CN110907372B - In-vitro evaluation model and method for anti-inflammatory performance of toothpaste containing bletilla striata extract - Google Patents
In-vitro evaluation model and method for anti-inflammatory performance of toothpaste containing bletilla striata extract Download PDFInfo
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Abstract
The invention discloses a toothpaste anti-inflammatory performance in vitro evaluation model and method comprising bletilla striata extract, wherein the evaluation index comprises at least one of cytokine IL-10, IL-6, IL-1 beta and TNF-alpha content in plasma, the optimum concentration of a toothpaste sample is determined through cell culture and activity detection, cells with logarithmic phase growth state are taken, LPS is added to induce HEGCs in the cells to be in an inflammatory state, the experimental sample and the control sample with the optimum concentration are respectively added, the culture is continued, the content of cell factors secreted by the cells is detected, and the toothpaste anti-inflammatory performance comprising the bletilla striata extract is evaluated through the change of the content of the cell factors. The invention provides a quick and reliable evaluation method for early verification of anti-inflammatory or injury repair efficacy of oral care products, and also provides a new idea for animal substitution experiments, oral care product development and efficacy verification in the oral care industry.
Description
Technical Field
The invention belongs to the technical field of oral care product performance tests, relates to evaluation of anti-inflammatory performance of toothpaste containing bletilla striata extracts, and particularly relates to construction of an in-vitro evaluation model for evaluating anti-gingivitis performance of the toothpaste.
Background
At present, the raw material screening and product anti-inflammatory efficacy testing methods of oral care products are mainly in vivo models, including animal models and clinical experimental models. Related research reports that a mouse gingival mucositis model caused by formaldehyde injection is adopted for carrying out toothpaste anti-inflammatory experiments in groups and the like, and the clinical experiment of treating oral ulcer by matching with mouthwash is also carried out; jiangcui and the like adopt xylene to cause mouse auricle swelling and Freund complete adjuvant to cause a rat foot swelling model and are used for evaluating the anti-inflammatory effect of toothpaste; the volunteers were tested by Loe and Silness gum index by chenqiuyan et al to evaluate the condition of gingival inflammation before and after use of toothpaste; the effect of the toothpaste on improving gingivitis symptoms is evaluated by adopting clinic experiments of department of stomatology in hospitals. However, both animal models and clinical test models have the limitations of high cost, high risk, complex operation, unstable test result and the like for early-stage raw material screening or product efficacy verification, and have certain blindness.
Human Gingival Epithelial Cells (HGECs) are polygonal, fusion presents a typical appearance of 'paving stones', and immunocytochemical detection shows that the cells are positive in keratin dyeing and negative in vimentin dyeing, prove that the cells are cells derived from ectoderm, are also cell activating factors and play an important role in generating periodontitis. However, the majority of cells currently used in anti-inflammatory models employ RAW264.7 macrophages, and there is less research on first layer oral surface HGECs against pathogenic bacteria.
The pathogenic bacteria endotoxin (LPS) is a glycolipid substance composed of lipoid A, core oligosaccharide and O-specific polysaccharide side chain, is the main component of gram-negative bacteria outer membrane, and can directly act on periodontal tissue cells to stimulate corresponding cells such as monocyte and macrophage, and induce inflammation reaction to cause destruction of periodontal tissue. At present, no in vitro model for LPS-induced HEGCs has been reported for evaluating the anti-inflammatory efficacy of toothpaste containing bletilla striata extract.
Disclosure of Invention
Aiming at the problem that no in-vivo and in-vitro gingivitis model is used for comprehensively evaluating the gingivitis resisting and gingivitis repairing effects of the bletilla striata extract and toothpaste containing the bletilla striata extract in the prior art, an in-vitro evaluation model is constructed, a first barrier HGECs resisting invasion of pathogenic microorganisms in an oral cavity is selected to be used in the model, and the anti-inflammatory performance of the toothpaste containing the bletilla striata extract is comprehensively evaluated.
The invention can also quantitatively evaluate the anti-gingivitis effect of the toothpaste containing the bletilla striata extract through the in-vitro evaluation model.
The technical scheme adopted by the invention for solving the technical problems is as follows:
an in-vitro evaluation model of the anti-inflammatory performance of the toothpaste containing the bletilla striata extract comprises at least one of the contents of cytokines IL-10, IL-6, IL-1 beta and TNF-alpha in plasma;
the method for constructing the in vitro evaluation model comprises the following steps:
pretreating a toothpaste sample; and
(ii) HGECs cell culture, and
(iii) determining the optimum concentration of the toothpaste sample containing the bletilla striata extract by cell activity assay, comprising the steps of:
a) Taking the cells with the logarithmic growth phase in step (ii), detecting the blank absorbance OD 0 ；
b) 100uL of experimental sample and control sample containing 10uL of 10 ug/mLLPS were added to the culture medium and incubated for 24h; wherein: the experimental samples comprise a plurality of toothpaste samples containing bletilla striata extracts with different concentration gradients, and the control sample is a culture medium solution without adding any sample;
c) Adding CCK-8 reagent, incubating for 6h, and measuring absorbance OD of each sample at 550nm t ；
d) Screening to obtain the optimal concentration of the test sample with the cell survival rate within the range of 80-100%;
(iv) taking the cells with the growth state in the logarithmic phase in the step (ii), adding 10uL of HEGCs in the cells induced by 10 mug/mLLPS to enable the cells to be in an inflammatory state, then adding 100uL of the experimental sample with the optimal concentration in the step (iii) and the same control sample respectively, culturing for 24h, and detecting the content of the secreted cytokines IL-10, IL-6, IL-1 beta and TNF-alpha respectively.
Further comprising the step of verifying the in vitro assessment model consistency by an animal model.
In the step (i), the toothpaste is dissolved in equivalent absolute ethyl alcohol, a small amount of anhydrous sodium sulfate is added at 60 ℃ and stirred to be fully dissolved, centrifugation is carried out, supernatant fluid is taken and then is subjected to rotary evaporation (40 ℃ and 75 rpm) to the volume of the initial toothpaste, vacuum drying is carried out for 6h, freezing and storing are carried out for 12h at-80 ℃ and freeze drying is carried out for 20h, so as to obtain the alcohol extract of the toothpaste, the alcohol extract is gradually diluted by 0.9% of normal saline or DMEM culture medium, centrifugation is carried out, filtering is carried out by a 0.22 mu m filter membrane, and the alcohol extract is stored at 4 ℃.
In step (ii), the method of cell culture comprises: HGECs were cultured in a DMEM high-sugar medium containing 10% heat-inactivated bovine serum, 1% of penicillin and streptomycin 100. Mu.g/mL were added, 37 ℃ and 5% of CO 2 And timely replacing the culture solution in the incubator with saturated humidity and carrying out subculture.
In the step (iii), the concentration gradient range of the bletilla striata extract in the toothpaste sample is 0.05-0.80 ug/mL.
In step (iii), cell survival = (OD of test sample) t -OD 0 ) /(OD of control sample) t -OD 0 ) X 100%. The optimal concentration of the splendid achnatherum extract in the toothpaste sample is 0.5ug/mL when the cell survival rate is within the range of 80-100%.
On the other hand, the in vitro evaluation model is used for a method for evaluating the anti-inflammatory performance of the toothpaste containing the bletilla striata extract, the contents of cytokines IL-10, IL-6, IL-1 beta and TNF-alpha of the toothpaste sample and the control sample are obtained through the in vitro evaluation model, and the anti-inflammatory performance of the toothpaste containing the bletilla striata extract is evaluated according to the content change of the cytokines IL-10, IL-6, IL-1 beta and TNF-alpha. Further, the evaluation criteria thereof include:
the anti-inflammatory factor IL-10 is increased, which shows that the toothpaste has better anti-inflammatory effect; and/or
The inflammatory factors IL-1 beta and IL-6 are reduced, which indicates that the toothpaste has better anti-inflammatory effect; and/or
The tumor necrosis factor TNF-alpha is reduced, which indicates that the toothpaste has better anti-inflammatory effect.
According to the in-vitro evaluation model disclosed by the invention, through the changes of the expression level of inflammatory factors and the cell migration, the optimal concentration screening and the efficacy verification of the anti-inflammatory performance of the toothpaste containing the bletilla striata extract are carried out in a short time, the limitations of high cost, high risk, complex operation, unstable test result and the like caused by blind animal and clinical experiments are avoided, the test time is greatly shortened, the cost is saved, a quick and reliable evaluation method is provided for the early-stage verification of the anti-inflammatory or injury repairing efficacy of an oral care product, and a new idea is provided for animal substitution experiments, oral care product development and efficacy verification in the oral care industry.
Drawings
FIG. 1 shows the cell viability change of different samples at different concentrations; wherein, the A-bletilla striata No. I toothpaste; b-bletilla striata toothpaste II; c-negative toothpaste; d-positive toothpaste (tranexamic acid toothpaste); e-bletilla striata extract.
FIG. 2 is a graph showing the effect of different samples on the stimulation of the secretion of the cytokines TNF-. Alpha.IL-1. Beta., IL-10 and IL-6 by HGECs by LPS; wherein, the A-bletilla striata No. I toothpaste; b-bletilla striata toothpaste II; c-negative toothpaste; d-positive toothpaste (tranexamic acid toothpaste); e-bletilla striata extract, control-blank model of inflammation of HGECs induced by LPS, P <0.05 indicated by an x; p <0.01 is indicated by x; p <0.001 is indicated by x.
FIG. 3 is a graph showing the effect of different samples in the examples on the secretion of the cytokines TNF- α, IL-1 β, IL-10 and IL-6 in the serum of a rat model of gingivitis; wherein, the A-bletilla striata No. I toothpaste; b-bletilla striata toothpaste II; c-negative toothpaste; d-positive toothpaste (tranexamic acid toothpaste); e-bletilla striata extract; f-gingivitis group; g-healthy group ( # P<0.05； ## P<0.01； ### P<0.001vs.F groups；*P<0.01；**P<0.01；***P<0.001vs.G groups)。
FIG. 4 is a graph of the effect of different samples on the gingival index of a gingivitis rat animal model in examples; wherein, the A-bletilla striata No. I toothpaste; b-bletilla striata toothpaste II; c-negative toothpaste; d-positive toothpaste (tranexamic acid toothpaste); e-bletilla striata extract; control-rat model with severe gingivitis, P <0.05 by x; p <0.01 is denoted by x; p <0.001 is indicated by x.
FIG. 5 is a photograph of an in vivo model of rat gingivitis in one example.
FIG. 6 is an X-ray image of rat maxillary lateral periodontal tissue of various samples in the examples; wherein, the A-bletilla striata No. I toothpaste; b-bletilla striata toothpaste II; c-negative toothpaste; d-positive toothpaste (tranexamic acid toothpaste); e-bletilla striata extract; f-gingivitis group; it: alveolar bone resorption; : (ii): repairing alveolar bone.
Detailed Description
The invention is further illustrated by the following examples in conjunction with the drawings.
< materials and methods >
Gingivalis (ATCC 33277), f.nuclearum (ATCC 49256), HGECs (shanghai seqiq biotechnology); wistar rats, female new zealand rabbits (SPF grade, shanghai jessie laboratory animals ltd); adenosine diphosphate (ADP, sigma-Aldrich, USA, batch A2754), ELISA kit (Nanjing institute of bioengineering); DMEM high-glucose medium, 0.25% pancreatin, fetal bovine serum, penicillin and streptomycin (Gibco); other analytical reagents are purchased from domestic reagent suppliers such as Chinese medicine and pharmaceutical industry.
And (5) preparing a bacterial liquid. Porphyromonas gingivalis (P.g) and Fusobacterium nucleatum (F.n) were inoculated into nutrient broth, cultured at 37 deg.C under anaerobic condition for 2-5 d, single colony on plate was picked and prepared into 5mL of bacterial suspension with sterile PBS (50 mmol/L, pH 7.4), and adjusted to 0.5 McLeod turbidity concentration (i.e., 1X 10) 8 CFU/mL), then adding carboxymethyl cellulose, filtering with a filter membrane with the aperture of 0.22 mu m, and preparing fresh bacterial liquid containing 2% carboxymethyl cellulose.
< construction of a model for in vitro evaluation of anti-inflammatory Properties of toothpaste >
The method comprises the following steps of (1) constructing an in-vitro evaluation model of the anti-inflammatory performance of the toothpaste, and specifically comprising the following steps:
the method comprises the following steps: and (4) pretreating a toothpaste sample. Dissolving toothpaste in equal amount of anhydrous ethanol, adding small amount of anhydrous sodium sulfate at 60 deg.C, stirring to dissolve completely, centrifuging, collecting supernatant, rotary steaming (40 deg.C, 75 rpm) to initial toothpaste volume, vacuum drying for 6 hr, freezing at-80 deg.C for 12 hr, lyophilizing for 20 hr to obtain toothpaste alcohol extract, diluting with 0.9% physiological saline or DMEM medium, centrifuging, filtering with 0.22 μm filter membrane, and storing at 4 deg.C (one week).
Step two: and (5) culturing the cells. HGECs were cultured in a 10% heat-inactivated bovine serum-containing DMEM high-glucose medium, and 1% penicillin and streptomycin were added at a concentration of 100. Mu.g/mL, at 37 ℃ and 5% CO 2 And timely replacing liquid in an incubator with saturated humidity, subculturing, and testing cells with growth states of 5-6 generations in a logarithmic phase.
Step three: cell activity detection and determination of bletilla striata extractThe optimum concentration of the extracted toothpaste sample. The method comprises the following specific steps: a) Taking the cells with logarithmic growth state, and detecting the absorbance OD of the cells in blank 0 (ii) a b) 100uL of experimental sample and control sample containing 10uL of 10 ug/mLLPS were added to the culture medium and incubated for 24h; wherein: the experimental samples comprise a plurality of toothpaste samples containing bletilla striata extracts with different concentration gradients, and the control sample is a culture medium solution without adding any sample; c) Adding CCK-8 reagent, incubating for 6h, and measuring absorbance OD of each sample at 550nm t (ii) a d) Screening to obtain the optimal concentration of the experimental sample with the cell survival rate within the range of 80-100%.
Step four: and detecting the content of the secreted cytokines IL-10, IL-6, IL-1 beta and TNF-alpha in the sample. Taking cells with logarithmic growth state, adding 10uL of HEGCs with concentration of 10 mug/mLLPS to induce the HEGCs in the cells to be in an inflammatory state, then respectively adding 100uL of an experimental sample with optimal concentration and the same control sample, culturing for 24h, and respectively detecting the contents of secreted cytokines IL-10, IL-6, IL-1 beta and TNF-alpha.
Example one model for in vitro evaluation of anti-inflammatory Properties of toothpaste
CCK-8 reagent can be used for detecting the cell proliferation and toxicity analysis of the toothpaste samples on HGECs, and the optimal toothpaste sample concentration value of the HGECs is screened according to the cell survival rate after the toothpaste samples are added, wherein:
cell viability = (OD of experimental sample) t -OD 0 ) /(OD of control sample) t -OD 0 )×100％。
ELISA for the detection of IL-6, IL-1. Beta., IL-10 and TNF-. Alpha.: HGECs cell suspension (5.0X 10) was added to each well in 96-well plates 4 cells/mL) 100uL, at 37 ℃ 5% CO 2 And (3) incubating in an incubator for 24h, discarding the culture solution, adding 100uL of fresh DMEM (high-sugar) culture solution containing toothpaste samples with different concentrations, continuing to incubate for 24h, and adding DMEM (high-sugar) culture solution containing LPS to enable the final concentration to be 10ug/mL.37 ℃ and 5% of CO 2 Culturing in an incubator for 24h, collecting cell supernatant, and determining the content according to the ELISA kit specification of IL-6, IL-1 beta, IL-10 and TNF-alpha.
Taking IL-1 beta as an example, an antigen IL-1 beta monoclonal antibody is coated on an enzyme label plate, IL-1 beta in a standard substance and a sample is combined with the monoclonal antibody, biotinylated antigen IL-1 beta is added to form an immune complex which is connected on the plate, horseradish peroxidase label is combined with biotin, an enzyme substrate OPD is added to generate blue, stop solution sulfuric acid is added to change the color to yellow, the absorbance A value is measured at 450nm, the concentration of IL-1 beta is in direct proportion to the A value, and the concentration of IL-1 beta in the sample can be calculated by drawing a standard curve.
The CCK-8 reagent is used for screening the optimum concentration of the toothpaste added with the bletilla extract or tranexamic acid, and has the advantages of high brightness, low cytotoxicity, better result than MTT and the like. And selecting the concentration of the toothpaste sample with the cell survival rate of 80-100% to detect the content of the HGECs secreting cytokines, and evaluating the anti-inflammatory effect of each group of toothpaste samples by adopting the concentration of 0.5ug/mL toothpaste sample (P is less than 0.05) as shown in figure 1.
IL-10 is an anti-inflammatory factor and can achieve the effect of inhibiting inflammation by releasing immune mediators, IL-1 beta and IL-6 are inflammatory factors, when an organism is inflamed, the two cytokines are correspondingly increased, and TNF-alpha is a tumor necrosis factor and can cause hemorrhagic necrosis of tumor cells by causing some inflammatory reactions. As shown in FIG. 2, the highest IL-10 secretion from HGECs was achieved in the tranexamic acid toothpaste group at 98.95 ng/L. + -. 2.42ng/L (P < 0.001); the contents of IL-1 beta, IL-6 and TNF-alpha are the lowest, and are respectively 6.12ng/L +/-0.02 ng/L,6.36ng/L +/-0.04 ng/L (P is less than 0.01) and 13.09ng/L +/-0.01 ng/L (P is less than 0.01); wherein the bletilla striata extract, the bletilla striata No. I toothpaste group and the bletilla striata No. II toothpaste group have significant influence on an HGECs inflammation model (P is less than 0.05).
EXAMPLE two rat animal experiments
When a rat model with filament ligation and gingivitis grinding is constructed, a gingivitis rat animal model is established by using porphyromonas gingivalis (P.g) and fusobacterium nucleatum (F.n) in an inducing manner, and the method comprises the following specific steps:
the total number of SPF Wistar rats is 20, female, 200 +/-20 g in body weight, good in activity, free of caries and periodontal disease. As the maxillary teeth of the rat are tiny and difficult to ligate, after the rat is anesthetized by injecting 10% chloral hydrate into the abdominal cavity by 3.5mg/kg, the second maxillary teeth on both sides are selected to ligate the neck of the tooth by using 5-0 medical silk thread, the ligature silk thread is put into the gingival sulcus as much as possible, and the ligature needs to be ligated again when the ligature falls off, as shown in figure 5. After ligation, the feed was fed with 10% sugar water instead of drinking water, and 0.5mL of the prepared bacterial solution was inoculated every other day. After 28 days, the toothpaste sample 15 days after successful modeling was used to detect the immunoglobulin IgG, igM, igA factors, and gingival index GI and plaque index PI of the rat saliva, and morphological analysis of the inflammatory factors TNF-alpha, IL-1 beta, IL-10 and IL-6 in the rat plasma and X-ray was performed. Wherein, igA is secretory immunoglobulin A, which is the first immunization defense line of the body against pathogens, and effectively prevents harmful substances in the oral cavity from entering epithelial cells; igM is immunoglobulin M which is an antibody appearing at the earliest in an immune response, and the repairing effect of gingivitis in an oral cavity can be evaluated according to the content of the IgM; igG is immunoglobulin G, which is the main antibody component in serum and has very low content in saliva. The gum index GI evaluates the health condition of the oral cavity by checking the change of gum color, the plaque index PI evaluates the control effect of periodontal disease in the oral cavity according to the thickness of dental plaque on the tooth surface, and the gum bleeding index BOP evaluates the pathological condition of the gum of the oral cavity by stimulating the gum part through a probe.
As shown in Table 1, the IgA changed most obviously after the treatment of tranexamic acid toothpaste, and increased by 1.18mg/L (P < 0.01), the IgM increased by 410ng/L (P < 0.01) after the treatment of bletilla striata I toothpaste, while IgG in the saliva of the oral cavity became a large amount of IgG in the serum due to easy bleeding of gum when the oral cavity inflammation of rats was severe in the third week, but the normal value was recovered after the treatment of the toothpaste in the sixth week.
Table 1: comparison of IgG, igM and IgA in saliva of rats of each group
As shown in fig. 3, in the rat gingivitis model, GI, PI, and BOP in the oral cavity of the rat treated with the bletilla striata extract are significantly reduced (P < 0.001), and then the treated group of the bletilla striata toothpaste (P < 0.01), which indicates that the bletilla striata extract and the toothpaste thereof have good oral treatment effect. As shown in FIG. 4, the IL-10 content in the treated group of bletilla striata extract in plasma was the highest, and was 27.98 ng/L. + -. 1.22ng/L (P < 0.05); the contents of IL-1 beta, IL-6 and TNF-alpha are the lowest, and are respectively 12.26ng/L +/-0.30 ng/L (P is less than 0.001), 2.62ng/L +/-0.01 ng/L (P is less than 0.001) and 103.99ng/L +/-4.37 ng/L (P is less than 0.001); the addition of bletilla striata extract or tranexamic acid toothpaste has significant repairing effect on a rat gingivitis model (P is less than 0.05).
As shown in fig. 6, mercury bullion from the control rat mercury tissue showed significant mercury papilla recession and inflammatory cell invasion, where the mercury papilla was located below the enamel cementum boundary, the alveolar tent height decreased, and the distance from the alveolar ridge to the enamel cementum boundary significantly increased. Compared with the control group, the teeth of each toothpaste treatment group are closely arranged, and the teeth are closely attached to the tooth sockets; the gap between the second molar of the rat in the medicine group and the adjacent molar is obviously reduced, and the gap between the tooth and the alveolar space is reduced. The toothpaste groups can obviously improve the gaps between the teeth and the alveoli of the rats with periodontitis. Wherein the absorption rate of rhizoma Bletillae I alveolus is higher than that of rhizoma Bletillae II group.
The study of LPS tolerance has mainly focused on immune cells such as monocytes/macrophages and dendritic cells. There is also no direct report of immune tolerance to HGECs and studies have demonstrated that virulence factors such as LPS can be recognized in gingival tissues of patients with chronic periodontitis. Selecting HGECs most close to the outermost layer and susceptible to infection for testing, wherein CCK-8 detection shows that 0.5ug/mL of sample concentration is selected for detecting inflammatory factors, bletilla striata No. I toothpaste, bletilla striata No. II toothpaste, negative toothpaste, tranexamic acid toothpaste and bletilla striata extract are very obviously increased (P is less than 0.001) for IL-10 in supernatant of LPS-stimulated HEGCs cells, and compared with a control group, the contents of the toothpaste are respectively increased by 36.62ng/L, 27.70ng/L, 60.73ng/L, 20.26ng/L and 90.02ng/L; TNF-alpha is reduced in generality (P < 0.05), 13.03ng/L, 8.80ng/L, 21.95ng/L, 2.59ng/L and 23.35ng/L are reduced compared with a control group, IL-1 beta is not obviously different, 4.83ng/L, 1.62ng/L, 6.19ng/L, 0.66ng/L and 7.48ng/L are reduced respectively compared with the control group, and IL-6 is reduced obviously (P < 0.01), 16.75ng/L,11.61ng/L, 45.08ng/L, 0.62ng/L and 45.94ng/L are reduced respectively compared with the control group. Four groups of test results show that in vitro inflammatory cell models, the in vitro anti-inflammatory effect is as follows: bletilla striata extract > tranexamic acid toothpaste > bletilla striata No. I toothpaste group > bletilla striata No. II toothpaste group > negative toothpaste group.
In periodontal disease, gingivitis and gingival bleeding occur at a higher rate. The two methods of microbial test evaluation and animal test evaluation are combined to improve the efficiency of product research work and reduce the cost of clinical test. An in-vitro model for comprehensively evaluating the anti-gingivitis repair effect of the bletilla striata extract and the toothpaste thereof is established, and the treatment analysis of the toothpaste on periodontitis is comprehensively evaluated according to X-ray tissue observation, probe gingival diagnosis (GI, PI), immunoglobulin (IgA, igM, igG) detection in saliva and cytokine IL-10, IL-6, IL-1 beta and TNF-alpha indexes in plasma. Wherein GI index and PI index are obviously lower than those of the toothpaste processed after the model is successfully made, X-ray images after the toothpaste is processed show that the gap between adjacent molars is obviously reduced compared with that of the toothpaste processed after the model is successfully made, the gap between the posterior tooth and the alveolar ridge is obviously reduced, the alveolar ridge is slightly absorbed, and alveolar bone is increased. Further proves that the inhibition effect of the toothpaste on inflammatory mediators and inflammatory cytokines leads to experimental results showing better anti-inflammation and is consistent with the results of in vitro HGECs inflammation models.
Claims (2)
1. A method for evaluating the anti-inflammatory performance of toothpaste containing bletilla striata extracts is characterized in that the contents of cell factors IL-10, IL-6, IL-1 beta and TNF-alpha of the toothpaste samples and control samples are obtained through an in-vitro evaluation model, and the anti-inflammatory performance of the toothpaste containing the bletilla striata extracts is evaluated according to the content change of the cell factors IL-10, IL-6, IL-1 beta and IL-6, wherein the anti-inflammatory factor IL-10 is increased, the anti-inflammatory effect of the toothpaste is better, the anti-inflammatory effects of the inflammation factors IL-1 beta and IL-6 are reduced, the anti-inflammatory effect of the toothpaste is better, and the tumor necrosis factor TNF-alpha is reduced, the anti-inflammatory effect of the toothpaste is better;
the toothpaste anti-inflammatory performance in-vitro evaluation model containing the bletilla striata extract is obtained by a construction method comprising the following steps:
pretreatment of a toothpaste sample: dissolving toothpaste in equivalent absolute ethyl alcohol, adding a small amount of anhydrous sodium sulfate at 60 ℃, stirring until the toothpaste is fully dissolved, centrifuging, taking supernate, carrying out rotary evaporation at 40 ℃ and 75rpm until the volume of the toothpaste is initial, carrying out vacuum drying for 6h, carrying out frozen storage at-80 ℃ for 12h, carrying out freeze-drying for 20h to obtain an alcohol extract of the toothpaste, gradually diluting the alcohol extract with 0.9% of physiological saline or a DMEM culture medium, centrifuging, filtering with a 0.22 mu m filter membrane, and storing at 4 ℃;
(ii) HGECs cell culture: HGECs were cultured in a DMEM high-glucose medium containing 10% heat-inactivated bovine serum, and penicillin and streptomycin were added in an amount of 1% to 100 μ g/mL, at 37 ℃ and 5% 2 Timely replacing liquid in an incubator with saturated humidity and carrying out subculture;
(iii) determining the optimum concentration of the toothpaste sample containing the bletilla striata extract by cell activity assay, comprising the steps of:
a) Taking the cells with the growth state in the logarithmic phase in the step (ii), and detecting the absorbance OD of the cells in the blank 0 ；
b) Adding 100 muL of an experimental sample containing 10 muL of 10 mug/mLLPS and a control sample into the mixture respectively, and culturing for 24h, wherein the experimental sample comprises a plurality of toothpaste samples containing bletilla striata extracts with different concentration gradients, and the control sample is a culture medium solution without any sample;
c) Adding CCK-8 reagent, incubating for 6h, and measuring absorbance OD of each sample at 550nm t ；
d) Screening to obtain the optimal concentration of the test sample with the cell survival rate within the range of 80-100%;
the concentration gradient of the bletilla striata extract in the toothpaste sample is 0.05-0.80 mug/mL, and the cell survival rate = (experiment sample OD) t -OD 0 ) /(control sample OD t -OD 0 )×100%；
(iv) taking the cells with the growth state in the logarithmic phase in the step (ii), adding 10 muL of LPS (lipopolysaccharide) with the concentration of 10 mug/mL to induce HEGCs in the cells to be in an inflammation state, then respectively adding an experimental sample with the optimal concentration and a control sample with the same concentration in the step (iii) of 100 muL, culturing for 24h, and respectively detecting the contents of cell secretion cytokines IL-10, IL-6, IL-1 beta and TNF-alpha;
further comprising the step of verifying the in vitro assessment model consistency by an animal model.
2. The method for evaluating the anti-inflammatory performance of the toothpaste containing the bletilla striata extract according to claim 1, wherein the optimal concentration of the bletilla striata extract in the toothpaste sample within a range of 80% -100% of a cell survival rate is 0.5 mug/mL.
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