A paris polyphylla traditional chinese medicine composition and application thereof in preparation of oral cavity pathogenic bacteria activity inhibitor

Abstract

The application discloses a traditional Chinese medicine composition of paris polyphylla and application of the traditional Chinese medicine composition in preparation of an oral cavity pathogenic bacterium activity inhibitor. The traditional Chinese medicine extract and extract of the application have the advantages of being natural, efficient, inexpensive, low in toxicity, easy to obtain and the like, and have obvious inhibiting effects on formation of oral cavity pathogenic bacterium Streptococcus mutans biofilm. The biofilm inhibition rate of the paris polyphylla alcohol extract is above 90%, the bacteriostatic rate of the Coptis deltoidea water extract is above 90%, and the bacteriostatic rate and the biofilm inhibition rate of the combination of the two are above 99%. The active ingredient acquisition method of the application is simple, safe and low in cost, and is convenient to popularize and use. The application further provides a natural medicine combination caries prevention method. The combination of drugs can not only enhance the treatment effect, but also serve as an effective prevention means to reduce drug resistance of oral cavity pathogenic bacteria.

Description

(I) Technical Field

[0001] This invention relates to a Paris polyphylla herbal composition and its application in the preparation of an active inhibitor of oral pathogenic bacteria. (II) Background Technology

[0002] Oral health is a global public health concern, with dental caries being one of the most prevalent and widespread chronic infectious diseases. Dental caries and halitosis are common oral diseases in clinical practice, with a prevalence exceeding 50%. These diseases not only destroy the hard tissues of teeth, causing pain and infection, but also severely impact patients' nutritional intake and quality of life. *Streptococcus mutans*, as a major cariogenic bacterium, plays a crucial role in the occurrence and development of dental caries. Studies have shown that *Streptococcus mutans* promotes tooth demineralization and caries formation by forming biofilms and creating an acidic environment. The cariogenic mechanism of *Streptococcus mutans* includes its ability to form biofilms in the oral cavity. These biofilms possess strong antimicrobial and anti-host immune capabilities, allowing *Streptococcus mutans* to survive long-term in the oral cavity and cause dental caries. Therefore, *Streptococcus mutans* exacerbates tooth damage by forming highly acidic biofilms that are difficult to remove.

[0003] Biofilms are membrane-like aggregates with complex three-dimensional structures formed by the aggregation of microorganisms on living or non-living surfaces, composed of extracellular polymers such as extracellular polysaccharides, proteins, and nucleic acids. The formation of dental biofilms is a complex process. *Streptococcus mutans* first adheres to the tooth surface, then colonizes and aggregates with other oral bacteria to form dental plaque. During this process, *Streptococcus mutans* synthesizes water-insoluble dextran via glucosyltransferases, providing binding sites for bacterial adhesion to the tooth surface and promoting biofilm formation. Biofilms provide protection for bacteria, but also increase their drug resistance, adhesion, and pathogenicity, leading to recurrent and difficult-to-treat infectious diseases. Therefore, inhibiting the formation of *Streptococcus mutans* biofilms is one of the main strategies for preventing dental caries.

[0004] The occurrence of oral diseases is closely related to the imbalance of the oral microbiota. Clinically, antibiotics and antifungal drugs are commonly used for treatment, but long-term or excessive use can easily disrupt the oral microbial ecosystem and even lead to the emergence of drug-resistant strains. Furthermore, oral pathogens often exist in the form of biofilms, and biofilm structures can significantly increase the tolerance of bacteria to drugs and the host's immune system, thereby reducing the efficacy of traditional antibacterial drugs. Fluoride and chlorhexidine are currently commonly used caries prevention drugs. Fluoride is effective in inhibiting the development of caries, but high-concentration application may cause cytotoxicity problems; while chlorhexidine has antibacterial effects, long-term or frequent use may lead to tartar buildup and oral irritation. Therefore, developing new, low-toxicity, and sustainable caries prevention strategies has become an urgent need for the prevention and treatment of oral diseases. In recent years, the potential of natural medicines in the prevention and treatment of oral diseases has gradually attracted attention. my country has a rich variety of traditional Chinese medicines, which have advantages in regulating the oral microecology, inhibiting the growth of pathogenic bacteria, and preventing infection. Multiple studies have shown that extracts of traditional Chinese medicine have significant inhibitory effects on oral pathogens such as Streptococcus mutans, and generally exhibit characteristics such as low toxicity, low drug resistance, and environmental friendliness, making them more suitable for long-term or preventative use compared to chemical drugs. However, systematic research on the effects of natural medicines on biofilms, the synergistic anti-caries effects between different drugs, and their combined application with existing caries prevention measures remains insufficient.

[0005] This invention explores the inhibitory effects of Paris polyphylla and other traditional Chinese medicine components on oral pathogens and biofilms by screening them, and further investigates the anti-caries effects of combined use of natural drugs, providing a new strategy for the prevention and treatment of oral diseases. This research not only helps enrich the application of traditional Chinese medicine in oral care, but also provides a feasible alternative to reduce antibiotic use and lower the risk of drug resistance. (III) Summary of the Invention

[0006] The purpose of this invention is to provide a *Paris polyphylla* herbal composition and its application in the preparation of an active inhibitor of oral pathogenic bacteria, solving the problems of antibiotic overuse, high risk of drug resistance, and limited antibacterial effect of single components in existing oral care products. The herbal composition used in this invention can synergistically inhibit the growth of *Streptococcus mutans* and the formation of biofilms, thereby effectively reducing the incidence of oral diseases and providing a new technical means and feasible solution for developing safe and effective natural oral care products.

[0007] The technical solution adopted in this invention is:

[0008] This invention provides a Paris polyphylla herbal composition for inhibiting oral pathogenic bacteria, the Paris polyphylla herbal composition comprising one of the following: Paris polyphylla alcohol extract (preferably ethanol extract) or an organic solvent extract phase of the alcohol extract, and Coptis chinensis water extract or an organic solvent extract phase of the water extract.

[0009] Furthermore, the organic solvent is n-butanol or ethyl acetate.

[0010] Furthermore, the Paris polyphylla herbal composition consists of the n-butanol extract phase of the Paris polyphylla alcohol extract and the n-butanol extract phase of the Coptis chinensis water extract.

[0011] Furthermore, the mass ratio of the n-butanol extract phase of the Paris polyphylla alcohol extract to the n-butanol extract phase of the Coptis chinensis water extract is 1:0.01-4, preferably 1:0.05-0.5.

[0012] Furthermore, the Paris polyphylla alcohol extract is prepared according to the following method:

[0013] The dried Paris polyphylla was pulverized and passed through a 50-mesh sieve to obtain Paris polyphylla powder. The Paris polyphylla powder was added to anhydrous ethanol and subjected to a first extraction by heating and reflux for 1-3 hours (preferably 3 hours). After cooling, it was filtered through three layers of gauze to obtain the first filtrate and the first residue. The first residue was then subjected to a second extraction by heating and reflux with anhydrous ethanol for 1-3 hours (preferably 2 hours). After cooling, it was filtered through three layers of gauze to obtain the second filtrate. The first and second filtrates were combined and vacuum filtered through filter paper (diameter: 12.5 cm). The filtrate was then concentrated to 1 / 10 of its original volume using a rotary evaporator to obtain the crude extract. The crude extract was freeze-dried at 4°C for 12 hours using a vacuum freeze concentrator to obtain Paris polyphylla alcohol extract powder.

[0014] Furthermore, the volume of anhydrous ethanol used in the first or second heating reflux is 2-8 mL / g (preferably 5 mL / g) based on the mass of Paris polyphylla powder.

[0015] Furthermore, the organic solvent extract phase of the Paris polyphylla alcohol extract is prepared according to the following method:

[0016] The Paris polyphylla alcohol extract was fully dissolved in anhydrous ethanol. Organic solvent was added to a separatory funnel, and the mixture was inverted and mixed thoroughly. After standing for 30 min, the upper layer was transferred. An equal volume of organic solvent was added to the lower layer solution, and the mixture was inverted and mixed thoroughly. After standing for 30 min, the upper layer was transferred again until the upper layer was colorless. All the upper layers were mixed together and concentrated under vacuum using a rotary evaporator to 1 / 10 of the original volume to obtain the crude extract. The crude extract was freeze-dried at 4°C for 12 h using a vacuum freeze concentrator to obtain the organic solvent extract phase powder of the Paris polyphylla alcohol extract.

[0017] Furthermore, the volume of anhydrous ethanol used is 5-15 mL / g (preferably 10 mL / g) based on the mass of the Paris polyphylla ethanol extract. The volume ratio of the organic solvent to anhydrous ethanol is 1:1.

[0018] Furthermore, the Coptis chinensis water extract is prepared according to the following method:

[0019] The dried Coptis chinensis was pulverized and passed through a 50-mesh sieve to obtain Coptis chinensis powder. The Coptis chinensis powder was added to deionized water and subjected to a first extraction by heating and reflux for 1-3 hours (preferably 3 hours). After cooling, it was filtered through three layers of gauze to obtain a first filtrate and a first residue. The first residue was then subjected to a second extraction by heating and reflux with deionized water for 1-3 hours (preferably 2 hours). After cooling, it was filtered through three layers of gauze to obtain a second filtrate. The first and second filtrates were combined and vacuum filtered through filter paper (diameter: 12.5 cm). The filtrate was then concentrated to 1 / 10 of its original volume using a rotary evaporator to obtain a crude extract. The crude extract was freeze-dried at 4°C for 12 hours using a vacuum freeze concentrator to obtain Coptis chinensis water extract powder.

[0020] Furthermore, the volume of deionized water used in the first or second heating reflux is 2-8 mL / g (preferably 5 mL / g) based on the mass of Coptis chinensis powder.

[0021] Furthermore, the organic solvent extract phase of the Coptis chinensis aqueous extract is prepared according to the following method:

[0022] The aqueous extract of Coptis chinensis was fully dissolved in deionized water. Organic solvent was added to a separatory funnel, and the mixture was inverted and mixed thoroughly. After standing for 30 min, the upper layer was transferred. An equal volume of organic solvent was added to the lower layer solution, and the mixture was inverted and mixed thoroughly. After standing for 30 min, the upper layer was transferred again until the upper layer was colorless. All the upper layers were mixed together and concentrated under vacuum to 1 / 10 of the original volume using a rotary evaporator to obtain the crude extract. The crude extract was freeze-dried at 4°C for 12 h using a vacuum freeze concentrator to obtain the organic solvent extract phase powder of the aqueous extract of Coptis chinensis.

[0023] Furthermore, the volume of deionized water used is 5-15 mL / g (preferably 10 mL / g) based on the mass of the Coptis chinensis water extract. The volume ratio of the organic solvent to the deionized water is 1:1.

[0024] The present invention also provides the application of the aforementioned Paris polyphylla herbal composition in the inhibition of oral pathogenic bacteria.

[0025] Furthermore, the oral pathogens include Streptococcus mutans.

[0026] Furthermore, the inhibitors include inhibitors of oral pathogenic bacteria activity or inhibitors of oral pathogenic bacteria forming biofilms.

[0027] Furthermore, among the inhibitors, the effective antibacterial concentration of the n-butanol extract phase of the Paris polyphylla ethanol extract is 6.25 mg / ml-12.5 mg / ml, and the effective antibacterial concentration of the n-butanol extract phase of the Coptis chinensis water extract is 6.25 mg / ml-12.5 mg / ml.

[0028] The present invention also provides the application of the aforementioned Paris polyphylla herbal composition in the preparation of dental caries prevention and treatment drugs.

[0029] Compared with existing methods, the beneficial effects of this invention are mainly reflected in:

[0030] The herbal extracts and extracts of this invention have advantages such as being natural, highly efficient, inexpensive, low in toxicity, and readily available. They exhibit significant inhibitory effects on the formation of biofilms of oral pathogens such as Streptococcus mutans. Specifically, the biofilm inhibition rate of the Paris polyphylla alcohol extract exceeds 90%, and the antibacterial rate of the Coptis chinensis water extract exceeds 90%. When used in combination, the antibacterial and biofilm inhibition rates reach over 99%. The method for obtaining the active ingredients of this invention is simple, safe, and low-cost, facilitating widespread use. This invention also provides a method for combined use of natural drugs to prevent dental caries. Combined use not only enhances the therapeutic effect but also serves as an effective preventative measure, reducing the occurrence of drug resistance in oral pathogens. (iv) Description of the attached drawings

[0031] Figure 1 Effects of Paris polyphylla ethanol extract and Coptis chinensis water extract on the biofilm of Streptococcus mutans GDMCC1.369. A. 24 h of culture; B. 48 h of culture; C. OD of Streptococcus mutans GDMCC1.369 after treatment with the three herbal extracts. 540 nm value.

[0032] Figure 2 The effect of different extraction times of Paris polyphylla ethanol extract on the biofilm formation of Streptococcus mutans GDMCC1.369. A. 24 h of culture; B. 48 h of culture; C. OD of Paris polyphylla ethanol extract relative to Streptococcus mutans GDMCC1.369 treatment. 540 nm value. (V) Detailed Implementation Methods

[0033] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0034] The Paris polyphylla of this invention is a plant belonging to the Liliaceae family. Anemarrhena asphodeloides The dried rhizome of *Magnolia officinalis*. *Magnolia officinalis* is a plant of the Magnoliaceae family. Magnolia officinalis The bark of Rehd. et Wils. Coptis chinensis is a plant belonging to the Ranunculaceae family. Coptis chinensis The dried rhizome of Franch.

[0035] Example 1: Preparation of Chinese herbal extracts and their extracts

[0036] 1. Preparation of extracts from traditional Chinese medicine

[0037] The dried Paris polyphylla was pulverized using a pulverizer and passed through a 50-mesh sieve to obtain Paris polyphylla powder. 200 g of the powder was weighed and added to 1 L of anhydrous ethanol. The mixture was refluxed for 3 h, cooled, and filtered through three layers of gauze to obtain the first filtrate and the first residue. The first residue was then refluxed again with 1 L of anhydrous ethanol for 2 h, cooled, and filtered through three layers of gauze to obtain the second filtrate. The first and second filtrates were combined and vacuum filtered through filter paper (12.5 cm in diameter). The extract was then concentrated to 1 / 10 of its original volume using a rotary evaporator to obtain the crude extract. The crude extract was transferred to a 50 mL centrifuge tube and freeze-dried at 4°C for 12 h using a vacuum freeze-drying apparatus to obtain 22 g of Paris polyphylla alcohol extract powder.

[0038] Using the same method, Paris polyphylla was replaced with Magnolia officinalis (as a control group, the extract of the Chinese herbal medicine that did not inhibit growth and biofilm formation), resulting in 17 g of Magnolia officinalis alcohol extract powder.

[0039] Using the same method, Paris polyphylla was replaced with Coptis chinensis, and anhydrous ethanol was replaced with deionized water, resulting in 29 g of Coptis chinensis water extract powder.

[0040] 2. Preparation of extracts from traditional Chinese medicine (Magnolia officinalis was used as a control group, and only the extract was tested).

[0041] Weigh 10 g of the obtained Paris polyphylla alcohol extract and dissolve it thoroughly in 100 ml of anhydrous ethanol. Add petroleum ether to the separatory funnel at a 1:1 (v:v) ratio, mix by inverting, and let stand for 30 min. Transfer the upper layer (i.e., the petroleum ether phase). Add an equal volume of petroleum ether to the lower layer solution, mix by inverting, and let stand for 30 min. Transfer the upper layer until the petroleum ether phase (upper layer) is colorless. Mix all the petroleum ether phases together and concentrate them under vacuum to 1 / 10 of the original volume using a rotary evaporator to obtain the crude extract. Transfer the crude extract to a 50 mL centrifuge tube and freeze-dry it at 4℃ for 12 h using a vacuum freeze concentrator to obtain 0.2 g of Paris polyphylla alcohol extract petroleum ether phase powder (referred to as Paris polyphylla petroleum ether phase).

[0042] Using the same method, petroleum ether was replaced with ethyl acetate to obtain 2.3 g of ethyl acetate phase of Paris polyphylla alcohol extract (hereinafter referred to as Paris polyphylla ethyl acetate phase) powder.

[0043] Using the same method, petroleum ether was replaced with n-butanol to obtain 5.2 g of n-butanol phase of Paris polyphylla extract (referred to as Paris polyphylla n-butanol phase) powder.

[0044] Using the same method, Paris polyphylla was replaced with Coptis chinensis, and anhydrous ethanol was replaced with deionized water, resulting in 0.1 g, 2.3 g, and 4.5 g of petroleum ether phase (for detecting petroleum ether phase of Coptis chinensis), ethyl acetate phase (for short, ethyl acetate phase of Coptis chinensis), and n-butanol phase (for short, n-butanol phase of Coptis chinensis) powders of Coptis chinensis water extract, respectively.

[0045] Example 2: Antibacterial activity test of traditional Chinese medicine extracts and their various extract phases

[0046] 1. Test strains and their culture

[0047] Streptococcus mutans GDMCC1.369 ( Streptococcus mutans ATCC25175 (purchased from the Guangdong Institute of Microbiology's Culture Preservation Center) was streaked onto BHI plates at -80℃ and activated in a 37℃ incubator for 24 h. Three single colonies were picked and inoculated into test tubes containing 5 mL of BHI liquid medium, and incubated overnight at 37℃. The OD concentration of the bacterial culture was then measured. 600 nm The bacterial concentration was diluted to OD using fresh BHI liquid medium. 600 nm =0.1, used as a dilution for the bacterial solution.

[0048] Brain Heart Infusion Broth (BHI) medium (purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.) consisted of: 10.0 g tryptone, 17.5 g beef heart extract powder, 5.0 g sodium chloride, 2.0 g glucose, 2.5 g disodium hydrogen phosphate, 1.0 L distilled water, and natural pH. BHI plates were prepared by adding 15 g / L agar to BHI liquid medium.

[0049] 2. Determination of minimum inhibitory concentration (MIC) using 96-well plate bacterial culture method.

[0050] Weigh 0.2 g of the Coptis chinensis aqueous extract or each extract phase powder obtained in Example 1, and dissolve it in 1 mL of sterile deionized water to obtain a Coptis chinensis aqueous extract with a concentration of 200 mg / mL and its mother liquor for each extract phase. Then, the Coptis chinensis aqueous extract and each extract phase were serially diluted twice to eight concentrations using BHI liquid medium to obtain diluted solutions of Coptis chinensis aqueous extract and its extract phase with concentrations of 100 mg / mL, 50 mg / mL, 25 mg / mL, 12.5 mg / mL, 6.25 mg / mL, 3.125 mg / mL, 1.5625 mg / mL, and 0.78125 mg / mL.

[0051] 100 μl of diluted bacterial culture and 100 μl of different concentrations of Coptis chinensis aqueous extract or dilutions of each extract phase were added to the wells of a 96-well plate and mixed well. This served as the experimental group, with three replicates for each concentration. 100 μl of BHI liquid culture medium and 100 μl of diluted bacterial culture were added to the wells of a 96-well plate and mixed well. This served as the positive control group. 200 μl of BHI liquid culture medium was added to the wells of a 96-well plate. This served as the negative control group, with three replicates for each group. After incubating the 96-well plates at 37℃ for 24 h, the OD was measured using a microplate reader. 600 nm Numerical values ​​are recorded. When antimicrobial substances inhibit bacterial growth, OD... 600 nm The levels were significantly lower than the negative control group. Combined with the MIC (minimum inhibitory concentration) of the extract, it was found that the aqueous extract of Coptis chinensis and its various extracts had significant antibacterial effects against Streptococcus mutans. The MIC values ​​were: 12.5 mg / mL for the aqueous extract, 6.25 mg / mL for the n-butanol phase of the aqueous extract, and 12.5 mg / mL for the ethyl acetate phase of the aqueous extract. The petroleum ether phase of the aqueous extract showed no significant antibacterial effect. Therefore, the n-butanol phase of the aqueous extract was selected as the subsequent antibacterial material.

[0052] Using the same method, the water extract of Coptis chinensis and its various extraction phases were replaced with the alcohol extract of Paris polyphylla, and sterile deionized water was replaced with anhydrous ethanol. The results showed that the alcohol extract of Paris polyphylla had no significant antibacterial effect on Streptococcus mutans.

[0053] Example 3: Biofilm Activity Test of Traditional Chinese Medicine Extracts

[0054] Following step 1 in Example 2, Streptococcus mutans GDMCC1.369 was cultured and diluted to obtain OD. 600 nm=0.1 dilution of bacterial suspension. Weigh 0.2 g of the Paris polyphylla ethanol extract powder obtained in Example 1, dissolve it in 1 mL of anhydrous ethanol to obtain a Paris polyphylla ethanol extract stock solution with a concentration of 200 mg / mL. Serially dilute the Paris polyphylla ethanol extract stock solution to 25 mg / mL using BHI liquid medium. Add 100 μl of diluted bacterial suspension and 100 μl of diluted Paris polyphylla ethanol extract to each well of a 96-well plate (200 μl per well, final drug concentration of 12.5 mg / mL), with 6 replicates (one column each), as the experimental group. Add 100 μl of BHI liquid medium and 100 μl of diluted Paris polyphylla ethanol to each well of a 96-well plate, with 6 replicates (one column each), as the negative control to avoid experimental errors caused by the color of the drug itself. Add 100 μl of BHI liquid medium containing an equal volume of anhydrous ethanol of the above-mentioned Paris polyphylla ethanol extract and 100 μl of diluted bacterial suspension to each well of a 96-well plate, with 6 replicates (one column each), as positive control 1. Using the same method, a 96-well plate was repeatedly loaded to analyze the effect of the drug on the biofilm of *Streptococcus mutans* at different incubation times. The 96-well plates were incubated at 37°C, and samples were taken at 24 h and 48 h to measure OD. 620 nm After recording the values, discard the bacterial culture and wash the 96-well plate 2-3 times with distilled water. Then, add 220 μl of 0.1% crystal violet solution and stain for 20 min. Discard the liquid and wash away excess dye with distilled water (2-3 times). Finally, add 220 μl of anhydrous ethanol solution to each sample well to dissolve the crystal violet, let stand for 5 min, and then measure the OD. 540 nm Value, OD 540 nm A higher value indicates a greater amount of biofilm formed. The quantitative formula for biofilm formation is OD. 540 nm =sample's OD 540 nm - Negative control OD 540 nm Biofilm activity tests revealed that the ethanol extract of Paris polyphylla significantly inhibited the formation of Streptococcus mutans biofilm, with the inhibitory effect lasting up to 48 hours. Figure 1 ).

[0055] Using the same method, the ethanol extract of Paris polyphylla was replaced with the aqueous extract of Coptis chinensis, and the anhydrous ethanol was replaced with sterile deionized water. Simultaneously, 100 μl of BHI liquid medium containing an equal volume of deionized water and 100 μl of diluted bacterial suspension were added to a 96-well plate, with 6 replicates (i.e., one column) as positive control 2. The results showed that the aqueous extract of Coptis chinensis significantly inhibited the formation of Streptococcus mutans biofilm. Figure 1The inhibitory effect mainly comes from its antibacterial activity. Considering that the n-butanol phase of the Coptis chinensis water extract used later has significant antibacterial activity, it will also inhibit the formation of biofilm. Therefore, it is not necessary to measure the biofilm inhibitory activity of the n-butanol phase of the Coptis chinensis water extract.

[0056] Example 4: Biofilm activity test of each extract phase of Paris polyphylla alcohol extract (the extract phase of Coptis chinensis has obvious antibacterial activity, so biofilm activity was not tested).

[0057] Following step 1 in Example 2, Streptococcus mutans GDMCC1.369 was cultured and diluted to obtain OD. 600nm =0.1 dilution of bacterial suspension. Weigh 0.1 g of each extract phase of the Paris polyphylla ethanol extract obtained in Example 1 (hereinafter referred to as each Paris polyphylla extract phase) powder, dissolve in 0.5 mL of anhydrous ethanol to obtain a stock solution of each Paris polyphylla extract phase with a concentration of 200 mg / mL. Serially dilute each Paris polyphylla extract phase stock solution to 25 mg / mL using BHI liquid medium. Add 100 μl of diluted bacterial suspension and 100 μl of diluted Paris polyphylla extract phase to each well of a 96-well plate (200 μl per well, final drug concentration of 12.5 mg / mL), with 6 replicates (i.e., one column) for each well, as the experimental group. Add 100 μl of BHI liquid medium and 100 μl of diluted Paris polyphylla extract phase to each well of a 96-well plate, with 6 replicates (i.e., one column), as the negative control group to avoid experimental errors caused by the color of the drug itself. Add 100 μl of BHI liquid medium and 100 μl of diluted bacterial suspension to each well of a 96-well plate, with 12 replicates (i.e., two columns), as the positive control group. Using the same method, a 96-well plate was repeatedly loaded to analyze the effect of the drug on the biofilm of *Streptococcus mutans* at different incubation times. The 96-well plates were incubated at 37°C, and samples were taken at 24 h and 48 h to measure OD. 620 nm After recording the values, discard the bacterial culture and wash the 96-well plate 2-3 times with distilled water. Then, add 220 μl of 0.1% crystal violet solution to stain for 20 min, discard the liquid, and wash away excess dye with distilled water (2-3 times). Finally, add 220 μl of anhydrous ethanol solution to each sample well to dissolve the crystal violet, let stand for 5 min, and then measure the OD. 540 nm Value, OD 540 nm A higher value indicates a greater amount of biofilm formed. The quantitative formula for biofilm formation is OD. 540 nm =sample's OD 540 nm - Negative control OD 540 nm Biofilm activity tests revealed that the ethyl acetate and n-butanol phases of Paris polyphylla significantly inhibited the formation of Streptococcus mutans biofilms, with the inhibitory effect lasting up to 48 hours; the petroleum ether phase of Paris polyphylla showed no significant inhibitory effect. Figure 2 ).

[0058] Example 5: Antibacterial activity test of synergistic interaction between the n-butanol phase of Coptis chinensis and the n-butanol phase of Paris polyphylla

[0059] 1. Dilution of the n-butanol extract phase of traditional Chinese medicine extracts

[0060] Following step 1 in Example 2, Streptococcus mutans GDMCC1.369 was cultured and diluted to obtain OD. 600nm =0.1 dilution of bacterial suspension. 0.2 g of the Coptis chinensis n-butanol phase powder obtained in Example 1 was weighed and dissolved in 1 mL of sterile deionized water to obtain a 200 mg / mL Coptis chinensis n-butanol phase stock solution. Then, the Coptis chinensis n-butanol phase was serially diluted eight times using BHI liquid medium to obtain Coptis chinensis n-butanol phase dilutions with concentrations of 100 mg / mL, 50 mg / mL, 25 mg / mL, 12.5 mg / mL, 6.25 mg / mL, 3.125 mg / mL, 1.5625 mg / mL, and 0.78125 mg / mL.

[0061] Using the same method, the Coptis chinensis n-butanol phase was replaced with the Paris polyphylla n-butanol phase and diluted to 25 mg / mL with BHI liquid medium.

[0062] 2. Antibacterial activity test of synergistic interaction between the n-butanol phase of Coptis chinensis and the n-butanol phase of Paris polyphylla

[0063] 100 μl of diluted bacterial suspension, 50 μl of different concentrations of the n-butanol phase of Coptis chinensis, and 50 μl of the n-butanol phase of Paris polyphylla (25 mg / mL) were added to 96-well plates and mixed well, forming experimental groups with three replicates for each concentration. 100 μl of diluted bacterial suspension and 100 μl of different concentrations of the n-butanol phase of Coptis chinensis served as positive control group 1. 100 μl of diluted bacterial suspension and 100 μl of the n-butanol phase of Paris polyphylla (25 mg / mL) served as positive control group 2. 100 μl of BHI liquid culture medium and 100 μl of diluted bacterial suspension served as negative control groups, and 200 μl of BHI liquid culture medium served as blank control group, with three replicates for each group. After incubating the 96-well plates at 37℃ for 24 h, the OD600 nm value was measured using a microplate reader, and the results were recorded.

[0064] The results showed that the butanol phase of *Paris polyphylla* in positive control group 2 had no antibacterial activity, the MIC of the butanol phase of *Coptis chinensis* in positive control group 1 was 6.25 mg / mL, and the MIC of the butanol phase of *Coptis chinensis* in the experimental group with added butanol phase was 3.125 mg / mL. These results indicate that synergistic use can significantly enhance the antibacterial effect of *Coptis chinensis* extract and promote its antibacterial activity.

Claims

1. A Chinese herbal composition containing Paris polyphylla, characterized in that, The Paris polyphylla herbal composition includes one of the following: Paris polyphylla alcohol extract or an organic solvent extract phase of the alcohol extract, and Coptis chinensis water extract or an organic solvent extract phase of the water extract.

2. The Paris polyphylla herbal composition as described in claim 1, characterized in that, The organic solvent is n-butanol or ethyl acetate.

3. The Paris polyphylla herbal composition as described in claim 1, characterized in that, The Paris polyphylla herbal composition consists of the n-butanol extract phase of the Paris polyphylla alcohol extract and the n-butanol extract phase of the Coptis chinensis water extract.

4. The Paris polyphylla herbal composition as described in claim 3, characterized in that, The mass ratio of the n-butanol extract phase of the Paris polyphylla alcohol extract to the n-butanol extract phase of the Coptis chinensis water extract is 1:0.01-4.

5. The Paris polyphylla herbal composition as described in claim 1, characterized in that, The Paris polyphylla alcohol extract and extract phase are prepared as follows: (1) The dried Paris polyphylla is pulverized and passed through a 50-mesh sieve to obtain Paris polyphylla powder; the Paris polyphylla powder is added to anhydrous ethanol and heated under reflux for 1-3 hours for the first extraction. After cooling, it is filtered through three layers of gauze to obtain the first filtrate and the first residue. The first residue is then heated under reflux for 1-3 hours for the second extraction with anhydrous ethanol. After cooling, it is filtered through three layers of gauze to obtain the second filtrate. The first filtrate and the second filtrate are combined and vacuum filtered. The filtrate is then concentrated to 1 / 10 of its original volume using a rotary evaporator to obtain the crude extract. The crude extract is freeze-dried at 4°C for 12 hours using a vacuum freeze concentrator to obtain Paris polyphylla alcohol extract powder; (2) The Paris polyphylla alcohol extract is fully dissolved in anhydrous ethanol. Organic solvent is added to a separatory funnel, and the mixture is mixed by inverting the funnel and allowed to stand for 30 minutes. After 30 minutes, transfer the upper layer; add an equal volume of organic solvent to the lower layer solution, mix by inverting, and let stand for 30 minutes. Transfer the upper layer until it is colorless; mix all the upper layers together and concentrate them to 1 / 10 of the original volume using a rotary evaporator to obtain the crude extract; freeze-dry the crude extract at 4°C for 12 hours using a vacuum freeze concentrator to obtain the organic solvent extract phase powder of the Paris polyphylla alcohol extract.

6. The Paris polyphylla herbal composition as described in claim 5, characterized in that, In step (1), the volume of anhydrous ethanol used in the first or second heating reflux is 2-8 mL / g based on the mass of Paris polyphylla powder; in step (2), the volume of anhydrous ethanol used is 5-15 mL / g based on the mass of Paris polyphylla alcohol extract, and the volume ratio of the organic solvent to anhydrous ethanol is 1:

1.

7. The Paris polyphylla herbal composition as described in claim 1, characterized in that, The Coptis chinensis water extract and extract phase are prepared as follows: (1) The dried Coptis chinensis is pulverized and passed through a 50-mesh sieve to obtain Coptis chinensis powder; the Coptis chinensis powder is added to deionized water and heated under reflux for 1-3 hours for the first time. After cooling, it is filtered with three layers of gauze to obtain the first filtrate and the first residue. The first residue is then heated under reflux for 1-3 hours for the second time with deionized water. After cooling, it is filtered with three layers of gauze to obtain the second filtrate. The first filtrate and the second filtrate are combined and vacuum filtered. The filtrate is then concentrated to 1 / 10 of its original volume using a rotary evaporator to obtain the crude extract. The crude extract is freeze-dried at 4°C for 12 hours using a vacuum freeze concentrator to obtain Coptis chinensis water extract powder; (2) The Coptis chinensis water extract is fully dissolved in deionized water. Organic solvent is added to the separatory funnel, and the mixture is mixed by inverting the funnel and then allowed to stand for 30 minutes. After 30 minutes, transfer the upper layer; add an equal volume of organic solvent to the lower layer solution, mix by inverting, and let stand for 30 minutes. Transfer the upper layer until it is colorless; mix all the upper layers together and concentrate them to 1 / 10 of the original volume using a rotary evaporator to obtain the crude extract; freeze-dry the crude extract at 4°C for 12 hours using a vacuum freeze concentrator to obtain the organic solvent extract phase powder of Coptis chinensis water extract.

8. The use of the Paris polyphylla composition of claim 1 in an oral pathogenic bacteria inhibitor.

9. The application as described in claim 8, characterized in that, The inhibitors include inhibitors of oral pathogenic bacteria activity or inhibitors of oral pathogenic bacteria forming biofilms.

10. The use of the Paris polyphylla composition of claim 1 in the preparation of a caries prevention and treatment drug.

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