A traditional Chinese medicine composition and its application in oral cavity

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a traditional Chinese medicine composition and application thereof in oral cavity. The application provides a traditional Chinese medicine composition with a brand-new formula, wherein the traditional Chinese medicine composition comprises extracts of Ficus hirta, Radix Paeoniae Alba, Glycyrrhiza uralensis and Sophora japonica prepared by alcohol extraction. Research results show that the obtained traditional Chinese medicine composition has excellent inhibition on formation of oral cavity biofilm, and the inhibition rate is more than 90%, which is significantly higher than that of single formula or other traditional Chinese medicine compositions formed by different kinds and / or different proportions, and the traditional Chinese medicine composition can be used for treatment of dental bacterial diseases including dental caries, and has important application value in preparation of oral cavity products.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a traditional Chinese medicine composition and its application in oral health. Background Technology

[0002] The oral cavity is a bacterial environment directly connected to the outside world. If microorganisms are not removed in time, they can easily accumulate in the oral cavity and form biofilms, leading to oral diseases such as tooth decay. Using oral care products is an important measure to prevent biofilm formation. Traditional Chinese medicine, due to its advantages of low toxicity, mild effects, and abundant resources, has been widely used in mouthwash, toothpaste, and other products.

[0003] However, most existing studies focus on planktonic bacteria, evaluating efficacy by measuring inhibition zone diameter, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC). This approach fails to accurately reflect the actual inhibitory effect of drugs on biofilms. In the oral environment, bacteria primarily exist in biofilm form, and their morphology, physiological characteristics, drug resistance, and pathogenicity differ significantly from those of planktonic bacteria. Therefore, herbal extracts screened using traditional antibacterial methods may not effectively inhibit oral biofilm formation.

[0004] Due to their pleasant aroma, good taste, and high safety, medicinal and edible plants have been added to the products of many toothpaste brands. However, systematic research on the inhibitory effects of these traditional Chinese medicines on biofilms is still relatively lacking. Ancient books record a variety of traditional Chinese medicine prescriptions for oral diseases. For example, the "Shengji Zonglu" records that licorice, apricot kernel, and ginger are widely used in prescriptions for various oral diseases; the "Five Fragrance Pill" for treating halitosis recorded in "Beiji Qianjin Yaofang" contains medicinal and edible ingredients such as chuanxiong, angelica, tangerine peel, cinnamon, and jujube; and the "Yuchi Powder" recorded in "Taiping Huimin Heji Jufang" mainly uses angelica, supplemented with angelica and licorice.

[0005] However, the aforementioned medicinal and edible herbs have the following problems: first, traditional antibacterial evaluation methods are disconnected from the actual oral biofilm environment; second, there is insufficient systematic research on the inhibition of oral biofilms by medicinal and edible herbs, and a lack of mature compositions with clear inhibitory effects. Therefore, developing a traditional Chinese medicine composition that can effectively inhibit the formation of oral biofilms has significant application value. Summary of the Invention

[0006] The technical problem to be solved by this invention is to overcome the shortcomings and deficiencies of traditional antibacterial evaluation methods being disconnected from the actual oral biofilm environment, insufficient systematic research on the inhibition of oral biofilm by traditional Chinese medicines that are both food and medicine, and the lack of mature compositions with clear inhibitory effects. The primary objective is to provide a traditional Chinese medicine composition.

[0007] A second objective of the present invention is to provide the use of the traditional Chinese medicine composition in the preparation of products having any of the following uses: (1) inhibiting and / or killing oral microorganisms; (2) inhibiting and / or removing biofilms formed by oral microorganisms.

[0008] A third objective of this invention is to provide the use of the aforementioned traditional Chinese medicine composition in the preparation of medicaments for the prevention and / or treatment of bacterial dental diseases.

[0009] The fourth objective of this invention is to provide an oral care product.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution:

[0011] This invention protects a traditional Chinese medicine composition comprising extracts obtained by alcohol extraction of Terminalia chebula, Paeonia lactiflora, Glycyrrhiza uralensis, and Sophora japonica; wherein the mass ratio of Terminalia chebula, Paeonia lactiflora, Glycyrrhiza uralensis, and Sophora japonica is (2.6~3.5):(2~2.5):(2.6~3.5):1.

[0012] Through extensive research, the inventors discovered that by adding Sophora japonica flowers to a formula containing Chinese medicinal herbs such as Terminalia chebula, Paeonia lactiflora, and Glycyrrhiza uralensis, the resulting herbal composition, when formulated in a specific ratio, can effectively inhibit the formation of oral biofilms, especially those caused by Streptococcus mutans, with an inhibition rate exceeding 90%, significantly higher than that of single herbs or other herbal compositions formed by different types and / or ratios.

[0013] The aforementioned green fruit is the dried, ripe fruit of the olive tree, a plant belonging to the Burseraceae family.

[0014] Preferably, the mass ratio of the green fruit, white peony root, licorice root and sophora flower is (2.8~3):(2~2.2):(2.8~3):1, more preferably 3:2:3:1.

[0015] Furthermore, the alcohol extraction can be performed by first mixing raw materials such as green plum, white peony root, licorice root, and sophora flower together for alcohol extraction, or by separately extracting raw materials such as green plum, white peony root, licorice root, and sophora flower and then mixing them together.

[0016] Further, as a preferred embodiment, the alcohol extraction includes the following steps: fully extracting a mixed powder containing green fruit, white peony root, licorice root and sophora flower in an ethanol solution to obtain an extract, and concentrating and drying the obtained extract to obtain the traditional Chinese medicine composition;

[0017] Wherein, the concentration of the ethanol solution is ≥50 vol.

[0018] Preferably, the concentration of the ethanol solution is 50 vol% to 80 vol%, more preferably 55 vol% to 65 vol.

[0019] Furthermore, the conditions for sufficient extraction include the use of ultrasound-assisted extraction.

[0020] Preferably, the power of the ultrasound is 80~120 kW, more preferably 90~110 kW.

[0021] Preferably, the ultrasound duration is 20-40 min, more preferably 25-35 min.

[0022] Furthermore, the extraction is performed multiple times, preferably 1 to 3 times.

[0023] Furthermore, the mixing ratio of the mixed powder to the ethanol solution is 1g:(8~15)mL, preferably 1g:(8~12)mL, and more preferably 1g:10mL.

[0024] Furthermore, the mixed powder is made by mixing and pulverizing various Chinese medicinal materials, or by mixing the pulverized raw material powders.

[0025] Furthermore, the concentration is carried out under reduced pressure to extract.

[0026] Furthermore, the temperature for the vacuum concentration is 40~60 ℃.

[0027] Furthermore, the drying process is freeze-drying, and the freeze-drying conditions are -60 to -80 ℃ for 2 to 3 days. These conditions can be adjusted flexibly according to actual circumstances.

[0028] This invention protects the use of the traditional Chinese medicine composition in the preparation of products having any of the following uses:

[0029] (1) Inhibit and / or kill oral microorganisms;

[0030] (2) Inhibit and / or remove biofilms formed by oral microorganisms.

[0031] Furthermore, the oral microorganisms include at least one of Streptococcus mutans, Streptococcus distalis, Porphyromonas gingivalis, Staphylococcus aureus, Candida albicans, Prevotella intermedius, Actinomyces negrius, Actinomyces myxobolus, Actinomyces eluti, Actinomyces mesei, Actinomyces odontolyticus, and Rhizoctonia solani.

[0032] Furthermore, the biofilm is a biofilm formed by Streptococcus mutans.

[0033] This invention protects the use of the traditional Chinese medicine composition in the preparation of remedies for the prevention and / or treatment of bacterial dental diseases.

[0034] Furthermore, the bacterial dental diseases include one or more of dental caries, pulpitis, periodontitis, periapical periodontitis, and pericoronitis of wisdom teeth.

[0035] Furthermore, the dental caries is caused by at least one of Streptococcus mutans and Streptococcus pyogenes.

[0036] Furthermore, when the bacterial disease is dental caries, the prevention and / or treatment of dental bacterial diseases involves reducing the secretion of immunoglobulin A (IgA) and / or IgG in oral tissues.

[0037] This invention also protects an oral care product, wherein the active ingredient of the oral care product includes one or more of the traditional Chinese medicine composition.

[0038] Furthermore, the oral products include oral treatment drugs, oral care products, or oral health care products.

[0039] Furthermore, the dosage forms of the oral treatment drugs include ointments, gels, films, powders, lozenges, patches, mouthwashes, or sprays.

[0040] Furthermore, the oral care products include one or more of toothpaste, tooth powder, and mouthwash.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] This invention provides a novel traditional Chinese medicine composition comprising extracts obtained from *Clematis chinensis*, *Paeonia lactiflora*, licorice, and *Sophora japonica* through alcohol extraction. Research results show that the obtained traditional Chinese medicine composition exhibits excellent inhibition of oral biofilm formation, with an inhibition rate exceeding 90%, significantly higher than that of single-herb or other combinations of different types and / or proportions of traditional Chinese medicine. It can be used to treat dental bacterial diseases, including dental caries, and has significant application value in the preparation of oral care products. Attached Figure Description

[0043] Figure 1 The figures show the experimental results of the inhibitory effects of the traditional Chinese medicine compositions of Examples 1-12 (i.e., Examples 1-12) and the single herbs of the same concentration in Examples 13-18 (i.e., Examples 13-18) on the biofilm inhibition of Streptococcus mutans; wherein, ***: compared with Example 1 group, P< 0.001.

[0044] Figure 2 The image shows a staining diagram of caries lesions in rat molars. Control: blank group; Model: model group; CHX: chlorhexidine solution; L: low-dose group of Example 1; M: medium-dose group of Example 1; H: high-dose group of Example 1; Vertical: front view; Front: top view.

[0045] Figure 3The image shows a Mirco-CT image of rat molars, where Control: blank group; Model: model group; CHX: chlorhexidine solution; L: low-dose group of Example 1; M: medium-dose group of Example 1; and H: high-dose group of Example 1.

[0046] Figure 4 This is a statistical graph showing the effect of Example 1 on salivary IgA secretion in carious rats; where Control: blank group; Model: model group; L: low-dose group of Example 1, M: medium-dose group of Example 1, H: high-dose group of Example 1; ###: compared with the blank group, P< 0.001; *: Compared to the model group, P< 0.05; ***: Compared with the model group, P< 0.001.

[0047] Figure 5 This is a statistical graph showing the effect of Example 1 on salivary IgG secretion in carious rats; where Control: blank group; Model: model group; L: low-dose group of Example 1, M: medium-dose group of Example 1, H: high-dose group of Example 1; ###: compared with the blank group, P< 0.001; ***: Compared with the model group, P< 0.001. Detailed Implementation

[0048] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0049] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0050] The raw materials for the Chinese medicinal herbs, including green plum, white peony root, licorice root, sophora japonica flower, angelica dahurica, and honeysuckle, were all purchased from the Nanjing Traditional Chinese Medicine Clinic.

[0051] Example 1: Preparation of Traditional Chinese Medicine Composition

[0052] The raw materials of the Chinese medicinal herbs are composed of the following parts by weight: 3 parts of green plum; 2 parts of white peony root; 3 parts of licorice root; and 1 part of sophora japonica flower.

[0053] Preparation method: The above-mentioned Chinese medicinal materials were mixed and pulverized, then mixed with 60 vol% ethanol solution (material-to-liquid ratio of 1 g:10 mL). Ultrasonic extraction was performed at 100 kW. After the first extraction, the mixture was filtered. The residue was then added to 60 vol% ethanol solution for a second extraction under the same conditions. The mixture was filtered again to obtain the filtrate. Each extraction lasted 30 minutes. After extraction, the filtrates from both extractions were combined and concentrated under reduced pressure (40 °C) to form an extract. This extract was then freeze-dried (-80 °C for 3 days) to obtain the aforementioned Chinese medicinal composition.

[0054] The above material-to-liquid ratio indicates that 1g of Chinese medicinal material is added to 10mL of ethanol solution for extraction.

[0055] Example 2 Preparation of Traditional Chinese Medicine Composition

[0056] The difference from Example 1 is that Sophora japonica flowers were not added, as detailed below:

[0057] Raw material composition by weight: 3 parts green plum; 2 parts white peony root; 3 parts licorice root;

[0058] Preparation method: Same as in Example 1.

[0059] Example 3 Preparation of Traditional Chinese Medicine Composition

[0060] The difference from Example 1 is that Angelica dahurica is used to replace Sophora japonica in the formula, as follows:

[0061] The raw materials of the Chinese medicinal herbs are composed of the following parts by weight: 3 parts of Terminalia chebula; 2 parts of Paeonia lactiflora; 3 parts of Glycyrrhiza uralensis; and 1 part of Angelica dahurica.

[0062] Preparation method: Same as in Example 1.

[0063] Example 4 Preparation of Traditional Chinese Medicine Composition

[0064] The difference from Example 1 is that honeysuckle is used to replace sophora japonica in the formula, as follows:

[0065] Composition of Chinese medicinal materials by weight: 3 parts green fruit; 2 parts white peony root; 3 parts licorice root; 1 part honeysuckle;

[0066] Preparation method: Same as in Example 1.

[0067] Example 5: Preparation of the Traditional Chinese Medicine Composition

[0068] The difference from Example 1 lies in the different weight composition of the Chinese medicinal materials, as detailed below:

[0069] The raw materials of the Chinese medicinal herbs are composed of the following parts by weight: 3 parts of green fruit; 3 parts of white peony root; 1 part of licorice root; and 2 parts of sophora japonica flower.

[0070] Preparation method: Same as in Example 1.

[0071] Example 6 Preparation of Traditional Chinese Medicine Composition

[0072] The difference from Example 1 lies in the different weight composition of the Chinese medicinal materials, as detailed below:

[0073] The raw materials of the Chinese medicinal herbs are composed of the following parts by weight: 2 parts of green fruit; 1 part of white peony root; 3 parts of licorice root; and 2 parts of sophora japonica flower.

[0074] Preparation method: Same as in Example 1.

[0075] Example 7 Preparation of Traditional Chinese Medicine Composition

[0076] The difference from Example 1 lies in the different weight composition of the Chinese medicinal materials, as detailed below:

[0077] The raw materials of the Chinese medicinal herbs are composed of the following parts by weight: 2 parts of green fruit; 3 parts of white peony root; 2 parts of licorice root; and 1 part of sophora japonica flower.

[0078] Preparation method: Same as in Example 1.

[0079] Example 8 Preparation of Traditional Chinese Medicine Composition

[0080] The difference from Example 1 lies in the different weight composition of the Chinese medicinal materials, as detailed below:

[0081] The raw materials of the Chinese medicinal herbs are composed of the following parts by weight: 2 parts of green fruit; 2 parts of white peony root; 1 part of licorice root; and 3 parts of sophora japonica flower.

[0082] Preparation method: Same as in Example 1.

[0083] Example 9 Preparation of Traditional Chinese Medicine Composition

[0084] The difference from Example 1 lies in the different weight composition of the Chinese medicinal materials, as detailed below:

[0085] The raw materials of the Chinese medicinal herbs are composed of the following parts by weight: 1 part of green plum; 3 parts of white peony root; 3 parts of licorice root; and 3 parts of sophora flower.

[0086] Preparation method: Same as in Example 1.

[0087] Example 10 Preparation of Traditional Chinese Medicine Composition

[0088] The difference from Example 1 lies in the different weight composition of the Chinese medicinal materials, as detailed below:

[0089] The raw materials of the Chinese medicinal herbs are composed of the following parts by weight: 1 part of green fruit; 1 part of white peony root; 1 part of licorice root; and 1 part of sophora japonica flower.

[0090] Preparation method: Same as in Example 1.

[0091] Example 11 Preparation of Traditional Chinese Medicine Composition

[0092] The difference from Example 1 lies in the different weight composition of the Chinese medicinal materials, as detailed below:

[0093] The raw materials of the Chinese medicinal herbs are composed of the following parts by weight: 3 parts of green fruit; 1 part of white peony root; 2 parts of licorice root; and 3 parts of sophora japonica flower.

[0094] Preparation method: Same as in Example 1.

[0095] Example 12 Preparation of Traditional Chinese Medicine Composition

[0096] The difference from Example 1 lies in the different weight composition of the Chinese medicinal materials, as detailed below:

[0097] Composition of Chinese medicinal materials by weight: 1 part green fruit; 2 parts white peony root; 2 parts licorice root; 2 parts sophora japonica flower;

[0098] Preparation method: Same as in Example 1.

[0099] Example 13 Preparation of ethanol extract from green plums

[0100] The raw materials of the Chinese medicinal herbs consist of: 1 part by weight of green fruit;

[0101] Preparation method: Same as in Example 1.

[0102] Example 14 Preparation of Paeonia lactiflora alcohol extract

[0103] The raw materials of Chinese medicinal herbs are composed of: 1 part white peony root;

[0104] Preparation method: Same as in Example 1.

[0105] Example 15 Preparation of Licorice Alcohol Extract

[0106] The raw materials of Chinese medicinal herbs are composed of: 1 part licorice root;

[0107] Preparation method: Same as in Example 1.

[0108] Example 16 Preparation of Sophora japonica flower alcohol extract

[0109] The raw materials of the Chinese medicinal herbs consist of: 1 part Sophora japonica flower;

[0110] Preparation method: Same as in Example 1.

[0111] Example 17 Preparation of Angelica dahurica alcohol extract

[0112] The raw materials of the Chinese medicinal herbs are composed of: Angelica dahurica 1 part by weight;

[0113] Preparation method: Same as in Example 1.

[0114] Example 18 Preparation of honeysuckle alcohol extract

[0115] Composition of Chinese medicinal materials by weight: 1 part honeysuckle;

[0116] Preparation method: Same as in Example 1.

[0117] Experimental Example 1: Experiment on the inhibition of biofilm formation by the traditional Chinese medicine composition of the present invention

[0118] Experimental method: In a 96-well plate, 20 μL of artificial saliva was added to each well. Then, 180 μL of culture medium was added to the blank control group, and 160 μL of culture medium and 20 μL of 1×10⁻⁶ solution were added to the model group. 6 CFU / mL Streptococcus mutans (ATCC 25175) bacterial suspension; the experimental group was given 160 μL of drug-containing culture medium and 20 μL of 1×10⁻⁶ CFU / mL Streptococcus mutans suspension. 6 A CFU / mL suspension of *Streptococcus mutans* was prepared and incubated at 37 °C for 24 h in an anaerobic incubator. After incubation, the supernatant was discarded, and the plate was gently washed three times with PBS to remove airborne bacteria and residual liquid was aspirated. 200 μL of 4% paraformaldehyde was added to each well for fixation for 10 min, followed by drying. 200 μL of 1% crystal violet was added for staining for 5 min, and the stain was removed. The plate was then washed three times with PBS, and residual PBS was aspirated. The plate was dried, and 200 μL of 33% glacial acetic acid solution was added to each well. The plate was incubated at 37 °C for 30 min, and the OD was measured using a microplate reader. 570nm The absorbance value is calculated according to the formula "I 生物膜抑制率 = (A 模型组 -A 样品 ) / (A 模型组 -A 空白组 The biofilm inhibition rate was calculated by multiplying the result by 100%; the test results are shown in Table 1. Each experiment was independently repeated 3 times, with 6 replicates per group, and the average of the multiple measurements was taken.

[0119] The culture medium used in this experiment was prepared as follows: 3.85 g of brain heart extract (BHI) broth and 0.600 g of sucrose were accurately weighed and dissolved in 100 mL of distilled water. The mixture was heated and stirred, cooled to room temperature, and the pH was adjusted to 6.5. The mixture was then autoclaved at 121 °C for 30 min to obtain BHI culture medium containing 6% sugar (w / v).

[0120] The method for preparing the drug-containing culture medium is as follows: the sample to be tested is added to the BHI culture medium containing 6% sugar (W / V) prepared above to prepare a drug-containing culture medium with a drug content of 0.5 mg / mL.

[0121] Table 1. Experimental results of the traditional Chinese medicine composition of the present invention inhibiting biofilm formation.

[0122]

[0123] From Table 1 and Figure 1 The experimental results show that the biofilm inhibition rate of the traditional Chinese medicine composition prepared in Example 2 is 71.97%. This indicates that the traditional Chinese medicine composition prepared by the present invention using Chinese medicinal herbs such as green plum, white peony root, and licorice has the effect of inhibiting the formation of oral biofilm caused by Streptococcus mutans, but the inhibitory effect is poor, with an inhibition rate of less than 90%.

[0124] The traditional Chinese medicine composition prepared in Example 1 showed a biofilm inhibition rate of 96.70%, which was significantly higher than that of the traditional Chinese medicine composition prepared in Example 2. It exhibited excellent inhibitory effects on oral biofilm formation caused by Streptococcus mutans. This indicates that adding Sophora japonica flowers to the formula made from traditional Chinese medicinal materials such as Terminalia chebula, Paeonia lactiflora, and Glycyrrhiza uralensis can significantly enhance the inhibitory effect of the traditional Chinese medicine composition made from these materials on oral biofilm formation caused by Streptococcus mutans.

[0125] The herbal compositions prepared in Examples 3 and 4 did not show an increase or significant increase in biofilm inhibition rate compared to Example 2; their biofilm inhibition rate was significantly lower than that of the herbal composition prepared in Example 1. This indicates that in formulations using herbs such as *Clerodendrum trichotomum*, *Paeonia lactiflora*, and licorice as raw materials, not all arbitrarily added herbs can significantly improve the inhibition rate of oral biofilm formation. The above experimental results show that in formulations using herbs such as *Clerodendrum trichotomum*, *Paeonia lactiflora*, and licorice as raw materials, only the addition of *Sophora japonica* can significantly improve the inhibitory effect of the herbal compositions prepared using these herbs on oral biofilm formation caused by *Streptococcus mutans*.

[0126] The traditional Chinese medicine compositions prepared in Examples 5-12 showed significantly lower inhibition rates against oral biofilm formation compared to the composition prepared in Example 1. This indicates that the weight ratio of the four components is crucial when adding Sophora japonica flowers to a formula using Chinese medicinal herbs such as Terminalia chebula, Paeonia lactiflora, and Glycyrrhiza uralensis. Different ratios of these four components result in significant differences in the inhibitory effect of the prepared traditional Chinese medicine compositions on oral biofilm formation caused by Streptococcus mutans. Only traditional Chinese medicine compositions prepared with the weight ratios described in this invention exhibit excellent inhibitory effects against oral biofilm formation caused by Streptococcus mutans.

[0127] Experimental Example 2: Validation of Drug Efficacy in Animals

[0128] 1. Experimental Materials

[0129] 1.1 Instruments and Consumables

[0130] Table 2 Instruments and Consumables

[0131]

[0132] 1.2 Reagents:

[0133] Table 3. Sources of some reagents

[0134]

[0135] 1.3 Laboratory animals and bacterial strains:

[0136] Twenty male, 19-day-old weaned Wistar rats were purchased from Qinglongshan Animal Breeding Farm in Jiangning District, Nanjing City. After purchase, they were housed in an SPF-grade animal room with a 12-hour diurnal cycle, relative humidity of 40-60%, and temperature of 22-26°C.

[0137] Streptococcus mutans ATCC 25175, Streptococcus pyogenes BNCC 353916, and Streptococcus salivarius ATCC 7073 were purchased from Beina Chuanglian Biotechnology Co., Ltd.; Streptococcus mutans UA 159 was purchased from the China Industrial Microbial Culture Collection Center (CICC).

[0138] 2. Experimental Methods

[0139] 2.1 Preparation of reagents and culture media

[0140] 2.1.1 Preparation of Brain Heart Extract (BHI) Solid Culture Medium:

[0141] Accurately weigh 3.85 g of BHI solid powder, heat and stir to dissolve in 100 mL of double-distilled water. After complete dissolution, add 2 g of agar, autoclave at 121 ℃ for 30 min, and cool to an appropriate temperature after sterilization to prepare plate culture medium for later use.

[0142] 2.1.2 Preparation of BHI liquid culture medium:

[0143] Accurately weigh 3.85 g of BHI solid powder, heat and stir to dissolve in 100 mL of double-distilled water, autoclave at 121 ℃ for 30 min, and cool before use.

[0144] 2.1.3 Preparation of sugar-containing BHI liquid culture medium:

[0145] Accurately weigh 3.85 g of BHI solid powder and 0.600 g of sucrose, dissolve them in 100 mL of distilled water, heat and stir, cool to room temperature, adjust the pH to 6.5, and autoclave at 121 ℃ for 30 min to obtain BHI medium with 6% sugar (w / v) (sucrose concentration of 6 mg / mL), for later use.

[0146] 2.1.4 Preparation of sterile water:

[0147] Measure 1 L of double-distilled water, dispense it into small bottles, autoclave at 121 °C for 30 min, and cool before use.

[0148] 2.1.5 Preparation of MSB plate culture medium.

[0149] Accurately weigh 9.008 g of Streptococcus salivarius (ATCC 7073), heat to boiling and completely dissolve in 100 mL of Wahaha water, dispense into 200 mL Erlenmeyer flasks, autoclave at 121 ℃ for 30 min, cool to 50~55 ℃, add 0.1 mL of sterile 1% potassium tellurite solution and 20 U bacitracin, mix well, pour into sterile petri dishes, and cool for later use.

[0150] 2.2 Culture of microbial strains

[0151] The activation and culture conditions for distant streptococci BNCC 353916 were the same as those for variant streptococci ATCC 25175.

[0152] 2.3.1 Activation and culture of Streptococcus mutans

[0153] After wiping the lyophilized tube containing *Streptococcus mutans* (ATCC 25175) with alcohol for sterilization, open it in a clean bench and add 0.5 mL of BHI liquid medium to the lyophilized tube. After complete dissolution, add the solution to a test tube containing 5 mL of BHI liquid medium and mix well. Add 0.2 mL of the bacterial suspension to a BHI plate and spread evenly. Repeat twice to obtain two plates. Place the liquid test tubes and plates in an anaerobic incubator (80% N2-10% CO2-10% H2) at 37 ℃ for 48 hours. Observe the bacterial morphology on the plates to ensure a pure culture. Pick a single colony of *Streptococcus mutans* from the plate and dissolve it in 3 mL of BHI liquid medium. Incubate anaerobicly at 37 ℃ for 24 hours to obtain an enrichment broth for the standard strain of *Streptococcus mutans*.

[0154] 2.3.2 Preparation of bacterial suspension

[0155] Add the Streptococcus mutans bacterial suspension to a sterile test tube, dilute with physiological saline, and adjust the bacterial suspension concentration to 0.5 McFarland turbidity (1.5 × 10⁻⁶) using a McFarland turbidity tube. 8 (CFU / mL), then diluted with physiological saline to obtain 1.0 × 10⁻⁶ CFU / mL. 6 Prepare a bacterial suspension of CFU / mL for later use.

[0156] 2.3.3 Preservation of microbial strains

[0157] 1.0×10 9The bacterial suspension of CFU / mL was added to the cell cryopreservation tube in equal proportion to 33% glycerol, mixed well, and pre-cooled at -20°C for 24 h, and then frozen at -80°C.

[0158] 2.3 In vivo efficacy verification

[0159] Thirty-six 18-day-old Wistar rats were randomly divided into six groups (n=6 per group): Control, Model, CHX (chlorhexidine solution), L (low dose of Example 1), M (medium dose of Example 1), and H (high dose of Example 1). The rats were given SPF-grade maintenance diet and sterile water containing 4000 U / mL potassium penicillin and 0.1% carbenicillin for 3 days to eliminate oral bacteria. Starting on day 4, they were given Diet2000 caries model diet and 5% sucrose solution for 5 consecutive days, with 100 μL of 1×10⁻⁶ solution injected twice daily. 9 A mixed bacterial suspension of *Streptococcus mutans* (ATCC 25175) and *Streptococcus distantly related* (BNCC 353916) (with a viable bacterial concentration ratio of 1:1) was applied evenly to the crowns of the rat molars using a dental brush. Colonization was confirmed using MSB medium. The Control and Model groups were administered 100 μL of physiological saline, the CHX group was administered 100 μL of chlorhexidine solution, and the L, M, and H groups were administered 100 μL of the traditional Chinese medicine composition from Example 1 at concentrations of 1.00 mg / mL, 2.0 mg / mL, and 4.0 mg / mL (solvent: ultrapure water), respectively. The solution was then applied topically to the rat molars using a camel hair brush for 5 minutes each time, twice daily (morning and evening) until day 60.

[0160] 2.4 Morphological observation of dental caries lesions in rats

[0161] On day 61, rats were sacrificed, and their upper and lower molars were collected and disinfected at 121 °C for 15 min. The remaining tissue was removed and the teeth were rinsed with physiological saline. The teeth were then soaked in 2 wt% ammonium hydroxide solution for 30 min, followed by soaking in bone violet staining solution for 24 h. The crown and neck of the molars were observed under a stereomicroscope to observe the caries lesions. The Keyes classic scoring method was used for evaluation. Under the microscope, the caries lesions were pink and the normal tooth body was unstained or very lightly stained.

[0162] 2.5 Determination of biochemical indicators in rat saliva and serum

[0163] Rat saliva (supernatant) and serum were collected, and the contents of IgA and IgG were determined by ELISA, following the instructions of the ELISA kit. 40 μL of sample diluent was added to the enzyme-labeled plate, followed by 10 μL of saliva or serum. The plate was shaken well, sealed with a sealing film, and incubated at 37 ℃ for 30 min. The plate was washed five times with washing buffer and patted dry. Then, under dark conditions, 50 μL of chromogenic reagent A and chromogenic reagent B were added sequentially, shaken well, and incubated at 37 ℃ for 15 min. After incubation, 50 μL of stop solution was added, the plate was shaken well, and the absorbance was measured at 450 nm. The absorbance was then substituted into the standard curve to calculate the content.

[0164] 2.6 Data Processing

[0165] Graphpad Prism 9.5 statistical software was used to perform statistical analysis on the experimental results and to generate bar charts. t-tests were used to compare the data from two groups, and ANOVA analysis was used to analyze the variance of multiple groups.

[0166] 3. Experimental Results

[0167] 3.1 Changes in dental caries lesions in rats

[0168] Depend on Figure 2 It can be seen that there are no obvious caries in the blank group and the chlorhexidine solution group. The front and top views of the model group show that the first, second and third molars have very obvious caries. The low-dose group of Example 1 has more obvious caries, but the depth of caries is shallower than that of the model group. Caries can also be seen in the medium-dose group of Example 1, and the caries is mainly concentrated in the second and third molars. The high-dose group of Example 1 also has shallow caries on the surface of its molars.

[0169] Based on Mirco-CT imaging observations, such as Figure 3 As shown, the enamel of the teeth in the blank group, CHX group, medium-dose group of Example 1 and high-dose group of Example 1 was relatively intact without caries; the enamel thickness of the molars in the model group and low-dose group of Example 1 was significantly reduced, and the caries in the pits and fissures of the three molars penetrated into the dentin, causing moderate and deep caries.

[0170] 3.2 Keyes score for caries lesions in rats

[0171] Table 4 Keyes Scoring Table for Caries in Rats

[0172]

[0173] Note: Surface: smooth surface caries; E: enamel caries; Ds: superficial dentin caries; Dm: caries within 3 / 4 of the dentin thickness; Dx: caries within 4 / 3 or the entire dentin depth.

[0174] The scoring results are shown in Table 4. Compared with the model group, all groups in Example 1 significantly improved the Keyes score for caries, especially the high-dose group, whose effect was similar to or even better than the positive control group.

[0175] 3.3 Results of the determination of biochemical indicators in rat saliva

[0176] Experimental results are as follows Figures 4-5 As shown, immunoglobulin A (IgA) levels in the saliva of normal rats and rats with dental caries are... Figure 4 ) and immunoglobulin G (IgG, Figure 5 The differences were significant; when treated with the traditional Chinese medicine composition obtained in Example 1 at three different dosage concentrations, the secretion of IgA and IgG in saliva was significantly different from that in the model group.

[0177] The above results indicate that stimulating the oral microecological environment through inoculation with pathogenic bacteria can promote increased secretion of IgA and IgG in oral tissues. With the use of the drug, the secretion of related immunoglobulins decreased significantly, indicating that the traditional Chinese medicine composition obtained in this invention has the effect of improving the oral microecology of caries-ridden rats.

[0178] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A traditional Chinese medicine composition, characterized in that, The extract is composed of an alcohol-extracted compound obtained from green plum, white peony root, licorice root, and sophora flower; wherein the mass ratio of green plum, white peony root, licorice root, and sophora flower is (2.6~3.5):(2~2.5):(2.6~3.5):

1. The solvent for the alcohol extraction is an ethanol solution; The concentration of the ethanol solution is ≥50 vol%. The alcohol extraction was performed using ultrasound-assisted extraction.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The mass ratio of the green fruit, white peony root, licorice root, and sophora flower is (2.8~3):(2~2.2):(2.8~3):

1.

3. The traditional Chinese medicine composition according to claim 1, characterized in that, The mass ratio of the green fruit, white peony root, licorice root, and sophora flower is 3:2:3:

1.

4. The traditional Chinese medicine composition according to any one of claims 1 to 3, characterized in that, The alcohol extraction is performed by mixing the raw materials of green plum, white peony root, licorice root and sophora flower and then performing alcohol extraction together, or by extracting the raw materials of green plum, white peony root, licorice root and sophora flower separately and then mixing them.

5. The traditional Chinese medicine composition according to any one of claims 1 to 3, characterized in that, The alcohol extraction includes the following steps: fully extracting a mixed powder containing green fruit, white peony root, licorice root and sophora flower in an ethanol solution to obtain an extract, and concentrating and drying the obtained extract to obtain the traditional Chinese medicine composition; Wherein, the concentration of the ethanol solution is ≥50 vol.

6. The use of the traditional Chinese medicine composition according to any one of claims 1 to 5 in the preparation of an oral therapeutic medicament having any of the following uses: (1) Inhibit and / or kill oral microorganisms; (2) Inhibit and / or remove biofilms formed by oral microorganisms; The oral microorganisms are Streptococcus mutans and / or distant Streptococcus.

7. The use of the traditional Chinese medicine composition according to any one of claims 1 to 5 in the preparation of oral care daily chemical products having any of the following uses: (1) Inhibit and / or kill oral microorganisms; (2) Inhibit and / or remove biofilms formed by oral microorganisms; The oral microorganisms are Streptococcus mutans and / or distant Streptococcus.

8. The use of the traditional Chinese medicine composition according to any one of claims 1 to 5 in the preparation of a drug for the prevention and / or treatment of dental caries.

9. An oral treatment drug, characterized in that, The active ingredient of the oral treatment drug includes the traditional Chinese medicine composition according to any one of claims 1 to 5.

10. A daily oral care product, characterized in that, The active ingredient of the oral care daily chemical product includes the traditional Chinese medicine composition according to any one of claims 1 to 5.

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