A spray for improving oral inflammation in pets and its preparation method.

Abstract

This invention provides a spray for improving oral inflammation in pets and its preparation method, belonging to the field of pet oral sprays. The preparation method of the spray for improving oral inflammation in pets includes the following steps: preparing deoxycholic acid-chitosan, preparing a first component, preparing a second component, and mixing the components. The spray for improving oral inflammation in pets and its preparation method of this invention can effectively mask the unpleasant taste of the herbal ingredients, improving palatability; simultaneously, the gel system used has good compatibility with the herbal ingredients, effectively protecting the herbal ingredients within the system; furthermore, it can further improve the gel film-forming strength of the gel system containing herbal ingredients in the pet's oral cavity, effectively prolonging the residence time of the active ingredients in the pet's oral cavity, and through the combination of the gel system and the herbal ingredients, further improving the nursing effect on pet oral inflammation.

Description

Technical Field

[0001] This invention relates to the field of oral sprays for pets, and in particular to a spray for improving oral inflammation in pets and its preparation method. Background Technology

[0002] As people's living standards improve, more and more people are finding happiness in raising pet dogs and cats, and the relationship between humans and pets is becoming increasingly close. However, because pet food and treats that pets consume over a long period of time are easily left in their mouths, the chances of pets developing oral inflammation have increased significantly, and there is a risk of transmission when humans come into contact with pets.

[0003] In the existing technology, traditional pet oral care solutions are generally compound chemical preparations with poor palatability. After use, pet dogs and cats will continue to lick the solution, which not only results in a short residence time of the active ingredients in the mouth and low bioavailability, but also leads to problems such as dry oral mucosa and bleeding in pets. It is difficult to meet the functional requirements of antibacterial and stomatitis improvement.

[0004] To improve the oral care effect for pets, existing technologies disclose pet oral care solutions with added Chinese herbal ingredients. However, the added Chinese herbal ingredients often have bitter or spicy irritating tastes, which not only further reduces the palatability of the care solution, but may also irritate stomatitis or oral ulcers. At the same time, the added Chinese herbal ingredients are not stable and are easily oxidized and degraded, and cannot maintain their intended care performance for a long time.

[0005] To prolong the residence time of active ingredients in a pet's mouth, existing technologies disclose pet oral care solutions that combine a gel system with traditional Chinese medicine ingredients. These solutions, when sprayed into the pet's mouth, gel and form a film, thereby extending the residence time of the active ingredients and ensuring the full effectiveness of the traditional Chinese medicine ingredients. However, because the traditional Chinese medicine ingredients require multiple formulations, and the existing gel system differs significantly in properties from various traditional Chinese medicine ingredients, the compatibility between the gel system and multiple traditional Chinese medicine ingredients is poor. This results in poor stability of the traditional Chinese medicine ingredients in the gel system, making them prone to precipitation or oil droplets during long-term storage, thus affecting the spraying and usage effects. Furthermore, existing gel systems (such as poloxamer) typically rely on the physical accumulation of micelles in the pet's mouth, resulting in poor gel strength. Under conditions of frequent licking and saliva rinsing, the formed gel film is easily broken or detached, limiting the effective residence time of the gel film in the pet's mouth.

[0006] Furthermore, the gel system used in existing pet oral care solutions is usually just a physical carrier for the herbal ingredients, which has limited protective function for the herbal ingredients, and the stability of the herbal ingredients in the gel system needs to be further improved; in addition, the performance of the gel system is relatively simple and cannot be combined with the herbal ingredients to further improve the care effect on pet oral inflammation.

[0007] Based on this, a method for preparing a spray for improving oral inflammation in pets is provided. This method effectively masks the unpleasant taste of the herbal ingredients, improves palatability, and reduces irritation to the oral cavity. Simultaneously, the gel system used has good compatibility with the herbal ingredients, effectively protecting them and preventing loss during gel formation or long-term storage. Furthermore, it can further enhance the gel film strength of the herbal-containing gel system in the pet's oral cavity, prolonging the residence time of the active ingredients. Through the synergy between the gel system and the herbal ingredients, the method further improves the nursing effect on oral inflammation in pets, demonstrating significant technical importance and research value. Summary of the Invention

[0008] To address the technical problems existing in the prior art, this invention provides a spray for improving oral inflammation in pets and its preparation method. This spray effectively masks the unpleasant taste of the herbal ingredients, improving palatability and reducing irritation to the oral cavity. Simultaneously, the gel system used has good compatibility with the herbal ingredients, effectively protecting them and preventing loss during gel formation or long-term storage. Furthermore, it enhances the gel film strength of the herbal-containing gel system in the pet's oral cavity, effectively prolonging the residence time of the active ingredients. Through the synergy between the gel system and the herbal ingredients, the therapeutic effect on oral inflammation in pets is further improved.

[0009] To solve the above technical problems, the technical solution adopted by the present invention is as follows: A method for preparing a spray for improving oral inflammation in pets includes the following steps: preparing deoxycholic acid-chitosan, preparing a first component, preparing a second component, and mixing the formulation; The method for preparing deoxycholic acid-chitosan is as follows: chitosan is dissolved in an aqueous acetic acid solution to obtain a chitosan solution. Then, in the presence of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, the chitosan is reacted with deoxycholic acid at room temperature and purified to obtain deoxycholic acid-chitosan. The method for preparing the first component is as follows: deoxycholic acid-chitosan is added to acetate-sodium acetate buffer solution and mixed evenly to obtain a dispersion; under stirring conditions, Angelica dahurica additive solution and Cyperus rotundus additive solution are simultaneously added dropwise to the dispersion; after the addition is completed, ethanol is removed to obtain the first component. The method for preparing the second component is as follows: acetylated sodium hyaluronate is added to deionized water and mixed evenly to obtain a dispersion; trehalose is added to the dispersion and mixed evenly; then, honeysuckle additive solution and hibiscus bark additive solution are added dropwise in sequence to obtain the second component. The method for preparing the mixed formulation involves mixing the first component and the second component evenly, adjusting the pH to 7.0, and obtaining a spray for improving oral inflammation in pets.

[0010] Furthermore, the Angelica dahurica additive solution is prepared by reflux extraction of Angelica dahurica with ethanol solution, followed by evaporation and desolvation of the extract, and then dissolving it in anhydrous ethanol and adjusting the imperatorin content to 0.25-0.3wt%. Preferably, after drying and pulverizing Angelica dahurica, it is added to an ethanol solution with a volume concentration of 80-90%, and extracted by reflux for 3-4 hours. After separating and removing the solids, the extract is evaporated to dryness. The evaporation residue is dissolved in anhydrous ethanol and the imperatorin content is adjusted to 0.25-0.3 wt% to obtain Angelica dahurica additive solution.

[0011] Furthermore, the Cyperus rotundus additive solution is prepared by steam distilling Cyperus rotundus with deionized water, collecting the oil phase in the distillate, dissolving it with anhydrous ethanol, and adjusting the oil phase content to 0.5-0.6 wt%. Preferably, after drying and pulverizing Cyperus rotundus, deionized water is used to perform steam distillation on the Cyperus rotundus, and the heating and distillation time is controlled to be 4-5 hours. After collecting the oil phase in the distillate, anhydrous ethanol is used to dissolve and adjust the oil phase content to 0.5-0.6 wt% to obtain Cyperus rotundus additive solution.

[0012] Furthermore, the honeysuckle additive solution is prepared by reflux extraction of honeysuckle with ethanol solution, followed by evaporation to remove ethanol from the extract, and adjustment of the chlorogenic acid content to 0.2-0.3 wt% with deionized water. Preferably, honeysuckle is dried and pulverized, then added to an ethanol solution with a volume concentration of 35-45%, refluxed for 3-4 hours, and the solids are separated and removed. The ethanol in the extract is then removed by evaporation, and the chlorogenic acid content is adjusted to 0.2-0.3 wt% with deionized water to obtain honeysuckle additive solution.

[0013] Furthermore, the hibiscus bark additive solution is prepared by soaking hibiscus bark in an ethanol solution, reflux extraction, evaporating and removing the ethanol from the extract, concentrating the extract, and then filtering the concentrate. Preferably, the hibiscus bark is dried, pulverized, and then added to an ethanol solution with a volume concentration of 50-60%. After soaking at room temperature for 10-12 hours, it is refluxed for 2-3 hours. After separating and removing solids, the extract is evaporated to remove ethanol, concentrated to 20-25% of its original volume, and then filtered to obtain the hibiscus bark additive solution.

[0014] Preferably, in the preparation of deoxycholic acid-chitosan, the molar ratio of chitosan, deoxycholic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 1:0.25-0.3:0.3-0.35:0.24-0.26.

[0015] Furthermore, the method for preparing deoxycholic acid-chitosan involves dissolving chitosan in an aqueous acetic acid solution to obtain a chitosan solution; adding deoxycholic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide to methanol to obtain a premix; adding the premix to the chitosan solution dropwise under stirring at room temperature, and reacting at room temperature for 40-42 hours after the addition is complete to obtain a reaction solution; purifying the reaction solution to obtain deoxycholic acid-chitosan.

[0016] Furthermore, the purification method is as follows: after adjusting the pH of the reaction solution to 8.5-9 and precipitating, the solid is separated and collected. After washing the solid with methanol, it is dialyzed using a dialysis bag with a molecular weight cutoff of 10-12 kDa and then dried to obtain deoxycholic acid-chitosan.

[0017] Preferably, in the preparation of deoxycholic acid-chitosan, the mass concentration of deoxycholic acid in the premix is ​​controlled to be 0.2-0.25 wt%.

[0018] Preferably, in the preparation of the first component, the pH of the acetate-sodium acetate buffer solution is 5.0; The mass-to-volume ratio of deoxycholic acid-chitosan to acetate-sodium acetate buffer is 1 g: 29-31 mL; The Angelica dahurica extract and Cyperus rotundus extract should be added simultaneously within 2-3 minutes.

[0019] Preferably, in the preparation of the second component, the mass-to-volume ratio of acetylated sodium hyaluronate to deionized water is 1g:58-60mL; The dropping rate of the hibiscus bark additive solution was controlled at 1-1.5 mL / min; The weight of trehalose added is 5-6 times that of acetylated sodium hyaluronate.

[0020] Preferably, in the preparation of the first component, the volume ratio of the dispersion, the Angelica dahurica additive solution, and the Cyperus rotundus additive solution is 29-31:1-1.2:1-1.2; In the preparation of the second component, the volume ratio of the dispersion, honeysuckle additive solution, and hibiscus bark additive solution is 58-60:5.5-6:1.2-1.4.

[0021] Preferably, in the mixed formulation, the volume ratio of the first component to the second component is 1:1-1.2.

[0022] A spray for improving oral inflammation in pets is prepared using the aforementioned method.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: In the preparation method of the spray for improving oral inflammation in pets according to the present invention, after extracting active ingredients from hibiscus bark, honeysuckle, angelica, and cyperus as raw materials to prepare additive solutions, in the preparation of the first component, deoxycholic acid-chitosan in aqueous solution can self-assemble hydrophobic segments to form hydrophobic nanomicelles. The hydrophobic active ingredients in the angelica and cyperus additive solutions are effectively encapsulated and fixed within the aforementioned micelle core through hydrophobic interactions, achieving stable drug loading of the active ingredients of angelica and cyperus, masking unpleasant tastes, and improving palatability. In the preparation of the second component, acetylated sodium hyaluronate and deionized water are used to provide a water-based hyaluronic acid network environment. The adhesiveness and hydrophobic interactions of acetylated sodium hyaluronate improve its gel performance after subsequent complexation with chitosan. At the same time, after the honeysuckle additive solution is added, the trehalose protects the active ingredients, preventing loss of active ingredients during gelation or storage. In this mixed formulation, after the first and second components are mixed, the hydrophobic segments of deoxycholic acid aggregate under the temperature-driven action of the pet's oral cavity, forming a dense, temperature-sensitive dual-gel network in the oral cavity in conjunction with acetylated sodium hyaluronate. The hydrophobic cross-linking of deoxycholic acid and chitosan enhances the strength and toughness of the gel network, while the electrostatic cross-linking network formed by chitosan and acetylated sodium hyaluronate improves the stability of the gel network, prolonging its adhesion time in the pet's oral cavity and thus extending the residence time of the active ingredients within the gel network. Furthermore, by forming a temperature-sensitive gel film in the pet's oral cavity, external stimuli are isolated. The sustained release of active ingredients from angelica and cyperus encapsulated in nanomicelles within the gel network, and from honeysuckle and hibiscus bark protected by trehalose, achieves analgesic, anti-inflammatory, and astringent effects on oral inflammation. Combined with the antibacterial and wound-healing properties of chitosan and the moisturizing and repairing functions of hyaluronic acid, this provides comprehensive care for pets with oral inflammation, from symptom relief (analgesia and anti-inflammation) to tissue repair (promoting healing). Detailed Implementation

[0024] To provide a clearer understanding of the technical features, objectives, and effects of this invention, specific embodiments are now described. It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0025] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, "first," "second," etc., are used to distinguish similar objects and are not used to describe a particular order or sequence. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0026] This invention provides a method for preparing a spray for improving oral inflammation in pets, comprising the following steps: preparing a plant extract, preparing deoxycholic acid-chitosan, preparing a first component, preparing a second component, and preparing a mixed formulation.

[0027] The preparation of the plant component extract includes the following steps: preparing hibiscus bark additive solution, preparing honeysuckle additive solution, preparing angelica dahurica additive solution, and preparing cyperus rotundus additive solution.

[0028] The method for preparing the hibiscus bark additive solution is as follows: hibiscus bark is washed, dried, and pulverized to obtain hibiscus bark powder; the hibiscus bark powder is added to 9-10 times its weight of an ethanol solution (volume concentration 50-60%), soaked at room temperature for 10-12 hours, then heated to reflux and extracted under reflux for 2-3 hours; the solids are removed by filtration, and the filtrate is concentrated to 20-25% of its original volume by rotary evaporation under reduced pressure to obtain a concentrated solution; the concentrated solution is filtered through a microporous membrane with a filtration precision of 0.4-0.45 μm to obtain the hibiscus bark additive solution for later use.

[0029] In this embodiment of the invention, the selected hibiscus bark has the effects of clearing heat and dampness, killing insects and relieving itching. When used in pet oral inflammation, it helps to clear heat and astringe, and has a certain auxiliary conditioning effect on the oral mucosa. By using a 50-60% ethanol solution for reflux extraction of hibiscus bark, combined with microporous membrane filtration, the effective components such as tannins in hibiscus bark are effectively enriched while removing impurities that may subsequently cause chitosan precipitation.

[0030] The method for preparing the honeysuckle additive solution is as follows: honeysuckle is washed, dried, and pulverized to obtain honeysuckle powder; the honeysuckle powder is added to 10-12 times its weight of ethanol solution (volume concentration 35-45%), stirred evenly, heated to reflux, and extracted under reflux for 3-4 hours. After filtration to remove solids, the filtrate is subjected to vacuum rotary evaporation to remove ethanol, and the chlorogenic acid content is adjusted to 0.2-0.3 wt% with deionized water to obtain the honeysuckle additive solution for later use.

[0031] In this embodiment of the invention, the selected honeysuckle has the effects of clearing heat and detoxifying, antibacterial and anti-inflammatory, and can inhibit pathogenic microorganisms in the pet's oral cavity and relieve oral inflammation. After reflux extraction of honeysuckle using an ethanol solution with a volume concentration of 35-45%, a honeysuckle additive solution with a predetermined chlorogenic acid content is prepared in an aqueous environment. This facilitates the uniform distribution of the active ingredients of honeysuckle (such as chlorogenic acid) in the hydrophilic acetylated hyaluronic acid network, thereby achieving stable drug loading.

[0032] The method for preparing the Angelica dahurica additive solution is as follows: Angelica dahurica is washed, dried, and pulverized to obtain Angelica dahurica powder; the Angelica dahurica powder is added to 18-20 times its weight of ethanol solution (volume concentration 80-90%), stirred evenly, heated to reflux, and extracted under reflux for 3-4 hours. After filtration to remove solids, the filtrate is evaporated to dryness under reduced pressure. The evaporation residue is dissolved in anhydrous ethanol and the imperatorin content is adjusted to 0.25-0.3 wt% to obtain the Angelica dahurica additive solution for later use.

[0033] In this embodiment of the invention, the selected Angelica dahurica has the effects of dispelling wind and dampness, reducing swelling and draining pus, relieving pain, and strengthening teeth. It can alleviate damage, swelling and pain of the oral mucosa and promote pus drainage. After reflux extraction of honeysuckle using an ethanol solution with a volume concentration of 80-90%, an Angelica dahurica additive solution with a predetermined imperatorin content is prepared using anhydrous ethanol. This facilitates the subsequent entry of hydrophobic active ingredients into the hydrophobic micelle core of the deoxycholic acid-chitosan aqueous solution through hydrophobic interactions, thereby achieving stable drug loading and avoiding precipitation and loss of active ingredients in the aqueous environment.

[0034] The method for preparing Cyperus rotundus additive solution is as follows: Cyperus rotundus is washed, dried, and pulverized to obtain Cyperus rotundus powder; Cyperus rotundus powder and deionized water are added to a distillation apparatus at a mass ratio of 1:6-7, and steam distillation is carried out for 4-5 hours. The oil phase in the distillate is collected; the oil phase is dissolved in anhydrous ethanol and the oil phase content is adjusted to 0.5-0.6 wt% to obtain Cyperus rotundus additive solution for later use.

[0035] In this embodiment of the invention, Cyperus rotundus has the effects of soothing the liver and regulating qi, calming the nerves, reducing inflammation and relieving pain. It can be combined with other raw materials to assist in pain relief and alleviate the emotional irritability of pets caused by stomatitis. The oil phase component in Cyperus rotundus is collected by steam distillation and anhydrous ethanol is used to prepare Cyperus rotundus additive solution with a predetermined oil phase content. This facilitates the entry of the active ingredients into the hydrophobic core of the deoxycholic acid-chitosan aqueous solution, thereby achieving stable drug loading.

[0036] The method for preparing deoxycholic acid-chitosan is as follows: chitosan is added to an aqueous acetic acid solution (volume concentration 1-2%), stirred and dissolved to prepare a chitosan solution with a chitosan concentration of 0.9-1 wt%; deoxycholic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) are added to methanol and stirred until homogeneous to obtain a premix; then, at room temperature, the premix is ​​added dropwise to the chitosan solution at a dropping rate of 2-2.5 mL / min, and after the addition is complete, the mixture is stirred and reacted for 40-42 h to obtain a reaction solution; the pH of the reaction solution is adjusted to 8.5-9 with ammonia, and the solid is collected by centrifugation. The solid is washed with methanol and placed in a dialysis bag with a molecular weight cutoff of 10-12 kDa. After dialysis for 150-170 h, the product in the dialysis bag is collected and freeze-dried to obtain deoxycholic acid-chitosan.

[0037] In this embodiment of the invention, chitosan is hydrophobically modified by an amidation reaction of deoxycholic acid and chitosan, covalently linking hydrophobic deoxycholic acid to chitosan to form deoxycholic acid-chitosan. In aqueous solution, the hydrophobic segments of the deoxycholic acid-chitosan self-assemble to form hydrophobic nanomicelles. The hydrophobic active ingredients in the Angelica dahurica and Cyperus rotundus extracts are effectively encapsulated and fixed within the aforementioned micelle core through hydrophobic interactions, achieving stable drug loading of the active ingredients of Angelica dahurica and Cyperus rotundus and preventing the precipitation and loss of these hydrophobic components in the aqueous environment. Simultaneously, the aforementioned hydrophobic modification process retains the amino groups of chitosan, which not only facilitates subsequent electrostatic adsorption onto the pet's oral mucosa but also allows for the synergistic construction of a dual gel network through both hydrophobic and electrostatic interactions after its subsequent combination with hyaluronic acid. Furthermore, the introduction of deoxycholic acid not only improves the drug-carrying capacity of chitosan, but also enables the aggregation of its hydrophobic segments under pet body temperature conditions to achieve a temperature-sensitive gel that can be sprayed into the pet's mouth, thus promoting the temperature-sensitive construction of the gel network.

[0038] Preferably, in the preparation of deoxycholic acid-chitosan, the molar ratio of chitosan, deoxycholic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC, and N-hydroxysuccinimide NHS is 1:0.25-0.3:0.3-0.35:0.24-0.26 (the molar amount of chitosan is calculated based on its repeating unit).

[0039] The mass concentration of deoxycholic acid in the premixed solution is 0.2-0.25 wt%.

[0040] The method for preparing the first component is as follows: Deoxycholic acid-chitosan is added to an acetate-sodium acetate buffer solution at pH 5.0 at a mass-to-volume ratio of 1g:29-31mL, and stirred at 400-500rpm for 30-40min to obtain a dispersion; under stirring at 400-500rpm, Angelica dahurica extract and Cyperus rotundus extract are simultaneously added dropwise to the dispersion, and the addition of Angelica dahurica extract and Cyperus rotundus extract is completed simultaneously within 2-3min; after the addition is completed, the ethanol is removed by nitrogen purging under stirring at room temperature to obtain the first component.

[0041] In this embodiment of the invention, deoxycholic acid-chitosan is first added to an acetate-sodium acetate buffer solution at pH 5.0. In the aqueous solution, the hydrophobic segments of deoxycholic acid-chitosan self-assemble to form hydrophobic nanomicelles. Then, Angelica dahurica and Cyperus rotundus extracts are added. The hydrophobic active ingredients in these extracts are effectively encapsulated and fixed within the aforementioned micelle core through hydrophobic interactions with the deoxycholic acid-chitosan, forming hydrophobic drug-loaded micelles. This achieves stable drug loading of the active ingredients of Angelica dahurica and Cyperus rotundus while masking their unpleasant taste. Furthermore, subsequent thermosensitive gel film formation in the pet's oral cavity prevents direct contact between these active ingredients and the pet's taste buds, improving palatability. Moreover, the hydrophobic drug-loaded micelles of the first component are uniformly dispersed in the thermosensitive gel network. After film formation in the pet's oral cavity, the physical barrier effect of the thermosensitive gel film further isolates unpleasant taste ingredients from the pet's taste buds, further improving palatability.

[0042] Preferably, in the preparation of the first component, the volume ratio of the dispersion, the Angelica dahurica additive solution, and the Cyperus rotundus additive solution is 29-31:1-1.2:1-1.2.

[0043] The method for preparing the second component is as follows: Sodium acetylated hyaluronic acid is added to deionized water at a mass-to-volume ratio of 1g:58-60mL, and stirred at 80-100rpm for 40-60min to obtain a dispersion; under stirring at 80-100rpm, trehalose is added to the dispersion, and after stirring for 20-30min, honeysuckle extract is added dropwise. After the honeysuckle extract is completely added, the mixture is stirred for 10-15min; then, hibiscus bark extract is added dropwise at a rate of 1-1.5mL / min. After the hibiscus bark extract is completely added, the mixture is stirred for 10-15min to obtain the second component.

[0044] In this embodiment of the invention, acetylated sodium hyaluronate and deionized water are used to provide a water-based hyaluronic acid network environment, forming a hydrophilic protective matrix. The adhesiveness and hydrophobic interaction of acetylated sodium hyaluronate enhance its subsequent electrostatic recombination with chitosan, thereby strengthening the temperature-sensitive gel network. During the preparation of the second component, trehalose is added beforehand to ensure its uniform distribution within the hyaluronic acid network. Then, honeysuckle extract is added to ensure the honeysuckle's active ingredients are uniformly distributed within the hyaluronic acid network. Next, hibiscus bark extract is added, where the hibiscus bark's active ingredients (such as tannins) are pre-bonded with trehalose via hydrogen bonds, shielding the active sites of the hibiscus bark's active ingredients and preventing precipitation with chitosan during mixing with the first component, thus improving the stability of the gel system. Simultaneously, trehalose also forms a protective layer around the honeysuckle and hibiscus bark's active ingredients, isolating them from the external environment and preventing oxidative degradation of the active ingredients during gelation or storage.

[0045] Preferably, in the preparation of the second component, the weight of trehalose added is 5-6 times the weight of acetylated sodium hyaluronate; The volume ratio of the dispersion, honeysuckle additive solution, and hibiscus bark additive solution is 58-60:5.5-6:1.2-1.4.

[0046] The method for preparing the mixed formulation is as follows: under stirring conditions, the second component is added to the first component at a volume ratio of 1:1-1.2. After stirring for 10-20 minutes, the pH of the material is adjusted to 7.0 using triethanolamine under stirring conditions. After vacuum degassing, the mixture is sterilized and filled into an opaque spray bottle to obtain a spray for improving oral inflammation in pets.

[0047] In this embodiment of the invention, the first and second components are mixed, and the pH is adjusted to 7.0 to weaken the positive charge of chitosan while retaining its adhesive properties. Negatively charged hyaluronic acid electrostatically crosslinks with chitosan to form a spray solution. When sprayed into a pet's mouth, the hydrophobic segments of deoxycholic acid aggregate under the temperature-driven action of the pet's oral cavity, forming a dense, temperature-sensitive dual-gel network with acetylated sodium hyaluronate. The hydrophobic crosslinking of deoxycholic acid and chitosan enhances the strength and toughness of the gel network, while the electrostatic crosslinking between chitosan and acetylated sodium hyaluronate improves the stability of the gel network, prolonging its adhesion time in the pet's oral cavity and thus extending the residence time of the active ingredients within the gel network. Furthermore, by forming a temperature-sensitive gel film inside the pet's mouth to isolate external stimuli, and through the sustained release of effective ingredients such as Angelica dahurica and Cyperus rotundus encapsulated in nano micelles within the gel network, and effective ingredients such as Lonicera japonica and Hibiscus syriacus bark protected by trehalose, analgesic, anti-inflammatory, and astringent effects are achieved for oral inflammation. In addition, the antibacterial and wound-healing functions of chitosan and the moisturizing and repairing functions of hyaluronic acid provide comprehensive care for pets' oral inflammation, from symptom relief (pain relief and anti-inflammation) to tissue repair (promoting healing).

[0048] The present invention also provides a spray for improving oral inflammation in pets prepared using the aforementioned method.

[0049] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described below in conjunction with some specific embodiments.

[0050] Example 1 This embodiment provides a spray for improving oral inflammation in pets and its preparation method, the specific steps of which are as follows: 1. Preparation of plant component extract (1) Preparation of Hibiscus bark additive solution After washing, drying, and pulverizing the hibiscus bark, obtain hibiscus bark powder. Add the hibiscus bark powder to 10 times its weight of ethanol solution (50% by volume), soak at room temperature for 12 hours, then heat to reflux and extract under reflux for 3 hours. Filter to remove solids, remove ethanol from the filtrate by rotary evaporation under reduced pressure, and concentrate to 20% of the original volume to obtain a concentrated solution. Filter the concentrated solution through a microporous membrane with a filtration precision of 0.45 μm to obtain the hibiscus bark additive solution for later use.

[0051] (2) Preparation of honeysuckle additive solution Honeysuckle flowers were washed, dried, and pulverized to obtain honeysuckle powder. The honeysuckle powder was added to 10 times its weight of ethanol solution (volume concentration 40%), stirred evenly, heated to reflux, and extracted under reflux for 3 hours. The solids were removed by filtration, and the filtrate was subjected to rotary evaporation under reduced pressure to remove ethanol. The chlorogenic acid content was adjusted to 0.25 wt% with deionized water to obtain honeysuckle additive solution for later use.

[0052] (3) Preparation of Angelica dahurica additive solution After washing, drying, and pulverizing Angelica dahurica, obtain Angelica dahurica powder. Add Angelica dahurica powder to 18 times its weight of ethanol solution (volume concentration 85%), stir evenly, heat to reflux, keep warm and reflux for 3 hours, filter to remove solids, evaporate the filtrate to dryness under reduced pressure, dissolve the evaporated residue with anhydrous ethanol and adjust the imperatorin content to 0.25 wt% to obtain Angelica dahurica additive solution for later use.

[0053] (4) Preparation of Cyperus rotundus additive solution After washing, drying, and pulverizing Cyperus rotundus, obtain Cyperus rotundus powder. Add Cyperus rotundus powder and deionized water to a distillation apparatus at a mass ratio of 1:6.5, and heat and distill for 5 hours using steam distillation. Collect the oil phase in the distillate. Dissolve the oil phase with anhydrous ethanol and adjust the oil phase content to 0.5 wt% to obtain Cyperus rotundus additive solution for later use.

[0054] 2. Preparation of deoxycholic acid-chitosan Chitosan was added to an aqueous acetic acid solution (1.1% by volume) and stirred to dissolve, preparing a chitosan solution with a chitosan concentration of 1 wt%. Deoxycholic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were added to methanol and stirred until homogeneous to obtain a premix. Then, at room temperature, the premix was added dropwise to the chitosan solution at a dropping rate of 2 mL / min. After the addition was complete, the mixture was stirred for 40 h to obtain a reaction solution. The pH of the reaction solution was adjusted to 9 with ammonia, and the solid was collected by centrifugation. The solid was washed with methanol and placed in a dialysis bag with a molecular weight cutoff of 10 kDa. After dialysis for 150 h, the product in the dialysis bag was collected and freeze-dried to obtain deoxycholic acid-chitosan.

[0055] The molar ratio of chitosan, deoxycholic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) is 1:0.25:0.3:0.24 (the molar amount of chitosan is calculated based on its repeating unit).

[0056] The mass concentration of deoxycholic acid in the premixed solution is 0.2 wt%.

[0057] 3. Preparation of the first component Deoxycholic acid-chitosan was added to an acetate-sodium acetate buffer solution at pH 5.0 at a mass-to-volume ratio of 1 g: 29 mL, and stirred at 400 rpm for 30 min to obtain a dispersion. Under stirring at 400 rpm, Angelica dahurica extract and Cyperus rotundus extract were simultaneously added dropwise to the dispersion, and the addition of Angelica dahurica extract and Cyperus rotundus extract was completed simultaneously within 2 min. After the addition was completed, the ethanol was removed by nitrogen purging under stirring at room temperature to obtain the first component.

[0058] The volume ratio of the dispersion, Angelica dahurica additive solution, and Cyperus rotundus additive solution is 29:1:1.

[0059] 4. Preparation of the second component Acetylated sodium hyaluronate was added to deionized water at a mass-to-volume ratio of 1 g: 58 mL and stirred at 80 rpm for 40 min to obtain a dispersion. Trehalose was added to the dispersion under stirring at 80 rpm, and after stirring for 20 min, honeysuckle extract was added dropwise. After the honeysuckle extract was added, the mixture was stirred for 10 min. Then, hibiscus bark extract was added dropwise at a rate of 1 mL / min. After the hibiscus bark extract was added, the mixture was stirred for 10 min to obtain the second component.

[0060] The weight of trehalose added is 5 times that of acetylated sodium hyaluronate.

[0061] The volume ratio of the dispersion, honeysuckle additive solution, and hibiscus bark additive solution was 58:5.5:1.2.

[0062] 5. Mixed preparations Under stirring conditions, the second component was added to the first component at a volume ratio of 1:1.1. After stirring for 10 minutes, the pH of the material was adjusted to 7.0 using triethanolamine under stirring conditions. After vacuum degassing, the material was sterilized and filled into an opaque spray bottle to obtain a spray for improving oral inflammation in pets.

[0063] This embodiment also provides a spray for improving oral inflammation in pets, prepared using the aforementioned method.

[0064] Example 2 This embodiment provides a spray for improving oral inflammation in pets and its preparation method, the specific steps of which are as follows: 1. Preparation of plant component extract (1) Preparation of Hibiscus bark additive solution After washing, drying, and pulverizing the hibiscus bark, obtain hibiscus bark powder. Add the hibiscus bark powder to 10 times its weight of ethanol solution (50% by volume), soak at room temperature for 11 hours, then heat to reflux and extract under reflux for 3 hours. Filter to remove solids, remove ethanol from the filtrate by rotary evaporation under reduced pressure, and concentrate to 20% of the original volume to obtain a concentrated solution. Filter the concentrated solution through a microporous membrane with a filtration precision of 0.45 μm to obtain the hibiscus bark additive solution for later use.

[0065] (2) Preparation of honeysuckle additive solution Honeysuckle flowers were washed, dried, and pulverized to obtain honeysuckle powder. The honeysuckle powder was added to an ethanol solution of 11 times its weight (40% by volume), stirred evenly, heated to reflux, and extracted under reflux for 3.5 hours. The solids were removed by filtration, and the filtrate was subjected to rotary evaporation under reduced pressure to remove ethanol. The chlorogenic acid content was adjusted to 0.25 wt% with deionized water to obtain honeysuckle additive solution for later use.

[0066] (3) Preparation of Angelica dahurica additive solution After washing, drying, and pulverizing Angelica dahurica, obtain Angelica dahurica powder. Add Angelica dahurica powder to 19 times its weight of ethanol solution (volume concentration 85%), stir evenly, heat to reflux, and extract under reflux for 3.5 hours. Filter to remove solids, evaporate the filtrate to dryness under reduced pressure, dissolve the evaporation residue with anhydrous ethanol and adjust the imperatorin content to 0.25 wt% to obtain Angelica dahurica additive solution for later use.

[0067] (4) Preparation of Cyperus rotundus additive solution After washing, drying, and pulverizing Cyperus rotundus, obtain Cyperus rotundus powder. Add Cyperus rotundus powder and deionized water to a distillation apparatus at a mass ratio of 1:6.5, and heat and distill for 5 hours using steam distillation. Collect the oil phase in the distillate. Dissolve the oil phase with anhydrous ethanol and adjust the oil phase content to 0.5 wt% to obtain Cyperus rotundus additive solution for later use.

[0068] 2. Preparation of deoxycholic acid-chitosan Chitosan was added to an aqueous acetic acid solution (1.1% by volume) and stirred to dissolve, preparing a chitosan solution with a chitosan concentration of 1 wt%. Deoxycholic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were added to methanol and stirred until homogeneous to obtain a premix. Then, at room temperature, the premix was added dropwise to the chitosan solution at a dropping rate of 2.3 mL / min. After the addition was complete, the mixture was stirred for 41 h to obtain a reaction solution. The pH of the reaction solution was adjusted to 9 with ammonia, and the solid was collected by centrifugation. The solid was washed with methanol and placed in a dialysis bag with a molecular weight cutoff of 10 kDa. After dialysis for 160 h, the product in the dialysis bag was collected and freeze-dried to obtain deoxycholic acid-chitosan.

[0069] The molar ratio of chitosan, deoxycholic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) is 1:0.28:0.32:0.25 (the molar amount of chitosan is calculated based on its repeating unit).

[0070] The mass concentration of deoxycholic acid in the premixed solution is 0.23 wt%.

[0071] 3. Preparation of the first component Deoxycholic acid-chitosan was added to an acetate-sodium acetate buffer solution at pH 5.0 at a mass-to-volume ratio of 1 g: 30 mL, and stirred at 500 rpm for 40 min to obtain a dispersion. Under stirring at 500 rpm, Angelica dahurica extract and Cyperus rotundus extract were simultaneously added dropwise to the dispersion, and the addition of Angelica dahurica extract and Cyperus rotundus extract was completed within 2 min. After the addition was completed, the ethanol was removed by nitrogen purging under stirring at room temperature to obtain the first component.

[0072] The volume ratio of the dispersion, Angelica dahurica additive solution, and Cyperus rotundus additive solution is 30:1.1:1.1.

[0073] 4. Preparation of the second component Acetylated sodium hyaluronate was added to deionized water at a mass-to-volume ratio of 1 g: 60 mL, and stirred at 100 rpm for 50 min to obtain a dispersion. Trehalose was added to the dispersion under stirring at 100 rpm, and stirred for 30 min. Honeysuckle extract was then added dropwise. After the honeysuckle extract was added, the mixture was stirred for 15 min. Then, hibiscus bark extract was added dropwise at a rate of 1.2 mL / min. After the hibiscus bark extract was added, the mixture was stirred for 10 min to obtain the second component.

[0074] The weight of trehalose added is 5.2 times that of acetylated sodium hyaluronate.

[0075] The volume ratio of the dispersion, honeysuckle additive solution, and hibiscus bark additive solution was 60:5.8:1.3.

[0076] 5. Mixed preparations Under stirring conditions, the second component is added to the first component at a volume ratio of 1:1. After stirring for 15 minutes, the pH of the material is adjusted to 7.0 using triethanolamine under stirring conditions. After vacuum degassing, the material is sterilized and filled into opaque spray bottles to obtain a spray for improving oral inflammation in pets.

[0077] This embodiment also provides a spray for improving oral inflammation in pets, prepared using the aforementioned method.

[0078] Example 3 This embodiment provides a spray for improving oral inflammation in pets and its preparation method, the specific steps of which are as follows: 1. Preparation of plant component extract (1) Preparation of Hibiscus bark additive solution After washing, drying, and pulverizing the hibiscus bark, obtain hibiscus bark powder. Add the hibiscus bark powder to 10 times its weight of ethanol solution (50% by volume), soak at room temperature for 10 hours, then heat to reflux and extract under reflux for 3 hours. Filter to remove solids, remove ethanol from the filtrate by rotary evaporation under reduced pressure, and concentrate to 20% of the original volume to obtain a concentrated solution. Filter the concentrated solution through a microporous membrane with a filtration precision of 0.45 μm to obtain the hibiscus bark additive solution for later use.

[0079] (2) Preparation of honeysuckle additive solution Honeysuckle flowers were washed, dried, and pulverized to obtain honeysuckle powder. The honeysuckle powder was added to 12 times its weight of ethanol solution (40% by volume), stirred evenly, heated to reflux, and extracted under reflux for 4 hours. The solids were removed by filtration, and the filtrate was subjected to rotary evaporation under reduced pressure to remove ethanol. The chlorogenic acid content was adjusted to 0.25 wt% with deionized water to obtain honeysuckle additive solution for later use.

[0080] (3) Preparation of Angelica dahurica additive solution After washing, drying, and pulverizing Angelica dahurica, obtain Angelica dahurica powder. Add Angelica dahurica powder to 20 times its weight of ethanol solution (volume concentration 85%), stir evenly, heat to reflux, keep warm and reflux for 4 hours, filter to remove solids, evaporate the filtrate to dryness under reduced pressure, dissolve the evaporation residue with anhydrous ethanol and adjust the imperatorin content to 0.25 wt% to obtain Angelica dahurica additive solution for later use.

[0081] (4) Preparation of Cyperus rotundus additive solution After washing, drying, and pulverizing Cyperus rotundus, obtain Cyperus rotundus powder. Add Cyperus rotundus powder and deionized water to a distillation apparatus at a mass ratio of 1:6.5, and heat and distill for 5 hours using steam distillation. Collect the oil phase in the distillate. Dissolve the oil phase with anhydrous ethanol and adjust the oil phase content to 0.5 wt% to obtain Cyperus rotundus additive solution for later use.

[0082] 2. Preparation of deoxycholic acid-chitosan Chitosan was added to an aqueous acetic acid solution (1.1% by volume) and stirred to dissolve, preparing a chitosan solution with a chitosan concentration of 1 wt%. Deoxycholic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were added to methanol and stirred until homogeneous to obtain a premix. Then, at room temperature, the premix was added dropwise to the chitosan solution at a dropping rate of 2.5 mL / min. After the addition was complete, the mixture was stirred for 42 h to obtain a reaction solution. The pH of the reaction solution was adjusted to 9 with ammonia, and the solid was collected by centrifugation. The solid was washed with methanol and placed in a dialysis bag with a molecular weight cutoff of 10 kDa. After dialysis for 170 h, the product in the dialysis bag was collected and freeze-dried to obtain deoxycholic acid-chitosan.

[0083] The molar ratio of chitosan, deoxycholic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) is 1:0.3:0.35:0.26 (the molar amount of chitosan is calculated based on its repeating unit).

[0084] The mass concentration of deoxycholic acid in the premixed solution is 0.25 wt%.

[0085] 3. Preparation of the first component Deoxycholic acid-chitosan was added to an acetate-sodium acetate buffer solution at pH 5.0 at a mass-to-volume ratio of 1 g: 31 mL, and stirred at 500 rpm for 40 min to obtain a dispersion. Under stirring at 500 rpm, Angelica dahurica extract and Cyperus rotundus extract were simultaneously added dropwise to the dispersion, and the addition of Angelica dahurica extract and Cyperus rotundus extract was completed within 3 min. After the addition was completed, the ethanol was removed by nitrogen purging under stirring at room temperature to obtain the first component.

[0086] The volume ratio of the dispersion, Angelica dahurica additive solution, and Cyperus rotundus additive solution is 31:1.2:1.2.

[0087] 4. Preparation of the second component Acetylated sodium hyaluronate was added to deionized water at a mass-to-volume ratio of 1 g: 59 mL, and stirred at 100 rpm for 60 min to obtain a dispersion. Trehalose was added to the dispersion under stirring at 100 rpm, and stirred for 30 min. Honeysuckle extract was then added dropwise. After the honeysuckle extract was added, the mixture was stirred for 15 min. Hibiscus bark extract was then added dropwise at a rate of 1.5 mL / min. After the hibiscus bark extract was added, the mixture was stirred for 15 min to obtain the second component.

[0088] The weight of trehalose added is 6 times that of acetylated sodium hyaluronate.

[0089] The volume ratio of the dispersion, honeysuckle additive solution, and hibiscus bark additive solution was 59:6:1.4.

[0090] 5. Mixed preparations Under stirring conditions, the second component was added to the first component at a volume ratio of 1:1.2. After stirring for 20 minutes, the pH of the material was adjusted to 7.0 using triethanolamine under stirring conditions. After vacuum degassing, the material was sterilized and filled into opaque spray bottles to obtain a spray for improving oral inflammation in pets.

[0091] This embodiment also provides a spray for improving oral inflammation in pets, prepared using the aforementioned method.

[0092] Comparative Example 1 Comparative Example 1 adopts the technical solution of Example 2, with the following changes: 1) The step of preparing deoxycholic acid-chitosan is omitted, and chitosan is used instead of deoxycholic acid-chitosan in the subsequent preparation of the first component; 2) Sodium hyaluronate is used instead of acetylated sodium hyaluronate in the preparation of the second component.

[0093] Comparative Example 2 Comparative Example 2 adopted the technical solution of Example 2, with the following changes: 1) the steps of preparing deoxycholic acid-chitosan and preparing the first component were omitted; 2) in the step of preparing the second component, the addition of trehalose was omitted, and after the addition of hibiscus bark solution was completed, angelica root solution and cyperus root solution were added simultaneously; and the volume ratio of dispersion, honeysuckle solution, hibiscus bark solution, angelica root solution, and cyperus root solution was controlled to be 91:5.8:1.3:1.1:1.1; finally, the second component was pH adjusted, vacuum degassed, and sterilized to obtain the spray of Comparative Example 2.

[0094] The temperature-sensitive gel properties of the sprays prepared in each embodiment were determined. At 37°C, a rheometer was used to detect the stable values ​​of the storage modulus and loss modulus of the sprays in Examples 1-3 and Comparative Examples 1-2 when they reached the plateau period. Furthermore, within a temperature range of 25-45°C, the rheometer was used to detect the changes in storage modulus and loss modulus of each spray as a function of temperature. The gel temperature was determined by taking the intersection of the storage modulus and loss modulus curves as the gel temperature. Simultaneously, using fresh porcine oral mucosa as a model, each spray was applied to the oral mucosa and allowed to stand for 1 minute. The maximum adhesion of the formed gel film on the oral mucosa was then detected using a texture analyzer. Specific results are shown in the table below:

[0095] Furthermore, the residence time of the spray prepared in the examples after gelation and film formation in the oral cavity of pets was measured. Nine healthy adult dogs of similar age and weight were selected as test dogs and randomly divided into three groups. Sodium fluorescein was added to the sprays of Examples 2 and Comparative Examples 1-2 at a ratio of 0.02 wt%, and then sprayed onto the oral mucosa of the test dogs in groups 1-3 respectively. The fluorescence intensity in the oral cavity of the test dogs in each group was observed every 1 hour until the fluorescence completely disappeared. The duration of fluorescence was recorded and the average value was taken. The specific results are shown in the table below:

[0096] As can be seen, in Comparative Example 1, after replacing deoxycholic acid-chitosan with chitosan and acetylated sodium hyaluronate with sodium hyaluronate, the lack of hydrophobic aggregation of the hydrophobic segments of deoxycholic acid-chitosan at the temperature of the pet's oral cavity, as well as the lack of water and adhesive properties of acetylated sodium hyaluronate, prevented chitosan and sodium hyaluronate from forming a dense temperature-sensitive dual gel network in the pet's oral cavity. This resulted in a significant deterioration in the mechanical and adhesive properties of the gel, and a significant reduction in the residence time of the gel film in the pet's oral cavity.

[0097] Comparative Example 2 omitted the steps of preparing deoxycholic acid-chitosan and the first component, and added all the plant additive solution in the second component preparation step. Due to the lack of hydrophobic aggregation and electrostatic cross-linking of deoxycholic acid-chitosan, it was almost impossible to form an effective gel network structure. The mechanical properties, adhesive properties, and residence time of the film formed in the pet's oral cavity were all significantly reduced. Simultaneously, due to the lack of hydrophobic segments in deoxycholic acid-chitosan responding to the temperature driven by the pet's oral cavity, the gel temperature also decreased significantly, severely affecting the subsequent storage and application performance of the spray.

[0098] Furthermore, the palatability of the sprays prepared in the examples was determined. An electronic tongue sensor array (taste sensor) was used to detect the bitterness response values ​​in the sprays of Example 2 and Comparative Examples 1-2, respectively. The bitterness response value of the control group was used as the baseline response value, and the percentage reduction in the detected response value of each spray compared to the baseline response value was calculated. Specifically, the calculation method was [(baseline response value - detected response value) / baseline response value] × 100%.

[0099] The control group was prepared by mixing deionized water, honeysuckle extract, hibiscus bark extract, angelica extract, and cyperus extract in a volume ratio of 91:5.8:1.3:1.1:1.1.

[0100] The specific results are shown in the table below:

[0101] As can be seen, in Comparative Example 1, by replacing deoxycholic acid-chitosan with chitosan and acetylated sodium hyaluronate with sodium hyaluronate, the active ingredients with unpleasant tastes were directly exposed to the aqueous phase during subsequent mixing due to the lack of effective encapsulation of the active ingredients of Angelica dahurica and Cyperus rotundus by nanomicelles. Consequently, the unpleasant tastes of these active ingredients could not be effectively masked. Furthermore, because chitosan and sodium hyaluronate could not form a dense, temperature-sensitive dual gel network in the pet's mouth, their physical shielding effect was reduced, failing to further isolate the unpleasant taste ingredients from the pet's taste buds. Similarly, Comparative Example 2, by omitting deoxycholic acid-chitosan and lacking effective encapsulation of the active ingredients of Angelica dahurica and Cyperus rotundus by nanomicelles, and relying solely on acetylated sodium hyaluronate, failed to form an effective gel network, resulting in poor physical shielding against unpleasant tastes and further deteriorating the palatability of the spray.

[0102] Furthermore, the antibacterial properties and biocompatibility of the spray prepared in the examples were determined. Staphylococcus aureus and Candida albicans were inoculated into agar medium and cultured at a constant temperature to obtain bacterial suspensions. These suspensions were then diluted to a viable bacterial concentration of 1000 CFU / mL to obtain test bacterial suspensions. One mL of either the Staphylococcus aureus or Candida albicans test bacterial suspension was evenly coated onto an LB nutrient agar plate. The spray from Example 2 was then sprayed onto the LB nutrient agar plate and incubated at 37°C for 12 hours. The viable bacterial concentration was then measured, and the inhibition rate was calculated. The specific calculation method was [(initial viable bacterial concentration of the test bacterial suspension - viable bacterial concentration after 12 hours of incubation) / initial viable bacterial concentration of the test bacterial suspension] × 100%.

[0103] Meanwhile, the relative cell viability and cytotoxicity level of the spray in Example 2 were determined according to the relevant provisions of the MTT method in GB / T16886.5-2017 "Biological evaluation of medical devices - Part 5: In vitro cytotoxicity test".

[0104] The specific results are shown in the table below:

[0105] Furthermore, the long-term storage performance of the sprays prepared in the examples was determined. The sprays from Examples 2 and Comparative Examples 1-2 were placed in a constant temperature environment of 25°C for static storage. High-performance liquid chromatography (HPLC) was used to sample and detect the contents of imperatorin (Angelica dahurica), α-cyperone (Cyperus rotundus), and chlorogenic acid (Lonicera japonica) in each spray at storage days 0, 30, 60, 90, and 180, respectively. Using the contents of imperatorin, α-cyperone, and chlorogenic acid at day 0 as a baseline, the retention rate of the active ingredients (imperatorin, α-cyperone, and chlorogenic acid) in each spray after 180 days of storage was calculated. The specific calculation method was (active ingredient content at day 180 / active ingredient content at day 0) × 100%. The specific results are shown in the table below:

[0106] Furthermore, the sprays from Examples 2 and Comparative Examples 1-2 were stored at a constant temperature of 25°C. The DPPH free radical scavenging rate of each spray was measured using the DPPH free radical scavenging method on days 0, 30, 60, 90, and 180 of storage. Using the DPPH free radical scavenging rate on day 0 as a baseline, the antioxidant activity retention rate of each spray after 180 days of storage was calculated. The specific calculation method was (DPPH free radical scavenging rate on day 180 / DPPH free radical scavenging rate on day 0) × 100%. The specific results are shown in the table below:

[0107] It can be seen that in Comparative Example 1, after replacing deoxycholic acid-chitosan with chitosan and acetylated sodium hyaluronate with sodium hyaluronate, the active ingredients of Angelica dahurica and Cyperus rotundus were easily precipitated or deactivated due to the lack of effective encapsulation of the active ingredients by nanomicelles and the direct dispersion of the active ingredients in the aqueous environment. Furthermore, the network structure formed by chitosan and sodium hyaluronate was loose and could not effectively isolate the external environment, thus failing to effectively protect the active ingredients. As a result, the spray of Comparative Example 1 lost a significant amount of active ingredients after long-term storage.

[0108] Comparative Example 2 omitted deoxycholic acid-chitosan, resulting in the lack of effective encapsulation of the active ingredients of Angelica dahurica and Cyperus rotundus by nanomicelles, thus reducing the protective performance of the active ingredients of Angelica dahurica and Cyperus rotundus. At the same time, the second component lacked trehalose to protect the active ingredients of Lonicera japonica and Hibiscus syriacus. Furthermore, the acetylated sodium hyaluronate alone could not form an effective gel network, failing to effectively isolate the external environment, leading to significant loss of active ingredients in the spray of Comparative Example 2 after long-term storage.

[0109] Furthermore, the therapeutic effect of the spray prepared in the examples on oral ulcers was determined. Sixteen healthy male mice of similar age and weight were randomly divided into four groups. Circular filter paper with a diameter of 6 mm was soaked in glacial acetic acid, and then the circular filter paper was gently applied to the buccal mucosa of the mice for 45 seconds to form oral ulcer lesions with a diameter of 6 mm. After photographing the oral ulcer lesions, the area of ​​the oral ulcer lesions was calculated using image analysis software.

[0110] The sprays from Examples 2 and 1-2 were sprayed into the oral cavity of mice in groups 1-3, twice daily (morning and evening). Group 4 served as a blank control group and received no treatment. The area of ​​oral ulcers was measured on days 0, 2, 4, 6, and 8 after spray administration. The healing rate was calculated on day 6, and the average value was taken. The specific calculation method was [(initial ulcer area - ulcer area on the current day) / initial ulcer area] × 100%. Simultaneously, the complete healing time of the oral ulcers in each group was recorded, and the average value was taken. The specific results are shown in the table below:

[0111] As can be seen, in the preparation method of the spray for improving oral inflammation in pets according to the present invention, after extracting active ingredients from hibiscus bark, honeysuckle, angelica, and cyperus as raw materials to prepare additive solutions, in the preparation of the first component, the hydrophobic segments of deoxycholic acid-chitosan in aqueous solution can self-assemble to form hydrophobic nanomicelles. The hydrophobic active ingredients in the angelica and cyperus additive solutions are effectively encapsulated and fixed within the aforementioned micelle core through hydrophobic interactions, achieving stable drug loading of the active ingredients of angelica and cyperus, masking unpleasant tastes, and improving palatability. In the preparation of the second component, acetylated sodium hyaluronate and deionized water are used to provide a water-based hyaluronic acid network environment, and the adhesiveness and hydrophobic interactions of acetylated sodium hyaluronate improve its gel performance after subsequent complexation with chitosan; at the same time, after the honeysuckle additive solution is added, the trehalose protects the active ingredients, preventing loss of active ingredients during gelation or storage. In this mixed formulation, after the first and second components are mixed, the hydrophobic segments of deoxycholic acid aggregate under the temperature-driven action of the pet's oral cavity, forming a dense, temperature-sensitive dual-gel network in the oral cavity in conjunction with acetylated sodium hyaluronate. The hydrophobic cross-linking of deoxycholic acid and chitosan enhances the strength and toughness of the gel network, while the electrostatic cross-linking network formed by chitosan and acetylated sodium hyaluronate improves the stability of the gel network, prolonging its adhesion time in the pet's oral cavity and thus extending the residence time of the active ingredients within the gel network. Furthermore, by forming a temperature-sensitive gel film in the pet's oral cavity, external stimuli are isolated. The sustained release of active ingredients from angelica and cyperus encapsulated in nanomicelles within the gel network, and from honeysuckle and hibiscus bark protected by trehalose, achieves analgesic, anti-inflammatory, and astringent effects on oral inflammation. Combined with the antibacterial and wound-healing properties of chitosan and the moisturizing and repairing functions of hyaluronic acid, this provides comprehensive care for pets with oral inflammation, from symptom relief (analgesia and anti-inflammation) to tissue repair (promoting healing).

[0112] Unless otherwise stated, all percentages used in this invention are mass percentages.

[0113] 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 method for preparing a spray for improving oral inflammation in pets, characterized in that, The process includes the following steps: preparing deoxycholic acid-chitosan, preparing the first component, preparing the second component, and mixing the formulation. The method for preparing deoxycholic acid-chitosan is as follows: chitosan is dissolved in an aqueous acetic acid solution to obtain a chitosan solution. Then, in the presence of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, the chitosan is reacted with deoxycholic acid at room temperature and purified to obtain deoxycholic acid-chitosan. The method for preparing the first component is as follows: deoxycholic acid-chitosan is added to acetate-sodium acetate buffer solution and mixed evenly to obtain a dispersion; under stirring conditions, Angelica dahurica additive solution and Cyperus rotundus additive solution are simultaneously added dropwise to the dispersion; after the addition is completed, ethanol is removed to obtain the first component. The method for preparing the second component is as follows: acetylated sodium hyaluronate is added to deionized water and mixed evenly to obtain a dispersion; trehalose is added to the dispersion and mixed evenly; then, honeysuckle additive solution and hibiscus bark additive solution are added dropwise in sequence to obtain the second component. The method for preparing the mixed formulation involves mixing the first component and the second component evenly, adjusting the pH to 7.0, and obtaining a spray for improving oral inflammation in pets.

2. The method for preparing the spray for improving oral inflammation in pets according to claim 1, characterized in that, The Angelica dahurica additive solution is prepared by reflux extraction of Angelica dahurica with ethanol solution, followed by evaporation and desolvation of the extract, and then dissolving it in anhydrous ethanol and adjusting the content of imperatorin to 0.25-0.3 wt%. The Cyperus rotundus additive solution is prepared by steam distilling Cyperus rotundus with deionized water, collecting the oil phase in the distillate, dissolving it with anhydrous ethanol, and adjusting the oil phase content to 0.5-0.6 wt%.

3. The method for preparing the spray for improving oral inflammation in pets according to claim 1, characterized in that, The honeysuckle additive solution is prepared by reflux extraction of honeysuckle with ethanol solution, followed by evaporation to remove ethanol from the extract, and adjustment of the chlorogenic acid content to 0.2-0.3 wt% with deionized water. The hibiscus bark additive solution is obtained by soaking hibiscus bark in an ethanol solution, reflux extraction, evaporation to remove ethanol, concentration of the extract, and filtration of the concentrate.

4. The method for preparing a spray for improving oral inflammation in pets according to claim 1, characterized in that, In the preparation of deoxycholic acid-chitosan, the molar ratio of chitosan, deoxycholic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 1:0.25-0.3:0.3-0.35:0.24-0.

26.

5. The method for preparing a spray for improving oral inflammation in pets according to claim 1, characterized in that, In the preparation of deoxycholic acid-chitosan, the reaction time of chitosan and deoxycholic acid at room temperature is 40-42 h. The purification method is as follows: after adjusting the pH of the reactants to 8.5-9 and precipitating, the solids are separated and collected. The solids are washed with methanol, dialyzed using a dialysis bag with a molecular weight cutoff of 10-12 kDa, and then dried.

6. The method for preparing a spray for improving oral inflammation in pets according to claim 1, characterized in that, In the preparation of the first component, the pH of the acetate-sodium acetate buffer solution is 5.0; The mass-to-volume ratio of deoxycholic acid-chitosan to acetate-sodium acetate buffer is 1g:29-31mL; The Angelica dahurica extract and Cyperus rotundus extract should be added simultaneously within 2-3 minutes.

7. The method for preparing a spray for improving oral inflammation in pets according to claim 1, characterized in that, In the preparation of the second component, the mass-to-volume ratio of acetylated sodium hyaluronate to deionized water is 1g:58-60mL. The dropping rate of the hibiscus bark additive solution was controlled at 1-1.5 mL / min; The weight of trehalose added is 5-6 times that of acetylated sodium hyaluronate.

8. The method for preparing a spray for improving oral inflammation in pets according to claim 1, characterized in that, In the preparation of the first component, the volume ratio of the dispersion, the Angelica dahurica additive solution, and the Cyperus rotundus additive solution is 29-31:1-1.2:1-1.2; In the preparation of the second component, the volume ratio of the dispersion, honeysuckle additive solution, and hibiscus bark additive solution is 58-60:5.5-6:1.2-1.

4.

9. The method for preparing a spray for improving oral inflammation in pets according to claim 1, characterized in that, In the mixed formulation, the volume ratio of the first component to the second component is 1:1-1.

2.

10. A spray for improving oral inflammation in pets, characterized in that, It is prepared by the preparation method according to any one of claims 1-9.

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