An extract of Rosa davurica with a function of preventing or relieving inflammation, a preparation method and application thereof

Abstract

The application discloses an extract of Pyrus pyrifolia, a preparation method and application thereof, and has the functions of preventing or relieving inflammation, and comprises the following steps: low-temperature crushing and sieving of Pyrus pyrifolia dried fruits to obtain Pyrus pyrifolia powder; adding cellulase and deionized water according to a material-liquid ratio of 1:15 to obtain a first mixed solution; adjusting the pH of the mixed solution to 5-5.5 for enzymolysis; the temperature of the enzymolysis is 50-60 DEG C, and the time of the enzymolysis is 50-60 min; adding 70% ethanol solution to obtain a second mixed solution; performing first ultrasonic extraction on the second mixed solution, performing second ultrasonic extraction on the filtrate, mixing the extractives obtained by the two times of extraction, filtering to remove the filtrate, and obtaining a crude extract; concentrating the crude extract to obtain the extract of Pyrus pyrifolia; the temperature of the reduced-pressure concentration is 35-45 DEG C; the total flavonoid content of the extract is greater than or equal to 25 mg / mL, and the vitamin C content is greater than or equal to 50 mg / mL. The extract has better active ingredient reservation and better antibacterial and anti-inflammatory effects.

Description

Technical Field

[0001] This invention relates to the field of plant extract technology, specifically to an extract of the fruit of the golden prickly pear that has the function of preventing or relieving inflammation, its preparation method and application. Background Technology

[0002] Golden prickly pear (Rosa roxburghii Tratt.), also known as prickly pear, is the fruit of the Rosa roxburghii plant, belonging to the genus Rosa in the Rosaceae family. It is mainly produced in Guizhou, Yunnan, and Sichuan provinces of my country, and possesses both edible and medicinal properties, making it a unique Chinese plant with both medicinal and edible uses. In recent years, with the continuous expansion of golden prickly pear cultivation and the rapid development of its deep processing industry, its industrial scale has been steadily increasing, making it one of the important pillar industries of the characteristic agricultural economy in Southwest my country.

[0003] However, current extraction technologies for the active ingredients of *Prickly Pear* have significant shortcomings, resulting in problems such as significant damage to active ingredients and low extraction efficiency. Furthermore, existing extraction processes suffer from common issues such as cumbersome procedures, high energy consumption, large amounts of organic solvents, and long production cycles, hindering the development and industrial application of its high-value-added products. For example, traditional extraction methods (such as hot reflux extraction and water bath extraction) involve high extraction temperatures (usually above 80℃) and long extraction times, leading to varying degrees of oxidative degradation or thermal decomposition of active ingredients during extraction, resulting in a significant reduction in component content and making it difficult to maintain their natural efficacy. Existing single extraction methods cannot simultaneously and efficiently extract multiple active ingredients from *Prickly Pear*: alcohol extraction is effective in extracting small-molecule flavonoids, but its extraction efficiency for large-molecule active ingredients such as polysaccharides and pectin is low; water extraction, while beneficial for polysaccharide dissolution, is insufficient to fully release fat-soluble components such as flavonoids and polyphenols encapsulated within the cell walls. Summary of the Invention

[0004] The purpose of this invention is to provide an extraction process that can fully protect the active ingredients of Prickly Pear, enabling the simultaneous and efficient extraction of multiple active ingredients, thereby achieving synergistic effects of multiple components and resource utilization, and thus improving the overall economic and environmental benefits of the entire Prickly Pear industry chain.

[0005] To achieve the above objectives, the present invention provides a method for preparing an extract of *Pyracantha fortuneana* fruit with anti-inflammatory or anti-inflammatory functions, comprising the following steps: Step 1: The dried fruit of the golden prickly pear is pulverized at low temperature and then sieved to obtain golden prickly pear fruit powder; Step 2: Add cellulase to the golden prickly pear fruit powder, and add deionized water at a material-to-liquid ratio of 1:15 to obtain a first mixed solution; adjust the pH of the mixed solution to 5-5.5, and place the mixed solution in an enzymatic hydrolysis tank for enzymatic hydrolysis; the enzymatic hydrolysis temperature is 50℃-60℃, and the enzymatic hydrolysis time is 50min-60min; Step 3: Add 70% ethanol solution to the mixed solution after enzymatic hydrolysis, adjust the material-liquid ratio to 1:20 to obtain a second mixed solution, place the second mixed solution in an ultrasonic extraction device for a first ultrasonic extraction, perform a second ultrasonic extraction on the filter residue after the first ultrasonic extraction, mix the extracts from the first and second ultrasonic extractions, filter to remove the filter residue, and obtain a crude extract. Step 4: The crude extract is concentrated in a vacuum concentration device to obtain an extract of golden prickly pear fruit; the vacuum concentration temperature is 35℃~45℃. The active ingredients of the extract of the golden prickly pear fruit include at least total flavonoids and vitamin C, wherein the content of total flavonoids is ≥25mg / mL and the content of vitamin C is ≥50mg / mL.

[0006] Preferably, in step 2, the cellulase activity is 5000 U / g, and the ratio of cellulase to golden prickly pear fruit powder is 1:200 by mass.

[0007] Preferably, in step 3, the temperature of the first ultrasonic extraction and the second ultrasonic extraction are both 25℃~35℃, and the ultrasonic power of the first ultrasonic extraction and the second ultrasonic extraction is 300W~500W.

[0008] Preferably, the active ingredients in the extract of the golden prickly pear fruit also include tannic acid and zinc.

[0009] The present invention also provides an extract of Prickly pear fruit with anti-inflammatory or anti-inflammatory functions, wherein the active ingredients of the extract of Prickly pear fruit include at least: total flavonoids, vitamin C, tannic acid, and zinc; wherein the content of total flavonoids is ≥25mg / mL, and the content of vitamin C is ≥50mg / mL.

[0010] Preferably, the extract of the golden prickly pear fruit has an inhibitory effect on canine oral porphyria, Staphylococcus aureus, and Escherichia coli.

[0011] The present invention also provides an application of an extract of the fruit of the golden prickly pear that has the function of preventing or relieving inflammation. The extract of the golden prickly pear fruit is used to prepare health food beverages or oral care agents with antioxidant, anti-inflammatory or anti-inflammatory functions.

[0012] Preferably, the oral care agent is formulated with: extract of golden prickly pear fruit, gum arabic, xylitol, glycerin, and deionized water.

[0013] Preferably, the concentration of the extract of Prickly pear fruit in the oral care agent is 50% to 60%.

[0014] The present invention also provides an application of an extract of the fruit of the golden prickly pear that has the function of preventing or relieving inflammation, wherein the extract of the golden prickly pear fruit is used to prepare drugs or cosmetics with antibacterial and anti-inflammatory effects.

[0015] Compared to the prior art, the beneficial effects of the present invention include at least the following: This invention uses the fruit of *Prickly Pear*, which has a higher concentration of active ingredients, as raw material for extraction. It employs a synergistic extraction method combining low-temperature ultrasonic extraction and low-temperature vacuum concentration. The ultrasonic extraction temperature is 25℃~35℃, and the vacuum concentration temperature is 35℃~45℃. During ultrasonic extraction, cavitation and mechanical shear force are used to break down cell walls at room temperature or lower, preventing the rapid oxidation of phenolic substances by activated oxidases released from cells at high temperatures. Low-temperature vacuum concentration significantly lowers the boiling point of the solvent by reducing system pressure, allowing water or ethanol to evaporate rapidly at low temperatures. This not only avoids secondary damage from high temperatures but, more importantly, reduces the residence time of active ingredients at high temperatures, preventing their destruction after extraction. Furthermore, ultrasound increases the dispersion of the extract. While ultrasound can enhance the extraction of vitamin C, prolonged sonication may cause localized heating of the solution. The solute-solvent complex structure formed in the extract is more stable at low temperatures. During vacuum concentration, as the solvent evaporates and the system concentration increases, the intermolecular distance shortens, making polymerization or precipitation more likely. If carried out at high temperatures, these reactions will erupt instantly. Low-temperature vacuum concentration provides a mild enrichment environment, allowing molecules to arrange themselves orderly and precipitate as the solvent decreases, preserving their original bioactive conformation. Furthermore, the ultrasonic cavitation effect introduces air into the liquid during cell disruption, increasing the dissolved oxygen concentration, which is detrimental to vitamin C extraction. Vacuum concentration removes dissolved oxygen from the liquid during vacuuming, resulting in a low-oxygen, low-temperature, and highly active final concentrate, thus preserving the active ingredients more completely. The extraction process constructs a complete low-temperature protection chain, overcoming the physical characteristics of *Rhizophora stylosa* components being easily oxidized and heat-sensitive. The final extract contains ≥25 mg / mL of total flavonoids and ≥50 mg / mL of vitamin C. Products prepared using this extract have better antibacterial, anti-inflammatory, and antioxidant effects, and have a wider range of applications. Attached Figure Description

[0016] Figure 1 This diagram illustrates the effect of the golden prickly pear extract of the present invention on the number of neutrophils throughout the body of zebrafish; where A is a visual graph and B is a statistical graph. Compared with the normal group, the model group showed a P-value < 0.001; compared with the model group, the 0.1% golden prickly pear extract group showed a P-value < 0.001. Detailed Implementation

[0017] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments. Unless otherwise specified, all experimental materials and reagents used in this invention are commercially available, and all experimental methods not specifically described herein are conventional experimental methods in the art.

[0018] This invention provides a method for preparing an extract of *Pyracantha fortuneana* fruit with anti-inflammatory or anti-inflammatory functions, comprising the following steps: Step 1, Raw Material Pretreatment: Raw material pretreatment ensures the purity and extraction efficiency of the extract, and clean, standardized operations are performed throughout the process. High-quality dried prickly pear fruit free from mold, insect infestation, and rot is selected. Fruit stems, dirt, branches, leaves, and damaged or spoiled fruit are thoroughly removed manually to ensure the raw material is free of impurities. The pretreated dried fruit is then fed into a specialized low-temperature pulverizing device for uniform crushing. The pulverized material is then passed through a 40-mesh standard inspection sieve to obtain prickly pear fruit powder with uniform particle size and good flowability. This particle size maximizes the contact area between the material and the enzymatic hydrolysate and extraction solvent, providing a stable material basis for subsequent cell wall breakdown and dissolution of active ingredients.

[0019] Based on the fundamental principle of solid-liquid mass transfer, the rate at which active ingredients diffuse from the solid to the solvent during extraction is directly proportional to the solid-liquid contact area. A 40-mesh sieve ensures that the golden prickly pear fruit powder particles are fine and uniform, resulting in an exponential increase in specific surface area. This allows the solvent to penetrate the particles more quickly, significantly improving both the dissolution rate and amount of active ingredients. Simultaneously, enzymes such as cellulase require sufficient contact with the cellulose in the substrate cell wall for effective catalysis. The 40-mesh particle size avoids both excessively coarse particles that allow cellulase to act only on the surface and not reach the interior, and excessively fine particles (e.g., exceeding 100 mesh) that form clumps after absorbing water, hindering enzyme penetration. Furthermore, the uniform particle size ensures that the enzymatic hydrolysis rate of each particle in the same batch is essentially synchronized, avoiding the unevenness caused by incomplete enzymatic hydrolysis of large particles and over-enzymatic hydrolysis of small particles. In addition, the sieved powder exhibits good flowability and uniform bulk density, preventing clumping or sedimentation during subsequent stirring. Precise metering is achieved during feeding and conveying, providing a uniform material system for subsequent solid-liquid separation, thus laying a material foundation for the stability and repeatability of the entire extraction process.

[0020] Step 2, Cellulase Pretreatment: Cellulase hydrolysis is a key step in efficiently breaking down the cell walls of *Pyracantha fortuneana* fruit, significantly improving the release efficiency of intracellular active ingredients. Weigh out *Pyracantha fortuneana* fruit powder according to the specified ratio, and add cellulase with an enzyme activity of 5000 U / g. The amount of cellulase added is strictly controlled at 0.5g / 100g of *Pyracantha fortuneana* fruit powder. Add purified water at a material-to-liquid ratio of 1:15 and mix thoroughly. Stabilize the pH of the system to 5.0 using an acidity adjuster, then transfer to a constant-temperature enzymatic hydrolysis tank. Control the enzymatic hydrolysis temperature at a constant 50℃ and continuously hydrolyze for 60 minutes, maintaining a low-speed, uniform stirring throughout the process to ensure full contact between the enzyme and the material, continuously degrading the cellulose structure and creating a loose, porous material state for subsequent ultrasonic extraction.

[0021] It should be noted that if the cellulase activity is too low, the hydrolysis of cellulose in the cell wall is insufficient, failing to effectively break down the cellulose structure of the prickly pear pomace. This results in the inability to fully release a large amount of active ingredients such as polysaccharides, flavonoids, and vitamin C bound within the cell wall, leading to low extraction efficiency and a significantly low yield of active ingredients. Conversely, if the cellulase activity is too high, the more residual enzyme protein remains, placing a heavier burden on the subsequent deproteinization process, and impurities will affect the purity of the extract.

[0022] Step 3, Ultrasonic-Assisted Extraction: Ultrasonic-assisted extraction further enhances the dissolution of active ingredients based on enzymatic hydrolysis, achieving efficient, gentle, and high-retention extraction. After enzymatic hydrolysis, 70% ethanol solution is added to the system to adjust the overall material-to-liquid ratio to 1:20. After thorough stirring, the mixture is transferred to an ultrasonic extraction device. The extraction temperature is set to 60℃ and the ultrasonic power to 300W, and continuous ultrasonic extraction is performed for 40 minutes. After the first round of extraction, the filter residue is subjected to a second ultrasonic extraction to ensure complete dissolution of residual active ingredients. The two extracts are combined, filtered to remove solid residue, and a crude extract with sufficient content of total flavonoids, vitamin C, and other components is obtained, providing a qualified feed solution for subsequent concentration and purification.

[0023] Step 4, concentration and index control, are used to stabilize the quality of the extract and ensure consistent product efficacy. The combined crude extract is transferred to a vacuum concentration apparatus, with the concentration temperature controlled at 35℃~45℃, and distilled under negative pressure until the system is completely free of alcohol odor. Purified water is added to the alcohol-free concentrate for precise dilution, and key indicators are strictly controlled to ensure that the final extract contains ≥25mg / mL total flavonoids and ≥50mg / mL vitamin C. The regulated golden prickly pear fruit extract has stable physicochemical properties and sufficient active ingredients, and can be directly used as a core active ingredient in the preparation of various products, ensuring stable and reliable antibacterial, anti-inflammatory, and repairing effects.

[0024] It should be noted that the active ingredients in the extract of the golden prickly pear fruit of this invention not only retain the total flavonoids and vitamin C well, but also the active ingredients such as triterpenoids, polyphenols and antioxidant enzymes (such as flavonols, dihydroflavones, tannins, chlorogenic acid, quercetin and ellagic acid). In addition to antibacterial and anti-inflammatory effects, it also has antioxidant capacity, which can help to eliminate free radicals, resist inflammatory damage and protect human cells.

[0025] The golden prickly pear fruit of this invention has antibacterial, anti-inflammatory and repairing effects, and can be used to prepare health food beverages, medicines, cosmetics and oral care agents.

[0026] (1) Antibacterial effect of extract from golden prickly pear fruit: The tannins in *Pyracantha fortuneana* fruit can specifically bind to the cell wall proteins of *Porphyromonas canis*, disrupting the cell wall integrity and membrane permeability, leading to leakage of intracellular substances and bacterial dehydration and inactivation. Simultaneously, it exhibits sustained inhibitory effects on common opportunistic pathogens in pet oral tracts, such as *Staphylococcus aureus* and *Escherichia coli*, achieving synergistic control of multiple strains. In a 100 mg / mL *Pyracantha fortuneana* fruit extract system, the inhibition zone diameter against *Porphyromonas canis* and *Staphylococcus aureus* reached over 18 mm, with the minimum inhibitory concentration consistently controlled below 45 mg / mL. The antibacterial effect is long-lasting and balanced, effectively controlling the proliferation of pathogenic bacteria in the oral cavity and creating a stable microecological environment for inflammation resolution and mucosal repair.

[0027] (2) Anti-inflammatory effect of extract from golden prickly pear fruit: The vitamin C and flavonoids in the fruit of *Prickly Pear* work synergistically to significantly inhibit the bioactivity of cyclooxygenase-2, reduce the synthesis and release of prostaglandin E2, and block inflammatory signal transduction pathways from upstream. The polyphenols abundant in the fruit can effectively clear excess reactive oxygen species accumulated at the site of inflammation, reduce oxidative stress damage, and downregulate the expression levels of pro-inflammatory factors IL-1β and TNF-α, alleviating symptoms of mucosal congestion, edema, and pain. In a rat model of oral ulcers, intervention with *Prickly Pear* fruit extract resulted in a reduction of IL-1β by more than 55% and TNF-α by more than 58%, significantly controlling the inflammatory response.

[0028] (3) The restorative effect of extract from golden prickly pear fruit: The zinc in the fruit of the golden prickly pear can directly participate in the DNA synthesis and proliferation of oral mucosal epithelial cells, accelerating tissue regeneration and structural reconstruction of ulcer wounds. B vitamins can regulate normal mucosal metabolic function, enhancing the integrity of epithelial tissue and barrier resistance. The natural pectin in the fruit can form a continuous and dense physical protective film on the surface of damaged mucosa, isolating it from food friction, saliva erosion, and microbial stimulation, providing a stable environment for wound healing. In animal models of oral ulcers, the wound healing rate reached over 96% after 7 days of intervention. After healing, the mucosal structure remained intact, barrier function was well restored, and the risk of recurrence was significantly reduced.

[0029] It should be noted that the reason for selecting the fruit of the golden prickly pear in this invention is that the fruit has significant exclusive advantages over the flower in terms of active ingredient composition: (1) The rutin content is significantly higher than that of golden prickly pear flowers, which can have a stronger inhibitory effect on a variety of mixed pathogenic bacteria in the oral cavity. It can accurately adapt to the clinical characteristics of cross-infection of multiple strains in the oral cavity, continuously maintain the balance of the oral microecology, and improve the long-term stability of the oral environment.

[0030] (2) The vitamin C content and tannin content of golden prickly pear fruit are higher than those of golden prickly pear flower. The combination of high concentration of active ingredients can form stronger antibacterial and anti-inflammatory effects, while accelerating the repair speed of mucosal tissue, effectively reducing the possibility of repeated stomatitis symptoms, reducing the probability of wound recurrence from the basis of ingredients, and improving the durability of nursing effect.

[0031] (3) The active ingredients of golden prickly pear fruit are highly compatible with the physiological structure and metabolic characteristics of the oral cavity. Flavonoids, tannins, vitamin groups, minerals and natural pectin together form a multi-target action system, which can simultaneously achieve antibacterial, anti-inflammatory, tissue repair and mucosal protection functions. Compared with traditional single-component oral care raw materials, it has more comprehensive functions and longer-lasting effects.

[0032] (4) The supply of raw materials of golden prickly pear fruit is stable and the processing adaptability is outstanding. The enzymatic hydrolysis and ultrasonic combined extraction process can fully break the cell wall and greatly improve the release efficiency of intracellular active ingredients. The total flavonoid yield and vitamin C retention rate are significantly higher than those of single extraction process, and the component utilization rate is higher. It can stably meet the needs of industrialized and large-scale production of various products.

[0033] The extract prepared in this invention was tested and confirmed to have anti-inflammatory effects on live animals.

[0034] I. Reference Standards 1. International Organization for Standardization (ISO) Standard (ISO 15088:2007): Water quality - Acute toxicity determination of zebrafish (Danio rerio) embryos.

[0035] 2. Organization for Economic Cooperation and Development (OECD) (OECD 236-2013): Acute Fetish Toxicity (FET) Tests of Cosmetics in Fish Embryos.

[0036] 3. National Standard (GB / T 39649-2020): Quality of Laboratory Animals (Laboratory Fish).

[0037] II. Detection Principle Zebrafish, as an important vertebrate model, has been widely used in research on immunology, developmental biology, and disease mechanisms due to its highly conserved genome, short reproductive cycle, and transparent embryos. Bacterial inflammation is an important immune response to pathogen infection, but excessive inflammation can lead to tissue damage and disease. Lipopolysaccharide (LPS) is a major component of the cell wall of Gram-negative bacteria. Neutrophils, as key effector cells of innate immunity, have migration and aggregation that are important indicators for assessing the degree of inflammation.

[0038] III. Experimental Instruments and Reagents 1. Main instruments: Fluorescence microscope, stereo microscope, vortex mixer, biochemical incubator.

[0039] 2. Main reagents: LPS.

[0040] IV. Testing System Zebrafish strain: Neutrophil fluorescent transgenic zebrafish; Zebrafish age: 2 days post-fertilization (2 dpf); Culture conditions: Adult zebrafish were raised in a culture system with 10 hours of darkness and 14 hours of light, a water temperature of 28 ± 0.5℃, and a pH of 7.0-7.5. They were fed twice a day with brine shrimp and once a day with compound feed. Embryos were raised in a biochemical incubator at 28 ± 0.5℃ (10 hours of darkness and 14 hours of light), with fresh E3 culture water changed every 24 hours.

[0041] V. Experimental Procedure 1. Maximum tolerated concentration (MTC) determination: Two dpf healthy wild-type AB strain zebrafish embryos were randomly selected and placed in 12-well cell culture plates, 10 embryos / well. Golden prickly pear extract solutions with volume concentrations of 0.0125%, 0.025%, 0.05%, 0.1%, 0.2%, and 0.4% were prepared using E3 culture water. The control group was treated with E3 culture water, while the other groups were treated with the corresponding concentrations of golden prickly pear extract solution. The plates were incubated at 28.5℃ for one day. The median toxicity (MTC) was determined based on the zebrafish malformation rate and mortality rate.

[0042] 2. Evaluation of anti-inflammatory efficacy: (1) Experimental groups: Normal group, model group, and 0.1% golden prickly pear extract group.

[0043] (2) Model construction and subject intervention: Two dpf healthy neutrophil fluorescent transgenic zebrafish were randomly selected and placed on agar plates. The normal group was microinjected with PBS, while the model group and the 0.1% golden prickly pear extract group were microinjected with LPS. After microinjection, the zebrafish were placed in 6-well cell culture plates. E3 culture water was added to the normal and model groups, and golden prickly pear extract solution was added to the 0.1% golden prickly pear extract group. The plates were then incubated at 28.5℃ for several days.

[0044] (3) Detection of neutrophil count in zebrafish: After the intervention, the zebrafish were placed under a fluorescence microscope to observe and photograph the number of neutrophils throughout their bodies.

[0045] VI. Experimental Data All data are expressed as mean ± sem. Data analysis was performed using GraphPad Prism software. t-tests were used for analysis: Compared with the normal group: ### P < 0.001; Compared with the model group: *** P < 0.001 (the smaller the P value, the more significant the difference).

[0046] VII. Judgment Basis Compared with the normal group, the number of neutrophils in the zebrafish of the model group was significantly increased (P<0.05), indicating that the model was successfully constructed.

[0047] Compared with the model group, the number of neutrophils in the zebrafish in the test group was significantly reduced (P<0.05), indicating that it has anti-inflammatory effects.

[0048] VIII. Experimental Results After zebrafish were treated with 0.0125%, 0.025%, 0.05%, 0.1%, 0.2%, and 0.4% solutions of *Rhizophora stylosa* extract, the mortality rates of each group were 0%, 0%, 0%, 0%, 0%, and 0%, respectively. Subsequent experiments used 0.1% as an example.

[0049] 2. Evaluation of anti-inflammatory efficacy: Based on the above testing methods, the effect of *Rhizophora stylosa* extract on the number of neutrophils throughout zebrafish is as follows: Figure 1 As shown: Depend on Figure 1 The results showed that, compared with the normal group, the number of neutrophils in the zebrafish in the model group was significantly increased (P<0.001), indicating that the zebrafish bacterial inflammation model was successfully established in this test. Compared with the model group, the number of neutrophils in the zebrafish in the 0.1% golden prickly pear extract group was significantly decreased (P<0.001).

[0050] The experimental results are shown in Table 1. The extract of Prickly Pear can significantly inhibit the aggregation of neutrophils in zebrafish at a concentration of 0.1%, and has anti-inflammatory effects.

[0051] Table 1. Anti-inflammatory effects of Prickly Pear Extract anti-inflammatory 0.1 ＜0.001 efficient Example

[0052] This embodiment provides a method for preparing pet stomatitis products using an extract of the fruit of the golden prickly pear, comprising the following steps: (1) Pretreatment: Wash the fresh golden prickly pear fruit, dry it in a 40℃ forced-air drying oven for 36 hours, pulverize it with a pulverizer and pass it through a 40-mesh standard inspection sieve to obtain golden prickly pear fruit powder with uniform particle size and good flowability.

[0053] (2) Enzymatic hydrolysis pretreatment: Take the golden prickly pear fruit powder, add deionized water at a material-to-liquid ratio of 1:15 (g / mL), adjust the pH to 5, add 5000U / g of cellulase (dissolve in 1:1 distilled water before use, activate in a 40℃ water bath for 5 min), and enzymatically hydrolyze in a 50℃ enzymatic hydrolysis tank for 50 min.

[0054] (3) Ultrasonic-assisted extraction: Add 70% ethanol solution to the enzymatically hydrolyzed residue, adjust the overall material-to-liquid ratio to 1:20 (g / mL), stir thoroughly, and transfer to an ultrasonic extraction device. Set the extraction temperature to 30℃, ultrasonic power to 300W, and ultrasonic frequency to 40kHz, and perform continuous ultrasonic extraction for 40 minutes. Filter and collect the filtrate. Repeat the ultrasonic extraction conditions on the residue once more. Combine the two filtrates, filter to remove solid residue, and obtain crude extract of Prickly Pear fruit.

[0055] (4) Purification: The crude extract was concentrated under reduced pressure at a temperature of 30°C. It was purified using AB-8 macroporous resin with a loading volume of 0.5 times the column volume and a flow rate of 1 mL / min. The sample was first washed with 8 times the column volume of distilled water and then desorbed with 70% ethanol. The desorbed liquid was collected, concentrated by rotary evaporation, and then freeze-dried to obtain the extract of golden prickly pear fruit.

[0056] Using a 100mL pet stomatitis spray as a quantitative benchmark, 50mL of golden prickly pear fruit extract is precisely added as the sole core active ingredient to ensure the stable performance of its three major functions: antibacterial, anti-inflammatory, and repairing. This extract undergoes rigorous enzymatic-ultrasonic extraction and indicator control, resulting in a total flavonoid content of no less than 25mg / mL and a vitamin C content of no less than 50mg / mL. It can continuously inhibit various pathogenic bacteria in the pet's oral cavity, reduce mucosal inflammation, accelerate ulcer healing, and provide stable and efficient functional support for the spray, guaranteeing the product's clinical efficacy.

[0057] Various pharmaceutical excipients are precisely added according to the formulation standards to form a stable and suitable auxiliary system for pet oral health. 2g of gum arabic is added to the system to increase liquid viscosity and form a uniform protective film on the mucous membrane surface, prolonging the residence time of the active ingredients and enhancing their efficacy. 1g of xylitol is added to improve the spray's taste, reduce resistance in dogs and cats, and improve medication compliance. Simultaneously, 5mL of glycerin is added to provide moisturizing, soothing, and film-protecting effects, reducing oral dryness and irritation, while also improving the overall physical stability of the system and preventing stratification and sedimentation.

[0058] Using high-purity purified water as the final solvent, the extract of *Rhizophora stylosa* fruit, gum arabic, xylitol, and glycerol were thoroughly mixed and dispersed in sequence, continuously stirred until the solids were completely dissolved and the system was homogeneous and transparent. Then, purified water was added to bring the volume to 100 mL. After volume adjustment, the pH of the system was precisely adjusted to maintain a stable range of 6.0-7.0, closely matching the physiological environment of a pet's oral cavity, avoiding discomfort such as mucosal irritation and redness, and ensuring safety and gentleness for long-term use.

[0059] The product scale-up process includes the following steps: Step 1: Following the fixed formula for pet stomatitis spray, precisely add golden prickly pear fruit extract, gum arabic, xylitol, glycerin, and purified water sequentially into a sealed mixing tank. Strictly control the mixing temperature to maintain at 55℃. Start the stirring device for continuous, uniform stirring at a set speed for 30 minutes to ensure complete dissolution of solid excipients such as gum arabic and xylitol, and that all components are fully dispersed in the liquid phase without agglomeration or clumping. After stirring, use a low-speed homogenizer to homogenize the mixture, eliminating phase separation and particle aggregation within the system. The result is a uniform, sediment-free, non-stratified, and highly fluid spray semi-finished product, providing a stable liquid for subsequent filtration and filling, and ensuring consistent spray particle size.

[0060] Step 2: The semi-finished spray is filtered stage by stage using a multi-stage precision filtration device to remove residual tiny insoluble particles, plant debris, and other visible impurities, ensuring the final solution is clear, transparent, and free of suspended matter and turbidity. After filtration, the qualified solution is sealed and transported to a fully automated filling machine, where it is quantitatively filled into pet-specific oral spray bottles according to standardized metering. Throughout the process, the spray particle size is controlled to remain stable within the range of 15μm–25μm, allowing the spray to adhere evenly to the pet's oral mucosa after spraying, prolonging the residence time of the active ingredients, improving the uniformity of action, and enhancing clinical efficacy.

[0061] Step 3: The filled and sealed spray product is placed into a high-pressure steam sterilizer. The sterilization temperature is set to 121℃ and maintained for 15 minutes. This ensures that the product's microbial limits are fully met and that it complies with pet care product safety regulations, while maximizing the protection of heat-sensitive active ingredients such as vitamin C and flavonoids in the golden prickly pear fruit extract from being destroyed by high temperatures. After the sterilization process, the product is allowed to cool naturally and slowly to room temperature in a clean environment, and then stored in a dark place. The final product is a golden prickly pear fruit pet stomatitis spray with stable physicochemical properties, intact active ingredients, and ready for direct use. As shown in Table 2: Table 2 Key Components and Physicochemical Indicators of Golden Prickly Pear Pet Stomatitis Spray Total flavonoid content 30mg / mL Ultraviolet-Vis Spectrophotometry Vitamin C content 55mg / mL High-performance liquid chromatography (HPLC) gum arabic addition amount 2g / 100mL Gravimetric determination Xylitol addition amount 1g / 100mL High-performance liquid chromatography (HPLC) Glycerin addition amount 5mL / 100mL Volumetric quantitative determination pH value 6.0-7.0 pH meter measurement spray particle size 15μm-25μm Laser particle size analyzer measurement sterilization conditions 121℃, 15min High-pressure steam sterilization Microbial limits Meets cosmetic safety standards Plate counting method The efficacy verification of the Golden Prickly Pear Fruit Pet Stomatitis Product included tests on broad-spectrum antibacterial properties, anti-inflammatory repair, palatability, and safety. All tests employed standardized methods and control systems to complete data determination. The antibacterial test selected *Porphyromonas canis*, *Neisseria canis*, *Staphylococcus aureus*, and *Escherichia coli* as test strains. The Oxford cup method was used to determine the diameter of the inhibition zone at different concentrations of Golden Prickly Pear Fruit extract to establish the minimum inhibitory concentration (MIC).

[0062] The anti-inflammatory and repair tests used a rat oral ulcer model to measure the reduction in inflammatory factors by the extract, record the number of ulcers healed and the healing cycle, and continuously observe the recurrence of the wound after healing. The palatability test used healthy dogs and cats as subjects, spraying the prepared spray and recording the number of cases without resistance. Safety tests included an acute oral toxicity test in mice and an ocular mucosal irritation test in rabbits, measuring toxicity-related values ​​and mucosal reaction status.

[0063] The experimental results showed that the extract of *Rhizophora rubra* fruit formed a stable inhibitory zone against various oral pathogens in pets, with a low minimum inhibitory concentration (MIC). It significantly reduced inflammatory factor levels, shortened ulcer healing time, and decreased the number of recurrences. Pets showed high tolerance, and there were no toxic reactions upon oral or mucosal contact. The overall effect was superior to conventional pet oral care products, meeting the practical application needs for the prevention and treatment of pet stomatitis. (See Table 3.) Table 3. Product efficacy verification of Golden Prickly Pear Pet Stomatitis Spray in Example 1 Antibacterial test Diameter of the inhibition zone of *Porphyromonas canis* in canine oral cavity (mm) 19.8 16.5 Antibacterial test Diameter of the inhibition zone of Staphylococcus aureus (mm) 18.5 17.0 Anti-inflammatory test IL-1β reduction rate corresponding value 56.2 45.1 Repair test Corresponding value for 7-day ulcer healing rate 96.3 88.2 Recurrence monitoring 14-day recurrence rate corresponding value 4.8 25.3 Palatability test Pet acceptance rate corresponding value 86 60 Antibacterial ability Minimum inhibitory concentration (mg / mL) 45 - Example 2 The difference between Example 2 and Example 1 is that the extraction temperature in step (3) is 35°C and the vacuum concentration temperature in step (4) is 45°C. The remaining steps are the same as in Example 1.

[0064] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the raw materials are different. Comparative Example 1 uses golden prickly pear flowers as raw materials for extraction, while the other steps are the same as those in Example 1.

[0065] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the extraction temperature in step (3) is 40°C and the vacuum concentration temperature in step (4) is 50°C. The remaining steps are the same as in Example 1.

[0066] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that the extraction temperature in step (3) is 50°C and the vacuum concentration temperature in step (4) is 60°C. The remaining steps are the same as in Example 1.

[0067] The extracts obtained from Examples 2 and Comparative Examples 1-3 were used to prepare pet stomatitis products, and the product efficacy was verified.

[0068] It is evident that the extraction process of this invention results in better retention of active ingredients, with the highest content of total flavonoids and vitamin C, reaching 30 mg / mL and 55 mg / mL respectively. The corresponding inhibition zone diameters for *Porphyromonas canis* and *Staphylococcus aureus* are the largest, and the corresponding L-1β reduction rate is also greater, demonstrating superior antibacterial and anti-inflammatory effects. According to Comparative Examples 1-3, the antibacterial and anti-inflammatory effects achievable by the extract of *Pyracantha fortuneana* flowers are less than those achieved by the fruit. Furthermore, with increasing ultrasonic extraction and reduced-pressure concentration temperatures, the active substances are significantly damaged, especially total flavonoids and vitamin C. The molecular structure of heat-sensitive active substances changes at high temperatures, further reducing their antibacterial and anti-inflammatory effects.

[0069] In summary, the method for preparing the extract of *Pyracantha fortuneana* fruit provided by this invention includes the following steps: Step 1: Low-temperature pulverization and sieving of dried *Pyracantha fortuneana* fruit to obtain *Pyracantha fortuneana* fruit powder; Step 2: Adding cellulase to the *Pyracantha fortuneana* fruit powder, and adding deionized water at a material-to-liquid ratio of 1:15 to obtain a first mixed solution; adjusting the pH of the mixed solution to 5-5.5, and placing the mixed solution in an enzymatic hydrolysis tank for enzymatic hydrolysis; the enzymatic hydrolysis temperature is 50℃-60℃, and the enzymatic hydrolysis time is 50-60 min; Step 3: Adding 70% ethanol solution to the mixed solution after enzymatic hydrolysis, adjusting the material-to-liquid ratio to 1:20 to obtain a second mixed solution, placing the second mixed solution in an ultrasonic extraction device for a first ultrasonic extraction, performing a second ultrasonic extraction on the filter residue after the first ultrasonic extraction, mixing the extracts from the first and second ultrasonic extractions, filtering to remove the filter residue, and obtaining a crude extract; Step 4: Concentrating the crude extract in a vacuum concentration device to obtain the extract of *Pyracantha fortuneana* fruit; the vacuum concentration temperature is 35℃-45℃. The extract prepared by this method has a total flavonoid content ≥25mg / mL and a vitamin C content ≥50mg / mL. It has achieved good antibacterial and anti-inflammatory effects in the preparation of anti-inflammatory products and can be used in health food beverages, oral care agents, pharmaceuticals, and cosmetics.

[0070] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A method for preparing an extract of *Pyracantha fortuneana* fruit with anti-inflammatory or anti-inflammatory functions, characterized in that, Includes the following steps: Step 1: The dried fruit of the golden prickly pear is pulverized at low temperature and then sieved to obtain golden prickly pear fruit powder; Step 2: Add cellulase to the golden prickly pear fruit powder, and add deionized water at a material-to-liquid ratio of 1:15 to obtain a first mixed solution; adjust the pH of the mixed solution to 5-5.5, and place the mixed solution in an enzymatic hydrolysis tank for enzymatic hydrolysis; the enzymatic hydrolysis temperature is 50℃-60℃, and the enzymatic hydrolysis time is 50min-60min; Step 3: Add 70% ethanol solution to the mixed solution after enzymatic hydrolysis, adjust the material-liquid ratio to 1:20 to obtain a second mixed solution, place the second mixed solution in an ultrasonic extraction device for a first ultrasonic extraction, perform a second ultrasonic extraction on the filter residue after the first ultrasonic extraction, mix the extracts from the first and second ultrasonic extractions, filter to remove the filter residue, and obtain a crude extract. Step 4: The crude extract is concentrated in a vacuum concentration device to obtain an extract of golden prickly pear fruit; the vacuum concentration temperature is 35℃~45℃. The active ingredients of the extract of the golden prickly pear fruit include at least total flavonoids and vitamin C, wherein the content of total flavonoids is ≥25mg / mL and the content of vitamin C is ≥50mg / mL.

2. The preparation method according to claim 1, characterized in that, In step 2, the cellulase activity is 5000 U / g, and the ratio of cellulase to golden prickly pear fruit powder is 1:200 by mass.

3. The preparation method according to claim 1, characterized in that, In step 3, the temperature of the first and second ultrasonic extractions is 25℃~35℃, and the ultrasonic power of the first and second ultrasonic extractions is 300W~500W.

4. The preparation method according to claim 1, characterized in that, The active ingredients in the extract of the golden prickly pear fruit also include tannic acid and zinc.

5. An extract of *Pyracantha fortuneana* fruit with anti-inflammatory or anti-inflammatory properties, characterized in that... The active ingredients of the extract of the golden prickly pear fruit include at least: total flavonoids, vitamin C, tannic acid, and zinc; wherein the content of total flavonoids is ≥25mg / mL, and the content of vitamin C is ≥50mg / mL.

6. The extract of *Pyracantha fortuneana* fruit as described in claim 5, characterized in that, The extract of the golden prickly pear fruit has an inhibitory effect on canine oral porphyria, Staphylococcus aureus, and Escherichia coli.

7. The application of an extract of *Pyracantha fortuneana* fruit with anti-inflammatory or anti-inflammatory functions as described in any one of claims 1 to 4, characterized in that... The extract of the golden prickly pear fruit is used to prepare health food beverages or oral care agents with antioxidant, anti-inflammatory, or anti-inflammatory functions.

8. The application as described in claim 7, characterized in that, The oral care agent is formulated with: extract of golden prickly pear fruit, gum arabic, xylitol, glycerin, and deionized water.

9. The application as described in claim 8, characterized in that, The concentration of the extract of golden prickly pear fruit in the oral care agent is 50%~60%.

10. The application of an extract of *Pyracantha fortuneana* fruit with anti-inflammatory or anti-inflammatory functions as described in any one of claims 1 to 4, characterized in that... The extract of the golden prickly pear fruit is used to prepare drugs or cosmetics with antibacterial and anti-inflammatory effects.

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