A composite antibacterial pet external use spray containing plant extracts and a preparation method thereof

Abstract

The application discloses a composite antibacterial pet external spray containing plant extracts and a preparation method thereof, and belongs to the technical field of pet care products. The external spray contains, in percentage by mass, 0.5-1.0% of cortex dictamni root extract, 0.75-1.5% of lycopersicum fruit extract, 0.75-1.5% of artemisia argyi leaf extract and 5-8% of chrysanthemum parthenium pure distillate, and can be further combined with auxiliary components such as emulsifiers and preservatives. The cortex dictamni root extract is prepared by using a supercritical CO2 extraction method combined with a specific composite entrainer, and active components are fully reserved; the spray is prepared by using a water phase and an oil phase in steps and is formed by high-shear emulsification, and has good stability. The product shows high bacteriostatic effects on staphylococcus aureus, salmonella and candida albicans, can significantly reduce the contents of inflammatory mediators NO and proinflammatory factors IL-1beta, has the effects of bacteriostasis, sterilization and anti-inflammation, is suitable for the preparation of pet skin care related products, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the technical field of pet care products, specifically relating to a compound antibacterial pet topical spray containing plant extracts and its preparation method. Background Technology

[0002] With the increasing pet ownership rate, pet skin diseases (such as fungal dermatitis and inflammation caused by bacterial infections) are frequent, causing trouble for pets' health and their owners' lives. Most existing topical antibacterial products for pets rely on chemical antibacterial agents, which can easily lead to skin irritation and drug resistance in pets with long-term use. In addition, some products have a narrow antibacterial spectrum, making it difficult to deal with both bacterial and fungal problems at the same time.

[0003] Plant extracts, due to their gentleness, safety, and good biocompatibility, are gradually becoming the preferred raw materials for pet care products. However, existing related products have significant shortcomings: First, the antibacterial and anti-inflammatory effects of single plant extracts are limited, lacking synergistic design; second, the extraction technology of plant active ingredients is not optimized enough, such as the low extraction rate and easy destruction of active ingredients by traditional solvent extraction methods, and the difficulty in fully releasing fat-soluble active ingredients by supercritical extraction with a single entrainer; third, some compound formulations have poor stability, and active ingredients are easily lost, affecting the effect of use. Therefore, developing a compound antibacterial pet topical product with synergistic ingredients, high extraction efficiency, good stability, and gentle safety has become an urgent need for the industry to address. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a compound antibacterial pet topical spray with natural plant extracts as its core, which combines highly effective antibacterial, bactericidal, and anti-inflammatory effects to meet the needs of pets for daily skin care and infection prevention.

[0005] To achieve the above objectives, the present invention discloses the following technical solutions:

[0006] In a first aspect, the present invention provides a compound antibacterial pet external spray containing plant extracts, wherein the external spray contains, by mass percentage, 0.5-1.0% Dictamnus dasycarpus root bark extract, 0.75-1.5% tomato fruit extract, 0.75-1.5% Artemisia argyi extract and 5-8% Chrysanthemum indicum hydrosol.

[0007] Preferably, the external spray further contains at least one of an emulsifier, a cosolvent, a stabilizer, a preservative, and a solvent.

[0008] Preferably, the Dictamnus dasycarpus root bark extract is prepared by the following method:

[0009] Step 1-1. Crush and sieve the dried Dictamnus dasycarpus root bark to obtain raw material powder, and then load it into the feed cylinder of the supercritical CO2 extraction vessel;

[0010] Step 1-2. Take Brazilian fruit seed oil, coconut oil alcohol-octanoate / capric acid ester and anhydrous ethanol and mix them evenly at a volume ratio of (2-3):(0.5-1.3):10 to obtain a composite entrainer;

[0011] Steps 1-3. Set the parameters of the extraction vessel: extraction pressure is 25-30 MPa, extraction temperature is 45-55℃, CO2 flow rate is 20-30 L / min, start the entrainer pump to synchronously inject the composite entrainer, the entrainer accounts for 15%-25% (v / v) of the CO2 flow rate, and perform dynamic extraction for 2.5-4.0 h. After extraction, the fluid enters the separator through the pressure reducing valve, and the extract is obtained by pressure reduction separation.

[0012] Steps 1-4. Transfer the collected extract to a vacuum distillation apparatus to remove ethanol by vacuum distillation, and obtain Dictamnus dasycarpus root bark extract.

[0013] Secondly, the present invention provides a method for preparing a compound antibacterial pet topical spray containing plant extracts, the preparation method comprising the following steps:

[0014] Step 2-1: Add approximately 80% of the formula amount of deionized water to a container, heat to 45-50℃, add tomato fruit extract, artemisia extract, potassium sorbate and sodium citrate in sequence, stir until completely dissolved, add glycerin and 1,3-propanediol, stir evenly to obtain the aqueous phase;

[0015] Step 2-2: In another container, mix PEG-40 hydrogenated castor oil with Dictamnus dasycarpus root bark extract and stir at 45-50°C until a uniform and transparent mixture is formed to obtain the oil phase.

[0016] Steps 2-3: Gradually add the oil phase to the aqueous phase at 300-500 rpm and 45-50℃. After the addition is complete, increase the stirring speed to 800-1000 rpm and continue stirring for 15-20 minutes to obtain the emulsion.

[0017] Steps 2-4: Add the white chrysanthemum hydrosol to the emulsion, stir evenly, rinse the container with the remaining deionized water and add it to the total formula volume, stir thoroughly, cool the mixture to 25-30℃, transfer the cooled liquid to a high-shear emulsifier, process it at 10000-15000 rpm for 5-10 minutes, then perform vacuum degassing, and fill it to obtain the composite antibacterial pet external spray.

[0018] Preferably, the amount of each raw material added in the preparation method is as follows:

[0019] Dictamnus dasycarpus root bark extract 0.5-1.0%, tomato fruit extract 0.75-1.5%, Artemisia argyi leaf extract 0.75-1.5%, chamomile hydrosol 5-8%, PEG-40 hydrogenated castor oil 0.5-1.5%, 1,3-propanediol 1-3%, glycerin 0.5-5%, sodium citrate 0.1-0.5%, potassium sorbate 0.05-0.15%, and deionized water 77.85-90.85%.

[0020] Thirdly, the present invention provides the application of the compound antibacterial pet topical spray containing plant extracts described in the first aspect in the preparation of pet care products with antibacterial, bactericidal and anti-inflammatory effects.

[0021] The beneficial effects of this invention are:

[0022] 1. The extracts of Dictamnus dasycarpus root bark, tomato fruit extract, Artemisia argyi extract and Chrysanthemum indicum hydrosol provided by this invention form a synergistic antibacterial system that achieves high antibacterial activity against common pathogens on pet skin. This is superior to products with single ingredients or those that deviate from the formula ratio, and can effectively inhibit mixed infections of pet skin caused by bacteria and fungi.

[0023] 2. The compound antibacterial external spray provided by this invention can significantly inhibit cellular inflammatory responses, reduce the content of inflammatory mediators and pro-inflammatory factors, effectively relieve skin inflammation and redness in pets, accelerate skin repair, and meet the needs of pet skin care.

[0024] 3. The Dictamnus dasycarpus root bark extract provided by this invention uses supercritical CO2 extraction combined with a composite entrainer composed of Brazilian fruit seed oil, coconut oil alcohol-caprylate / caprylate and anhydrous ethanol, which significantly improves the extraction rate of active ingredients. The spray preparation is carried out through stepwise emulsification and high shear treatment, resulting in good product stability. Detailed Implementation

[0025] Those skilled in the art can refer to the content of this document to appropriately improve the process parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention.

[0026] The products and methods of the present invention have been described through preferred embodiments. Those skilled in the art will be able to make modifications or appropriate alterations and combinations to the products and methods described herein without departing from the content, spirit and scope of the present invention, so as to realize and apply the technology of the present invention.

[0027] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Materials, reagents, etc., used are commercially available unless otherwise specified. Unless otherwise stated, substances described in this invention are calculated as percentages and parts by mass.

[0028] I. In this invention

[0029] Dictamnus dasycarpus root bark: The root bark of the plant Dictamnus dasycarpus (family Rutaceae), commercially available;

[0030] Tomato fruit extract: Tomato (SOLANUM LYCOPERSICUM) fruit extract, commercially available;

[0031] Artemisia argyi extract: Artemisia argyi leaf extract, commercially available;

[0032] White Chrysanthemum Nobilis Hydrosol: White chrysanthemum (ANTHEMIS NOBILIS) hydrosol is obtained by water distillation and is commercially available.

[0033] All the original materials used in the other embodiments are commercially available.

[0034] II. Dictamnus dasycarpus root bark extract

[0035] The preparation method of Dictamnus dasycarpus root bark extract is as follows:

[0036] Step 1. Crush the dried Dictamnus dasycarpus root bark with a pulverizer, pass it through a 60-mesh sieve to obtain a uniform raw material powder, weigh 1 kg and put it into the material cylinder of the supercritical CO2 extraction vessel, ensuring that the packing is uniform and tight.

[0037] Step 2. Mix Brazilian fruit seed oil, coconut oil alcohol-caprylate / caprylate and anhydrous ethanol in a volume ratio of 3:1:10 to form a uniform and transparent composite entrainer.

[0038] Step 3. Set the parameters of the extraction vessel: extraction pressure is 28MPa, extraction temperature is 50℃, CO2 flow rate is 22L / min, start the entrainer pump to inject the composite entrainer synchronously, the entrainer accounts for 20% (v / v) of the CO2 flow rate, and perform dynamic extraction for 3.5h. After extraction, the fluid is introduced into the separator through the pressure reducing valve. CO2 fluid and most of the anhydrous ethanol are removed by pressure reduction separation to obtain an extract containing Brazilian rosehip seed oil, coconut oil alcohol-octanoate / caprylate and a small amount of anhydrous ethanol.

[0039] Step 4. Transfer the collected extract to a vacuum distillation apparatus and perform vacuum distillation for 50 min at a temperature of 50℃ and a pressure of 0.005MPa to remove ethanol and obtain Dictamnus dasycarpus root bark extract.

[0040] The preparation method of Dictamnus dasycarpus root bark extract ① is as follows:

[0041] The preparation method of Dictamnus dasycarpus root bark extract was followed, except that the volume ratio of Brazil nut seed oil, coconut oil alcohol-caprylate / capric acid ester and anhydrous ethanol in step 2 was adjusted to 1:3:10, while the rest remained unchanged.

[0042] The preparation method of Dictamnus dasycarpus root bark extract ② is as follows:

[0043] The preparation method of Dictamnus dasycarpus root bark extract was followed, except that the volume ratio of Brazil nut seed oil, coconut oil alcohol-caprylate / caprylate and anhydrous ethanol in step 2 was adjusted to 1:0.3:10, while the rest remained unchanged.

[0044] The preparation method of Dictamnus dasycarpus root bark extract ③ is as follows:

[0045] The preparation method of Dictamnus dasycarpus root bark extract is followed, except that Brazil seed oil in step 2 is replaced with macadamia seed oil. That is, macadamia seed oil, coconut oil ester-caprylate / caprylate and anhydrous ethanol are mixed evenly in a volume ratio of 3:1:10 to obtain a composite entrainer, and the rest remains unchanged.

[0046] The preparation method of Dictamnus dasycarpus root bark extract ④ is as follows:

[0047] The preparation method of Dictamnus dasycarpus root bark extract was followed, except that in step 2, coconut oil alcohol-caprylate / capric acid ester was replaced with caprylate-capric acid triglyceride. That is, Brazil nut seed oil, caprylate-capric acid triglyceride and anhydrous ethanol were mixed evenly in a volume ratio of 3:1:10 to obtain a composite entrainer, and the rest remained unchanged.

[0048] The preparation method of Dictamnus dasycarpus root bark extract ⑤ is as follows:

[0049] The preparation method of Dictamnus dasycarpus root bark extract was followed, except that Brazil seed oil and coconut oil alcohol-caprylate / caprylate were omitted in step 2, and only anhydrous ethanol was used as an entrainer, while the rest remained unchanged.

[0050] III. Compound Antibacterial Pet Topical Spray

[0051] 1. Examples 1-3

[0052] Step 1: Add approximately 80% of the formula amount of deionized water to preparation tank A, heat to 45-50℃, add tomato fruit extract, artemisia extract, potassium sorbate and sodium citrate in sequence, stir until completely dissolved, add glycerin and 1,3-propanediol, stir evenly to obtain the aqueous phase;

[0053] Step 2: In another container, mix PEG-40 hydrogenated castor oil with Dictamnus dasycarpus root bark extract and stir at 45-50°C until a uniform and transparent mixture is formed to obtain the oil phase;

[0054] Step 3: Gradually add the oil phase prepared in Step 2 to the aqueous phase at 300-500 rpm and 45-50℃. After the addition is complete, increase the stirring speed to 800-1000 rpm and continue stirring for 15-20 minutes to form a preliminary homogeneous emulsion.

[0055] Step 4: Add the white chrysanthemum hydrosol to the above emulsion, stir well, rinse the container with the remaining deionized water and add it to the total formula volume, stir thoroughly, cool the mixture to 25-30℃, transfer the cooled liquid to a high shear emulsifier, process it at 10000-15000 rpm for 5-10 minutes, then perform vacuum degassing, and finally fill it into a light-proof spray bottle to obtain the composite antibacterial pet external spray of Examples 1-3.

[0056] The amount of raw materials added in the above preparation method is shown in Table 1.

[0057] Table 1. Percentage of raw materials by mass in the examples (unit: %)

[0058] Raw material name Example 1 Example 2 Example 3 Dictamnus dasycarpus root bark extract 0.5 0.8 1.0 Tomato fruit extract 0.75 1.0 1.5 Artemisia argyi extract 0.75 1.0 1.5 White Chrysanthemum Hydrosol 5 7 8 PEG-40 hydrogenated castor oil 1.0 1.0 1.0 1,3-Propanediol 3.0 3.0 3.0 glycerin 2.0 2.0 2.0 Sodium citrate 0.2 0.2 0.2 Potassium sorbate 0.1 0.1 0.1 Deionized water Add to 100 Add to 100 Add to 100

[0059] 2. Comparative Examples 1-4

[0060] Comparative Example 1: The preparation method of Example 3 was the same, except that the Dictamnus dasycarpus root bark extract was replaced with Dictamnus dasycarpus root bark extract①, and the rest remained the same;

[0061] Comparative Example 2: The preparation method of Example 3 was the same, except that the Dictamnus dasycarpus root bark extract was replaced with Dictamnus dasycarpus root bark extract ②, and the rest remained the same;

[0062] Comparative Example 3: The preparation method of Example 3 is the same, except that the Dictamnus dasycarpus root bark extract is replaced with Dictamnus dasycarpus root bark extract ③, and the rest remains the same;

[0063] Comparative Example 4: The preparation method of Example 3 was the same, except that the Dictamnus dasycarpus root bark extract was replaced with Dictamnus dasycarpus root bark extract ④, and the rest remained the same;

[0064] Comparative Example 5: The preparation method of Example 3 was the same, except that the Dictamnus dasycarpus root bark extract was replaced with Dictamnus dasycarpus root bark extract ⑤, and the rest remained the same;

[0065] For details on the amount of the above-mentioned raw materials to be added, please refer to Table 2.

[0066] Table 2. Raw material mass percentage for Comparative Examples 1-5 (unit: %)

[0067] Raw material name Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Dictamnus dasycarpus root bark extract ① 1.0 / / / / Dictamnus dasycarpus root bark extract ② / 1.0 / / / Dictamnus dasycarpus root bark extract ③ / / 1.0 / / Dictamnus dasycarpus root bark extract ④ / / / 1.0 / Dictamnus dasycarpus root bark extract ⑤ / / / / 1.0 Tomato fruit extract 1.5 1.5 1.5 1.5 1.5 Artemisia argyi extract 1.5 1.5 1.5 1.5 1.5 White Chrysanthemum Hydrosol 8 8 8 8 8 PEG-40 hydrogenated castor oil 1.0 1.0 1.0 1.0 1.0 1,3-Propanediol 3.0 3.0 3.0 3.0 3.0 glycerin 2.0 2.0 2.0 2.0 2.0 Sodium citrate 0.2 0.2 0.2 0.2 0.2 Potassium sorbate 0.1 0.1 0.1 0.1 0.1 Deionized water Add to 100 Add to 100 Add to 100 Add to 100 Add to 100

[0068] Note: " / " in the table indicates no addition.

[0069] 3. Comparative Examples 6-9

[0070] Comparative Examples 6-9 were prepared according to the preparation method of Example 3, wherein the raw materials of Comparative Examples 6-9 were added as listed in Table 3, as follows:

[0071] Table 3. Raw material mass percentage for Comparative Examples 6-9 (unit: %)

[0072] Raw material name Comparative Example 6 Comparative Example 7 Comparative Example 8 Comparative Example 9 Dictamnus dasycarpus root bark extract / 1.6 1.6 1.0 Tomato fruit extract 2.0 / 2.4 1.5 Artemisia argyi extract 2.0 2.4 / 1.5 White Chrysanthemum Hydrosol 8 8 8 / Chamomile Rectuta Hydrosol / / / 8 PEG-40 hydrogenated castor oil 1.0 1.0 1.0 1.0 1,3-Propanediol 3.0 3.0 3.0 3.0 glycerin 2.0 2.0 2.0 2.0 Sodium citrate 0.2 0.2 0.2 0.2 Potassium sorbate 0.1 0.1 0.1 0.1 Deionized water Add to 100 Add to 100 Add to 100 Add to 100

[0073] Note: " / " in the table indicates no addition.

[0074] IV. Performance Testing

[0075] 1. Antibacterial test

[0076] 1.1 Test Objective

[0077] To evaluate the in vitro antibacterial activity of compound antibacterial pet topical spray against common pathogens (Staphylococcus aureus, Salmonella) and fungi (Candida albicans), to clarify its antibacterial and antifungal effects and the rationality of its formulation, and to provide data support for the practical application of the product.

[0078] 1.2 Test Materials

[0079] Test samples: The compound antibacterial pet topical sprays of Examples 1-3 and Comparative Examples 1-9 were tested directly using the undiluted solution.

[0080] Tested bacterial strains: Staphylococcus aureus, Salmonella, Candida albicans.

[0081] Main reagents: nutrient broth, nutrient agar (for bacterial culture), Sabouraud agar medium (for fungal culture), 0.85% sterile physiological saline, sterile distilled water; positive control reagents: enrofloxacin (0.01 mg / mL, for bacteria), fluconazole (0.01 mg / mL, for fungi).

[0082] Instruments and equipment: clean bench, constant temperature incubator (37℃ for bacteria, 28℃ for fungi), constant temperature water bath (50±1℃), autoclave, 9cm sterile petri dishes, Oxford cups (6mm inner diameter), pipettes (20~1000μL), sterile pipette tips, test tubes, vernier calipers, vortex mixer.

[0083] 1.3 Testing Method (Refer to the Oxford Cup Method)

[0084] 1.3.1. Strain Cultivation and Preparation of Bacterial Solution

[0085] Bacterial culture: Staphylococcus aureus and Salmonella bacterial colonies were picked and inoculated into 5 mL nutrient broth test tubes. The cultures were incubated at 37°C and 180 rpm on a shaker for 12–16 h until the logarithmic growth phase. The cultures were then serially diluted with sterile physiological saline, and the bacterial concentration was adjusted to 1 × 10⁻⁶ using turbidimetric methods. 6～7 CFU / mL, for later use.

[0086] Fungal culture: Pick Candida albicans mycelial growths and inoculate into 5 mL of Sabouraud medley medium. Incubate at 28°C with shaking at 150 rpm for 24 h. Dilute to 1×10⁻⁶. 6～7 CFU / mL, for later use.

[0087] 1.3.2. Culture medium preparation and plate inoculation

[0088] Prepare nutrient agar and Saburg agar separately, autoclave at 121℃ for 20 min, cool to 50-55℃, pour into sterile petri dishes (15-20 mL per dish), place horizontally to solidify, and prepare sterile culture medium plates.

[0089] Take 0.1 mL of the adjusted bacterial solution and spread it evenly on the surface of the corresponding culture medium plate. After spreading, let it stand in a clean bench for 15 minutes to allow the bacterial solution to be fully absorbed.

[0090] 1.3.3 Placement and Sample Addition of Oxford Cups

[0091] Using sterile forceps, gently place the sterilized Oxford cups onto the plate coated with bacterial solution. Place 4 cups on each plate, with appropriate spacing between the cups and the distance between the cups and the edge of the plate (≥2cm) to avoid overlapping inhibition zones.

[0092] Add 100 μL of the test sample (example / comparative example), positive control reagent (enrofloxacin for bacterial group, fluconazole for fungal group), and negative control (0.85% sterile saline) to each Oxford cup. Add the samples gently to avoid spilling.

[0093] After the sample is added, the plate is left to stand in the clean bench for 20 minutes to allow the sample to fully diffuse into the culture medium.

[0094] 1.3.4. Cultivation and Measurement of Inhibition Zones

[0095] Bacterial plates: invert and place in a 37℃ incubator for 18–24 hours; Fungal plates: invert and place in a 28℃ incubator for 48 hours.

[0096] After incubation, remove the plates and use calipers to measure the diameter of the inhibition zone around each Oxford cup using the cross-hatching method (accurate to 0.01 mm). Take the average of the two measurements as the diameter of the inhibition zone for that sample. Set up 3 replicate plates for each sample and take the average of the 3 plates as the final result.

[0097] 1.3.5 Evaluation Criteria for Antibacterial Performance

[0098] Inhibition zone diameter <10mm: no antibacterial effect; 10mm ≤ Inhibition zone diameter <15mm: moderate antibacterial effect; Inhibition zone diameter ≥15mm: high antibacterial effect.

[0099] 1.4 Test Results

[0100] Table 4. Diameter of inhibition zone against target bacteria in test samples (average, mm)

[0101] Group Staphylococcus aureus salmonella Candida albicans negative control 0.00 0.00 0.00 Positive control (enrofloxacin / fluconazole) 19.85 18.62 17.34 Example 1 16.32 15.78 14.86 Example 2 17.59 16.94 15.97 Example 3 18.27 17.63 16.75 Comparative Example 1 14.58 13.96 13.24 Comparative Example 2 13.87 13.42 12.89 Comparative Example 3 15.13 14.35 13.67 Comparative Example 4 15.46 14.72 14.03 Comparative Example 5 12.35 11.89 11.56 Comparative Example 6 11.76 11.23 10.98 Comparative Example 7 12.14 11.67 11.32 Comparative Example 8 11.92 11.45 11.15 Comparative Example 9 14.28 13.75 13.06

[0102] 1.5 Results Analysis

[0103] The negative control showed no inhibition zone, while the positive control exhibited high inhibitory activity against all three bacterial species, indicating that the experimental system was stable and reliable. Examples 1-3 showed high inhibitory activity against Staphylococcus aureus, Salmonella, and Candida albicans (inhibition zone diameter ≥14.8 mm), and the diameter of the inhibition zone gradually increased with the increase of the amount of core components such as Dictamnus dasycarpus root bark extract and tomato fruit extract.

[0104] Comparative Examples 1-4 showed weaker antibacterial effects than Example 3 because the entrainer formulation of the Dictamnus dasycarpus root bark extract deviated from the optimized range, indicating that the composite entrainer can maximize the extraction of antibacterial active ingredients from Dictamnus dasycarpus root bark. Comparative Example 5 used only anhydrous ethanol as an entrainer, and had the worst antibacterial effect, further verifying the importance of the composite entrainer.

[0105] Comparative Examples 6-8 lacked Dictamnus dasycarpus root bark extract, tomato fruit extract, or Artemisia argyi extract, resulting in insufficient core antibacterial components and an antibacterial zone that only reached a moderate level. Comparative Example 9, which replaced Chrysanthemum indicum hydrosol with Chamomile hydrosol, showed a decrease in antibacterial effect, indicating that the flavonoids and volatile oils in Chamomile hydrosol have a synergistic antibacterial effect with other plant extracts and cannot be replaced.

[0106] 2. Anti-inflammatory test

[0107] 2.1 Test Objective

[0108] To evaluate the protective effect of a compound antibacterial pet topical spray against lipopolysaccharide (LPS)-induced inflammatory damage in RAW264.7 cells, its anti-inflammatory efficacy was verified by detecting the levels of inflammatory mediator NO and pro-inflammatory factor IL-1β.

[0109] 2.2 Test Materials

[0110] Test samples: The compound antibacterial pet topical sprays of Examples 1-3 and Comparative Examples 1-9 were diluted with serum-free DMEM medium to the corresponding mass percentage concentration before use.

[0111] Test cells: RAW264.7 cells (purchased from Wuhan Pronosai).

[0112] Main reagents: LPS, cell counting kit (CCK8), NO detection kit, IL-1β ELISA detection kit, DMEM culture medium, fetal bovine serum, penicillin-streptomycin bispecific antibody, etc., all of which are commercially available qualified products.

[0113] Instruments and equipment: cell culture incubator, enzyme-linked immunosorbent assay (ELISA) reader, fluorescence inverted microscope, etc.

[0114] 2.3 Test Method

[0115] 2.3.1 Cell Culture

[0116] RAW264.7 cells were cultured in DMEM complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics at 37°C in a 5% CO2 cell culture incubator. After cell resuscitation, the cells were passaged twice, and cells in the logarithmic growth phase were used to prepare a concentration of 5 × 10⁻⁶ cells / cells. 4 pcs·mL -1 The cell suspension is prepared for use.

[0117] 2.3.2 Cytotoxicity assay (CCK8 assay)

[0118] Concentration settings: mass percentage gradients of 0.1%, 0.2%, 0.5%, 1%, and 2% were used, with three replicates for each concentration and three repetitions for each experiment.

[0119] Procedure: Seed 100 μL of cell suspension into each well of a 96-well plate and incubate overnight until adherence. Then, treat with different concentrations of sample for 12 hours. Follow the CCK8 kit instructions and measure absorbance (OD value) at 450 nm. Calculate relative cell viability using the following formula: Cell Viability = Drug-Added Blank + Drug-Added Blank

[0120] Result determination: Screen for safe concentrations with cell viability ≥90% to determine the concentrations for subsequent experiments.

[0121] Test results: When the mass percentage was ≤1%, the sample did not significantly inhibit the viability of RAW264.7 cells, and the inhibition rate was greater than 90%; at a concentration of 2%, cell viability decreased significantly. Therefore, 1% was determined as the working concentration for subsequent anti-inflammatory tests.

[0122] 2.3.3 Cellular Inflammation Damage Protection Test

[0123] Group setup: Control group (normal culture, no treatment), LPS group (normal culture followed by the addition of 1 μg / mL) -1 LPS treatment for 24 h), sample group (pretreated with 1% mass of sample for 12 h after normal culture, then added with 1 μg·mL⁻¹) -1 LPS treatment for 24 hours, with 3 replicates per group and 3 replicates per experiment.

[0124] NO content determination: The Griess reagent method was used. 50 μL of cell supernatant was taken, and an equal volume of Griess reagent was added and mixed. After the reaction was carried out at room temperature, the OD value was measured at a wavelength of 540 nm. The NO concentration was calculated according to the standard curve.

[0125] IL-1β content determination: The ELISA method was used. Cell supernatant was taken and the IL-1β content was determined according to the kit instructions.

[0126] 2.4 Test Results

[0127] The results of the inflammatory marker tests are shown in Table 5;

[0128] Table 5. Effects of test samples on the levels of cellular inflammatory factors (mean ± standard deviation)

[0129] Group <![CDATA[NO / μmol·L -1 ]]> Significance (compared to Example 3) <![CDATA[IL-1β / pg·mL -1 ]]> Significance (compared to Example 3) control group 12.3±2.5 / 18.7±4.3 / LPS Group 45.7±7.2 / 89.4±10.1 / Example 1 23.6±4.7 / 42.8±6.5 / Example 2 19.8±3.1 / 35.6±5.9 / Example 3 18.9±2.5 / 31.2±3.7 / Comparative Example 1 27.4±2.5 P＜0.01 48.5±5.3 P＜0.01 Comparative Example 2 29.1±4.0 P＜0.01 51.3±6.4 P＜0.01 Comparative Example 3 26.7±2.3 P＜0.01 46.9±4.8 P＜0.01 Comparative Example 4 25.9±3.8 P＜0.01 44.7±5.6 P＜0.01 Comparative Example 5 32.4±3.1 P＜0.01 59.8±7.2 P＜0.01 Comparative Example 6 35.2±4.2 P＜0.01 64.3±6.9 P＜0.01 Comparative Example 7 33.7±3.3 P＜0.01 61.5±8.3 P＜0.01 Comparative Example 8 34.5±2.7 P＜0.01 63.1±7.1 P＜0.01 Comparative Example 9 28.6±3.4 P＜0.01 50.2±7.5 P＜0.01

[0130] 2.5 Results Analysis

[0131] Compared with the control group, the NO concentration and IL-1β content in the LPS group were significantly increased, indicating that LPS successfully induced an inflammatory response in RAW264.7 cells.

[0132] After pretreatment with 1% concentration, the NO concentration and IL-1β content of all sample groups (Examples 1-3, Comparative Examples 1-9) were significantly lower than those of the LPS group, indicating that the compound antibacterial pet topical spray has clear anti-inflammatory activity.

[0133] The anti-inflammatory effect of Example 3 was significantly better than that of the comparative group. Comparative examples 1-5 all had weaker anti-inflammatory effects than Example 3 because the entrainer formulation of the Dictamnus dasycarpus root bark extract deviated from the optimized range. Specifically, Comparative example 1 adjusted the compound entrainer ratio to 1:3:10 (Brazilian argan oil: coconut oil-caprylate / caprylate: anhydrous ethanol), which disrupted the synergistic solubilizing effect between oils and esters, resulting in a decrease in the purity of the extracted anti-inflammatory active ingredients. Comparative example 2 had an entrainer ratio of 1:0.3:10, with insufficient coconut oil-caprylate / caprylate content, reducing the extraction efficiency of fat-soluble active ingredients. Comparative example 3 replaced Brazilian argan oil with macadamia seed oil, and Comparative example 4 replaced coconut oil-caprylate / caprylate with caprylic / caprylate triglycerides. Because the polarity and solubility of the replaced components did not match the target active ingredients, the content of effective anti-inflammatory ingredients in the extract was reduced. Comparative example 5 used only anhydrous ethanol as a single entrainer, lacking the auxiliary extraction effect of oil components, and thus failing to fully release the fat-soluble anti-inflammatory substances in the Dictamnus dasycarpus root bark.

[0134] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A compound antibacterial pet topical spray containing plant extracts, characterized in that, By weight percentage, the external spray contains 0.5-1.0% Dictamnus dasycarpus root bark extract, 0.75-1.5% tomato fruit extract, 0.75-1.5% Artemisia argyi extract, and 5-8% Chrysanthemum indicum hydrosol; The Dictamnus dasycarpus root bark extract was prepared by the following method: Step 1-1. Crush and sieve the dried Dictamnus dasycarpus root bark to obtain raw material powder, and then load it into the feed cylinder of the supercritical CO2 extraction vessel; Step 1-2. Take Brazilian fruit seed oil, coconut oil alcohol-octanoate / capric acid ester and anhydrous ethanol and mix them evenly at a volume ratio of (2-3):(0.5-1.3):10 to obtain a composite entrainer; Steps 1-3. Set the parameters of the extraction vessel: extraction pressure is 25-30 MPa, extraction temperature is 45-55℃, CO2 flow rate is 20-30 L / min, start the entrainer pump to synchronously inject the composite entrainer, the entrainer accounts for 15%-25% (v / v) of the CO2 flow rate, and perform dynamic extraction for 2.5-4.0 h. After extraction, the fluid enters the separator through the pressure reducing valve, and the extract is obtained by pressure reduction separation. Steps 1-4. Transfer the collected extract to a vacuum distillation apparatus to remove ethanol by vacuum distillation, and obtain Dictamnus dasycarpus root bark extract.

2. The composite antibacterial pet topical spray according to claim 1, characterized in that, The external spray also contains at least one of the following: emulsifier, cosolvent, stabilizer, preservative, and solvent.

3. The preparation method of the composite antibacterial pet topical spray according to claim 1, characterized in that, The preparation method includes the following steps: Step 2-1: Add 80% of the formula amount of deionized water to a container, heat to 45-50℃, add tomato fruit extract, artemisia extract, potassium sorbate and sodium citrate in sequence, stir until completely dissolved, add glycerin and 1,3-propanediol, stir evenly to obtain the aqueous phase; Step 2-2: In another container, mix PEG-40 hydrogenated castor oil with Dictamnus dasycarpus root bark extract and stir at 45-50°C until a uniform and transparent mixture is formed to obtain the oil phase. Steps 2-3: Gradually add the oil phase to the aqueous phase at 300-500 rpm and 45-50℃. After the addition is complete, increase the stirring speed to 800-1000 rpm and continue stirring for 15-20 minutes to obtain the emulsion. Steps 2-4: Add the white chrysanthemum hydrosol to the emulsion, stir evenly, rinse the container with the remaining deionized water and add it to the total formula volume, stir thoroughly, cool the mixture to 25-30℃, transfer the cooled liquid to a high-shear emulsifier, process it at 10000-15000 rpm for 5-10 minutes, then perform vacuum degassing, and fill it to obtain the composite antibacterial pet external spray.

4. The preparation method according to claim 3, characterized in that, In the preparation method, the amount of each raw material added is as follows by mass percentage: Dictamnus dasycarpus root bark extract 0.5-1.0%, tomato fruit extract 0.75-1.5%, Artemisia argyi leaf extract 0.75-1.5%, chamomile hydrosol 5-8%, PEG-40 hydrogenated castor oil 0.5-1.5%, 1,3-propanediol 1-3%, glycerin 0.5-5%, sodium citrate 0.1-0.5%, potassium sorbate 0.05-0.15%, and deionized water 77.85-90.85%.

5. The application of the compound antibacterial pet topical spray containing plant extracts as described in claim 1 or 2 in the preparation of pet care products with antibacterial, bactericidal, and anti-inflammatory effects.