Use of a vinca alkaloid lactam in the preparation of a medicament for the treatment of periodontitis

Abstract

The application belongs to the technical field of medicine, and particularly relates to application of isovincadiffine lactam in preparation of a drug for treating periodontitis, wherein the isovincadiffine lactam is extracted and separated from Nauclea officinalis medicinal materials, a periodontitis animal model is constructed, the model is intervened and treated by using the isovincadiffine lactam, periodontal tissues are taken for TRAP staining, Micro-CT detection and HE staining detection, it is proved that the isovincadiffine lactam can specifically regulate activation of osteoclasts, effectively inhibit pathological alveolar bone absorption caused by periodontitis, reduce alveolar bone loss, significantly reduce damage of periodontal tissues, promote bone repair, and fundamentally block the disease progression of periodontitis. The experimental results of the application have clinical significance, and provide a new scheme for clinical treatment of periodontitis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of isocinolone lactone in the preparation of drugs for treating periodontitis. Background Technology

[0002] Periodontitis is a common chronic inflammatory disease of the oral cavity with a high prevalence, affecting approximately 10% of the global population. The heavy epidemiological burden of periodontitis has caused significant economic losses in various countries. Periodontitis not only severely impacts oral health but also increases the incidence of many systemic diseases, including cardiovascular disease, diabetes, and Alzheimer's disease. The pathological process of this disease is complex, mainly manifested as gingival redness and bleeding, periodontal pocket formation, inflammatory destruction of periodontal soft tissues, and progressive alveolar bone resorption, ultimately leading to loss of periodontal attachment and irreversible damage to the tooth's supporting structures. Therefore, periodontitis is a major public health challenge faced by countries worldwide.

[0003] Although periodontitis is a common and frequently occurring disease in oral clinics, its etiology is still unclear. Current clinical treatments are mostly symptomatic, such as removing gingival calculus and relieving oral inflammation. These treatments lack effective regulation of alveolar bone resorption and periodontal tissue repair and regeneration, which are crucial in the progression of periodontitis. However, these treatments not only fail to completely eliminate periodontal pathogens but also exacerbate the patient's immune response, leading to the gradual loss of tooth tissue. Controlling the continuous deterioration of periodontitis is a problem that urgently needs to be solved in clinical practice.

[0004] Strictosamide (STR) is an indole alkaloid extracted from *Gnaphalium affine* and is one of its core active components. It possesses certain activities such as antitumor, antipyretic, analgesic, antitussive, antiasthmatic, and antipigmentation effects, and also boasts advantages such as low toxicity, high safety, and few side effects. A search revealed no reports on the use of strictosamide for the treatment of periodontitis. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention proposes the application of isocinolone lactone in the preparation of drugs for treating periodontitis. Isocinolone lactone extracted from *Gnaphalium affine* can regulate osteoclasts, inhibit alveolar bone resorption, repair periodontal tissue damage, and alleviate periodontitis symptoms.

[0006] The technical solution adopted in this invention:

[0007] Application of a vinblastine lactone in the preparation of a drug for treating periodontitis.

[0008] The isovinca lactone described in this invention is extracted from plants, Chinese medicinal materials, or prepared slices of Chinese medicinal herbs containing this component.

[0009] In one optional embodiment, the active ingredient of the periodontitis treatment drug includes isovinctin lactam, and the active ingredient is formulated with pharmaceutically common excipients into a clinically common dosage form.

[0010] In one optional embodiment, the commonly used pharmaceutical excipients are one or more of the following: fillers, diluents, binders, disintegrants, emulsifiers, lubricants, and sweeteners.

[0011] In one optional embodiment, the commonly used clinical dosage form is an effervescent tablet, powder, tablet, capsule, spray, drop, or suspension.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] This invention provides a novel application of isocinolone lactone, a chemical component of *Gnaphalium affine*, in the preparation of drugs for treating periodontitis. It primarily alleviates periodontitis symptoms by regulating osteoclast activation, inhibiting alveolar bone resorption, and repairing periodontal tissue damage, offering a new approach to periodontitis treatment. Furthermore, isocinolone lactone is derived from the natural plant *Gnaphalium affine*, exhibiting high safety, few side effects, ease of extraction, and controllable quality, demonstrating significant clinical application value and industrialization potential. Attached Figure Description

[0014] Figure 1 TRAP staining image of mouse maxillary periodontal tissue;

[0015] Figure 2 Micro-CT 3D reconstruction of the mouse maxilla;

[0016] Figure 3 HE staining image of mouse maxillary periodontal tissue. Detailed Implementation

[0017] The technical solutions of the embodiments of this application will be further described clearly and completely below with reference to the accompanying drawings. It should be noted that the described embodiments are only some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0018] Example 1: Extraction of isoclavillarin lactone

[0019] Coarse separation: Take 50 kg of *Gnaphalium affine* stems, coarsely crush them, add 150 L of water, heat and reflux to extract three times, combine the extracts, concentrate under reduced pressure to obtain about 750 g of extract, take the extract and evenly disperse it in distilled water, extract with dichloromethane (5000 mL × 3 times) and ethyl acetate (5000 mL × 3 times) respectively, to obtain 147 g of dichloromethane extract and 166 g of ethyl acetate extract respectively.

[0020] Fine separation: 147g of dichloromethane extract was loaded onto a silica gel column (1500g, 200-300 mesh) using a wet method. Gradient elution was performed with dichloromethane-ethyl acetate as the eluent at volume ratios of 9:1, 8:2, 7:3, 6:4, and 1:9, with eluent volumes of 5000, 5000, 10000, 10000, and 7500 mL respectively. Each 750 mL eluent was considered a fraction, concentrated to 25 mL, and analyzed by thin-layer chromatography. Similar fractions were combined to obtain 10 fractions, designated Fr.1 to Fr.10. Approximately 15.0g of Fr.4 was diluted with methanol to 500 mg / mL and subjected to preparative liquid chromatography. Elution was performed with 50% methanol, and the eluent was collected for 36.8–40.0 min. The eluent was rotary evaporated to 9–10 mL and dried under nitrogen to obtain approximately 4300 mg of isocynoplasmin.

[0021] Example 2: The therapeutic effect of isocinolone lactone on periodontitis

[0022] Fifty male C57BL / 6J mice were randomly and equally divided into the following five groups:

[0023] Normal control group: No treatment was given;

[0024] Model group: Mice with second molars ligated with silk sutures;

[0025] Metronidazole treatment group (positive control group): After ligating the second molars of mice with silk thread, they were treated with metronidazole (60 mg / kg);

[0026] Low-dose vinblastine lactone treatment group (STR L group): Mice were treated with vinblastine lactone (100 mg / kg) after their second molars were ligated with silk thread.

[0027] High-dose vinblastine lactone treatment group (STR H group): Mice were treated with vinblastine lactone (200 mg / kg) after their second molars were ligated with silk thread.

[0028] Two weeks after the mice were drugged to establish the model (fasting for 12 hours before sampling), the mice were anesthetized with 1.25% tribromoethanol. The right and left maxillae were dissected and fixed in 4% paraformaldehyde for Micro-CT detection and hematoxylin-eosin (HE) and tartrate-resistant acid phosphatase (TRAP) staining detection, respectively.

[0029] 1. Effects of isocinolone lactone on osteoclast activation

[0030] The TRAP working solution was prepared according to the instructions of the TRAP staining kit (Saiwell G1050-50T). Left maxillae from mice were decalcified, dehydrated, paraffin-embedded, and sectioned. The sections were stained with the TRAP working solution, and the nuclei were stained with hematoxylin. The sections were then mounted with neutral resin and observed using an optical microscope. Images were then taken using a microscopic imaging system. The results are shown below. Figure 1 As shown, it is used for TRAP staining analysis.

[0031] Depend on Figure 1 The results showed that, compared with the normal control group, the number and staining intensity of osteoclasts in the model group were significantly increased, indicating that osteoclasts were overactivated and mainly distributed in the alveolar bone margin and periodontal space, consistent with the bone metabolism characteristics of periodontitis, thus the periodontitis model was successfully established. Compared with the model group, the number of osteoclasts in the positive control, STR L, and STR H groups was significantly reduced, and the red signal was sparse, indicating that all three intervention methods could inhibit osteoclast activity and reduce bone resorption. Among them, the number and staining intensity of osteoclasts in the STR H group were close to those in the normal control group, almost completely inhibiting osteoclast activity, significantly blocking bone resorption, resulting in smooth and continuous alveolar bone margins, near disappearance of bone resorption lacunae, and restoration of bone structural integrity. The results indicate that isocynoplasmin intervention can regulate osteoclast activation and thus reduce the bone resorption process, with higher doses showing more significant effects.

[0032] 2. Effects of isovincinolactam on alveolar bone resorption

[0033] Right maxillae of mice from each group were numbered, fixed, and then subjected to Micro-CT scanning. Scanning parameters were set as follows: X-ray tube current 134uA, voltage 60kV, scanning the entire object at a resolution of 15.00μm. The original images were obtained after scanning. Selected regions were then reconstructed using 3D reconstruction software. The results are shown below. Figure 2 As shown in Table 1. Next, the bone biological parameters were analyzed. First, the distance from the cemento-cementum junction (CEJ) to the alveolar ridge crest (AC) was measured to reflect the degree of alveolar bone resorption. Then, the bone volume fraction was calculated by the ratio of bone volume (BV) to tissue volume (TV) to reflect the bone mass level. The results are shown in Table 1.

[0034] Table 1 Analysis of bone biological parameters of mouse maxilla

[0035]

[0036] Note: a) Compared with the normal control group, p<0.05; b) Compared with the model group, p<0.05.

[0037] As shown in Table 1, compared with the normal control group, the CEJ-AC value of the maxilla in the model group mice was significantly increased (P<0.05), and the BV / TV value was significantly decreased (P<0.05), indicating that the periodontitis model was successfully established. Compared with the model group, the CEJ-AC value of the positive control, STR L, and STR H groups was significantly decreased, and the BV / TV value was significantly increased (P<0.05). Among them, the STR H group showed the most significant improvement, with CEJ-AC decreasing to 174.06±16.06 mm and BV / TV increasing to 0.72±0.07, close to the level of the normal control group, indicating that isocinolone lactone can dose-dependently inhibit alveolar bone resorption and preserve bone mass.

[0038] Depend on Figure 2 The results showed that the alveolar bone structure around the second molar region of mice in the normal control group was intact, with minimal root exposure and normal periodontal space morphology, and no obvious bone resorption was observed. Compared with the normal control group, the alveolar ridge height around the molar region of the model group was significantly reduced, and root exposure was increased, exhibiting typical pathological features of alveolar bone resorption, indicating that the periodontitis model was successfully established. Compared with the model group, the positive control, STR L, and STR H groups showed significantly reduced bone resorption and some recovery in bone height. Among them, the STR H group showed the most significant effect, with alveolar bone morphology and bone mass approaching those of the normal control group, significantly reduced root exposure, and significantly inhibited bone resorption. The results indicate that isocinolone lactone intervention can reduce alveolar bone resorption and promote bone repair.

[0039] 3. Effects of isocinolone lactone on periodontal tissues

[0040] Left maxillae of mice from each group were decalcified, dehydrated, paraffin-embedded, embedded, sectioned, stained with hematoxylin and eosin (HE), dehydrated, mounted, and observed under an optical microscope. The stained images were then captured using a microscopic imaging system for pathological analysis.

[0041] The result is Figure 3 It was found that the periodontal tissue structure of mice in the normal control group was clear, the gingival epithelium was intact, there was no loss of attachment, no obvious inflammatory cell infiltration, the collagen fibers in the connective tissue were arranged in an orderly manner, and the alveolar crest height was normal. Compared with the normal control group, the model group showed a large number of inflammatory cell infiltrations, apical displacement of the junctional epithelium, disordered collagen fiber arrangement, reduced alveolar crest height, and obvious alveolar bone resorption, indicating that the mouse periodontitis model was successfully established. Compared with the model group, the positive control, STR L, and STR H groups all showed reduced periodontal lesions, reduced inflammatory cell infiltration, reduced collagen fiber degradation, and reduced alveolar bone resorption.

[0042] The STR H group showed the most significant effect, with both inflammatory infiltration and periodontal tissue structural integrity approaching those of the normal control group. The results indicate that isocinolone lactone intervention can reduce the degree of periodontal tissue damage.

[0043] The results showed that intervention with isocinolone lactone can regulate osteoclast activation, reduce bone resorption, and alleviate the degree of damage to periodontal tissues, thus fundamentally relieving the symptoms of periodontitis.

[0044] Although preferred embodiments of this application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this application.

[0045] Obviously, those skilled in the art can make various modifications and variations to this application without departing from the spirit and scope of this application. Therefore, if such modifications and variations fall within the scope of the claims of this application and their equivalents, this application also intends to include such modifications and variations.

Claims

1. The application of isocinolone lactone in the preparation of a drug for treating periodontitis, characterized in that, The active ingredient of the drug for treating periodontitis is isovinctin lactam; the isovinctin lactam is extracted from plants, Chinese medicinal materials or prepared slices of Chinese medicinal materials containing the ingredient; the active ingredient is combined with commonly used pharmaceutical excipients to form a commonly used clinical dosage form.

2. The application according to claim 1, characterized in that, The commonly used pharmaceutical excipients are one or more of the following: fillers, diluents, binders, disintegrants, emulsifiers, lubricants, and sweeteners.

3. The application according to claim 1, characterized in that, The commonly used clinical dosage forms are effervescent tablets, powders, tablets, capsules, sprays, pellets, or suspensions.

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