Application of isovitexin in preparation of medicine for treating oral submucosal fibrosis

Abstract

The invention discloses an application of isovitexin in preparation of a medicine for treating oral submucosal fibrosis (OSF). Experiments prove that isovitexin improves OSF pathological changes in a dose-dependent manner, and the effect of isovitexin is superior to that of a positive control drug tanshinone II A. The mechanism is to up-regulate Mfn2 and STX17 protein expression, enhance mitochondrial and lysosome co-localization and STX17-VDAC1 combination, down-regulate mitochondrial DNA copy number and ROS generation, and regulate mitochondrial autophagy; meanwhile, the level of p-Smad3 is reduced, the secretion of type I collagen is reduced, the activity of MMP-1 is improved, collagen deposition is reduced, and the mitochondrial membrane potential is improved. The oral coating particle prefabricated tablet (prepared from isovitexin, hydroxypropyl methyl cellulose and the like) and the gel (containing isovitexin, carbomer 940 and the like) provided by the invention are administrated through local smearing or focus injection, the mucous membrane morphology can be effectively recovered, the mouth opening degree can be improved, the safety is good, and a new choice is provided for OSF treatment.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of isovitexin in the preparation of drugs for treating oral submucosal fibrosis. Background Technology

[0002] Oral submucosal fibrosis (OSF) is a precancerous lesion of the oral cavity closely related to arecoline exposure, with a global prevalence of 0.1%–2.3%, particularly high in Southeast Asia and southern China. The malignancy rate can reach 7%–15%, seriously threatening patients' oral health and lives. OSF has a clear pathological progression: after arecoline enters oral mucosal fibroblasts, it leads to a significant decrease in the frequency of mitochondrial-lysosome contact (MALM); reduced MALM further induces mitochondrial dysfunction, manifested as a decrease in mitochondrial membrane potential and an increase in reactive oxygen species (ROS) production. Damaged mitochondria cannot be effectively cleared through autophagy, thereby activating the TGF-β1 / Smad3 signaling pathway, prompting oral mucosal fibroblasts to excessively secrete collagen I, with secretion levels reaching 2–3 times that of normal tissue, ultimately leading to oral mucosal fibrosis and clinical manifestations such as difficulty opening the mouth.

[0003] Currently, clinical treatment for OSF focuses on "symptomatic relief + risk control," with no radical cure. Specific treatment methods and limitations are as follows: 1) Etiological intervention: The main approach is mandatory abstinence from betel nut, which only slows OSF progression and cannot reverse existing fibrosis. 2) Anti-inflammatory treatment: Local injection of dexamethasone is commonly used, which can temporarily alleviate mucosal redness and swelling, but long-term use can lead to oral mucosal atrophy and exacerbate collagen deposition, hindering disease control. 3) Collagen inhibition: Oral colchicine can reduce collagen synthesis by inhibiting fibroblast proliferation, but its gastrointestinal side effects are as high as 30%, and it cannot improve mitochondrial damage in OSF patients. 4) Physical therapy: Hyperbaric oxygen therapy can improve oxygen supply to the oral mucosa, but it has no effect on MALM and mitochondrial function. 5) Combination therapy: Triamcinolone acetonide combined with tanshinone II. A (one of the tanshinone monomers) injection is a commonly used combination therapy in clinical practice. Its mechanism of action is to reduce the levels of TGF-β1 and IL-6 in serum. It can only achieve single-target intervention and cannot improve the pathological root cause of OSF, namely the reduction of mitochondrial-lysosome contact and mitochondrial dysfunction. At the same time, it does not activate mitochondrial autophagy and cannot eliminate the continuous stimulation of damaged mitochondria on fibroblasts. The therapeutic effect is limited to "delaying the progression" and cannot achieve pathological reversal.

[0004] In summary, existing treatments for OSF and research on related active ingredients have significant technical shortcomings: 1) Single target: Existing drugs such as tanshinone IIA and colchicine only target a single pathological link in "collagen synthesis" or "inflammatory response," failing to intervene in the core pathological chain of OSF: "arecoline → reduced MALM → mitochondrial dysfunction → excessive collagen deposition." This results in irreversible mucosal fibrosis after treatment and a recurrence rate as high as 35%-40%. 2) Lack of specific MALM regulation: Existing mitochondrial autophagy regulators such as aconitine only clear damaged mitochondria through pathways such as Parkin and PINK1. However, the key pathological problem in OSF is the impaired initiation of mitochondrial autophagy caused by reduced MALM, not an abnormality in the autophagy pathway itself. Therefore, existing drugs cannot restore MALM frequency and cannot improve mitochondrial function from the source. 3) Difficulty in balancing safety and efficacy: Among existing drugs, long-term use of dexamethasone can easily cause oral mucosal atrophy, colchicine has significant gastrointestinal side effects, and tanshinone IIA... While drug A has a high safety profile, its therapeutic efficacy is limited, failing to meet the clinical needs of OSF for "long-term intervention, pathological reversal, and low side effects." Furthermore, research on the application scenarios and mechanisms of promising active ingredients also has limitations. For example, current mitophagy regulators such as aconitine for fibrosis mainly promote the degradation of damaged mitochondria through pathways like Parkin and PINK1, without involving the regulation of mitophagy pathways (MALM). This makes them unsuitable for the specific pathological feature of OSF—the impaired initiation of mitophagy due to reduced MALM. Therefore, there is an urgent need in this field for a therapeutic drug that can target the core pathological chain of OSF, achieve MALM regulation and mitochondrial function repair, while also possessing high safety and suitability for local administration, to fill the gap in radical treatment options for OSF.

[0005] Isovitexin is a natural flavonoid compound derived from hawthorn, honeysuckle, and other plants. It has been shown to possess three main pharmacological effects: first, it exerts anti-inflammatory effects by inhibiting the NF-κB pathway, reducing the release of inflammatory factors such as IL-6 and TNF-α; second, it achieves antioxidant and ROS scavenging effects by upregulating SOD and GSH-Px activities; and third, it exhibits tissue-protective effects by inhibiting collagen deposition in diseases such as gastric ulcers and liver fibrosis. However, current research on isovitexin has not yet included OSF treatment, nor has its role in regulating the mitochondrial-lysosomal contact (MALM) or Mfn2-STX17 pathway been reported. Summary of the Invention

[0006] The present invention aims to provide the application of isovitexin in the preparation of medicaments for treating oral submucosal fibrosis.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: Application of isovitexin in the preparation of drugs for treating oral submucosal fibrosis; Furthermore, the administration methods of the drug include topical application to the oral cavity and / or injection around oral fibrotic lesions; When the administration method is topical application to the oral cavity, the above-mentioned drugs are pre-formed granules for oral application.

[0008] A pre-formed granule for oral application in the treatment of oral submucosal fibrosis, wherein the raw materials for preparing the pre-formed granule include isovitexin; Furthermore, the raw materials for preparing the oral granule preform also include hydroxypropyl methylcellulose, microcrystalline cellulose, crospovidone XL and magnesium stearate, with the mass ratio of isovitexin:hydroxypropyl methylcellulose:microcrystalline cellulose:crospovidone XL:magnesium stearate being 20:150:100:30:5.

[0009] The above-mentioned method for preparing oral granule preforms includes the following steps: 1) Mix hydroxypropyl methylcellulose, microcrystalline cellulose, crospovidone XL and magnesium stearate evenly to obtain a mixture; 2) Add hydroxypropyl methylcellulose ethanol solution to the mixture, mix well, and then perform wet granulation to obtain wet granules; 3) The wet particles are fluidized and dried to obtain dry particles; 4) The dried granules are granulated and then compressed into tablets to obtain uncoated tablets; 5) Hydroxypropyl methylcellulose and triacetin are mixed evenly as coating material to coat the uncoated tablets with a film to obtain pre-made granular tablets for oral application.

[0010] When administered via injection around oral fibrosis lesions, the above-mentioned drugs are in the form of gels.

[0011] A gel for treating oral submucosal fibrosis, wherein the raw materials for preparing the gel include isovitexin; Furthermore, by weight percentage, the gelling agent comprises: 0.5% isovitexin, 1.5% carbomer 940, 5% glycerin, 0.05% methylparaben, 0.5% triethanolamine, and the balance being water.

[0012] The above-mentioned method for preparing a gelling agent includes the following steps: 1) Add carbomer 940 to water to swell, then add glycerin and methylparaben in sequence, stir well to obtain a gel matrix; 2) While stirring, slowly add triethanolamine to the gel matrix to adjust the pH value; 3) Add isovitexin to the pH-adjusted gel matrix, stir continuously until completely dissolved, and add water to the specified total amount; 4) After the obtained mixture is aseptically filtered through a microporous membrane, it is dispensed under aseptic conditions to obtain a gel.

[0013] The beneficial effects of this invention are as follows: This invention provides a novel use of isovitelline in the preparation of drugs for treating oral submucosal fibrosis (OSF), and discloses two local dosage forms: pre-made granules and gels for oral application. These can be administered via topical application or injection into the lesion, precisely targeting the affected area and improving drug utilization. Experiments have demonstrated that isovitelline can dose-dependently improve the pathological state of OSF, with medium and high doses showing superior effects compared to the positive control drug tanshinone IIA. It can significantly reduce collagen deposition, restore mitochondrial membrane potential, and downregulate abnormally elevated mitochondrial DNA copy number and ROS production, bringing them closer to normal levels. Its mechanism of action is clear: it upregulates the expression of Mfn2 and STX17 proteins, promotes the binding of STX17 to VDAC1 and mitochondrial-lysosomal co-localization, and regulates the recovery of mitochondrial autophagy; simultaneously, it downregulates p-Smad3 to reduce type I collagen secretion, increases MMP-1 activity to promote collagen degradation, bidirectionally balances collagen metabolism, improves fibrosis, and restores mouth opening in rats. Isovitelline has good safety, with no oral mucosal irritation and minimal impact on liver function. This invention provides new, highly effective, and low-toxicity drugs, suitable dosage forms, and well-defined mechanisms for OSF, and has significant clinical translational value. Attached Figure Description

[0014] Figure 1 Effects of high-dose vitexin on Mfn2 protein expression and mitochondrial-lysosome contact (MALM). (A) Western blot analysis of Mfn2 protein expression in each group. (B) Immunofluorescence staining showing the colocalization of mitochondria (Tom20, green) and lysosomes (LAMP1, red) (yellow in the merged image represents the colocalization region). The vitexin group restored the contact level between mitochondria and lysosomes (scale bar: 10 μm).

[0015] Figure 2 High-dose isovitexin mediates mitophagy and improves mitochondrial function by promoting STX17-VDAC1 binding. (A) Immunoprecipitation assay to detect the interaction between STX17 and VDAC1. (B) Binding amount of STX17 to VDAC1. (C) Mitochondrial DNA copy number detection. (D) Detection of reactive oxygen species production. (E) JC-1 staining to detect mitochondrial membrane potential.

[0016] Figure 3 High-dose vitexin inhibits p-Smad3 expression and regulates collagen metabolism pathways. (A) Western blot analysis of p-Smad3 and total Smad3 expression levels in each group. (B) ELISA analysis of type I collagen content in cell supernatant. (C) Gelatin zymography analysis of relative MMP-1 activity. Detailed Implementation

[0017] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0018] The isovitexin involved in the embodiments of this invention has CAS number 29702-25-8 and molecular formula C. 21 H 20 O 10 The structural formula is as follows:

[0019] Example 1: Preparation of pre-formed granules for oral application of isovitelline 1) Mix isovitexin, hydroxypropyl methylcellulose (Aladdin, item number H108815), microcrystalline cellulose (Maclean, item number M909921), crospovidone XL (Yuanye Biotechnology, item number S25115) and magnesium stearate evenly to obtain a mixture. 2) Add hydroxypropyl methylcellulose ethanol solution to the mixture obtained in step 1), mix evenly, and then perform wet granulation to obtain wet granules; 3) The wet particles obtained in step 2) are fluidized and dried to obtain dry particles; 4) The dried granules obtained in step 3) are granulated and then compressed into tablets to obtain uncoated tablets; 5) Hydroxypropyl methylcellulose and triacetin are mixed evenly as coating material, and the tablets obtained in step 4) are coated with a film to obtain isovitexin oral granule pre-prepared tablets. In step 1), the mass ratio of isovitexin: hydroxypropyl methylcellulose: microcrystalline cellulose: crospovidone XL: magnesium stearate is 20:150:100:30:5; In step 2), the ratio of the mixture to the hydroxypropyl methylcellulose ethanol solution is 10:1 (g:mL); the concentration of the hydroxypropyl methylcellulose ethanol solution is 5wt%; the wet granulation conditions are: stirring speed 150rpm, cutting speed 600rpm, and granulation time 4min. In step 3), the fluidized bed drying conditions are: inlet air temperature 60℃, drying time 2h; In step 4), the weight of the unprocessed tablet is 30 mg; In step 5), the ratio of hydroxypropyl methylcellulose to triacetin is 10:1 (g:mL); the weight gain of the film coating layer is 3%. Comparative Example 1: Preparation of blank oral smear pre-formed granules

[0020] 1) Hydroxypropyl methylcellulose (Aladdin, catalog number H108815), microcrystalline cellulose (Maclean, catalog number M909921), crospovidone XL (Yuanye Biotechnology, catalog number S25115) and magnesium stearate are mixed evenly to obtain a mixture. 2) Add hydroxypropyl methylcellulose ethanol solution to the mixture obtained in step 1), mix evenly, and then perform wet granulation to obtain wet granules; 3) The wet particles obtained in step 2) are fluidized and dried to obtain dry particles; 4) The dried granules obtained in step 3) are granulated and then compressed into tablets to obtain uncoated tablets; 5) Hydroxypropyl methylcellulose and triacetin are mixed evenly as coating material, and the uncoated tablets obtained in step 4) are coated with a film to obtain pre-made granular tablets for oral application; In step 1), the mass ratio of hydroxypropyl methylcellulose: microcrystalline cellulose: crospovidone XL: magnesium stearate is 150:100:30:5; In step 2), the ratio of the mixture to the hydroxypropyl methylcellulose ethanol solution is 10:1 (g:mL); the concentration of the hydroxypropyl methylcellulose ethanol solution is 5wt%; the wet granulation conditions are: stirring speed 150rpm, cutting speed 600rpm, and granulation time 4min. In step 3), the fluidized bed drying conditions are: inlet air temperature 60℃, drying time 2h; In step 4), the weight of the unprocessed tablet is 30 mg; In step 5), the ratio of hydroxypropyl methylcellulose to triacetin is 10:1 (g:mL); the weight gain of the film coating layer is 3%. Example 2: Preparation of isovitexin gel

[0021] 1) Add carbomer 940 to an appropriate amount of distilled water and let it swell for 24 hours. Then add glycerin and methylparaben in sequence and stir evenly to obtain a gel matrix. 2) While stirring, slowly add triethanolamine to the gel matrix obtained in step 1) to adjust the pH value; 3) Add isovitexin to the gel matrix after pH adjustment in step 2), stir continuously until it is completely dissolved, and then add distilled water to the specified total amount; 4) The mixture obtained in step 3) was aseptically filtered through a 0.22 μm microporous membrane and then dispensed under aseptic conditions to obtain isovitexin gel. The concentrations of each component in this vitexin gel are as follows: Carbomer 940 1.5wt%, glycerin 5wt%, methylparaben 0.05wt%, triethanolamine 0.5wt%, vitexin 0.5wt%, and the remainder is distilled water. Example 3:

[0022] Oral mucosal fibroblasts were obtained from healthy individuals (Wuhan Pronosei Life Sciences Co., Ltd., product number CP-H205) at a concentration of 5 × 10⁻⁶. 4 Cells were seeded at a density of [number] cells / well in 6-well plates using DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics. The cells were incubated at 37°C with 5% CO2 for 24 hours until 80% confluence was achieved, at which point further treatment was performed. The normal control group (healthy human oral mucosal fibroblasts) maintained the above basic culture conditions, while the remaining cells used for modeling were replaced with fresh DMEM medium containing 200 μmol / L arecoline (10% fetal bovine serum and 1% penicillin-streptomycin antibiotics). The medium was changed daily for 72 hours to construct an in vitro cell model of oral submucosal fibrosis (OSF). Successful model construction was determined by: type I collagen secretion in the model group reaching twice that of the normal control group (healthy human oral mucosal fibroblasts), and the number of mitochondrial-lysosomal (MALM) colocalization sites being less than 50% of that in the normal control group. The experiment consisted of 6 groups, with 3 replicates per group: a normal control group, an OSF model group, a low-dose isovitelline group (10 μmol / L), a medium-dose isovitelline group (30 μmol / L), a high-dose isovitelline group (50 μmol / L), and a positive control group (tanshinone IIA, 20 μmol / L). The normal control group and the OSF model group were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics. For each drug intervention group, the successfully constructed OSF in vitro cell model was replaced with DMEM medium containing the corresponding concentration of the test drug and 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics. All cells were incubated at 37°C in a 5% CO2 incubator for 48 hours, after which various experimental indicators were measured.

[0023] Western blot analysis: Expression levels of mitochondrial fusion protein 2 (Mfn2), synaptic fusion protein 17 (STX17), p-Smad3 protein, and total Smad3 protein were detected (GAPDH protein was used as the internal control). Primary antibody was incubated overnight at 4°C, followed by secondary antibody incubation at room temperature for 1 hour. Subsequent imaging and analysis were performed according to standard procedures.

[0024] Immunofluorescence colocalization assay: The contact between mitochondria and lysosomes was observed. The mitochondrial probe used was Tom20 (CST, catalog number 42406S, dilution 1:500), and the lysosomal probe used was LAMP1 (Abcam, catalog number ab25245, dilution 1:500). The secondary antibody was labeled with Alexa Fluor 488 / 594 (dilution 1:1000). Cells were fixed with 4% paraformaldehyde for 20 min, permeated with 0.1% Triton X-100 for 10 min, blocked with 5% BSA for 30 min, incubated with primary antibody overnight at 4°C, incubated with secondary antibody at room temperature for 1 h, and stained the nuclei with DAPI for 5 min. The number of mitochondrial-lysosomal colocalization sites was calculated using ImageJ software.

[0025] Immunoprecipitation assay: to detect the binding level of Stx17 protein to mitochondrial membrane protein VDAC1 and to clarify their interaction.

[0026] Real-time quantitative PCR (qRT-PCR) was used to detect mitochondrial DNA (mtDNA) copy number: Target gene ND1 primer sequences: forward 5'-CCCTAAAACCCGCCACATCT-3', reverse 5'-GAGCGATGGTGAGAGCTAAGGT-3'; internal reference gene GAPDH primer sequences: forward 5'-GCACCGTCAAGGCTGAGAAC-3', reverse 5'-TGGTGAAGACGCCAGTGGA-3'. Reaction system (20 μl): 10 μl of 2×SYBR Green Mix, 0.5 μl each of forward and reverse primers, 2 μl of cDNA template, and 7 μl of ddH2O; Reaction conditions: 95℃ pre-denaturation for 3 min, followed by 95℃ for 10 s and 60℃ for 30 s, repeated 40 times. -ΔΔCt The relative expression level is calculated using this method.

[0027] Determination of reactive oxygen species (ROS) generation: The DCFH-DA fluorescent probe method was used to detect the ROS levels of cells in each group according to the kit instructions.

[0028] Mitochondrial membrane potential detection: JC-1 staining method was used. After incubating cells with 5 μmol / L JC-1 for 20 min, the red / green fluorescence ratio was detected by flow cytometry. The ratio of the normal control group was used as the baseline (100%) to calculate the relative recovery rate of mitochondrial membrane potential in each group.

[0029] Type I collagen content detection: The ELISA method (Macklin, catalog number I773782-48T / EA) was used, with the detection wavelength set at 450nm and the reference wavelength at 570nm. The concentration of type I collagen in the cell supernatant was calculated by fitting a four-parameter curve.

[0030] Collagenase 1 (MMP-1) activity assay: The gelatin zymography method was used. A 10% SDS-PAGE gel containing 0.1% gelatin was prepared. Electrophoresis was performed on an 80V stacking gel for 20 min and a 120V separating gel for 60 min. After electrophoresis, the gel was incubated at 37℃ for 18 h. After Coomassie brilliant blue staining and destaining, the MMP-1 activity was analyzed.

[0031] Quantitative analysis of intracellular collagen deposition: Type I collagen immunofluorescence staining (antibody dilution ratio 1:200) was used, and the proportion of fluorescent positive areas to the total cell area was quantitatively analyzed using ImageJ software to reflect the level of intracellular collagen deposition.

[0032] The results show: Compared with the normal control group, the oral submucosal fibrosis (OSF) model group showed significant pathological changes: the collagen deposition area in the normal control group was 8.2±1.3%, the mitochondrial membrane potential level (red / green fluorescence ratio) was 2.5, and the expression levels of Mfn2 and STX17 proteins were both 1.0±0.1; while in the OSF model group, the collagen deposition area was significantly increased to 35.6±2.8%, the mitochondrial membrane potential recovery rate was 32.1±3.5%, and the expression levels of Mfn2 and STX17 proteins were downregulated to 0.4±0.1 and 0.3±0.1, respectively. Isovitexin showed a significant dose-dependent ameliorative effect on the pathological changes in the OSF model. After intervention with low, medium, and high doses of vitexin, the collagen deposition area in each group was 24.3±2.1%, 15.7±1.8%, and 12.3±1.5%, respectively; the mitochondrial membrane potential recovery rate increased to 51.2±4.2%, 68.5±3.8%, and 78.3±4.5%, respectively; the Mfn2 protein expression level increased to 0.7±0.1, 1.5±0.2, and 2.3±0.2, respectively; and the STX17 protein expression level increased to 0.6±0.1, 1.4±0.2, and 2.1±0.2, respectively. The tanshinone IIA group, serving as a positive control, showed a weaker intervention effect, with a collagen deposition area of ​​22.5±2.3%, a mitochondrial membrane potential recovery rate of only 38.6±3.2%, and relative Mfn2 and STX17 protein expression levels of 0.5±0.1 and 0.4±0.1, respectively. Overall, the effect was inferior to that of the medium and high dose vitexin groups. Further investigation into its mechanism of action revealed that, compared to the normal control group, the colocalization signal between mitochondria and lysosomes was significantly reduced in the OSF model group; however, after high-dose isovitexin intervention, the colocalization signal between mitochondria and lysosomes significantly increased, causing their contact levels to revert to those of the normal control group. Compared to the OSF model group, the binding amount of STX17 protein to VDAC1 protein was significantly increased in the high-dose group. Compared to the normal control group, the mitochondrial DNA copy number and ROS production were significantly increased in the OSF model group; however, after high-dose isovitexin intervention, the mitochondrial DNA copy number and ROS production were significantly decreased, both reverting to the levels of the normal control group. Compared to the normal control group, the expression level of p-Smad3 protein and the secretion of type I collagen were significantly increased in the OSF model group; however, after high-dose isovitexin intervention, the expression level of p-Smad3 protein and the secretion of type I collagen were significantly decreased, reverting to the levels of the normal control group. Furthermore, compared to the normal control group, the MMP-1 activity in the OSF model group was significantly reduced; however, after intervention with high-dose isovitexin, the MMP-1 activity significantly increased, returning to the level of the normal control group. This indicates that isovitexin can reduce collagen deposition and improve oral submucosal fibrosis by restoring mitochondrial function (promoting mitochondrial-lysosomal interaction, increasing membrane potential, and reducing ROS and mtDNA copy number), upregulating Mfn2 and STX17 protein expression, and inhibiting the p-Smad3 signaling pathway. Its effect is superior to the positive control tanshinone IIA and is dose-dependent. Example 4:

[0033] SPF-grade SD rats weighing 200-220g were selected to establish an oral submucosal fibrosis (OSF) rat model using a combined approach: 0.5ml of 2% arecoline solution was applied to the oral cavity of each rat daily, while arecoline was administered intraperitoneally at a dose of 1mg / kg body weight weekly for 8 consecutive weeks. Successful model establishment was determined by: oral mucosal thickness in the model group not less than 1.5 times that of the normal control group, mouth opening not exceeding 60% of the normal control group, and typical collagen hyperplasia visible in pathological sections. The experimental animals were randomly divided into 6 groups of 10 rats each, with each group receiving the treatment for 8 weeks. The normal control group consisted of normal rats that had not undergone OSF modeling. The specific administration methods were as follows: The normal control group received an equal volume of blank excipient (prepared by crushing pre-made blank oral granules and mixing with physiological saline), applied to the oral mucosa of the rats three times daily; the OSF model group consisted of successfully modeled rats, administered the same blank excipient as the normal control group, applied to the oral lesions of the rats three times daily; the low-dose isovitillin group (total isovitillin dose 1 mg / kg) and the medium-dose isovitillin group (total isovitillin dose 3 mg / kg) both used successfully modeled rats, and the corresponding dose of isovitillin oral granules was crushed and mixed with physiological saline, applied to the oral lesions of the rats three times daily; the high-dose isovitillin group (total isovitillin dose 5 mg / kg) used successfully modeled rats, administered via local injection, with isovitillin gel injected twice weekly at 2-3 points around the oral fibrosis lesions; the positive control group (tanshinone II)... A total dosage of 2 mg / kg), select rats with successful modeling, dissolve the corresponding dose of tanshinone IIA in physiological saline, and administer intraperitoneally once a day. After drug administration, various tests and scores were conducted simultaneously: the Draize mucosal irritation rating scale was used to assess oral mucosal reaction, which is divided into 3 levels: 0 (no reaction), 1 (mild erythema), and 2 (significant erythema), with a score ≤1 indicating no irritation; a 5-level scale was used to determine the degree of mucosal fibrosis: 0 (normal mucosal structure, loose and evenly distributed collagen fibers), 1 (mild collagen fiber hyperplasia, slightly dense arrangement in some areas), 2 (moderate collagen fiber hyperplasia, thickening of the lamina propria, disordered arrangement), 3 (severe collagen fiber hyperplasia, significant thickening of the lamina propria, involving the submucosa), and 4 (extremely severe collagen fiber hyperplasia, disordered structure of all mucosal layers, and fusion of fiber bundles into clumps); rat serum was collected, and serum collagen I content and alanine aminotransferase (ALT) level were measured using appropriate test kits to assess collagen metabolism and liver function. Mouth opening was measured using a vernier caliper with an accuracy of 0.1 mm. Before measurement, rats were lightly anesthetized by intraperitoneal injection of 30 mg / kg of 1% sodium pentobarbital. The head was fixed and the mouth was kept in a naturally open state. The maximum distance between the upper and lower incisors was measured. The measurement was repeated 3 times and the average value was taken as the final data.

[0034] The preparation methods of blank oral granule preforms, isovitelline oral granule preforms, and isovitelline gel are described in Comparative Example 1, Example 1, and Example 2.

[0035] The results show: In the normal control group, the mucosal fibrosis score of rats was 0.3±0.1, the mouth opening was 10.5±0.8 mm, the serum collagen I content was 125.3±10.2 ng / mL, and the ALT level was 26.5±3.1 U / L. In the successfully constructed OSF model group, all of the above indicators showed significant deterioration: fibrosis score was 4.2±0.3, mouth opening was 5.8±0.6 mm, serum collagen I content was 386.7±25.4 ng / mL, and ALT level was 59.8±6.2 U / L. After intervention with isovitexin, all indicators showed a clear dose-dependent improvement: in the low, medium and high dose groups, the fibrosis score decreased to 3.1±0.2, 2.0±0.2 and 1.3±0.1 respectively, the mouth opening increased to 7.2±0.7mm, 8.8±0.5mm and 9.4±0.6mm respectively, the serum collagen I content gradually decreased to 289.5±20.3ng / mL, 201.4±15.6ng / mL and 156.8±12.4ng / mL, and the ALT level also recovered to 35.2±4.5U / L, 29.8±3.8U / L and 27.6±3.3U / L respectively. The high-dose group showed particularly significant efficacy, with a 69.0% decrease in fibrosis score compared to the model group and a recovery of mouth opening to 89.5% of the normal control group. This improvement was superior to the positive control group (fibrosis score 3.3±0.3, mouth opening 6.9±0.5mm, serum collagen I 295.6±18.7ng / mL, ALT 36.5±4.1U / L). Regarding safety, the ALT levels in all isovitelline dose groups were close to those in the normal control group, and no side effects such as oral mucosal irritation or gastrointestinal damage were observed throughout the experiment, demonstrating good safety.

Claims

1. Application of isovitexin in the preparation of drugs for treating oral submucosal fibrosis.

2. The application according to claim 1, characterized in that: The drug can be administered via topical application to the oral cavity and / or injection around oral fibrotic lesions.

3. The application according to claim 2, characterized in that: When the administration method is topical application to the oral cavity, the drug is a pre-formed granular tablet for oral application.

4. A pre-formed granular sheet for oral application in the treatment of oral submucosal fibrosis, characterized in that: The raw materials for preparing the oral granule preform include isovitexin.

5. The pre-formed granular sheet for oral application according to claim 4, characterized in that: The raw materials for preparing the oral granule preform also include hydroxypropyl methylcellulose, microcrystalline cellulose, crospovidone XL and magnesium stearate, with the mass ratio of isovitexin:hydroxypropyl methylcellulose:microcrystalline cellulose:crospovidone XL:magnesium stearate being 20:150:100:30:

5.

6. The method for preparing pre-formed granules for oral application as described in claim 5, characterized in that: Includes the following steps: 1) Mix hydroxypropyl methylcellulose, microcrystalline cellulose, crospovidone XL and magnesium stearate evenly to obtain a mixture; 2) Add hydroxypropyl methylcellulose ethanol solution to the mixture, mix well, and then perform wet granulation to obtain wet granules; 3) The wet particles are fluidized and dried to obtain dry particles; 4) The dried granules are granulated and then compressed into tablets to obtain uncoated tablets; 5) Hydroxypropyl methylcellulose and triacetin are mixed evenly as coating material to coat the uncoated tablets with a film to obtain pre-made granular tablets for oral application.

7. The application according to claim 2, characterized in that: When administered via injection around the oral fibrosis lesions, the drug is a gel.

8. A gel for treating oral submucosal fibrosis, characterized in that: The raw materials for preparing the gel include isovitexin.

9. The gelling agent according to claim 8, characterized in that: The gelling agent comprises, by weight percentage: 0.5% isovitexin, 1.5% carbomer 940, 5% glycerin, 0.05% methylparaben, 0.5% triethanolamine, and the balance being water.

10. The method for preparing the gelling agent as described in claim 9, characterized in that: Includes the following steps: 1) Add carbomer 940 to water to swell, then add glycerin and methylparaben in sequence, stir well to obtain a gel matrix; 2) While stirring, slowly add triethanolamine to the gel matrix to adjust the pH value; 3) Add isovitexin to the pH-adjusted gel matrix, stir continuously until completely dissolved, and add water to the specified total amount; 4) After the obtained mixture is aseptically filtered through a microporous membrane, it is dispensed under aseptic conditions to obtain a gel.

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