Application of honokiol combined with sulfonamides in preparation of vibrio parahaemolyticus inhibitor

Abstract

The application belongs to the technical field of disease prevention and treatment of aquaculture, and particularly relates to application of honokiol combined with sulfisoxazole sodium in preparation of a Vibrio parahaemolyticus inhibitor, characterized by that the mass ratio of honokiol to sulfisoxazole sodium in the composition is 1:0.5-2, the concentration of honokiol in the composition is 32-64 µg / mL, and the concentration of sulfisoxazole sodium in the composition is 32-128 µg / mL, and the application further provides a medicine for improving sensitivity of Vibrio parahaemolyticus to sulfisoxazole sodium and treating acute hepatopancreas necrosis disease of prawns, and the mass ratio of honokiol to sulfisoxazole sodium in the medicine is 1:0.125-0.5; the composition can significantly reverse drug resistance of Vibrio parahaemolyticus to SMM-Na, reduce the use amount of SMM-Na, and produce a rapid sterilization effect through combined action, and meanwhile, the composition exhibits excellent protection efficacy in prawn bodies.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture disease prevention and control technology, specifically involving the application of magnolol in combination with sulfamethoxypyrimidine sodium in the preparation of Vibrio parahaemolyticus inhibitors. Background Technology

[0002] Acute hepatopancreatic necrosis disease (AHPND) is a bacterial disease that seriously threatens the global shrimp farming industry. Its main pathogen is *Vibrio parahaemolyticus* carrying the pVA1 virulence plasmid (encoding PirA / PirB toxins). Vibrio parahaemolyticus This disease has a rapid onset and high mortality rate, causing huge economic losses to shrimp farming. Currently, sulfonamide antibiotics such as sulfamonomethoxine sodium (SMM-Na) remain one of the main chemical drugs for controlling this disease. However, long-term and irregular use of antibiotics has led to increasingly serious drug resistance in Vibrio parahaemolyticus, reducing the clinical efficacy of antibiotics such as SMM-Na and posing risks of drug residues and environmental pollution. Therefore, developing new synergistic treatment strategies that can enhance the efficacy of existing antibiotics and reduce their dosage is of great significance for the green and sustainable development of aquaculture.

[0003] Plant-derived active ingredients have become a research hotspot for potential antibiotic adjuvants or alternatives due to their wide availability, low toxicity, and low likelihood of inducing drug resistance. Honokiol (HKL), a natural biphenyl lignan extracted from the traditional Chinese medicine Magnolia officinalis, has been reported to possess various biological activities, including antibacterial, anti-inflammatory, and antioxidant properties. However, research on the combined use of HKL and antibiotics for the prevention and control of aquatic pathogens, particularly for reversing bacterial resistance and treating AHPND, remains lacking. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an application of magnolol in combination with sulfamethoxypyrimidine sodium in the preparation of Vibrio parahaemolyticus inhibitors. This composition can significantly reverse the resistance of Vibrio parahaemolyticus to SMM-Na, reduce the amount of SMM-Na used, and produce a rapid bactericidal effect through synergistic action. At the same time, it exhibits excellent protective efficacy in shrimp, including improving survival rate, clearing pathogens, reducing tissue damage, enhancing immune response and improving intestinal microecology.

[0005] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows: This invention provides the use of a composition of magnolol and sulfamethoxypyrimidine sodium in the preparation of Vibrio parahaemolyticus inhibitors.

[0006] Preferably, the mass ratio of honokiol to sodium sulfamethoxypyrimidine in the composition is 1:0.5~2.

[0007] Preferably, the concentration of magnolol in the composition is 32-64 µg / mL, and the concentration of sulfamethoxypyrimidine sodium is 32-128 µg / mL.

[0008] More preferably, the composition is a combination of honokiol 64 μg / mL and sulfamethoxypyrimidine sodium 32 μg / mL, or a combination of honokiol 32 μg / mL and sulfamethoxypyrimidine sodium 128 μg / mL.

[0009] The present invention also provides the use of a composition of magnolol and sulfamethoxypyrimidine sodium in the preparation of a formulation that enhances the susceptibility of Vibrio parahaemolyticus to sulfamethoxypyrimidine sodium.

[0010] The present invention also provides a drug for treating acute hepatopancreatic necrosis in shrimp, the drug comprising a composition of magnolol and sulfamethoxypyrimidine sodium, wherein the drug is a composition of magnolol and sulfamethoxypyrimidine sodium.

[0011] Preferably, the mass ratio of magnolol to sulfamethoxypyrimidine sodium in the composition is 1:0.125~0.5, and the drug is administered orally (mixed with feed), wherein the dosage of the drug added to the feed is: 8~16 mg / kg feed of sulfamethoxypyrimidine sodium and 32~64 mg / kg feed of magnolol.

[0012] More preferably, the dosage of the drug added to the feed is: 8 mg / kg feed of sulfamethoxypyrimidine sodium and 64 mg / kg feed of magnolol, or 16 mg / kg feed of sulfamethoxypyrimidine sodium and 32 mg / kg feed of magnolol, or 16 mg / kg feed of sulfamethoxypyrimidine sodium and 64 mg / kg feed of magnolol.

[0013] Compared with existing technologies, the advantages of this invention are as follows: This invention discloses for the first time the application of magnolol as an antibiotic adjuvant in combination with sulfamethoxypyrimidine sodium in the preparation of Vibrio parahaemolyticus and in the treatment of acute hepatopancreatic necrosis disease (AHPND) in shrimp caused by Vibrio parahaemolyticus. It can effectively reverse the resistance of Vibrio parahaemolyticus to SMM-Na, producing a significant synergistic bactericidal effect. In vivo experiments have demonstrated that this composition can significantly improve the survival rate of shrimp infected with Vibrio parahaemolyticus, effectively reduce the pathogen load in the hepatopancreas, alleviate histopathological damage, and simultaneously enhance the host's humoral and mucosal immunity, reshaping a healthy intestinal flora structure. This invention provides a multi-target, highly efficient, and reduced-dosage combined prevention and control strategy for AHPND in shrimp, offering a new solution for the green control of AHPND in shrimp. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings required in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the accompanying drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0015] Figure 1 The graph shows the synergistic antibacterial effect of magnolol and sulfamethoxypyrimidine sodium against Vibrio parahaemolyticus DX190406 as determined by the checkerboard method. Figure 2 The graph shows the synergistic antibacterial effect of magnolol and sulfamethoxypyrimidine sodium against Vibrio parahaemolyticus DX190406 determined by the checkerboard method. Figure 3 The figure shows the effect of the combination of magnolol and sulfamethoxypyrimidine sodium on the time-growth curve of Vibrio parahaemolyticus DX190406. In the figure, A is the combination of magnolol 64 μg / mL and sulfamethoxypyrimidine sodium 32 μg / mL, and B is the combination of magnolol 32 μg / mL and sulfamethoxypyrimidine sodium 128 μg / mL. Figure 4 The effect of the combination of magnolol and sulfamethoxypyrimidine sodium on the time-growth curve of Vibrio parahaemolyticus DX190406 is shown in the figure. Figure 5 Time-bactericidal curves of different drug treatments on Vibrio parahaemolyticus DX190406; Figure 6 Figure showing the effect of different drug treatments on biofilm formation of Vibrio parahaemolyticus DX190406; Figure 7 This is a diagram showing the effect of different drug treatments on the cell membrane permeability of Vibrio parahaemolyticus observed under a fluorescence microscope. PI: propidium iodide staining, Hoechst: Hoechst 33342 nuclear dye, Merged: overlay image. Figure 8 Figure showing the effect of different drug treatments on the cell morphology of Vibrio parahaemolyticus as observed by scanning electron microscopy. Figure 9 Figure showing the effect of different drug treatments on intracellular ATP levels in Vibrio parahaemolyticus cells; Figure 10 The cumulative survival rate curves of Litopenaeus vannamei after infection with Vibrio parahaemolyticus and treatment with different drugs are shown. Figure 11 This graph shows the changes in Vibrio parahaemolyticus load in the hepatopancreas of shrimp in each experimental group at different time points after infection. Figure 12The graph shows the changes in total hemocytocyte count in shrimp of each experimental group at different time points after infection. ** indicates p < 0.01, *** indicates p < 0.001, and ns indicates no significant difference. Figure 13 This graph shows the changes in the expression levels of immune-related genes in the hepatopancreas of shrimp in each experimental group at different time points after infection, where A represents... Alf Gene, B is Tlr Gene, C is Lec Gene, D is Crustin Gene, E is Lzm Gene, F is CatB Gene; Figure 14 This graph shows the changes in the expression levels of mucosal barrier-related genes in the intestines of shrimp in each experimental group at different time points after infection. A represents... Muc-1 Gene, B is Muc-4 Gene, C is Muc-5AC Gene, D is Muc-19 Gene; Figure 15 Histopathological sections (H&E staining) of shrimp hepatopancreas and intestinal tissue from each experimental group on day 10 of infection. Figure 16 The image shows a comparison of the α diversity index of the gut microbiota of shrimp in each experimental group on day 10 of infection. A is the dilution curve based on the Shannon-Wiener index; B is the species accumulation curve; C is the Venn diagram of OTU distribution; and D is the rank-abundance curve. Each sample was a mixture of gut samples from 3 shrimp in the same container, and each group contained 3 biological replicates. Figure 17 The diagram shows the composition and distribution of the intestinal flora of shrimp in each experimental group at the phylum level on day 10 of infection. Figure 18 The graph shows the relative abundance of key genera in the gut microbiota of shrimp in each experimental group at the genus level on day 10 after infection. A represents the relative abundance of the microbiota at the genus level; B represents the relative abundance of some dominant genera. Data are expressed as mean ± standard deviation (n = 3), and different letters indicate significant differences between groups (p < 0.05). Figure 19 This is a heatmap of the KEGG pathway (Level 2) function of shrimp gut microbiota in each experimental group based on PICRUSt2 prediction. Detailed Implementation

[0016] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0017] 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 obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0018] 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.

[0019] 1. Test drug Sulfamonomethoxine Sodium (SMM-Na): effective content ≥98%, purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; sulfamonomethoxine sodium was prepared into a stock solution with an initial concentration of 5120 μg / mL using sterile deionized water, and after sterilization by filtration using a sterile syringe and a 0.22 μm filter membrane, it was aliquoted and stored at -20℃ for later use.

[0020] Honokiol (HKL): effective content ≥98%, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; Magnool (MGL): effective content ≥98%, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; Honokiol and Magnool were prepared into stock solutions with an initial concentration of 5120 μg / mL using dimethyl sulfoxide (DMSO) and stored at -20℃ protected from light for later use.

[0021] 2. Culture medium The culture media used in the experiment included 2216E liquid medium and 2216E agar medium, which were purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd. Preparation of 2216E solid medium (1000 mL system): Weigh 52.4 g of 2216E agar medium, add 1000 mL of distilled water, mix well, autoclave at 121℃ for 15 min, let cool to 50℃, pour into plates and cool to room temperature. Preparation of 2216E liquid culture medium (1000 mL system): Weigh 37.4 g of 2216E liquid culture medium, add 1000 mL of distilled water, mix well, autoclave at 121℃ for 15 min, and let cool for later use.

[0022] 3. Experimental strains The strain used in this study was Vibrio parahaemolyticus (Vibrio parahaemolyticus). Vibrio parahaemolyticusDX190406, this strain was isolated from the hepatopancreatic tissue of Litopenaeus vannamei shrimp suffering from typical acute hepatopancreatic necrosis disease (AHPND) at a shrimp farm in Ninghai County, Ningbo City, Zhejiang Province. It was identified as carrying the PirA and PirB virulence genes. The complete genome sequence of this strain has been submitted to the NCBI GenBank database (accession numbers: CP187480-CP187485). The strain was provided by the Zhejiang Provincial Marine Fisheries Research Institute.

[0023] The bacterial strain, stored in glycerol tubes at -80℃, was streaked onto 2216E solid medium and incubated at 28℃ for 18-24 hours. A single colony was picked and inoculated into 5 mL of 2216E liquid medium and cultured at 28℃ with shaking at 180 rpm for 12 hours until the logarithmic growth phase. The bacterial concentration was adjusted to approximately 1.5 × 10⁻⁶ using fresh 2216E liquid medium via McFarland turbidimetric assay. 6 CFU / mL, used for subsequent experiments.

[0024] 4. Laboratory animals Healthy Litopenaeus vannamei ( Penaeus vannamei The shrimp were purchased from the Yongxing Aquaculture Base of the Zhejiang Provincial Marine Fisheries Research Institute. They measured 4.0 ± 0.33 cm in length and 1.05 ± 0.27 g in weight. Before the experiment, they were temporarily held in 300 L fiberglass tanks for one week, fed commercial feed twice daily (09:00 and 21:00) at 0.5% of their body weight. The aquaculture water was sand-filtered natural seawater, with 50% of the water changed daily. Water quality parameters were maintained as follows: temperature 28 ± 0.5℃, pH 7.8–8.2, salinity approximately 21‰, and dissolved oxygen approximately 6 mg / L. All aquaculture containers were disinfected before use. Before the experiment, shrimp were randomly sampled, and their absence of Vibrio parahaemolyticus was verified by TCBS agar culture and 16S rRNA gene PCR.

[0025] Example 1: Determination of the minimum inhibitory concentration of sulfamethoxypyrimidine sodium, honokiol, and magnolol against Vibrio parahaemolyticus DX190406.

[0026] 1. Experimental Methods: The micro-broth dilution method was used for determination. In a sterile 96-well plate, 100 μL of 2216E liquid medium was added to wells 2 through 11 of each column. 100 μL of SMM-Na stock solution (diluted with medium) at a concentration of 512 μg / mL was added to each well in columns 1 and 2. Starting from column 2, continuous two-fold dilutions were performed up to column 10. The 100 μL dilution in column 11 was discarded. Two more 96-well plates were prepared using the same method, with HKL and MGL dilution series respectively. Then, 100 μL of 1.5 × 10⁻⁶ SMM-Na stock solution was added to each well. 6Vibrio parahaemolyticus DX190406 bacterial solution at CFU / mL (the 11th column is the positive growth control, only adding the bacterial solution and the medium; another well containing only the medium is set as the negative control). Place the 96-well plate in an incubator at 28°C and incubate it statically for 18 h, then observe the results. The lowest drug concentration in the wells without visible bacterial growth observed by the naked eye is the minimum inhibitory concentration (MIC) of this drug. All experiments are independently repeated three times.

[0027] 2. Experimental results: As shown in Table 1, the MIC of sulfamonomethoxine sodium against Vibrio parahaemolyticus DX190406 > 1024 μg / mL, the MIC of honokiol against this bacterium is 128 μg / mL, and the MIC of magnolol against this bacterium > 256 μg / mL. According to the provisions of the CLSI M45 document, for Vibrio spp., when the MIC of sulfamonomethoxine sodium ≥ 512 μg / mL, it can be determined as a sulfamonomethoxine sodium-resistant bacterium.

[0028] Table 1 Minimum inhibitory concentrations of SMM-Na and HKL against Vibrio parahaemolyticus DX190406

[0029] Example 2. Evaluation of the synergistic effect of the combined application of honokiol and sulfamonomethoxine sodium (checkerboard method).

[0030] 1. Experimental method: Refer to the CLSI M27-A3 guideline and use the checkerboard method. In a 96-well plate, dilute the SMM-Na solution in a two-fold dilution gradient, and also dilute the HKL and MGL solutions in a two-fold dilution gradient respectively. Then, add 100 μL of the bacterial solution at a concentration of 1.5 × 10 6 CFU / mL to each well to make the final volume 200 μL. Set up bacterial solution control wells and medium control wells. After incubating at 28°C for 18 h, read the growth status of each well. Calculate the fractional inhibitory concentration index (FICI), and the formula is: FICI = (MIC of SMM-Na when used in combination / MIC of SMM-Na when used alone) + (MIC of HKL or MGL when used in combination / MIC of HKL or MGL when used alone). Judgment criteria: FICI ≤ 0.5 indicates a synergistic effect; 0.5 < FICI ≤ 1 indicates an additive effect; 1 < FICI ≤ 4 indicates an irrelevant effect; FICI > 4 indicates an antagonistic effect. The experiment is independently repeated three times.

[0031] 2. Experimental results: As Figure 1As shown in Table 2, when HKL (32 μg / mL or 64 μg / mL) was used in combination with SMM-Na at sub-inhibitory concentrations, the MIC of SMM-Na was significantly reduced from >1024 μg / mL to 32-128 μg / mL, a reduction of 8-32 times. The calculated FICI values ​​were 0.53 and 0.375, respectively, indicating that HKL and SMM-Na had a significant combined antibacterial effect against Vibrio parahaemolyticus DX190406. The MIC of MGL was >256 μg / mL, and the MIC of SMM-Na was >1024 μg / mL, even under conditions where both were used at relatively high test concentrations (MGL 256 μg / mL, SMM-Na 256 μg / mL) (e.g. Figure 2 As shown in the figure, the combined treatment still could not effectively inhibit bacterial proliferation. Given that further increasing the concentration of SMM-Na would not yield a significant reduction in antibiotic resistance and enhancement of efficacy, this study did not further evaluate the combined antibacterial effect of MGL and SMM-Na.

[0032] Table 2. MIC and FICI index of HKL combined with SMM-Na against Vibrio parahaemolyticus DX190406

[0033] Example 3: Effects of magnolol and sulfamethoxypyrimidine sodium compositions on bacterial growth curves.

[0034] 1. Experimental Methods: Logarithmic growth phase Vibrio parahaemolyticus DX190406 bacterial suspension was diluted with fresh 2216E medium to approximately 1.5 × 10⁶ CFU / mL. The experimental groups for the combination of magnolol and sulfamethoxypyrimidine sodium were set up as follows: SMM-Na (32 μg / mL) + HKL (64 μg / mL) combination group; SMM-Na (32 μg / mL) single drug group; HKL (64 μg / mL) single drug group; SMM-Na (128 μg / mL) + HKL (32 μg / mL) combination group; SMM-Na (128 μg / mL) single drug group; HKL (32 μg / mL) single drug group; SMM-Na (1024 μg / mL) single drug group; bacterial growth control group without drugs; blank control group without bacteria. The bacterial culture from each treatment group was added to a 96-well plate, 200 μL per well. The plates were incubated at 28℃, and the absorbance (OD600) at 600 nm was measured every hour using a microplate reader for 24 hours. The experiment was repeated three times. The experimental groups for the combination of magnolol and sulfamethoxypyrimidine sodium were set up as follows: SMM-Na (256 μg / mL) + MGL (64 μg / mL) combination group; SMM-Na (512 μg / mL) + MGL (64 μg / mL) combination group; MGL (64 μg / mL) single drug group; SMM-Na (1024 μg / mL) single drug group; and bacterial growth control group without drugs.

[0035] 2. Experimental results: such as Figure 3 A and Figure 3 As shown in Figure B, compared with the bacterial growth control group, the SMM-Na (32 μg / mL) + HKL (64 μg / mL) combination completely inhibited bacterial growth within 24 hours, and its OD... 600 The values ​​were similar to those in the blank control group of the culture medium. Although the combination of SMM-Na (128 μg / mL) + HKL (32 μg / mL) did not completely inhibit growth, it significantly prolonged the lag phase of bacterial growth, and the final bacterial count was much lower than that of the single-drug group and the control group. The use of SMM-Na (32 μg / mL, 128 μg / mL, 1024 μg / mL) or HKL (32 μg / mL, 64 μg / mL) alone only slowed down the bacterial growth rate to a certain extent, but failed to completely inhibit it. Figure 4As shown, compared with the bacterial growth control group, treatment with SMM-Na (1024 μg / mL) or MGL (64 μg / mL) alone only showed a certain degree of antibacterial effect. Although the combined treatment groups SMM-Na (256 μg / mL) + MGL (64 μg / mL) and SMM-Na (512 μg / mL) + MGL (64 μg / mL) had a stronger growth inhibitory effect than MGL (64 μg / mL) alone, the bacteria still maintained a significant proliferative capacity. This indicates that the combined treatment failed to achieve effective growth control and therefore could not achieve the ideal infection inhibition effect, and thus has no practical significance for production application.

[0036] Example 4: Time-bacterial curve of the combination of magnolol and sulfamethoxypyrimidine sodium.

[0037] 1. Experimental method: The concentration was approximately 1.5 × 10⁻⁶. 6 Bacterial suspensions at CFU / mL were prepared by adding SMM-Na, HKL, or combinations thereof to achieve final concentrations of: SMM-Na (128 μg / mL), HKL (32 μg / mL), SMM-Na (128 μg / mL) + HKL (32 μg / mL), SMM-Na (32 μg / mL), HKL (64 μg / mL), SMM-Na (32 μg / mL) + HKL (64 μg / mL), HKL (128 μg / mL) (positive control). A control without added drugs was also provided. The suspensions were incubated at 28°C in a shaker, and samples were taken at 0, 10, and 30 min, and at 1, 3, 6, 9, 12, and 24 h. The samples were serially diluted 10-fold, and 100 μL of each diluted suspension was plated onto 2216E agar plates and incubated at 28°C for 24 h. Colony forming units (CFU) were then counted. The final concentrations were calculated as the logarithm of CFU / mL. 10 Plot the CFU / mL count against time to create a time-bacterial kill curve. The kill curve is defined as a reduction in bacterial count of ≥3 log₂CFU / mL in the combination therapy group compared to the most effective single-drug group at 24 h. 10 CFU / mL indicates a bactericidal effect.

[0038] 2. Experimental results: such as Figure 5 As shown, using HKL (32 μg / mL, 64 μg / mL) or SMM-Na (128 μg / mL, 32 μg / mL) alone resulted in a decrease of less than 3 logs of bacterial count within 24 h. 10 The combination of SMM-Na (128 μg / mL) and HKL (32 μg / mL) reduced bacterial count by more than 3 logs after 24 h. 10It exhibits a bactericidal effect. Notably, the combination of SMM-Na (32 μg / mL) + HKL (64 μg / mL) showed no detectable viable bacteria after 10 min of treatment and remained sterile throughout the 24 h observation period, demonstrating rapid and thorough bactericidal activity.

[0039] Example 5: The effect of the combination of magnolol and sulfamethoxypyrimidine sodium on the formation of Vibrio parahaemolyticus biofilm.

[0040] 1. Experimental Method: Crystal violet staining was used. 100 μL of a 1.5 × 10⁻⁶ concentration of crystal violet was added to each well of a 96-well cell culture plate. 6 CFU / mL bacterial suspension was added, followed by 100 μL of culture medium containing different drugs (final concentrations: SMM-Na (32 μg / mL), HKL (64 μg / mL), SMM-Na (32 μg / mL) + HKL (64 μg / mL), SMM-Na (1024 μg / mL), HKL (128 μg / mL)). Wells containing only bacterial suspension and culture medium served as biofilm formation controls. The wells were incubated at 28 °C for 24 h to allow biofilm formation. The airborne bacteria and culture medium were carefully aspirated from the wells, and the well walls were gently washed twice with sterile PBS and air-dried. 150 μL of 0.4% (w / v) crystal violet solution was added to each well, and staining was performed at room temperature for 20 min. The staining solution was discarded, and unbound dye was washed away with sterile water and air-dried. 200 μL of 33% (v / v) glacial acetic acid was added to each well to dissolve the bound crystal violet, and the wells were shaken at room temperature for 10 min. Pipette the dissolving solution into a new 96-well plate and measure the absorbance (OD) at 570 nm using a microplate reader. 570 This reflects the biomass of the biofilm. The experiment was conducted in triplicate.

[0041] 2. Experimental results: such as Figure 6 As shown, in the control group without any treatment, bacteria formed a distinct biofilm. The biofilm production of bacteria treated with HKL (64 μg / mL) or SMM-Na (32 μg / mL) alone was not significantly different from the control group; however, the biofilm production of bacteria treated with 128 μg / mL HKL alone was significantly affected (p<0.01); the biofilm production of bacteria treated with SMM-Na (1024 μg / mL) alone also decreased significantly (p<0.05); and the biofilm production of the group treated with SMM-Na (32 μg / mL) + HKL (64 μg / mL) was significantly decreased (p<0.01).

[0042] Example 6: The effect of the combination of magnolol and sulfamethoxypyrimidine sodium on bacterial cell membrane permeability.

[0043] 1. Experimental Methods: Logarithmic growth phase bacterial cultures were collected and, after adjusting the concentration, treated with SMM-Na (32 μg / mL), HKL (64 μg / mL), and SMM-Na (32 μg / mL) + HKL (64 μg / mL) for 10 min each. A control group without treatment was also included. After treatment, the bacterial cells were collected by centrifugation and washed once with PBS. The cells were resuspended in PBS containing Hoechst 33342 (final concentration 10 μg / mL) and stained in the dark for 20 min. After centrifugation, the supernatant was discarded, and the cells were resuspended in PBS containing propidium iodide (PI, final concentration 10 μg / mL) and stained in the dark for 20 min. After staining, the cells were washed with PBS to remove excess dye, resuspended, and dropped onto a glass slide, which was then covered with a coverslip. Observation and photography were performed under a fluorescence microscope. All cell nuclei stained with Hoechst showed blue fluorescence, while cells with impaired membrane integrity showed red fluorescence due to PI uptake. Changes in membrane permeability were assessed by measuring the intensity of red fluorescence and the number of cells.

[0044] 2. Experimental results: such as Figure 7 As shown, the untreated control group bacteria showed only blue fluorescence (Hoechst) and almost no red fluorescence (PI), indicating good cell membrane integrity. Bacteria treated with SMM-Na alone showed a small amount of red fluorescence, indicating good cell membrane integrity. Bacteria treated with HKL alone showed significant red fluorescence, indicating increased cell membrane permeability and significantly impaired cell membrane integrity. Bacteria treated with the SMM-Na + HKL combination were mostly stained with PI, showing strong red fluorescence, indicating that the combined treatment severely damaged bacterial cell membrane integrity, leading to a significant increase in permeability.

[0045] Example 7: Effect of the combination of magnolol and sulfamethoxypyrimidine sodium on bacterial cell membranes (scanning electron microscopy observation).

[0046] 1. Experimental method: Approximately 1.5 × 10 6 CFU / mL bacterial suspensions were treated with HKL (64 μg / mL) and SMM-Na (32 μg / mL) for 30 min, or a combination of SMM-Na (32 μg / mL) + HKL (64 μg / mL) for 10 min. Bacterial cells were collected by centrifugation and washed twice with PBS. Fixation was performed with 2.5% glutaraldehyde (prepared in 0.1 M PBS) at 4°C for at least 4 h. After PBS washing, the cells were sequentially dehydrated with 30%, 50%, 70%, 80%, 90%, 95%, and 100% ethanol, 10-15 min each time. Samples were dropped onto clean coverslips, dried, and then sputter-coated with gold. Bacterial morphology was observed using a scanning electron microscope (Hitachi S-3400).

[0047] 2. Experimental results: such as Figure 8 As shown, the untreated control group bacteria had smooth surfaces and intact morphology. The morphological changes of bacteria treated with SMM-Na alone were similar to those of the control group. Bacteria treated with HKL alone showed obvious cell membrane damage and collapse, with a significant decrease in cell integrity. However, bacteria treated with the SMM-Na + HKL combination showed severe cell surface collapse and shrinkage, significant cell structure destruction, and leakage of contents, demonstrating that the combined drug treatment severely damaged the bacterial cell membrane.

[0048] Example 8: The effect of the combination of magnolol and sulfamethoxypyrimidine sodium on intracellular ATP levels in bacteria.

[0049] 1. Experimental Methods: An enhanced ATP assay kit was used. Bacterial culture was treated with the reagents (final concentrations: SMM-Na (32 μg / mL), HKL (64 μg / mL) and a combination of SMM-Na (32 μg / mL) + HKL (64 μg / mL)) and incubated at 28°C. 100 μL samples were taken at 0, 5, 10, 15, and 20 min, and immediately placed on ice. The bacterial cells were collected by centrifugation, and lysis buffer was added and lysed on ice. The supernatant was collected by centrifugation and mixed with an equal volume of the ATP assay working solution in a white 96-well plate. The chemiluminescence value was immediately detected using a microplate reader. The experiment was repeated three times.

[0050] 2. Experimental results: such as Figure 9 As shown, SMM-Na alone had no significant effect on intracellular ATP levels within 10 min. HKL alone caused a significant decrease in ATP at 10 min (p<0.05). However, the combined treatment of SMM-Na and HKL caused a significantly greater decrease in ATP than either single treatment (p<0.05), and led to near-complete depletion of ATP after 20 min, indicating that the combined treatment severely interfered with bacterial energy metabolism.

[0051] Example 9: The protective effect of the combination of magnolol and sulfamethoxypyrimidine sodium on infected Litopenaeus vannamei.

[0052] 1. Infection model establishment and dose screening: The 120-hour median lethal concentration (LD50) of Vibrio parahaemolyticus DX190406 against Litopenaeus vannamei was determined through preliminary experiments. 50 Approximately 6.0 × 10 7 CFU / mL (Reed-Muench method). In the formal experiment, the shrimp were first placed at 6.0 × 10⁻⁶. 8 Immerse in a high-concentration bacterial solution of CFU / mL for 15 min, then transfer to a solution maintaining the bacterial concentration in the water at LD50. 50 Horizontal (6.0 × 10) 7The infection persisted in a 30 L breeding tank (CFU / mL).

[0053] 2. Experimental Design and Drug Administration: 630 shrimp were randomly divided into 7 groups, with 3 replicates per group and 30 shrimp per replicate. The groups were as follows: NC group (uninfected, fed commercial feed coated with peanut oil); PC group (infected, fed commercial feed coated with peanut oil); SMM-Na group (infected, fed feed containing 80 mg / kg SMM-Na); HKL group (infected, fed feed containing 64 mg / kg HKL); Combination A (infected, fed feed containing 8 mg / kg SMM-Na + 64 mg / kg HKL); Combination B (infected, fed feed containing 16 mg / kg SMM-Na + 32 mg / kg HKL); Combination C (infected, fed feed containing 16 mg / kg SMM-Na + 64 mg / kg HKL). The drug was dissolved in peanut oil and sprayed evenly onto the surface of the feed. Medicated feed was administered immediately after infection, twice daily for 10 consecutive days. The daily mortality rate was recorded, and the survival rate was calculated.

[0054] 3. Experimental results: such as Figure 10 As shown, on day 10 after infection, the survival rate of the negative control group (uninfected and not given medication) was 100%, while the survival rate of the positive control group (infected only with bacteria) was 26.7%. After infection, the survival rate of shrimp fed SMM-Na (80 mg / kg) was 66.7%, and the survival rate of shrimp fed HKL (64 mg / kg) was 50%. The survival rate of shrimp fed with SMM-Na 8 mg / kg + HKL 64 mg / kg reached 66.7%, which was comparable to the survival rate of shrimp fed with SMM-Na alone (80 mg / kg). The survival rate of shrimp fed with SMM-Na 16 mg / kg + HKL 64 mg / kg reached 80%, and the survival rate of shrimp fed with SMM-Na 16 mg / kg + HKL 32 mg / kg reached 90%. The survival rate was significantly improved even when the amount of SMM-Na used was reduced by 5 times (from 80 mg / kg to 16 mg / kg). The mass ratio of HKL to SMM-Na was found to be 1:0.125 ~ 0.5.

[0055] Example 10: Comprehensive evaluation of the therapeutic efficacy and mechanism of the optimal composition (SMM-Na 16 mg / kg + HKL 32 mg / kg).

[0056] 1. Experimental Design: The optimal combination (SMM-Na 16 mg / kg + HKL 32 mg / kg) selected in Example 9 was used to comprehensively evaluate its therapeutic efficacy and mechanism. Four groups were established: NC group (uninfected, fed peanut oil diet), PC group (infected, fed peanut oil diet), HKL monotherapy group (infected, fed HKL 64 mg / kg diet), and the optimal combination group (infected, fed SMM-Na 16 mg / kg + HKL 32 mg / kg diet). Infection and feeding conditions were the same as in Example 9.

[0057] 2. Sample Collection and Testing: On days 2, 4, 6, 8, and 10 post-infection, three shrimp were randomly selected from each container (nine shrimp per group / time point) for sampling. Hemolymph was aseptically collected for total blood cell count (THC). Hepatopancreas and intestinal tissue were aseptically dissected. A portion was used for plate count to determine the bacterial load (CFU / g) in the hepatopancreas; another portion was flash-frozen in liquid nitrogen and stored at -80°C for RNA extraction and qRT-PCR analysis of immunogene expression; and the remaining portion was fixed with Davidson's fixative for histopathological examination (H&E staining). Intestinal contents were collected on day 10 for 16S rRNA gene sequencing analysis of the gut microbiota.

[0058] 3. Experimental Results: (1) Bacterial load in the hepatopancreas: such as Figure 11 As shown, the viral load of Vibrio parahaemolyticus in the liver and pancreas of the PC group remained at a high level, exceeding 10 after 8 days of infection. 7 CFU / g. The HKL monotherapy group reduced the viral load to about 5% of the PC group, but it was still above 10. 5 CFU / g. The optimal combination therapy significantly reduced the number of Vibrio parahaemolyticus in the hepatopancreas at all time points, with the bacterial load as low as 10 on day 10. 5 The concentration of CFU / g was below 0.3% of that in the PC group.

[0059] (2) Total blood cell count (THC): such as Figure 12 As shown, THC in the PC group dropped sharply on day 2 post-infection, but recovery was incomplete in the later stages. THC in the HKL monotherapy group also dropped rapidly from day 2, then gradually recovered, but by day 10, it had not yet returned to NC levels. THC in the optimal combination group began to drop on day 2 post-infection, but the drop was very gradual, much less pronounced than in the PC and HKL monotherapy groups, and by day 10, it had recovered to NC levels.

[0060] (3) Immunogenetic gene expression: qRT-PCR results showed that, compared with the PC group, the optimal combination group significantly upregulated the expression levels of immune-related genes in the hepatopancreas, including those involved in pathogen recognition. Tlr , Lec Participating in direct antibacterial Alf , Crustin , Lzm and immune regulation CatB And the expression levels were significantly higher than those of the HKL monotherapy group during the same period (within 2-8 days). Figure 13 AF)(p<0.05); compared with the PC group, the optimal combination group significantly upregulated genes involved in the mucosal barrier in the intestine (AF) (p<0.05); Muc-1 , Muc-4 , Muc-5AC , Muc-19 The expression of [the drug] was significantly higher than that of the HKL monotherapy group during the same period (2-8 days) (p<0.05). Figure 14 AD).

[0061] (4) Histopathology: such as Figure 15 As shown, in the PC group, the hepatopancreas of shrimp exhibited extensive shedding of hepatopancreatic tubular epithelial cells, tubular dilation, and necrosis, as well as destruction of the intestinal mucosal epithelial structure. The damage was somewhat reduced in the HKL monotherapy group. The optimal combination group showed significantly reduced histopathological damage, with the hepatopancreatic tubular structure remaining largely intact, showing only focal mild epithelial shedding, and the intestinal mucosal structure was also more intact.

[0062] (5) Gut microbiota analysis: 16S rRNA gene sequencing was performed on the gut contents on day 10 after infection. Alpha diversity analysis showed that ( Figure 16 (AD, Table 3) The optimal combination group significantly increased the Chao1, ACE, Shannon, and Simpson indices of the gut microbiota, indicating that it restored the decreased microbiota diversity caused by infection. Principal component analysis showed clear separation of the microbiota structure in each group. At the phylum level ( Figure 17 The optimal composition group reduced Proteobacteria and Bacteroidota The relative abundance increased Desulfobacterota , Chloroflexi and Actinobacteriota At the genus level ( Figure 18 A and Figure 18 B), the optimal combination group significantly reduced pathogenic bacteria. Vibrio and potential pathogens Motilimonas The abundance increased Marinobacter and Woeseia Species such as *Isochrysis*. KEGG functional prediction ( Figure 19 The results showed that the optimal combination partially restored the function of pathways related to amino acid metabolism and exogenous degradation metabolism, and downregulated the overactive immune pathways associated with bacterial infection diseases.

[0063] Table 3 Comparison of gut microbiota α diversity index on day 10 after infection in shrimp (n=3, mean ± SD)

[0064] Note: Values ​​with significant differences in the same column are labeled with different lowercase letters (p<0.05).

[0065] In summary, this invention, through a series of in vitro and in vivo experiments, demonstrates that the combination of magnolol (HKL) and sulfamethoxypyrimidine sodium (SMM-Na), particularly at a mass ratio of approximately 1:0.5, significantly enhances the ability to kill Vibrio parahaemolyticus and significantly reduces the amount of SMM-Na required, restoring bacterial sensitivity to SMM-Na. For example, the combination of SMM-Na (32 μg / mL) + HKL (64 μg / mL) in in vitro experiments effectively inhibits and kills drug-resistant Vibrio parahaemolyticus through mechanisms such as disrupting bacterial membrane structure, increasing membrane permeability, and depleting cellular energy (ATP). The combination of SMM-Na (16 mg / kg) + HKL (32 mg / kg) in in vitro experiments significantly improves shrimp survival rates through multiple effects, including significantly reducing the pathogen load in infected tissues, alleviating histopathological damage, enhancing host immune responses (including humoral and mucosal immunity), and reshaping a healthy gut microbiota. This combined strategy provides a new, efficient, and sustainable approach for the prevention and control of acute hepatopancreatic necrosis disease in shrimp, which can significantly reduce antibiotic usage.

[0066] This invention, through systematic research, reveals for the first time that plant-derived lignans and magnolol (HKL) can act as effective antibiotic adjuvants, exhibiting a significant synergistic effect with sulfamethoxypyrimidine sodium (SMM-Na) and reversing the resistance of Vibrio parahaemolyticus to SMM-Na. Checkerboard assays show that sub-inhibitory concentrations of HKL (32-64 μg / mL) can reduce the minimum inhibitory concentration (MIC) of SMM-Na against resistant Vibrio parahaemolyticus DX190406 by 8-32 times, confirming a synergistic antibacterial effect. Time-kill curves demonstrate that the combined use of SMM-Na (32 μg / mL) and HKL (64 μg / mL) can completely kill bacteria within 10 minutes, exhibiting a rapid bactericidal effect. The significant antibacterial effect of this composition is mainly achieved through severely disrupting bacterial cell membrane integrity, inducing severe depletion of intracellular ATP, and reducing bacterial biofilm formation. In a challenge experiment on Litopenaeus vannamei, oral administration of a diet containing a combination of SMM-Na (16 mg / kg) and HKL (32 mg / kg) increased the highest survival rate of infected shrimp to approximately 90%, significantly reduced bacterial load in the hepatopancreas, alleviated pathological damage to hepatopancreas and intestinal tissues, and promoted the recovery of total blood cell counts. Furthermore, this drug combination significantly upregulated the expression of hepatopancreas immune-related genes (Alf, Tlr, Lec, Crustin, Lzm, CatB) and intestinal mucosal barrier genes (Muc-1, Muc-4, Muc-5AC, Muc-19), and restored host intestinal health by reshaping the gut microbiota structure, increasing α-biota diversity, reducing the abundance of potentially harmful genera, and partially restoring predicted metabolic function. Therefore, the combination of magnolol and sulfamethoxypyrimidine sodium provided by this invention offers a novel and highly effective combined treatment strategy for treating acute hepatopancreatic necrosis in shrimp caused by Vibrio parahaemolyticus, which can reduce the amount of antibiotics used and contributes to promoting the green and healthy development of aquaculture.

[0067] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. The application of the combination of magnolol and sulfamethoxypyrimidine sodium in the preparation of Vibrio parahaemolyticus inhibitors.

2. The application according to claim 1, characterized in that, The mass ratio of magnolol to sodium sulfamethoxypyrimidine in the composition is 1:0.5~2.

3. The application according to claim 2, characterized in that, The concentration of magnolol in the composition is 32-64 µg / mL, and the concentration of sulfamethoxypyrimidine sodium is 32-128 µg / mL.

4. The application according to claim 3, characterized in that, The composition is a combination of honokiol 32 µg / mL and sulfamethoxypyrimidine sodium 128 µg / mL, or the composition is a combination of honokiol 64 µg / mL and sulfamethoxypyrimidine sodium 32 µg / mL.

5. The use of any one of claims 1-4 and the composition of magnolol and sulfamethoxypyrimidine sodium in the preparation of a formulation for improving the susceptibility of Vibrio parahaemolyticus to sulfamethoxypyrimidine sodium.

6. A drug for treating acute hepatopancreatic necrosis in shrimp, characterized in that: The drug comprises a combination of magnolol and sulfamethoxypyrimidine sodium.

7. A drug for treating acute hepatopancreatic necrosis in shrimp according to claim 6, characterized in that, The drug is a combination of magnolol and sulfamethoxypyrimidine sodium.

8. The drug according to claim 6 or 7, characterized in that, In the composition, magnolol and sodium sulfamethoxypyrimidine are present in a mass ratio of 1:0.125 to 0.

5.

9. The medicament according to claim 8, characterized in that, The drug is administered orally or as a feed additive. The dosage of the drug in the feed is: 8-16 mg / kg feed of sulfamethoxypyrimidine sodium and 32-64 mg / kg feed of magnolol.

10. The medicament according to claim 9, characterized in that, The dosage of the drug added to the feed is as follows: 8 mg / kg feed of sulfamethoxypyrimidine sodium and 64 mg / kg feed of magnolol, or 16 mg / kg feed of sulfamethoxypyrimidine sodium and 32 mg / kg feed of magnolol, or 16 mg / kg feed of sulfamethoxypyrimidine sodium and 64 mg / kg feed of magnolol.

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