Composition, application thereof and product for preventing and treating periodontal diseases

Abstract

The invention relates to the technical field of medical health, in particular to a composition, application thereof and a product for preventing and treating periodontal diseases. The invention provides a composition capable of promoting alveolar bone regeneration, promoting fibroblast osteogenic differentiation and collagen synthesis, inhibiting and killing periodontitis pathogenic bacteria and stopping bleeding, the composition is composed of vanillic acid (VA) and cuttlebone, the mass ratio of the composition is 1: 1-1: 10, and the composition can be added into toothpaste and mouth wash to be used for preventing and treating periodontal diseases, and has the functions of preventing and treating periodontal diseases. The composition is especially used for preventing and treating periodontitis.

Description

Technical Field

[0001] The present invention relates to the field of medical health technology, and in particular to a composition and application thereof, and a product for preventing and treating periodontal diseases. Background Art

[0002] Periodontal disease is a common chronic inflammatory disease that affects nearly half of the world's population over 30 years old. In my country, 80% to 90% of adults suffer from periodontal disease to varying degrees. Among them, periodontitis has been identified by the World Health Organization as the third largest killer threatening human health after malignant tumors and cardiovascular and cerebrovascular diseases. Most periodontitis patients do not receive reasonable treatment.

[0003] Periodontal tissue regeneration is the ultimate goal of periodontitis prevention and treatment. Currently, there are few drugs for the treatment of periodontitis. There are only 6 types of oral drugs in the 2023 National Medical Insurance Catalogue, and there are only 3 drugs for periodontitis: oral regular release of furfural sterol, metronidazole and chlorhexidine topical liquid, which are rare. These drugs have bactericidal, anti-inflammatory and anti-gingival bleeding effects, and have a certain preventive and alleviating effect on periodontal diseases, but have no therapeutic effect on damaged periodontal tissues and alveolar bones.

[0004] In recent years, there are very few new drugs for the treatment of periodontitis on the market worldwide, with the exception of OraPharma Inc's minocycline ointment Arestin, which was approved by the US FDA on February 16, 2001. Other minocycline preparations on sale are Atridox, a gel from Atrix Pharmaceuticals in the United States, PerioChip from Astra Pharmaceuticals, and PerioStat from CollaGenex. In 2015, there was only one periodontitis drug that entered the top 500 best-selling drugs in the world, which was the periodontitis targeted drug minocycline ointment (Arestin) sold by OraPharma Inc / Valeant, with sales of US$151 million. According to the CFDA official website, on August 29, 2017, the minocycline ointment developed by Hainan Jianke Pharmaceuticals has obtained clinical approval (2017L03197) and entered the clinical trial stage. In addition, Hainan Jianke has developed a microcapsule made of sustained-release material to encapsulate minocycline. All of these new drugs on sale and in development are based on minocycline, a broad-spectrum antibiotic with pharmacological properties of anti-inflammatory, inhibition of collagenase activity, and promotion of periodontal tissue regeneration. However, minocycline has allergic reactions such as itching, urticaria, and exfoliative dermatitis, as well as gastrointestinal reactions such as vomiting and diarrhea. In addition, it has certain ototoxicity and can cause pigmentation involving the skin, nails, bones, and teeth, and these pigmentations are irreversible.

[0005] Therefore, the prior art still needs to be improved and developed. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a composition and its application, a product for preventing and treating periodontal diseases, aiming to solve the problem that existing drugs for treating periodontitis can cause allergic reactions, gastrointestinal reactions, etc.

[0007] The technical solution of the present invention is as follows:

[0008] A first aspect of the present invention provides a composition comprising vanillic acid and cuttlebone.

[0009] Optionally, the mass ratio of the vanillic acid to the cuttlebone is 1:1 to 1:10.

[0010] Optionally, the cuttlebone is a cuttlebone extract.

[0011] Optionally, the cuttlebone extract is obtained by extraction by the following method:

[0012] Washing and drying the cuttlebone, then crushing and sieving to obtain cuttlebone powder;

[0013] After soaking the cuttlebone powder in deionized water, heat reflux extraction for 1 to 4 hours, filtering, repeating the extraction 1 to 3 times, and combining the extracts;

[0014] The combined extracts are concentrated under reduced pressure and then freeze-dried to obtain a cuttlebone extract.

[0015] The second aspect of the present invention provides a use of the vanillic acid of the present invention in the preparation of a product for preventing and treating periodontal diseases.

[0016] Optionally, the product comprises the composition of the present invention.

[0017] Optionally, the periodontal disease is periodontitis.

[0018] Optionally, the product is toothpaste or mouthwash.

[0019] A third aspect of the present invention provides a product for preventing and treating periodontal diseases, wherein the product comprises vanillic acid.

[0020] Optionally, the product comprises the composition of the present invention.

[0021] Optionally, the periodontal disease is periodontitis.

[0022] Optionally, the product is toothpaste or mouthwash.

[0023] Beneficial effects: The present invention provides a composition that can promote alveolar bone regeneration, promote osteogenic differentiation of fibroblasts and collagen synthesis, inhibit and kill periodontitis pathogens, and has hemostatic function. The composition consists of vanillic acid (VA) and cuttlebone, and can be added to toothpaste and mouthwash for the prevention and treatment of periodontal diseases, especially for the treatment of periodontitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a test result of the antibacterial effect of vanillic acid on the periodontitis pathogen Fusobacterium nucleatum (Fn).

[0025] Figure 2 This is a test result of the bactericidal effect of vanillic acid on the periodontitis pathogen Fusobacterium nucleatum (Fn).

[0026] Figure 3 This is a graph showing the antibacterial effect test results of vanillic acid on the periodontitis pathogen Porphyromonas gingivalis (Pg).

[0027] Figure 4 This is a graph showing the bactericidal effect test results of vanillic acid on the periodontitis pathogen Porphyromonas gingivalis (Pg).

[0028] Figure 5 This is a graph showing the antibacterial effect test results of vanillic acid on the periodontitis pathogen Actinomyces viscosus (Av).

[0029] Figure 6 This is a graph showing the results of a test on the activity of fibroblast L929 cells in which vanillic acid reverses LPS inflammatory damage.

[0030] Figure 7 This is a graph showing the test results of fibroblast collagen I synthesis at different times.

[0031] Figure 8 This is a graph showing the results of alkaline phosphatase activity testing of fibroblasts after osteogenic differentiation at different times.

[0032] Fig. 9 This is the result of periodontal pocket depth test.

[0033] Fig.10 This is the alveolar bone parameter test result diagram. DETAILED DESCRIPTION

[0034] The present invention provides a composition and its application, and a product for preventing and treating periodontal diseases. In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0035] The embodiment of the present invention provides a composition, which includes vanillic acid and cuttlebone. Preferably, the composition consists of vanillic acid and cuttlebone.

[0036] In an embodiment of the present invention, the composition has the following technical effects:

[0037] 1) Vanillic acid has antibacterial and bactericidal activity against periodontitis pathogens;

[0038] 2) Vanillic acid promotes the transformation of fibroblasts into osteoblasts;

[0039] 3) Vanillic acid promotes collagen synthesis in fibroblasts;

[0040] 4) Vanillic acid promotes alveolar bone regeneration and reverses alveolar bone loss;

[0041] 5) The combination of vanillic acid and cuttlebone (VS) has a rapid hemostatic effect.

[0042] Among them, vanillic acid and cuttlebone are both commercially available.

[0043] In one embodiment, the mass ratio of vanillic acid to cuttlebone is 1:1 to 1:10, such as 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, etc. The composition can effectively promote alveolar bone regeneration, promote osteogenic differentiation of fibroblasts and collagen synthesis, inhibit and kill periodontitis pathogens, and significantly reduce coagulation time, having a significant hemostatic effect.

[0044] In one embodiment, the cuttlebone is a cuttlebone extract.

[0045] In one embodiment, the cuttlebone extract is obtained by the following method:

[0046] Washing and drying the cuttlebone, then crushing and sieving to obtain cuttlebone powder;

[0047] After soaking the cuttlebone powder in deionized water, extracting it by heat reflux for 1 to 4 hours (such as 1 hour, 2 hours or 4 hours), filtering, repeating the extraction for 1 to 3 times (such as 1 time, 2 times or 3 times), and combining the extracts;

[0048] The combined extracts are concentrated under reduced pressure and then freeze-dried to obtain a cuttlebone extract.

[0049] The embodiment of the present invention provides an application of vanillic acid in preparing a product for preventing and treating periodontal diseases.

[0050] In one embodiment, the product comprises the composition, that is, the composition comprises vanillic acid and cuttlebone. Other details of the composition are as described above and will not be repeated here.

[0051] In one embodiment, the periodontal disease is periodontitis.

[0052] In one embodiment, the product is a toothpaste or a mouthwash.

[0053] An embodiment of the present invention provides a product for preventing and treating periodontal diseases, wherein the product includes vanillic acid.

[0054] In one embodiment, the product comprises the composition, that is, the composition comprises vanillic acid and cuttlebone. Other details of the composition are as described above and will not be repeated here.

[0055] In the embodiments of the present invention, the vanillic acid small molecule has the effect of inhibiting and killing periodontitis pathogens, and also has the activity of promoting osteogenic differentiation of fibroblasts and collagen synthesis, and promoting the regeneration of periodontal tissues. Cuttlebone has a hemostatic effect. After using the product containing vanillic acid and cuttlebone, it can play a good preventive and therapeutic role in the prevention stage before the occurrence of periodontitis and in the treatment stage after the occurrence.

[0056] In one embodiment, the periodontal disease is periodontitis.

[0057] In one embodiment, the product is toothpaste or mouthwash. When the product is toothpaste, the mass content of the composition in the toothpaste is 0.1% to 50%, such as 0.1%, 0.5%, 1%, 10%, 20%, 30%, 40%, 50%, etc. When the product is mouthwash, the mass content of the composition in the mouthwash is 0.1% to 30%, such as 0.1%, 0.5%, 1%, 10%, 20%, 30%, etc.

[0058] The present invention will be further described below by means of specific examples.

[0059] The activated periodontitis pathogen Fusobacterium nucleatum (Fn, BNCC 280188) was cultured to a McFarland turbidity of 0.5, and then diluted 100 times to obtain a bacterial suspension for use. Vanillic acid was dissolved in liquid thioglycollate medium by a two-fold dilution method to make the final concentration of vanillic acid reach 10, 8, 6, 5, 4, 3, 2, 1, 0.5, 0.2, and 0.1 mg / mL. The bacterial suspension and vanillic acid (VA) solution of different concentrations were inoculated in a 96-well plate at a volume ratio of 1:1 (V:V), and 100 μL of each was inoculated in the plate. The plate was cultured at 95% N2, 5% CO2, and 37°C for 48 hours. The OD600nm was measured by an enzyme reader. According to the standards of the American Clinical Laboratory Standards Institute (CLSI), the minimum concentration at which the bacteria in the experimental group were reduced by more than 80% compared with the blank group was the minimum inhibitory concentration (MIC). MIC is the lowest concentration of an antibacterial agent that prevents visible growth of microorganisms. Figure 1 The experimental results showed that compared with the blank group, VA concentrations higher than 0.25 mg / mL could significantly inhibit the proliferation of Fn bacteria. Compared with the blank group, the minimum inhibitory concentration of VA to reduce Fn bacteria by 80% was 1.5 mg / mL.

[0060] The culture of the experimental group with a concentration greater than MIC after 48 hours of culture was spread on a solid plate of Fn bacteria and cultured under 95% N2, 5% CO2, 37°C for 48 hours. The number of colonies was observed. The lowest drug concentration corresponding to less than 5 colonies was the minimum bactericidal concentration MBC. MBC reflects the lowest level of antimicrobial concentration that causes microbial death. If MBC does not exceed four times the MIC, the antimicrobial is generally considered to have a bactericidal effect. Figure 2 The results showed that the MBC of VA against Fn was 2.5 mg / mL, which was less than four times the MIC. Therefore, it was determined that VA had a bactericidal effect on the periodontitis pathogen Fn.

[0061] The same method as that for Fn bacteria was used to culture the periodontitis pathogen Porphyromonas gingivalis (Pg, ATCC33277) and determine the MIC. Figure 3 The results showed that the minimum inhibitory concentration of VA to reduce Pg bacteria by 80% was 2 mg / mL.

[0062] The minimum bactericidal concentration (MBC) of Pg was tested using the same method as that for Fn bacteria. Figure 4 The results showed that the MBC of VA against Pg was 3 mg / mL, which was less than four times the MIC, confirming that VA had a bactericidal effect against the periodontitis pathogen Pg.

[0063] The same method as for Fn bacteria was used to culture Actinomyces viscosus (Av, ATCC19246), the pathogenic bacteria of periodontitis, but the culture time was changed to 24 hours, and the MIC was determined. Figure 5 The results showed that the MIC of VA against Av was 2.5 mg / mL.

[0064] Fibroblast L929 is a cell line used to study the function of gingival cells. Lipopolysaccharide (LPS) can induce cell inflammation. L929 cells (6×10 4 Cells were inoculated in 24-well plates, and then cultured in MEM medium + 10% serum + 1% double antibody basal medium for 5 hours. After starvation treatment for 24 hours (replaced with serum-free basal medium), the basal medium + LPS 100 μg / mL and different concentrations of VA were replaced and cultured for 48 hours. The cell proliferation activity was determined using the CCK8 kit. The test results are as follows Figure 6 As shown, Figure 6 NC refers to the negative control (without LPS and VA), LPS was added to the LPS group but not VA, and LPS and VA were added to the VA group. Figure 6 As shown, LPS inhibited the proliferation of L929 cells, and vanillic acid (VA) was administered for 10 -9 and 10 -8 M can significantly reverse the damage caused by LPS and increase the proliferation activity of L929 cells, indicating that vanillic acid has regenerative activity on gingival cells damaged by inflammation.

[0065] The same culture, starvation treatment and drug administration as above were used. After 48 hours, the cells were collected and RNA was extracted according to the operating method of the Trizol instruction manual. The concentration and purity of the RNA were recorded. 1μg of RNA was transcribed into cDNA. The Real-time PCR reaction mixture consisted of 10μl SsoFastTM Eva Green supermix, 0.5μl cDNA, primers and water. The amplification reaction was carried out in the following process: 98℃ for 2 minutes, 98℃ for 10 seconds, 60℃ for 15 seconds, 72℃ for 15 seconds, 35 cycles, and final extension at 72℃ for 5 minutes. The experimental results are shown in Figure 7 LPS inhibited the production of type I collagen (COL1) in L929 cells, and vanillic acid (VA) 10 -8 and 10 -7 M can significantly increase the production of COL1, indicating that vanillic acid has regenerative activity on periodontal collagen fibers damaged by inflammation.

[0066] Fibroblast L929 cells were cultured and starved as described above. After 24 hours of starvation, they were co-cultured with osteogenic induction medium + LPS and VA for 7, 14, and 21 days, respectively. Alkaline phosphatase (ALP) activity was measured using a kit. ALP is an indicator of osteoblast differentiation. Figure 8 As shown, the NC group was the negative control (without LPS and VA), the LPS group was added with LPS, and the VA group was added with different concentrations of VA and LPS. LPS-induced inflammation has the effect of inhibiting ALP activity. -8 and 10 -7 M can promote ALP activity, indicating that VA has the effect of reversing inflammation-inhibited osteogenesis and promoting bone formation.

[0067] The cuttlebone was washed, dried, crushed, and passed through a 40-mesh sieve to obtain cuttlebone powder. The cuttlebone powder was soaked in deionized water, extracted by hot reflux for 2 hours, filtered, and extracted once, the extracts were combined, concentrated under reduced pressure, and freeze-dried to obtain a cuttlebone extract. Vanillic acid and cuttlebone extract were mixed in a mass ratio of 1:1, 1:5, and 1:10 to prepare a composition (VS). Fresh blood was collected from healthy rats to prepare sodium citrate anticoagulated blood (V / V, blood: sodium citrate = 9:1). First add 0.2mL of VS solution of different concentrations (preheated at 37°C) to the centrifuge tube, then add 0.2mL of calcium chloride solution (preheated at 37°C), mix well, then add 0.5mL of sodium citrate anticoagulated blood (preheated at 37°C), and start timing immediately after mixing. Observe whether there is coagulation every 5 seconds, stop timing when the blood coagulates, determine the coagulation time, repeat 3 times, and take the average value. Use physiological saline as blank control and Yunnan Baiyao as positive control. The experimental results are shown in Table 1. The composite VS can significantly reduce the coagulation time at doses of 1:1, 1:5 and 1:10, and has a significant hemostatic effect.

[0068] Table 1. Effect of the composition on coagulation time

[0069]

[0070] Drug-loaded hydrogel:

[0071] (1) Preparation of hydrogel

[0072] 2-Methacrylic acid and heptane were uniformly mixed at a volume ratio of 1:1, and then 50% (w / v) NaOH solution was added at 55°C. After the reaction was fully dissolved, an appropriate amount of hydroquinone was added to the system, and then purified by vacuum distillation. The purified product was mixed with pre-dried hydroquinone, tetrabutylammonium bromide and propylene oxide, reacted at 90°C for 4 hours, and then cooled to room temperature. The crude reaction mixture was filtered, and the filtrate was further purified by vacuum distillation until a colorless and transparent flowing liquid was obtained, that is, component A.

[0073] Component A and hydroxypropyl methylcellulose (HPMC) were added to the DMSO solution and reacted under inert gas for 72 hours. Afterwards, the reaction mixture was dialyzed with deionized water to remove impurities, and the dry product was obtained by freeze-drying, that is, the hydrogel, and stored at room temperature for subsequent use.

[0074] (2) Preparation of drug-loaded hydrogel

[0075] 1 mg of vanillic acid was dissolved in DMSO, and then water was added to dilute the vanillic acid aqueous solution with a DMSO concentration of 1 mg / mL, and then the cuttlebone extract was added (the mass ratio of vanillic acid to cuttlebone extract was 1:1), and mixed evenly to obtain a VS aqueous solution; the hydrogel prepared in step (1) was dissolved in the VS aqueous solution to make the mass concentration of the hydrogel 50 mg / mL, and the drug-loaded hydrogel was obtained after gelation and filtration.

[0076] (3) Pharmacological effects of drug-loaded hydrogel on rats with periodontitis

[0077] Sprague-Dawley (SD) rats were anesthetized with Zota 50 (75 mg / kg, intraperitoneal injection), and a ligature was used to wrap around the neck of the left maxillary second molar. The ligature was pressed into the gingival sulcus. On the first day (D1), the third day (D3), and the fifth day (D5), 10 mL of LPS (1 mg / mL) was injected into the gingival sulcus on the buccal and palatal sides between the left maxillary first and second molars of the rats. The model was established for 2 weeks, and the animals were given 5% glucose drinking water during the modeling period. The success of the model was determined based on periodontal probing. After the periodontitis model was successfully established, saline (Control), blank gel (BG), positive control minocycline (MC, 20 mg / mL), VS hydrogel low-dose group (VSL, 1 mg / mL), and VS hydrogel high-dose group (VSH, 2.5 mg / mL) were injected into the periodontal pocket, respectively, and the drug was administered 2 days a week for 4 weeks.

[0078] After the medication was completed, the periodontal pocket depth (Pocket Depth, PD) was measured. The test results are shown in Fig. 9 ,from Fig. 9 It can be seen that VSL and VSH can significantly reduce the depth of periodontal pockets, and their effects are stronger than the positive control minocycline.

[0079] After the administration, micro-CT was used to measure various parameters of the alveolar bone. The test results are shown in Fig.10 , Fig.10BMD is bone mineral density, BV / TV refers to bone volume / tissue volume, and CEJ-ABC distance refers to the distance from the alveolar bone ridge to the cementum junction. The positive control (MC) and VSH groups can significantly improve the bone loss caused by modeling, increase alveolar bone density and bone volume, and reduce CEJ-ABC distance. Its effect is close to or stronger than minocycline. This shows that VS hydrogel has the pharmacological effect of regenerating lost alveolar bone.

[0080] In summary, the present invention provides a composition and its application, and a product for preventing and treating periodontal diseases. The present invention provides a composition that can promote alveolar bone regeneration, promote fibroblast osteogenic differentiation and collagen synthesis, inhibit and kill periodontitis pathogens, and has hemostatic function. The composition is composed of vanillic acid (VA) and cuttlebone, and the mass ratio of the composition is 1:1 to 1:10. The composition can be added to toothpaste (0.1% to 50%) and mouthwash (0.1% to 30%) for the prevention of periodontal diseases. It can also be prepared with drug-carrying materials such as hydrogels for the treatment of periodontal diseases, especially for the treatment of periodontitis.

[0081] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A composition, characterized in that Includes vanillic acid and cuttlefish.

2. The composition according to claim 1, characterized in that The mass ratio of the vanillic acid to the cuttlebone is 1:1 to 1:

10.

3. The composition according to claim 1, characterized in that The cuttlebone is a cuttlebone extract.

4. The composition according to claim 3, characterized in that The cuttlebone extract is obtained by the following method: Washing and drying the cuttlebone, then crushing and sieving to obtain cuttlebone powder; After soaking the cuttlebone powder in deionized water, heat reflux extraction for 1 to 4 hours, filtering, repeating the extraction 1 to 3 times, and combining the extracts; The combined extracts were concentrated under reduced pressure and then freeze-dried to obtain a cuttlebone extract.

5. Use of vanillic acid in the preparation of products for preventing and treating periodontal diseases.

6. The use according to claim 5, characterized in that: The product comprises the composition according to any one of claims 1 to 4.

7. The use according to claim 5, characterized in that: The periodontal disease is periodontitis.

8. The use according to claim 5, characterized in that: The product is toothpaste or mouthwash.

9. A product for preventing and treating periodontal diseases, characterized in that: The product includes vanillic acid.

10. The product for preventing and treating periodontal diseases according to claim 9, characterized in that: The product comprises the composition according to any one of claims 1 to 4; The periodontal disease is periodontitis; The product is toothpaste or mouthwash.

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