Composition for improving lung injury of dog after bacterial infection as well as preparation method and application of composition

Abstract

The invention discloses a composition for improving lung injury of dogs after bacterial infection as well as a preparation method and application thereof, and relates to the technical field of animal health care products. The composition is prepared from a honeysuckle flower extract, fish collagen peptide, a herba houttuyniae extract, L-carnitine, isoleucine, valine, taurine, glutamine, L-glutamic acid, L-lysine, L-threonine, L-histidine, succinic acid, L-proline, methionine, cysteine and glycine. The composition can effectively relieve dog lung epithelial cell injury caused by streptococcus pneumoniae infection, reduce intracellular lactic dehydrogenase release, improve cellular morphology and reduce inflammatory response and oxidative stress level. The composition disclosed by the invention can improve respiratory tract discomfort symptoms, such as high cough frequency, increased mucus secretion, poor mental state and the like, which still exist after the treatment of the dog bacterial pneumonia through a multi-target effect. The health recovery level of the dogs after conventional antibiotic treatment can be improved.

Description

Technical Field

[0001] The present invention relates to the technical field of animal health products, and in particular to a composition for improving lung damage after bacterial infection in dogs, a preparation method and an application thereof. Background Art

[0002] Streptococcus pneumoniae (SP) is a major cause of respiratory infections in companion animals, including dogs and cats. It is particularly susceptible to invasive pneumococcal disease under conditions of seasonal stress, immunosuppression, or high-density housing. In clinical practice, SP infection can lead to acute lung injury, manifested by epithelial barrier disruption, exaggerated inflammatory responses, and disrupted tissue metabolism.

[0003] Currently, veterinary clinics often use antibiotics (such as doxycycline) to eliminate pneumococcal infections. However, a significant number of animals continue to experience symptoms such as runny nose, increased secretions, and mucosal swelling even after bacterial clearance. Studies have shown that this post-infectious respiratory syndrome (PIRS) is primarily caused by unrepaired epithelial damage, residual inflammation, and immune dysfunction. Further research has revealed that even in the absence of viable bacteria, pneumococcal surface proteins, such as choline-binding proteins (CBPs) and hemolysin, can continue to induce cell adhesion, proinflammatory responses, and membrane damage, potentially contributing to chronic damage.

[0004] However, existing veterinary Chinese herbal compositions are primarily used to alleviate general respiratory symptoms such as bronchitis, cough, and wheezing, lacking functional compositions specifically designed to repair the respiratory system during the post-bacterial respiratory syndrome (PIRS) phase. For example, patent CN104688920A discloses an oral Chinese herbal solution for bronchitis in dogs and cats; CN114133563A discloses a canine cough relief composition containing traditional herbs such as Houttuynia cordata; and CN110284858A proposes a Chinese herbal preparation for treating animal respiratory diseases. While these approaches are effective in alleviating symptoms, they lack targeted research into the mechanisms of bacterial infection and do not address the reconstruction and repair of epithelial cells during the post-antibiotic phase.

[0005] In research on natural functional ingredients, chlorogenic acid, a rich source of honeysuckle extract, has excellent antioxidant, anti-inflammatory, and tissue-protective effects; the active ingredients in Houttuynia cordata extract (primarily houttuynia cordata) have inhibitory effects on a variety of respiratory pathogens and can regulate the host's inflammatory response; fish collagen peptides, as small molecule active peptides, have been shown to promote cell repair and antioxidant defense. Although the above ingredients have shown potential therapeutic effects in other diseases, no studies have combined them for the mechanistic repair and metabolic intervention of epithelial damage after SP infection in companion animals, nor have functional combination strategies been established that are consistent with the post-antibiotic stage of the disease.

[0006] Therefore, it is of great significance to develop a multifunctional composite composition that can target and repair epithelial damage after bacterial infection in dogs and has anti-inflammatory, antioxidant and metabolic regulation effects. Summary of the Invention

[0007] To address the above problems, the present invention first constructed a Streptococcus pneumoniae infection model using canine type II lung epithelial (CAE) cells, and performed metabolomics analysis using liquid chromatography-mass spectrometry (LC-MS). It was found that after SP infection combined with antibiotic treatment, multiple key metabolites related to amino acid metabolism, membrane lipid synthesis, anti-oxidation and methyl cycle in the cells were significantly downregulated, including taurine, glutamate, glutamine, lysine, proline, S-adenosylmethionine (SAM), oxidized glutathione, etc.; enrichment analysis suggested that functional disorders involving pathways such as glutamate metabolism, purine metabolism, glutathione metabolism, and amino acid biosynthesis may cause oxidative stress, immune disorders, and decreased cell damage repair ability.

[0008] In view of this, the present invention screened functional ingredients with metabolic repair, anti-oxidation, immunomodulation and tissue repair functions based on the above-mentioned metabolic imbalance mechanism targets, and constructed a functional composition that can be used to improve lung damage after bacterial infection in dogs.

[0009] Based on the above findings, the present invention provides a composition for improving lung injury after bacterial infection in dogs, which comprises the following components in parts by mass:

[0010] Honeysuckle extract: 200-400 parts; Fish collagen peptide: 1000-1500 parts; Houttuynia cordata extract: 0.5-3 parts; L-carnitine: 30-60 parts; Isoleucine: 20-40 parts; Valine: 20-40 parts; Taurine: 30-50 parts; L-glutamic acid: 20-40 parts; Glutamine: 30-50 parts; L-lysine: 20-40 parts; L-threonine: 10-30 parts; L-histidine: 10-30 parts; Succinic acid: 10-20 parts; L-proline: 20-40 parts; Methionine: 10-20 parts; Cysteine: 5-15 parts; Glycine: 5-15 parts.

[0011] Preferably, the composition is composed of the following components in the following mass ratios:

[0012] Honeysuckle extract: 320 parts; Fish collagen peptide: 1280 parts; Houttuynia cordata extract: 2.5 parts; L-carnitine: 50 parts; Isoleucine: 30 parts; Valine: 30 parts; Taurine: 40 parts; L-glutamic acid: 30 parts; Glutamine: 40 parts; L-lysine: 30 parts; L-threonine: 20 parts; L-histidine: 20 parts; Succinic acid: 15 parts; L-proline: 30 parts; Methionine: 15 parts; Cysteine: 10 parts; Glycine: 10 parts.

[0013] The chlorogenic acid content of the honeysuckle extract in the composition accounts for more than 70% of the total mass of the extract.

[0014] After model verification and metabolic mechanism research, the effects of each raw material in the present invention are as follows:

[0015] Honeysuckle extract is rich in chlorogenic acid, which has anti-inflammatory and antioxidant effects and can alleviate cellular stress caused by SP infection;

[0016] Fish collagen peptides promote epithelial cell repair;

[0017] Houttuynia cordata extract has certain antibacterial and immunomodulatory effects;

[0018] L-carnitine and branched-chain amino acids (valine and isoleucine) contribute to energy metabolism and mitochondrial function recovery;

[0019] Glutamic acid, glutamine, proline, etc. promote cell metabolism and structural protein synthesis;

[0020] Cysteine, glycine, etc. participate in anti-oxidation and enhance the cell's ability to resist stress.

[0021] The present invention also provides the composition for preparing functional feed, functional health food, health preparation or nutritional supplement.

[0022] The present invention also provides that the composition can be processed together with acceptable feed bases or food supplements to prepare dosage forms such as powders, granules, liquid additives, freeze-dried preparations, coated granules or tablets.

[0023] The present invention also provides a method for preparing tablets of the composition, which is carried out according to the following steps:

[0024] (1) Weighing and preparing materials: weighing the raw materials of each component of the composition, hydroxypropyl methylcellulose, magnesium stearate, sodium carboxymethylcellulose and microcrystalline cellulose;

[0025] (2) Granulation and drying: the composition and part of the auxiliary materials are mixed, water is added to moisten the mixture to form wet granules, the granules are extruded, dried in a fluidized bed dryer to a suitable moisture content, and the granules are sieved;

[0026] (3) Total mixing: Mix the dry granules with the remaining excipients, and finally add magnesium stearate and mix well;

[0027] (4) Tableting: Pressing the mixed material into tablets;

[0028] (5) Packaging: Packaging and encapsulating the tableted preparation.

[0029] The mass fractions of the excipients in the tablets are: 1% to 3% of hydroxypropyl methylcellulose, 0.3% to 1% of magnesium stearate, 0.5% to 2% of sodium carboxymethyl cellulose, and 0.5% to 2% of microcrystalline cellulose, calculated based on the proportion to the total mass of the composition.

[0030] Compared with the prior art, the present invention has the following advantages:

[0031] (1) Target clarification: Screening of functional components based on the metabolic mechanism after SP infection treated with antibiotics, and precise supplementation and downregulation of metabolites;

[0032] (2) Multi-pathway synergy: covering multiple levels such as anti-oxidation, anti-inflammation, and tissue repair, enhancing the overall therapeutic effect;

[0033] (3) Wide applicability: It is suitable for auxiliary rehabilitation of dogs in the late stage of treatment or recovery period of bacterial lung infection and has practical conversion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The accompanying drawings, which constitute part of the present invention, are provided to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are provided to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0035] Figure 1 Changes in lactate dehydrogenase (LDH) release in CAE cells following infection with Streptococcus pneumoniae (SP) and treatment with doxycycline hydrochloride. LDH release remained significantly elevated even after treatment with doxycycline hydrochloride (3.75 μg / mL), indicating persistent cell damage. Different letters indicate significant differences (P < 0.05).

[0036] Figure 2 Effects of honeysuckle extract, fish collagen peptide and Houttuynia cordata extract on CAE cell viability (CCK-8 method).

[0037] Figure 3 Effects of the three functional ingredients alone on LDH release induced by SP infection. NC represents the uninfected control group, and SP represents the untreated group after infection. The HC group was treated with Houttuynia cordata extract (1.25 and 2.5 μg / mL), the CA group was treated with Honeysuckle extract (160 and 320 μg / mL), and the FP group was treated with fish collagen peptide (640 and 1280 μg / mL).

[0038] Figure 4 Effects of different functional ingredient combinations on LDH release induced by SP infection. NC represents the uninfected control group, and SP represents the untreated group after infection. The HC group was treated with Houttuynia cordata extract, the CA group was treated with honeysuckle extract, and the FP group was treated with fish collagen peptide.

[0039] Figure 5 Effects of a combination of three functional ingredients on the release of inflammatory factors induced by SP infection. IL-1β and IL-8 levels in the cell culture supernatant were measured. NC: uninfected control group; SP: group infected with Streptococcus pneumoniae; SP+CAFP: group treated with a combination of honeysuckle extract and fish collagen peptide; SP+CAFPHC: group treated with a combination of honeysuckle extract, fish collagen peptide, and Houttuynia cordata extract.

[0040] Figure 6 The effect of the combination of three functional ingredients on oxidative stress-related indicators. Cellular indicators such as MDA (malondialdehyde), T-AOC / TOS (total antioxidant capacity to total oxidative status ratio), and GPX (glutathione peroxidase) were measured. NC: uninfected control group; SP: infected group; SP+CAFP: honeysuckle extract + fish collagen peptide group; SP+CAFPHC: three-component combination group.

[0041] Figure 7 Effects of the three-component combination on junctional protein expression. Expression levels of tight junction and adherens junction-associated proteins, including claudin, ZO-1, occludin, MUC1, and E-cadherin, were measured. NC: uninfected control; SP: infected group; SP+CAFP: honeysuckle extract + fish collagen peptide combination group; SP+CAFPHC: three-component combination group.

[0042] Figure 8 Effects of the three-component combination on cell morphology and apoptosis in SP-infected cells. Crystal violet staining was used to observe cell morphology, and Annexin V-FITC fluorescence staining was used to detect cell apoptosis. NC: uninfected control group; SP: infected group; SP_CA: honeysuckle extract alone; SP_CAFP: honeysuckle extract plus fish collagen peptide; SP_CAFPHC: three-component combination group. Scale bar: 100 μm.

[0043] Figure 9 Cough frequency score time curve.

[0044] Figure 10 Time curve of nasal secretion scoring.

[0045] Figure 11 Time curve of mental state and physical condition scores. DETAILED DESCRIPTION

[0046] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0047] The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments.

[0048] The honeysuckle extract used in the present invention was purchased from Xi'an Mixianer Biotechnology Co., Ltd.

[0049] Fish collagen peptides were purchased from Hainan Huapeptide Biotechnology Co., Ltd.

[0050] Houttuynia cordata extract was purchased from Xi'an Jushengyuan Biotechnology Co., Ltd.

[0051] Experimental Example 1 Metabolic Mechanism of Canine Pulmonary SP Infection after Antibiotic Treatment

[0052] 1. Test method

[0053] (1) Canine lung epithelial cell culture

[0054] Canine lung type II epithelial (CAE) cells were cultured in modified DMEM medium containing 12% fetal bovine serum and 1% double antibody in a 37°C, 5% CO2 incubator. Cell passage and experiment were performed when the cells grew to 90% confluence.

[0055] (2) Establishment of Streptococcus pneumoniae infection model

[0056] An in vitro infection model was used to simulate canine Streptococcus pneumoniae (SP) infection. SP strain (ATCC 6305) was inoculated into brain heart infusion (BHI) medium and incubated at 37°C for 24 hours. The culture was centrifuged at 5000 rpm for 10 minutes and then resuspended in MEM to prepare the bacterial suspension for infection. The concentration was adjusted to 0.05 McFarland using a McFarland turbidimeter. CAE cells were plated at 5×10 3 Cells were seeded at 100 μL / well in a 96-well plate. After 24 hours of adherence, 100 μL of SP suspension was added and infected for 2-6 hours to induce cell damage. After infection, MEM containing 7.5 μg / mL doxycycline hydrochloride (2× concentration) was added to inhibit further bacterial growth and cultured for another 18 hours.

[0057] (3) Cytotoxicity assay

[0058] The degree of cell damage was assessed by lactate dehydrogenase (LDH) release. Cell supernatant (100 μL) was collected and centrifuged at 3000 rpm for 5 min. The supernatant was reacted with LDH reagent (Beyotime, C0017) for 30 min, and the absorbance was measured at 490 nm using a microplate reader.

[0059] (4) Metabolomics sample processing and detection process

[0060] A negative control group (NC) was set up: no infection, only treated with culture medium for 2h+18h; SP infection group: SP infection for 2h and then antibiotic treatment for 18h. An equal volume of cell lysate from each group was mixed and set as the quality control sample (QC), and the blank group was treated with 53% methanol solution. All samples were added with 300μL of 80% methanol aqueous solution, quickly frozen in liquid nitrogen, and then thawed on ice, ultrasonicated for 6min, centrifuged at 5000rpm and 4℃, and the supernatant was freeze-dried and re-dissolved with 10% methanol. A Vanquish UHPLC system (Thermo Fisher) was used with QExactive TM HF high-resolution mass spectrometer was used for quantitative detection of metabolites.

[0061] (5) Data processing and differential metabolite screening

[0062] Principal component analysis (PCA) was used to assess intergroup variability, and variable importance projection (VIP) values ​​were extracted. Differential metabolites were identified using a univariate t-test (P value) combined with fold change (FC). The screening criteria were: VIP > 1, P < 0.05, and FC ≥ 1.2 or FC ≤ 0.83. Metabolic pathway analysis was performed using the KEGG database for annotation and enrichment. Hypergeometric P values ​​were used, with P < 1 indicating enriched pathways and P < 0.05 indicating significant enrichment.

[0063] 2. Test results

[0064] (1) Changes in LDH after antibiotic treatment of bacterially infected lung cells

[0065] To verify the extent of damage caused by Streptococcus pneumoniae infection to canine alveolar epithelial cells and to simulate the actual clinical status after antibiotic intervention, the present invention established a cell damage model of SP infection + doxycycline hydrochloride treatment. Figure 1 shown.

[0066] In the uninfected control group (SP not added), the release rate of lactate dehydrogenase (LDH) in the cell supernatant was maintained at a low level regardless of whether antibiotics were added, indicating that the culture medium and antibiotics themselves had no significant toxicity to the cells. However, after infection with SP (0.05McF), even after antibiotic intervention was added 2 hours after infection, the LDH release rates in the subsequent 2h, 4h and 6h treatment groups were significantly increased (approximately 700-800% of the original level), suggesting that after SP infection, while antibiotics control bacterial proliferation, cells still suffer significant structural damage. The results show that in the in vitro cell model established by the present invention, Streptococcus pneumoniae can induce typical cell damage and still have significant cytotoxicity release after antibiotic treatment. This model can be used to further simulate the prolonged lung injury period (PIRS) after bacterial infection and provide a repeatable and stable experimental basis for the protective effect of the functional composition.

[0067] (2) Metabolic changes in lung cells infected by SP

[0068] As shown in Table 1, SP infection significantly inhibited the following key metabolic pathways:

[0069] I. Disorders of branched-chain amino acid metabolism and lipid metabolism: Levels of 2-methylbutyrylcarnitine, isobutyrylcarnitine, and propionylcarnitine were significantly decreased, suggesting disorders of branched-chain amino acid metabolism such as isoleucine and valine, and decreased mitochondrial energy conversion capacity.

[0070] II. Impaired antioxidant system function: Important antioxidant substances such as taurine, L-glutamate and oxidized glutathione were significantly reduced, indicating that SP infection can induce persistent oxidative stress and lead to disturbances in intracellular redox balance.

[0071] III. Decreased protein synthesis and structural repair capabilities: The levels of proline, lysine, glutamine, and their related dipeptides (such as prolyl-glutamine) were downregulated to varying degrees, suggesting that cells lost their normal collagen synthesis and barrier repair capabilities after infection.

[0072] IV. Dysfunction of methyl donors and purine synthesis: The levels of S-adenosylmethionine (SAM, FC=0.59) and succinyladenosine (FC=0.56) were reduced, suggesting that methyl cycle and DNA repair synthesis were limited.

[0073] The above results indicate that even under antibiotic control, pneumococcal infection can still lead to damage to multiple pathways of the cellular metabolic system, including energy supply, antioxidant capacity, barrier construction and repair mechanisms.

[0074] Therefore, the present invention selects carnitine, branched-chain amino acids (such as isoleucine and valine), antioxidant amino acids (glutamic acid, taurine, glycine, cysteine), glutamine, proline and methyl donors (such as methionine) as functional components, and has a clear theoretical basis and metabolic support for targets such as post-infection energy metabolism disorders, oxidative stress, cell repair and DNA synthesis disorders.

[0075] Table 1 Changes in key metabolites based on which the composition of the present invention is designed

[0076]

[0077]

[0078] Experimental Example 2 Effect of the Main Functional Components on Lung Injury in Dogs

[0079] 1. Test method

[0080] (1) Determination of the concentration of functional ingredients

[0081] Honeysuckle extract (CA), fish collagen peptide (FP) and Houttuynia cordata extract (HC) were prepared into different concentrations of working solution, and CAE cells were treated for 24 hours. Cell viability was detected by CCK-8 assay to determine the non-toxic concentration range. Among them, the cell seeding density was 5×10 4 cells / mL, and the final concentration gradient range was: CA 80-640 μg / mL, FP 320-2560 μg / mL, and HC 0.5-4 μg / mL.

[0082] (2) Evaluation of the mitigating effect of functional ingredients on cytotoxicity after SP infection

[0083] In a 96-well plate, 5 × 10 3 After inoculating cells at 400 μg / well and culturing for 24 hours, SP suspension (0.05 McF) was added for infection for 2 hours. MEM medium containing 7.5 μg / mL doxycycline and various concentrations of functional components was then added for a further 18 hours. The cell supernatant was used to measure LDH release.

[0084] (3) Cell morphology observation and apoptosis analysis:

[0085] After plating and treatment in 6-well plates, cells were fixed with 4% paraformaldehyde and stained with crystal violet to assess morphology. Apoptosis was assessed by Annexin V-FITC / PI double staining and observed under a fluorescence microscope to analyze the proportion of early and late apoptotic cells.

[0086] (4) Detection of oxidative stress indicators

[0087] Cell lysates were used to measure indicators such as T-AOC, TOS, MDA, and GPX. Protein concentrations were quantified using the BCA assay, and indicators were measured using commercial kits. Detection wavelengths were: T-AOC (593 nm), TOS (560 nm), MDA (532 nm), and GPX (412 nm).

[0088] (5) Determination of cell junction protein content

[0089] Canine-specific ELISA kits were used to quantitatively measure the levels of cell junction proteins, including E-cadherin, MUC1, occludin, ZO-1, and claudin, in cell lysates. 50 μL of sample was added to each well, incubated at 37°C for 1 hour, and then the plates were washed. HRP-conjugated antibodies and TMB colorimetric solution were added, and the absorbance was measured at 450 nm after the reaction was terminated.

[0090] (6) Detection of inflammatory factor levels

[0091] A canine ELISA kit was used to detect the concentrations of IL-1β and IL-8 in the cell culture supernatant. The operating procedures were consistent with those of the connexin ELISA test.

[0092] 2. Test results

[0093] (1) Screening of effective concentrations of functional ingredients

[0094] The CCK-8 assay was used to evaluate the safety of functional ingredients at different concentrations on CAE cells. Figure 2 As shown in the results, honeysuckle extract did not affect cell viability at concentrations between 160 and 320 μg / mL, but significantly inhibited cell viability above 640 μg / mL. Fish collagen peptide showed no toxicity at concentrations between 10 and 1280 μg / mL, demonstrating good biocompatibility. Houttuynia cordata extract showed no toxicity to cells at concentrations between 1.25 and 2.5 μg / mL, but significantly inhibited cell proliferation at concentrations above 10 μg / mL. Therefore, the above concentration ranges were determined to be safe dosages for each component, suitable for subsequent functional validation.

[0095] (2) Single component has a repairing effect on lung cell membrane damage

[0096] like Figure 3As shown, SP infection increased the LDH release rate of CAE cells to 778.1%, significantly higher than the negative control group (100%), indicating severe cell membrane damage. After 18 hours of treatment alone, honeysuckle extract (320 μg / mL) reduced LDH release to 161.2%, demonstrating the most significant mitigating effect. Fish collagen peptide (1280 μg / mL) and Houttuynia cordata extract (2.5 μg / mL) also showed some protective effects, reducing LDH release to 582.0% and 719.7%, respectively. This suggests that all three agents can alleviate SP-induced cell damage to some extent.

[0097] (3) The combined use of the three components has a synergistic effect

[0098] like Figure 4 As shown, the protective effects of different combinations were evaluated through orthogonal experiments. The results showed that when honeysuckle extract (320μg / mL), fish collagen peptide (1280μg / mL) and Houttuynia cordata extract (2.5μg / mL) were used in combination (CAFPHC combination), the LDH release rate was significantly reduced to 119.8%, which was better than any single component or two-component combination; the CA+FP combination also showed a good effect (131.5%), further verifying the dominant role of CA in membrane protection and showing the synergistic potential of FP.

[0099] (4) The combined use of the three components significantly inhibits the release of inflammatory factors

[0100] like Figure 5 As shown, the SP group significantly induced the release of IL-1β and IL-8. The two-component combination (SP+CAFP) had a certain anti-inflammatory effect, while the three-component combination (SP+CAFPHC) further significantly suppressed the levels of inflammatory factors, indicating that the functional component combination has a synergistic anti-inflammatory effect, which is superior to a single combination.

[0101] (5) The combined use of the three components significantly inhibited oxidative stress

[0102] like Figure 6 As shown, infection with Streptococcus pneumoniae (SP group) significantly increased MDA and decreased T-AOC / TOS and GPX levels, suggesting intense cellular oxidative stress. The two-component combination (SP + CAFP) partially alleviated this stress, while the three-component combination (SP + CAFPHC) further significantly reduced MDA and increased antioxidant markers, demonstrating a more pronounced restoration of antioxidant capacity.

[0103] (6) The combined use of the three components significantly enhanced the connexin

[0104] like Figure 7As shown, SP infection significantly inhibited the expression of multiple cell junction proteins. The two-component combination (SP+CAFP) partially restored the expression, while the three-component combination (SP+CAFPHC) significantly increased the levels of claudin, occludin, ZO-1, MUC1, and E-cadherin, with a recovery effect superior to other groups, suggesting that this combination can effectively improve epithelial cell barrier structure.

[0105] (7) The three-component combination significantly improved cell morphology and reduced apoptosis

[0106] like Figure 8 As shown, SP infection significantly caused cell shrinkage and rupture, increased intercellular spaces, decreased intercellular junctions, and increased apoptosis. Honeysuckle extract and its two-component combination partially alleviated the damage. Treatment with the three-component combination (SP_CAFPHC) enhanced intercellular junctions, improved cell morphology and integrity, and significantly reduced apoptosis, suggesting its repair and anti-apoptotic abilities.

[0107] Examples 1 to 3

[0108] Examples 1 to 3 were prepared according to the formulations shown in Table 2 and in the following manner:

[0109] (1) Weighing and preparing materials:

[0110] Accurately weigh the active ingredients, including honeysuckle extract, fish collagen peptide, houttuynia cordata extract, L-carnitine, isoleucine, valine, taurine, L-glutamic acid, glutamine, L-lysine, L-threonine, L-histidine, succinic acid, L-proline, methionine, cysteine, and glycine, as well as the excipients hydroxypropyl methylcellulose, magnesium stearate, sodium carboxymethylcellulose, and microcrystalline cellulose, for later use. All raw materials used are commercially available feed-grade or pharmaceutical-food-grade products and meet standards for animal nutritional supplements.

[0111] (2) Granulation and drying:

[0112] Mix fish collagen peptide, part of hydroxypropyl methylcellulose and sodium carboxymethyl cellulose in a mixer, slowly add appropriate amount of pure water to moisten the powder, stir to form wet granules, extrude and send into fluidized bed dryer to dry until the moisture content is controlled within the appropriate range (about ≤5%), then sieve and granulate.

[0113] (3) Total mixing:

[0114] The dried whole granules, honeysuckle extract, houttuynia cordata extract, amino acid components, and remaining excipients (hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, and microcrystalline cellulose) are put into a total mixer for mixing, and finally magnesium stearate is added and mixed thoroughly.

[0115] (4) Tableting:

[0116] The mixture was fed into a rotary tablet press and tableted using a round die. The weights of the tablets obtained in the three groups of examples were:

[0117] Example 1: 0.75g / tablet;

[0118] Example 2: 1.03 g / tablet;

[0119] Example 3: 1.25 g / tablet.

[0120] (5) Packaging:

[0121] The obtained tablets are packaged in aluminum-plastic blister packaging or sealed bottles and stored at room temperature away from light.

[0122] Table 2 Formulas used in each embodiment

[0123]

[0124]

[0125] Test Example 3 Case Observation Test

[0126] 1. Test Method

[0127] Twelve dogs diagnosed with bacterial pneumonia by a veterinarian were recruited from a pet hospital. All dogs presented with clinical symptoms such as coughing, tachypnea, and mucous nasal discharge. They had received antibiotic treatment (doxycycline hydrochloride) for more than one day and had residual mild to moderate respiratory symptoms. The dogs' daily diet remained unchanged. The 12 dogs were randomly divided into three groups of four:

[0128] Control group: continued with normal diet and medication, without other interventions;

[0129] Example 2 Intervention Group: Based on the original treatment plan, the tablets prepared in Example 2 were orally fed, 2 times a day, 2 tablets each time, for 7 consecutive days.

[0130] Example 3 Intervention Group: Based on the original treatment plan, the tablets prepared in Example 3 were orally fed, twice a day, 2 tablets each time, for 7 consecutive days.

[0131] From pre-intervention (Day 0) to Day 7 post-intervention, veterinarians or pet owners assessed daily symptom improvement, including cough frequency, nasal discharge, mental state, and physical recovery. Each symptom was scored on a scale of 0-3 (0 = no abnormality, 3 = severe abnormality). The average score for each group was recorded daily and calculated. A time-dependent curve was plotted to comprehensively evaluate the potential of the combination to improve the symptoms of bacterial lung infection in dogs.

[0132] 2. Test Results

[0133] like Figure 9-11 As shown, after 7 consecutive days of intervention, the dogs in Example 2 and Example 3 groups showed a trend of being better than the control group in multiple indicators:

[0134] (1) Cough frequency score ( Figure 9 ): The score of the control group decreased to a limited extent, with some residual score remaining on day 7. In contrast, the scores of the intervention groups of Example 2 and Example 3 decreased significantly from day 3 onwards, reaching the lowest level on day 7, indicating that the composition has a good auxiliary regulatory effect on the respiratory tract manifestations of dogs after infection.

[0135] (2) Nasal secretion score ( Figure 10 ):The nasal secretion score of the intervention group decreased more rapidly, almost reaching 0 on the 6th to 7th day, while the control group still showed a certain secretion.

[0136] (3) Mental state and physical condition score ( Figure 11 The intervention group's score continued to decline, reflecting a more significant improvement in the dogs' overall condition. The control group's score declined more slowly, remaining higher than the intervention group on day 7.

[0137] In general, it is shown that supplementing the composition of the present invention on the basis of standard antibiotic treatment can help promote the improvement of respiratory system-related indicators in the recovery period of dog infection and support the regulation of its overall health status.

[0138] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A composition for improving lung damage after bacterial infection in dogs, characterized in that: The composition comprises the following components by weight: honeysuckle extract: 200-400 parts; fish collagen peptide: 1000-1500 parts; houttuynia cordata extract: 0.5-3 parts; L-Carnitine: 30-60 parts; Isoleucine: 20-40 parts; Valine: 20-40 parts; Taurine: 30-50 parts; L-Glutamic acid: 20-40 parts; Glutamine: 30-50 parts; L-Lysine: 20-40 parts; L-Threonine: 10-30 parts; L-Histidine: 10-30 parts; Succinic acid: 10-20 parts; L-proline: 20-40 parts; Methionine: 10-20 parts; Cysteine: 5-15 parts; Glycine: 5-15 parts.

2. A composition for improving lung damage after bacterial infection in dogs according to claim 1, characterized in that: The composition comprises the following components in proportions by weight: 320 parts of honeysuckle extract; 1280 parts of fish collagen peptide; 2.5 parts of houttuynia cordata extract; 50 parts of L-carnitine; 30 parts of isoleucine; 30 parts of valine; 40 parts of taurine; 30 parts of L-glutamic acid; 40 parts of glutamine; 30 parts of L-lysine; 20 parts of L-threonine; 20 parts of L-histidine; 15 parts of succinic acid; 30 parts of L-proline; and 15 parts of methionine. Cysteine: 10 parts; Glycine: 10 parts.

3. A composition for improving lung damage after bacterial infection in dogs according to any one of claims 1 to 2, characterized in that: The chlorogenic acid content of the honeysuckle extract in the composition accounts for more than 70% of the total mass of the extract.

4. A composition for improving lung damage after bacterial infection in dogs according to any one of claims 1 to 2, characterized in that: The composition is used for preparing functional feed, functional health food, health preparation or nutritional supplement.

5. A composition for improving lung damage after bacterial infection in dogs according to any one of claims 1 to 2, characterized in that: The composition can be processed together with acceptable feed bases or food supplements to prepare dosage forms such as powders, granules, liquid additives, freeze-dried preparations, coated granules or tablets.

6. A method for preparing tablets of the composition for improving lung injury after bacterial infection in dogs according to any one of claims 1 to 2, characterized in that: Follow the steps below: (1) Weighing and preparing materials: weighing the raw materials of each component of the composition, hydroxypropyl methylcellulose, magnesium stearate, sodium carboxymethylcellulose and microcrystalline cellulose; (2) Granulation and drying: the composition and part of the auxiliary materials are mixed, water is added to moisten the mixture to form wet granules, the granules are extruded, dried in a fluidized bed dryer to a suitable moisture content, and the granules are sieved; (3) Total mixing: Mix the dry granules with the remaining excipients, and finally add magnesium stearate and mix well; (4) Tableting: Pressing the mixed material into tablets; (5) Packaging: Packaging and encapsulating the tableted preparation.

7. The method of preparing a tablet composition for improving lung injury after bacterial infection in dogs according to claim 6, characterized in that: The mass fractions of the excipients in the tablets are: 1% to 3% of hydroxypropyl methylcellulose, 0.3% to 1% of magnesium stearate, 0.5% to 2% of sodium carboxymethylcellulose, and 0.5% to 2% of microcrystalline cellulose, calculated based on the proportions of the total mass of the composition.

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