Application of daphnetin in oral treatment medicine

Abstract

The invention provides application of daphnetin in oral treatment medicines, relates to the field of medicines, in particular to the technical field of new application of medicines, discloses new application of daphnetin in medicines, and particularly discloses application of daphnetin in oral treatment medicines. The daphnetin can be applied as a promising candidate medicine for treating oral diseases.

Description

[0001] This disclosure claims priority to Chinese Patent Application No. 2025108534096, filed on June 24, 2025, entitled “Use of Daphne in Oral Therapeutic Drugs”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of medicine, particularly to the field of new uses of pharmaceuticals, and especially to the use of daphne in oral therapeutics. Background Technology

[0003] Oral diseases include tooth decay, periodontitis, and oral ulcers, among others. They are caused by a variety of factors. One type of cause is local trauma to the oral cavity (trauma can be mechanical, such as from brushing too hard or friction from braces, or chemical, such as from consuming excessively hot or irritating foods) or a weakened immune system. This allows pathogenic bacteria and microorganisms to invade the oral mucosa and periodontal tissues, leading to pain, infection, and inflammation. Periodontitis and oral ulcers are examples.

[0004] Periodontitis is a chronic infectious disease of the periodontal supporting tissues caused by microorganisms in dental plaque, characterized by progressive destruction of these tissues. Endotoxins from periodontal-specific pathogens such as *Porphyromonas gingivalis*, *Prevotella intermedius*, and *Actinomyces actinomyces* can induce the production of certain inflammatory factors in the local area and peripheral blood, such as IL-6, TNF-α, and CRP. Detecting the levels of inflammatory factors in the gingival crevicular fluid can sensitively reflect the local periodontal health status.

[0005] Oral ulcers, commonly known as canker sores, are a common ulcerative lesion of the oral mucosa, most frequently occurring on the inner lip, tongue, ventral surface of the tongue, buccal mucosa, vestibule, and soft palate. Tongue ulcers refer to oral ulcers occurring on the tongue or ventral surface of the tongue. Drug treatment involves both topical and systemic medications. Topical medications include oral anti-inflammatory drugs, analgesics, healing promoters, or corticosteroids to relieve pain and inflammation. Recurrent aphthous stomatitis is a recurring oral ulcer with an unclear cause, and current clinical treatments are not very effective.

[0006] Daphne, chemically named 7,8-dihydroxycoumarin, also known as daphne acetonide, with the chemical formula C9H6O4, is a natural coumarin derivative. It is currently mainly used as an adjunct treatment for thromboangiitis obliterans and other occlusive vascular diseases, as well as coronary heart disease. In addition, daphne is also used to treat rheumatoid arthritis. Currently, there are no reports of daphne being found to be effective in treating oral diseases such as oral ulcers and periodontitis. Summary of the Invention

[0007] The main objective of this invention is to provide new pharmaceutical uses for daphne.

[0008] To achieve the above objectives, the use of daphne as a pharmaceutical active ingredient in oral disease treatment drugs, wherein the oral diseases are selected from periodontitis or oral ulcers.

[0009] Daphne is a natural coumarin-type active ingredient, mainly derived from plants in the Thymelaeaceae family, such as Daphne genkwa and Edgeworthia chrysantha, as well as other plants in the Thymelaeaceae family. Its extraction typically involves several steps: raw material pretreatment, extraction, separation and purification, and component identification, ultimately yielding daphne as the active pharmaceutical ingredient.

[0010] Through experimental research, this invention has found that daphne exhibits good therapeutic effects on periodontitis and oral ulcers, and can be used as a promising drug for the treatment of oral diseases.

[0011] Preferably, the structural formula of daphne is: .

[0012] The present invention also provides a drug for treating oral diseases.

[0013] Specifically, oral medications contain daphne and pharmaceutically acceptable salts, and the oral diseases are selected from periodontitis or oral ulcers.

[0014] Or, more specifically, oral medications contain daphne and pharmaceutically acceptable excipients, and the oral diseases are selected from periodontitis or oral ulcers.

[0015] More preferably, the effective dose of the oral treatment drug containing the active ingredient daphne is 150–1350 mg / day.

[0016] Further preferably, the dosage form of the oral therapeutic drug is selected from any one of the following: oral dosage form, local sustained-release formulation, local controlled-release formulation, adhesive topical formulation, and traditional topical formulation.

[0017] Specifically, traditional topical preparations are selected from any one of the following: mouthwash, rinse, ointment, and cream.

[0018] Specifically, the oral dosage form is selected from any one of the following: tablets, capsules, pills, granules, powders, drops, oral films, and oral liquid preparations. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The images show the gross morphological recovery of gingival tissue in rats with periodontitis after intervention with daphne (macroscopic observation). Figure 2 Image showing HE staining results of periodontal tissue pathological changes in rats of each group; Figure 3 Image showing the Micro-CT three-dimensional reconstruction results and bone biological parameters of the maxilla of rats in each group; Figure 4 This is a graph showing the mRNA expression levels of inflammation-related factors in periodontal tissues of rats in different groups after daphne intervention (Note: **** indicates...). p <0.0001, ** indicates p <0.01); Figure 5 Figure 1 shows the change in oral ulcer healing over time in each group of rats (Note: **** indicates...). p <0.0001, *** indicates p <0.001, ** indicates p <0.01); Figure 6 Graphs showing the pathological changes of oral mucosal tissue in each group of rats over time (×100x). Figure 7 Map showing the expression of VEGF-A and inflammation-related factor mRNA in rat tissues of each group (Note: **** indicates...) p <0.0001, *** indicates p <0.001, ** indicates p <0.01, * indicates p <0.05); Figure 8 The graph shows the expression of NF-κB p65, IκBα and p38 MAPK signaling pathway proteins in rat tissues of each group.

[0021] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0023] This application provides oral disease treatment drugs for treating the following oral diseases: periodontitis or oral ulcers.

[0024] In specific embodiments, the oral disease treatment drug includes daphne and pharmaceutically acceptable excipients. Excipients involved in oral dosage forms typically include: diluents, wetting agents, binders, disintegrants, lubricants, antioxidants, coating materials, flavoring agents, coloring agents, etc. Excipients involved in local sustained-release formulations, local controlled-release formulations, adhesive local formulations, and traditional local formulations typically include: matrix materials, controlled-release polymers, coating or microcapsule materials, plasticizers, pore-forming agents, solvents, co-solvents, stabilizers, preservatives, osmotic pressure regulators, etc. Different excipients are selected and combined according to different dosage forms, and appropriate proportions are adjusted during production. The dosage form of the oral disease treatment drug can be in vivo or external, and the specific dosage form selection is not limited. The oral disease treatment drug is prepared using known preparation methods.

[0025] In a specific embodiment, the oral disease treatment drug includes daphne and a pharmaceutically acceptable salt, such as using metal hydroxides such as sodium or potassium or alkaline substances such as organic bases to neutralize the acidic groups of daphne to form the corresponding salt, so as to improve the physicochemical properties or pharmacokinetic characteristics.

[0026] In a specific embodiment, the dosage form of the oral therapeutic drug is selected from any one of the following: oral dosage form, local sustained-release agent, local controlled-release preparation, adhesive local preparation, and traditional local preparation.

[0027] Specifically, traditional topical preparations are selected from any one of the following: mouthwash, rinse, ointment, and cream.

[0028] Specifically, the oral dosage form is selected from any one of the following: tablets, capsules, pills, granules, powders, drops, oral films, and oral liquid preparations.

[0029] In a preferred embodiment, the dosage form of the oral disease treatment drug is selected from: capsules.

[0030] In a preferred embodiment, the effective dose range of the active ingredient daphne contained in the oral disease treatment drug is 150–1350 mg / day. In specific embodiments, the effective dose can be 150 mg / day, 175 mg / day, 200 mg / day, 225 mg / day, 250 mg / day, 275 mg / day, 300 mg / day, 325 mg / day, 350 mg / day, 375 mg / day, 400 mg / day, 425 mg / day, 450 mg / day, 475 mg / day, 500 mg / day, 525 mg / day, 550 mg / day, 575 mg / day, 600 mg / day, 625 mg / day, 650 mg / day, 675 mg / day, 700 mg / day, 725 mg / day, 750 mg / day, 775 mg / day, 800 mg / day, 825 mg / day, 850 mg / day, 875 mg / day, 900 mg / day, 925 mg / day, 950 mg / day, 975 mg / day, etc. mg / day, 1000 mg / day, 1025 mg / day, 1050 mg / day, 1075 mg / day, 1100 mg / day, 1125 mg / day, 1150 mg / day, 1175 mg / day, 1200 mg / day, 1225 mg / day, 1250 mg / day, 1275 mg / day, 1300 mg / day, 1325 mg / day, or 1350 mg / day.

[0031] This application provides an example of the use of daphne as a pharmaceutical active ingredient in a drug for treating oral diseases.

[0032] In the following specific embodiments, the daphne used was purchased from Jilin Xidian Pharmaceutical Technology Development Co., Ltd. (CAS No.: 486-35-1). The structural formula of the daphne used in this application embodiment is: .

[0033] Example 1 This example demonstrates that daphne is effective in treating periodontitis.

[0034] Periodontitis is a chronic, non-infectious inflammatory disease that damages the connective tissue around the teeth and causes destructive resorption of the alveolar bone, ultimately leading to tooth loss and affecting the patient's quality of life. Statistics show that the prevalence of mild periodontitis is approximately 62%, and about 23.6% of patients suffer from severe periodontitis, making it the seventh most common disease in humans. In the pathological process of periodontitis, the ulcers extending into the periodontal pockets become the gateway for oral pathogens and their metabolites to enter the systemic circulation. Pathogenic components activate a systemic immune response, leading to a sustained increase in serum levels of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and C-reactive protein (CRP). This project uses SPF-grade SD rats as experimental animals to study the therapeutic effects of daphnetin at different doses on rats with periodontitis. By observing the changes in the pathological and biochemical indicators of periodontitis in rats after treatment with different doses, the therapeutic effect of daphnetin on periodontitis is evaluated.

[0035] 1.1 Establishment of a rat model of periodontitis Thirty-six male SD mice aged 4–6 weeks, weighing 200–280 g, were randomly divided into 6 groups (n=6 per group). The groups included a blank control group, a model group, a high-dose daphne administration group, a medium-dose daphne administration group, a low-dose daphne administration group, and a minocycline hydrochloride ointment administration group. Rats were anesthetized by intraperitoneal injection of 1.25% aphrodisiac (10 mL / kg) and placed on a surgical table in a supine position. The oral cavity was opened using a mouth opener, and the interdental space between the first and second molars was separated using a dental probe. A 0.2 mm diameter orthodontic stainless steel wire was used to ligate the cervical region of both maxillary first molars. The wire was held in place by a needle holder and inserted from the distal palatal side of both maxillary first molars, circling the first molars once before ligating and fixing at a mesial position on the palatal side. The end of the ligature was left with a 3 mm bend and placed subgingivally to prevent scratching of the oral soft tissues and to avoid damaging the junctional epithelium of the gingiva. The rats were closely monitored post-surgery. They were fed sugar water from the day of the experiment, followed by regular food 12 hours later, and fed a high-sugar diet daily (drinking water was 10% glucose solution, and the feed was regular feed softened by soaking in 10% glucose solution). Except for the blank control group and the model group, which received no treatment, the other groups were treated with different concentrations of daphne via gavage or minocycline hydrochloride ointment on day 0 after successful model establishment. Four weeks post-surgery, teeth and periodontal tissues from the left maxillary side of the rats, including the area between the first and second molars, were collected.

[0036] Table 1. Dosage of drugs administered to rats in each group 1.2 Experimental Materials 1.2.1 Laboratory Animals Thirty-six male SPF-grade SD rats (4–6 weeks old, weighing 200–280 g) were housed under the following conditions: temperature 22–24℃, humidity 40%–70%, with a 12-hour light / dark cycle to simulate a normal diurnal cycle. The rats were acclimatized for one week prior to the intervention, with free access to food and water. All animal experimental procedures were strictly performed in accordance with the "Animal Ethics and Welfare Requirements of the School of Basic Medical Sciences, Jilin University".

[0037] 1.2.2 Experimental Instruments and Reagents Electronic balance; Multifunctional ELISA reader; Low-temperature high-speed centrifuge; Paraffin slicer; Pathological slide scanning imaging system; Routine equipment for histopathology (dehydrator, embedding machine, etc.); High-speed refrigerated centrifuge; Vortex oscillator; Real-time quantitative PCR instrument; Micro CT scanner; Skyscan software (image reconstruction); CTan software (bone parameter analysis); CTvox software (3D visualization); Micropipette; Ultra-low temperature freezer; HE staining kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd. (Catalog No.: C0105S). RNA extraction was performed using Trizol reagent from Invitrogen (catalog number: 15596018CN). Reverse transcription uses Takara's PrimeScript TM RT reagent kit with gDNA Eraser (Catalog No.: RR047A); qPCR amplification was performed using TB Green from Takara. ® Premix Ex Taq TM II (Tli RNaseH Plus) (Item No.: RR820A); Primers were synthesized by Thermo Fisher Scientific. The primer sequences for each gene in qPCR are as follows (5′→3′): 1.3 Experimental Methods 1.3.1 Periodontal histopathological examination Separated rat gingival and dental tissue samples were fixed in 10% neutral formalin at 4°C for at least 48 hours. After fixation, they were rinsed with PBS buffer and placed in 10% EDTA decalcification solution (pH 7.2–7.4) at 4°C in the dark for 7–10 days (the decalcification solution was changed daily, and the endpoint of decalcification was reached when a dental probe could gently pierce the dentin without resistance). After decalcification, the samples were rinsed thoroughly with PBS. Subsequently, they were dehydrated with a series of ethanol solutions, cleared with xylene, and embedded in paraffin at 60°C to prepare 5–6 μm serial sections, which were then baked at 60°C for 2–3 hours. After dewaxing with xylene and rehydration with a series of ethanol solutions, the sections were stained with hematoxylin for 5–10 minutes, differentiated with 1% hydrochloric acid ethanol, and blued with 0.5% ammonia. They were then stained with eosin for 30 seconds to 1 minute, dehydrated with a series of ethanol solutions, cleared with xylene, mounted with neutral resin, and air-dried for pathological observation. The entire process was performed gently, with strict control over decalcification, staining time, and temperature to ensure the integrity of tissue morphology and staining effect. After completion, the pathological changes of the gingiva and tooth tissue in each group were observed and photographed under a microscope.

[0038] 1.3.2 Micro-CT Scan Rat teeth and periodontal tissue samples were collected and scanned using micro-computed tomography (Micro-CT). The raw scan data were then reconstructed into three-dimensional images using Skyscan software to obtain the three-dimensional structural images of the samples. The reconstructed images were then analyzed and processed using CTan and CTvox software.

[0039] The distance from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC) was measured at six sites on the buccal and palatal sides of the first molar (mesial, central, and distal) to quantitatively assess the degree of alveolar bone resorption. Bone mineral density (BMD), tissue mineral density (TMD), and bone volume / tissue volume (BV / TV) were also measured. BMD was used to analyze the overall bone density and bone mass changes in the left maxillary first and second molar region; TMD was used to assess the mineralization quality of the bone tissue itself; and BV / TV reflected the proportion of bone tissue in the total volume of this region. These three measurements were used synergistically to analyze changes in bone mass and the level of bone mineralization in this region.

[0040] 1.3.3 Inflammation-related gene detection Total RNA extraction: Approximately 50 mg of rat oral mucosa tissue was homogenized thoroughly in 1 mL of pre-chilled Trizol reagent and allowed to stand at room temperature for 5 min to promote the dissociation of nucleic acid-protein complexes. 200 μL of chloroform was added, and the mixture was vigorously shaken for 15 s, then allowed to stand at room temperature for 3 min. The mixture was then centrifuged at 12000 × g for 15 min at 4 ℃. The supernatant was transferred to a new centrifuge tube, and an equal volume of isopropanol was added. The mixture was incubated at -20 ℃ for 30 min to precipitate RNA. The RNA precipitate was collected by centrifugation at 12000 × g for 10 min at 4 ℃. The precipitate was washed twice with 75% RNase-free ethanol, air-dried at room temperature, and dissolved in an appropriate amount of RNase-free water. RNA concentration and purity were determined using a NanoDrop 2000 (A260 / A280 ratio of 1.8–2.0 was considered acceptable) to ensure good RNA integrity.

[0041] RNA reverse transcription to synthesize cDNA: A reverse transcription kit containing genomic DNA removal enzyme was used to construct a 20 μL reaction system containing 4 μL 5× buffer, 1 μL reverse transcriptase mixture, 1 μL genomic DNA removal enzyme, and 1 μg total RNA. RNase-free water was added to the system volume. The reaction conditions were: reverse transcription at 42 ℃ for 15 min, followed by heating at 85 ℃ for 5 s to terminate the reaction, yielding cDNA.

[0042] qPCR amplification: β-actin was used as an internal reference gene, and the target genes for detection were tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), and vascular endothelial growth factor A (VEGF-A). A quantitative real-time PCR kit was used for amplification. A 20 μL reaction system was constructed, containing 10 μL of quantitative real-time PCR premix, 0.8 μL each of 10 μmol / L upstream and downstream primers for each gene, 2 μL of cDNA template, and RNase-free water to a final volume of 20 μL. The amplification program was: 95 ℃ pre-denaturation for 30 s; 95 ℃ denaturation for 5 s, 60 ℃ annealing and extension for 30 s, for 40 cycles; subsequent melting curve analysis was performed (95 ℃ 15 s, 60 ℃ 1 min, 95 ℃ 15 s).

[0043] 1.3.4 Statistical Analysis Statistical analysis was performed using SPSS 20.0 software, and t-tests were used to compare data between two groups. p A value <0.05 was considered statistically significant. GraphPad Prism 9.0 software was used for plotting.

[0044] 1.4 Experimental Results 1.4.1 The therapeutic effects of different doses of daphne on periodontitis in rats 1.4.1.1 Healing of periodontitis in rats of different groups after treatment with different doses of daphne The gross morphological recovery of gingival tissue in rats with periodontitis after intervention with daphne is shown in the attached figure. Figure 1 As shown in the figure, after successful modeling, compared with the blank control group, rats in the model group and other groups showed obvious signs of periodontitis, mainly manifested as varying degrees of tooth loosening, widening of interdental spaces, and gingival redness and swelling, spontaneous or probing bleeding in some animals. The gingiva was soft and lacked normal toughness and color. Then, different concentrations of daphne or minocycline hydrochloride were administered for treatment. As shown in the figure, after intervention with daphne and positive control drugs, the periodontal clinical signs of rats in each group were improved to varying degrees. Among them, the gingival redness and bleeding symptoms in the positive control group basically disappeared, the gingival texture returned to near normal, the tooth loosening was significantly reduced, and the widening of interdental spaces was significantly improved. The different dose groups of daphne showed a dose-dependent improvement trend. Among them, the low-dose and medium-dose groups showed relief of gingival bleeding and swelling, significant reduction in tooth mobility, and narrowing of interdental space. The high-dose group showed the most significant improvement in periodontal clinical signs. The gingival morphology, texture, and color were close to those of the positive drug control group, tooth mobility was basically restored to normal, and the interdental space was restored to near physiological level.

[0045] 1.4.1.2 Changes in HE staining of rats in different groups before and after treatment with different doses of daphne The results of HE staining are shown in the attached figure. Figure 2 As shown, the periodontal tissue structure in the blank control group was basically intact, with no obvious inflammatory lesions. The periodontal tissue in the model group showed significant pathological damage, manifested as local tissue structural disorder and extensive inflammatory cell infiltration. Compared with the model group, the low- and medium-dose daphne groups showed varying degrees of reduction in periodontal inflammation, decreased inflammatory cell infiltration, and improved tissue damage; the high-dose daphne group and the positive control group also showed significant improvement. The results suggest that daphne can alleviate the inflammatory damage of periodontal tissue in rats with experimental periodontitis to a certain extent.

[0046] 1.4.1.3 Effects of different doses of daphne on alveolar bone repair in rats with periodontitis The results of the Micro-CT sagittal scan and buccal and lingual 3D reconstruction are shown in the attached figure. Figure 3 As shown, in the blank control group, the alveolar bone structure around the maxillary first molar of rats was intact, with continuous cortical bone and dense, orderly trabecular bone arrangement. In contrast, the model group showed significant alveolar bone resorption, interruption of cortical bone continuity, and sparse, disordered trabecular bone, demonstrating a significant effect in establishing a periodontitis model. Low, medium, and high doses of daphne significantly improved the aforementioned pathological changes in a dose-dependent manner. The high-dose group showed a significant reduction in the alveolar bone defect area and a marked restoration of trabecular bone continuity and density, with effects approaching those of the positive control group. Bone biological parameters for each group are attached. Figure 3 As shown, quantitative analysis of bone biological parameters further validated the morphological observation results: compared with the blank control group, the bone mineral density (BMD), bone volume fraction (BV / TV), and trabecular thickness (Tb.Th) of the maxilla in the model group rats were significantly reduced, while the intertrabecular spacing (Tb.Sp) and the distance between the cementoenamel junction and the alveolar ridge crest (CEJ-ABC) were significantly increased (all... p <0.001 or p <0.0001), while there was no statistically significant difference in total mineral density (TMD) among the groups. p >0.05); After intervention with daphne, BMD, BV / TV, and Tb.Th were significantly increased in the medium and high dose groups compared with the model group, while Tb.Sp and CEJ-ABC were significantly decreased ( p <0.01、 p <0.001 or p <0.0001), and the therapeutic effect of high-dose daphne was comparable to that of the positive control group.

[0047] 1.4.1.4 Effects of different doses of daphne on the levels of periodontal inflammatory cytokines in rats: tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) The mRNA expression levels of inflammation-related factors (IL-1β, IL-6, and TNF-α) in the periodontal tissues of rats in each group were detected by qPCR. The results are shown in the attached figure. Figure 4 As shown, compared with the blank control group, the relative expression levels of mRNA of pro-inflammatory factors TNF-α, IL-1β and IL-6 in the maxillary bone tissue of rats in the model group were significantly increased (all... p <0.0001 indicates a significant local inflammatory response in the periodontitis model. After intervention with daphne, the expression of the above-mentioned inflammatory factors in each dose group decreased in a dose-dependent manner: among them, the mRNA expression levels of TNF-α, IL-1β, and IL-6 in the medium and high dose daphne groups were significantly downregulated compared with the model group ( p <0.001 or p The value of daphne was <0.0001), and the high-dose daphne showed no significant difference in its inhibitory effect on inflammatory factors compared to the positive control group, indicating that daphne can effectively inhibit the local inflammatory response in rats with experimental periodontitis.

[0048] 1.4 Summary This study systematically explored the therapeutic effects of different doses of daphne on periodontitis by constructing a rat experimental periodontitis model. Its therapeutic value was verified from three core aspects: clinical signs, alveolar bone microstructure repair, and local inflammation regulation.

[0049] At the whole animal level, through dynamic observation of periodontal clinical signs and microscopic findings... CT three-dimensional reconstruction and quantitative analysis of bone biological parameters clarified that daphne can improve periodontitis-related symptoms in a dose-dependent manner, inhibit alveolar bone resorption, and promote bone microstructure repair. At the molecular level, qPCR technology confirmed that daphne can significantly downregulate local periodontal pro-inflammatory factors (ILs). 1β, IL 6. TNF The expression of α) mRNA can reduce the damage of excessive inflammatory response to periodontal tissues.

[0050] The above results confirm that daphne has a significant therapeutic effect on experimental periodontitis in rats, and its effect is dose-dependent. The efficacy of high-dose daphne is comparable to that of minocycline hydrochloride, a commonly used clinical drug. The therapeutic mechanism of daphne may be closely related to inhibiting the expression of local pro-inflammatory factors in the periodontium, alleviating inflammatory damage, and thus promoting the repair of alveolar bone microstructure. This study provides experimental evidence for the clinical application of daphne in the treatment of periodontitis and also provides new ideas for the development of periodontitis treatment drugs.

[0051] Example 2 This embodiment demonstrates that daphne is effective in treating oral ulcers.

[0052] Oral ulcers (OU) are among the most common benign lesions of the oral mucosa, with a global prevalence of 5%–25%, and a tendency to recur. They are particularly prevalent in individuals with weakened immunity, high stress levels, vitamin deficiencies, and local trauma. Pathologically, OU is characterized by the destruction of the oral mucosal epithelium, resulting in localized, superficial ulcerative lesions. Etiology involves multiple factors, including immune dysfunction, genetic susceptibility, endocrine disorders (such as fluctuations in estrogen levels), nutritional deficiencies (such as B vitamin and iron deficiencies), local microenvironment imbalance, and psychological factors. Clinically, most OU patients experience localized pain and burning sensation from the ulcer, affecting eating and speech. Recurrent oral ulcers can lead to decreased appetite and malabsorption, severely impacting patients' quality of life and oral health.

[0053] This project uses SPF-grade SD rats as the research subject to study the therapeutic effect of daphnetin at different doses on an experimental oral ulcer model in rats. The therapeutic efficacy of daphnetin on oral ulcers was evaluated by observing changes in the healing process of oral ulcers, the pathological morphology of the oral mucosa, and changes in blood immune-related biochemical indicators after treatment with different doses.

[0054] 2.1 Establishing a rat model of oral ulcers Forty-two SD rats were randomly divided into six groups of seven each. The groups included a blank control group, a model group, a high-dose daphne group, a medium-dose daphne group, a low-dose daphne group, and a dexamethasone acetate oral patch (positive control).

[0055] Rats were anesthetized by intraperitoneal injection of 1.25% aphthylamine (10 mL / kg), placed on a control table, and fixed in a supine position. The oral cavity was opened using a mouth gag to fully expose the buccal mucosa, providing a good field of vision for subsequent procedures. A 5 mm diameter circular piece of filter paper was cut, soaked in 50% glacial acetic acid for 5 seconds, and then pressed onto the buccal mucosa for 40 seconds. Excess glacial acetic acid was then wiped away with a cotton swab dipped in physiological saline. The pressed area was observed to be damaged and bright red. After 24 hours, the presence of local redness, swelling, and ulceration at the treated buccal mucosa indicated successful modeling, recorded as day 0 (D0). Different concentrations of daphne were prepared using hydrogel: high-dose group 100 mg / kg, medium-dose group 50 mg / kg, and low-dose group 25 mg / kg. Except for the blank control group and the model group, which did not receive corresponding treatment, the rats in the other groups were treated with different concentrations of daphne hydrogel or dexamethasone acetate oral patches on day 0 (D0) after successful modeling, once daily. On days 0, 4, and 8 (i.e., D0, D4, and D8), two rats from each group were sacrificed, and the healing of the oral ulcers was photographed, fixed, and stained with hematoxylin and eosin (HE).

[0056] 2.2 Experimental Materials 2.2.1 Laboratory Animals Healthy SD rats, SPF grade, half male and half female, 6-8 weeks old, weighing approximately 180-220 g, were used in the experiment. They were purchased from Beijing Vital River Laboratory Animal Co., Ltd., and were acclimatized in the barrier environment facility of the Animal Experiment Center of the School of Basic Medical Sciences, Jilin University, before subsequent experiments were conducted.

[0057] 2.2.2 Experimental Instruments and Reagents Electronic balance; Multifunctional ELISA reader; Low-temperature high-speed centrifuge; Paraffin slicer; Pathological slide scanning imaging system; Routine equipment for histopathology (dehydrator, embedding machine, etc.); High-speed refrigerated centrifuge; Vortex oscillator; Electrophoresis apparatus; Transfer apparatus; Decolorizing shaker; Chemiluminescence imaging system; Real-time quantitative PCR instrument; Physiological sodium chloride solution (Sichuan Kelun Pharmaceutical Co., Ltd.); Dexamethasone acetate oral patches (Shenzhen Taitai Pharmaceutical Co., Ltd.); Glacial acetic acid was purchased from Aladdin (product number: A116166). 10% neutral formalin was purchased from Sigma (item number: F5554); Tribromoethanol powder was purchased from Sigma (item number: T4840). HE staining kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd. (Catalog No.: C0105S). RNA extraction was performed using Trizol reagent from Invitrogen (catalog number: 15596018CN). Reverse transcription uses Takara's PrimeScript TM RT reagent kit with gDNA Eraser (Catalog No.: RR047A); qPCR amplification was performed using TB Green from Takara. ® Premix Ex Taq TM II (Tli RNaseH Plus) (Item No.: RR820A); Primers were synthesized by Thermo Fisher Scientific. The primer sequences for each gene in qPCR are as follows (5′→3′): All antibodies used in the Western blotting experiments were purchased from Sigma-Aldrich, including P-NF-κB p65 (catalog number: SAB5700363), NF-κB p65 (catalog number: SAB4502610), P-IκBα (catalog number: ZRB1554), P-p38 MAPK (catalog number: SAB4504498), and the internal control β-Actin (catalog number: A5441); the secondary antibodies were HRP-labeled goat anti-rabbit IgG (catalog number: AP106P) and HRP-labeled rabbit anti-mouse IgG (catalog number: AP160P). Protein extraction was performed using RIPA lysis buffer containing protease and phosphatase inhibitors (catalog number: P0013B). Protein quantification was performed using the BCA Protein Quantification Kit (catalog number: P0010). The SDS-PAGE gel preparation kit (catalog number: P0012A), PVDF membrane (catalog number: FFP20), 5×SDS-PAGE electrophoresis buffer (catalog number: P0015), wet transfer buffer (catalog number: P0021) and 10×TBST buffer (catalog number: P0023B) were all purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0058] 2.3 Experimental Methods 2.3.1 Preparation of Daphne hydrogel Weigh an appropriate amount of PF-127 powder, pre-cool it to 4℃, and dissolve it in dPBS buffer (pH 7.4) pre-cooled to 4℃ to prepare a 24% (w / w) PF-127 hydrogel stock solution. Place the solution on a magnetic stirrer at 4℃ and stir continuously overnight (≥12 h) until the powder is completely dissolved. Filter through a 0.22 μm microporous membrane to remove impurities, obtaining a clear, particle-free PF-127 hydrogel stock solution. Accurately weigh daphne powder and dissolve it in the above stock solution to prepare daphne hydrogels with concentrations of 100 mg / mL, 50 mg / mL, and 25 mg / mL.

[0059] 2.3.2 Healing status of oral ulcers Rat ulcer photographs were taken using a digital camera on days D0, D4, and D8. A calibrated periodontal probe was placed at the ulcer edge for reference during photography. Each photograph was analyzed using image analysis software (Image J) to quantitatively measure the ulcer area. The degree of ulcer healing was calculated as follows: Ulcer healing degree = (A0 - A...) n ) / A0*100%, where A0 is the original ulcer area on day D0, A n This represents the area of ​​the ulcer that has not healed each day after day D0.

[0060] 2.3.3 Histopathological examination of rat oral mucosa Isolated rat oral mucosal tissue was fixed in 10% neutral formalin for at least 48 hours. After fixation, it was embedded in paraffin and paraffin sections were prepared. The paraffin sections were then dewaxed in xylene for 5–10 min, and the process was repeated with fresh xylene for 5–10 min to ensure complete dewaxing. Following this, the sections were sequentially rehydrated by soaking in anhydrous ethanol for 5 min, 90% ethanol for 2 min, 80% ethanol for 2 min, and 70% ethanol for 2 min. After rehydration, the sections were stained with hematoxylin for 5–10 min, rinsed in tap water for 10 min to remove excess staining, and then washed once with distilled water. Eosin staining was then performed, followed by soaking in distilled water for 2 min. Finally, the sections underwent sequential dehydration with ethanol, clearing with xylene, and mounting with neutral resin. The pathological changes in the oral mucosal tissues of each group were observed and photographed under a microscope.

[0061] 2.3.4 qPCR detection of inflammation-related genes in rat oral mucosa tissue Total RNA extraction: Approximately 50 mg of rat oral mucosa tissue was homogenized in 1 mL of pre-cooled Trizol reagent and allowed to stand at room temperature for 5 min to promote nucleic acid production. The protein complex was dissociated; 200 μL of chloroform was added, and the mixture was vigorously shaken for 15 s, then allowed to stand at room temperature for 3 min. The mixture was then centrifuged at 12,000 × g for 15 min at 4 °C. The supernatant was transferred to a new centrifuge tube, and an equal volume of isopropanol was added. The mixture was incubated at -20 °C for 30 min to precipitate RNA. The RNA precipitate was collected by centrifugation at 12,000 × g for 10 min at 4 °C. The precipitate was washed twice with 75% RNase-free ethanol, air-dried at room temperature, and then dissolved in an appropriate amount of RNase-free water. RNA concentration and purity were measured using a NanoDrop 2000 (A260 / A280 ratio of 1.8–2.0 was considered acceptable) to ensure good RNA integrity.

[0062] RNA reverse transcription to synthesize cDNA: A reverse transcription kit containing genomic DNA removal enzyme was used to construct a 20 μL reaction system containing 4 μL 5× buffer, 1 μL reverse transcriptase mixture, 1 μL genomic DNA removal enzyme, and 1 μg total RNA. RNase-free water was added to the total volume. The reaction conditions were: reverse transcription at 42℃ for 15 min, followed by heating at 85℃ for 5 s to terminate the reaction, yielding cDNA.

[0063] qPCR amplification: β-actin was used as an internal reference gene, and the target genes for detection were tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), and vascular endothelial growth factor A (VEGF-A). A quantitative real-time PCR kit was used for amplification. A 20 μL reaction system was constructed, containing 10 μL of quantitative real-time PCR premix, 0.8 μL each of 10 μmol / L upstream and downstream primers for each gene, 2 μL of cDNA template, and RNase-free water to the total volume. The amplification program was 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 60℃ annealing and extension for 30 s, for 40 cycles; subsequent melting curve analysis was performed (95℃ 15 s, 60℃ 1 min, 95℃ 15 s).

[0064] 2.3.5 Western Blotting Detection of the Expression of Inflammation Pathway-Related Proteins in Rat Oral Mucosal Tissue Approximately 50 mg of rat oral mucosal tissue was collected and added to pre-chilled RIPA lysis buffer containing protease and phosphatase inhibitors. The mixture was homogenized thoroughly on ice and allowed to stand for 30 min to allow for complete protein extraction. The tissue was then centrifuged at 12,000 × g for 15 min at 4°C, and the supernatant was collected as the total protein extract. The total protein concentration was determined using a BCA protein quantification kit. Based on the determination results, loading buffer was added, and the sample was heated in a boiling water bath for 5 min to denature the protein. The loading volume was calculated according to the protein concentration, and the denatured protein sample was added to the wells of an SDS-PAGE gel for electrophoresis separation (stacking gel voltage 80 V, separating gel voltage 120 V, until the bromophenol blue indicator migrated to the bottom of the gel). After electrophoresis, the protein on the gel was transferred to a PVDF membrane using a wet transfer method (transfer voltage 100 V, transfer time 90 min). After transfer, the PVDF membrane was placed in 5% skim milk blocking buffer and incubated on a shaker at room temperature for 2 h to block non-specific binding.

[0065] After blocking, the PVDF membrane was incubated overnight at 4°C with dilution buffers for P-NF-κB p65, NF-κB p65, P-IκBα, P-p38 MAPK, and β-Actin primary antibodies, respectively. The next day, the primary antibodies were recovered, and the membrane was washed three times with 1×TBST buffer on a shaker at room temperature for 10 min each time. Then, the corresponding HRP-labeled secondary antibody dilution buffer was added, and the membrane was incubated on a shaker at room temperature for 1.5 h. After incubation, the membrane was washed three times with 1×TBST buffer on a shaker at room temperature for 10 min each time.

[0066] After washing, chemiluminescent reagent was added to the PVDF membrane, and protein band images were acquired using a gel imaging camera. ImageJ software was used to quantitatively analyze the gray values ​​of each target protein and internal reference protein band, and the relative expression level of the target protein was expressed as the ratio of the gray value of the target protein band to the gray value of the internal reference β-Actin band.

[0067] 2.3.6 Statistical Analysis Statistical analysis was performed using GraphPad Prism 10.4. All data are expressed as mean ± standard error. One-way ANOVA was used for comparisons among multiple groups. p A value <0.05 is considered statistically significant.

[0068] 2.4 Experimental Results 2.4.1 The therapeutic effects of different doses of daphne on oral ulcers in rats 2.4.1.1 Healing of oral ulcers in rats of different groups over time after treatment with different doses of daphne. like Figure 5As shown, macroscopic observation revealed well-defined ulcerative lesions on the buccal mucosa of rats in all groups except the blank control group on day 0, indicating successful model establishment. With the progression of treatment, the ulcer healing process varied significantly among the groups. Specifically, on day 4, the ulcer area in the medium-dose and high-dose daphne groups and the positive control group significantly decreased, and the wound surface became smoother; by day 8, the ulcers were basically healed, with only a small amount of scar tissue remaining, while the model group still showed obvious unhealed wounds.

[0069] The results of quantitative analysis were basically consistent with the results of gross morphological observation: on day 4, compared with the model group, the degree of ulcer healing was significantly increased in the high, medium, and low dose groups of daphne and the positive control group. p <0.01), among which, the healing effects of the medium-dose and high-dose groups of daphne were similar, while the positive control group showed the best degree of healing. Specifically, the ulcer healing rate in the model group was only about 15%, while the healing rates in the high-dose, medium-dose, and low-dose groups of daphne and the positive control group increased to about 45%, 36%, 24%, and 57%, respectively, indicating that daphne can dose-dependently accelerate the early healing of ulcers.

[0070] By day 8, the degree of ulcer healing in all groups had further improved. The ulcer healing rate in the high-dose daphne group (approximately 76%) and the medium-dose group (approximately 74%) was even slightly better than that in the positive control group (approximately 67%), and both were significantly higher than those in the model group (approximately 40%) and the low-dose daphne group (approximately 51%). Meanwhile, the healing effect in the medium-dose daphne group was also significantly better than that in the model group and the low-dose group. p The result (<0.01) further clarifies that daphne has a good therapeutic effect on oral ulcers, and its prognostic effect on the later stage of ulcers is even better than that of dexamethasone acetate oral patches.

[0071] 2.4.1.2 Changes in staining of oral ulcer lesions in rats after treatment with different doses of daphne The results of HE staining are shown in the attached figure. Figure 6 In the blank control group, the oral mucosal epithelium of rats was intact and clearly layered, with regular arrangement of connective tissue in the lamina propria and no obvious inflammatory cell infiltration. In all other groups, epithelial continuity was interrupted at day 0 after modeling, with abundant lymphocytes and neutrophils infiltrating the lamina propria and submucosa. After intervention with daphne, by day 4, except for the model group, inflammatory infiltration in all other groups was reduced to varying degrees, and the continuity of epithelial and tissue structures began to recover. By day 8, inflammatory cell infiltration was significantly reduced in the medium- and high-dose daphne groups and the positive control group, collagen fiber arrangement became more regular, and tissue morphology approached normal; the improvement effect in the high-dose group was comparable to that in the positive control group and slightly better than that in the medium-dose group.

[0072] 2.4.1.3 Effects of different doses of daphnetin on the transcription of inflammation-related genes in rat oral mucosa, specifically the mRNA expression level of TNF-α. Results showed that, compared with the control group, the expression levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α in the model group were significantly higher. The mRNA expression levels of VEGF-A and inflammation-related factors (IL-1β, IL-6, IL-10, and TNF-α) in the oral mucosa tissues of rats in each group were detected by qPCR. The results are shown in the attached figure. Figure 7 Compared with the control group, the expression of pro-inflammatory factors IL-1β, IL-6, and TNF-α was significantly increased in the model group. p <0.0001), while the expression of anti-inflammatory factor IL-10 and angiogenesis-related factor VEGF-A were also upregulated. After intervention with thymol, all dose groups significantly reduced the expression levels of IL-1β, IL-6, and TNF-α ( p <0.0001), while the expression of IL-10 and VEGF-A was upregulated in a dose-dependent manner. Overall, the high-dose group of daphne showed the most prominent anti-inflammatory and angiogenic effects compared to the positive control group. Furthermore, the anti-inflammatory and oral mucosal tissue repair effects of daphne in regulating the balance of pro-inflammatory / anti-inflammatory factors and promoting VEGF-A expression in oral ulcers were verified.

[0073] 2.4.1.4 Effects of different doses of daphne on the expression of proteins related to the inflammatory pathway in rat oral mucosa Western blot analysis was performed on NF-κB p65 and p-IκBα to detect the phosphorylation levels of NF-κB p65, IκBα, and p38 MAPK in the oral mucosa tissue of rats in each group. The experimental results are shown in the attached figure. Figure 8 Compared with the control group, the protein expression levels of P-NF-κB p65, P-IκBα, and P-p38 MAPK were significantly increased in the model group. p The value <0.0001 indicates that the NF-κB, IκBα, and p38 MAPK signaling pathways are significantly activated in the model group during the development of oral ulcers. After treatment with daphne hydrogel, the low-dose group showed no significant effect, while the medium- and high-dose groups showed significantly reduced expression levels of PP-NF-κB p65, P-IκBα, and P-p38 MAPK, approaching those of the positive control group. This suggests that daphne can block the transduction of inflammatory signals by inhibiting the overactivation of the NF-κB and p38 MAPK signaling pathways, providing a molecular mechanism basis for its anti-inflammatory effect.

[0074] 2.5 Summary This study systematically evaluated the therapeutic effects and potential molecular mechanisms of different doses of daphne on oral ulcers by constructing an oral ulcer model in SD rats.

[0075] At the whole animal level, dynamic observation of the oral ulcer wound healing process and histopathological staining clarified that daphne can promote oral ulcer wound healing in a dose-dependent manner and reduce local inflammatory response and tissue damage. At the molecular level, qPCR technology confirmed that daphne can significantly downregulate the mRNA expression of pro-inflammatory factors (IL-1β, IL-6, TNF-α) while upregulating the transcriptional levels of anti-inflammatory factors (IL-10) and angiogenesis-related factors (VEGF-A). Furthermore, Western blot experiments revealed that daphne can effectively inhibit the overactivation of NF-κB p65, IκBα and p38 MAPK signaling pathways and reduce the phosphorylation level of key proteins.

[0076] The above results indicate that low-dose daphne has no significant therapeutic effect, while medium- and high-dose daphne may exert anti-inflammatory and tissue-repair-promoting effects in oral ulcers by regulating the expression of inflammatory factors and the NF-κB / p38 MAPK pathway, providing experimental evidence for its further development as a drug for the treatment of oral ulcers.

[0077] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention under the inventive concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. The use of daphne as an active pharmaceutical ingredient in oral disease treatment drugs, wherein the oral disease is selected from periodontitis or oral ulcers.

2. The use in a medicament for treating oral diseases as described in claim 1, characterized in that, The structural formula of the daphne is: .

3. An oral treatment drug, characterized in that, The oral treatment medication contains daphne and a pharmaceutically acceptable salt, and the oral disease is selected from periodontitis or oral ulcers.

4. An oral treatment drug, characterized in that, The oral treatment medication contains daphne and pharmaceutically acceptable excipients, and the oral disease is selected from periodontitis or oral ulcers.

5. The oral treatment drug as described in claim 3 or 4, characterized in that, The structural formula of the daphne is: .

6. The oral treatment drug as described in claim 5, characterized in that, The effective dose of the active ingredient daphne contained in the oral treatment drug is 150–1350 mg / day.

7. The oral treatment drug as described in claim 5, characterized in that, The dosage form of the oral therapeutic drug is selected from any one of the following: oral dosage form, local sustained-release preparation, local controlled-release preparation, adhesive local preparation, and traditional local preparation.

8. The oral treatment drug as described in claim 7, characterized in that, The oral dosage form is selected from any one of the following: tablets, capsules, pills, granules, powders, drops, oral films, and oral liquid preparations.

9. The oral treatment drug as described in claim 7, characterized in that, The conventional topical preparations are selected from any one of the following: mouthwash, rinse, ointment, and cream.

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