A composition for enhancing disease resistance in frogs and its application

Abstract

This invention discloses a composition for enhancing the disease resistance of frogs and its applications. The composition mainly consists of the core active peptide Peptide-3, astragalus polysaccharide, ginseng polysaccharide, prepared rehmannia root, and rosmarinic acid. Targeting "tiltosis and cataracts" in frogs caused by Elizabethanella miltiorrhiza, this invention works based on a synergistic mechanism of "targeted sterilization—immune warning—neural repair": Peptide-3 destroys the cell membrane of drug-resistant bacteria through a physical perforation mechanism to reduce pathogen load; plant polysaccharides induce non-specific immune warning in the body; prepared rehmannia root and rosmarinic acid synergistically scavenge free radicals, enhance SOD activity, and inhibit the expression of the pro-inflammatory factor TNF-α, precisely repairing damage to the central nervous system and eye tissues. Experiments show that the composition of this invention can increase the survival rate of diseased frogs to 92.4%, significantly reduce the incidence of tilted head and cataracts, and achieve a technological leap from single sterilization to comprehensive protection of the body.

Description

Technical Field

[0001] This invention belongs to the fields of aquaculture and biomedicine, specifically relating to a composition for improving the disease resistance of frogs, and the application of this composition in preventing and treating symptoms of "head tilt and cataracts" in frogs caused by Elizabethkingia miricola. Background Technology

[0002] In intensive black-spotted frog farming, with increasing stocking density, various bacterial diseases frequently occur. Among them, "head tilting and cataract disease" caused by Elizabethan mil has become a major killer threatening the frog farming industry. This pathogen is highly invasive, not only causing severe septicemia in frogs but also crossing the blood-brain barrier and localizing in the eye tissue, causing infected frogs to exhibit obvious neurological and ocular damage symptoms such as head tilting, circling, cloudy eyes, and even cataracts. Because this disease is usually accompanied by extremely high mortality and disability rates, once it breaks out, it often brings devastating economic losses to farmers.

[0003] Currently, traditional control methods for Elizabethan mil mainly rely on chemical antibiotics such as florfenicol and enrofloxacin. However, clinical practice has shown that Elizabethan mil exhibits strong multidrug resistance, and conventional drugs have low bactericidal efficiency in aquatic applications, and are difficult to achieve effective therapeutic concentrations in the brain and eye tissues through blood circulation. More seriously, while antibiotics can inhibit the reproduction of some pathogens, they cannot repair the irreversible damage to the central nervous system and optic nerve caused by the intense "cytokine storm" induced by the pathogen. Therefore, even under drug control, the survival quality and feeding ability of recovered frogs are still difficult to restore to normal levels.

[0004] Therefore, the concept of this invention lies in breaking through the limitations of single antibiotic prevention and treatment through a synergistic three-pronged strategy of "targeted sterilization, immune early warning, and neural repair." First, the antimicrobial peptide Peptide-3, with its strong cationic properties, directly kills drug-resistant bacteria through a physical perforation mechanism, circumventing chemical resistance. Second, Astragalus membranaceus and ginseng polysaccharides are used to construct a non-specific immune defense network, enhancing lysozyme activity and achieving early pathogen blocking. Finally, Rehmannia glutinosa and rosmarinic acid are creatively introduced to precisely target brain and eye inflammation. By scavenging free radicals, enhancing SOD activity, and inhibiting the overexpression of the pro-inflammatory factor TNF-α, neuroinflammation is fundamentally suppressed, achieving a technological leap from simple sterilization to functional protection of the body's organs. Summary of the Invention

[0005] This invention aims to address the lack of effective prevention and treatment methods for "head tilt and cataract disease" in frogs caused by Elizabethkingia miricola, and the technical bottleneck of traditional antibiotics' inability to penetrate the blood-brain barrier and repair nerve damage. This invention provides a composite composition with synergistic effects of "targeted bactericidal action, immune warning, and nerve repair," aiming to significantly reduce the mortality and disability rates of diseased frogs.

[0006] On one hand, the present invention provides a composition for improving the disease resistance of frogs, which comprises the following components in parts by weight: Core active peptide Peptide-3: 20 portions, the amino acid sequence of which is shown in SEQ ID NO:3; Immune enhancers: Astragalus polysaccharide 30 parts, Ginseng polysaccharide 20 parts; Rehmannia glutinosa 15 parts; Anti-stress repair agent: 10 parts rosmarinic acid; Stabilizing carrier: 5 parts of nano-sized montmorillonite powder.

[0007] Preferably, the core active peptide Peptide-3 has strong cationic properties, with a theoretical isoelectric point (pI) of around 10.5. This property enables it to specifically bind to the negatively charged cell membrane of Elizabethanella miltiorrhiza via electrostatic attraction and destroy the pathogen through a physical perforation mechanism, thereby effectively circumventing the problem of chemical resistance.

[0008] Preferably, the composition is micronized to a particle size range of 50-80 μm; the ultra-micronized particle size facilitates the rapid release and efficient absorption of the active ingredient in the frog's intestine.

[0009] Preferably, the composition is encapsulated using soybean lecithin via a vacuum spraying process; this encapsulation process can effectively reduce the loss of the composition components in water, and improve feed utilization and efficacy stability.

[0010] On the other hand, the present invention provides the application of the above composition in the preparation of feed to improve the disease resistance of frogs; in practical applications, it is preferred to add the composition to the basal feed at a ratio of 2.5 g / kg.

[0011] Preferably, the application is specifically targeted at preventing and treating symptoms in frogs caused by Elizabethan miltiorrhiza, such as head tilting, circling movements, cloudy eyes, and cataracts. The prevention and control logic of the composition of this invention is based on a three-in-one synergistic mechanism: Targeted sterilization: Using Peptide-3 as a "physical scavenger", the pathogen load in the frog's body is rapidly reduced, creating a critical window for subsequent tissue repair.

[0012] Immune warning: By inducing non-specific immunity through astragalus polysaccharide and ginseng polysaccharide, serum lysozyme (LZM) activity is enhanced, blocking pathogens from invading deep tissues.

[0013] Tissue Repair: Targeting the inflammatory storm caused by Elizabethan miltiorrhiza, this treatment utilizes Rehmannia glutinosa (processed) to clear heat and cool the blood, combined with rosmarinic acid to scavenge free radicals. The synergistic effect of these two ingredients significantly enhances SOD activity in brain tissue, reduces MDA content, and precisely inhibits the expression of the pro-inflammatory factor TNF-α, thereby suppressing neuroinflammation and protecting the central nervous system and eye tissues from irreversible damage.

[0014] Compared with the prior art, the present invention has at least the following beneficial effects: The survival rate of frogs using the composition of this invention reached 92.4%, and the overall clinical score was significantly better than that of the antibiotic control group. The application of this composition not only achieved highly efficient eradication of drug-resistant Elizabethan miltiorrhiza, but also successfully overcame the technical challenge of reversing head tilt and cataracts in frogs, significantly improving the survival quality and economic value of farmed frogs. Attached Figure Description

[0015] Figure 1 The image shows the mass spectrometry identification of the antimicrobial peptide Peptide-1, whose amino acid sequence is shown in SEQ ID NO:1. The image also shows the secondary mass spectrometry fragment ion information of each peptide fragment, which is used to confirm the specific sequence and molecular weight distribution of the peptide.

[0016] Figure 2 The image shows the mass spectrometry identification of the antimicrobial peptide Peptide-2, whose amino acid sequence is shown in SEQ ID NO:2. The image also shows the secondary mass spectrometry fragment ion information of each peptide fragment, which is used to confirm the specific sequence and molecular weight distribution of the peptide.

[0017] Figure 3 The image shows the mass spectrometry identification of the antimicrobial peptide Peptide-3, whose amino acid sequence is shown in SEQ ID NO:3. The image also shows the secondary mass spectrometry fragment ion information of each peptide fragment, which is used to confirm the specific sequence and molecular weight distribution of the peptide.

[0018] Figure 4 The image shows the mass spectrometry identification of the antimicrobial peptide Peptide-4, whose amino acid sequence is shown in SEQ ID NO:4. The image also shows the secondary mass spectrometry fragment ion information of each peptide fragment, which is used to confirm the specific sequence and molecular weight distribution of the peptide.

[0019] Figure 5The image shows the mass spectrometry identification of the antimicrobial peptide Peptide-5, whose amino acid sequence is shown in SEQ ID NO:5. The image also shows the secondary mass spectrometry fragment ion information of each peptide fragment, which is used to confirm the specific sequence and molecular weight distribution of the peptide. Detailed Implementation

[0020] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0021] Example 1: A method for preparing and screening the activity of a frog skin-derived anti-disease and antimicrobial peptide. This embodiment provides a method for preparing highly active antimicrobial peptides from the skin of the black-spotted frog. Through precise pretreatment and a compound enzyme-directed enzymatic hydrolysis process, polypeptide components with highly efficient antibacterial activity are obtained.

[0022] 1. Experimental Materials and Reagents The raw material used in this embodiment is fresh frog skin, a byproduct of black-spotted frog processing. Information on the main enzyme preparations and reagents involved is shown in the table below:

[0023] 2. Frog skin pretreatment process Cleaning and Grinding: Take fresh black-spotted frog skin, soak it in 0.1% KMnO4 solution for 5 minutes for disinfection, then rinse repeatedly with deionized water until neutral. Use a tissue homogenizer to grind the frog skin into 1-3 mm pieces. 2 Fragments of skin.

[0024] Degreasing treatment: Place the skin fragments in 4 times their volume of 95% ethanol solution and stir at a constant temperature of 4°C for 4 hours to degrease. Filter out the solvent. This step aims to remove sebaceous gland secretions and adipose tissue from the frog skin to prevent them from embedding collagen and affecting enzymatic reactions.

[0025] Desalting and swelling: The defatted frog skin was placed in a 0.5 mol / L acetic acid solution and soaked for 12 hours at a material-to-liquid ratio of 1:10 (w / v) to allow it to fully absorb water and swell, exposing the protein peptide bond cleavage sites.

[0026] 3. Composite enzyme targeted enzymatic hydrolysis process The multi-enzyme stepwise enzymatic hydrolysis method is adopted to fully utilize the advantages of the cleavage sites of different proteases: Primary enzymatic hydrolysis: Adjust the pH of the system to 2.0-2.5, add pepsin (2.0% of the dry weight of frog skin), and hydrolyze with shaking at 37°C for 3 hours. Pepsin preferentially cleaves aromatic amino acid residues, initially opening the structure of large protein molecules.

[0027] Secondary enzymatic hydrolysis: Adjust the pH of the system to 7.5, add trypsin and neutral protease in a 1:1 ratio (total addition of 3.0%), and continue enzymatic hydrolysis at 45°C for 4 hours.

[0028] Inactivation and centrifugation: After enzymatic digestion, the mixture was placed in a 95°C water bath for 10 minutes to inactivate the protease. After cooling, it was centrifuged at 8000 r / min for 20 minutes, and the supernatant was collected.

[0029] Ultrafiltration fractionation: The supernatant was fractionated using ultrafiltration membranes with molecular weight cutoffs of 3 kDa and 1 kDa. Peptide fractions with molecular weights less than 3 kDa were collected, freeze-dried, and stored for later use.

[0030] 4. Mass spectrometry identification and sequence analysis of antimicrobial peptides Sample desalination and pretreatment: using C 18 ZipTip microcolumns are used to desalt the peptide fractions obtained by ultrafiltration. Elution is performed using a 0.1% formic acid solution to ensure a pure ionization environment for the sample and avoid interference from inorganic salts with the mass spectrometry signal.

[0031] High-performance liquid chromatography (Nano-LC): The desalted sample was injected into the Easy-nLC 1200 nano-liquid chromatography system (Thermo Fisher, catalog number: LC140). C140 was used for separation. 18 A reverse-phase column (75 μm × 15 cm) was used at a flow rate of 300 nL / min to separate complex peptides online using an acetonitrile gradient elution method.

[0032] Tandem mass spectrometry (MS / MS): Connected to a Q Exactive Plus orbital trap mass spectrometer (Thermo Fisher). Full scan (Full MS) and high-energy collisional dissociation (HCD) modes were used. The secondary mass spectrometer resolution was set to 17,500, and the scan range was 150–1800 m / z.

[0033] Data search and de novo sequencing: Raw mass spectrometry data (.raw) were analyzed using PEAKS Studio X software and matched against the Pelophylax nigromaculatus protein database (see Table 1). Figure 1-5 .

[0034] Table 1: Sequence Analysis of 5 Antimicrobial Peptides The results showed that, identified by LC-MS / MS, peptides as shown in SEQ ID NO. 1-5 were obtained from components with a molecular weight of less than 3 kDa. The sequence length was distributed between 10 and 15 amino acid residues, and the sequence was rich in lysine (Lys) and arginine (Arg). Calculated using BioEdit software, the theoretical isoelectric point (pI) of this peptide was approximately 10.5, exhibiting a typical cationic surface charge. This type of positively charged short peptide is expected to specifically bind to the negatively charged cell membrane of Elizabethanella miltiorrhiza through electrostatic attraction, causing membrane perforation. This provides a molecular basis for subsequent combination with immunomodulators such as ginseng polysaccharides.

[0035] Example 2: Screening of antimicrobial peptide activity and determination of dominant components based on mass spectrometry sequence synthesis This embodiment aims to artificially synthesize the five candidate antimicrobial peptides (Peptide 1-5) obtained by mass spectrometry identification in Example 1, and to conduct precise antimicrobial activity evaluation against Elizabethkingiamiricola, the pathogen causing wry neck cataracts in frogs, thereby screening out the core active components for subsequent compound formulations.

[0036] 1. Experimental Materials and Sample Preparation Peptide synthesis: Peptides 1-5 were synthesized artificially using a solid-phase synthesis method (Fmoc method), and the purity of all peptides was greater than 98% as determined by HPLC. Each synthesized peptide was dissolved in sterile phosphate-buffered saline (PBS) to prepare a stock solution with a concentration of 10.0 mg / mL.

[0037] Test strain: Elizabethan bacillus mil (isolated from brain tissue of a black-spotted frog exhibiting symptoms of torticollis and cataracts), inoculated into MH broth and cultured with shaking at 30°C until the logarithmic growth phase, then the bacterial suspension concentration was adjusted to 1×10⁻⁶. 7 CFU / mL.

[0038] Main equipment: Oxford cup (inner diameter 6.0 mm, outer diameter 8.0 mm), constant temperature incubator, digital vernier caliper.

[0039] 2. Experimental Procedure Plate preparation: Spread 100 μL of Elizabethan mil suspension evenly on a 90 mm diameter MH solid medium plate and let stand for 10 minutes.

[0040] Oxford cup placement: Place 6 sterile Oxford cups equidistantly on each plate.

[0041] Sample addition design: 100 μL of each peptide solution (concentration uniformly 5.0 mg / mL) was added to an Oxford cup. PBS was set as the negative control group and florfenicol (10 μg / mL) was set as the positive control group. Each group was set up with 3 replicates.

[0042] Incubation and Measurement: Incubate the plates at 28°C for 24 hours. Measure the diameter of the inhibition zone (transparent area) using digital calipers and take the average value.

[0043] 3. Experimental Results and Analysis The results of the inhibition zone diameter tests for each synthetic peptide against Elizabethan mil are shown in Table 2: Table 2: Comparison of inhibition zone diameters of 5 candidate antimicrobial peptides against Elizabethan mil. Table 2 shows that all five synthetic peptides exhibited antibacterial activity against *Elizabeth mil*. Due to the multidrug resistance of *Elizabeth mil*, traditional antibiotics often fail to achieve ideal inhibitory effects. Peptide-3 (FAKLFRKLRKGL) demonstrated the strongest inhibitory activity, with an inhibition zone diameter of 23.84±0.22 mm, significantly higher than other peptide components (P<0.05), and superior to the florfenicol control group. This further confirms that Peptide-3's extremely high isoelectric point and strong cationic properties allow it to penetrate the complex cell membrane structure of *Elizabeth mil* through more efficient electrostatic interactions, directly killing the pathogen physically. Therefore, this invention identifies Peptide-3 as the core anti-disease component against deviated head cataracts, for subsequent synergistic studies with Rehmannia glutinosa, plant polysaccharides, and rosmarinic acid.

[0044] Example 3: Verification of the preventive and therapeutic effects of a compound composition for enhancing disease resistance in frogs on "crooked head cataract disease". This embodiment aims to verify the clinical protective efficacy of a compound composition based on the superior antimicrobial peptide Peptide-3, combined with Astragalus polysaccharide, ginseng polysaccharide, Rehmannia glutinosa and rosmarinic acid, against Elizabethanella miltiorrhiza (a major pathogen causing head tilt and cataracts) in black-spotted frogs.

[0045] 1. Experimental Materials and Preparation Process 1.1 Experimental Formulation of this Invention: Based on the pathogenic characteristics of Elizabethan mil (invading the nervous system and easily developing drug resistance), the core formulation (by mass parts) is determined as follows: Core active peptide: Peptide-3 (amino acid sequence as shown in SEQ ID NO:3) 20 parts; Immune enhancer: Astragalus polysaccharide (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: P664535-10mg) 30 parts, Ginseng polysaccharide (Shanghai Yuanye Biotechnology Co., Ltd., catalog number: S27806-25mg) 20 parts; Rehmannia glutinosa (Tongrentang quality Rehmannia glutinosa) 15 parts; Anti-stress repair agent: Rosmarinic acid (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: R109804-20mg) 10 parts; Stabilizing carrier: Nano-grade montmorillonite powder (Aladdin, catalog number: M141491) 5 parts.

[0046] Control Group A (Component Deletion Group - Peptide Removal): Based on the pathogenic characteristics of Elizabethan mil (invasion of the nervous system, easy development of drug resistance), the core formula was determined (by mass parts): This formula removed Peptide-3, i.e., immune enhancer: Astragalus polysaccharide (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: P664535-10mg) 30 parts, Ginseng polysaccharide (Shanghai Yuanye Biotechnology Co., Ltd., catalog number: S27806-25mg) 30 parts; Rehmannia glutinosa (Tongrentang quality Rehmannia glutinosa) 25 parts; Anti-stress repair agent: Rosmarinic acid (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: R109804-20mg) 10 parts; Stabilizing carrier: Nano-grade montmorillonite powder (Aladdin, catalog number: M141491) 5 parts.

[0047] 1.2 Preparation of experimental feed Micronization: The above full-formula group and control group A were processed to a particle size of 50-80 μm using a high-energy ball mill to ensure ultra-micro absorption of the active ingredients.

[0048] Preparation and encapsulation: The composition was added to the basic feed at a ratio of 2.5 g / kg, and soybean lecithin was encapsulated by vacuum spraying to reduce water loss.

[0049] 2. Experimental Design and Model Building 2.1 Experimental Subjects Healthy black-spotted frog tadpoles with an average weight of 30.0 ± 1.5 g were selected, and the water temperature was maintained at 26-28℃.

[0050] 2.2 Animal Grouping The 800 experimental frogs were randomly divided into 4 groups (200 frogs in each group): Blank control group (CK): fed with basal feed.

[0051] Model control group: fed with basic feed + challenged with the virus.

[0052] Positive drug group: Florfenicol (0.2 g / kg) was added to the basal feed and challenged with the drug.

[0053] Control group A (component missing group - peptide removed): Peptide-3 was removed from the formula, and only polysaccharides, Rehmannia glutinosa and rosmarinic acid were contained, plus the drug was challenged.

[0054] Experimental composition of the present invention: feeding with feed containing the composition of the present invention + challenge with poison.

[0055] 2.3 Establishment of a model for head tilting disease After 14 days of continuous pre-feeding, all groups except the control group received intraperitoneal injection of Elizabethan bacillus suspension (concentration 1×10⁻⁶). 8 An acute head tilt-cataract model was established (CFU / mL, injection volume 0.1 mL / animal).

[0056] 3. Detection Indicators and Experimental Methods 3.1 Comprehensive Clinical Signs Assessment: Clinical manifestations of the black-spotted frogs in each group were observed daily at regular intervals, with a focus on quantitative scoring of head tilting symptoms, eye condition, and motor ability (total score 100 points, higher scores indicate milder symptoms). Head tilt angle (40 points): 40 points for no head tilt; 20 points for head tilt angle < 45 degrees; angle > A score of 0 is given if the angle is 45 degrees or if there is continuous circular motion.

[0057] Ocular opacity (30 points): 30 points for clear and transparent eyes; 15 points for slight opacity in one or both eyes; 0 points for obvious cataracts, protruding eyes, or ulceration.

[0058] Reaction ability (30 points): 30 points for jumping quickly and in the correct direction after being startled; 0 points for slow reaction, loss of balance or rolling to the side.

[0059] 3.2 Survival rate and feeding recovery records Survival rate: The total number of deaths within 10 days after the injection challenge experiment is counted. The formula is: Survival rate (%) = (Number of surviving animals / Total number of animals in the experiment) × 100%.

[0060] Feeding recovery time: The feeding behavior of each frog group after feeding was continuously observed. The number of days required for the feed intake of the infected group to recover to more than 80% of the normal level (blank group) was taken as the recovery time.

[0061] 3.3 Serum Lysozyme (LZM) Activity Assay: 48 hours after the challenge experiment, blood was collected from the hearts of 12 randomly selected black-spotted frogs from each group. Serum was collected by centrifugation at 4°C (3500 r / min, 15 min). Serum LZM activity was determined using a turbidimetric method. Using *Micrococcus lysodeoxycholica* suspension as a substrate, the enhancement effect of the composition on nonspecific humoral immunity was evaluated by measuring changes in absorbance.

[0062] 3.4 Detection of oxidative stress indicators in brain tissue (SOD and MDA): After blood collection, the frogs were quickly dissected to remove brain tissue, which was then washed in ice-cold saline to remove residual blood. Physiological saline was added at a weight-to-volume ratio (1:9), and the tissue was homogenized by ultrasonication. After centrifugation at 4000 r / min for 10 min, the supernatant was collected. Superoxide dismutase (SOD) activity: The WST-8 assay was used to evaluate the ability of Rehmannia glutinosa and rosmarinic acid in the composition to scavenge free radicals in the brain and alleviate nerve damage.

[0063] Malondialdehyde (MDA) content: determined by the TBA (thiobarbituric acid) method. The degree of oxidative damage to brain tissue by Elizabethan mil was assessed by lipid peroxidation level, and the protective efficacy of the composition was evaluated.

[0064] 3.5 Detection of pro-inflammatory factor TNF-α expression in brain tissue: The relative mRNA expression level of tumor necrosis factor TNF-α in brain tissue was detected by RT-qPCR: RNA extraction and reverse transcription: Total RNA was extracted from brain tissue using the Trizol method, and after treatment with DNase, it was reverse transcribed into cDNA; β-actin was used as an internal reference gene, and the relative abundance of TNF-α was calculated to evaluate the inhibitory effect of the composition on the inflammatory storm of the nervous system.

[0065] 4. Experimental Results and Analysis The following data were obtained by observing the experimental frogs for 10 days and sampling them 48 hours after the challenge experiment.

[0066] Table 3: Clinical signs and survival rates of black-spotted frogs after the challenge experiment in each group. Table 3 shows that the survival rate of the composition group of this invention (92.4%) was significantly higher than that of the traditional antibiotic group (65.4%). More importantly, regarding the neurological and ocular lesions easily caused by Elizabethan miltiorrhiza, the incidence rate in the experimental group was only 6.5%, and the clinical scores were close to healthy levels. The survival rate of control group A (peptide-free group) was only 42.5%, far lower than that of the full-formulation group. This indicates that Peptide-3 is the vanguard of the entire defense system, rapidly reducing the pathogen load through physical perforation, which is a prerequisite for subsequent immune regulation and tissue repair. This demonstrates that this invention not only utilizes the highly efficient physical bactericidal properties of Peptide-3 to reduce the pathogen load, but also, through the synergistic effect of Rehmannia glutinosa components and rosmarinic acid, blocks the pathogen's invasion of the blood-brain barrier and aqueous humor circulation system in the early stages of infection.

[0067] Table 4: Comparison of serum and brain tissue immune and biochemical indicators of black-spotted frogs in different groups Table 4 shows that the LZM activity in the experimental group reached 224.6 U / mL, far exceeding that of other groups. This demonstrates that Peptide-3 and Astragalus / Ginseng polysaccharide produced a strong synergistic effect, inducing the frog to produce endogenous defense substances and putting the body in a highly efficient early warning state. The model group showed a sharp decrease in SOD activity and a significant increase in MDA (oxidative damage marker) in brain tissue, indicating severe neurological damage. In contrast, the experimental group's MDA content basically recovered to the level of the blank group, and its SOD activity was slightly higher than that of the blank group. This is attributed to the strong free radical scavenging ability of Rehmannia glutinosa and rosmarinic acid, which effectively protected the central nervous system from secondary damage caused by inflammatory storms. The experimental group significantly inhibited the overexpression of the pro-inflammatory factor TNF-α. This regulatory mode proves that the composition does not blindly stimulate the immune system, but rather guides it to a balanced and orderly anti-infective response, thereby effectively preventing symptoms such as head tilt and cataracts caused by "inflammatory storms," ​​achieving a technological leap from "simple sterilization" to "comprehensive protection of the body."

[0068] Although the serum lysozyme (LZM) activity in control group A was higher than that in the model group, reaching approximately 145.5 U / mL, this reflects the inducing effect of Astragalus polysaccharides and ginseng polysaccharides. However, due to the lack of Peptide-3 to rapidly reduce pathogen load, the proliferation rate of Elizabethan mil exceeded the defense limit of the body's immune system, leading to severe LZM depletion and an inability to maintain the high levels seen in the full-formula group. In terms of brain tissue indicators, the SOD activity in control group A was significantly lower than that in the full-formula group, while the MDA content (a marker of oxidative damage) remained at a high level. This indicates that when drug-resistant bacteria massively breach the blood-brain barrier and induce a strong "cytokine storm," even the presence of Rehmannia glutinosa and rosmarinic acid cannot reverse the neurotoxicity and oxidative stress damage continuously generated by a large number of pathogens. The data from control group A directly supports the conceptual sequence of this invention—bactericidal action is the prerequisite, and repair is the core. This proves that Peptide-3 is not just an additive, but a prerequisite for the entire composition to perform "targeted repair". Without the physical perforation mechanism of Peptide-3, the composition degenerates into an ordinary nutritional supplement and cannot cope with clinical-grade head tilt and cataracts.

[0069] In summary, the prevention and treatment logic of the composition of this invention is based on a synergistic mechanism of "targeted sterilization - immune early warning - nerve repair". Through the precise formulation of multiple components, it overcomes the problem of preventing and treating "head tilt and cataracts" caused by Elizabethan miltiorrhiza. First, the core component, the antimicrobial peptide Peptide-3, acts as a "physical scavenger." Targeting the extremely strong antibiotic resistance of Elizabethan mil, Peptide-3, with its strong cationic properties, specifically disrupts the pathogen's cell membrane through a physical perforation mechanism. This non-chemical resistance-based bactericidal method rapidly reduces the pathogen load in the frog's body, creating a critical window for subsequent tissue repair.

[0070] Secondly, astragalus and ginseng polysaccharides form an "immune defense network" for the body. Experimental data showed a significant increase in serum lysozyme (LZM) activity, indicating that the polysaccharide components do not directly kill bacteria, but rather induce non-specific immunity, putting the frog in a state of high alert and effectively blocking the invasion of pathogens into the brain and eyes.

[0071] Finally, the inventive logic of this invention lies in the "precise tissue repair" effect of Rehmannia glutinosa and rosmarinic acid. The key to the lethality and disability caused by Elizabethan miltiorrhiza lies in the oxidative damage to the central nervous system and eye tissues caused by its inflammatory storm. This invention introduces Rehmannia glutinosa for its heat-clearing and blood-cooling properties, combined with rosmarinic acid to scavenge free radicals, which is strongly evidenced by the increase in SOD activity and the decrease in MDA content. This mechanism effectively downregulates the expression of the pro-inflammatory factor TNF-α, suppressing brain inflammation, logically explaining why the experimental group was able to control the incidence of "head tilt and cataracts" at an extremely low level.

[0072] This composition represents a technological leap from simple physical sterilization to comprehensive protection of the body. Through the logical synergy between its components, it not only eliminates drug-resistant bacteria but also repairs critical damaged organs, significantly improving the survival quality and growth potential of diseased frogs.

[0073] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A composition for improving the disease resistance of frogs, characterized in that, It is composed of the following components in parts by weight: 20 parts of core active peptide Peptide-3, 30 parts of Astragalus polysaccharide, 20 parts of ginseng polysaccharide, 15 parts of Rehmannia glutinosa, 10 parts of rosmarinic acid, and 5 parts of nano-sized montmorillonite powder; wherein, the amino acid sequence of Peptide-3 is as shown in SEQ ID NO:

3.

2. The composition according to claim 1, characterized in that, The particle size of each component in the composition is 50-80 μm.

3. The composition according to claim 1, characterized in that, The composition is encapsulated with soybean lecithin.

4. The use of the composition according to any one of claims 1 to 3 in the preparation of feed to improve the disease resistance of frogs.

5. The application according to claim 4, characterized in that, The disease resistance specifically refers to the prevention and treatment of head tilting and cataract symptoms in frogs caused by Elizabethan miltiorrhiza.

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