Use of a citrus peel extract in the preparation of a product for the relief and / or treatment of diabetes periodontitis

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to the application of a citrus peel extract in the preparation of products for relieving and / or treating diabetic periodontitis. This invention efficiently enriches a citrus peel extract containing specific polymethoxyflavonoid components from citrus peel. This extract can be used to relieve and / or treat diabetic periodontitis. Its core objective is to utilize the multiple synergistic pharmacological effects of the natural active ingredients (such as polymethoxyflavonoids) in this extract, including anti-inflammatory, antioxidant, gut microbiota regulation, and insulin resistance improvement, to target and regulate the systemic pathological network driving the development of diabetic periodontitis from upstream. In particular, by regulating the "gut-periodontal axis" to reduce systemic inflammatory load, it compensates for the shortcomings of existing therapies that only treat the symptoms, providing a novel, multi-targeted, safe, and easily adhered-to adjunctive solution for relieving and treating diabetic periodontitis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of a citrus peel extract in the preparation of products for relieving and / or treating diabetic periodontitis. Background Technology

[0002] Diabetic periodontitis (DP) refers to periodontitis occurring in patients with diabetes. It is a specific type of periodontitis that develops and progresses in the context of diabetes, interacting with and exacerbating the condition. DP is not merely a simple coexistence of two diseases; rather, it is based on the systemic metabolic disorders caused by diabetes. By significantly altering the periodontal tissues' response to local plaque stimulation, it makes periodontitis more likely to occur, progresses more rapidly, and becomes more severe, making clinical treatment more difficult. DP has an extremely high prevalence among diabetic patients. A 2021 clinical study involving 1613 patients with type 2 diabetes showed that the overall prevalence of periodontitis in this population was as high as 80.8%, with 21.8% having progressed to severe periodontitis. This data fully confirms that diabetes is a significant risk factor for the occurrence and development of periodontitis. There is a bidirectional promoting pathological association between diabetes and periodontitis. Studies have shown that the risk of developing periodontitis in diabetic patients is 1.9 times that of non-diabetic patients; conversely, the proportion of undiagnosed diabetes is also significantly higher among patients with severe periodontitis. This dual effect is particularly pronounced in diabetic patients with poor glycemic control; for example, elevated glycated hemoglobin (HbA1c) levels are an independent risk factor for diabetes mellitus (DP).

[0003] The pathogenesis of diabetic dysplasia (DP) is a complex regulatory network driven by hyperglycemia and involving both local and systemic factors. Gut microbiota dysbiosis serves as a key hub, bridging the pathological link between metabolic disorders and periodontal immune damage. Its core pathway begins with systemic immunometabolic reprogramming induced by long-term hyperglycemia and the abnormal accumulation of advanced glycation end products (AGEs). These changes not only weaken the physiological functions of immune cells such as neutrophils but also overactivate pro-inflammatory signaling pathways such as the RAGE receptor pathway, thereby exacerbating the destructive inflammatory response of periodontal tissues to plaque biofilms. Ultimately, this manifests as accelerated alveolar bone resorption and a significant decrease in periodontal tissue repair capacity. Recent studies have revealed that gut microbiota dysbiosis in diabetes (e.g., reduced butyrate-producing beneficial bacteria and increased opportunistic pathogens) leads to disruption of the intestinal barrier integrity, allowing endotoxins (such as lipopolysaccharide) and pro-inflammatory mediators to reach periodontal tissues via the bloodstream, systematically amplifying the periodontal inflammatory response. Simultaneously, the disordered profile of gut microbiota metabolites (such as short-chain fatty acid imbalance) directly affects the host's immune homeostasis. Therefore, DP can be regarded as a special pathological process initiated by hyperglycemia and characterized by a vicious cycle of the "gut-periodontal axis". In this process, gut microbiota dysbiosis not only exacerbates the systemic inflammatory load, but also directly deteriorates the local microenvironment of the periodontium.

[0004] Related research indicates that regulating the gut-periodontal axis can provide new treatment strategies for periodontal disease. The gut microbiota participates in food digestion, vitamin synthesis, and the production of metabolites such as short-chain fatty acids and bile acids. These metabolites can influence the inflammatory immune response in periodontium through the RAGE receptor pathway. As research into the mechanism linking gut microbiota and periodontitis deepens, researchers have discovered that gut microbiota can affect blood glucose homeostasis through the production of specific metabolites and the regulation of intestinal barrier function, leading to abnormally high blood glucose levels and thus inducing diabetic periodontitis (DP). This finding opens up a new direction for the clinical treatment of diabetic periodontitis. Although research on the relationship between gut microbiota and diabetic periodontitis has made some progress, our understanding in this area remains limited. Future research needs to further explore the specific mechanisms by which gut microbiota affects DP and periodontitis prevention and treatment strategies based on gut microbiota. Furthermore, due to the significant differences in periodontal damage caused by different modeling methods, current specific drug treatments for DP are still very limited. Therefore, there is an urgent need to develop new and effective drugs for treating DP. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention efficiently enriches citrus peel extract containing specific polymethoxyflavonoid components from citrus peel. This extract can provide a novel adjunctive solution for relieving and treating diabetic periodontitis through multi-target anti-inflammatory, antioxidant and unique "gut-periodontal axis" synergistic mechanism.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides the use of citrus peel extract in the preparation of products for relieving and / or treating diabetic periodontitis.

[0007] Preferably, the preparation method of the citrus peel extract is as follows: freeze-dry and pulverize the citrus peel, add 60%-80% ethanol aqueous solution, extract by hot reflux, collect the filtrate and remove the solvent, and then purify it with macroporous adsorption resin to wash away impurities, thereby obtaining the extract.

[0008] This invention employs specific solvents and purification processes to efficiently enrich citrus peel extracts containing specific polymethoxylated flavonoid components from citrus peels. These extracts can effectively intervene in systemic pathological processes that are difficult to reach with existing local therapies through multi-target anti-inflammatory, antioxidant, and uniquely modulating "gut-periodontal axis" synergistic mechanisms, thereby alleviating and / or treating diabetic periodontitis. This provides a novel adjunctive solution for alleviating and treating diabetic periodontitis that is multi-targeted, safe, and easy to adhere to.

[0009] More preferably, the citrus peel is selected from the peel of Xinhui citrus from Jiangmen, Guangdong.

[0010] More preferably, the volume ratio of the citrus peel to the aqueous ethanol solution is 1:20-30.

[0011] More preferably, the ethanol-water solution is a 60% ethanol-water solution, and the temperature of the hot reflux extraction is 77–83°C (the atmospheric boiling point of 60% ethanol is 82–85°C, and the actual operating temperature is usually controlled 2–5°C lower than the atmospheric boiling point); or the ethanol-water solution is a 70% ethanol-water solution, and the temperature of the hot reflux extraction is 75–80°C (the atmospheric boiling point of 70% ethanol is 80–82°C, and the actual operating temperature is usually controlled 2–5°C lower than the atmospheric boiling point). C); or the ethanol-water solution is an 80% ethanol-water solution, and the temperature of the hot reflux extraction is 74–79°C (the atmospheric boiling point of 80% ethanol is 79–81°C, and the actual operating temperature is usually controlled 2–5°C lower than the atmospheric boiling point); or the ethanol-water solution is a 95% ethanol-water solution, and the temperature of the hot reflux extraction is 73–76°C (the atmospheric boiling point of 95% ethanol is 78°C, and the actual operating temperature is usually controlled 2–5°C lower than the atmospheric boiling point).

[0012] More preferably, the hot reflux extraction time is 1-4 h.

[0013] More preferably, the freeze-drying time is 40-60 h.

[0014] More preferably, the macroporous adsorption resin is selected from AB-8 resin columns.

[0015] More preferably, the citrus peel is freeze-dried and then pulverized to 20-60 mesh.

[0016] Preferably, the product for relieving and / or treating diabetic periodontitis is a medicament for relieving and / or treating diabetic periodontitis, wherein the medicament has citrus peel extract as the main active ingredient and also includes pharmaceutically acceptable excipients.

[0017] More preferably, the excipients include at least one of the following: solubilizer, emulsifier, colorant, binder, disintegrant, lubricant, wetting agent, osmotic pressure regulator, stabilizer, flow aid, flavoring agent, preservative, coating material, pH adjuster, absorbent, and antioxidant.

[0018] More preferably, the dosage form of the drug includes an oral formulation, an injectable formulation, an inhaled formulation, or a transdermal formulation.

[0019] Compared with the prior art, the beneficial effects of the present invention are: Compared with existing treatment strategies that mainly target local debridement and systemic blood glucose control, the citrus peel extract provided by this invention exhibits excellent multi-dimensional synergistic effects in the treatment of diabetic periodontitis (DP): (1) In terms of mechanism of action, it goes beyond simple antibacterial or blood glucose control. Through its rich content of polymethoxyflavonoids and other active ingredients, it can simultaneously exert multi-target effects of anti-inflammatory, antioxidant, immune regulation and improvement of insulin resistance, thereby intervening in the core pathology of DP, "systemic metabolic-immune disorder", from upstream; (2) In terms of therapeutic target, it innovatively acts on the emerging "gut-dental" pathway. "Zhouzhou" is expected to reduce the systemic inflammatory load that drives periodontal destruction from the source by regulating the homeostasis of gut microbiota and enhancing the intestinal barrier function. This is something that existing local therapies and traditional nutritional interventions cannot achieve precisely. (3) In terms of application attributes, as a natural extract derived from food by-products, it has high biosafety and few potential side effects, avoiding the drug resistance and dysbiosis problems that may be caused by long-term use of antibiotics. It is easier for patients to adhere to it for a long time, providing a new adjunctive treatment option for DP management that can be combined with other therapies, has the potential to "treat both the symptoms and the root cause" and has good compliance. Attached Figure Description

[0020] Figure 1 The results show the fasting blood glucose and periodontal CT scans of mice; (A) Changes in fasting blood glucose in mice; (B) Blood glucose levels in mice at different stages; (C) CT scan of the maxilla in mice (the yellow area indicates the extent of periodontitis); (D) BV / TV (%); (E) Tb.n; (F) Tb.Sp; (G) Tb.Th; Student T test. <0.05, <0.01, <0.001.

[0021] Figure 2 Staining and scoring of mouse periodontal pathology; (A) Eosin hematoxylin staining; (B) Periodontal inflammation score; (C) Periodontal epithelial migration score; (D) Alveolar bone loss score; (E) Masson staining; (F) Relative proportion of collagen region; (G) IL-6 fluorescent staining (yellow arrows indicate fluorescent stained areas); (H) Relative fluorescence area of ​​IL-6; Student T test, <0.05, <0.01, <0.001.

[0022] Figure 3 For mouse microbial α-diversity; (A) Shannon index; (B) Shannon index curve; (C) Simpson index; (D) Simpson index curve; nonparametric t-test, <0.05, <0.01, <0.001.

[0023] Figure 4 For mouse microbial β-diversity; (A) PCoA2; (B) X-variate; (C) between-group distance; nonparametric t-test, <0.05, <0.01, <0.001.

[0024] Figure 5 Relative abundance of mouse species at the genus level; (A) Stacked bar chart of relative abundance of species; Relative abundance of mouse species in (B) Akkemansia, (C) Allobaculum, (D) Bacterides, (E) Eubacterium, (F) Lactobacillus, (G) Alistipes_A_871400, (H) Anaerorotruncus, (I) Parasutterella, (J) Streptococcus, and (K) Emergencia; Nonparametric t-test, <0.05, <0.01, <0.001.

[0025] Figure 6 Principal component analysis (PCA) plots of mouse periodontal tissue transcriptomes; (A) Transcriptome differences between the model group and the control group and (B) clustering; (C) Transcriptome differences between the treatment group and the model group and (D) clustering; (E) Transcriptome differences between the treatment group and the control group and (F) clustering.

[0026] Figure 7 For functional analysis prediction; (A) Bubble chart of GO enrichment analysis results; (B) Bubble chart of KEGG pathway enrichment analysis. Detailed Implementation

[0027] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0028] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0029] Current mainstream treatments for diabetic periodontitis include mechanical debridement, antibiotic use, and strict blood glucose control. However, these methods all have significant limitations: they primarily focus on controlling local infection or regulating systemic blood glucose, lacking direct and effective intervention strategies for the deeper pathological links connecting diabetes and periodontitis, such as systemic hyperinflammatory states, immune metabolic disorders, and the "gut-periodontal axis" imbalance proposed in emerging research. This often results in unsatisfactory treatment outcomes for complex cases and a high recurrence rate. Therefore, developing novel therapeutic products that can alleviate or even cure diabetic periodontitis has significant clinical value.

[0030] Therefore, this invention provides the application of citrus peel extract in the preparation of products related to diabetic periodontitis. Its core objective is to utilize the multiple synergistic pharmacological effects of natural active ingredients (such as polymethoxyflavones) in the extract, including anti-inflammatory, antioxidant, gut microbiota regulation, and improvement of insulin resistance, to target and regulate the systemic pathological network driving the development of diabetic periodontitis from upstream. In particular, by regulating the "gut-periodontal axis" to reduce the systemic inflammatory load, it compensates for the shortcomings of existing therapies that "treat the symptoms but not the root cause," providing a novel adjunctive solution for alleviating and treating diabetic periodontitis that is multi-targeted, safe, and easy to adhere to.

[0031] To fully and clearly present the technical solution and significant advantages of the present invention, the present invention will be described in detail below with reference to specific embodiments.

[0032] Example 1: Preparation of Citrus Peel Extract Its preparation method includes the following steps: (1) Preparation of dried citrus peel: Peel the citrus peel from the citrus (Xinhui citrus from Jiangmen, Guangdong) and freeze dry it in a freeze dryer at -40℃ for 48 h to make dried citrus peel; (2) Processing of citrus peel: Cut the citrus peel into small pieces and grind them into fine powder (20-60 mesh) using a high-speed blender. (3) After mixing the citrus peel powder with 60% ethanol aqueous solution at a volume ratio of 1:30, the mixture was extracted by hot reflux at 77°C for 1 hour. The filtrate was then collected and concentrated to 1 / 4 of the original volume to obtain the extract. (4) Under reduced pressure (e.g., rotary evaporation), most of the organic solvent in the extract is recovered and removed to obtain a concentrated extract. Subsequently, the extract is purified using a macroporous adsorption resin (AB-8 resin column). By utilizing the selective adsorption characteristics of the resin, the target components can be efficiently enriched, and some impurities such as sugars and pigments can be separated simultaneously. The specific operation process is as follows: first, distilled water is used for elution to remove water-soluble impurities such as sugars and pigments from the system; then, 70% ethanol solution is used as the eluent to elute and collect the effective components such as flavonoids.

[0033] (5) The eluent was further concentrated under reduced pressure and then freeze-dried to obtain the extract powder: The ethanol eluent from the macroporous adsorption resin was transferred to the rotating flask of a rotary evaporator. The water bath temperature was controlled at 50-55 °C, the vacuum degree was -0.09 to -0.1 MPa, and the concentration was carried out under reduced pressure at a speed of 50-80 r / min. During the concentration process, the ethanol vapor was recovered to the collection bottle through the condenser. When the liquid in the rotating flask became thick, the fluidity decreased significantly, and there was no obvious alcohol smell, heating and rotation were stopped, the vacuum was released, and the concentrated liquid was obtained.

[0034] Example 2: Preparation of Citrus Peel Extract Its preparation method includes the following steps: (1) Preparation of dried citrus peel: Peel the citrus peel from the citrus (Xinhui citrus from Jiangmen, Guangdong) and freeze dry it in a freeze dryer at -40℃ for 48 h to make dried citrus peel; (2) Processing of citrus peel: Cut the citrus peel into small pieces and grind them into fine powder (20-60 mesh) using a high-speed blender. (3) The citrus peel powder and 70% ethanol aqueous solution were mixed at a volume ratio of 1:25, and then extracted by hot reflux at 77°C for 1 h. The filtrate was collected and concentrated to 1 / 5 of the original volume to obtain the hot water extract. (4) Under reduced pressure (e.g., rotary evaporation), most of the organic solvent in the extract is recovered and removed to obtain a concentrated extract. Subsequently, the extract is purified using a macroporous adsorption resin (AB-8 resin column). By utilizing the selective adsorption characteristics of the resin, the target components can be efficiently enriched, and some impurities such as sugars and pigments can be separated simultaneously. The specific operation procedure is as follows: first, distilled water is used for elution to remove water-soluble impurities such as sugars and pigments from the system; then, 70% ethanol solution is used as the eluent to elute and collect the effective components such as flavonoids.

[0035] (5) The eluent was further concentrated under reduced pressure and then freeze-dried to obtain the extract powder: The ethanol eluent from the macroporous adsorption resin was transferred to the rotating flask of a rotary evaporator. The water bath temperature was controlled at 50-55 °C, the vacuum degree was -0.09 to -0.1 MPa, and the concentration was carried out under reduced pressure at a speed of 50-80 r / min. During the concentration process, the ethanol vapor was recovered to the collection bottle through the condenser. When the liquid in the rotating flask became thick, the fluidity decreased significantly, and there was no obvious alcohol smell, heating and rotation were stopped, the vacuum was released, and the concentrated liquid was obtained.

[0036] Experimental Example 1: Identification of Flavonoid Content in Citrus Peel Extract Using the citrus peel extract powder from Example 1 as the test sample, the flavonoid content was identified using a plant flavonoid content detection kit (Solepro, Cat: BC1330). The specific detection steps are as follows: (1) Preparation of standard curve: Rutin was used as the standard for the determination of total flavonoids, with a purity ≥98%. The rutin standard solution was diluted with double-distilled water to prepare solutions with concentration gradients of 1.5, 1.25, 0.625, 0.3125, 0.15625, 0.078, 0.039, and 0.02 mg / mL. Each concentration of standard solution was taken and reagents 1, 2, and 3 were added according to the instructions. The solutions were allowed to stand for color development. The standard curve was plotted on graph paper or using software such as Excel with the standard solution concentration as the abscissa (x) and the corresponding absorbance value as the ordinate (y). The regression equation (y=0.5325x-0.0043) was obtained.

[0037] (2) Sample processing: Dissolve the sample to be tested in the reagent kit extract, perform color development according to the procedure, and calculate the flavonoid content. The formula is: flavonoid content (mg / mg prot) = concentration of standard tube × volume of added extract ÷ sample mass.

[0038] (3) Results: 40 mg of citrus peel extract was accurately weighed, dissolved, and tested according to the experimental procedure. The absorbance difference was calculated as ΔA. 测定 =A 测定 -A 对照 ΔA 测定 Substituting the values ​​into the standard curve equation "y = 0.5325x - 0.0043", the mass x of the total flavonoids in the extract was calculated. The content was then calculated using the formula "Total flavonoid content (wt%) = x ÷ Sample mass". After three parallel experiments, the average total flavonoid content of the citrus peel extract obtained by this extraction method was 38.59 wt%.

[0039] Table 1: Identification results of flavonoid content in citrus peel extract Experiment Example 2: In vivo improvement of diabetic periodontitis (1) Experimental materials 1) Mice: 18 male C57 mice aged 7 weeks. Housing environment parameters: temperature maintained at 20-25℃, relative humidity 40%-50%, natural diurnal light rhythm; mice had free access to food and water.

[0040] 2) Sample: Citrus peel extract from Example 1.

[0041] 3) Instruments: blood glucose meter, No. 8 gavage needle, sterile syringe, etc.

[0042] (2) Experimental methods 1) Grouped administration Eighteen mice were randomly divided into a control group, a model group, and a treatment group, with six mice in each group. After one week of acclimatization, mice in the model and treatment groups were intraperitoneally injected once daily for four consecutive days with streptozotocin (STZ) at a dose of 60 mg / kg; the control group was intraperitoneally injected with an equal volume of sodium citrate solution during the same period. Three days after the injection, fasting blood glucose was measured in the mice, and a fasting blood glucose level ≥11.1 mmol / L was used as the criterion for successful establishment of the diabetes model. After successful modeling, all mice underwent maxillary periodontal ligation to establish a periodontitis model; at the same time, drug intervention was initiated. The treatment group was administered the citrus peel extract of Example 1 by gavage, while the control and model groups were administered an equal volume of 0.5% sodium carboxymethyl cellulose (CMC) solution by gavage, for two consecutive weeks, once daily by gavage. During the experiment, mice had free access to food and water, and fasting blood glucose was monitored weekly, and body weight, water intake, and food intake were recorded. Specific grouping and drug administration are shown in Table 2 below: Table 2: Group Dosing Table 2) Recording of mouse weight, water intake, and food consumption. During the experiment, the mice's body weight, food intake, and water intake were recorded four times to observe their activity status.

[0043] 3) Fasting blood glucose level test Fasting blood glucose was measured on the 1st and 5th of each week, as follows: Mice were fasted for 10 hours starting at 10 PM on the last and fourth day of each week (feed and bedding removed, but water was allowed free access). Fasting blood glucose was measured at 8 AM the following morning. Immediately after the blood glucose measurement, the mice were given medication and feed. Fasting blood glucose testing began in week 0 and was conducted twice a week for 12 consecutive days.

[0044] 4) Mouse Micro-CT Scans and Bone Microstructure Analysis After pre-scan cleaning, the fixed maxilla was imaged using a Micro-CT scanner. Scanning parameters: voltage 50 kV, current 200 μA, resolution 9 μm. Three-dimensional images were reconstructed using software after scanning, and the palatal interroot region of the maxillary second molar was selected as the Region of Interest (ROI). After automatic thresholding, the following bone microstructure parameters were calculated: BV / TV (bone volume fraction, %), Tb.N (trabecular bone number, 1 / mm), Tb.Th (trabecular bone thickness, μm), and Tb.Sp (trabecular bone separation, μm). Six specimens were analyzed in each group.

[0045] 5) 16S rRNA gene sequencing Total DNA was extracted from the contents of the ileocecal junction of mice. The full-length 16S rRNA gene was amplified using the universal bacterial primers 27F / 1492R. After constructing a library from the PCR products, third-generation sequencing was performed on a sequencing platform. The data were quality-controlled and noise-reduced to obtain ASV / OTUs. Species annotation was performed by comparison with databases, and Alpha(α) diversity (Shannon and Chao1 indices) and Beta(β) diversity (PCoA analysis) were calculated.

[0046] Simultaneously, based on the species annotation results of ASV / OTU, the abundance of all ASV sequences belonging to the same genus is merged, and the percentage of reads from each genus in each sample is calculated to obtain the relative abundance of species at the genus level. This process is implemented using the phyloseq package in R language, and finally generates a genus-level relative abundance matrix for subsequent stacked bar charts, heatmaps, and intergroup difference analysis.

[0047] 6) Periodontal tissue transcriptome sequencing Total RNA was extracted from maxillary periodontal tissues of mice. Transcriptome sequencing was performed using the Illumina NovaSeq 6000 platform. After quality control, alignment, and quantification, differentially expressed genes were screened using DESeq2 (|log2FC|≥1, P<0.05), and GO and KEGG enrichment analyses were performed using clusterProfiler.

[0048] 7) Periodontal tissue pathology examination Mouse maxillae were collected, fixed, decalcified, and then paraffin-embedded sections were prepared for HE and Masson staining. HE staining was used to assess the degree of periodontal inflammation (specific assessment methods are shown in Table 3-5). The relative collagen area ratio in the Masson staining was quantified, and the results were statistically analyzed using a nonparametric t-test between groups.

[0049] Table 3: Inflammation Score Continued from the previous table: Table 4: Epithelial Migration Score Table 5: Alveolar Bone Loss Score Continued from the previous table: 8) Immunofluorescence detection of IL6 in periodontal tissues Mouse maxillary bone tissue was fixed in 4% paraformaldehyde and decalcified with EDTA, then frozen sections (8-10 μm thick) were prepared. Sections were permeabilized with 0.3% Triton X-100 membrane, blocked with 10% goat serum, and incubated overnight at 4°C with rabbit anti-mouse IL-6 primary antibody (1:200, Abcam). The next day, goat anti-rabbit secondary antibody (1:500) labeled with Alexa Fluor 488 was added, and the sections were incubated at room temperature in the dark for 1 h. Cell nuclei were counterstained with DAPI, and the sections were mounted with anti-fluorescence quenching mounting medium. Images were observed and acquired under a fluorescence microscope. IL-6 positive expression was observed as green fluorescence, and the positive area was quantified and compared.

[0050] (3) Experimental results Depend on Figure 1 It can be seen that the blood glucose level of the treatment group mice was not significantly different from that of the model group in the middle of the experiment, but was significantly lower than that of the model group at the end of the experiment. Figure 1 AB). CT scans of the mouse maxillae were performed, and the results are as follows. Figure 1 As shown in Figure C: The control group mice did not show any pathological manifestations of periodontitis, the model group mice had significantly larger lesions of periodontitis, while the treatment group mice had significantly smaller lesions of periodontitis compared to the model group. Quantitative analysis results of bone microstructure ( Figure 1The DG (Digital Grafting) showed that the treatment group had significantly improved bone microstructure parameters (BV / TV, Tb.N, Tb.Sp, Tb.Th) compared to the model group. BV / TV, Tb.N, Tb.Sp, and Tb.Th are core morphometric parameters for assessing bone microstructure, especially the state of cancellous bone such as alveolar bone. Among them, BV / TV (bone volume fraction) directly reflects bone mass, that is, the relative volume ratio of bone tissue in a specific area. A decrease in this index indicates overall bone loss. Tb.N (trabecular bone number) and Tb.Th (trabecular bone thickness) explain the bone loss mechanism at the microstructural level, representing the number and average thickness of trabeculae per unit length, respectively. A decrease in either indicates that the trabeculae are becoming sparse and thin. Tb.Sp (trabecular bone separation) is used to quantify the average spacing between trabeculae. An increase in this index directly reflects a loose bone structure and widened gaps. In the pathological context of diabetic periodontitis, the above parameters can synergistically reveal the severity of alveolar bone resorption: typical pathological features include decreased BV / TV, Tb.N, and Tb.Th, while Tb.Sp increases. Therefore, the key to evaluating the effectiveness of treatments like citrus peel extract lies in whether it can reverse this pathological trend at the data level—that is, significantly increase BV / TV, Tb.N, and Tb.Th, while decreasing Tb.Sp, thereby confirming its structural therapeutic effect of inhibiting bone resorption and improving bone quality.

[0051] Depend on Figure 2 It can be seen that the periodontitis symptoms in the treatment group were significantly milder than those in the model group. Figure 2 A); Pathological scoring analysis showed that the periodontal inflammation score, periodontal epithelial migration score, and alveolar bone loss score in the treatment group were significantly lower than those in the model group ( Figure 2 BD). Masson staining results showed that the model group had reduced collagen fiber content and decreased bone quality, while the treatment group had significantly higher collagen fiber content and bone quality compared to the model group. Figure 2 EF). Furthermore, IL-6 fluorescence staining results showed that the periodontal tissues in the model group exhibited stronger fluorescence signals, while the treatment group effectively reduced the intensity of this fluorescence signal. Figure 2 The above results confirm that citrus peel extract can improve the pathological state of diabetic periodontitis by reducing periodontal tissue inflammation.

[0052] In microbiome studies based on 16S rRNA gene sequencing, the Shannon index and the Simpson index are two of the most commonly used and core alpha diversity indices, used to quantify the diversity of the microbial community within a single sample. Figure 3The Shannon and Simpson indices showed that the microbial diversity in the model group was significantly higher than that in the control group, suggesting that the increase in low-abundance bacterial species in the disease state led to microbial complexity. The diversity in the treatment group decreased to near-healthy levels, indicating that citrus peel extract intervention can effectively inhibit disease-related microbiota and promote microbial ecological restoration.

[0053] PCoA2, X-variate, and between-group distance are core indicators used to assess overall microbial community differences (β-diversity) between groups. Figure 4 As can be seen from the PCA and PLS-DA analyses, the control group samples clustered tightly, the model group deviated significantly, while the treatment group partially recovered to a near-healthy state. Figure 4 AB). Further β-diversity analysis showed that the distance between the control group and the model group was the largest, while the treatment group fell in between, suggesting that citrus peel extract intervention can partially improve the periodontitis-related flora imbalance (AB). Figure 4 C). The above results indicate that there are significant differences in the bacterial community structure among different groups, and that citrus peel extract has a certain regulatory effect on the periodontal bacterial community structure.

[0054] Species relative abundance is a core quantitative indicator used in microbial ecology, especially in microbiome studies based on high-throughput sequencing (such as 16S rRNA gene sequencing), to describe the proportion of a specific microbial species (or other taxonomic units, such as genus or phylum) in a community. Figure 5 The data shows significant differences in the relative abundance of species at the genus level among the three groups. Figure 5 A). Further analysis revealed significant differences in the abundance of Akkemansia (Verruciformis), Allobaculum (Firmwallis), Bacterides (Bacteroides), Eubacterium (Firmwallis), Lactobacillus (Firmwallis), Alistipes_A_871400 (Bacteroides), Anaerorotruncus (Firmwallis), Parasutterella (Proteobacteria), Streptococcus (Firmwallis), and Emergencia (Firmwallis) among the three groups. Figure 5(BK). The aforementioned differentially expressed bacterial genera encompass multiple types, including clearly beneficial bacteria (such as Akkermansia and Eubacterium), opportunistic pathogens, and bacteria with unknown functions. Among them, the abundance of beneficial genera such as Akkermansia, Eubacterium, Allobaculum, and Emergencia increased significantly, while the abundance of genera associated with inflammation or disease progression (such as some pathogenic strains in Bacteroides, Parasutterella, and Streptococcus) showed a decreasing trend. This trend provides crucial microbiological evidence for the intervention efficacy of citrus peel extract.

[0055] RNA-Seq analysis was performed on periodontal tissues. RNA-Seq, a core technology, enables comprehensive, high-throughput detection and analysis of the types and expression levels of all transcriptomic RNAs (primarily messenger RNA) in specific cells, tissues, or organisms under specific conditions. This technology, by reverse transcribing RNA into cDNA and then performing high-throughput sequencing, can unbiasedly capture almost all transcript information, thereby accurately revealing gene expression levels and their variations. Figure 6 As shown, the point clouds of the model group and the control group are clearly separated in space. This intuitively indicates that the disease state triggers large-scale, systematic gene expression reprogramming across the entire genome, with fundamental differences in the transcriptional profiles between the two groups. Furthermore, after intervention with citrus peel extract, the expression of some genes showed a trend reverting to that of the control group. These results demonstrate that citrus peel extract can regulate gene expression at the systemic level and reverse disease-related transcriptional disorders.

[0056] Figure 7 The results of functional enrichment analysis of differentially expressed genes are presented, aiming to reveal which biological processes these genes are mainly involved in, where they are located in the cell, and which key pathways they are involved in. Figure 7The significantly enriched entries were concentrated in "steroid metabolism," "response to exogenous stimuli," "hormone metabolism," and "glial cell differentiation." This strongly suggests that the therapeutic intervention significantly affected the body's metabolic reprogramming (especially steroid and hormone metabolism). The presence of glial cell-related processes may indicate that citrus peel extract intervention has an impact on glial cells (associated with neuroinflammation). Genes were significantly enriched in the "vacuole proton transporter type V ATPase complex" and its related structures. The main function of type V ATPase is to acidify organelles (such as lysosomes and osteoclast folds), which is crucial for osteoclast bone resorption. This result strongly suggests that citrus peel extract treatment may inhibit osteoclast bone resorption activity by affecting the function of acid-secreting organelles in osteoclasts, which is directly related to improving the alveolar bone loss phenotype in diabetic periodontitis. Molecular functions were enriched in "cytokine activity," "chemokine activity," and various "ATPase activities." This validates the core regulatory role of citrus peel extract in the treatment of immune inflammatory responses (cytokines / chemokines) and ion transport energy metabolism (ATPases). Figure 7 B is a KEGG pathway enrichment analysis plot, showing signaling pathways with significantly enriched differentially expressed genes. Column length represents the proportion of enriched genes (Gene Ratio), and column color also indicates significance. The most significantly enriched pathways include "retinol metabolism," "drug metabolism-cytochrome P450," "steroid hormone biosynthesis," and "exogenous substance metabolism." This again and more systematically confirms that citrus peel extract treatment intervention profoundly affects the body's metabolic network, especially pathways related to vitamin A (retinol) metabolism, drug / foreign substance detoxification, and endogenous hormone synthesis. The enrichment of "cytokine-cytokine receptor interaction," "chemokine signaling pathway," and "rheumatoid arthritis" clearly indicates that citrus peel extract treatment significantly regulates key immune and inflammatory pathways. The enrichment of the "rheumatoid arthritis" pathway is often used as a molecular marker of chronic inflammation and bone destruction. In summary, the multi-target molecular effect network of citrus peel extract in treating diabetic periodontitis can be summarized in the following three aspects: First, it inhibits bone resorption by significantly acting on the key mechanism of osteoclast function—the "vacuole proton transporter type V ATPase complex"—blocking the pathological bone resorption process; second, it regulates immune inflammation by significantly downregulating cytokine / chemokine-related signaling pathways, elucidating its mechanism of action in alleviating periodontal tissue inflammation at the molecular level; and third, it remodels systemic and local metabolism by broadly regulating the "steroid / hormone metabolism," "retinol metabolism," and "exogenous substance metabolism" pathways, demonstrating its potential value in intervening in the pathological process of diabetes and periodontitis from a metabolic perspective.

[0057] In summary, at the intestinal end, AGEs (advanced glycation end products) damage intestinal epithelial cell junction proteins, leading to impaired intestinal barrier function. This damage makes it easier for bacterial toxins such as LPS (lipopolysaccharide) in the intestine to enter the bloodstream, thereby triggering systemic inflammation. The decreased abundance of beneficial bacteria such as Akkermansia (which helps maintain the intestinal mucus barrier) and the increased abundance of potentially pathogenic bacteria are important markers of this pathological process (diabetic periodontitis).

[0058] At the periodontal level, systemic inflammation driven by both LPS and AGEs amplifies the destructive immune response to plaque in the periodontal region, exacerbating alveolar bone resorption. The enrichment of the "cytokine-cytokine receptor interaction" and "osteoclast type V ATPase" pathways in transcriptomic analysis is a molecular manifestation of this amplified inflammatory effect. Ultimately, periodontitis, as a chronic inflammatory focus, continuously produces inflammatory mediators such as TNF-α and IL-1β into the bloodstream, further aggravating systemic inflammation and AGE formation, while simultaneously worsening the gut microecological environment, forming a self-reinforcing vicious triangle of "hyperglycemia / AGEs-gut dysbiosis-periodontal destruction."

[0059] The mechanism of action of this invention can be understood as acting simultaneously on multiple nodes of this vicious triangle, thereby breaking its vicious cycle: the polymethoxyflavonoids in citrus peel extract have direct anti-inflammatory and antioxidant activities, which can inhibit the AGEs-RAGE signaling pathway and downregulate inflammatory pathways (such as cytokine pathways) enriched in the transcriptome, thereby reducing pathological driving factors from the source; in addition, it can increase the abundance of beneficial bacteria such as Akkermansia and Eubacterium (as shown in the previous list of bacterial genera), enhance intestinal barrier function, reduce LPS entry into the blood, and thus reduce the systemic inflammatory load driving periodontal destruction; at the same time, by regulating osteoclast-related pathways (such as type V ATPase) and improving bone microstructure parameters (increased BV / TV, decreased Tb.Sp), it directly enhances the alveolar bone's anti-resorption capacity.

[0060] This invention does not treat oral or intestinal lesions in isolation, but rather, through its multi-functional active ingredients, simultaneously intervenes in the AGEs-induced systemic inflammation that connects diabetes, gut microbiota, and periodontitis, thereby breaking the vicious cycle of the "gut-periodontal axis." This constructs a complete evidence system from multiple levels—metabolites (AGEs), microbial ecology (microbiota structure), host gene expression (transcriptome), and histopathology (bone microstructure)—clearly elucidating its unique advantages and underlying mechanisms in treating diabetic periodontitis.

[0061] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. Application of citrus peel extract in the preparation of products for relieving and / or treating diabetic periodontitis.

2. The application according to claim 1, characterized in that, The preparation method of the citrus peel extract is as follows: freeze-dry and pulverize the citrus peel, add 60%-95% ethanol aqueous solution, extract by hot reflux, collect the filtrate and remove the solvent, and then purify it with macroporous adsorption resin to wash away impurities, thereby obtaining the extract.

3. The application according to claim 2, characterized in that, The citrus peel used is from Xinhui citrus trees in Jiangmen, Guangdong.

4. The application according to claim 2, characterized in that, The volume ratio of the citrus peel to the ethanol aqueous solution is 1:20-30.

5. The application according to claim 2, characterized in that, The ethanol-water solution is a 60% ethanol-water solution, and the hot reflux extraction temperature is 77–83°C; or the ethanol-water solution is a 70% ethanol-water solution, and the hot reflux extraction temperature is 75–80°C; or the ethanol-water solution is an 80% ethanol-water solution, and the hot reflux extraction temperature is 74–79°C; or the ethanol-water solution is a 95% ethanol-water solution, and the hot reflux extraction temperature is 73–76°C.

6. The application according to claim 2, characterized in that, The hot reflux extraction time is 1-4 h.

7. The application according to claim 2, characterized in that, The freeze-drying time is 40-60 h.

8. The application according to claim 2, characterized in that, The macroporous adsorption resin used is AB-8 resin column.

9. The application according to claim 1, characterized in that, The products mentioned above are medications for relieving and / or treating diabetic periodontitis, with citrus peel extract as the main active ingredient and also including pharmaceutically acceptable excipients.

10. The application according to claim 1, characterized in that, The dosage forms of the drug include oral formulations, injectable formulations, inhaled formulations, or transdermal formulations.

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