[0059] The above description of the embodiments is for the convenience of those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. Application of matrine in preparing a preparation against Vibrio anguillarum.

2. The application according to claim 1, wherein The preparation against Vibrio anguillarum is a Vibrio anguillarum biofilm inhibitor.

3. The application according to claim 1, characterized in that, The preparation against Vibrio anguillarum is a Vibrio anguillarum biofilm inhibitor that does not affect the growth of Vibrio anguillarum.

4. An anti-Vibrio anguillarum preparation, characterized in that, The Vibrio anguillarum inhibitor is a Vibrio anguillarum biofilm inhibitor that does not affect the growth of Vibrio anguillarum, and it is composed of matrine and pharmaceutically acceptable adjuvants.

5. The vibrio anguillarum-resistant preparation according to claim 4, wherein The concentration of matrine is ≥0.5 mg / mL and <10 mg / mL.

6. The vibrio anguillarum-resistant preparation according to claim 5, characterized in that, The concentration of matrine is 1 mg / mL.

7. The vibriosis anguillarum-resistant preparation according to claim 4, wherein The preparation against Vibrio anguillarum is composed of matrine, DMSO, and TSB culture medium.

8. The vibriosis anguillarum-resistant preparation according to claim 7, wherein, The concentration of matrine is ≥0.5 mg / mL and <10 mg / mL, and the volume ratio of DMSO is (0.1 - 2)%.

9. The vibrio anguillarum-resistant preparation according to claim 8, wherein, The concentration of matrine is 1 mg / mL.

10. A method for inhibiting the formation of Vibrio anguillarum biofilm by using matrine, characterized in that, It includes the following steps: Inoculate the Vibrio anguillarum liquid into the TSB liquid medium containing matrine, and statically culture it at (20 - 30)°C for at least 24 h, where the final concentration of matrine is ≥0.5 mg / mL and <10 mg / mL, and the inoculation amount of Vibrio anguillarum is 5% of the TSB liquid medium.

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