Macleaya cordata recovery prescription medicine for preventing and treating fish nocardia disease as well as preparation method and application of macleaya cordata recovery prescription medicine

Abstract

The invention discloses a macleaya cordata recovery prescription medicine for preventing and treating fish nocardia disease as well as a preparation method and application of the macleaya cordata recovery prescription medicine. The compound medicine consists of 10 to 30 percent of macleaya cordata alkaloid hydrochloride compound, 5 to 15 percent of chitosan quaternary ammonium salt, 10 to 20 percent of mannan oligosaccharide and a medicinal carrier. The preparation method comprises the following steps: carrying out freezing and heat shock pretreatment on a macleaya cordata raw material, and carrying out ultrasonic-flash synergistic extraction by adopting acidified ethanol; performing concentration, macroporous resin purification and high-speed counter-current chromatography separation on the extracting solution to obtain a high-purity macleaya cordata alkaloid hydrochloride compound; and finally, mixing the compound with chitosan quaternary ammonium salt and a carrier, granulating, and carrying out enteric coating. According to the method, active alkaloids such as sanguinarine and chelerythrine in macleaya cordata can be efficiently extracted and refined. According to the compound medicine, through scientific compatibility, for example, macleaya cordata alkaloid is antibacterial, chitosan quaternary ammonium salt and mannan oligosaccharide are used for enhancing immunity and targeting, the bioavailability is improved by utilizing a modern preparation technology, and the compound medicine has a good effect of preventing and treating fish nocardia disease.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture disease prevention and control and veterinary drug preparation technology, specifically relating to a Boraluo Fuxing formula for the prevention and treatment of nocardiosis in fish, its preparation method and application. Background Technology

[0002] Nocardiosis is a globally distributed and serious chronic bacterial disease caused by *Nocardia amberjack*, primarily affecting high-value fish species such as largemouth bass, grouper, and snakehead. The pathogen is a Gram-positive, aerobic filamentous bacillus whose pathogenicity lies in its ability to invade the host's internal organs, such as the liver, spleen, and kidneys, forming persistent granulomatous structures. This pathological change not only leads to organ failure but also constitutes a physical and immune barrier, making it difficult for most antibacterial drugs to effectively penetrate the core of the lesions, thus causing difficulties in clinical treatment. Currently, prevention and treatment mainly rely on antibiotics such as florfenicol and sulfonamides; however, long-term use has led to the widespread emergence of drug-resistant strains, and there are risks of drug residues and food safety.

[0003] Therefore, the inventors published an article in the journal Microorganisms entitled "Serene and Chelidonium Alkyl ...

[0004] However, the direct use of sanguisorbin and celandine as treatments for nocardiosis in fish has the following drawbacks: (1) Poor targeting: Nocardia amberjack is a facultative intracellular parasite. It can be phagocytosed by the host's macrophages, but instead of being killed, it can survive and multiply within the cells and migrate throughout the body with the macrophages, forming granulomas. This survival mode provides the pathogen with a natural physical barrier, allowing it to effectively evade the host's humoral immunity and the attack of many conventional drugs. Although sanguisorbin and celandine are small molecule alkaloids with a certain degree of membrane permeability, their distribution, metabolism, and efficiency in reaching lesions (especially intracellular infection sites) are difficult to control. After direct oral or injectable administration, the drugs may be rapidly metabolized and excreted, or widely distributed in non-target organs, resulting in insufficient effective concentrations within infected macrophages or granulomatous lesions. This is one of the key reasons for the limited efficacy of treating facultative intracellular parasites like Nocardia.

[0005] (2) Single mechanism of action, easily inducing drug resistance or failing to eradicate infection: Although studies show that sanguinarine and celandine can target multiple sites such as disrupting cell membranes and inhibiting energy metabolism, as single chemical components, long-term use may still screen out drug-resistant strains. More importantly, for granulomas and intracellular parasitic states formed by Nocardia, simply relying on antibacterial compounds to kill bacteria is insufficient to address the problems of bacterial immune escape and physical barriers. Drugs are difficult to penetrate to the core of granulomas and cannot effectively activate the host's immune system to actively clear pathogens.

[0006] (3) Low efficiency of active ingredient extraction and purification: Traditional extraction processes (such as hot reflux and percolation) have low extraction yield of borax alkaloids, and the crude extract obtained has complex components (containing a large number of tannins, pigments, polysaccharides and other impurities), resulting in low purity and unstable quality of active ingredients, making it difficult to conduct accurate efficacy evaluation and preparation quality control.

[0007] In summary, current technologies lack a systematic solution that integrates formulation technologies from obtaining high-purity active ingredients to designing synergistic compound formulations targeting intracellular bacterial infections, and finally to achieving efficient oral delivery. Therefore, developing a dedicated compound drug with clearly defined components, a well-defined mechanism, significant efficacy, and safe use is of great theoretical and practical value for overcoming the challenge of controlling nocardiosis in fish. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide a compound drug for the prevention and treatment of nocardiac disease in fish, which can efficiently extract and purify active alkaloids from *Botrytis cinerea*, enhance the effect on intracellular parasites through scientific compound design, and improve bioavailability using modern formulation technology.

[0009] The objective of this invention is achieved through the following technical solution: A compound drug for the prevention and treatment of nocardiosis in fish, comprising the following components: 10%-30% of borax alkaloid hydrochloride complex, 5%-15% of chitosan quaternary ammonium salt, 10%-20% of mannan oligosaccharide, with the remainder being a pharmaceutical carrier.

[0010] Preferably, the purslane alkaloid hydrochloride complex comprises sanguisorbin hydrochloride and celandine hydrochloride, wherein the mass ratio of sanguisorbin hydrochloride to celandine hydrochloride is 1:(0.5-2).

[0011] A key step was combining sanguisorbin and celandine in a specific, experimentally validated ratio (1:0.5-1:2). Studies have confirmed that this combination can exert a synergistic effect on the "antibacterial-anti-inflammatory-repair" chain. This synergy not only reduces the required effective concentrations of each drug, thereby broadening the therapeutic window and reducing toxicity risks, but also reduces the possibility of bacteria developing single-drug resistance through multi-target attacks (such as sanguisorbin disrupting the membrane and celandine inhibiting key enzymes).

[0012] Meanwhile, chitosan quaternary ammonium salt is a cationic polysaccharide derivative with excellent film-forming properties, antibacterial activity, and biocompatibility. In formulations, it plays multiple roles: a) its positive charge allows it to bind to negatively charged bacterial cell membranes, interfering with the initial colonization of Nocardia in the intestinal mucosa or wounds, reducing the chance of infection at its source. b) it forms a physical barrier in the intestines or wounds and continuously releases antibacterial components, increasing local drug concentration. c) chitosan quaternary ammonium salt also has the potential to serve as a carrier material, potentially improving the solubility and stability of sanguisorbin / chelidonine through ion interactions or encapsulation, achieving sustained release and prolonging the duration of action.

[0013] Furthermore, the addition of manno-oligosaccharides can address immunosuppression and gut microbiota disruption. Manno-oligosaccharides are prebiotics that cannot be digested by the host but can be utilized by beneficial bacteria. They selectively promote the proliferation of beneficial bacteria such as Bifidobacteria and Lactobacillus, competitively inhibiting the colonization of pathogenic bacteria, thereby maintaining or restoring the balance of intestinal flora and counteracting the negative impact of antibacterial components on the gut microbiota. Moreover, manno-oligosaccharides themselves are an immune enhancer (immune adjuvant). They can activate macrophages, enhance their phagocytic capacity, and stimulate the body to produce an immune response.

[0014] Preferably, the pharmaceutical carrier is a mixture of microcrystalline cellulose, pregelatinized starch and magnesium stearate.

[0015] As a preferred dosage form, enteric-coated granules are preferred.

[0016] This invention also provides a method for preparing the Boluo Fuxing formula, which includes the following steps: (1) Freezing and short-term high-temperature heat shock treatment of the raw materials from the Boluo River; (2) The treated Boluhui raw material was subjected to ultrasonic-flash synergistic extraction with acidified ethanol to obtain the extract; (3) The extract is concentrated and purified by macroporous adsorption resin. The target eluent is collected and concentrated to obtain an extract. (4) The extract was separated by high-speed countercurrent chromatography to obtain sanguisorbine hydrochloride and celandine hydrochloride, respectively; (5) Combine sanguisorbin hydrochloride and celandine hydrochloride in a certain proportion, then mix with chitosan quaternary ammonium salt, mannan oligosaccharide, microcrystalline cellulose, pregelatinized starch and magnesium stearate, granulate, and enteric coating to obtain the aforementioned Boluo Fuxing formula.

[0017] Preferably, in step (1), the freezing temperature is -40°C to -60°C, and the time is 24-48 h.

[0018] Preferably, in step (1), the heat shock temperature is 105℃-120℃ and the time is 3-5 min.

[0019] As a preferred embodiment, in step (2), the ultrasonic-flash synergistic extraction step is as follows: add an 82% ethanol solution with concentrated hydrochloric acid adjusted to pH 3.5 to the raw material powder, set the ultrasonic power to 420 W, the high-speed shearing speed to 7200 r / min, the water bath temperature to 53℃, extract for 20 min, filter, and after the filter residue is re-extracted, combine the two filtrates, and concentrate under reduced pressure at 52℃ and -0.085 MPa until there is no alcohol odor, thus obtaining the extract.

[0020] Preferably, in step (4), the two-phase solvent system used in the high-speed countercurrent chromatography is chloroform-methanol-0.2mol / L hydrochloric acid aqueous solution with a volume ratio of 4:2:2.

[0021] The present invention also provides applications of the above-mentioned Boluo Fuxing formula, the applications of which include (1), (2), (3) and (4): (1) Disrupts the integrity of the cell wall / membrane structure of Nocardia; (2) Inhibition of Nocardia amberjack growth based on disrupting the integrity of Nocardia cell wall / membrane structure; (3) To prepare drugs for the prevention and treatment of nocardiosis in fish; (4) Prepare feed additives for the prevention and treatment of Nocardia disease in fish.

[0022] Compared with the prior art, the advantages or beneficial effects of the technical solution of this application include: (1) Scientifically designed compound with a clear and multifaceted synergistic mechanism: This invention is not a simple formulation, but rather constructs a triple synergistic system of "direct sterilization + colonization inhibition + immune regulation". The core is a high-purity alkaloid hydrochloride complex, whose specific ratio (1:0.5-1:2) has been experimentally proven to have a synergistic antibacterial effect; supplemented by chitosan quaternary ammonium salt to interfere with pathogen adhesion, and mannan oligosaccharides to improve the intestinal immune microenvironment. This design targets the characteristics of chronic Nocardia infection, providing comprehensive intervention through multiple pathways, and its effect is significantly better than that of a single component or a simple mixture.

[0023] (2) Advanced dosage form design significantly improves bioavailability and drug safety: The final product is designed as enteric-coated granules, which realizes targeted drug delivery. This dosage form can protect the acid-labile active ingredients and safely pass through the fish's stomach, where they are concentrated and released in the neutral intestinal environment, thus improving the drug's bioavailability and efficacy.

[0024] (3) Innovative extraction and purification processes result in high product purity and controllable quality: The innovative "freezing-heat shock" pretreatment combined with ultrasonic-flash synergistic extraction significantly improves the extraction efficiency of the target alkaloids. Subsequent enrichment with macroporous resin and purification by high-speed countercurrent chromatography (HSCCC) forms an efficient and continuous purification pathway, which can stably prepare sanguisorbin and chelidonine hydrochloride complexes with a total purity ≥95%, overcoming the defects of complex composition and unstable quality of traditional crude extracts. Attached Figure Description

[0025] Figure 1 This is a liquid chromatogram of the main alkaloids sanguisorbin and celandine from the present invention.

[0026] Figure 2 The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the main alkaloids of salicylate (A) and celandine (B) against Nocardia amberjack were determined.

[0027] Figure 3 The ultrastructure of Nocardia amberjack cells in the drug-treated and control groups as observed by transmission electron microscopy (TEM).

[0028] Figure 4 The changes in β-galactosidase activity and total protein content, which are related indicators of cell membrane permeability, were observed in each experimental group.

[0029] Figure 5 The survival rate of largemouth bass in each experimental group during the Nocardia purpureus challenge experiment.

[0030] Figure 6 Microscopic images of the head kidney tissue of largemouth bass in each experimental group at the end of the experiment. Detailed Implementation

[0031] The following detailed description of the embodiments of this application, in conjunction with the accompanying drawings, will provide a thorough understanding of how this application uses technical means to solve technical problems and achieve corresponding technical effects, enabling its implementation. The embodiments of this application and the various features within them can be combined with each other without conflict, and all resulting technical solutions are within the protection scope of this application.

[0032] It should be clearly stated that the embodiments described below are only some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0033] Example 1: Extraction of the alkaloid hydrochloride complex from *Botrytis cinerea* (1) Raw material pretreatment: Take 5.0 kg of dried Bodhi leaves and crush them through a 40-mesh sieve. Spread the powder evenly on a stainless steel tray and freeze it in an ultra-low temperature freezer at -40℃ to -60℃ for 36 h. Quickly transfer the frozen powder to a short-time hot air treatment device preheated to 105℃-120℃, treat it for 3-5 min, and then immediately take it out and place it in a desiccator to cool to room temperature.

[0034] (2) Ultrasonic-flash synergistic extraction: Weigh 1.0 kg of the raw material powder after the above treatment and place it in a 10 L ultrasonic extraction vessel. Add 9.0 L of 82% ethanol solution with pH adjusted to 3.5 by concentrated hydrochloric acid. Set the ultrasonic power to 420 W (intermittent mode), the high-speed shear speed to 7200 r / min, and the water bath temperature to 53℃, and extract for 20 min. Filter, and extract the residue again with 7.0 L of the same solvent using the same method. Combine the two filtrates and concentrate under reduced pressure at 52℃ and -0.085 MPa until no alcohol odor remains, to obtain a dark reddish-brown concentrate.

[0035] (3) Gradient purification with macroporous resin: The concentrate was loaded onto a pretreated HPD-100 macroporous resin column at a flow rate of 1.8 BV / h. The column was washed sequentially with 5 BV of deionized water and 2 BV of 25% ethanol to remove impurities. Finally, the target component was eluted with 5 BV of 82% ethanol at a flow rate of 0.8 BV / h. The eluent was collected, concentrated under reduced pressure, and dried under vacuum to obtain a brownish-red alkaloid-enriched powder.

[0036] (4) High-speed countercurrent chromatography (HSCCC) fine separation: The determination was performed using a TBE-300A HSCCC instrument. The solvent system was: chloroform:methanol:0.2 mol / L HCl = 4:2:2 (v / v), and the phases were separated after full equilibration. The upper phase was the stationary phase, and the lower phase was the mobile phase. The main unit speed was 480 r / min, and the mobile phase flow rate was 9 mL / min. Take 3.0 g of the above powder, dissolve it in the mixture of the upper and lower phases, filter it, and then inject it. According to the 270 nm UV detection spectrum, peak I (sanguisorbine hydrochloride) was collected at 125-142 min, and peak II (chelidonine hydrochloride) was collected at 195-210 min. Figure 1 The collected solutions were concentrated and freeze-dried to obtain sanguisorbine hydrochloride and celandine hydrochloride crystals. UPLC analysis showed purities of 99.6% and 99.2%, respectively.

[0037] (5) Preparation of complexes with different ratios: The refined sanguisorbine hydrochloride (S) and celandine hydrochloride (C) powders were accurately weighed and mixed in mass ratios of S:C = 2:1, 1:1, and 1:2 to prepare complexes SC-2:1, SC-1:1, and SC-1:2, respectively, for subsequent ratio screening tests.

[0038] Example 2: Evaluation of the synergistic antibacterial effect of compound drugs in vitro (1) Strains culture and drug preparation The test strain was *Nocardia amberjack* strain 20811 (this strain is the same as the *Nocardia amberjack* strain experimental number 21811 in the published article titled "Borneoline and Chelidonium Alkyl ...

[0039] (2) Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) The MICs of sanguisorbin and chelidonine against Nocardia amberjack were determined using a modified resazurin microwell dilution method. In short, the bacterial suspension (1 × 10⁻⁶) was diluted using this method. 5 CFU / mL of the drug was added to 96-well plates containing sanguisorbin and chelidonine in serially diluted two-fold gradients. Wells containing 1% DMSO in BHI broth served as negative controls, while wells containing only bacterial suspension and no drug served as positive controls. After incubation at 28°C for 5 days, 50 μL of resveratrol solution (0.01%) was added to each well, and incubation continued for 24 h to observe color changes. MIC was defined as the lowest drug concentration that prevented the color from changing from blue to pink. To determine MBC, 100 μL of culture from wells without bacterial growth was plated onto BHIA plates and incubated for 5 days. MBC was defined as the lowest drug concentration at which no colonies grew on the plate. Results are as follows: Figure 2As shown, when the sanguinarine concentration > 62.5 μg / mL and the chelerythrine concentration > 7.8 μg / mL, the bacterial suspension in the 96-well plate turns blue (indicating no viable bacteria), indicating that these concentrations are the MICs of the two substances respectively. The MBC was determined by subculturing from the wells without growth (≥ MIC). For the treatment groups with sanguinarine concentration > 250.0 μg / mL or chelerythrine concentration > 62.5 μg / mL, no colony formation was observed after culturing, and these concentrations were defined as the corresponding MBCs.

[0040] (3)Determination of the fractional inhibitory concentration (FIC) of sanguinarine and chelerythrine The FIC was determined by the checkerboard method. An 8×8 checkerboard of the sanguinarine (S), chelerythrine (C) complex SC was designed. The solutions of S and C were serially diluted 2-fold along the rows and columns, covering a concentration range from 1 / 8 to 2 times their respective MICs. 50 μL of different concentrations of S solution and 50 μL of different concentrations of C solution were added to each well, and then 100 μL of the bacterial suspension was added. The culture conditions were the same as above. After the culture ended, the bacterial growth in each well of the checkerboard was read. Calculate the fractional inhibitory concentration index: FIC index = (MIC of S in combination / MIC of S alone) + (MIC of C in combination / MIC of C alone). FIC ≤ 0.5 indicates strong synergy, 0.5 < FIC ≤ 1.0 indicates synergy, 1.0 < FIC ≤ 2.0 indicates no interaction, and FIC > 2.0 indicates antagonism. The results showed that: within the tested ratio range, SC-1:1 showed the strongest synergistic antibacterial activity (the lowest MIC and the smallest FIC index), and SC-2:1 and SC-1:2 also showed synergistic effects, confirming the effectiveness of the ratio range from 1:0.5 to 1:2.

[0041] (4)Observation by transmission electron microscopy (TEM) Nocardia in the logarithmic growth phase was taken and treated with the SC-1:1 complex at a concentration of 1 / 2 MIC for 6 h. A drug-free control was set simultaneously. The bacteria were collected by centrifugation, fixed with 2.5% glutaraldehyde, dehydrated by conventional ethanol gradient, embedded in epoxy resin, ultrathin sectioned, double-stained with uranyl acetate-lead citrate, and then observed under a transmission electron microscope. The results are as Figure 3 shown: The bacteria in the control group had a complete morphology, clear cell wall and membrane structures, and uniform cytoplasm; the bacteria in the treatment group showed obvious morphological abnormalities, with the cell wall shrunk and blurred, the cell membrane separated from the wall to form gaps, and damaged membrane structures and leakage of intracellular contents were visible in local areas. It was proved that the complex could seriously damage the integrity of the bacterial cell wall / membrane structure.

[0042] (5)Determination of cell membrane permeability To quantitatively assess the drug's disruptive effect on cell membranes, extracellular β-galactosidase activity and total protein content were measured. *Nocardia amberjack* was treated with different concentrations (1 / 2 MIC, MIC) of the SC-1:1 complex for 4 h, and the supernatant was collected by centrifugation. A control group (CK) with an equal volume of PBS was included. The supernatant was then reacted with ONPG as a substrate at 37℃ for 30 min, and the absorbance (OD) at 420 nm was measured. 420 The β-galactosidase activity was obtained by taking another portion of the supernatant and measuring the absorbance (OD) at 595 nm using the Bradford method. 595 The total protein content is obtained by calculating the total protein content. The results are as follows: Figure 4 As shown, with the increase of SC-1:1 drug concentration, the OD in the supernatant... 420 and OD 595 All values ​​showed a significant dose-dependent increase ( P The result was <0.01, indicating that the treatment led to increased bacterial cell membrane permeability and a large leakage of intracellular enzymes and proteins, further quantitatively confirming the damaging effect of the drug on the cell membrane.

[0043] Example 3: Preparation and in vivo protective effect evaluation of compound drug enteric-coated granules (1) Preparation of compound enteric-coated granules Weigh the following ingredients (based on 1 kg of granules): 200 g of the SC-1:1 complex obtained in Example 1, 100 g of chitosan quaternary ammonium salt (degree of substitution ≥95%), 150 g of mannan oligosaccharide (DP2-10), 450 g of microcrystalline cellulose, 80 g of pregelatinized starch, and 20 g of magnesium stearate. Mix all powder components evenly using an equal-volume incremental method. Compress, crush, and granulate the mixed powder using a dry granulator to obtain pellets with a particle size of 0.8-1.2 mm. Feed the pellets into a fluidized bed coating machine, using acrylic resin II (Utec L100-55) aqueous dispersion (12% solid content) as the coating solution, and perform enteric coating using a bottom-spray process, controlling the coating weight gain to approximately 9%. After coating, dry and mature the granules to obtain the enteric-coated granules of this invention.

[0044] (2) Laboratory animals and experimental design Healthy largemouth bass (average weight 10.5 ± 1.3 g) were purchased from a fish farm in Huzhou. Before the experiment, the fish were acclimatized for two weeks in a 300 L fiberglass tank with continuous aeration, maintaining a water temperature of 25 ± 1 °C, pH 7.2 ± 0.2, and dissolved oxygen > 5.0 mg / L. During the acclimatization period, they were fed a commercially available basal diet (crude protein ≥ 45%, crude fat ≥ 8%) twice daily. After acclimatization, the fish were randomly divided into four groups (90 fish per group), with three replicates per group. Group A: No virus challenge + basal diet (blank control group); Group B: Virus challenge + basal diet + 500 mg / kg of the compound enteric-coated granules of this invention (SC drug group); Group C: Virus challenge + basal diet + 500 mg / kg of *Polygonum hydropiper* crude extract granules (control drug group); Group D: Virus challenge + basal diet (virus challenge control group). After being fed in groups for 4 days, the fish were artificially infected via intraperitoneal injection with Nocardia amberjack suspended in sterile phosphate-buffered saline (PBS) at a concentration of 1 × 10⁻⁶. 7 CFU / tail. The control group was injected with an equal volume of sterile PBS.

[0045] (3) Sample collection and analysis The protective effect of drug treatment after 35 days was evaluated using multiple indicators, including relative protection rate (RPS), bacterial load, clinical symptoms, and histopathological changes. At the experimental endpoint, liver tissue was aseptically collected, and the bacterial load of *Nocardia amberjack* was quantitatively analyzed using the TaqMan qPCR specific method. Mortality was recorded daily throughout the 35-day experiment, and the relative protection rate (RPS) was calculated using the following formula: [1 − (mortality rate in the treatment group / mortality rate in the control group)] × 100. For pathological evaluation, gross lesions in the liver and spleen were examined, with particular attention to characteristic white granulomatous nodules. Subsequently, the tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for histological examination to assess granulomatous inflammation and necrosis.

[0046] (4) Experimental results At the end of the trial, the survival rates of groups A, B, C, and D were 100.0%, 54.4%, 34.4%, and 0%, respectively. Figure 5 This indicates that both drug SC and the control drug, *Polygonum hydropiper* extract, have certain protective effects; among them, the RPS of drug SC reached 54.4%, which was significantly better than that of the control drug group (34.4%). P<0.01). Histopathological observation of the head kidney tissue revealed that group D fish were covered with typical granulomatous nodules, group C had fewer nodules, while group B only occasionally showed small early granuloma rudiments, with basically normal tissue structure; group A showed no lesions. These results indicate that drug SC can alleviate tissue damage caused by Nocardia amberjack challenge. qPCR quantitative analysis of liver bacterial load showed that group D had (3.22±0.48)×10⁻⁶. 6 Copies / mg, Group C was (4.35±1.18)×10 4 In group C, the number of copies / mg decreased significantly to (3.31±0.85)×10⁻⁶. 3 The number of copies / mg was nearly three orders of magnitude lower than in group A, and significantly lower than in group B. P <0.01).

[0047] The above embodiments fully demonstrate that the Boruo Fuxing formula provided by the present invention obtains high-purity active ingredients through its innovative preparation process, and achieves efficient and safe prevention and treatment of nocardiosis in fish through scientific compound and dosage form design.

Claims

1. A botanical drug for controlling Nocardia disease of fish, characterized in that, It is composed of the following components: 10%-30% of the phytoalkaloid hydrochloride complex, 5%-15% of the chitosan quaternary ammonium salt, 10%-20% of the mannan oligosaccharide, and the remainder is a pharmaceutical carrier.

2. The Plumbago reverse drug of claim 1, wherein, The *Boluocybe salsa* alkaloid hydrochloride complex comprises sanguisorbin hydrochloride and celandine hydrochloride, wherein the mass ratio of sanguisorbin hydrochloride to celandine hydrochloride is 1:(0.5-2).

3. The Plumbago reverse drug of claim 1, wherein, The pharmaceutical carrier is a mixture of microcrystalline cellulose, pregelatinized starch and magnesium stearate.

4. The Plumbago reverse-phase medicine according to any one of claims 1-3, characterized in that, The dosage form is enteric-coated granules.

5. A method for preparing a purgative formula, characterized in that, Includes the following steps: (1) Freezing and short-term high-temperature heat shock treatment of the raw materials from the Boluohui; (2) The treated Boluhui raw material was subjected to ultrasonic-flash synergistic extraction with acidified ethanol to obtain the extract; (3) The extract is concentrated and purified by macroporous adsorption resin. The target eluent is collected and concentrated to obtain an extract. (4) The extract was separated by high-speed countercurrent chromatography to obtain sanguisorbine hydrochloride and celandine hydrochloride, respectively; (5) Combine sanguisorbin hydrochloride and celandine hydrochloride in a certain proportion, then mix with chitosan quaternary ammonium salt, mannan oligosaccharide, microcrystalline cellulose, pregelatinized starch and magnesium stearate, granulate, and enteric coating to obtain the aforementioned Boluo Fuxing formula.

6. The production method according to claim 5, wherein In step (1), the freezing temperature is -40℃ to -60℃, and the time is 24-48 h.

7. The preparation method according to claim 5, characterized in that, In step (1), the heat shock temperature is 105℃-120℃ and the time is 3-5 min.

8. The preparation method according to claim 5, characterized in that, In step (2), the ultrasonic-flash synergistic extraction step is as follows: add 82% ethanol solution with concentrated hydrochloric acid to the raw material powder, adjust the pH to 3.5, set the ultrasonic power to 420 W, the high-speed shearing speed to 7200 r / min, the water bath temperature to 53℃, extract for 20 min, filter, and after the filter residue is re-extracted, combine the two filtrates, and concentrate under reduced pressure at 52℃ and -0.085 MPa until there is no alcohol odor, thus obtaining the extract.

9. The preparation method according to claim 5, characterized in that, In step (4), the two-phase solvent system used in the high-speed countercurrent chromatography is chloroform-methanol-0.2 mol / L hydrochloric acid aqueous solution, with a volume ratio of 4:2:

2.

10. The application of the Boluo Fuxing formula as described in claims 1-4 and the Boluo Fuxing formula prepared according to claims 5-9, characterized in that, Including (1), (2), (3) and (4): (1) Disrupts the integrity of the cell wall / membrane structure of Nocardia; (2) Inhibition of Nocardia amberjack growth based on disrupting the cell wall / membrane structure of Nocardia amberjack; (3) To prepare drugs for the prevention and treatment of nocardiosis in fish; (4) Prepare feed additives for the prevention and treatment of Nocardia disease in fish.

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