A complex synergist of neomycin sulfate and its preparation method and application

Abstract

The present application belongs to the technical field of aquaculture, and discloses a compound synergist of neomycin sulfate as well as a preparation method and application thereof. The present application discloses, for the first time, the application of Wedelia prostrata extract, Gastrodia extract and Picrasma extract in inhibiting the growth of Vibrio in vitro and in preventing and treating the glass fry disease of South American white shrimp caused by Vibrio infection. Experimental verification shows that the Wedelia prostrata extract, Gastrodia extract and Picrasma extract have good effects in inhibiting the growth of Vibrio in vitro when used alone, in combination with each other or simultaneously. Meanwhile, in view of the treatment of Vibrio infection causing the glass fry disease, the above extracts are used in combination with neomycin sulfate, so as to overcome the objective problem of low or weak efficacy of neomycin sulfate at a recommended dose, and to simultaneously achieve the enhancement of the efficacy of neomycin sulfate after being used in combination with neomycin sulfate, and the reduction of the usage amount of neomycin sulfate.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture technology, specifically relating to a compound synergist of neomycin sulfate, its preparation method, and its application. Background Technology

[0002] TPD (transmissible leukemia) is a highly contagious disease in Litopenaeus vannamei that causes rapid onset, high mortality, and is caused by highly lethal Vibrio. Highly lethal Vibrio does not refer to a specific species, but rather to a class of Vibrio that secrete extremely potent Tc toxins. These Tc toxins are encoded by three virulence genes (TcA, TcB, and TcC) located on two virulence plasmids, pHLB and pHLC. The HLVA and HLVB sites are commonly used to indicate these two virulence plasmids.

[0003] From May to September 2023, the inventor's team collected 72 samples of shrimp exhibiting suspected "glass seedling" symptoms from major Litopenaeus vannamei farming areas such as Zhuhai, Fujian, Jiangsu, and Shandong. From these diseased shrimp, 65 highly lethal Vibrio bacteria carrying the Tc toxin gene and 14 Vibrio bacteria not carrying the Tc toxin gene were isolated. Susceptibility testing was conducted on these 79 Vibrio strains using four compliant aquatic antibiotics: enrofloxacin, florfenicol, doxycycline hydrochloride powder, and neomycin sulfate. The results showed that both types of Vibrio were relatively sensitive to enrofloxacin and doxycycline, with sensitivity rates of 80.0% and 78.6%, and 61.5% and 64.3%, respectively. Both strains showed high resistance to florfenicol, with resistance rates of 95.3% and 100%. However, significant differences were observed in neomycin sulfate resistance; the resistance rate of Vibrio carrying the Tc toxin gene reached 100%, while the resistance rate of those without the Tc toxin gene was only 21.4%. Neomycin sulfate is a compliant drug permitted for use in the control of Vibrio in Litopenaeus vannamei farming. However, given the high resistance of currently collected Vibrio strains to highly lethal Vibrio carrying the Tc toxin gene, the application of neomycin sulfate for the control of these strains presents several problems: First, maintaining the recommended dosage cannot reduce the mortality rate caused by this pathogen, not only failing to produce the desired therapeutic effect and delaying optimal treatment, but also increasing losses and economic benefits for aquaculture. Second, while increasing the dosage several times the recommended level may reduce losses to some extent, high-dose use can easily lead to excessive drug residues in shrimp and the aquatic environment, preventing shrimp from entering the market. It is particularly noteworthy that, based on the pathological examination and prognosis results of practical cases, highly lethal Vibrio is extremely virulent, often causing damage to organs such as the hepatopancreas and intestines once infected. Using neomycin sulfate alone to treat diseases caused by this type of Vibrio has very limited effectiveness. Therefore, an effective solution is urgently needed for controlling neomycin sulfate-resistant highly lethal Vibrio. Summary of the Invention

[0004] To address one of the problems existing in the prior art, this invention provides a technical solution for targeting highly lethal Vibrio resistant to neomycin sulfate. It provides a component that has a synergistic effect when used in combination with neomycin sulfate, which can overcome the objective problem that neomycin sulfate is weak or ineffective at the recommended dosage. It also simultaneously enhances the efficacy of neomycin sulfate when used in combination with it, while reducing the amount of neomycin sulfate used. This provides a practical and innovative solution for the production end to overcome the problem that neomycin sulfate alone cannot effectively solve the problem of vitreous larvae disease in shrimp.

[0005] The first aspect of the present invention is to provide the application of extracts of Wedelia candel, Gastrodia elata, and Quercus mongolica.

[0006] The second objective of this invention is to provide a neomycin sulfate synergist for inhibiting the growth of Vibrio.

[0007] The third objective of this invention is to provide an anti-Vibrio drug.

[0008] The fourth aspect of this invention aims to provide the application of the neomycin sulfate combined with the synergist for inhibiting Vibrio growth according to the second aspect of this invention or the anti-Vibrio drug according to the third aspect of this invention.

[0009] The fifth aspect of this invention aims to provide a method for inhibiting Vibrio.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] In a first aspect of the present invention, the use of Wedelia candel extract, Gastrodia elata extract and Paeonia lactiflora extract in any one of (1) to (4) is provided:

[0012] (1) Inhibits Vibrio growth in vitro;

[0013] (2) Prepare products that inhibit the growth of Vibrio;

[0014] (3) Preparation of a synergist for the combined use of neomycin sulfate to inhibit the growth of Vibrio;

[0015] (4) Prepare drugs for the prevention and / or treatment of diseases caused by Vibrio infection.

[0016] In some embodiments of the present invention, the wedelia candel extract is prepared by a method comprising: mixing wedelia candel with a eutectic solvent and extracting to obtain the wedelia candel extract.

[0017] In some embodiments of the present invention, the mass-to-volume ratio of the wedelia triloba to the eutectic solvent is 1:(15-25); preferably 1:(15-25); more preferably 1:20.

[0018] In some embodiments of the present invention, the eutectic solvent includes choline chloride, malic acid, and water.

[0019] In some embodiments of the present invention, the molar ratio of choline chloride, malic acid and water is 1:(1-3):(4-7); preferably 1:(2-3):(4-5), and more preferably 1:2:5.

[0020] In some embodiments of the present invention, before the *Wedelia trilobata* is mixed with the eutectic solvent, the *Wedelia trilobata* is pretreated, including: pulverizing *Wedelia trilobata* with stems containing ≤8% moisture content at low temperature and passing it through a 90-110 mesh sieve.

[0021] In some embodiments of the present invention, the extraction is ultrasonic extraction.

[0022] In some embodiments of the present invention, the extraction conditions are ultrasonic extraction at 50-65°C for 30-40 minutes.

[0023] In some embodiments of the present invention, the preparation method further includes purification and drying steps, including concentration, passing through a macroporous resin column, further concentration, and drying.

[0024] In some embodiments of the present invention, the purification and drying steps include extraction completion, solid-liquid separation, collection of supernatant, filtration, vacuum concentration of the filtrate to obtain a concentrate; adsorption of the concentrate using a macroporous resin column, followed by sequential adsorption with 2 volume fractions of pure water and 1.5 volume fractions of 30%–40% ethanol aqueous solution, collection of the 30%–40% ethanol aqueous solvent eluent, vacuum concentration to one-fifth of the original volume, and vacuum freeze-drying to obtain a solid powder with a moisture content of 4%–6%, which is the Wedelia candel extract.

[0025] In some embodiments of the present invention, the wedelia trilobata includes the South American wedelia trilobata.

[0026] In some embodiments of the present invention, the gastrodia extract is an enzymatic hydrolysis extract of gastrodia.

[0027] In some embodiments of the present invention, the Gastrodia elata extract is prepared by a method comprising the following steps: mixing Gastrodia elata with a compound enzyme preparation and a buffer solution, enzymatically hydrolyzing, inactivating the enzyme, separating and collecting the precipitate; and subjecting the precipitate to supercritical CO2 extraction to obtain the Gastrodia elata extract.

[0028] In some embodiments of the present invention, the compound enzyme preparation includes cellulase, pectinase and xylanase.

[0029] In some embodiments of the present invention, the mass ratio of cellulase, pectinase and β-glucanase is (3-5):(2-3):1; preferably (3-4):(2-3):1; more preferably 3:2:1.

[0030] In some embodiments of the present invention, the mass of the compound enzyme preparation is 0.2% to 0.8% of the mass of Gastrodia elata; preferably 0.4% to 0.5%; more preferably 0.5%.

[0031] In some embodiments of the present invention, the buffer solution is an acetate-sodium acetate buffer solution with a pH of 4.2.

[0032] In some embodiments of the present invention, the mass-to-volume ratio of the gastrodia elata and the buffer solution is 1:(8-15); preferably 1:(8-10); more preferably 1:10.

[0033] In some embodiments of the present invention, before mixing Gastrodia elata with the compound enzyme preparation, the Gastrodia elata undergoes pretreatment, including low-temperature pulverization of Gastrodia elata with a moisture content ≤12% and passing it through a 50-70 mesh sieve. To rapidly pulverize the Gastrodia elata, it can be sliced ​​thinly and then frozen with liquid nitrogen until completely brittle.

[0034] In some embodiments of the present invention, the enzymatic hydrolysis includes pre-activating the mixture at 30-40°C for 15-25 min, placing it in a constant temperature shaker at 35-50°C and 100-150 rpm for 50-70 min, and shaking at 45-55°C and 160-200 rpm for 25-40 min to promote deep cell wall disruption.

[0035] In some embodiments of the present invention, the enzyme inactivation includes placing the enzymatically hydrolyzed solution at 95–100°C for 15–20 min to inactivate the enzyme.

[0036] In some embodiments of the present invention, the precipitate is freeze-dried to a water content of less than 5% before supercritical CO2 extraction.

[0037] In some embodiments of the present invention, the conditions for supercritical CO2 extraction are as follows: ethyl acetate as the entrainer, CO2 flow rate of 20-40 kg / h, extraction pressure of 20-35 MPa, extraction temperature of 40-50°C, and extraction time of 80-110 min.

[0038] In some embodiments of the present invention, the preparation method further includes purification and drying steps, including collecting the extract after extraction, passing it through a macroporous resin column, concentrating it, and drying it to a water content of (4-8)%.

[0039] In some embodiments of the present invention, the Gastrodia elata includes black Gastrodia elata.

[0040] In some embodiments of the present invention, the extract of *Paspalum notatum* is an enzymatic hydrolysis extract of *Paspalum notatum*.

[0041] In some embodiments of the present invention, the *Paspalum notatum* extract is prepared by the following method: mixing *Paspalum notatum* with a buffer solution and a complex enzyme preparation, enzymatically hydrolyzing, inactivating the enzyme, and obtaining an enzymatic hydrolysate; mixing the enzymatic hydrolysate with ethanol, extracting, and separating the solid and liquid to obtain supernatant A and a precipitate; mixing the precipitate with ethyl acetate, extracting, and separating the solid and liquid to obtain supernatant B; combining supernatant A and supernatant B to obtain the *Paspalum notatum* extract.

[0042] In some embodiments of the present invention, the compound enzyme preparation includes cellulase, pectinase and xylanase.

[0043] The mass ratio of cellulase, pectinase and xylanase is (4-6):(2-4):1; preferably (5-6):(2-3):2; more preferably 5:3:2.

[0044] In some embodiments of the present invention, the mass of the compound enzyme preparation is 0.5% to 2% of the mass of the bitter tree; preferably 1% to 2%; more preferably 1%.

[0045] In some embodiments of the present invention, the buffer solution is an acetate-sodium acetate buffer solution with a pH of 5.0.

[0046] In some embodiments of the present invention, the mass-to-volume ratio of the gastrodia elata and the buffer solution is 1:(8-15); preferably 1:(8-10); more preferably 1:10.

[0047] In some embodiments of the present invention, before mixing the *Paspalum notatum* with the compound enzyme preparation, the *Paspalum notatum* undergoes pretreatment, including low-temperature pulverization of *Paspalum notatum* with a moisture content ≤10% and passing it through a 90-100 mesh sieve. To rapidly pulverize the *Paspalum notatum*, it can be frozen with liquid nitrogen until completely brittle.

[0048] In some embodiments of the present invention, the enzymatic hydrolysis includes placing the mixture in a constant temperature shaker at 35-50°C and 100-150 rpm for 1-3 hours.

[0049] In some embodiments of the present invention, the enzyme inactivation includes placing the enzymatically hydrolyzed solution at 95–100°C for 15–25 minutes to inactivate the enzyme.

[0050] In some embodiments of the present invention, the extraction is ultrasonic extraction.

[0051] In some embodiments of the present invention, the preparation method further includes a concentration and drying step, which includes filtration of the combined supernatant A and supernatant B through a filter membrane, collecting the filtrate, concentrating it under vacuum to one-third of its original volume, and vacuum drying it to a water content of 4% to 8%.

[0052] In some embodiments of the present invention, the Vibrio described in (1) to (3) includes Vibrio carrying a gene encoding Tc toxin.

[0053] In some embodiments of the present invention, the Vibrio includes Vibrio carrying a gene encoding Tc toxin and resistant to neomycin sulfate.

[0054] In some embodiments of the present invention, the Vibrio includes at least one of Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, and Vibrio erwinii.

[0055] In a second aspect, the present invention provides a neomycin sulfate synergist for inhibiting the growth of Vibrio, comprising the extracts of Wedelia candel, Gastrodia elata, and Quercus mongolica from the first aspect of the present invention.

[0056] In some embodiments of the present invention, the mass ratio of the *Wedelia trifoliata* extract, *Gastrodia elata* extract, and *Paspalum notatum* extract is 1:2:(2-4).

[0057] The combination of neomycin sulfate and a potentiator has a broad-spectrum antibacterial synergistic effect on clinically highly lethal Vibrio strains that carry the Tc toxin gene and are resistant to acid neomycin. This effectively reduces the dosage of neomycin sulfate and enhances the antibacterial effect.

[0058] When neomycin sulfate is used in combination with a synergist at a ratio of 20% to 60%, its efficacy in controlling glassy larvae disease in Litopenaeus vannamei is significantly better than that of neomycin sulfate alone, with the optimal substitution ratio being 40%. Replacing a portion of neomycin sulfate with this synergist at the recommended ratio overcomes the objective problem of weak or low efficacy of neomycin sulfate at recommended dosages. Simultaneously, it enhances the efficacy of neomycin sulfate when used in combination, while reducing the amount of neomycin sulfate used. This provides a practical and innovative solution for the production sector to overcome the challenge of effectively addressing glassy larvae disease in shrimp when neomycin sulfate is used alone.

[0059] A third aspect of the present invention provides an anti-Vibrio drug comprising the extracts of Wedelia candel, Gastrodia elata, and Quercus mongolica from the first aspect of the present invention.

[0060] In some embodiments of the present invention, the anti-vibrio drug further includes neomycin sulfate.

[0061] In some embodiments of the present invention, the Vibrio includes Vibrio carrying a gene encoding Tc toxin.

[0062] In some embodiments of the present invention, the Vibrio includes Vibrio carrying a gene encoding Tc toxin and resistant to neomycin sulfate.

[0063] In some embodiments of the present invention, the Vibrio includes at least one of Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, and Vibrio erwinii.

[0064] A fourth aspect of the present invention provides the use of the neomycin sulfate synergist of the second aspect of the present invention or the anti-vibrio drug of the third aspect of the present invention in any one of (a1) to (a2):

[0065] (a1) Inhibits the growth of Vibrio in vitro;

[0066] (a2) Prepare drugs for the prevention and / or treatment of diseases caused by Vibrio infection.

[0067] In some embodiments of the present invention, the Vibrio includes Vibrio carrying a gene encoding Tc toxin.

[0068] In some embodiments of the present invention, the Vibrio includes Vibrio carrying a gene encoding Tc toxin and resistant to neomycin sulfate.

[0069] In some embodiments of the present invention, the Vibrio includes at least one of Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, and Vibrio erwinii.

[0070] In some embodiments of the present invention, the disease includes glassy seed disease in Litopenaeus vannamei.

[0071] A fifth aspect of the present invention provides a method for inhibiting Vibrio, comprising the step of treating Vibrio with an anti-Vibrio agent according to the third aspect of the present invention.

[0072] The beneficial effects of this invention are:

[0073] This invention provides the first public disclosure of the in vitro inhibitory effect of extracts of *Wedelia trifoliata*, *Gastrodia elata*, and / or *Schisandra chinensis* on Vibrio growth, and their application in the prevention and treatment of vibrio-induced glassy larvae disease in shrimp. Experimental verification shows that the extracts of *Wedelia trifoliata*, *Gastrodia elata*, or *Schisandra chinensis*, used alone, in combination of two, or simultaneously, have a good in vitro inhibitory effect on Vibrio growth. Furthermore, for the treatment of Vibrio infection causing glassy larvae disease, the aforementioned synergist is used in combination with neomycin sulfate. This overcomes the objective problem of weak or low efficacy of neomycin sulfate at recommended doses, simultaneously enhancing the efficacy of neomycin sulfate while reducing the amount of neomycin sulfate required.

[0074] The neomycin sulfate synergist provided by this invention has good safety for oral administration and, when used in combination with neomycin sulfate, achieves both enhanced efficacy and reduced dosage of neomycin sulfate. Specifically, the neomycin sulfate synergist, when used in combination with neomycin sulfate, exhibits a significant and broad-spectrum antibacterial synergistic effect against 65 clinically isolated highly lethal Vibrio strains carrying the Tc toxin gene and resistant to neomycin sulfate. It also shows an additive inhibitory effect against two clinically isolated Vibrio strains that do not carry the Tc toxin gene and are sensitive to neomycin sulfate. When the neomycin sulfate synergist is used to replace neomycin sulfate at a ratio of 20%–60%, the protective effect against Vibrio hygroscopicis is superior to that of neomycin sulfate alone, with the optimal replacement ratio being 20%–40%. Similarly, when the neomycin sulfate synergist is used to replace neomycin sulfate at a ratio of 20%–60%, the therapeutic effect against Vibrio hygroscopicis is superior to that of neomycin sulfate alone, with the optimal replacement ratio being 40%. Attached Figure Description

[0075] Figure 1 To prevent shrimp mortality after Vp32 challenge in each experimental group.

[0076] Figure 2 The mortality rate of shrimp treated with Vp32 in each experimental group was determined.

[0077] Figure 3 The appearance of shrimp that survived Vp32 challenge as a positive control group.

[0078] Figure 4 The appearance of surviving shrimp treated with Vp32-infected shrimp was improved by combining a compound synergist with neomycin sulfate. Detailed Implementation

[0079] The present invention will be further described in detail below through specific embodiments.

[0080] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0081] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0082] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0083] Example 1

[0084] The preparation method of purified *Wedelia chinensis* extract includes the following steps: Weigh 100 parts by weight of *Wedelia chinensis* with stems and a water content ≤8%, cut them into 1-2 cm segments, and then pulverize them into 100-mesh powder using a low-temperature pulverizer. Add the pulverized raw material to a eutectic solvent DES (choline chloride, D-malic acid, and ultrapure water in a molar ratio of 1:2:5) at a ratio of 1:20 (w / v). Extract using ultrasound at 58℃ for 40 min. After extraction, centrifuge at 10000 rpm for 10 min, collect the supernatant, and extract the remaining precipitate twice more using the same ultrasonic extraction method. Combine all extracts. Filter the combined extract through a ceramic membrane filtration system with the following process parameters: membrane pore size 0.1 μm, crossflow velocity 4 m / s, and operating pressure 0.25 MPa, to obtain the filtrate. The filtrate was concentrated under vacuum using a rotary evaporator at a vacuum of -0.09 MPa and a temperature of 45°C until a relative density of 1.15 was obtained, yielding a concentrated solution. The concentrated solution was then adsorbed onto an AB-8 macroporous resin column. After adsorption, it was sequentially eluented with 2 volumes of pure water and 1.5 volumes of 30% ethanol aqueous solution, collecting the 30% ethanol aqueous solvent eluent. The eluent was then concentrated under vacuum using a rotary evaporator to one-fifth of its original volume. The concentrated solution was then dried in a vacuum freeze dryer for 40 hours to obtain a solid powder with a moisture content of (4–6)%, yielding the purified *Wedelia chinensis* extract. This purified extract was stored under low-temperature drying conditions (18°C, 45% RH) for later use. The purified *Wedelia chinensis* extract will be referred to as "Drug A" below.

[0085] The preparation method of the enzymatic hydrolysis purified Gastrodia elata includes the following steps: Weigh 100 parts by weight of Gastrodia elata with a water content ≤12%, cut it into thin slices, and put it into a liquid nitrogen quick-freezing tank for 10 minutes until it is completely brittle. Immediately transfer it to a low-temperature pulverizer and pulverize it into 60-mesh powder. Add pH 4.2 acetate-sodium acetate buffer and 0.5 w / w% compound enzyme preparation (a mixture of cellulase, pectinase and β-glucanase powders in a mass ratio of 3:2:1) at a material-to-liquid ratio of 1:10 (w / v). Pre-activate at 40℃ for 20 minutes. Place the above mixture in a constant temperature shaker and shake at 40℃ and 120 rpm for 60 minutes, and then shake at 50℃ and 180 rpm for 30 minutes to promote deep cell wall disruption. After enzymatic hydrolysis, the sample was placed in a 100℃ water bath for 18 minutes to inactivate the enzyme. It was then cooled to room temperature and centrifuged at 10,000 rpm for 20 minutes to collect the precipitate. The precipitate was then freeze-dried under vacuum until the moisture content was ≤6%, yielding enzymatically hydrolyzed Gastrodia elata powder. The obtained enzymatically hydrolyzed Gastrodia elata powder was passed through a 60-mesh sieve and extracted using a supercritical CO2 extraction system with 1% ethyl acetate as the entrainer. The CO2 flow rate was 30 kg / h, the extraction pressure was 25 MPa, and the extraction temperature was 45℃ for 100 minutes. After extraction, the extract flowed into a collection tank and cooled to room temperature to obtain the extract. The obtained extract was then passed through an AB-8 macroporous resin column for adsorption. After complete adsorption, it was sequentially eluented with 2 volumes of pure water and 1.5 volumes of 50% ethanol aqueous solution, collecting the 50% ethanol aqueous solvent eluent. The eluent was concentrated to one-fifth of its original volume by rotary evaporation under vacuum. The concentrated solution was then dried in a vacuum freeze dryer for 40 hours to form a solid powder with a moisture content of (4-8)%, which yielded the Gastrodia elata enzymatic hydrolysis purified product. This purified product was stored at low temperature (18°C, 45% RH) for later use. The Gastrodia elata enzymatic hydrolysis purified product will be referred to as "Drug B" below.

[0086] The preparation method of the enzymatic hydrolysis purified *Quercus variabilis* includes the following steps: Weigh 100 parts by weight of *Quercus variabilis* with a moisture content ≤10%, treat with liquid nitrogen quick-freezing machine for 15 min until brittle, and then pulverize into 100-mesh powder in an airflow cryogenic pulverizer to obtain *Quercus variabilis* powder. Add pH 5.0 acetate-sodium acetate buffer and 1 w / w% of a compound enzyme preparation (a mixture of cellulase, pectinase, and xylanase powders in a mass ratio of 5:3:2) at a material-to-liquid ratio of 1:10 (w / v). Place the mixture in a constant temperature shaker at 45℃ and 150 rpm for 2 h of enzymatic hydrolysis. After hydrolysis, place it in a 100℃ water bath for 20 min to inactivate the enzyme, and then cool to room temperature. Add 1.5 times the volume of 60% ethanol solution to the cooled hydrolysate, and then extract using an ultrasonic extractor at 50℃ and 200 rpm for 30 min. After extraction, centrifuge at 10000 rpm to collect the supernatant a. Add 2 volumes of ethyl acetate (purity ≥99.5%) to the centrifuged precipitate and extract ultrasonically at 35℃ and 180 rpm for 25 min. After extraction, collect the supernatant b by centrifugation at 10000 rpm. Combine supernatant a and supernatant b, filter through a 0.22 μm filter membrane (to remove interfering substances), and collect the clear extract. Concentrate the obtained extract under vacuum using a rotary evaporator to one-third of its original volume. Then, freeze-dry the concentrated extract in a vacuum freeze dryer for 40 h to form a solid powder with a moisture content of (4-8)%, which is the purified *Panicum esculentum* enzymatic hydrolysis product. Store it under low-temperature drying (18℃, 45% RH) for later use. The purified *Panicum esculentum* enzymatic hydrolysis product will be referred to as "Drug C" below.

[0087] Example 2

[0088] The preparation method of water extract of Wedelia candidae includes the following steps: Weigh 100 parts by weight of Wedelia candidae with stems and a water content ≤8%, cut them into 1-2 cm pieces, and then pulverize them into 100-mesh powder using a low-temperature pulverizer. Add the pulverized raw material to purified water at a ratio of 1:20 (w / v), and extract ultrasonically at 58℃ for 40 min. After extraction, centrifuge at 10000 rpm for 10 min, collect the supernatant, and extract the remaining precipitate twice more using the same ultrasonic extraction method. Combine all extracts. Filter the combined extract through a ceramic membrane filtration system with the following process parameters: membrane pore size 0.1 μm, cross-flow velocity 4 m / s, and operating pressure 0.25 MPa to obtain the filtrate. Concentrate the filtrate under vacuum using a rotary evaporator at a vacuum degree of -0.09 MPa and a temperature of 45℃ until the relative density reaches 1.15 to obtain the concentrated solution. The concentrated solution was adsorbed onto a macroporous resin column. After adsorption, it was sequentially eluented with 2 volumes of pure water and 1.5 volumes of 30% ethanol aqueous solution, and the 30% ethanol aqueous solution was collected. The eluent was concentrated to one-fifth of its original volume by rotary evaporation under vacuum. The concentrated solution was then dried in a vacuum freeze dryer for 40 hours to obtain a solid powder with a moisture content of (4-6)%, which yielded the purified Wedelia candel extract. It was stored at low temperature (18℃, 45% RH) for later use. The purified Wedelia candel aqueous extract will be referred to as "Drug A Reference".

[0089] The preparation method of non-enzymatic hydrolysis purified Gastrodia elata includes the following steps: Weigh 100 parts by weight of Gastrodia elata with a water content ≤12%, cut it into thin slices, and put it into a liquid nitrogen quick-freezing tank for 10 minutes until it is completely brittle. Immediately transfer it to a low-temperature pulverizer and pulverize it into 60-mesh powder. Add pH 4.2 acetate-sodium acetate buffer at a material-liquid ratio of 1:10 (w / v), pre-activate at 40℃ for 20 minutes, and place the above mixture in a constant temperature shaker. Shake at 40℃ and 120 rpm for 60 minutes, and then shake at 50℃ and 180 rpm for 30 minutes to promote deep cell wall disruption. After cell wall disruption, centrifuge at 10,000 rpm for 20 minutes to collect the precipitate, and then freeze-dry it under vacuum until the water content is less than ≤6% to obtain non-enzymatic hydrolysis powder of Gastrodia elata. The obtained non-enzymatic hydrolysate powder of Gastrodia elata was passed through a 60-mesh sieve and extracted using a supercritical CO2 extraction system with 1% ethyl acetate as the entrainer. The CO2 flow rate was 30 kg / h, the extraction pressure was 25 MPa, the extraction temperature was 45℃, and the extraction time was 100 min. After extraction, the extract was collected in a collection tank and cooled to room temperature. The extract was then passed through a macroporous resin column for adsorption. After complete adsorption, it was sequentially eluented with 2 volumes of pure water and 1.5 volumes of 50% ethanol aqueous solution, and the 50% ethanol aqueous solution was collected. The eluent was concentrated to one-fifth of its original volume by rotary evaporation under vacuum. The concentrated solution was then placed in a vacuum freeze dryer and dried for 40 h to form a solid powder with a moisture content of (4-8)%, which was the purified non-enzymatic hydrolysate of Gastrodia elata. It was stored at low temperature (18℃, 45% RH) for later use. The purified non-enzymatic hydrolysate of Gastrodia elata is referred to as "Drug B Control".

[0090] The preparation method of the non-enzymatic hydrolysis purified product of *Mallotus quassinensis* includes the following steps: Weigh 100 parts by weight of *Mallotus quassinensis* with a water content ≤10%, treat with liquid nitrogen quick-freezing machine for 15 min until brittle, and then pulverize into 100-mesh powder in a low-temperature airflow pulverizer to obtain *Mallotus quassinensis* powder. Add pH 5.0 acetate-sodium acetate buffer at a material-to-liquid ratio of 1:10 (w / v), place the mixture in a constant temperature shaker at 45℃ and 150 rpm for 2 h. After shaking, add 1.5 times the volume of 60% ethanol solution to the mixture, and then extract using an ultrasonic extractor at 50℃ and 200 rpm for 30 min. After extraction, collect the supernatant (a) by centrifugation at 10000 rpm. Add 2 times the volume of ethyl acetate (≥99.5%) to the centrifuged precipitate, and extract ultrasonically at 35℃ and 180 rpm for 25 min. After extraction, collect the supernatant (b) by centrifugation at 10000 rpm. Supernatant a and supernatant b were combined and filtered through a 0.22 μm filter membrane, and the clear extract was collected. The obtained extract was concentrated under vacuum using a rotary evaporator to one-third of its original volume. The concentrated extract was then placed in a vacuum freeze dryer and dried for 40 hours to form a solid powder with a moisture content of (4-8)%, which is the non-enzymatic hydrolysis purified product of *Paeonia lactiflora*. It was stored under low-temperature drying (18℃, 45% RH) for later use. The enzymatic hydrolysis purified product of *Paeonia lactiflora* will be referred to as "Drug C Reference Material" below.

[0091] Example 3

[0092] A neomycin sulfate compound synergist, by weight, comprises 20 parts of purified Wedelia chinensis extract, 40 parts of purified Gastrodia elata extract, and 40 parts of purified Sterculia lychnophora extract.

[0093] The purified extracts of Wedelia candel, Gastrodia elata, and Quercus mongolica were prepared in Example 1.

[0094] The preparation method of the above-mentioned neomycin sulfate compound synergist involves weighing the purified extracts of Wedelia chinensis, Gastrodia elata, and Quercus mongolica, and mixing them evenly in a predetermined ratio under a dry and sterile environment (40%–50% RH) to obtain the neomycin sulfate compound synergist.

[0095] Example 4: In vitro MIC determination of neomycin-resistant highly lethal Vibrio spp. using different treatment methods of purified traditional Chinese medicine. 1. Experimental consumables

[0096] Drugs: (1) Neomycin sulfate standard: purchased from China Institute of Veterinary Drug Control; (2) Three kinds of purified Chinese medicines and their references: Drug A (i.e., purified South American Wedelia candel), Drug A reference (i.e., water-extracted purified South American Wedelia candel), Drug B (i.e., enzymatically hydrolyzed purified Gastrodia elata), Drug B reference (i.e., non-enzymatically hydrolyzed purified Gastrodia elata), Drug C (enzymatically hydrolyzed purified Magnolia officinalis), Drug C reference (non-enzymatically hydrolyzed purified Magnolia officinalis), prepared from Examples 1 to 2.

[0097] Drug preparation: (1) Neomycin sulfate: Dissolve in sterile water before the experiment to prepare a stock solution of 10240 μg / mL for later use. (2) Three purified Chinese medicines and their references: Drug A, Drug B, Drug B reference, Drug C, Drug C reference, all were dissolved in DMSO before the experiment to prepare a stock solution of 40960 μg / mL for later use; Drug A reference, was dissolved in sterile water before the experiment to prepare a stock solution of 40960 μg / mL for later use.

[0098] Test strain: Vibrio parahaemolyticus Vp32 (disclosed in Chinese invention patent 2024118285220), a highly lethal Vibrio resistant to neomycin sulfate, was isolated from typical shrimp suffering from glassy seedling disease in a farm by the Aquatic Functional Evaluation and Development Technology Platform of the Animal Husbandry and Aquatic Research Center of Guangdong Haid Group Co., Ltd.

[0099] Culture media: (1) 1.5% sodium chloride TSB broth, 1.5% sodium chloride MH agar and 1.5% sodium chloride MH broth: TSB broth, MH agar and MH broth were all purchased from Qingdao Haibo Biotechnology Co., Ltd. A certain amount of solid sodium chloride was added to each of the three culture media until the final concentration of sodium chloride was 1.5%. (2) HLVBS agar: purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0100] 2. Experimental Methods

[0101] 2.1 Preparation of test strains

[0102] Dissolve Vibrio parahaemolyticus Vp32, stored in glycerol at -80℃, at room temperature. In a clean bench, add 1 mL of the bacterial suspension to sterilized TSB broth tubes containing 1.5% sodium chloride. Add one tube of each bacterium type to each tube and incubate at 28℃ and 200 rpm for 18–24 h to reactivate the strains. Using a sterile inoculating loop, inoculate the reactivated bacterial suspension onto HLVBS agar using the three-zone streak method in a clean bench. Incubate at 28℃ for 18–24 h. Pick typical single colonies and inoculate them into MH broth containing 1.5% sodium chloride. Incubate at 28℃ and 200 rpm for 8–10 h. Adjust the bacterial suspension to an OD of 0.2 (approximately 10⁻⁶). 8 (CFU / mL), dilute the bacterial solution 100 times with MH broth to achieve a bacterial concentration of 10. 6CFU / mL was used as the test bacterial solution.

[0103] 2.2 Determination of MIC for single-drug susceptibility

[0104] The MICs of Vibrio parahaemolyticus Vp32, drug A, drug A control, drug B, drug B control, drug C, drug C control, and neomycin sulfate were determined using the microbroth dilution method specified by CLSI. 100 μL of MH broth was added to each well of a 96-well plate. The drug stock solution was diluted 10-fold to prepare the working solution. 100 μL of the prepared working solution was added to the first well of each row. Then, the drug was diluted two-fold, and 100 μL was added to the second well, followed by thorough mixing with the broth. This process was repeated until the last well. 100 μL of the diluted bacterial suspension was discarded from the 12th column. 100 μL of the diluted bacterial suspension was then added to each well. Three replicates were performed for each drug. Simultaneously, a row of negative controls (MH broth only, no bacterial suspension) and a row of positive controls (bacterial suspension, no drug) were prepared on the same plate. After sample addition, the 96-well plate was incubated at 28°C for 20–24 h, and the results were observed.

[0105] Result interpretation: The lowest drug concentration that completely inhibits bacterial growth is defined as the drug MIC.

[0106] 3. Experimental Results

[0107] Table 1 shows that neomycin sulfate has a MIC of 128 μg / mL against highly lethal Vibrio parahaemolyticus Vp32, classifying it as a drug-resistant strain. Drugs A, B, and C all exhibited some in vitro antibacterial activity against Vibrio parahaemolyticus Vp32, with MICs of 256 μg / mL, 512 μg / mL, and 1024 μg / mL, respectively. Although their antibacterial effects were all weaker than those of neomycin sulfate, they were all stronger than their corresponding control drugs.

[0108] Table 1. MIC test results of the test drugs against Vibrio parahaemolyticus Vp32

[0109] Drug Name MIC (μg / mL) Drug A 256 Drug A control 2048 Drug B 512 Drug B control >2048 Drug C 1024 Drug C control >2048 Neomycin sulfate 128 MH Broth - MH broth + bacterial solution +

[0110] Note: The symbol "-" indicates that the well is clear and there is no bacterial growth; "+" indicates that the well is turbid and there is bacterial growth.

[0111] 4. Experiment Summary

[0112] (1) The purified extract of Wedelia chinensis extracted with DES showed better in vitro antibacterial effect against highly lethal Vibrio parahaemolyticus resistant to neomycin sulfate than its water extract.

[0113] (2) The in vitro antibacterial effect of the Gastrodia elata enzymatic hydrolysate obtained by enzymatic hydrolysis with compound enzyme preparation against highly lethal Vibrio parahaemolyticus resistant to neomycin sulfate is better than that of the Gastrodia elata purified without compound enzyme preparation.

[0114] (3) The in vitro antibacterial effect of the purified quassula hydrolysate obtained by enzymatic hydrolysis with compound enzyme preparation against highly lethal Vibrio parahaemolyticus resistant to neomycin sulfate is better than that of the purified quassula hydrolysate obtained without compound enzyme preparation.

[0115] Example 5: In vitro MIC determination of purified traditional Chinese medicine extracts, alone and in combination with neomycin sulfate, against neomycin sulfate-resistant highly lethal Vibrio spp.

[0116] 1. Experimental Consumables

[0117] Drugs: (1) Neomycin sulfate standard: purchased from China Institute of Veterinary Drug Control; (2) Three purified Chinese medicines: Drug A, Drug B and Drug C were all prepared from Example 1.

[0118] Drug preparation: (1) Neomycin sulfate: Dissolve in sterile water before the experiment to prepare a stock solution of 10240μg / mL for later use. (2) Three purified Chinese medicines: Drug A, Drug B and Drug C, all dissolved in DMSO before the experiment to prepare a stock solution of 40960μg / mL for later use.

[0119] Test strain: Vibrio parahaemolyticus Vp32, a highly lethal Vibrio resistant to neomycin sulfate, was isolated from typical shrimp suffering from glassy seedling disease in a farm by the Aquatic Functional Evaluation and Development Technology Platform of the Animal Husbandry and Aquatic Research Center of Guangdong Haid Group Co., Ltd.

[0120] Culture media: (1) 1.5% sodium chloride TSB broth, 1.5% sodium chloride MH agar and 1.5% sodium chloride MH broth: TSB broth, MH agar and MH broth were all purchased from Qingdao Haibo Biotechnology Co., Ltd. A certain amount of solid sodium chloride was added to each of the three culture media until the final concentration of sodium chloride was 1.5%. (2) TCBS agar: purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0121] 2. Test Methods

[0122] 2.1 Preparation of test strains

[0123] Dissolve Vibrio parahaemolyticus Vp32, stored in glycerol at -80℃, at room temperature. In a clean bench, add 1 mL of the bacterial suspension to sterilized TSB broth tubes containing 1.5% sodium chloride. Add one tube of each bacterium type to each tube and incubate at 28℃ and 200 rpm for 18–24 h to reactivate the strains. Using a sterile inoculating loop, inoculate the reactivated bacterial suspension onto TCBS agar medium using the three-zone streak method in a clean bench. Incubate at 28℃ for 18–24 h. Pick typical single colonies and inoculate them into MH broth containing 1.5% sodium chloride. Incubate at 28℃ and 200 rpm for 8–10 h. Adjust the bacterial suspension to an OD of 0.2 (approximately 10⁻⁶). 8(CFU / mL), dilute the bacterial solution 100 times with MH broth to achieve a bacterial concentration of 10. 6 CFU / mL was used as the test bacterial solution.

[0124] 2.2 Broth dilution checkerboard method combined with drug sensitivity testing

[0125] (1) Combined drug susceptibility results of drugs A, B, and C in combination with neomycin sulfate against neomycin sulfate-resistant, highly lethal Vibrio vp32.

[0126] Group design (Table 2): Using the 96-well micro-broth dilution checkerboard method, two drugs were designed to be combined at final concentrations of 2 times, 1 times, 1 / 2 times, 1 / 4 times, 1 / 8 times, 1 / 32 times, and 1 / 64 times the MIC.

[0127] Table 2. Experimental group design for single-drug combination with neomycin sulfate

[0128]

[0129]

[0130] Drug dilution: Each drug stock solution was diluted 10 times to obtain the concentration for the first test tube. Except for the first test tube, which contained 1.8 mL of MH broth, 1 mL of MH broth was added to each of the other test tubes. 0.2 mL of the antibacterial drug stock solution was added to the first test tube and mixed well. Then 1 mL was transferred to the second test tube, mixed well, and then 1 mL was transferred to the third test tube. This serial dilution was continued until the last test tube.

[0131] Sample loading procedure: 50 μL of neomycin sulfate is loaded into each well in the horizontal column. 50 μL of the purified herbal extract is loaded into each well in the vertical column. Finally, 100 μL of diluted bacterial suspension is added to each well. Three replicates are performed for each drug. Simultaneously, one row of negative controls (filled with blank broth only, without bacterial suspension) and one row of positive controls (filled with bacterial suspension, without drug) are prepared on the same plate. After sample loading, the 96-well plate is incubated at 28°C for 20–24 hours, and the results are observed.

[0132] Record the MICs of the drug alone and in combination with neomycin sulfate, and calculate the corresponding FIC (inhibition index).

[0133] Result Interpretation: There are four types.

[0134] Synergistic effect: The combined activity of the two antibacterial drugs is significantly greater than the sum of the antibacterial effects of the individual drugs (1+1>2).

[0135] b. Cumulative effect: The activity of two antibacterial drugs combined is slightly increased compared to either drug alone (1+1=2).

[0136] c. Irrelevant effect: The activity of the two antibacterial drugs is not affected by the other drug (1+1=1).

[0137] d. Antagonistic effect: The activity of one antibacterial drug is weakened by another drug (1+1<1).

[0138] In the laboratory, the calculation of partial inhibitory concentration is used as the basis for judgment in combined drug susceptibility testing.

[0139]

[0140] Judgment criteria: FIC index < 0.5 is considered synergistic; FIC index 0.5 to 1.0 is considered additive; FIC index 1.0 to 2.0 is considered irrelevant; FIC index > 2.0 is considered antagonistic.

[0141] (2) The combined drug susceptibility MIC of drugs A, B, and C in combination with neomycin sulfate against highly pathogenic Vibrio vp32 resistant to neomycin sulfate.

[0142] Group design (Table 3): The 96-well micro-broth dilution checkerboard method was used. The final concentrations of neomycin sulfate were 2 times, 1 times, 1 / 2 times, 1 / 4 times, 1 / 8 times, 1 / 32 times, and 1 / 64 times the MIC. The final concentrations of drug A, drug B, and drug C were 1 / 2 times and 1 / 4 times the MIC. The MICs of the single drugs combined with neomycin sulfate were also determined.

[0143] Table 3. Experimental group design for single-drug, pairwise, and combination therapies with neomycin sulfate.

[0144]

[0145]

[0146] Drug dilution: Each drug stock solution was diluted 10 times to obtain the concentration for the first test tube. Except for the first test tube, which contained 1.8 mL of MH broth, 1 mL of MH broth was added to each of the remaining test tubes. 0.2 mL of the antibacterial drug stock solution was added to the first test tube and mixed well. Then 1 mL was transferred to the second test tube, mixed well, and then 1 mL was transferred to the third test tube. This serial dilution was continued until the last test tube.

[0147] Sample loading procedure: 50 μL of neomycin sulfate is loaded into each well in the horizontal column. 25 μL of each drug is loaded into each well in the vertical column for the purified traditional Chinese medicine extract. Finally, 100 μL of diluted bacterial suspension is added to each well. Three replicates are performed for each drug. Simultaneously, one row of negative controls (filled with blank broth only, without bacterial suspension) and one row of positive controls (filled with bacterial suspension, without drug) are prepared on the same plate. After sample loading, the 96-well plate is incubated at 28°C for 20–24 hours, and the results are observed.

[0148] Record the minimum inhibitory concentrations (MICs) of neomycin sulfate in combination with other drugs and compare them with the MICs of neomycin sulfate alone.

[0149] (3) Combined drug susceptibility MIC of three purified Chinese herbal medicines in combination with neomycin sulfate against neomycin sulfate-resistant, highly lethal Vibrio vp32

[0150] Group design (Table 4): The 96-well micro-broth dilution checkerboard method was used. The final concentrations of neomycin sulfate were 2 times, 1 time, 1 / 2 times, 1 / 4 times, 1 / 8 times, 1 / 32 times, and 1 / 64 times the MIC. The final concentrations of drug A / drug B / drug C were 1 / 2 times the MIC and 1 / 4 times the MIC. The MIC of the combination of the three purified Chinese medicines with neomycin sulfate was also calculated.

[0151] Table 4. Experimental group design for single-drug, pairwise, and combination therapies with neomycin sulfate.

[0152]

[0153]

[0154] Drug dilution: Each drug stock solution was diluted 10 times to obtain the concentration for the first test tube. Except for the first test tube, which contained 1.8 mL of MH broth, 1 mL of MH broth was added to each of the remaining test tubes. 0.2 mL of the antibacterial drug stock solution was added to the first test tube and mixed well. Then 1 mL was transferred to the second test tube, mixed well, and then 1 mL was transferred to the third test tube. This serial dilution was continued until the last test tube.

[0155] Sample loading procedure: Horizontal column: Neomycin sulfate, 25 μL per well. Vertical column: Purified traditional Chinese medicine extracts, 25 μL per well for each drug. Finally, add 100 μL of diluted bacterial suspension to each well. Perform three replicates for each drug. Simultaneously, on the same plate, prepare one row of negative controls (blank broth only, no bacterial suspension) and one row of positive controls (bacterial suspension only, no drug). After sample loading, incubate the 96-well plate at 28℃ for 20–24 h and observe the results.

[0156] Record the minimum inhibitory concentration (MIC) of the combination of the drug trimethoprim-tri ...sulfate and neomycin sulfate, and compare it with the MIC of neomycin sulfate alone.

[0157] 2.3 MIC determination of compound synergists

[0158] The MICs of Vibrio parahaemolyticus Vp32, drugs A, B, C, and a combined potentiator (drug A:drug B:drug C = 1:2:2, w / w / w) were determined using the microbroth dilution method specified by CLSI (Table 5). 100 μL of MH broth was added to each well of a 96-well plate. The drug stock solution was diluted 10-fold to prepare the working solution. 100 μL of the prepared working solution was added to the first well of each row. Then, the drug was diluted two-fold, and 100 μL was added to the second well, followed by thorough mixing with the broth. This process was repeated until the last well. 100 μL of the diluted bacterial solution was discarded from the 12th column. 100 μL of the diluted bacterial solution was then added to each well. Three replicates were performed for each drug. Simultaneously, a row of negative controls (MH broth only, no bacterial solution) and a row of positive controls (bacterial solution, no drug) were prepared on the same plate. After adding the samples, place the 96-well plate in a 28℃ incubator and incubate for 20–24 hours, then observe the results.

[0159] Result interpretation: The lowest drug concentration that completely inhibits bacterial growth is defined as the drug MIC.

[0160] Table 5. MIC determination of compound synergists

[0161]

[0162] 2.4 Combined drug susceptibility results of the compound synergist combined with neomycin sulfate against neomycin sulfate-resistant, highly lethal Vibrio vp32

[0163] Group design: Using the 96-well micro-broth dilution checkerboard method, neomycin sulfate and a compound synergist (drug A: drug B: drug C = 1:2:2, w / w / w) were designed to be combined at final concentrations of 2, 1, 1 / 2, 1 / 4, 1 / 8, 1 / 32, and 1 / 64 times the MIC.

[0164] Drug dilution: Each drug stock solution was diluted 10 times to obtain the concentration for the first test tube. Except for the first test tube, which contained 1.8 mL of MH broth, 1 mL of MH broth was added to each of the other test tubes. 0.2 mL of the antibacterial drug stock solution was added to the first test tube and mixed well. Then 1 mL was transferred to the second test tube, mixed well, and then 1 mL was transferred to the third test tube. This serial dilution was continued until the last test tube.

[0165] Sample loading procedure: 50 μL of neomycin sulfate is loaded into each well in the horizontal column. 50 μL of the purified herbal extract is loaded into each well in the vertical column. Finally, 100 μL of diluted bacterial suspension is added to each well. Three replicates are performed for each drug. Simultaneously, one row of negative controls (filled with blank broth only, without bacterial suspension) and one row of positive controls (filled with bacterial suspension, without drug) are prepared on the same plate. After sample loading, the 96-well plate is incubated at 28°C for 20–24 hours, and the results are observed.

[0166] Record the MICs of the drug alone and in combination with neomycin sulfate, and calculate the corresponding FIC (inhibition index).

[0167] Result Interpretation: There are four types.

[0168] (1) Synergistic effect: The combined activity of the two antibacterial drugs is significantly greater than the sum of the antibacterial effects of each single drug (1+1>2).

[0169] (2) Cumulative effect: The activity of two antibacterial drugs combined is slightly increased compared to either drug alone (1+1=2).

[0170] (3) Irrelevant effect: The activity of the two antibacterial drugs is not affected by the other drug (1+1=1).

[0171] (4) Antagonistic effect: The activity of one antibacterial drug is weakened by another drug (1+1<1).

[0172] In the laboratory, the calculation of partial inhibitory concentration is used as the basis for judgment in combined drug susceptibility testing.

[0173]

[0174] Judgment criteria: FIC index < 0.5 is considered synergistic; FIC index 0.5 to 1.0 is considered additive; FIC index 1.0 to 2.0 is considered irrelevant; FIC index > 2.0 is considered antagonistic.

[0175] 3. Experimental Results

[0176] (1) Combined drug sensitivity results of three purified Chinese herbal medicines as single drugs in combination with neomycin sulfate

[0177] The MIC of drug A against the highly lethal Vibrio parahaemolyticus Vp32 resistant to neomycin sulfate was 256 μg / mL. When drug A was added at 1 / 2 MIC (128 μg / mL) and 1 / 4 MIC (64 μg / mL), the MIC of neomycin sulfate was 32 μg / mL and 64 μg / mL, respectively, representing a 4-fold and 2-fold decrease compared to neomycin sulfate alone (Table 6). The combined FIC of the two drugs was 0.75 and 0.75, demonstrating an additive effect.

[0178] The MIC of drug B against the highly lethal Vibrio parahaemolyticus Vp32 resistant to neomycin sulfate was 512 μg / mL. When 1 / 2 MIC (256 μg / mL) of drug B was added, the MIC of neomycin sulfate was 64 μg / mL, which was 2 times lower than the MIC of neomycin sulfate alone (Table 7). The FIC of the two drugs combined was 1, showing an additive effect.

[0179] The MIC of drug C against the highly lethal Vibrio parahaemolyticus Vp32 resistant to neomycin sulfate was 1024 μg / mL. When 1 / 2 MIC (512 μg / mL) of drug C was added, the MIC of neomycin sulfate was 64 μg / mL, which was 2 times lower than the MIC of neomycin sulfate alone (Table 8). The FIC of the two drugs combined was 1, showing an additive effect.

[0180] Table 6. MICs of combined drug susceptibility testing for neomycin sulfate and drug A against Vibrio parahaemolyticus Vp32.

[0181]

[0182]

[0183] Table 7. MICs of combined drug susceptibility testing for neomycin sulfate and drug B against Vibrio parahaemolyticus Vp32.

[0184]

[0185] Table 8. MICs of combined drug susceptibility testing for neomycin sulfate and drug C against Vibrio parahaemolyticus Vp32.

[0186]

[0187] Note: In Tables 6 to 8, the symbol "-" indicates that the well is clear and there is no bacterial growth; "+" indicates that the well is turbid and there is bacterial growth.

[0188] (2) The combined drug sensitivity MIC of three purified Chinese herbal medicines in pairs with neomycin sulfate

[0189] As shown in Table 9, the MIC of neomycin sulfate against neomycin sulfate-resistant, highly lethal Vibrio parahaemolyticus Vp32 is 128 μg / mL. When 1 / 2 MIC (128 μg / mL) of drug A and 1 / 2 MIC (256 μg / mL) of drug B were added, and when 1 / 2 MIC (128 μg / mL) of drug A and 1 / 2 MIC (512 μg / mL) of drug C were added, the MIC in both groups combined with neomycin sulfate was 32 μg / mL, a four-fold decrease compared to the MIC when neomycin sulfate was used alone. When 1 / 2 MIC (128 μg / mL) of drug A and 1 / 4 MIC (128 μg / mL) of drug B were added, and when 1 / 2 MIC (128 μg / mL) of drug A and 1 / 4 MIC (256 μg / mL) of drug C were added, and when 1 / 4 MIC (64 μg / mL) of drug A and 1 / 2 MIC (256 μg / mL) of drug B were added, and when 1 / 4 MIC (64 μg / mL) of drug A and 1 / 2 MIC (512 μg / mL) of drug C were added, and When 1 / 2 MIC (256 μg / mL) of drug B and 1 / 2 MIC (512 μg / mL) of drug C were added, and when 1 / 2 MIC (256 μg / mL) of drug B and 1 / 4 MIC (256 μg / mL) of drug C were added, and when 1 / 4 MIC (128 μg / mL) of drug B and 1 / 2 MIC (512 μg / mL) of drug C were added, the MIC of all seven groups combined with neomycin sulfate was 64 μg / mL, which was 2 times lower than the MIC when neomycin sulfate was used alone. When 1 / 4 MIC (64 μg / mL) of drug A and 1 / 4 MIC (128 μg / mL) of drug B were added, and when 1 / 4 MIC (64 μg / mL) of drug A and 1 / 4 MIC (256 μg / mL) of drug C were added, and when 1 / 4 MIC (128 μg / mL) of drug B and 1 / 4 MIC (256 μg / mL) of drug C were added, the MIC of neomycin sulfate in all three groups was 128 μg / mL, the same as the MIC when neomycin sulfate was used alone. In summary, the antibacterial synergistic effect of groups 1-5, 7, and 9-11 when combined with neomycin sulfate was better than that of neomycin sulfate alone, with groups 1 and 3 showing the best MIC when combined with neomycin sulfate.

[0190] Table 9. Combined MICs of neomycin sulfate in combination with two single drugs against Vibrio parahaemolyticus Vp32

[0191]

[0192] (3) MIC of combined drug sensitivity of three purified traditional Chinese medicines with neomycin sulfate

[0193] As shown in Table 10, the combination of drugs A, B, and C at their respective sub-MIC concentrations with neomycin sulfate can produce a synergistic antibacterial effect. Among them, the combination of 1 / 2 MIC (128 μg / mL) drug A, 1 / 2 MIC (256 μg / mL) drug B, and 1 / 2 MIC (512 μg / mL) drug C, and the combination of 1 / 2 MIC (128 μg / mL) drug A, 1 / 2 MIC (256 μg / mL) drug B, and 1 / 4 MIC (256 μg / mL) drug C with neomycin sulfate has the lowest MIC, both at 8 μg / mL, which is 16 times lower than the MIC when neomycin sulfate is used alone. The antibacterial effect is better than the effect of the combination of each drug. Based on the selection criteria of "good antibacterial and sensitizing effect with low drug concentration", the concentration of "1 / 2MIC (128μg / mL) drug A + 1 / 4MIC (256μg / mL) drug B + 1 / 4MIC (256μg / mL) drug C" was selected, that is, the mass ratio of drug A: drug B: drug C = 1:2:2 as the combination compound synergist ratio.

[0194] Table 10. MICs of combined drug susceptibility testing for Vibrio parahaemolyticus Vp32 against neomycin sulfate and three single-agent drugs.

[0195]

[0196]

[0197] (4) MIC determination of compound synergists

[0198] As shown in Table 11, the MIC of the compound synergist against the highly lethal Vibrio parahaemolyticus Vp32 resistant to neomycin sulfate was 128 μg / mL, which was 2 times, 4 times and 8 times lower than the MICs of drugs A, B and C, respectively.

[0199] Table 11 MIC test results of the compound synergist against Vibrio parahaemolyticus Vp32

[0200] Drug Name MIC (μg / mL) Drug A 256 Drug B 512 Drug C 1024 Compound synergist 128 MH Broth - MH broth + bacterial solution +

[0201] (5) Drug sensitivity results of the combination synergist and neomycin sulfate

[0202] Table 12 shows that the MIC of neomycin sulfate against neomycin sulfate-resistant, highly lethal Vibrio parahaemolyticus Vp32 is 128 μg / mL. When a synergist with 1 / 4 MIC (32 μg / mL) and 1 / 8 MIC (16 μg / mL) concentrations was added, the MICs of neomycin sulfate were 8 μg / mL and 16 μg / mL, respectively, representing a 16-fold and 8-fold decrease compared to neomycin sulfate alone. The FICs of the combined use were 0.375 and 0.25, both demonstrating a synergistic effect. Considering reducing antibiotic usage and lowering Vibrio parahaemolyticus resistance, a 1 / 8 MIC concentration of the synergist was chosen as the optimal concentration for combined use.

[0203] Table 12 MICs of combined neomycin sulfate and its synergistic agent against Vibrio parahaemolyticus Vp32

[0204]

[0205] 4. Experiment Summary

[0206] (1) The combination of the purified extracts of Wedelia cannabidiol, Gastrodia elata, and Quercus mongolica with neomycin sulfate can reduce the MIC of neomycin sulfate against strain Vp32, but they only have an additive effect and no synergistic effect.

[0207] (2) When the purified extracts of Wedelia candel, Gastrodia elata, and Quercus mongolica were combined with neomycin sulfate in a certain proportion and applied to strain Vp32, they produced a stronger antibacterial and sensitizing effect than when the three Chinese herbal medicines were combined with neomycin sulfate alone or any two of the three purified extracts were combined with neomycin sulfate.

[0208] (3) The purified extracts of Wedelia candel, Gastrodia elata, and Psoralea corylifolia were combined in a mass ratio of 1:2:2 and used in combination with neomycin sulfate to treat strain Vp32, achieving the effect of the least amount of compound synergist and the lowest MIC of neomycin sulfate.

[0209] (4) The purified extracts of Wedelia candel, Gastrodia elata, and Psoralea corylifolia in a mass ratio of 1:2:2, when combined with neomycin sulfate, have antibacterial and sensitizing effects, with an FIC of 0.25.

[0210] Example 6: In vitro antibacterial synergistic effect of neomycin sulfate combined with neomycin sulfate against clinical isolates of highly lethal Vibrio resistant to neomycin sulfate.

[0211] 1. Experimental Consumables

[0212] Drugs: (1) Neomycin sulfate standard: purchased from China Institute of Veterinary Drug Control; (2) Neomycin sulfate compound synergist (hereinafter referred to as compound synergist): from Example 3.

[0213] Drug preparation: (1) Neomycin sulfate: Dissolve in sterile water before the experiment to prepare a stock solution of 10240μg / mL for later use. (2) Compound synergist: Dissolve in DMSO before the experiment to prepare a stock solution of 10240μg / mL for later use.

[0214] Test strains: (1) 65 highly lethal Vibrio strains resistant to neomycin sulfate and carrying the Tc toxin gene were identified by physiological and biochemical methods and 16S RNA sequencing. These strains included: 53 Vibrio parahaemolyticus strains, numbered Vp1-53; 8 Vibrio harveyi strains, numbered Vh1-8; 3 Vibrio alginolyticus strains, numbered Va1-3; and 1 Vibrio erwinii strain, numbered Vo1. All 65 Vibrio strains were isolated from shrimp with typical vitreous disease in aquaculture farms by the Aquatic Function Evaluation and Development Technology Platform of the Animal Husbandry and Aquatic Research Center of Guangdong Haid Group Co., Ltd. (2) 2 Vibrio strains sensitive to neomycin sulfate and not carrying the Tc toxin gene were identified by physiological and biochemical methods and 16S RNA sequencing. These strains included: Vibrio parahaemolyticus Vp54 and Vibrio harveyi Vh9. The two Vibrio strains were isolated from shrimp with vibrio disease in aquaculture farms by the Aquatic Function Evaluation and Development Technology Platform of the Animal Husbandry and Aquatic Research Center of Guangdong Haid Group Co., Ltd.

[0215] Culture media: (1) 1.5% sodium chloride TSB broth, 1.5% sodium chloride MH agar and 1.5% sodium chloride MH broth: TSB broth, MH agar and MH broth were all purchased from Qingdao Haibo Biotechnology Co., Ltd. A certain amount of solid sodium chloride was added to each of the three culture media until the final concentration of sodium chloride was 1.5%. (2) HLVBS agar: purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0216] 2. Test Methods

[0217] 2.1 Preparation of test strains

[0218] The test strains preserved at -80℃ glycerol were dissolved at room temperature. The preparation method for each strain was as follows: In a clean bench, 1 mL of bacterial suspension was added to a sterile tube containing 1.5% sodium chloride TSB broth (5 mL / tube). One tube was added for each type of bacteria. The tubes were incubated at 28℃ and 200 rpm for 18–24 h to reactivate the strains. Using a sterile inoculating loop, the reactivated bacterial suspension was inoculated onto HLVBS agar using the three-zone streak method in a clean bench. The tubes were incubated at 28℃ inverted for 18–24 h. Typical single colonies were picked and inoculated into 1.5% sodium chloride MH broth. The tubes were incubated at 28℃ and 200 rpm for 8–10 h. The bacterial suspension was then adjusted to an OD of 0.2 (approximately 10⁻⁶). 8 (CFU / mL), dilute the bacterial solution 100 times with MH broth to achieve a bacterial concentration of 10. 6 CFU / mL was used as the test bacterial solution.

[0219] 2.2 Broth dilution checkerboard method combined with drug sensitivity testing

[0220] (1) To determine the antibacterial effect of the compound synergist combined with neomycin sulfate on highly lethal Vibrio resistant to neomycin sulfate, the checkerboard method was used to determine the antibacterial effect of 65 clinically highly lethal Vibrio strains. The bacterial suspension concentration was prepared to be 1.0 × 10⁻⁶. 6 CFU / mL, the stock solutions of neomycin sulfate and the combined synergist were serially diluted to the corresponding concentrations using MH broth. In a 96-well plate, neomycin sulfate was added in rows, 50 μL per well. The combined synergist was added in columns, 50 μL per well. Finally, 100 μL of diluted bacterial suspension was added to each well, resulting in a final neomycin sulfate concentration of 256 μg / mL to 2 μg / mL, and a combined synergist concentration of 32 μg / mL and 16 μg / mL, respectively. Each drug was tested in triplicate. Simultaneously, a row of negative controls (MH broth added only, no bacterial suspension) and a row of positive controls (bacterial suspension added, no drug) were prepared on the same plate. After sample addition, the 96-well plate was incubated at 28°C for 20–24 h, and the results were observed. The MICs of the combined synergist alone and in combination with neomycin sulfate were recorded, and the corresponding FICs were calculated.

[0221] (2) To determine the antibacterial effect of the compound synergist combined with neomycin sulfate on Vibrio strains sensitive to neomycin sulfate and not carrying the Tc toxin gene, the checkerboard method was used to determine the antibacterial effect on Vibrio strains (Vp54 and Vh9) at a concentration of 1.0 × 10⁻⁶. 6 The stock solutions of neomycin sulfate and the combined synergist were serially diluted with MH broth to the corresponding concentrations (CFU / mL). In a 96-well plate, neomycin sulfate was added in rows of 50 μL per well, and the combined synergist in columns of 50 μL per well. Finally, 100 μL of the diluted bacterial culture was added to each well, resulting in a final neomycin sulfate concentration of 16 μg / mL–0.25 μg / mL and a combined synergist concentration of 32 μg / mL and 16 μg / mL, respectively. Each drug was tested in triplicate. Simultaneously, a row of negative controls (MH broth added only, no bacterial culture) and a row of positive controls (bacterial culture added, no drug) were prepared on the same plate. After sample addition, the 96-well plate was incubated at 28°C for 20–24 hours, and the results were observed. The MICs of the combined synergist alone and in combination with neomycin sulfate were recorded, and the corresponding FICs were calculated.

[0222] Result interpretation: There are four types.

[0223] (1) Synergistic effect: The combined activity of the two antibacterial drugs is significantly greater than the sum of the antibacterial effects of each single drug (1+1>2).

[0224] (2) Cumulative effect: The activity of two antibacterial drugs combined is slightly increased compared to either drug alone (1+1=2).

[0225] (3) Irrelevant effect: The activity of the two antibacterial drugs is not affected by the other drug (1+1=1).

[0226] (4) Antagonistic effect: The activity of one antibacterial drug is weakened by another drug (1+1<1).

[0227] In the laboratory, the calculation of partial inhibitory concentration is used as the basis for judgment in combined drug susceptibility testing.

[0228]

[0229] Judgment criteria: FIC index < 0.5 is considered synergistic; FIC index 0.5 to 1.0 is considered additive; FIC index 1.0 to 2.0 is considered irrelevant; FIC index > 2.0 is considered antagonistic.

[0230] 3. Experimental Results

[0231] For strains resistant to neomycin sulfate and carrying the Tc toxin gene, as shown in Table 13, when the total concentration of the compound synergist was 32 μg / mL, the proportion of strains exhibiting synergistic effects when combined with neomycin was 72.3%. When the total concentration of the compound synergist was 16 μg / mL, the proportion of strains exhibiting synergistic effects when combined with neomycin was 75.4%. The experimental results indicate that when the total concentration of the compound synergist was 16 μg / mL and 32 μg / mL, combined with neomycin sulfate, it produced a significant and broad-spectrum antibacterial sensitization effect against clinically isolated highly lethal Vibrio bacteria resistant to neomycin sulfate and carrying the Tc toxin gene.

[0232] For strains that do not carry the Tc toxin gene and are sensitive to neomycin sulfate, as shown in Table 14, when the total concentration of the compound synergist was 32 μg / mL, the proportion of strains showing synergistic effects when combined with neomycin sulfate was 0%, while the proportion of strains showing additive effects was 100%. When the total concentration of the compound synergist was 16 μg / mL, the proportion of strains showing synergistic effects when combined with neomycin sulfate was 50%, and the proportion of strains showing additive effects was also 50%. The experimental results indicate that when the total concentration of the compound synergist was 16 μg / mL and 32 μg / mL, the combination with neomycin sulfate could also produce an additive effect on clinically isolated Vibrio strains that do not carry the Tc toxin gene and are sensitive to neomycin sulfate.

[0233] Table 13 Effects of the combined potentiator and neomycin sulfate on the antibacterial activity of 65 clinically isolated Vibrio cylindrica strains carrying the Tc toxin gene and resistant to neomycin sulfate.

[0234]

[0235]

[0236] Table 14. Effects of the combined synergist and neomycin sulfate on the antibacterial activity of two clinically isolated Vibrio filaments that do not carry the Tc toxin gene and are sensitive to neomycin sulfate.

[0237]

[0238] 4. Experiment Summary

[0239] The purified extracts of Wedelia candel, Gastrodia elata, and Quercus mongolica were scientifically formulated into a compound (i.e., the neomycin sulfate compound synergist of Example 3) in a ratio of 1:2:2 (w / w). When combined with neomycin sulfate, the compound synergist showed a significant and broad-spectrum antibacterial synergistic effect on 65 clinically isolated highly lethal Vibrio strains carrying the Tc toxin gene and resistant to neomycin sulfate. It also showed an additive antibacterial effect on 2 clinically isolated Vibrio strains that did not carry the Tc toxin gene and were sensitive to neomycin sulfate.

[0240] Example 7: In vivo prophylactic effect of compound synergist combined with neomycin sulfate against neomycin sulfate-resistant highly lethal Vibrio infections.

[0241] 1. Experimental animals and materials

[0242] Test strain: Vibrio parahaemolyticus Vp32, a strain carrying the Tc toxin gene and resistant to neomycin sulfate, was isolated from typical shrimp suffering from glassy seedling disease in a farm by the Aquatic Functional Evaluation and Development Technology Platform of the Animal Husbandry and Aquatic Research Center of Guangdong Haid Group Co., Ltd.

[0243] Test drugs: (1) Neomycin sulfate standard, purchased from China Institute of Veterinary Drug Control; (2) Neomycin sulfate compound synergist (hereinafter referred to as compound synergist): from Example 3.

[0244] Experimental feed: Beikesu (factory-specific) No. 2 microparticle compound feed for shrimp larvae, produced and sold by Qingyuan Haibei Co., Ltd.

[0245] Preparation of experimental medicated feed: (1) Design and dosage of medicated feed groups: The dosage of neomycin sulfate was 0.3g per kilogram of feed. The dosage of compound synergist was set as 0%, 20% replacement (0.24g neomycin sulfate + 0.6g compound synergist per kilogram of feed), 40% replacement (0.18g neomycin sulfate + 0.12g compound synergist per kilogram of feed) and 60% replacement (0.12g neomycin sulfate + 0.18g compound synergist per kilogram of feed). At the same time, the control group containing only the compound synergist dosage (0.3g compound synergist per kilogram of feed) was used as the drug control group, and the blank control group containing no neomycin sulfate and compound synergist was used as the shell fast feed. (2) Preparation of medicated feed: After the drug was mixed evenly with the raw materials of Beikesu feed in proportion (the drug replaced part of the soybean meal in the Beikesu formula), it was granulated according to the Beikesu production process of Qingyuan Haibei Co., Ltd. Beikesu production plant.

[0246] Experimental Litopenaeus vannamei shrimp larvae: purchased from Zhongshan Branch of Guangdong Haixingnong Group Co., Ltd., with a wet weight of 40-60 mg.

[0247] 2. Experimental plan:

[0248] Litopenaeus vannamei larvae were randomly divided into 9 groups of 40 shrimp each: control group (CK, no challenge, fed with shellfish feed), positive challenge group (challenge treatment, fed with shellfish feed), drug group 1 (no challenge treatment, fed with feed containing only neomycin sulfate), drug group 2 (no challenge treatment, fed with feed containing only compound synergist), control group 1 (challenge treatment, fed with feed containing only neomycin sulfate), control group 2 (challenge treatment, fed with feed containing only compound synergist), control group 3 (challenge treatment, fed with feed containing 20% ​​compound synergist), control group 4 (challenge treatment, fed with feed containing 40% compound synergist), and control group 5 (challenge treatment, fed with feed containing 60% compound synergist). Each group had 3 replicates. Each group was fed its corresponding feed daily for 3 consecutive days, with 3 meals per day. The daily feed amount was 5% of the shrimp's body weight. One hour after feeding each day, the water was cleaned and changed, with 25% of the water replaced each time. During the experiment, the water salinity was 20‰, the temperature was 28℃, the pH was 7.5–7.8, and oxygenation was provided for 24 hours. Three hours after the end of the drug administration, except for the blank control group, drug group 1, and drug group 2, the remaining four groups were added with challenge solution Vp32 at a final concentration of (1–2) × 10⁻⁶. 5 CFU / mL, soaked for 6 hours for virus challenge. After the challenge, each group was transferred to a clean culture system (with the same parameters as above) for 24 hours of observation, and the mortality rate of shrimp larvae in each group was recorded.

[0249]

[0250] 3. Experimental Results

[0251] As shown in Table 15 and Figure 1 As shown in the results, the analysis revealed that during the observation period after challenge, the positive control group had a 100% mortality rate and a survival rate of 0%, significantly lower than the groups treated with neomycin sulfate alone, the group treated with the combined synergist alone, and the group treated with neomycin sulfate and the combined synergist. Compared with the 50% survival rate of the neomycin sulfate alone group, the survival rates of the three dosage groups treated with neomycin sulfate in combination with the combined synergist were significantly higher by 35%, 32.5%, and 12.5%, respectively.

[0252] Therefore, it can be concluded that the compound synergist has a better protective effect against Vibrio hygroscopicis in glass seedlings than neomycin sulfate alone, replacing 20% ​​to 60% of neomycin sulfate.

[0253] Table 15. Effects of different drug groups on the prevention of Vp32 challenge.

[0254]

[0255]

[0256] 4. Experiment Summary

[0257] The combination of purified extracts of Wedelia candel, Gastrodia elata, and Quercus mongolica in a 1:2:2 ratio (i.e., the neomycin sulfate compound synergist of Example 3) has good safety when taken orally. Moreover, when it replaces neomycin sulfate at a ratio of 20% to 60%, the protective effect against Vibrio hygroscopicis pathogen challenge is better than that of neomycin sulfate alone. The optimal replacement ratio is 20% to 40%.

[0258] Example 8: In vivo therapeutic effect of compound synergist combined with neomycin sulfate against neomycin sulfate-resistant highly lethal Vibrio infections.

[0259] 1. Experimental animals and materials

[0260] Test strain: Vibrio parahaemolyticus Vp32, a strain carrying the Tc toxin gene and resistant to neomycin sulfate, was isolated from typical shrimp suffering from glassy seedling disease in a farm by the Aquatic Functional Evaluation and Development Technology Platform of the Animal Husbandry and Aquatic Research Center of Guangdong Haid Group Co., Ltd.

[0261] Test drugs: (1) Neomycin sulfate standard, purchased from China Institute of Veterinary Drug Control; (2) Neomycin sulfate compound synergist (hereinafter referred to as compound synergist): from Example 3.

[0262] Experimental feed: Beikesu (factory-specific) No. 2 microparticle compound feed for shrimp larvae, produced and sold by Qingyuan Haibei Co., Ltd.

[0263] Medicated feed: (1) Design and dosage of medicated feed groups: The dosage of neomycin sulfate was 0.3g per kilogram of feed. The dosage of compound synergist was set as 0%, 20% replacement (0.24g neomycin sulfate + 0.6g compound synergist per kilogram of feed), 40% replacement (0.18g neomycin sulfate + 0.12g compound synergist per kilogram of feed) and 60% replacement (0.12g neomycin sulfate + 0.18g compound synergist per kilogram of feed). At the same time, the control group containing only the compound synergist dosage (0.3g compound synergist per kilogram of feed) was used as the drug control group, and the shell fast feed containing no neomycin sulfate and compound synergist was used as the blank control. (2) Preparation of medicated feed: After the drug was mixed evenly with the raw materials of Beikesu feed in proportion (the drug replaced part of the soybean meal in the Beikesu formula), it was produced and granulated according to the Beikesu formula and process of Qingyuan Haibei Co., Ltd.

[0264] Experimental Litopenaeus vannamei shrimp larvae: purchased from Zhongshan Branch of Guangdong Haixingnong Group Co., Ltd., with a wet weight of 40-60 mg.

[0265] 2. Test Plan

[0266] 1000 shrimp were randomly selected, with a weight of (50±5) mg / shrimp, and placed in a 98cm×60cm×34cm white plastic tank containing 25L of artificial seawater with a salinity of 20‰, and temporarily held for 0.5 days. Fresh Vibrio parahaemolyticus Vp32 bacterial suspension was added to the tank until the final bacterial concentration reached (3~5)×10⁻⁶. 4 After soaking the shrimp larvae in cfu / mL solution for 1.5 hours to induce viral infection, the larvae were removed and placed in sterile artificial seawater with a salinity of 20‰ for 10 seconds to remove the bacterial solution adhering to the surface of the shrimp. After being challenged with the virus, shrimp larvae were randomly divided into 7 groups, with 40 shrimp in each group: a positive challenge group (challenged and fed with shellfish feed), control group 1 (challenged and fed with feed containing only neomycin sulfate), control group 2 (challenged and fed with feed containing only a compound synergist), control group 3 (challenged and fed with feed containing 20% ​​compound synergist), control group 4 (challenged and fed with feed containing 40% compound synergist), and control group 5 (challenged and fed with feed containing 60% compound synergist). A blank control (CK) (no challenge, fed with shellfish feed), drug group 1 (no challenge, fed with feed containing only neomycin sulfate), and drug group 2 (no challenge, fed with feed containing only a compound synergist) were also included, with 3 replicates per group. Each group was fed its corresponding feed 3 times a day, with the daily feed amount being 5% of the shrimp's body weight. During the experiment, the water salinity was 20‰, the temperature was 28℃, the pH was 7.5-7.8, and oxygenation was provided for 24 hours. Observations were conducted for 92 hours, and the mortality rate of shrimp larvae and changes in the appearance of the hepatopancreas were recorded for each group.

[0267]

[0268] 3. Experimental Results

[0269] As shown in Table 16 and Figure 2 As shown in the results, the survival rate of the positive control group during the post-challenge observation period was 5.0%, significantly lower than that of the neomycin sulfate monotherapy group, the combined synergist monotherapy group, and the neomycin sulfate combined synergist group. Compared with the 37.5% survival rate of the neomycin sulfate monotherapy group, the survival rates of the three dosage groups using neomycin sulfate in combination with the combined synergist were significantly higher by 25%, 35%, and 22.5%, respectively.

[0270] Based on the analysis of the hepatopancreatic health status of surviving shrimp in each group in Table 17, the hepatopancreas of surviving shrimp in the positive control group all showed vitreous bleaching, indicating they were near death. Figure 3 In the three groups (combined with the compound synergist and neomycin sulfate), >70% of the surviving shrimp had clear and plump hepatopancreas outlines, which is considered normal. Figure 4 The proportion of normal shrimp was much higher than that of the group treated with neomycin sulfate alone or the group treated with compound synergists, and significantly higher than that of the positive control group.

[0271] Therefore, it can be concluded that the combination of compound synergists with neomycin sulfate has a better therapeutic effect on Vibrio hygroscopicis than neomycin sulfate alone.

[0272] Table 16. Effects of different drug groups on Vp32 challenge therapy

[0273]

[0274] Table 17 Hepatopancreatic health status of surviving shrimp in different groups 92 hours after viral challenge.

[0275]

[0276]

[0277] 4. Experiment Summary

[0278] The combination of purified *Wedelia triloba*, enzymatically hydrolyzed *Gastrodia elata*, and enzymatically hydrolyzed *Quercus serrata* in a 1:2:2 ratio (the neomycin sulfate compound synergist of Example 3) has good safety when taken orally. Moreover, when it replaces neomycin sulfate at a ratio of 20% to 60%, the therapeutic effect on vitreous disease is better than that of neomycin sulfate alone or the compound synergist alone, with the optimal replacement ratio being 40%.

[0279] Example 9: Clinical application effect of compound synergist combined with neomycin sulfate

[0280] 1. Experimental Animals and Methods

[0281] A large-scale factory-style shrimp farm in Weifang, Shandong Province, recently experienced a decrease in feeding time (from 35 minutes to 50 minutes) in 14 ponds with a shrimp size of 80-100 per pond, with a daily mortality rate of 20-30 shrimp per pond. This phenomenon had only been present for one day. Observation of weak shrimp swimming near the ponds revealed leukoplakia symptoms in their hepatopancreas, suggesting vitreous leukoplakia disease. Hepatopancreas tissue samples (3 shrimp per pond, mixed sample) were taken from these 14 ponds and subjected to fluorescent PCR testing. The results showed that all the mixed shrimp samples from these 14 ponds were positive for HLVA and HLVB virulence genes, confirming a diagnosis of vitreous leukoplakia disease, requiring urgent drug intervention and treatment.

[0282] Experimental feed: High-grade factory-produced D-series No. 1 particle size shrimp feed, product number D6210 (composition content: crude protein ≥44.0%, crude fat ≥6.0%, crude fiber ≥5.0%, crude ash ≤15.0%, total phosphorus ≥1.0%, lysine ≥2.0%, moisture 12.0%), produced by Nantong Haida Biotechnology Co., Ltd.

[0283] Test drugs: (1) Neomycin sulfate standard, purchased from the China Institute of Veterinary Drug Control; (2) Neomycin sulfate compound synergist (hereinafter referred to as compound synergist): from Example 3. (3) Compound synergist combined with neomycin sulfate: compound synergist: neomycin sulfate = 2:3 (w:w).

[0284] Experimental groups: Nine pools were selected as validation pools, with three pools using neomycin sulfate (numbered A to C), three pools using a compound synergist (numbered D to F), and three pools using a compound synergist in combination with neomycin sulfate (numbered G to I).

[0285] Dosage and administration: Add 1g of neomycin sulfate or a compound synergist, or a combination of compound synergist and neomycin sulfate, per kilogram of shrimp feed. Administer for 5 consecutive days, 4 meals per day. Mix the medication with the feed. Add 0.15kg of tap water to each kilogram of feed, mix thoroughly, then mix with the feed. After mixing, allow to air dry in a cool, shaded place for 30 minutes before use.

[0286] Result judgment: If the mortality rate drops to ≤10 fish / pool during the medication period, it indicates that the drug is effective.

[0287] 2. Experimental Results

[0288] Table 18 shows the results: In the three application pools using neomycin sulfate, the mortality rate after 5 days of treatment ranged from 16 to 27 fish / pool, none of which reduced to ≤10 fish / pool, resulting in an effectiveness rate of 0%. In the three application pools using the compound synergist, the mortality rate after 5 days of treatment ranged from 9 to 15 fish / pool, with one pool reducing to ≤10 fish / pool, resulting in an effectiveness rate of 33%. In the three application pools using the compound synergist in combination with neomycin sulfate, the mortality rate after 5 days of treatment ranged from 6 to 8 fish / pool, all of which reduced to ≤10 fish / pool, resulting in an effectiveness rate of 100%.

[0289] Table 18 Clinical Application Effects of Compound Synergists

[0290]

[0291]

[0292] 3. Experiment Summary

[0293] Timely use of compound synergists in combination with neomycin sulfate can effectively solve the problem of glass seedling disease in aquaculture, and the treatment effect is better than using neomycin sulfate alone or compound synergists alone.

[0294] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. Use of a Wedelia prostrata extract, a Gastrodia elata extract and a Picrasma quassioides extract in the preparation of a neomycin sulfate combination synergist for inhibiting the growth of Vibrio: The mass ratio of the Wedelia prostrata extract, the Gastrodia elata extract and the Picrasma quassioides extract is 1:2:(2-4); The Wedelia prostrata extract is prepared by a preparation method comprising the following steps: mixing Wedelia prostrata with a deep eutectic solvent, and extracting to obtain the Wedelia prostrata extract; The Gastrodia elata extract is prepared by a preparation method comprising the following steps: mixing Gastrodia elata with a complex enzyme preparation and a buffer, and enzymatically hydrolyzing, and then inactivating the enzyme, and then separating and collecting the precipitate; and then performing supercritical CO2 extraction on the precipitate to obtain the Gastrodia elata extract; The Picrasma quassioides extract is prepared by a preparation method comprising the following steps: mixing Picrasma quassioides with a buffer and a complex enzyme preparation, and enzymatically hydrolyzing, and then inactivating the enzyme to obtain an enzymatic hydrolysate; mixing the enzymatic hydrolysate with ethanol, and extracting, and then performing solid-liquid separation to obtain supernatant A and precipitate; mixing the precipitate with ethyl acetate, and extracting, and then performing solid-liquid separation to obtain supernatant B; and then combining the supernatant A and the supernatant B to obtain the Picrasma quassioides extract; The deep eutectic solvent is composed of choline chloride, D-malic acid and ultrapure water in a molar ratio of 1:2:5, The complex enzyme preparation in the Gastrodia elata extract is composed of cellulase, pectinase and β-glucanase in a mass ratio of 3:2:1; The complex enzyme preparation in the Picrasma quassioides extract is composed of cellulase, pectinase and xylanase in a mass ratio of 5:3:2; The Vibrio is Vibrio parahaemolyticus carrying a Tc toxin gene-encoding gene and resistant to neomycin sulfate.

2. Use according to claim 1, characterized in that, The mass-volume ratio of the Wedelia prostrata to the deep eutectic solvent is 1:(15-25); and the Wedelia prostrata comprises Wedelia prostrata from South America.

3. Use according to claim 1, characterized in that, The mass of the complex enzyme preparation in the preparation process of the Gastrodia elata extract is 0.2%-0.8% of the mass of the Gastrodia elata; the Gastrodia elata comprises Gastrodia elata var. wilsonii; and the supercritical CO2 extraction is performed under the following conditions: ethyl acetate is used as a entrainer, the CO2 flow rate is 20-40 kg / h, the extraction pressure is 20-35 MPa, the extraction temperature is 40-50 ℃, and the extraction time is 80-110 min.

4. Use according to claim 1, characterized in that, The mass of the complex enzyme preparation in the preparation process of the Picrasma quassioides extract is 0.5%-2% of the mass of the Picrasma quassioides; the enzymatic hydrolysis is performed at 40-50 ℃ and 100-200 rpm for 1-3 h; the mass-volume ratio of the Picrasma quassioides to the buffer is 1:(8-15); and the extraction is ultrasonic extraction.

5. A neomycin sulfate combination synergist for inhibiting the growth of Vibrio, comprising the Wedelia prostrata extract, the Gastrodia elata extract and the Picrasma quassioides extract in any one of claims 1-4; The mass ratio of the Wedelia prostrata extract, the Gastrodia elata extract and the Picrasma quassioides extract is 1:2:(2-4).

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