Application of sea buckthorn extract in the preparation of periodontitis prevention and treatment products

Abstract

This invention relates to the field of pharmaceutical technology, specifically disclosing the application of sea buckthorn extract in the preparation of products for the prevention and treatment of periodontitis. The invention uses network pharmacology to predict the potential molecular mechanisms of sea buckthorn extract in preventing and treating periodontitis, and validates these mechanisms in vivo using a rat model of experimental periodontitis. Results show that sea buckthorn extract significantly improves periodontal probing depth and gingival bleeding index in rats with periodontitis, reduces the expression levels of IL-6, TNF-α, IL-1β, and MMP-9 in serum and gingival tissue, and alleviates periodontal inflammation. Simultaneously, sea buckthorn extract upregulates the expression of RUNX2, OCN, OPG, and ALP, downregulates the expression of RANKL and COX-2, inhibits osteoclast formation, improves alveolar bone resorption, and regulates bone homeostasis. This invention provides a new medical application for sea buckthorn extract, which can be used to prepare products for the prevention and / or treatment of periodontitis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to the application of sea buckthorn extract in the preparation of periodontitis prevention and treatment products. Background Technology

[0002] Periodontitis is a chronic inflammatory disease with an extremely high global prevalence. Its core pathology is an imbalance in the host immune inflammatory response triggered by dental plaque biofilm, leading to progressive destruction of periodontal tissues such as the gingiva, periodontal ligament, and alveolar bone. It is a leading cause of tooth loss in adults. While basic treatments, such as supragingival scaling, subgingival scaling, and root planing, can effectively remove plaque biofilm, their ability to remodel the microecology of deep periodontal pockets is limited, and they are insufficient to correct the already imbalanced host immune response. The use of adjuvant antibiotics easily leads to drug resistance and oral flora imbalance. Therefore, there is still a clinical need for novel adjuvant therapies that can synergistically inhibit pathogen virulence, reduce excessive inflammatory responses, and promote tissue repair.

[0003] Natural products have become a research hotspot due to their multi-target regulatory potential and good biosafety. Sea buckthorn (Hippophae rhamnoides L.), a traditional medicinal and edible plant, is rich in flavonoids (such as kaempferol and isorhamnetin), polyphenols, and polysaccharides, exhibiting anti-inflammatory, antioxidant, and immunomodulatory activities in various chronic inflammation and oxidative stress-related disease models. However, its precise application to periodontitis, a localized inflammatory condition with a unique microbial community and bone immune environment, still lacks convincing mechanistic studies and efficacy evidence.

[0004] A search revealed existing technologies that utilize sea buckthorn as one of the herbal components in the preparation of toothpaste for treating periodontitis. For example, Chinese patent application CN109852481A discloses a low-temperature herbal extraction process and the resulting toothpaste for treating periodontitis. The toothpaste contains over ten ingredients, including green plum, patchouli, peppermint, sesame, sea buckthorn, licorice, chrysanthemum, clove, astragalus, codonopsis, gastrodia, cornus, ganoderma, safflower, dandelion, honeysuckle, polygonatum, and prunella vulgaris. This technology uses a combination of various herbs, obtaining a complex essential oil through subcritical extraction, which is then added to the toothpaste for adjunctive treatment of periodontitis. However, this existing technology has the following shortcomings:

[0005] (1) The use of sea buckthorn in combination with a large number of other herbal ingredients failed to reveal whether sea buckthorn extract alone has a definite preventive and therapeutic effect on periodontitis and its specific active ingredients;

[0006] (2) The target and molecular mechanism of sea buckthorn extract in preventing and treating periodontitis have not been elucidated, especially its regulatory pathways on periodontal tissue inflammation and bone metabolism.

[0007] (3) The composition is complex and the batch-to-batch stability is difficult to control, which is not conducive to developing it into a standardized formulation with clear composition and mechanism.

[0008] Therefore, the technical problem that urgently needs to be solved in this field is to clarify the pharmacodynamic material basis and molecular mechanism of sea buckthorn extract alone in the prevention and treatment of periodontitis, and to provide a new use for sea buckthorn extract with clear components, clear targets, and the ability to exert anti-inflammatory and bone homeostasis regulation effects at the same time. Summary of the Invention

[0009] The purpose of this invention is to solve the technical problems mentioned in the background section and to provide the application of sea buckthorn extract in the preparation of periodontitis prevention and treatment products.

[0010] The above-mentioned objective of the present invention is achieved as follows:

[0011] Application of sea buckthorn extract in the preparation of periodontitis prevention and treatment products.

[0012] Furthermore, the sea buckthorn extract is sea buckthorn flavonoids.

[0013] Furthermore, the dosage form of the product is a topical administration formulation.

[0014] Furthermore, the topical medication is a preparation that is injected into the gingival sulcus.

[0015] Furthermore, the product is a systemic administration formulation.

[0016] Furthermore, the systemic administration formulation is an oral administration formulation.

[0017] Furthermore, the concentration of the sea buckthorn extract is 0.25% (w / v) to 4% (w / v).

[0018] Furthermore, the concentration of the sea buckthorn extract is 1% (w / v) to 4% (w / v).

[0019] Furthermore, the product is administered once daily.

[0020] Furthermore, the product is administered over a period of at least 14 days.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The present invention systematically reveals for the first time the network pharmacological mechanism of sea buckthorn extract in preventing and treating periodontitis. Through screening the TCMSP database, 33 active components of sea buckthorn were obtained, with 201 predicted targets. An intersection of 3697 targets related to periodontitis yielded 123 common targets. PPI network analysis identified 20 core targets, including IL-6, TNF, MMP9, PTGS2 (COX-2), AKT1, and CASP3. GO and KEGG enrichment analyses showed that sea buckthorn mainly exerts its regulatory effects through the TNF signaling pathway, IL-17 signaling pathway, PI3K-Akt signaling pathway, NF-κB signaling pathway, and osteoclast differentiation-related pathways. Molecular docking results showed that the main active components quercetin, β-sitosterol, isorhamnetin, and kaempferol have strong binding affinity to the core targets PTGS2, AKT1, IL-6, and TNF (binding affinity -6.4 to -9.7 kcal / mol).

[0023] 2. The solution of this invention can significantly improve clinical indicators of periodontitis. In a rat experimental periodontitis model, the periodontal probing depth (PD) and gingival bleeding index (GBI) of the sea buckthorn extract intervention groups (0.25%-4% local administration and 4% gavage) were significantly lower than those of the model group, showing a dose-dependent trend. Among them, the 4% local administration group showed the most significant improvement, with PD and GBI approaching the level of the normal group (see...). Figure 3 (B, C).

[0024] 3. The solution of this invention can effectively inhibit systemic and local inflammatory responses. ELISA results showed that sea buckthorn extract can dose-dependently reduce the levels of IL-1β, IL-6, TNF-α, and MMP-9 in the serum of rats with periodontitis. Specifically, in the 4% local administration group (SBE-HDG), the levels of the above inflammatory factors were significantly lower than those in the model group (EP group), and some indicators were close to the levels of the normal group (see...). Figure 14 (AD). HE staining results showed that after intervention with sea buckthorn extract, the degree of periodontal epithelial proliferation decreased, inflammatory cell infiltration decreased, connective tissue structure became denser, trabecular bone continuity increased, and inflammation score significantly decreased (see AD). Figure 5 A, 5B). Masson staining results showed that sea buckthorn extract could increase the surface area of ​​collagen fibers and improve disordered collagen fiber arrangement and breakage (see A, B). Figure 6 (A, B). Immunohistochemical results showed that sea buckthorn extract significantly downregulated the expression of the inflammation-related marker COX-2 (see A, B). Figure 13 (A and B).

[0025] 4. The solution of this invention can significantly improve alveolar bone microstructure and regulate bone homeostasis. Micro-CT analysis shows that alveolar bone loss is significantly reduced and bone microstructure parameters are significantly improved in the sea buckthorn extract intervention group. Compared with the model group, the bone volume fraction, trabecular thickness, and number of trabecular bones are significantly increased, trabecular separation is significantly reduced, and the distance from the cementoenamel junction to the alveolar bone apex (CEJ-ABC) is significantly shortened (see [link to original text]). Figure 4 (AF); TRAP staining results showed that sea buckthorn extract significantly reduced the number of TRAP-positive osteoclasts on the alveolar bone surface (see AF); Figure 7 Immunohistochemical results showed that sea buckthorn extract upregulated the expression of osteogenic markers ALP, OCN, and RUNX2, upregulated osteoprotegerin (OPG) expression, and downregulated receptor activator of nuclear factor-κB (RANKL) expression and the RANKL / OPG ratio (see AD). Figure 8-12 ).

[0026] 5. The method of the present invention has good systemic safety. HE staining results of major organs (heart, liver, spleen, lung, and kidney) showed that no significant pathological damage was observed in the intervention groups of sea buckthorn extract compared with the normal control group, indicating that the sea buckthorn extract used in the present invention has good systemic safety within the experimental dosage range (see...). Figure 5 (C). Attached Figure Description

[0027] Figure 1 This is a schematic diagram illustrating the construction of an experimental rat model of periodontitis in an embodiment of the present invention.

[0028] Figure 2 These are the network pharmacology analysis results in the embodiments of the present invention (A: Venn diagram of sea buckthorn chemical component targets and periodontitis targets; B: PPI network diagram; C: GO enrichment analysis and KEGG pathway analysis diagram; D: component-target-network diagram; E: molecular docking model diagram).

[0029] Figure 3 This invention relates to the effects of sea buckthorn extract on body weight, gingival bleeding index, and probing depth in rats with periodontitis (A: daily body weight changes in rats during the experimental period; B: daily probing depth trends in rats during the experimental period; C: daily gingival bleeding index (GBI: 0-5) trends in rats during the experimental period).

[0030] Figure 4 These are three-dimensional and cross-sectional Micro-CT images of the right maxilla in this embodiment of the invention and their impact on alveolar bone parameters (A: 3D images and sagittal, coronal, and horizontal plane images; B: CEJ-ABC distance; C: BV / TV (%); D: Tb.Th; E: Tb.N; F: Tb.Sp).

[0031] Figure 5 These are HE staining and inflammation scores in embodiments of the present invention. A: HE staining images of periodontal tissues in each group (×100 and ×200); B: Inflammation scores from HE staining; C: HE staining images of the heart, liver, spleen, lung, and kidney.

[0032] Figure 6 These are the Masson staining results in the embodiments of the present invention (A: Masson staining images of periodontal tissues in each group (×100); B: Analysis of the area ratio of collagen fibers).

[0033] Figure 7 This invention includes TRAP staining and TRAP-positive cell counts in different regions (A: TRAP staining in the mesial gingival sulcus region, interdental space, and root bifurcation region of M1 (×100 and ×200); B: osteoclast count on the mesial proximal surface of M1; C: osteoclast count in the root bifurcation region; D: osteoclast count in the interdental space).

[0034] Figure 8 The following are the immunohistochemical staining results and quantification of ALP in the embodiments of the present invention (A: staining results (×200 and ×400); B: quantitative analysis).

[0035] Figure 9 The following are the immunohistochemical staining results and quantification of OCN in the embodiments of the present invention (A: staining results (×200 and ×400); B: quantitative analysis).

[0036] Figure 10 The following are the immunohistochemical staining results and quantification of RUNX2 in the embodiments of the present invention (A: staining results (×200 and ×400); B: quantitative analysis).

[0037] Figure 11 These are the immunohistochemical staining results and quantification of OPG in this embodiment of the invention. A: Staining results (×200 and ×400); B: Quantitative analysis.

[0038] Figure 12 The following are the immunohistochemical staining results and quantification of RANKL in the embodiments of the present invention (A: staining results (×200 and ×400); B: RANKL and RANKL / OPG quantitative analysis).

[0039] Figure 13 The following are the immunohistochemical staining results and quantification of COX-2 in the embodiments of the present invention (A: staining results (×200 and ×400); B: quantitative analysis).

[0040] Figure 14This invention relates to the effects of sea buckthorn extract on serum IL-1β, TNF-α, IL-6, and MMP-9 levels in rats with periodontitis (A: IL-1β level; B: IL-6 level; C: MMP-9 level; D: TNF-α level). Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0042] The implementation of the present invention will be described in detail below with reference to specific embodiments.

[0043] Example 1: Preparation of Seabuckthorn Extract and Test Solution

[0044] The sea buckthorn extract used in this embodiment is sea buckthorn flavonoid (purchased from Solarbio Life Science, catalog number SVS1025). Using glycerol as a solvent, sea buckthorn extract glycerol solutions with concentrations of 4% (w / v), 2% (w / v), 1% (w / v), 0.5% (w / v), and 0.25% (w / v) were prepared. A separate 4% (w / v) sea buckthorn extract glycerol solution was prepared for oral administration. All solutions were thoroughly mixed by shaking after preparation and stored at 4°C for later use.

[0045] The positive control was a 2% (w / v) minocycline hydrochloride glycerol solution (20 mg minocycline hydrochloride dissolved in 1 mL glycerol). The solvent control was pure glycerol.

[0046] Example 2: Network Pharmacology and Molecular Docking Analysis

[0047] 2.1 Screening of active ingredients and targets of sea buckthorn

[0048] Using the TCMSP database (https: / / old.tcmsp-e.com / ), and with oral bioavailability (OB) ≥30% and drug similarity (DL) ≥0.18 as parameters, active ingredients of Hippophae rhamnoides L. were screened, resulting in 33 active ingredients. The target sites corresponding to each ingredient were imported into the UniProt database for gene name normalization, yielding a total of 201 chemical target sites.

[0049] 2.2 Periodontitis-related targets and common targets

[0050] Using "periodontitis" as the keyword, we searched for periodontitis-related disease targets in the GeneCards and OMIM databases. After merging and deduplicating, we obtained 3697 targets. Using the Venny 2.1 platform, we took the intersection of the sea buckthorn active ingredient targets and periodontitis targets, obtaining 123 common targets (see...). Figure 2 A).

[0051] 2.3 Construction of protein-protein interaction networks and screening of core targets

[0052] 123 common targets were imported into the STRING database (version 12.0), with the species selected as "Homosapiens" and the minimum interaction score set to 0.7, to construct a protein-protein interaction (PPI) network. The resulting tsv file was imported into Cytoscape 3.8.2 software, and the degree centrality (DC) was calculated using the CytoNCA plugin. The top 20 targets with the highest DC values ​​were selected as core targets, including IL-6, TNF, MMP9, PTGS2 (COX-2), AKT1, CASP3, etc. (see [link to relevant documentation]). Figure 2 B).

[0053] 2.4 GO Functional Annotation and KEGG Pathway Enrichment Analysis

[0054] The core targets were imported into the DAVID database for GO functional annotation (including biological processes, cellular components, and molecular functions) and KEGG signaling pathway enrichment analysis. The results showed that the targets were significantly enriched in biological processes such as inflammatory responses and cytokine-mediated signal transduction, as well as in the TNF signaling pathway, IL-17 signaling pathway, PI3K-Akt signaling pathway, NF-κB signaling pathway, and osteoclast differentiation-related pathways (see...). Figure 2 C).

[0055] 2.5 Construction and Molecular Docking Verification of Component-Target-Pathway Network

[0056] A "component-target-pathway" regulatory network was constructed using Cytoscape software (see...). Figure 2D). The 3D structures of key targets (IL-6, MAPK1, PTGS2, ADRA2A, TNF) were downloaded from the PDB database. Original ligands were removed using PyMOL, and dehydration and hydrogenation were performed using ADT software. The structural files of the main active components of sea buckthorn (β-sitosterol, β-carotene, quercetin, kaempferol, isorhamnetin) were downloaded from the PubChem database, converted to PDB format using OpenBabelGUI, and hydrogenated, ligands defined, and rotatable bonds set using ADT software. Semi-flexible molecular docking was performed using AutoDock Vina, and the results were visualized using PyMOL and DS software. The docking results showed that quercetin had the highest binding affinity to PTGS2 (-9.7 kcal / mol). β-sitosterol, isorhamnetin, and kaempferol also showed stable binding abilities to targets such as AKT1, IL-6, and TNF. Specific binding energy data are shown in Table 1, and the molecular docking model is shown in [Table 1]. Figure 2 E.

[0057] Table 1. Binding affinity of the main active ingredients of sea buckthorn to the core target sites (kcal / mol)

[0058] target PDB ID β-sitosterol beta-carotene Quercetin Kaempferol Rhamnetin PTGS2 5F12 -9.3 -9.0 -9.7 -8.9 -9.3 CASP3 1NME -6.5 -7.6 -7.8 -7.8 -7.8 AKT1 4GV1 -8.2 -8.9 -8.0 -7.7 -8.4 IL-6 4CNI -7.6 -8.4 -8.1 -7.8 -8.2 TNF 5UUI -6.4 -7.7 -7.0 -6.6 -6.7

[0059] Example 3: In vivo efficacy evaluation of sea buckthorn extract against experimental periodontitis in rats

[0060] 3.1 Laboratory Animals and Grouping

[0061] Sixty male SD rats, 6–8 weeks old and weighing 180–220 g, were purchased from the Medical Animal Experiment Center of Xinjiang Medical University. The experiment was approved by the center's animal ethics committee. The animals were acclimatized for one week under constant temperature (22±2℃), constant humidity (50%–60%), and 12-hour light-dark alternation environment. They were randomly divided into 10 groups (n=6 per group) using a random number table: blank group (NG), glycerol solvent group (G), periodontitis model group (EP), minocycline hydrochloride group (MC), 4% sea buckthorn extract topical administration group (SBE-HDG), 2% sea buckthorn extract topical administration group (SBE-MHDG), 1% sea buckthorn extract topical administration group (SBE-MDG), 0.5% sea buckthorn extract topical administration group (SBE-MLDG), 0.25% sea buckthorn extract topical administration group (SBE-LDG), and 4% sea buckthorn extract gavage group (SBE-GG).

[0062] 3.2 Establishment of an experimental periodontitis model

[0063] Except for the blank group, all other groups are referenced. Figure 1An experimental periodontitis model was established using the method shown. Specific procedures: Rats were completely anesthetized and fixed. A sterile 4-0 silk suture was used to ligate the neck of the right maxillary first molar, ensuring the suture was close to the tooth neck and placed within the gingival sulcus. Then, a suspension of *Porphyromonas gingivalis* was locally inoculated to enhance the periodontal inflammatory response. The control group received no treatment.

[0064] 3.3 Drug Intervention Program

[0065] Drug administration began on the day of model establishment, once daily for two consecutive weeks. Local administration group: Rats were fixed in a supine position with their mouths open. The corresponding drug solution (50 μL / rat) was slowly and evenly injected into the gingival sulcus of the right upper first molar using a microsyringe. The head was kept in the same position for 1 minute after injection to prevent drug leakage. Gavage group: 4% sea buckthorn extract glycerol solution was injected into the stomach using a special gavage needle at a dose of 1 mL / 100 g body weight. Glycerol solvent group: An equal volume of pure glycerol was administered. Minocycline hydrochloride group: 2% minocycline hydrochloride glycerol solution (50 μL / rat, injected into the gingival sulcus) was administered. All rats were fasted and deprived of water for 1 hour after each administration. The integrity of the ligation sutures in the model group was observed daily.

[0066] 3.4 Clinical Indicator Monitoring

[0067] The rats were weighed daily during the experiment, and the results were as follows: Figure 3 As shown in Figure A. Periodontal probing depth (PD) and gingival bleeding index (GBI) were measured before daily medication. PD measurement: A probe was used to measure three sites on the right maxillary first molar: mesobacterial, disbubacterial, and buccal. The probe tip was parallel to the long axis of the tooth, and the pressure was constant. The procedure was performed by the same technician. GBI scores were based on a scale of 1–5 (1 point: no bleeding with light probing; 2 points: pinpoint bleeding; 3 points: linear or patchy bleeding overflowing into the gingival sulcus; 4 points: significant bleeding with overflow; 5 points: spontaneous bleeding). Results are shown in Figure A. Figure 3 B and Figure 3 C. Compared with the NG group, the PD and GBI of the EP group and the G group were significantly increased (P<0.05), indicating that the model was successful; the PD and GBI of each sea buckthorn extract intervention group were significantly lower than those of the EP group (P<0.05), showing a dose-dependent effect, with the 4% local administration group showing the most significant improvement.

[0068] 3.5 Specimen Collection and Processing

[0069] After the experiment, rats were anesthetized and X-rays of the jawbone were taken to confirm the formation of periodontitis. Blood was collected via the abdominal aorta, and after coagulation, it was centrifuged at 1500 rpm for 15 min at 4°C. Serum was separated, aliquoted, and stored at -80°C. The heart, liver, spleen, lungs, and kidneys were isolated, rinsed with physiological saline, and fixed with 4% paraformaldehyde. The right maxilla was isolated, and the buccal and palatal gingiva and adjacent bone tissue of the first molar were gently dissected. After rinsing with physiological saline, some gingival tissue was frozen at -80°C for ELISA, and some maxilla tissue was fixed with 4% paraformaldehyde for 24 h and then transferred to 75% ethanol for Micro-CT scanning. After scanning, the maxilla was placed in 5% EDTA decalcification solution (changed weekly, until a fine needle could be inserted without resistance) for decalcification. After decalcification, it was rinsed with running water for 24 h, fixed and rinsed again, and finally stored in 75% ethanol for histopathological examination.

[0070] 3.6 Micro-CT Scan and Bone Parameter Analysis

[0071] The fixed maxillary bone specimen was scanned using a Micro-CT scanner with the following parameters: voltage 55 kVp, current 200 μA, and resolution 6.6 μm. Three-dimensional reconstructed images and sagittal, coronal, and horizontal plane images are shown below. Figure 4 A. The distance from the cementoenamel junction to the alveolar bone crest (CEJ-ABC) was measured, and the average value of three sites (mesial, central, and distal) was taken for each sample. NRecon software was used to analyze bone microstructural parameters within the region of interest (ROI): bone volume fraction (BV / TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp). Results are as follows... Figure 4 As shown in B-4F. In the EP group, CEJ-ABC was significantly increased compared to the NG group (P<0.05), BV / TV, Tb.Th, and Tb.N were significantly decreased, and Tb.Sp was significantly increased. All the above indicators were significantly improved in each sea buckthorn extract intervention group (P<0.05), with the SBE-HDG and SBE-GG groups showing the best effects, approaching the level of the NG group.

[0072] 3.7 ELISA detection of inflammatory factors

[0073] The levels of IL-1β, IL-6, TNF-α, and MMP-9 in rat serum and gingival tissue were determined using an ELISA kit. The procedure was as follows: Serum samples were slowly thawed at 4°C; gingival tissue was weighed, homogenized with pre-chilled lysis buffer, and centrifuged to collect the supernatant. Working solutions were prepared according to the kit instructions, with zero wells, standard wells, and sample wells. 100 μL of sample or standard was added to each well, and the mixture was incubated at 37°C for 2 h. After washing four times, 100 μL of HRP-labeled detection antibody was added to each well, followed by four more washes. 100 μL of TMB chromogenic buffer was added to each well, and the mixture was incubated at 37°C in the dark for 20 min. Stop solution was added, and the absorbance was immediately measured at 450 nm using a microplate reader. The concentrations were calculated based on the standard curve. Results are shown below. Figure 14 A-14D. The serum levels of all inflammatory factors in the EP and G groups were significantly higher than those in the NG group (P<0.05). After intervention with sea buckthorn extract, the levels of all inflammatory factors decreased in a dose-dependent manner (P<0.05): IL-1β showed the most significant decrease in the SBE-HDG and SBE-MHDG groups ( Figure 14 A); IL-6 was highest in the SBE-LDG group (higher than the EP group), but significantly lower in the SBE-HDG group than in the EP group ( Figure 14 B); MMP-9 and TNF-α were lowest in the SBE-HDG group ( Figure 14 C, 14D). The trends of changes in various factors in gingival tissue were consistent with those in serum.

[0074] 3.8 Histopathological Examination

[0075] 3.8.1 HE staining

[0076] After decalcification, the maxilla was embedded in paraffin and sectioned serially along the sagittal plane (4-5 μm thick) for HE staining. The integrity of the gingival epithelium, connective tissue density, inflammatory cell infiltration, and alveolar bone morphology were observed under a light microscope. Inflammation was scored according to a scale of 0-3 (0: no or very few inflammatory cells, intact bone; 3: abundant inflammatory cells, complete bone resorption). HE staining results are shown below. Figure 5 A, Inflammation score (see...) Figure 5 B. The NG group showed intact periodontal tissue structure, continuous epithelium, and dense connective tissue, resulting in the lowest score. The EP group showed epithelial thickening, loose connective tissue, extensive inflammatory cell infiltration, and trabecular bone resorption, leading to a significantly higher score (P<0.05). After intervention with sea buckthorn extract, inflammation was significantly reduced, and the score decreased (P<0.05), with the high-dose group approaching that of the NG group. HE staining of major organs (heart, liver, spleen, lung, and kidney) is shown in [image / description]. Figure 5 C. No obvious pathological damage was observed in any of them.

[0077] 3.8.2 Masson staining

[0078] Sections were stained with Masson's trichrome staining; collagen fibers appeared blue, while muscle fibers and cytoplasm appeared red. The distribution density, continuity, and orientation of collagen fibers in the periodontal ligament and alveolar ridge regions were observed under a ×100 magnification microscope, and image analysis software was used to calculate the percentage of blue collagen area relative to the total tissue area. Results are shown below. Figure 6 A (coloring diagram) and Figure 6 B (Area Percentage Analysis). In the NG group, collagen fibers were densely and orderly arranged, with a high area percentage; in the EP group, collagen fibers were significantly reduced, broken, and disordered, resulting in a significantly lower area percentage (P<0.05); the area percentage of collagen fibers in each sea buckthorn extract intervention group was significantly increased (P<0.05), showing a dose-dependent effect.

[0079] 3.8.3, TRAP staining

[0080] Sections were stained with TRAP. Osteoclast cytoplasm appeared purplish-red, and nuclei were blue (multinucleated). Under ×100 and ×200 magnification, the number of TRAP-positive osteoclasts per unit length of bone surface was counted in the bifurcation zone, mesial proximal surface, and interdental space of the first molar root. The staining results are shown in [Figure number missing]. Figure 7 A, the counting results for each region are shown in [reference]. Figure 7 B-7D. Only a small number of osteoclasts were observed in the NG group; the number of osteoclasts in all regions of the EP group increased significantly (P<0.05); the number of osteoclasts decreased significantly after intervention with sea buckthorn extract (P<0.05), with the SBE-HDG and SBE-GG groups showing the most significant effects.

[0081] 3.8.4 Immunohistochemical staining

[0082] Immunohistochemistry was used to detect the expression of osteogenic markers (ALP, OCN, RUNX2), bone homeostasis regulators (OPG, RANKL), and inflammatory markers (COX-2). After dewaxing and hydration, antigen retrieval, hydrogen peroxide blocking, and serum blocking, the corresponding primary antibodies (targeting RUNX2, OCN, ALP, OPG, RANKL, and COX-2) were added, and the mixture was incubated overnight at 4°C. The next day, secondary antibodies were added, and DAB staining was performed, followed by hematoxylin counterstaining. Positive expression was brownish-yellow, and the average optical density was measured using image analysis software. The results are as follows:

[0083] Osteogenesis markers ( Figure 8-10 The expression of ALP, OCN, and RUNX2 in the EP group was significantly lower than that in the NG group (P<0.05); after intervention with sea buckthorn extract, the expression of all three increased in a dose-dependent manner (P<0.05), and the high-dose group was close to the level of the MC group.

[0084] Bone homeostasis regulators ( Figure 11-12): In the EP group, OPG expression was decreased and RANKL expression was increased, and the RANKL / OPG ratio was significantly increased (P<0.05); after intervention with sea buckthorn extract, OPG increased, RANKL decreased, and the ratio decreased (P<0.05).

[0085] Inflammatory markers ( Figure 13 COX-2 expression was significantly increased in the EP group (P<0.05); COX-2 expression was significantly decreased after intervention with sea buckthorn extract (P<0.05), showing a dose-dependent effect.

[0086] 3.9 Statistical Processing

[0087] All experimental data were statistically analyzed using SPSS 26.0 software. Normality and homogeneity of variance tests were performed first. One-way ANOVA was used for comparisons among multiple groups, with P < 0.05 considered statistically significant. GraphPad Prism 10.2.1 was used for plotting.

[0088] As demonstrated in Examples 1-3 of this invention, this invention systematically elucidates for the first time the network pharmacological mechanism of sea buckthorn extract in preventing and treating periodontitis. Example 2 screened 33 active ingredients and 123 common targets, with the core targets enriched in key pathways such as TNF, IL-17, NF-κB, and osteoclast differentiation. Molecular docking verified the strong binding ability of the main active ingredients to the core targets (binding energy -6.4 to -9.7 kcal / mol). This provides a clear material basis and mechanism of action for the use of sea buckthorn extract alone in periodontitis, overcoming the shortcomings of existing technologies that use sea buckthorn as one of the components in compound preparations with unclear mechanisms. Furthermore, this invention can significantly improve clinical indicators and systemic / local inflammation in periodontitis. In Example 3, sea buckthorn extract dose-dependently reduced PD and GBI (glucose dinitrate). Figure 3 B, 3C), reduce serum and gingival levels of IL-1β, IL-6, TNF-α and MMP-9 (B, 3C), Figure 14 A-14D reduces inflammatory cell infiltration in periodontal tissues and increases the proportion of collagen fiber area. Figure 5 , Figure 6 Furthermore, this invention effectively improves alveolar bone microstructure and regulates bone homeostasis; sea buckthorn extract can significantly shorten CEJ-ABC, increase BV / TV, Tb.Th, and Tb.N, and decrease Tb.Sp (…). Figure 4 ), reduce the number of osteoclasts ( Figure 7 Upregulates osteogenic markers (ALP, OCN, RUNX2) and OPG, downregulates RANKL, RANKL / OPG ratio, and COX-2 ( Figure 8-13 This indicates that it can inhibit osteoclastosis, promote osteogenic formation, and reverse periodontal bone resorption. Simultaneously, this invention exhibits good safety; no abnormalities were observed in the HE staining of major organs. Figure 5 (C) indicates that the sea buckthorn extract has no significant systemic toxicity within the experimental dosage range.

[0089] In summary, existing technologies only use sea buckthorn as a compound ingredient in toothpaste, without providing any evidence of its effectiveness in preventing and treating periodontitis when used alone. Furthermore, they fail to reveal its target sites, signaling pathways, and specific regulation of alveolar bone metabolism. This invention, through integrating network pharmacology predictions and validation using a rat experimental periodontitis model, demonstrates for the first time that sea buckthorn extract alone can synergistically exert anti-inflammatory and bone homeostasis-regulating effects through multiple components, targets, and pathways, thereby effectively preventing and treating periodontitis. Moreover, the technical solution of this invention has clearly defined components, a clear mechanism, significant efficacy, and good safety, providing a novel standardized functional formulation for the adjunctive treatment of periodontitis.

[0090] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of sea buckthorn extract in the preparation of periodontitis prevention and treatment products.

2. The application according to claim 1, characterized in that, The sea buckthorn extract is sea buckthorn flavonoids.

3. The application according to claim 1, characterized in that, The product is a topical medication.

4. The application according to claim 3, characterized in that, The topical medication is an injection into the gingival sulcus.

5. The application according to claim 1, characterized in that, The product is a systemic drug delivery formulation.

6. The application according to claim 5, characterized in that, The systemic administration formulation is a gavage formulation.

7. The application according to claim 1, characterized in that, The concentration of the sea buckthorn extract is 0.25% (w / v) to 4% (w / v).

8. The application according to claim 7, characterized in that, The concentration of the sea buckthorn extract is 1% (w / v) to 4% (w / v).

9. The application according to claim 1, characterized in that, The product is to be administered once daily.

10. The application according to claim 1, characterized in that, The product is administered over a period of at least 14 days.

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