Composite synergist of neomycin sulfate as well as preparation method and application of composite synergist

Abstract

The invention belongs to the technical field of aquaculture, and discloses a neomycin sulfate composite synergist as well as a preparation method and application thereof. The invention discloses application of wedelia chinensis extract, gastrodia elata extract and picrasma quassioides extract in inhibiting vibrio growth in vitro and preventing and treating penaeus vannamei glass seedling disease caused by vibrio infection for the first time. Experimental verification shows that when the wedelia chinensis extract, the gastrodia elata extract and the picrasma quassioides extract are independently used, combined pairwise or used at the same time, the good effect of inhibiting vibrio growth in vitro is achieved. Meanwhile, aiming at the treatment of the problem of vibrio infection causing glass seedlings, the extract is combined with neomycin sulfate for use, so that the objective problem of weak or low efficiency of neomycin sulfate according to recommended dosage is solved, the effect of the neomycin sulfate is enhanced after the extract is combined with the neomycin sulfate, and the use amount of the neomycin sulfate is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture, and particularly relates to a composite synergist of neomycin sulfate and a preparation method and application thereof. Background Art

[0002] Penaeus vannamei glass seed disease (TPD) is a highly contagious disease of shrimp that causes rapid onset, high mortality, and is highly contagious. The pathogen is a highly lethal Vibrio species. Highly lethal Vibrio species do not refer to a specific species of Vibrio, but rather to a group of Vibrio species that secrete the highly virulent Tc toxin. This Tc toxin is encoded by three virulence genes (TcA, TcB, and TcC) located on two virulence plasmids, pHLB and pHLC. These plasmids are commonly identified using the HLVA and HLVB loci.

[0003] From May to September 2023, the inventor's team collected 72 samples of dying shrimp with suspected "glass seedling" symptoms from the main breeding areas of whiteleg shrimp such as Zhuhai, Fujian, Jiangsu and Shandong. From these diseased shrimps, 65 strains of highly lethal Vibrio carrying the Tc toxin encoding gene and 14 strains of Vibrio not carrying the Tc toxin encoding gene were isolated. The 79 strains of Vibrio were subjected to sensitivity tests against four compliant antibiotics for use in aquaculture, including enrofloxacin, florfenicol, doxycycline hydrochloride powder and neomycin sulfate. The results showed that the two types of Vibrio collected were relatively sensitive to enrofloxacin and doxycycline, with sensitivity rates of 80.0% and 78.6% and 61.5% and 64.3% respectively; they were highly resistant to florfenicol, with resistance rates of 95.3% and 100%; but there were significant differences in resistance to neomycin sulfate. The resistance rate of Vibrio carrying the gene encoding Tc toxin was 100%, while the resistance rate of those not carrying the gene encoding Tc toxin was only 21.4%. Neomycin sulfate is a legally permitted drug for the control of Vibrio species in whiteleg shrimp aquaculture. However, given that currently collected Vibrio species exhibit high resistance to highly lethal Vibrio species carrying genes encoding Tc toxins, its continued use in the control of these species presents several challenges. First, maintaining the recommended dosage fails to achieve the desired reduction in mortality from these pathogens. This not only fails to produce the desired therapeutic effect, delays optimal treatment, and increases losses, leading to economic losses in aquaculture. Second, while increasing the dosage several times the recommended dosage can somewhat reduce losses, high doses can easily lead to excessive drug residues in the shrimp and water environment, preventing the shrimp from entering the market. Of particular note, based on the pathological examination and prognosis of actual cases, highly lethal Vibrio species are extremely virulent, often causing damage to organs such as the hepatopancreas and intestines. Using neomycin sulfate alone to treat diseases caused by these species is therefore extremely ineffective. Therefore, an effective solution is urgently needed to combat highly lethal Vibrio species resistant to neomycin sulfate. Summary of the Invention

[0004] In response to one of the problems existing in the prior art, the present invention provides a technical solution for targeting highly lethal Vibrio resistant to neomycin sulfate, and provides a component that has synergistic effect when used in combination with neomycin sulfate. This component can not only overcome the objective problem that neomycin sulfate is weak or inefficient at the recommended dosage, but also simultaneously achieve the enhancement of the efficacy of neomycin sulfate after combined use with neomycin sulfate, while reducing the amount of neomycin sulfate used. This provides a practical and innovative solution for the production side to overcome the problem that neomycin sulfate alone cannot effectively solve the problem of shrimp seedling disease.

[0005] The first aspect of the present invention aims to provide applications of Wedelia chrysantha extract, Gastrodia elata extract and Quassinus chinensis extract.

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

[0007] The third aspect of the present invention aims to provide a Vibrio-inhibiting drug.

[0008] The fourth aspect of the present invention aims to provide the use of the neomycin sulfate combined synergist for inhibiting Vibrio growth according to the second aspect of the present invention or the Vibrio inhibitory drug according to the third aspect of the present invention.

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

[0010] In order to achieve the above object, the technical solution adopted by the present invention is:

[0011] The first aspect of the present invention provides the use of Wedelia chrysantha extract, Gastrodia elata extract and Quassinus chinensis extract in any one of (1) to (4):

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

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

[0014] (3) preparing a synergist for use with neomycin sulfate to inhibit Vibrio growth;

[0015] (4) Preparation of drugs for preventing and / or treating diseases caused by Vibrio infection.

[0016] In some embodiments of the present invention, the Wedelia chrysantha extract is prepared by the following preparation method: Wedelia chrysantha is mixed with a low eutectic solvent, and extracted to obtain the Wedelia chrysantha extract.

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

[0018] In some embodiments of the present invention, the deep eutectic solvent comprises 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 chrysanthemum is mixed with the deep eutectic solvent, the Wedelia chrysanthemum is pre-treated, comprising: low-temperature crushing the Wedelia chrysanthemum with stems having a moisture content of ≤8%, and passing the mixture 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 min.

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

[0024] In some embodiments of the present invention, the purification and drying steps include extraction completion, solid-liquid separation, collection of the supernatant, filtration, and vacuum concentration of the filtrate to obtain a concentrated solution; the concentrated solution is adsorbed on a macroporous resin column, and after adsorption, it is sequentially treated with 2 times the volume fraction of pure water and 1.5 times the volume fraction of 30% to 40% ethanol aqueous solution, and the 30% to 40% ethanol aqueous solution eluate is collected, vacuum concentrated to one-fifth of the original volume, and vacuum freeze-dried to obtain a solid powder with a moisture content of 4% to 6%, thereby obtaining the Wedelia extract.

[0025] In some embodiments of the present invention, the Wedelia includes Wedelia spp.

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

[0027] In some embodiments of the present invention, the Gastrodia elata extract is prepared by the following preparation method: mixing Gastrodia elata with a complex enzyme preparation and a buffer, enzymatic hydrolysis, enzyme inactivation, separation and collection of precipitates; and supercritical CO2 extraction of the precipitate to obtain the Gastrodia elata extract.

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

[0029] In some embodiments of the present invention, the mass ratio of the 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 complex 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 is acetic acid-sodium acetate buffer, pH 4.2.

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

[0033] In some embodiments of the present invention, before being mixed with the complex enzyme preparation, the Gastrodia elata is pre-treated, including low-temperature grinding of the Gastrodia elata with a moisture content of ≤12% and passing through a 50-70 mesh sieve. To quickly grind the Gastrodia elata, the Gastrodia elata can be sliced ​​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 minutes, placing it in a constant temperature oscillator at 35-50°C and 100-150 rpm for 50-70 minutes, and oscillating it at 45-55°C and 160-200 rpm for 25-40 minutes to promote deep cell wall breaking.

[0035] In some embodiments of the present invention, the enzyme inactivation comprises placing the enzymatically hydrolyzed solution at 95-100° C. for 15-20 minutes 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 the supercritical CO2 extraction.

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

[0038] In some embodiments of the present invention, the preparation method further comprises purification and drying steps, including collecting the extract after the extraction is completed, 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 Gastrodia elata.

[0040] In some embodiments of the present invention, the Quassima extract is an enzymatic extract of Quassima.

[0041] In some embodiments of the present invention, the quassin extract is prepared by the following preparation method: mixing quassin with a buffer solution and a complex enzyme preparation, enzymolysis, and enzyme inactivation to obtain an enzymatic hydrolyzate; mixing the enzymatic hydrolyzate with ethanol, extracting, and solid-liquid separation to obtain supernatant A and a precipitate; mixing the precipitate with ethyl acetate, extracting, and solid-liquid separation to obtain supernatant B; combining supernatant A and supernatant B to obtain the quassin extract.

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

[0043] The mass ratio of the 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 complex enzyme preparation is 0.5% to 2% of the mass of Quassima wood; preferably 1% to 2%; more preferably 1%.

[0045] In some embodiments of the present invention, the buffer is acetic acid-sodium acetate buffer, pH 5.0.

[0046] In some embodiments of the present invention, the mass volume ratio of Gastrodia elata to 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 quassia wood with the complex enzyme preparation, the quassia wood is pre-treated, including low-temperature pulverization of the quassia wood with a moisture content of ≤10% and passing through a 90-100 mesh sieve. To quickly pulverize the quassia wood, the quassia wood can be frozen with liquid nitrogen until it is completely brittle.

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

[0049] In some embodiments of the present invention, the enzyme inactivation comprises 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, comprising filtering the combined supernatant A and supernatant B through a filter membrane, collecting the filtrate, vacuum concentrating it 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 that carries a gene encoding Tc toxin and is 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 erwinia.

[0055] The second aspect of the present invention provides a neomycin sulfate combined synergist for inhibiting the growth of Vibrio, comprising the Wedelia chrysantha extract, Gastrodia elata extract and Quassinus sylvestris extract of the first aspect of the present invention.

[0056] In some embodiments of the present invention, the mass ratio of the Wedelia chrysantha extract, the Gastrodia elata extract and the Quassinus chinensis extract is 1:2:(2-4).

[0057] The use of neomycin sulfate in combination with a synergist has a broad antibacterial synergistic effect on the clinically highly lethal Vibrio isolated from the clinic that carries the gene encoding Tc toxin and is resistant to neomycin sulfate, effectively reducing the dosage of neomycin sulfate and improving the anti-Vibrio effect.

[0058] Neomycin sulfate combined with a synergist, when used in combination with neomycin sulfate at a 20% to 60% replacement ratio, is significantly more effective against glass seed disease in white shrimp than neomycin sulfate alone, with the optimal replacement ratio being 40%. Replacing part of neomycin sulfate at the recommended ratio overcomes the objective issue of weak or inefficient neomycin sulfate at the recommended dose. It also enhances the efficacy of neomycin sulfate when used in combination with neomycin sulfate, while reducing neomycin sulfate usage. This provides a practical and innovative solution for manufacturers, overcoming the ineffectiveness of neomycin sulfate alone in treating glass seed disease in shrimp.

[0059] The third aspect of the present invention provides a Vibrio inhibitory drug comprising the Wedelia chrysantha extract, Gastrodia elata extract and Quassinus chinensis extract of the first aspect of the present invention.

[0060] In some embodiments of the present invention, the Vibrio inhibitory drug further comprises 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 that carries a gene encoding Tc toxin and is 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 erwinia.

[0064] The fourth aspect of the present invention provides the use of the neomycin sulfate combined synergist of the second aspect of the present invention or the Vibrio inhibitory drug of the third aspect of the present invention in any one of (a1) to (a2):

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

[0066] (a2) preparing a drug for preventing and / or treating a disease 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 that carries a gene encoding Tc toxin and is 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 erwinia.

[0070] In some embodiments of the present invention, the disease comprises Penaeus vannamei glass seedling disease.

[0071] The fifth aspect of the present invention provides a method for inhibiting Vibrio, comprising the step of treating Vibrio with the Vibrio inhibitory drug according to the third aspect of the present invention.

[0072] The beneficial effects of the present invention are:

[0073] The present invention discloses for the first time the in vitro inhibition of Vibrio growth by Wedelia chrysantha extract, Gastrodia elata extract, and / or Quassia oleracea extract, as well as their use in preventing and treating Vibrio-infected shrimp seedling disease. Experimental verification shows that the use of Wedelia chrysantha extract, Gastrodia elata extract, or Quassia oleracea extract alone, in combination of two or all three, has a good in vitro inhibition of Vibrio growth. At the same time, in order to treat the problem of Vibrio infection in seedlings, the inventive synergist is used in combination with neomycin sulfate, which not only overcomes the objective problem of weak or low efficacy of neomycin sulfate at the recommended dose, but also simultaneously achieves enhanced efficacy of neomycin sulfate after combined use with neomycin sulfate, while reducing the amount of neomycin sulfate used.

[0074] The neomycin sulfate combination enhancer provided by the present invention has good safety for oral use, and when used in combination with neomycin sulfate, it can enhance the efficacy of neomycin sulfate while reducing the amount of neomycin sulfate used. Specifically, the use of the neomycin sulfate combination enhancer in combination with neomycin sulfate has a significant and extensive antibacterial synergistic effect on 65 clinically isolated highly lethal Vibrio species that carry genes encoding Tc toxins and are resistant to neomycin sulfate, and also has a cumulative antibacterial effect on 2 clinically isolated Vibrio species that do not carry genes encoding Tc toxins and are sensitive to neomycin sulfate. When the neomycin sulfate anti-vibrio enhancer is used to replace neomycin sulfate at a ratio of 20% to 60%, the protective effect against the pathogenic Vibrio vibrio of Vibrio viridis is better than that of neomycin sulfate alone, with the optimal replacement ratio being 20% ​​to 40%. When the neomycin sulfate is replaced at a ratio of 20% to 60%, the therapeutic effect against the pathogenic Vibrio vibrio viridis is better than that of neomycin sulfate alone, with the optimal replacement ratio being 40%. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0077] Figure 3 Appearance of surviving shrimps in the positive control group treated with Vp32 poisoning.

[0078] Figure 4 The appearance of surviving shrimps infected with Vp32 treated with a compound enhancer combined with neomycin sulfate. DETAILED DESCRIPTION

[0079] The present invention is further described in detail below through specific examples.

[0080] It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0081] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

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

[0083] Example 1

[0084] A method for preparing a purified Wedelia spp. product comprises the following steps: weighing 100 parts by weight of Wedelia spp. with stems and a moisture content of ≤8%, cutting the stems into 1-2 cm segments, and then pulverizing the segments into a 100-mesh powder using a low-temperature grinder. The pulverized material is then added to a deep eutectic solvent (DES) (choline chloride, D-malic acid, and ultrapure water in a molar ratio of 1:2:5) in a ratio of 1:20 (w / v). Ultrasonic extraction is then performed at 58°C for 40 minutes. After extraction, the mixture is centrifuged at 10,000 rpm for 10 minutes, the supernatant is collected, and the remaining precipitate is extracted twice more using the ultrasonic extraction method described above. All extracts are combined. The combined extracts are filtered through a ceramic membrane filtration system using the following process parameters: membrane pore size 0.1 μm, cross-flow velocity 4 m / s, and operating pressure 0.25 MPa to obtain a filtrate. The filtrate was vacuum concentrated using a rotary evaporator at a vacuum degree of -0.09 MPa and a temperature of 45°C, and vacuum concentrated to a relative density of 1.15 to obtain a concentrated solution. The concentrated solution was adsorbed using an AB-8 macroporous resin column, and after adsorption, 2 times the volume of pure water and 1.5 times the volume of a 30% ethanol aqueous solution were used in sequence to collect the 30% ethanol aqueous solvent eluent. The eluate was vacuum concentrated to one-fifth of its original volume by rotary evaporation, and the concentrated solution was placed in a vacuum freeze dryer and dried for 40 hours to form a solid powder with a moisture content of (4-6)%, thereby obtaining a purified Wedelia scabra, which was stored in a low-temperature drying environment (18°C, 45% RH) for future use. The purified Wedelia scabra is hereinafter referred to as "Drug A".

[0085] The preparation method of the enzymatically purified black gastrodia elata comprises the following steps: weighing 100 parts of black gastrodia elata with a water content of ≤12% by weight, cutting the slices into thin slices, placing the slices into a liquid nitrogen quick-freezing tank for 10 minutes until completely brittle, and immediately grinding the slices into a 60-mesh powder in a low-temperature grinder. A pH 4.2 acetic acid-sodium acetate buffer solution and 0.5 w / w% of a composite enzyme preparation (a mixture of cellulase, pectinase, and β-glucanase powders in a mass ratio of 3:2:1) are added at a material-liquid ratio of 1:10 (w / v), pre-activating the mixture at 40°C for 20 minutes, and then oscillating the mixture in a constant temperature oscillator at 40°C and 120 rpm for 60 minutes, and then at 50°C and 180 rpm for 30 minutes to promote deep cell wall breaking. After the enzymolysis is completed, the extract is placed in a 100°C water bath for 18 minutes to inactivate the enzyme, then cooled to room temperature and centrifuged at 10,000 rpm for 20 minutes to collect the precipitate, and then vacuum freeze-dried to a moisture content of ≤6% to obtain enzymolysis black gastrodia elata powder. The enzymolysis black gastrodia elata powder is passed through a 60-mesh sieve and extracted using a supercritical CO2 extraction system with 1% ethyl acetate as an entrainer, a CO2 flow rate of 30 kg / h, an extraction pressure of 25 MPa, and an extraction temperature of 45°C for 100 minutes. After the extraction is completed, the extract flows into a collection tank and is cooled to room temperature to obtain an extract. The obtained extract is adsorbed on an AB-8 macroporous resin column. After complete adsorption, 2 times the volume fraction of pure water and 1.5 times the volume fraction of 50% ethanol aqueous solution are sequentially added, and the 50% ethanol aqueous solution eluent is collected. The eluate was vacuum concentrated by rotary evaporation to one-fifth of its original volume, and the concentrated solution 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)%, thereby obtaining a purified enzymatic hydrolysis product of Gastrodia elata, which was stored in a low-temperature dry environment (18°C, 45% RH) until use. The purified enzymatic hydrolysis product of Gastrodia elata is hereinafter referred to as "Drug B."

[0086] The method for preparing a purified enzymatic hydrolysis product of quassia wood comprises the following steps: weighing 100 parts of quassia wood with a moisture content of ≤10% by weight, subjecting the product to a liquid nitrogen quick-freeze for 15 minutes until brittle, and then grinding the product into a 100-mesh powder in a low-temperature airflow mill to obtain quassia wood powder. A pH 5.0 acetic acid-sodium acetate buffer solution and 1 w / w% of a composite enzyme preparation (a mixture of cellulase, pectinase, and xylanase powders in a mass ratio of 5:3:2) are added to the product at a material-liquid ratio of 1:10 (w / v). The mixture is placed in a thermostatic oscillator at 45°C and 150 rpm for enzymatic hydrolysis for 2 hours. After the enzymatic hydrolysis is completed, the product is placed in a 100°C water bath for 20 minutes to inactivate the enzymes, and then cooled to room temperature. 1.5 times the volume of a 60% ethanol solution is added to the cooled enzymatic hydrolyzate, followed by extraction at 50°C and 200 rpm for 30 minutes using an ultrasonic extractor. After the extraction is completed, the supernatant is collected by centrifugation at 10,000 rpm. Add 2 times the volume of ethyl acetate (purity ≥ 99.5%) to the centrifugal precipitate, perform ultrasonic extraction at 35°C and 180rpm for 25 minutes, and collect supernatant b by centrifugation at 10,000rpm after the extraction. Combine supernatant a and supernatant b, filter with a 0.22μm filter membrane (to remove interfering substances), and collect the clarified extract. Use a rotary evaporator to vacuum concentrate the obtained extract to one-third of its original volume. Then place the concentrated concentrate in a vacuum freeze dryer and dry it for 40 hours to form a solid powder with a moisture content of (4-8)%, thereby obtaining the purified enzymatic hydrolysis product of Quassinus, which is stored in a low-temperature drying environment (18°C, 45% RH) for future use. The purified enzymatic hydrolysis product of Quassinus is hereinafter referred to as "Drug C".

[0087] Example 2

[0088] A method for preparing a purified aqueous extract of Wedelia serrata comprises the following steps: weighing 100 parts by weight of Wedelia serrata with stems and a water content of 8% or less, cutting the stems into 1-2 cm segments, and then pulverizing the segments into a 100-mesh powder using a low-temperature grinder. The pulverized material is added to purified water in a ratio of 1:20 (w / v), and ultrasonically extracted at 58°C for 40 minutes. After extraction, the mixture is centrifuged at 10,000 rpm for 10 minutes, and the supernatant is collected. The remaining precipitate is extracted twice more using the ultrasonic extraction method described above, and all extracts are combined. The combined extracts are filtered through a ceramic membrane filtration system using the following process parameters: membrane pore size 0.1 μm, cross-flow velocity 4 m / s, and operating pressure 0.25 MPa to obtain a filtrate. The filtrate is then vacuum concentrated using a rotary evaporator at a vacuum degree of -0.09 MPa and a temperature of 45°C to a relative density of 1.15, thereby obtaining a concentrated solution. The concentrate was adsorbed on a macroporous resin column. After adsorption, 2 volumes of pure water and 1.5 volumes of 30% ethanol aqueous solution were added in sequence, and the 30% ethanol aqueous solution eluent was collected. The eluent was vacuum concentrated to one-fifth of its original volume by rotary evaporation, and then the concentrated concentrate was placed in a vacuum freeze dryer and dried for 40 hours to form a solid powder with a moisture content of (4-6)%, thereby obtaining a purified Wedelia scabra. The purified Wedelia scabra water extract was stored in a low-temperature drying environment (18°C, 45% RH) for future use. The purified Wedelia scabra water extract is hereinafter referred to as "Drug A Control".

[0089] The method for preparing a non-enzymatically purified product of Gastrodia elata comprises the following steps: weighing 100 parts of Gastrodia elata with a water content of ≤12% by weight, cutting the slices into thin slices, placing the slices in a liquid nitrogen quick-freezing tank for 10 minutes until the slices are completely brittle, and immediately placing the slices in a low-temperature grinder to grind the slices into a 60-mesh powder; adding a pH 4.2 acetic acid-sodium acetate buffer solution at a material-liquid ratio of 1:10 (w / v), pre-activating the mixture at 40°C for 20 minutes, placing the mixture in a constant temperature oscillator and first oscillating at 40°C and 120 rpm for 60 minutes, then oscillating at 50°C and 180 rpm for 30 minutes to promote deep cell wall fragmentation; after the cell wall fragmentation is complete, centrifuging the mixture at 10,000 rpm for 20 minutes to collect the precipitate, and then vacuum freeze-drying the mixture until the water content is less than 6%, thereby obtaining a non-enzymatically purified product of Gastrodia elata powder. The non-enzyme hydrolysate powder of Gastrodia elata is obtained by passing through a 60-mesh sieve and adopting a supercritical CO extraction system, with 1% ethyl acetate as an entrainer, CO flow velocity 30kg / h, extraction pressure 25MPa, extraction temperature 45 ℃, extraction 100min, extraction ends, and the extract flows into a holding tank and is cooled to room temperature to obtain an extract. The extract obtained is adsorbed by a macroporous resin column, and after adsorption is complete, 2 times of volume pure water, 1.5 times of volume of 50% ethanol aqueous solution are used successively to collect the 50% ethanol aqueous solution eluent. The eluent is vacuum concentrated to 1 / 5th of the original volume by rotary evaporation, and the concentrated solution is put into a vacuum freeze dryer and dried for 40h to form a moisture content of (4~8)% solid powder after concentrating, to obtain the non-enzyme hydrolysate purified product of Gastrodia elata, and is preserved for standby use under a low-temperature drying (18 ℃, 45%RH) environment. The non-enzyme hydrolysate purified product of Gastrodia elata is hereinafter uniformly referred to as "drug B control".

[0090] The method for preparing a purified non-enzymatic hydrolysis product of quassinase comprises the following steps: weighing 100 parts by weight of quassin with a moisture content of ≤10%, subjecting the mixture to a liquid nitrogen quick freezer for 15 minutes until brittle, and then pulverizing the mixture into a 100-mesh powder in a low-temperature pulverizer with a propulsion airflow to obtain quassin powder. Acetic acid-sodium acetate buffer with a pH of 5.0 is added at a material-liquid ratio of 1:10 (w / v). The mixture is oscillated in a thermostatic oscillator at 45°C and 150 rpm for 2 hours. After oscillation, 1.5 times the volume of 60% ethanol solution is added to the mixture. Extraction is then performed using an ultrasonic extractor at 50°C and 200 rpm for 30 minutes. After extraction, supernatant (a) is collected by centrifugation at 10,000 rpm. Ethyl acetate (99.5%) is then added to the centrifuged precipitate, and ultrasonic extraction is performed at 35°C and 180 rpm for 25 minutes. After extraction, supernatant (b) is collected by centrifugation at 10,000 rpm. Combine supernatant a and supernatant b, filter with a 0.22 μm filter membrane, and collect the clarified extract. Use a rotary evaporator to vacuum concentrate the obtained extract to one-third of its original volume. Then place the concentrated concentrate in a vacuum freeze dryer and dry it for 40 hours to form a solid powder with a moisture content of (4-8)%, thereby obtaining a non-enzymatically purified product of Quassinus sylvatica, which is stored in a low-temperature dry environment (18°C, 45% RH) for future use. The enzymatically purified product of Quassinus sylvatica is hereinafter referred to as "Drug C Control".

[0091] Example 3

[0092] A neomycin sulfate composite synergist comprises, by weight, 20 parts of a purified Wedelia serrata, 40 parts of an enzymatically purified Gastrodia elata, and 40 parts of an enzymatically purified Quassima serrata.

[0093] The purified product of Wedelia scabra, the purified product by enzymatic hydrolysis of Gastrodia elata, and the purified product by enzymatic hydrolysis of Quassima were prepared according to Example 1.

[0094] The preparation method of the above-mentioned neomycin sulfate composite synergist is as follows: weigh the purified product of Wedelia serrata, the enzymatically purified product of Gastrodia elata and the enzymatically purified product of Quassinus chinensis, and mix the components in a predetermined proportion in a dry and sterile environment (40% to 50% RH) to obtain the neomycin sulfate composite synergist.

[0095] Example 4 In vitro MIC determination of highly lethal Vibrio resistant to neomycin sulfate by different treatment methods of purified traditional Chinese medicine 1 Test consumables

[0096] Drugs: (1) Neomycin sulfate standard: purchased from China Veterinary Drug Administration; (2) Three purified Chinese medicines and their controls: drug A (i.e., purified Wedelia chinensis), drug A control (i.e., purified Wedelia chinensis water extract), drug B (i.e., purified Gastrodia elata by enzymatic hydrolysis), drug B control (i.e., purified Gastrodia elata by non-enzymatic hydrolysis), drug C (purified Quassinus truncatus by enzymatic hydrolysis), and drug C control (purified Quassinus truncatus by non-enzymatic hydrolysis), prepared according to Examples 1 and 2.

[0097] Drug preparation: (1) Neomycin sulfate: Dissolve in sterile water to prepare a 10240 μg / mL stock solution before the experiment and set aside. (2) Three purified Chinese herbal medicines and their controls: Drug A, Drug B, Drug B control, Drug C, and Drug C control: Dissolve in DMSO to prepare a 40960 μg / mL stock solution before the experiment and set aside. Drug A control: Dissolve in sterile water to prepare a 40960 μg / mL stock solution before the experiment and set aside.

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

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

[0100] 2 Test methods

[0101] 2.1 Preparation of test strains

[0102] Dissolve the Vibrio parahaemolyticus Vp32 stored in glycerol at -80℃ at room temperature, draw 1mL of bacterial solution in a clean bench and add it to a TSB broth test tube containing 1.5% sodium chloride (5mL / tube, sterilized), add each bacterium to a test tube, and culture it on a shaker at 28℃ at 200rpm for 18-24 hours to revive the strain. In a clean bench, use a sterile inoculation loop to dip the revived bacterial solution in the three-zone streak method and inoculate it on the HLVBS agar medium, and culture it in an incubator at a constant temperature of 28℃ for 18-24 hours. Pick a typical single colony and inoculate it in MH broth containing 1.5% sodium chloride, and culture it on a shaker at 28℃ at 200rpm for 8-10 hours. Adjust the bacterial solution to OD 0.2 (concentration of about 10 8 CFU / mL), dilute the bacterial solution to 100 times with MH broth to make the bacterial solution concentration 10 6CFU / mL, as the test bacterial solution.

[0103] 2.2 Determination of single-drug susceptibility MICs

[0104] MICs of drug A, drug A control, drug B, drug B control, drug C, drug C control, and neomycin sulfate were determined using the CLSI-specified broth microdilution method for Vibrio parahaemolyticus Vp32. 100 μL of MH broth was added to a 96-well plate. The drug stock solution was diluted 10-fold to prepare the working solution. 100 μL of the prepared drug working solution was added to the first well of each row. The drug was then diluted two-fold, and 100 μL was pipetted into the second well and thoroughly mixed with the broth by pipetting. This process was repeated until the last well. 100 μL was aspirated from the 12th column and discarded. 100 μL of the diluted bacterial solution was then added to each well. Three replicates were performed for each drug. A negative control (MH broth alone without bacterial solution) and a positive control (bacteria solution without drug) were also performed 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.

[0105] Interpretation of results: The lowest drug concentration that completely inhibits bacterial growth is the drug MIC.

[0106] 3 Test results

[0107] As shown in Table 1, the MIC of neomycin sulfate against the highly lethal Vibrio parahaemolyticus Vp32 is 128 μg / mL, indicating that the strain is resistant. Drugs A, B, and C all exhibited some in vitro antibacterial activity against V. parahaemolyticus Vp32, with MICs of 256 μg / mL, 512 μg / mL, and 1024 μg / mL, respectively. Although their antibacterial activity was inferior to that of neomycin sulfate, they were all more potent than their corresponding control drugs.

[0108] Table 1 MIC test results of the tested 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 suspension +

[0110] Note: The symbol “-” indicates that the well is clear and no bacteria grow; the symbol “+” indicates that the well is turbid and bacteria grow.

[0111] 4 Experimental Summary

[0112] (1) The purified substance extracted from Wedelia serrata by 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 purified Gastrodia elata hydrolyzed with a composite enzyme preparation on the highly lethal Vibrio parahaemolyticus resistant to neomycin sulfate was better than that of the purified Gastrodia elata hydrolyzed without a composite enzyme preparation.

[0114] (3) The in vitro antibacterial effect of the purified product of Quassinus woody hydrolysis using a composite enzyme preparation on the highly lethal Vibrio parahaemolyticus resistant to neomycin sulfate was better than that of the purified product of Quassinus woody hydrolysis using no composite enzyme preparation.

[0115] Example 5 In vitro MIC determination of purified Chinese herbal medicine alone and in combination with neomycin sulfate against highly lethal Vibrio resistant to neomycin sulfate

[0116] 1Test consumables

[0117] Drugs: (1) Neomycin sulfate standard: purchased from China Veterinary Drug Administration; (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 to prepare a 10240 μg / mL stock solution before the experiment and set aside. (2) Three purified Chinese herbal medicines: Drug A, Drug B, and Drug C, all dissolved in DMSO to prepare a 40960 μg / mL stock solution before the experiment and set aside.

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

[0120] Culture media: (1) TSB broth with 1.5% sodium chloride, MH agar with 1.5% sodium chloride, and MH broth with 1.5% sodium chloride: TSB broth, MH agar, and MH broth were purchased from Qingdao Haibo Biotechnology Co., Ltd. A certain amount of solid sodium chloride was added to each of these three media to a final sodium chloride concentration of 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 the Vibrio parahaemolyticus Vp32 stored in glycerol at -80℃ at room temperature, draw 1mL of bacterial solution in a clean bench and add it to a TSB broth test tube containing 1.5% sodium chloride (5mL / tube, sterilized), add each bacterium to a test tube, and culture it at 28℃ with a shaker at 200rpm for 18-24 hours to revive the strain. In a clean bench, use a sterile inoculation loop to dip the revived bacterial solution into the TCBS agar medium according to the three-zone line method, and culture it upside down in an incubator at a constant temperature of 28℃ for 18-24 hours. Pick a typical single colony and inoculate it into MH broth containing 1.5% sodium chloride, and culture it at 28℃ with a shaker at 200rpm for 8-10 hours. Adjust the bacterial solution to OD 0.2 (concentration of about 10 8CFU / mL), dilute the bacterial solution to 100 times with MH broth to make the bacterial solution concentration 10 6 CFU / mL, as the test bacterial solution.

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

[0125] (1) Combined drug sensitivity results of drug A, drug B, and drug C combined with neomycin sulfate against neomycin sulfate-resistant and highly lethal Vibrio vp32

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

[0127] Table 2 Group design of the single drug combined with neomycin sulfate trial

[0128]

[0129]

[0130] Drug dilution: Dilute each drug stock solution 10-fold to the concentration in the first test tube. Add 1 mL of MH broth to each test tube, except for the first test tube, where 1.8 mL is added. Add 0.2 mL of the antibiotic stock solution to the first tube and mix thoroughly. Then pipette 1 mL into the second tube, mix thoroughly, and pipette 1 mL into the third tube. Repeat this process until the final test tube is reached.

[0131] Sample loading: Add 50 μL of neomycin sulfate to each well in the horizontal rows. Add 50 μL of purified Chinese herbal extract to each well in the vertical rows. Finally, add 100 μL of the diluted bacterial solution to each well. Perform three replicates for each drug. Simultaneously, perform a negative control (blank broth only, no bacterial solution) and a positive control (bacteria solution, no drug) on ​​the same plate. After loading, place the 96-well plate in a 28°C incubator for 20-24 hours and observe the results.

[0132] The MICs of the drugs alone and in combination with neomycin sulfate were recorded, and the corresponding inhibition index (FIC) was calculated.

[0133] Result interpretation: There are four types

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

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

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

[0137] d Antagonism: 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 judging the combined drug sensitivity test.

[0139]

[0140] Judgment criteria: FIC index <0.5 was judged as synergistic effect; FIC index 0.5-1.0 was judged as additive effect; FIC index 1.0-2.0 was judged as irrelevant effect; FIC index >2.0 was judged as antagonistic effect.

[0141] (2) Combined drug sensitivity MICs of drug A, drug B, and drug C in combination with neomycin sulfate against highly pathogenic Vibrio vp32 resistant to neomycin sulfate

[0142] Group design (Table 3): The 96-well microbroth 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 / drug C were 1 / 2 times and 1 / 4 times the MIC. The single drugs were combined in pairs and then combined with the MIC of neomycin sulfate.

[0143] Table 3 Group design of single drug and double drug combination of neomycin sulfate

[0144]

[0145]

[0146] Drug dilution: Dilute each drug stock solution 10-fold to the concentration in the first test tube. Add 1 mL of MH broth to each test tube, except for the first test tube, where 1.8 mL is added. Add 0.2 mL of the antibiotic stock solution to the first tube and mix thoroughly. Then pipette 1 mL into the second tube, mix thoroughly, and pipette 1 mL into the third tube. Repeat this process until the final test tube is reached.

[0147] Sample loading: 50 μL of neomycin sulfate is added to each well in the horizontal rows. 25 μL of each purified Chinese herbal extract is added to each well in the vertical rows. Finally, 100 μL of the diluted bacterial solution is added to each well. Three replicates are performed for each drug. Simultaneously, a negative control (blank broth only, no bacterial solution) and a positive control (bacteria solution, no drug) are performed on the same plate. After loading, place the 96-well plate in a 28°C incubator for 20-24 hours and observe the results.

[0148] The MICs of the drug combinations of neomycin sulfate were recorded and compared with the MICs of neomycin sulfate alone.

[0149] (3) Combined drug sensitivity MIC of three purified Chinese herbal medicines combined with neomycin sulfate against highly lethal Vibrio Vp32 resistant to neomycin sulfate

[0150] Group design (Table 4): The 96-well microbroth 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 / drug C were 1 / 2 times the MIC and 1 / 4 times the MIC. The MIC of the three purified Chinese medicines combined with neomycin sulfate was calculated.

[0151] Table 4 Group design of single drug and double combination neomycin sulfate test

[0152]

[0153]

[0154] Drug dilution: Dilute each drug stock solution 10-fold to the concentration in the first test tube. Add 1 mL of MH broth to each test tube, except for the first test tube, where 1.8 mL is added. Add 0.2 mL of the antibiotic stock solution to the first tube and mix thoroughly. Then pipette 1 mL into the second tube, mix thoroughly, and pipette 1 mL into the third tube. Repeat this process until the final test tube is reached.

[0155] Sample loading: 25 μL of neomycin sulfate is added to each well in the horizontal rows. 25 μL of each purified Chinese herbal extract is added to each well in the vertical rows. Finally, 100 μL of the diluted bacterial solution is added to each well. Three replicates are performed for each drug. Simultaneously, a negative control (blank broth only, no bacterial solution) and a positive control (bacteria solution, no drug) are performed on the same plate. After loading, place the 96-well plate in a 28°C incubator for 20-24 hours and observe the results.

[0156] The MICs of the drug Triple Tri combined with neomycin sulfate were recorded and compared with the MIC of neomycin sulfate alone.

[0157] 2.3 MIC determination of compound synergists

[0158] The MICs of drug A, drug B, drug C, and the combined potentiator (drug A: drug B: drug C = 1:2:2, w / w / w) were determined using the CLSI-specified broth microdilution method for Vibrio parahaemolyticus Vp32 (Table 5). 100 μL of MH broth was added to a 96-well plate. The drug stock solution was diluted 10-fold to prepare the working solution. 100 μL of the prepared drug working solution was added to the first well of each row. The drug was then diluted two-fold, and 100 μL was pipetted into the second well and thoroughly mixed with the broth by pipetting. This process was repeated until the last well. 100 μL was aspirated from the 12th column and discarded. 100 μL of the diluted bacterial solution was then added to each well. Three replicates were performed for each drug. A negative control (MH broth alone without bacterial solution) and a positive control (bacterial solution without drug) were also performed on the same plate. After the addition of samples, the 96-well plate was placed in a 28°C constant temperature incubator for 20 to 24 hours and the results were observed.

[0159] Interpretation of results: The lowest drug concentration that completely inhibits bacterial growth is the drug MIC.

[0160] Table 5 MIC determination of composite synergists

[0161]

[0162] 2.4 Combined drug sensitivity results of the composite enhancer combined with neomycin sulfate against highly lethal Vibrio Vp32 resistant to neomycin sulfate

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

[0164] Drug dilution: Dilute each drug stock solution 10-fold to the concentration in the first test tube. Add 1 mL of MH broth to each test tube, except for the first test tube, where 1.8 mL is added. Add 0.2 mL of the antibiotic stock solution to the first tube and mix thoroughly. Then pipette 1 mL into the second tube, mix thoroughly, and pipette 1 mL into the third tube. Repeat this process until the final test tube is reached.

[0165] Sample loading: Add 50 μL of neomycin sulfate to each well in the horizontal rows. Add 50 μL of purified Chinese herbal extract to each well in the vertical rows. Finally, add 100 μL of the diluted bacterial solution to each well. Perform three replicates for each drug. Simultaneously, perform a negative control (blank broth only, no bacterial solution) and a positive control (bacteria solution, no drug) on ​​the same plate. After loading, place the 96-well plate in a 28°C incubator for 20-24 hours and observe the results.

[0166] The MICs of the drugs alone and in combination with neomycin sulfate were recorded, and the corresponding inhibition index (FIC) was calculated.

[0167] Result interpretation: There are four types

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

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

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

[0171] (4) Antagonism: 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 judging the combined drug sensitivity test.

[0173]

[0174] Judgment criteria: FIC index <0.5 was judged as synergistic effect; FIC index 0.5-1.0 was judged as additive effect; FIC index 1.0-2.0 was judged as irrelevant effect; FIC index >2.0 was judged as antagonistic effect.

[0175] 3 Test results

[0176] (1) Combined drug sensitivity results of three purified Chinese herbal medicines combined with neomycin sulfate

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

[0178] The MIC of drug B against the highly lethal V. parahaemolyticus Vp32 strain resistant to neomycin sulfate was 512 μg / mL. When drug B was added at 1 / 2 its MIC (256 μg / mL), the MIC of neomycin sulfate was 64 μg / mL, a two-fold decrease compared to neomycin sulfate alone (Table 7). The combined FIC of the two agents was 1, demonstrating an additive effect.

[0179] The MIC of drug C against the highly lethal V. parahaemolyticus Vp32 strain resistant to neomycin sulfate was 1024 μg / mL. When drug C was added at 1 / 2 its MIC (512 μg / mL), the MIC of neomycin sulfate was 64 μg / mL, a two-fold decrease compared to neomycin sulfate alone (Table 8). The combined FIC of the two agents was 1, demonstrating an additive effect.

[0180] Table 6 Combined drug sensitivity MIC of neomycin sulfate combined with drug A against Vibrio parahaemolyticus Vp32

[0181]

[0182]

[0183] Table 7 Combined drug sensitivity MIC of neomycin sulfate combined with drug B against Vibrio parahaemolyticus Vp32

[0184]

[0185] Table 8 Combined drug sensitivity MIC of neomycin sulfate combined with drug C against Vibrio parahaemolyticus Vp32

[0186]

[0187] Note: In Tables 6 to 8, the symbol “-” indicates that the well is clear and no bacteria grow; the symbol “+” indicates that the well is turbid and bacteria grow.

[0188] (2) Combined drug sensitivity MIC of three purified Chinese herbal medicines in pairs and neomycin sulfate

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

[0190] Table 9 Combined drug sensitivity MIC of neomycin sulfate combined with two single drugs against Vibrio parahaemolyticus Vp32

[0191]

[0192] (3) Combined drug sensitivity MIC of three purified Chinese herbal medicines and neomycin sulfate

[0193] As shown in Table 10, the combination of drugs A, B, and C at their respective sub-MIC concentrations can produce an antibacterial synergistic effect with neomycin sulfate. When 1 / 2MIC (128 μg / mL) of drug A, 1 / 2MIC (256 μg / mL) of drug B, and 1 / 2MIC (512 μg / mL) of drug C were added, and when 1 / 2MIC (128 μg / mL) of drug A, 1 / 2MIC (256 μg / mL) of drug B, and 1 / 4MIC (256 μg / mL) of drug C were added, the MIC of these two groups combined with neomycin sulfate was the lowest, both 8 μg / mL, which was 16 times lower than the MIC of neomycin sulfate alone. The antibacterial effect was better than the effect of the two combinations. According to the selection criteria of "good antibacterial sensitization effect and 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 combined composite synergist ratio.

[0194] Table 10 Combined drug sensitivity MIC of neomycin sulfate combined with three single drugs against Vibrio parahaemolyticus Vp32

[0195]

[0196]

[0197] (4) MIC determination of composite synergists

[0198] As can be seen from Table 11, the MIC of the composite enhancer against the highly lethal Vibrio parahaemolyticus Vp32 resistant to neomycin sulfate is 128 μg / mL, which is 2 times, 4 times and 8 times lower than the MICs of drug A, drug B and drug C, respectively.

[0199] Table 11 MIC test results of composite synergists against Vibrio parahaemolyticus Vp32

[0200] Drug name MIC (μg / mL) Drug A 256 Drug B 512 Drug C 1024 Composite synergist 128 MH broth - MH broth + bacterial suspension +

[0201] (5) Combined drug sensitivity results of compound enhancer and neomycin sulfate

[0202] As shown in Table 12, the MIC of neomycin sulfate against the highly lethal neomycin-resistant Vibrio parahaemolyticus Vp32 is 128 μg / mL. When the compound synergist is added at 1 / 4 MIC (32 μg / mL) or 1 / 8 MIC (16 μg / mL), the MICs of neomycin sulfate are 8 μg / mL and 16 μg / mL, respectively. These decreases are 16-fold and 8-fold compared to the MICs of neomycin sulfate alone. The combined FICs of the two agents are 0.375 and 0.25, respectively, demonstrating synergistic effects. Considering the need to reduce antibiotic usage and mitigate the degree of resistance in Vibrio parahaemolyticus, a compound synergist concentration of 1 / 8 MIC was selected for combined use.

[0203] Table 12 Combined drug sensitivity MIC of neomycin sulfate combined with compound synergist against Vibrio parahaemolyticus Vp32

[0204]

[0205] 4 Experimental Summary

[0206] (1) The purified products of Wedelia serrata, the purified products hydrolyzed by Gastrodia elata, and the purified products hydrolyzed by Quassinus chinensis respectively combined with neomycin sulfate can reduce the MIC of neomycin sulfate against Vp32 strain, but they have only cumulative effects and no synergistic effects.

[0207] (2) When the purified products of Wedelia chinensis, Gastrodia elata enzymatically purified products, and Quassinus quinata enzymatically purified products were mixed in a certain proportion and applied in combination with neomycin sulfate to Vp32 strain, it could produce a stronger antibacterial sensitization effect than the combination of the three purified products alone with neomycin sulfate or the combination of any two of the three purified products with neomycin sulfate.

[0208] (3) The purified products of Wedelia serrata, the purified products by enzymatic hydrolysis of Gastrodia elata, and the purified products by enzymatic hydrolysis of Quassinus were mixed in a mass ratio of 1:2:2 and used in combination with neomycin sulfate to treat the Vp32 strain, achieving the effect of minimizing the dosage of the composite enhancer and achieving the lowest MIC of neomycin sulfate.

[0209] (4) The mass ratio of the purified product of Wedelia serrata, the purified product by enzymatic hydrolysis of Gastrodia elata, and the purified product by enzymatic hydrolysis of Quassinus chinensis was 1:2:2, and they were combined with neomycin sulfate to have an antibacterial sensitization effect, with an FIC of 0.25.

[0210] Example 6 In vitro antibacterial synergistic effect of neomycin sulfate composite synergist combined with neomycin sulfate on clinical isolates of highly lethal Vibrio resistant to neomycin sulfate

[0211] 1Test consumables

[0212] Drugs: (1) Neomycin sulfate standard product: purchased from China Veterinary Drug Administration; (2) Neomycin sulfate composite enhancer (hereinafter referred to as composite enhancer): from Example 3.

[0213] Drug preparation: (1) Neomycin sulfate: Dissolve in sterile water to prepare a 10240 μg / mL stock solution before the experiment and set aside. (2) Composite enhancers: Dissolve in DMSO to prepare a 10240 μg / mL stock solution before the experiment and set aside.

[0214] Test strains: (1) 65 highly lethal Vibrio strains that are resistant to neomycin sulfate and carry the gene encoding Tc toxin. These strains have been identified by physiological and biochemical methods and 16sRNA sequencing. They include: 53 strains of Vibrio parahaemolyticus, numbered Vp1-53; 8 strains of Vibrio harveyi, numbered Vh1-8; 3 strains of Vibrio alginolyticus, numbered Va1-3; and 1 strain of Vibrio erwinii, numbered Vo1. All 65 strains of Vibrio were isolated from shrimp with typical vibriosis in the farm 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 strains of Vibrio strains that are sensitive to neomycin sulfate and do not carry the gene encoding Tc toxin. These strains have been identified by physiological and biochemical methods and 16sRNA sequencing. They include: Vibrio parahaemolyticus Vp54 and Vibrio harveyi Vh9. The two strains of Vibrio were isolated from shrimp with vibriosis in the farm 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) TSB broth with 1.5% sodium chloride, MH agar with 1.5% sodium chloride, and MH broth with 1.5% sodium chloride: TSB broth, MH agar, and MH broth were purchased from Qingdao Haibo Biotechnology Co., Ltd. A certain amount of solid sodium chloride was added to each of these three media to a final sodium chloride concentration of 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 in -80℃ glycerol were dissolved at room temperature. The preparation method of each strain was as follows: 1mL of bacterial solution was drawn into a TSB broth test tube containing 1.5% sodium chloride (5mL / tube, sterilized) in a clean bench. Each bacterium was added to a test tube and cultured at 28℃ with a shaker at 200rpm for 18-24 hours to revive the strain. In the clean bench, a sterile inoculation loop was used to dip the revived bacterial solution into the HLVBS agar medium according to the three-zone line method. The culture was inverted and cultured at a constant temperature of 28℃ in an incubator for 18-24 hours. A typical single colony was picked and inoculated into MH broth containing 1.5% sodium chloride. The culture was shaken at 28℃ with a shaker at 200rpm for 8-10 hours. The bacterial solution was adjusted to OD 0.2 (concentration of about 10 8 CFU / mL), dilute the bacterial solution to 100 times with MH broth to make the bacterial solution concentration 10 6 CFU / mL, as the test bacterial solution.

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

[0220] (1) To determine the antibacterial effect of the composite synergist combined with neomycin sulfate on highly lethal Vibrio resistant to neomycin sulfate, the chessboard method was used to determine the antibacterial effect of 65 clinical highly lethal Vibrio as test strains. The bacterial solution concentration was prepared to 1.0×10 6 To determine the CFU / mL, the stock solutions of neomycin sulfate and the compound enhancer were serially diluted with MH broth to the corresponding concentrations. In a 96-well culture plate, add 50 μL of neomycin sulfate to each well in the horizontal rows and 50 μL of compound enhancer to each well in the vertical rows. Finally, add 100 μL of the diluted bacterial solution to each well, resulting in a final concentration of neomycin sulfate of 256 μg / mL to 2 μg / mL, and a final concentration of compound enhancer of 32 μg / mL and 16 μg / mL. Three replicates were performed for each drug. A negative control (MH broth alone without bacterial solution) and a positive control (bacteria solution without drug) were also performed on the same plate. After addition, the 96-well plate was incubated at 28°C for 20-24 hours and the results were observed. The MIC of the compound enhancer alone and in combination with neomycin sulfate was recorded, and the corresponding inhibitory index (FIC) was calculated.

[0221] (2) To determine the antibacterial effect of the composite enhancer combined with neomycin sulfate on Vibrio that is sensitive to neomycin sulfate and does not carry the gene encoding Tc toxin, the chessboard method was used to determine the antibacterial effect of the composite enhancer combined with neomycin sulfate on two clinical Vibrio strains (Vp54 and Vh9) as test strains. The bacterial solution concentration was prepared to 1.0×10 6 To determine the CFU / mL, the stock solutions of neomycin sulfate and the compound enhancer were serially diluted with MH broth to the corresponding concentrations. In a 96-well culture plate, add 50 μL of neomycin sulfate to each well in the horizontal rows and 50 μL of compound enhancer to each well in the vertical rows. Finally, add 100 μL of the diluted bacterial solution to each well, resulting in a final concentration of neomycin sulfate of 16 μg / mL to 0.25 μg / mL and a final concentration of compound enhancer of 32 μg / mL and 16 μg / mL. Three replicates were performed for each drug. A negative control (MH broth alone without bacterial solution) and a positive control (bacteria solution without drug) were also performed on the same plate. After addition, the 96-well plate was incubated at 28°C for 20-24 hours and the results were observed. The MIC of the compound enhancer alone and in combination with neomycin sulfate was recorded, and the corresponding inhibitory index (FIC) was calculated.

[0222] Result interpretation: There are four types.

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

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

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

[0226] (4) Antagonism: 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 judging the combined drug sensitivity test.

[0228]

[0229] Judgment criteria: FIC index <0.5 was judged as synergistic effect; FIC index 0.5-1.0 was judged as additive effect; FIC index 1.0-2.0 was judged as irrelevant effect; FIC index >2.0 was judged as antagonistic effect.

[0230] 3 Test results

[0231] As shown in Table 13, when the total concentration of the compound synergist was 32 μg / mL, 72.3% of the strains showed synergistic effects with neomycin. At a total concentration of 16 μg / mL, the proportion of strains showing synergistic effects with neomycin was 75.4%. These results demonstrate that when the compound synergist is present at both 16 and 32 μg / mL, it can produce a significant and broad antimicrobial sensitization effect in combination with neomycin against clinically isolated, highly lethal Vibrio spp. that are resistant to neomycin sulfate and carry the gene encoding the Tc toxin.

[0232] As shown in Table 14, for strains that do not carry genes encoding Tc toxins and are sensitive to neomycin sulfate, at a total concentration of 32 μg / mL, the proportion of strains exhibiting a synergistic effect with neomycin sulfate was 0%, and the proportion of strains exhibiting an additive effect was 100%. At a total concentration of 16 μg / mL, the proportion of strains exhibiting a synergistic effect with neomycin sulfate was 50%, and the proportion of strains exhibiting an additive effect was 50%. These experimental results demonstrate that at total concentrations of 16 μg / mL and 32 μg / mL, the combination of the compound synergist and neomycin sulfate also produces an additive effect against clinically isolated Vibrio species that do not carry genes encoding Tc toxins and are sensitive to neomycin sulfate.

[0233] Table 13 Effect of the antibacterial activity of the composite enhancer combined with neomycin sulfate on 65 clinically isolated Vibrio strains carrying genes encoding Tc toxins and resistant to neomycin sulfate

[0234]

[0235]

[0236] Table 14 Effect of the antibacterial activity of the composite enhancer combined with neomycin sulfate on two clinically isolated Vibrio strains that do not carry the gene encoding Tc toxin and are sensitive to neomycin sulfate

[0237]

[0238] 4 Experimental Summary

[0239] The purified product of Wedelia scabra, the enzymatically purified product of Gastrodia elata, and the enzymatically purified product of Quassinus chinensis were scientifically formulated into a compound agent (i.e., the neomycin sulfate composite synergist of Example 3) in a ratio of 1:2:2 (w / w). When combined with neomycin sulfate, the compound agent had a significant and extensive antibacterial synergistic effect on 65 clinically isolated highly lethal Vibrio spp. that carried the gene encoding Tc toxin and were resistant to neomycin sulfate, and also had a cumulative antibacterial effect on 2 clinically isolated Vibrio spp. that did not carry the gene encoding Tc toxin and were sensitive to neomycin sulfate.

[0240] Example 7 In vivo preventive effect of the compound synergist combined with neomycin sulfate on neomycin sulfate-resistant and highly lethal Vibrio infection

[0241] 1 Experimental animals and materials

[0242] The experimental strain: Vibrio parahaemolyticus Vp32, a strain carrying the gene encoding Tc toxin and resistant to neomycin sulfate, was isolated from shrimp with typical glass seedling disease in the farm by the Aquatic Function 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 product, purchased from China Veterinary Drug Administration; (2) Neomycin sulfate composite synergist (abbreviated as composite synergist): from Example 3.

[0244] Experimental feed: Microparticle compound feed for shrimp fry No. 2, Beikesu (for factory production only), produced and sold by Qingyuan Haibei Co., Ltd.

[0245] Preparation of experimental drug feed: (1) Design and dosage of drug feed groups: The dosage of neomycin sulfate was 0.3g per kilogram of feed, and 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, a drug control group containing only compound synergist (0.3g compound synergist per kilogram of feed) was used as the drug control group, and a shell speed feed that did not contain neomycin sulfate and compound synergist was used as the blank control. (2) Preparation of drug feed: After the drug was evenly mixed with the raw materials of Beikesu feed according to the proportion (the drug replaced part of the soybean meal in the Beikesu formula), granulation was carried out according to the Beikesu production process of the Beikesu production plant of Qingyuan Haibei Co., Ltd.

[0246] Experimental whiteleg shrimp fry: purchased from Zhongshan Branch of Guangdong Hisingnong Group Co., Ltd., wet weight 40-60 mg

[0247] 2. Experimental plan:

[0248] The whiteleg shrimp fry were randomly divided into 9 groups, with 40 tails in each group, namely blank control CK (not challenged, fed with shell speed feed), positive challenge group (challenge treated, fed with shell speed feed), drug group 1# (not challenged, fed with bait containing only neomycin sulfate), drug group 2# (not challenged, fed with bait containing only compound synergist), comparison group 1# (challenge treated, fed with bait containing only neomycin sulfate), comparison group 2# (challenge treated, fed with bait containing only compound synergist), comparison group 3# (challenge treated, fed with 20% alternative bait of compound synergist), comparison group 4# (challenge treated, fed with 40% alternative bait of compound synergist), comparison group 5# (challenge treated, fed with 60% alternative bait of compound synergist), with 3 parallel groups in each group. Each group was fed the corresponding feed every day for 3 consecutive days, with 3 meals a day. The daily feed intake was 5% of the shrimp body weight. The water was cleaned and replaced 1 hour after feeding, with each water change of 25%. During the experiment, the water salinity was 20‰, the temperature was 28℃, the pH value was 7.5-7.8, and oxygen was added for 24 hours. 3 hours after the end of the drug administration, except for the blank control group, drug 1# and drug group 2#, the venom solution Vp32 was added to the water of the remaining 4 groups at a final concentration of (1-2)×10 5 cfu / mL, and soaked for 6 hours. After the challenge, each challenge group was transferred to a clean culture system (culture system parameters are the same as above) and observed for 24 hours, and the mortality rate of shrimp seedlings in each group was recorded.

[0249]

[0250] 3 Test results

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

[0252] It was concluded that the replacement of 20% to 60% of neomycin sulfate with a composite synergist had a better protective effect against the pathogenic Vibrio viridis than neomycin sulfate alone.

[0253] Table 15 Preventive effects of different groups of drugs on Vp32 virus attack

[0254]

[0255]

[0256] 4 Experimental Summary

[0257] The purified product of Wedelia serrata, the enzymatically purified product of Gastrodia elata, and the enzymatically purified product of Quassima chinensis are compounded in a ratio of 1:2:2 (i.e., the neomycin sulfate composite synergist of Example 3) for oral use. The protective effect against the pathogenic Vibrio vibrio of Viola viridis after replacing neomycin sulfate at a ratio of 20% to 60% is better than that of neomycin sulfate alone, and the optimal replacement ratio is 20% to 40%.

[0258] Example 8 In vivo therapeutic effect of a compound potentiator combined with neomycin sulfate on highly lethal Vibrio infections resistant to neomycin sulfate

[0259] 1 Experimental animals and materials

[0260] The experimental strain: Vibrio parahaemolyticus Vp32, a strain carrying the gene encoding Tc toxin and resistant to neomycin sulfate, was isolated from shrimp with typical glass seedling disease in the farm by the Aquatic Function 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 product, purchased from China Veterinary Drug Administration; (2) Neomycin sulfate composite synergist (abbreviated as composite synergist): from Example 3.

[0262] Experimental feed: Microparticle compound feed for shrimp fry No. 2, Beikesu (for factory production only), produced and sold by Qingyuan Haibei Co., Ltd.

[0263] Medicated feed: (1) Design and dosage of medicated feed groups: The dosage of neomycin sulfate is 0.3g per kilogram of feed, and the dosage of compound synergist is 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, a compound synergist dose alone (0.3g compound synergist per kilogram of feed) is used as the drug control group, and a shell speed feed that does not contain neomycin sulfate and compound synergist is used as the blank control. (2) Preparation of medicated feed: After the drug is evenly mixed with the raw materials of Beikesu feed according to the proportion (the drug replaces part of the soybean meal in the Beikesu formula), it is produced and granulated according to the formula and process of Beikesu produced by Qingyuan Haibei Co., Ltd.

[0264] Experimental whiteleg shrimp fry: purchased from Zhongshan Branch of Guangdong Hisingnong Group Co., Ltd., wet weight 40-60 mg

[0265] 2 Experimental plan

[0266] 1000 shrimps were randomly selected, weighing (50±5) mg / tail, and placed in a 98 cm × 60 cm × 34 cm white plastic box filled with 25 L of artificial seawater with a salinity of 20‰ for 0.5 days. Fresh Vibrio parahaemolyticus Vp32 bacterial suspension was added to the box to a final concentration of (3-5) × 10 4 cfu / mL, after immersion for 1.5 h, the shrimp fry were fished out and placed in sterile artificial seawater with a salinity of 20‰ for 10 s to remove the bacterial solution adhering to the shrimp surface. After the challenge, the shrimp fry were randomly divided into 7 groups, 40 in each group, including the positive challenge group (challenged, fed with shell speed feed), control group 1# (challenged, fed with bait containing only neomycin sulfate), control group 2# (challenged, fed with bait containing only compound synergist), control group 3# (challenged, fed with 20% compound synergist alternative bait), control group 4# (challenged, fed with 40% compound synergist alternative bait), control group 5# (challenged, fed with 60% compound synergist alternative bait), and blank control CK (not challenged, fed with shell speed feed), drug group 1# (not challenged, fed with bait containing only neomycin sulfate) and drug group 2# (not challenged, fed with bait containing only compound synergist), with 3 replicates per group. Each group was fed the corresponding feed three times a day, and the daily feed intake was 5% of the shrimp body weight. During the experiment, the water had a salinity of 20‰, a temperature of 28°C, a pH of 7.5-7.8, and 24-hour oxygenation. The shrimp fry were observed for 92 hours, and the mortality rate and changes in the appearance of the hepatopancreas of each group were recorded.

[0267]

[0268] 3 Test results

[0269] As shown in Table 16 and Figure 2 As shown in the experimental results, analysis of the results showed that during the observation period after the challenge, the survival rate of the positive control group was 5.0%, which was significantly lower than that of the neomycin sulfate alone group, the compound synergist alone group, and the neomycin sulfate compound synergist combination group. Compared with the 37.5% survival rate of the neomycin sulfate alone group, the survival rates of the three dose groups of neomycin sulfate combined with the compound synergist were significantly higher by 25%, 35%, and 22.5%, respectively.

[0270] From the analysis of the health status of the hepatopancreas of the surviving shrimps in Table 17, the hepatopancreas of the surviving shrimps in the positive control group all showed the phenomenon of vitreous seedlings and were at the level of dying ( Figure 3 In the three groups of compound enhancer combined with neomycin sulfate, >70% of the shrimp survived and the hepatopancreas was clear and full, which was a normal state. Figure 4 ), the proportion of normal shrimps was much higher than that of the neomycin sulfate single group or the compound synergist group, and significantly higher than that of the positive control group.

[0271] It can be concluded that the combination of compound enhancer and neomycin sulfate has a better therapeutic effect on Vibrio vibrio than neomycin sulfate alone.

[0272] Table 16 Effects of different groups of drugs on Vp32 virus attack

[0273]

[0274] Table 17 Hepatopancreas health status of surviving shrimps in different groups 92 hours after infection

[0275]

[0276]

[0277] 4 Experimental Summary

[0278] The purified product of Wedelia scabra, the enzymatically purified product of Gastrodia elata, and the enzymatically purified product of Quassinus chinensis are compounded in a ratio of 1:2:2 (the neomycin sulfate composite synergist of Example 3) for oral use. The oral administration of the product has good safety, and the therapeutic effect on vitreous seedling disease after replacing neomycin sulfate at a ratio of 20% to 60% is better than that of neomycin sulfate alone or the composite 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] At a large-scale, factory-scale shrimp farm in Weifang, Shandong Province, 14 ponds housing 80 to 100 shrimp recently experienced a slowed feeding rate (down from 35 minutes to 50 minutes), with daily mortality rates of 20 to 30 shrimp per pond. This phenomenon had only occurred for one day. The hepatopancreas of the prawns in the ponds, which were found to be albinos, was suspected to be caused by vitreous seed disease. Fluorescence PCR analysis of the hepatopancreas tissue of 3 shrimp per pond from these 14 ponds revealed that all samples tested positive for the HLVA and HLVB virulence genes, confirming a diagnosis of vitreous seed disease and requiring urgent drug intervention.

[0282] Experimental feed: high-end factory-produced D series 1# particle size shrimp feed, product number D6210 (ingredient 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 China Veterinary Drug Administration; (2) Neomycin sulfate composite synergist (abbreviated as composite synergist): from Example 3. (3) Composite synergist combined with neomycin sulfate: composite synergist: neomycin sulfate = 2:3 (w:w).

[0284] Test grouping: 9 pools were selected as verification pools, of which 3 used neomycin sulfate, numbered A to C; 3 used compound synergists, numbered D to F; 3 used compound synergists combined with neomycin sulfate, numbered G to I.

[0285] Dosage and Administration: Add 1g of neomycin sulfate, a compound enhancer, or a compound enhancer combined with neomycin sulfate per kilogram of shrimp feed. Use for 5 consecutive days, four times a day. For each kilogram of feed, add 0.15kg of tap water. Add the drug to the tap water and mix thoroughly. Then mix thoroughly with the feed. After mixing thoroughly, place in a cool, dark place to dry for 30 minutes before use.

[0286] Result judgment: If the death rate drops to ≤10 per pool during the medication period, it means that the drug is effective.

[0287] 2 Test results

[0288] The results in Table 18 show that in the three pools using neomycin sulfate, the mortality rate ranged from 16 to 27 fish per pool after 5 days of use, with none of the pools reducing the mortality rate to ≤10 fish per pool, resulting in an effective rate of 0. In the three pools using the compound synergist, the mortality rate ranged from 9 to 15 fish per pool after 5 days of use, with one pool reducing the mortality rate to ≤10 fish per pool, resulting in an effective rate of 33%. In the three pools using the compound synergist combined with neomycin sulfate, the mortality rate ranged from 6 to 8 fish per pool after 5 days of use, with all of the pools reducing the mortality rate to ≤10 fish per pool, resulting in an effective rate of 100%.

[0289] Table 18 Clinical application effects of compound synergists

[0290]

[0291]

[0292] 3 Experimental Summary

[0293] Timely use of compound enhancers combined with neomycin sulfate can effectively solve the problem of glass seedling disease during the breeding process, and the treatment effect is better than using neomycin sulfate alone or compound enhancers alone.

[0294] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. Use of Wedelia officinalis extract, Gastrodia elata extract and Quassinus officinalis extract in any one of (1) to (4): (1) Inhibit Vibrio growth in vitro; (2) preparing products that inhibit the growth of Vibrio; (3) preparing a neomycin sulfate synergist for inhibiting Vibrio growth; (4) Preparation of drugs for preventing and / or treating diseases caused by Vibrio infection.

2. The use according to claim 1, characterized in that The Wedelia chrysantha extract is prepared by the following preparation method: mixing Wedelia chrysantha with a deep eutectic solvent, extracting, and obtaining the Wedelia chrysantha extract; Preferably, the mass volume ratio of Wedelia chrysantha to the deep eutectic solvent is 1:(15-25); Preferably, the deep eutectic solvent comprises choline chloride and malic acid; Preferably, the Wedelia includes Wedelia spp.

3. The use according to claim 1, characterized in that The Gastrodia elata extract is an enzymatic hydrolysis extract of Gastrodia elata; preferably, the Gastrodia elata extract is prepared by the following preparation method: mixing Gastrodia elata with a composite enzyme preparation and a buffer, enzymatic hydrolysis, enzyme inactivation, separation and collection of a precipitate; and supercritical CO2 extraction of the precipitate to obtain the Gastrodia elata extract; Preferably, the complex enzyme preparation comprises cellulase, pectinase and β-glucanase; Preferably, the mass of the complex enzyme preparation is 0.2% to 0.8% of the mass of Gastrodia elata; Preferably, the conditions for the supercritical CO2 extraction are: 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; Preferably, the Gastrodia elata includes Gastrodia elata.

4. The use according to claim 1, characterized in that The quassin extract is an enzymatic hydrolysis extract of quassin; preferably, the quassin extract is prepared by the following preparation method: mixing quassin with a buffer and a complex enzyme preparation, enzymatically hydrolyzing, inactivating the enzyme, and obtaining an enzymatic hydrolyzate; mixing the enzymatic hydrolyzate with ethanol, extracting, and performing solid-liquid separation to obtain a supernatant A and a precipitate; mixing the precipitate with ethyl acetate, extracting, and performing solid-liquid separation to obtain a supernatant B; and combining the supernatant A and the supernatant B to obtain the quassin extract; Preferably, the complex enzyme preparation comprises cellulase, pectinase and xylanase; Preferably, the mass of the complex enzyme preparation is 0.5% to 2% of the mass of Quassima; Preferably, the mass volume ratio of the Quassin to the buffer solution is 1:(8-15); Preferably, the enzymatic hydrolysis conditions are 40-50°C, 100-200 rpm for 1-3 hours; Preferably, the extraction is ultrasonic extraction.

5. The use according to any one of claims 1 to 4, characterized in that The Vibrio described in (1) to (3) includes Vibrio carrying a gene encoding Tc toxin; Preferably, the Vibrio includes Vibrio carrying a gene encoding Tc toxin and being resistant to neomycin sulfate; Preferably, the Vibrio includes at least one of Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, and Vibrio erwinia.

6. A neomycin sulfate combined synergist for inhibiting the growth of Vibrio, comprising the Wedelia chrysantha extract, Gastrodia elata extract, and Quassinus chinensis extract described in any one of claims 1 to 5; Preferably, the mass ratio of the Wedelia chrysantha extract, Gastrodia elata extract and Quassinus sylvestris extract is 1:2:(2-4).

7. A Vibrio-inhibiting drug comprising the Wedelia chrysantha extract, Gastrodia elata extract and Quassinus chinensis extract as claimed in claims 1 to 5; Preferably, the Vibrio includes Vibrio that carries a gene encoding Tc toxin and is resistant to neomycin sulfate.

8. The Vibrio inhibitory drug according to claim 7, characterized in that The antibacterial drug also includes neomycin sulfate.

9. Use of the neomycin sulfate combined synergist for inhibiting Vibrio growth according to claim 6 or the Vibrio inhibitory drug according to claim 7 or 8 in any one of (a1) to (a2): (a1) Inhibit Vibrio growth in vitro; (a2) preparing drugs for preventing and / or treating diseases caused by Vibrio infection; Preferably, the Vibrio includes a Vibrio carrying a gene encoding Tc toxin; Preferably, the Vibrio includes Vibrio carrying a gene encoding Tc toxin and being resistant to neomycin sulfate; Preferably, the Vibrio includes at least one of Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, and Vibrio erwinia.

10. A method for inhibiting Vibrio, comprising the step of treating Vibrio with the Vibrio inhibitory drug according to claim 7 or 8.

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