CN107496427B

CN107496427B - Application of evodiamine, mouthwash and preparation method of mouthwash

Info

Publication number: CN107496427B
Application number: CN201710932784.5A
Authority: CN
Inventors: 陈行平
Original assignee: Shenzhen Xinhongmei Medical Apparatus And Instruments Co ltd
Current assignee: Shenzhen Xinhongmei Medical Apparatus And Instruments Co ltd
Priority date: 2017-10-10
Filing date: 2017-10-10
Publication date: 2020-07-14
Anticipated expiration: 2037-10-10
Also published as: CN107496427A

Abstract

The invention provides application of evodiamine, mouthwash and a preparation method of the mouthwash, and belongs to the field of medicines. The application of the evodiamine comprises the following steps: the application of evodiamine in preparing a medicament for preventing or treating periodontitis; the application of evodiamine in preparing an inhibitor for periodontal ligament stem cell inflammatory reaction; application of evodiamine in preparing osteoclast differentiation and maturation inhibitor is provided. The mouthwash for treating periodontitis comprises the following raw materials: evodiamine, a surfactant, a hydrophilic dispersant and a lipophilic dispersant. The preparation method of the mouthwash comprises the following steps: mixing and dispersing evodiamine, a surfactant, a hydrophilic dispersant and a lipophilic dispersant in water, and stirring at 80-85 ℃. The invention provides a new idea for preventing or treating periodontitis and also expands the application range of evodiamine. In addition, the mouth wash prepared from the evodiamine can effectively prevent and treat periodontitis.

Description

Application of evodiamine, mouthwash and preparation method of mouthwash

Technical Field

The invention relates to the field of medicine and health care, and particularly relates to application of evodiamine, mouthwash and a preparation method of the mouthwash.

Background

Periodontitis is a chronic inflammation that invades the gingiva and periodontal tissue, and is a destructive disease mainly characterized by the formation of periodontal pockets and inflammation of pocket walls, alveolar bone resorption and gradual loosening of teeth, which are major causes of tooth loss in adults. The disease is mainly caused by bacterial plaque, dental calculus, food impaction, bad restoration, bite wound and the like, and the gingiva is inflamed and swollen, and meanwhile, the bacterial plaque is accumulated and extends from the upper part of the gingiva to the lower part of the gingiva. Because of the characteristics of the subgingival micro-ecological environment, a large number of periodontal pathogenic bacteria with high toxicity, such as gingival bacteroides, intermediate bacteroides, spirochetes and the like, are bred in the subgingival bacterial plaque, so that the inflammation of gingiva is aggravated and expanded, periodontal pockets are formed, alveolar bone is absorbed, and periodontitis is caused.

Periodontitis has been defined by medical theory as the third largest killer threatening human health, following cancer, cardiovascular and cerebrovascular diseases, and also as the "first killer" for oral health. Some of the initial symptoms of periodontitis also become a major feature of oral "sub-health" exacerbations.

Most of patients with periodontitis are accompanied by symptoms of gingival atrophy, and more than 90% of people over 45 years old have different degrees of gingival atrophy, and the gingival atrophy is also regarded as an irreversible physiological degeneration phenomenon by the oral medical community. It is important to note that the physiological degeneration of gingival atrophy tends to be age-reduced year by year due to genetic, environmental and food safety factors, and the youngest patients with gingival atrophy are 18 years old.

The existing treatment methods for periodontitis mainly comprise scaling treatment and antibiotic treatment, wherein the antibiotic treatment comprises systemic administration (oral administration and injection) and periodontal local administration, and has the disadvantages of great side effect and poor treatment effect. Based on this, there is an urgent need for a technique with strong universality for preventing or treating increasingly severe periodontitis.

Disclosure of Invention

The first purpose of the invention is to provide a new application of evodiamine, namely the application of the evodiamine in preparing a medicament for preventing or treating periodontitis; the application of evodiamine in preparing an inhibitor for periodontal ligament stem cell inflammatory reaction; the application of the evodiamine in preparing an osteoclast differentiation and maturation inhibitor provides a new idea for preventing or treating periodontitis and expands the application range of the evodiamine.

The second purpose of the invention is to provide a mouthwash for treating periodontitis and a preparation method thereof, wherein the mouthwash takes evodiamine as an effective component, can effectively prevent and treat periodontal diseases, avoids tooth loosening and has a good treatment effect.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

application of evodiamine in preparing medicine for preventing or treating periodontitis is provided.

Application of evodiamine in preparing inhibitor for periodontal ligament stem cell inflammatory reaction is provided.

Application of evodiamine in preparing osteoclast differentiation and maturation inhibitor is provided.

A mouthwash for treating periodontitis comprises the following raw materials: evodiamine, a surfactant, a hydrophilic dispersant and a lipophilic dispersant.

A method of preparing a mouthwash for the treatment of periodontitis, comprising:

mixing and dispersing evodiamine, a surfactant, a hydrophilic dispersant and a lipophilic dispersant in water, and stirring for 20-30 min at 80-85 ℃.

Compared with the prior art, the beneficial effects of the invention comprise:

the inventor researches and discovers that the evodiamine can be used for preventing and treating periodontitis mainly through the following two aspects that the evodiamine can effectively inhibit the generation of inflammatory reaction of periodontal ligament stem cells so as to inhibit the expression of interleukin-6, interleukin-1 and tumor necrosis factor- α, so that the evodiamine can be used for preparing the inhibitor of the inflammatory reaction of the periodontal ligament stem cells, and the evodiamine can effectively inhibit the differentiation and maturation of osteoclasts so as to remarkably inhibit the symptom of excessive absorption of alveolar bones caused by the activation of the osteoclasts, so that the evodiamine can be used for preparing the inhibitor of the differentiation and maturation of the osteoclasts.

The mouthwash for treating periodontitis and the preparation method thereof provided by the disclosure are prepared by uniformly dispersing evodiamine serving as a medicinal component by using a surfactant, a hydrophilic dispersant and a lipophilic dispersant, and can effectively prevent and treat periodontitis.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a graph showing the results of an experiment in which the concentration of evodiamine is dependent on the osteoclast formation inhibition;

FIG. 2 is a statistical graph of the number of osteoclasts in FIG. 1;

FIG. 3 is a statistical plot of the coverage area of osteoclasts in FIG. 1;

FIG. 4 is a graph showing the results of an evodiamine time-dependent osteoclast formation inhibition experiment;

FIG. 5 is a statistical graph of the number of osteoclasts in FIG. 2;

FIG. 6 is a statistical graph of the coverage area of osteoclasts in FIG. 2.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The embodiment provides an application of evodiamine in preparation of a medicine for preventing or treating periodontitis.

Stem cells in the healing and regeneration of periodontal tissue is mainly composed of gingiva, periodontal ligament, cementum and alveolar bone, and this complex tissue can heal in a special way after the periodontal tissue is injured. The periodontal ligament has important mission in the regeneration process, and research shows that a group of mesenchymal stem cells with repair function near the periodontal ligament have wide proliferation and differentiation potential and can be differentiated into new tooth bad bones and periodontal membranes. However, recent studies have found that this population of cells is called periodontal ligament stem cells (PDSC), a healthy double-edged sword. Can be differentiated into periodontal tissue under normal physiological condition for periodontal repair, and can also secrete large amount of inflammatory factors under pathological condition, activate periodontal mononuclear macrophage, promote excessive osteoclast formation, cause tooth bad bone absorption and periodontal pocket formation, and cause and aggravate periodontitis.

Based on this, the key point of preventing or treating periodontitis is how to effectively avoid excessive secretion of inflammatory factors by periodontal ligament stem cells under pathological conditions. The inventor finds that the evodiamine which is a natural monomer compound can effectively prevent or treat periodontitis in long-term scientific research work, so that the evodiamine can be used for preparing medicines for preventing or treating periodontitis.

Evodiamine (EVO) is an indole alkaloid and is colorless or light yellow crystal. The molecular formula of the evodiamine is C₁₉H₁₇N₃O, molecular weight 303.36, CAS No. 518-17-25. The literature reports that the evodiamine has the activity of resisting tumors and vascular proliferation.

The structural formula is as follows:

the inventor researches and discovers that the evodiamine is mainly used for preventing and treating periodontitis through the following two aspects:

in the first aspect, evodiamine can effectively inhibit the generation of inflammatory reaction of periodontal ligament stem cells.

The research shows that L PS can initiate the synthesis and release of various inflammatory mediators and cytokines, such as interleukin-1 (I L-1), interleukin-6 (I L-6), tumor necrosis factor- α (TNF- α) and the like, when human periodontal ligament stem cells (human periodontal ligament stem cells, hPD L SCs) are stimulated by L PS, the human periodontal ligament stem cells can participate in local immune response by secreting cytokines such as TNF- α, I L-6, I L-1 and the like as immune auxiliary cells and play an important role in the generation and development of periodontitis.

The inventor researches and discovers that evodiamine can inhibit the expression increase of mRNA (messenger ribonucleic acid) I L-6, I L-1 and TNF- α caused by L PS (Poly-N-phenylethanoid), and indicates that the evodiamine can inhibit inflammatory reaction of periodontal ligament stem cells by inhibiting inflammatory factors generated by the periodontal ligament stem cells, so that the evodiamine can be used for preparing the inhibitor of the inflammatory reaction of the periodontal ligament stem cells.

In a second aspect, evodiamine is effective in inhibiting osteoclast differentiation and maturation.

The periodontal ligament stem cell is stimulated by periodontal bacteria, and the generation of a large amount of inflammatory factors does not cause serious periodontal disease, and only after the generation of inflammatory factors activates osteoclasts in a large amount, the alveolar bone is excessively absorbed, so that serious periodontal disease and tooth loosening are caused.

The inventor researches and discovers that the number of mature osteoclast is obviously reduced after different dosages of evodiamine are added, and the evodiamine is indicated to remarkably inhibit the alveolar bone resorption symptom caused by osteoclast activation, so that the evodiamine can be used for preparing the osteoclast differentiation and maturation inhibitor.

The present embodiment also provides a mouthwash for treating periodontitis, which comprises the following raw materials: evodiamine, a surfactant, a hydrophilic dispersant and a lipophilic dispersant.

Further, the hydrophilic dispersant includes: dideethanol, sodium monofluorophosphate and chlorhexidine gluconate.

Further, the lipophilic dispersing agent includes: glycerol, polyoxyethylene chlorinated castor oil, and polyoxyethylene hydrogenated castor oil.

Further, the surfactant includes: sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, polysorbate, sorbitan fatty acid, fatty glyceride and lecithin.

Optionally, the mouthwash comprises the following raw materials in parts by weight: 4-6 parts of evodiamine, 0.5-1.5 parts of surfactant, 8.1-12.4 parts of hydrophilic dispersant and 3.5-8.5 parts of lipophilic dispersant.

Optionally, the mouthwash comprises the following raw materials in parts by weight: 4-6 parts of evodiamine, 0.5-1.5 parts of sodium dodecyl sulfate, 8-12 parts of dideoxyethanol, 0.03-0.07 part of sodium monofluorophosphate, 0.1-0.3 part of chlorhexidine gluconate, 3-7 parts of glycerol and 0.5-1.5 parts of polyoxyethylene chlorinated castor oil.

Or the mouthwash comprises the following raw materials in parts by weight: 5 parts of evodiamine, 1 part of lauryl sodium sulfate, 10 parts of didethanol, 0.05 part of sodium monofluorophosphate, 0.2 part of chlorhexidine gluconate, 5 parts of glycerol and 1 part of polyoxyethylene castor oil chloride.

The preparation method of the mouthwash for treating periodontitis comprises the following steps:

Optionally, the method further comprises adjusting the pH value of the reaction solution to 5.5-6.5 after the reaction is completed. Preferably, the pH is adjusted with baking soda or ammonia.

The mouthwash for treating periodontitis and the preparation method thereof are characterized in that evodiamine is used as an effective component and is uniformly dispersed by utilizing a surfactant, a hydrophilic dispersant and a lipophilic dispersant to prepare the mouthwash, so that periodontitis can be effectively prevented and treated.

The features and properties of the present invention are further described in detail below with reference to examples:

example 1

The embodiment provides a mouthwash for treating periodontitis, which is prepared by a method comprising the following steps:

a. preparing materials: 5g of evodiamine, 1g of lauryl sodium sulfate, 10g of dideethanol, 0.05g of sodium monofluorophosphate, 0.2g of chlorhexidine gluconate, 5g of glycerol and 1g of polyoxyethylene castor oil chloride.

b. And (3) emulsion reaction: mixing the above materials, dispersing in 80g purified water, stirring at 82 deg.C for 25min, adjusting pH to 6.0 with alkali, filtering, and making into collutory.

Example 2

a. preparing materials: 4g of evodiamine, 1.5g of polysorbate, 8g of didethanol, 0.03 g of sodium monofluorophosphate, 0.3g of chlorhexidine gluconate, 3g of glycerol and 0.5g of polyoxyethylene castor oil chloride.

b. And (3) emulsion reaction: mixing the above materials, dispersing in 80g purified water, stirring at 80 deg.C for 30min, adjusting pH to 6.5 with alkali, and filtering to obtain collutory.

Example 3

a. preparing materials: 6g of evodiamine, 1.5g of sorbitan fatty acid, 12g of dideoxyethanol, 0.03 g of sodium monofluorophosphate, 0.1g of chlorhexidine gluconate, 7g of glycerol and 1.5g of polyoxyethylene chlorinated castor oil.

b. And (3) emulsion reaction: mixing the above materials, dispersing in 80g purified water, stirring at 85 deg.C for 20min, adjusting pH to 5.5 with alkali, and filtering to obtain collutory.

Example 4

a. preparing materials: 5g of evodiamine, 1g of sodium dodecyl benzene sulfonate, 12g of dideethanol, 0.03 g of sodium monofluorophosphate, 0.36g of chlorhexidine gluconate, 5g of glycerol and 1g of polyoxyethylene hydrogenated castor oil.

Example 5

a. preparing materials: 5g of evodiamine, 1g of polysorbate, 8g of dideethanol, 0.03 g of sodium monofluorophosphate, 0.07g of chlorhexidine gluconate, 3g of glycerol and 1g of polyoxyethylene hydrogenated castor oil.

The following data are combined to evaluate the effect of evodiamine in preventing or treating periodontitis.

Experimental example 1

Effect of evodiamine on periodontitis:

firstly, an experimental process:

1.1 Primary reagents and instruments

250 g/L trypsin, collagenase, α -MEM medium (GibcoBR L, USA), Porphyromonas gingivalis L PS (Sigma, USA), reverse transcription polymerase chain reaction (RT-PCR) kit, cell total RNA extraction kit, reverse transcription kit (Takara, Japan), enzyme linked immunosorbent assay (BIO-TEK, USA), inverted microscope and photographic system (Olympus, Japan), RT-PCR instrument (Applied Biosystems, USA)

1.2 method for isolating periodontal ligament Stem cells

18 healthy premolar teeth extracted by orthodontic subtraction from 12-15-year-old volunteers (informed by both patients and parents) are collected, washed repeatedly with PBS buffer immediately, the periodontal membrane tissue of 1/3 in roots is scraped and transferred to a sterile culture dish with a proper amount of α -MEM culture solution, then the periodontal membrane tissue is cut into tissue blocks of 1mm × 1mm × 1mm under the soaking of α -MEM culture solution and digested for 15min with 250 g/L of trypsin.

After digestion is stopped, the supernatant is centrifuged and discarded, the separated tissue blocks are evenly spread in a 6-well plate, sterile cover glass is used for covering the tissue blocks to adhere to the walls, a proper amount of α -MEM culture solution containing 100m L/L fetal calf serum is added into each well, and the mixture is placed in a 50m L/L CO₂The culture was carried out in an incubator at 37 ℃. Changing the liquid every 3d for 1 time, adopting STRO-1 as a marker when the cells grow to 80 percent, separating and screening the human periodontal ligament stem cells by immunomagnetic beads, and carrying out conventional subculture.

Second, test method

2.1 MTT method for determining influence of evodiamine on proliferation of periodontal ligament stem cells

Collecting the 4 th generation human periodontal ligament stem cell with good growth state, digesting with trypsin, and making into the extract with α -MEM to density of 5 × 10⁷The cells are inoculated into a 96-well plate in 200 mu L per well after being suspended in L, after culturing for 24h under a standard environment, the original culture solution is discarded, and the cells are randomly divided into 5 groups of A, B, C, D and E, wherein L3-MEM containing evodiamine 1 mu g/m L0 + L1 PS 10ng/m L2 is added into the group A, L6-MEM containing evodiamine 3 mu g/m L4 + L5 PS 10ng/m L7 is added into the group B, L8-MEM containing evodiamine 6 mu g/m L9 + L PS 10ng/m L is added into the group C, α -MEM containing L PS 10ng/m L is added into the group D, and α -MEM (200 mu L per well) containing no L PS is added into the group E to continue culturing.

The cells of each group are taken at each time point of 12 hours, 24 hours, 48 hours and 72 hours of culture (4 holes are taken at each time point of each group), 20 mu L MTT solution is added into each hole for standing culture for 4 hours, supernatant in the holes is carefully absorbed, 150 mu L DMSO is added into each hole respectively, the mixture is shaken for 15 minutes at room temperature, then, the absorbance (A490) value at 490nm wavelength of each hole is detected by a microplate reader respectively, and the average value of the 4 holes is taken for statistical analysis.

2.2 inhibitory Effect of Evodiamine on L PS-induced inflammatory reaction of periodontal Membrane Stem cells

The well-grown 4 th generation human periodontal stem cells were collected and subjected to grouping treatment and culture in the same manner as in 2.1. After continuous culture for 72h, cellular RNA was extracted by one step using Trizol kit, and the RNA was reverse transcribed into cDNA by random primer method according to the reverse transcription kit instructions. Primers were designed using Primer 5.0 computer software according to GenBank database and Primer synthesis was performed by Shanghai Biotech, and the sequences of the primers are shown in Table 1.

TABLE 1 primer sequences

Then using cDNA as template and β -actin as internal reference, using RT-PCR instrument to make PCR reaction, its reaction condition is that pre-denaturation at 94 deg.C for 4min, denaturation at 94 deg.C for 20s, annealing at 60 deg.C for 30s and extension at 72 deg.C for 30s, and making 35 cycles, and finally detecting signal at 72 deg.C, after the PCR reaction is finished, directly obtaining threshold value Ct on PCR instrument, and adopting 2^-△△ctThe relative expression level of mRNA of I L-6, I L-1 β and TNF- α in each group was calculated, 5 parallel multiple wells were set for each group, and the average value was taken.

2.3 Effect of Evodiamine on osteoclast differentiation and maturation.

According to research, periodontal mononuclear macrophages can differentiate towards osteoclasts under the stimulation induction of a nuclear factor receptor activating factor ligand RANK L and a human macrophage colony stimulating factor.

The observation of whether the sophoridine can inhibit the expression of specific related genes of osteoclasts and the influence of the sophoridine on the osteoclast bone-eating capability is carried out, so that the application and the mechanism of the sophoridine in preparing the medicament for preventing and treating osteoporosis are explained.

To show that the sophoridine of the present invention can inhibit mouse bone marrow macrophages from differentiating into osteoclasts, mouse bone marrow macrophages are now replaced with 1 × 10⁴The cells/cell are inoculated in a 96-well plate, the culture medium is discarded after the cells grow full, evodiamine is added into each fresh culture medium at final concentrations of 0 (blank control group), 5 mu g/M L, 10 mu g/M L and 15 mu g/M L respectively, then the evodiamine is added into the 96-well plate, and simultaneously RANK L (50ng/M L) and M-CSF (50ng/M L) induce the mouse bone marrow macrophages to differentiate into osteoclasts, then the mouse macrophages are subjected to TRAP staining (figure 1), and the results are subjected to quantitative statistics (figure 2 and figure 3), and the mouse bone marrow macrophages are obviously inhibited from differentiating into the osteoclasts along with the increase of the concentration of the sophoridine.

As can be seen from the above experiments and fig. 1 to 3, the evodiamine of the present invention can inhibit differentiation of mononuclear macrophages into osteoclasts, inhibit bone resorption, and inhibit tooth loosening caused by periodontitis from the origin in a concentration-dependent manner.

To determine that the addition of evodiamine in that time period may better inhibit osteoclast formation, mouse bone marrow macrophages were now treated with 1 × 10⁴One cell/cell was seeded in 96-well plates, while RANK L (50ng/m L) induced mouse bone marrow macrophages to osteoclastsThe cells were differentiated and then culture medium was discarded at day 0, day 2, day 4 and day 5, respectively, and medium of evodiamine was added at a final concentration of 60 μ g/m L then mouse macrophages were TRAP stained (fig. 4) and the results were quantified (fig. 5 and fig. 6) indicating that evodiamine needs to occur at an early stage and throughout RANK L-induced differentiation in order to effectively inhibit osteoclast formation.

2.4 statistical analysis

Data were analyzed for variance using SPSS 19.0 software, and pairwise comparisons were checked using t-test, test level α ═ 0.01.

Third, experimental results

3.1 Effect of Evodiamine with different concentrations on the proliferation rate of periodontal ligament Stem cells

After different concentrations of evodiamine and L PS of 10ng/m L act on human periodontal ligament stem cells for 12 hours, 24 hours, 48 hours and 72 hours, MTT detection results show that the A490 value of each group at each time point is the lowest in a negative control group (group E), and the A490 values of A, B, C and D and E in each time point are statistically different (P is less than 0.05), but compared between A, B, C and D, the cell proliferation rate of the evodiamine is slightly reduced, but the statistical significance is not realized, which indicates that the evodiamine has no influence on the proliferation of the periodontal ligament stem cells at the concentration of 6 mu g/m L or less.

TABLE 2 Effect of Evodiamine on the proliferation of periodontal ligament Stem cells at different time points

In the experimental example, 10ng/m L L PS is adopted to stimulate human periodontal ligament stem cells to generate a periodontal ligament stem cell inflammation model, and meanwhile, evodiamine is used for co-culture to observe the influence of the evodiamine on the proliferation rate of the periodontal ligament stem cells.

The results show that the A490 value of the 10ng/m L L PS group is significantly increased at each time compared with the control group, and the A490 value at each time point is not significantly changed after the evodiamine is added, which indicates that L PS (10ng/m L) with low concentration has the promotion effect on the proliferation of the human periodontal ligament stem cells, and the evodiamine has no significant effect on the proliferation of the periodontal ligament stem cells.

3.2 Evodiamine inhibits L PS-induced inflammatory reaction of periodontal ligament Stem cells

Different concentrations of evodiamine (1, 3, 6 mug/m L) and 10ng/m L of L PS act on human periodontal ligament stem cells, and RT-PCR detection results show that compared with group E, the mRNA expression levels of I L-6, I L-1 β and TNF-L0 are all increased in A, B, C and D groups, which indicates successful modeling, and compared with group D, the mRNA expression levels of I L-6, I L-1 β and TNF- α in A, B and C groups are averagely decreased, and are more obvious in high-dose group C group, and the statistical difference (P < 0.01) exists between the C group and the D group (Table 3).

TABLE 3 Evodiamine inhibition of inflammatory factor expression in periodontal ligament Stem cells

In the experimental example, 10ng/m L L PS is adopted to stimulate human periodontal ligament stem cells to generate a periodontal ligament stem cell inflammation model, and meanwhile, evodiamine is used for co-culture to observe the inhibition effect of the evodiamine on the periodontal ligament stem cell inflammation.

The study shows that the L PS of the Porphyromonas gingivalis can promote the growth factors of the mononuclear cells and the liver cells to release interleukin, TNF- α and prostate element E2, and can obviously increase the secretion levels of I L-1 and I L-6 of the growth factors of the stem cells.

The result shows that compared with a control group (a group D), the mRNA expression levels of I L-6, I L-1 β and TNF- α of A, B and a group C (a group with low, medium and high doses of evodiamine respectively) are obviously reduced, the mRNA expression levels of the group C (a group with high dose) are reduced more obviously, and the statistic difference (P is less than 0.05) is formed between the group C and the group D in a pairwise way, so that the evodiamine can inhibit the mRNA expression increase of I L-6, I L-1 and TNF- α caused by L PS.

The results show that the evodiamine can effectively inhibit the inflammation of the periodontal ligament stem cells caused by L PS of the porphyromonas gingivalis, and the evodiamine can be used for preparing the inhibitor of the inflammatory reaction of the periodontal ligament stem cells.

3.3 Evodiamine can significantly inhibit osteoclast differentiation and maturation

The periodontal ligament stem cell is stimulated by periodontal bacteria, and the generation of a large amount of inflammatory factors does not cause serious periodontal disease, and only after the generation of inflammatory factors activates osteoclasts in a large amount, the alveolar bone is excessively absorbed, so that serious periodontal disease and tooth loosening are caused. As shown in fig. 1 to 6, the number of mature osteoclasts was significantly reduced (red is mature osteoclasts) after adding different doses of evodiamine. The evodiamine is shown to be capable of remarkably inhibiting alveolar bone resorption caused by osteoclast activation, and the evodiamine can be used for preparing an osteoclast differentiation and maturation inhibitor.

In conclusion, the evodiamine can effectively inhibit the inflammation of periodontal ligament stem cells caused by L PS of Porphyromonas gingivalis and inhibit the over-absorption of alveolar bone caused by the activation of osteoclasts, and can be used for preparing a medicament for preventing or treating periodontitis.

Experimental example 2

Therapeutic effect of evodiamine mouth wash on tooth loosening

76 users, male and female halves, with ages ranging from 46 to 59 years, were randomly selected and divided into healthy (19 people, PTV-8-9), light loose (20 people, PTV10-19), loose (17 people, PTV20-29) and heavy loose (20 people, PTV > 30) groups according to the tooth loosening index PTV. After half a year of treatment of all groups of users with the mouthwash provided in example 1 of the present invention, the loosening of the teeth of the users was recorded according to the loosening of teeth index PTV, and the results are shown in table 4:

TABLE 4 loosening of teeth of the user

	Before treatment (PTV + -SD)	After treatment (PTV + -SD)
			Health group	3±4	1±4
Slight loosening group	16±5	12±2^*
			Loosening group	25±3	15±4^**
Group of severe loosening	37±5	28±3

^*p≦0.05，^**p≦0.01

The experimental result shows that the mouthwash has no influence on the tooth loosening condition of healthy groups of people, and no side effect on teeth. The traditional Chinese medicine composition has significant treatment effect on a slight loosening group and a loosening group, but has general treatment effect on patients with severe loosening.

In addition, the mouthwash provided by other embodiments is used for performing the above experiment, and the result is substantially the same as that of embodiment 1, and therefore, the details are not repeated.

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims

1. The application of the evodiamine in preparing the medicine for preventing or treating periodontitis is characterized in that the only active ingredient of the medicine is the evodiamine.

2. The application of the evodiamine in preparing the inhibitor for the inflammatory reaction of the periodontal ligament stem cells is characterized in that the only active component of the inhibitor is the evodiamine;

the evodiamine can inhibit inflammatory reaction of periodontal ligament stem cells by inhibiting the periodontal ligament stem cells from generating inflammatory factors;

the inflammatory factors comprise interleukin-6, interleukin-1, tumor necrosis factor- α;

the periodontal ligament stem cell inflammatory response is induced by lipopolysaccharide of periodontal dominant bacteria.