Composition containing Polygonum cuspidatum extract for inhibiting oral malodor-causing bacteria and oral care product

Abstract

The present invention discloses a composition, which comprises a Polygonum cuspidatum extract and carvacrol, or comprises a Polygonum cuspidatum extract and cinnamaldehyde. The present invention also discloses the use of the composition in inhibiting oral odor-causing bacteria, and discloses an oral care product comprising the composition. The composition provided by the present invention has strong antibacterial activity against oral odor-causing bacteria, is easy to penetrate into bacterial cells, and can interfere with the mechanism of bacterial quorum sensing.

Description

Technical Field

[0001] The invention relates to the technical field of oral care, and in particular to a composition containing a knotweed extract for inhibiting oral odor-causing bacteria and an oral care product. Background Art

[0002] Microorganisms in the oral cavity are highly concentrated locally, especially in the tongue, teeth and periodontal tissues, forming biofilms, which are the main cause of oral halitosis. Biofilm is a type of organized bacterial community that can attach to biological or non-biological surfaces and is embedded in a protective extracellular polymer matrix (EPS) to form a stable three-dimensional structure. This three-dimensional structure can provide a refuge for bacteria to avoid oral saliva flow and the host's defense system. Compared with planktonic bacteria, mature biofilms have enhanced toxicity and prevent antibacterial agents from penetrating into the bacteria, that is, the bacterial population that forms biofilms has increased resistance to antibacterial agents and requires higher concentrations of antibacterial agents to be eliminated. Oral halitosis bacteria protected by three-dimensional biofilms continue to decompose sulfur-containing substrates in food or saliva, and the main volatile sulfur compounds (VSCs) produced in oral halitosis are generated.

[0003] For oral halitosis, oral care products containing antibacterial agents such as chlorhexidine and triclosan or antibiotics such as metronidazole used clinically, although they work quickly in the short term, cannot completely solve the problem; long-term use also has the risk of destroying the balance of oral microecology and developing drug resistance, making treatment more difficult. Among them, according to the description of the adverse reactions of chlorhexidine in the medical encyclopedia, its adverse reactions include: 1. It can cause contact dermatitis, and high-concentration solutions are highly irritating to the conjunctiva of the eyes; 2. Oral gargles can stain teeth, cause taste disorders, and cause painless superficial desquamation in the mouths of children and young people; 3. A solution with a concentration of 0.05 is used to disinfect skin, mucous membranes or wounds. The time for allergic reaction is 5 to 40 minutes after medication, and localized or systemic urticaria occurs. There may also be cough, asthma, dyspnea, abdominal pain, conjunctival edema, shock and other reactions.

[0004] Therefore, in the face of the problems of strong virulence and drug resistance of biofilms of pathogenic bacteria causing oral halitosis, patients with oral halitosis need a composition that has antibacterial activity, can easily penetrate into bacterial cells, and can interfere with the mechanism of bacterial quorum sensing. Summary of the invention

[0005] The first object of the present invention is to provide a composition that can effectively inhibit oral malodor-causing bacteria, the composition has strong antibacterial activity against oral malodor-causing bacteria, can easily penetrate into bacterial cells, and can interfere with the mechanism of bacterial quorum sensing.

[0006] The second object of the present invention is to provide an oral care product comprising the above composition.

[0007] Specifically, the technical solution provided by the present invention is as follows:

[0008] A first aspect of the present invention provides a composition comprising a knotweed extract and carvacrol.

[0009] Polygonum cuspidatum is the stem and root of Polygonum cuspidatum Sieb.etZucc, a plant of the Polygonaceae family. It is an important traditional Chinese medicine in my country. It has the effects of removing dampness and jaundice, clearing away heat and detoxifying, dispersing blood stasis and relieving pain, relieving cough and reducing phlegm. It can be used to treat damp-heat jaundice, stranguria, leucorrhea, rheumatic pain, carbuncle, burns, amenorrhea, abdominal mass, sprain, and lung heat cough. The main components of Polygonum cuspidatum extract are diphenyl glycosides, anthraquinones, and flavonoids, including polydatin, resveratrol, rheumatoid arthritis, and rheumatoid arthritis. It has a wide range of physiological activities.

[0010] Carvacrol is an aromatic phenolic monoterpene compound, commonly found in volatile oils of aromatic plants such as thyme and oregano. It can be used as a flavor additive in soaps, toothpastes, and cosmetics. Low concentrations of carvacrol can also be used as a food additive in various foods. Carvacrol has a variety of biological activities, such as antioxidant, antibacterial, anti-inflammatory, anti-tumor, and vasodilator effects.

[0011] The inventors found during the experiment that when the Polygonum cuspidatum extract and carvacrol were used in combination, the minimum inhibitory concentration (MIC) for Porphyromonas gingivalis could be greatly reduced. Specifically, the MIC value of the combined use of the Polygonum cuspidatum extract was only 1 / 17 to 1 / 18 of the MIC value of the Polygonum cuspidatum extract alone, and the MIC value of the combined use of carvacrol was only 1 / 3 to 1 / 4 of the MIC value of carvacrol alone. It can be seen that when the Polygonum cuspidatum extract and carvacrol are used in combination, a synergistic effect will occur, which improves the inhibitory activity against Porphyromonas gingivalis.

[0012] In addition, when the Polygonum cuspidatum extract is used in combination with carvacrol, the minimum inhibitory concentration (MBIC) for the biofilm formed by Porphyromonas gingivalis can be greatly reduced. Specifically, the MBIC value of the combined use of the Polygonum cuspidatum extract is only 1 / 3 to 1 / 4 of the MBIC value of the Polygonum cuspidatum extract alone, and the MBIC value of the combined use of carvacrol is only 1 / 3 of the MBIC value of carvacrol alone. Therefore, when the Polygonum cuspidatum extract is used in combination with carvacrol, the inhibitory activity against the biofilm formed by Porphyromonas gingivalis is also improved.

[0013] The polygonum cuspidatum extract of the present invention can be either a commercially available polygonum cuspidatum extract or a polygonum cuspidatum extract obtained by processing and extracting polygonum cuspidatum Chinese medicine. An exemplary extraction method is: placing polygonum cuspidatum medicinal material powder in an alcohol solution of 5 to 10 times its weight, soaking for 5 to 60 minutes; then heating and refluxing at 50°C to 80°C for 1 to 3 hours, extracting 1 to 3 times, combining the filtrate and concentrating to obtain the polygonum cuspidatum extract.

[0014] In the above extraction method, the alcohol solution can be methanol, ethanol, acetone or their aqueous solutions, preferably ethanol or ethanol aqueous solutions of different concentrations, more preferably 50% to 90% ethanol aqueous solution.

[0015] The method for extracting Polygonum cuspidatum described in the present invention is not limited to the above method, and other methods such as supercritical extraction and ultrasonic extraction can also be used for extraction.

[0016] It should be pointed out that the Polygonum cuspidatum extract in the present invention is not limited to the extract solution, but can also be in other forms such as pure extract powder or extract.

[0017] In a preferred embodiment, in the composition, the concentration of the Polygonum cuspidatum extract is 6.25 mg / mL≤6.88 mg / mL, and the concentration of carvacrol is 160.00 μg / mL≤266.50 μg / mL. The composition of the Polygonum cuspidatum extract and carvacrol at this concentration has a good inhibitory effect on planktonic Porphyromonas gingivalis.

[0018] In the present invention, the concentration of the Polygonum cuspidatum extract is the concentration calculated based on the amount of the raw drug of Polygonum cuspidatum medicinal material, that is, the concentration calculated using the Polygonum cuspidatum medicinal material as the solute.

[0019] In a preferred embodiment, in the composition, the concentration of the Polygonum cuspidatum extract is 58.33 mg / mL≤≤80.00 mg / mL, and the concentration of carvacrol is 373.33 μg / mL≤≤426.67 μg / mL. The composition of the Polygonum cuspidatum extract and carvacrol at this concentration has a good inhibitory effect on Porphyromonas gingivalis in a capsular state.

[0020] The present invention also provides another composition, which comprises a Polygonum cuspidatum extract and cinnamaldehyde. Cinnamaldehyde is an olefinic organic compound, a yellow viscous liquid, and exists in large quantities in plants such as cinnamon. Cinnamaldehyde has multiple pharmacological effects such as antipyretic and analgesic, anti-inflammatory, antibacterial, anti-tumor, hypoglycemic, anti-obesity and neuroprotective, and is commonly used in external medicines and synthetic medicines.

[0021] The inventors found that when the Polygonum cuspidatum extract and cinnamaldehyde are used in combination, the minimum inhibitory concentration (MIC) for Porphyromonas gingivalis can be greatly reduced. The MIC value of the combined use of the Polygonum cuspidatum extract is only 1 / 8 to 1 / 9 of the MIC value of the Polygonum cuspidatum extract alone, and the MIC value of the combined use of cinnamaldehyde is only 1 / 4 of the MIC value of cinnamaldehyde alone. It can be seen that when the Polygonum cuspidatum extract and cinnamaldehyde are used in combination, a synergistic effect will occur, which improves the inhibitory activity against Porphyromonas gingivalis.

[0022] In addition, when the Polygonum cuspidatum extract and cinnamaldehyde are used in combination, the minimum inhibitory concentration (MBIC) for the biofilm formed by Porphyromonas gingivalis can be greatly reduced. The MBIC value of the combined use of Polygonum cuspidatum extract is only 1 / 3 of the MBIC value of the Polygonum cuspidatum extract alone, and the MBIC value of the combined use of cinnamaldehyde is only 1 / 3 of the MBIC value of cinnamaldehyde alone. Therefore, when the Polygonum cuspidatum extract and cinnamaldehyde are used in combination, the inhibitory activity against the biofilm formed by Porphyromonas gingivalis is enhanced.

[0023] In a preferred embodiment, in the composition, the concentration of the Polygonum cuspidatum extract is 12.50 mg / mL≤≤16.92 mg / mL, and the concentration of cinnamaldehyde is 160.00 μg / mL≤≤256.00 μg / mL. The composition of the Polygonum cuspidatum extract and cinnamaldehyde at this concentration has a good inhibitory effect on planktonic Porphyromonas gingivalis.

[0024] In a preferred embodiment, in the composition, the concentration of the Polygonum cuspidatum extract is 66.67 mg / mL≤≤80.00 mg / mL, and the concentration of cinnamaldehyde is 426.67 μg / mL≤≤512.00 μg / mL. The composition of the Polygonum cuspidatum extract and cinnamaldehyde at this concentration has a good inhibitory effect on Porphyromonas gingivalis in a capsular state.

[0025] In a second aspect, the present invention provides the use of the composition in inhibiting oral malodor-causing bacteria and especially the biofilm formed.

[0026] Furthermore, the oral halitosis-causing bacteria include various types of microorganisms that can cause oral halitosis, including but not limited to Porphyromonas gingivalis.

[0027] In a third aspect, the present invention provides an oral care product, comprising an effective dose of the above composition.

[0028] Furthermore, the oral care products include but are not limited to toothpaste, tooth powder, mouthwash, chewing gum, breath freshener, tooth paint, etc. According to different products, different doses of the above composition can be added to enable the composition to exert its efficacy in inhibiting oral odor-causing bacteria and biofilm, and the present invention is not limited to this.

[0029] Compared with the prior art, the present invention has achieved the following technical effects:

[0030] 1. In the composition provided by the present invention, the two components of Polygonum cuspidatum extract and carvacrol produce a synergistic effect, which greatly reduces the minimum inhibitory concentration (MIC) and minimum inhibitory concentration (MBIC) of Polygonum cuspidatum extract and carvacrol on oral odor-causing bacteria. The composition has a high inhibitory effect on oral odor-causing bacteria and biofilms, and can easily penetrate into bacterial cells, which can interfere with the mechanism of bacterial quorum sensing, and achieve technical effects such as reducing dosage and increasing efficiency, and reducing the emergence of drug-resistant bacteria.

[0031] 2. In the composition provided by the present invention, the two components of Polygonum cuspidatum extract and cinnamaldehyde produce a synergistic effect, which greatly reduces the minimum inhibitory concentration (MIC) and minimum inhibitory concentration (MBIC) of Polygonum cuspidatum extract and cinnamaldehyde on oral odor-causing bacteria. The composition has a high inhibitory effect on oral odor-causing bacteria and biofilms, and is easy to penetrate into bacterial cells, which can interfere with the mechanism of bacterial quorum sensing, and achieve technical effects such as reducing dosage and increasing efficiency, reducing the emergence of drug-resistant bacteria, etc.

[0032] 3. The composition provided by the present invention can be applied to oral care products and can achieve the effect of reducing oral halitosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is the inhibitory effect of composition A on the metabolic activity of biofilm growth at each stage of Pg (n=3);

[0034] Figure 2 is the inhibitory effect of composition B on the metabolic activity of biofilm growth at each stage of Pg (n=3);

[0035] Figure 3 is the inhibitory effect of composition A on the content of Pg biofilm exopolysaccharide (n=3);

[0036] Figure 4 is the inhibitory effect of composition B on the content of Pg biofilm exopolysaccharide (n=3);

[0037] Figure 5 is the inhibitory effect of composition A on the content of extracellular protein in Pg biofilm (n=3);

[0038] Figure 6 is the inhibitory effect of composition B on Pg biomembrane extracellular protein (n=3);

[0039] Figure 7 is the inhibitory effect of composition A on DNA outside Pg biofilm (n=3);

[0040] Figure 8is the inhibitory effect of composition B on DNA outside Pg biofilm (n=3);

[0041] Among them: Control: growth group; CHX: chlorhexidine; Composition A: Polygonum cuspidatum extract 58.33 mg / mL + carvacrol 373.33 μg / mL; Composition B: Polygonum cuspidatum extract 66.67 mg / mL + cinnamaldehyde 426.67 μg / mL;

[0042] * indicates comparison with the growth group, P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; # indicates comparison with the combination, #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001; + indicates comparison with the positive drug CHX, +P<0.05, ++P<0.01, +++P<0.001, ++++P<0.0001. DETAILED DESCRIPTION

[0043] The technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0044] Materials and reagents used in the following examples:

[0045] 1. Polygonum cuspidatum was purchased from the Chinese Medicine Piece Factory of Guangdong Medicinal Materials Company, item number: SYYP141;

[0046] 2. Carvacrol was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., batch number: C10965851, 99.5%;

[0047] 3. Cinnamaldehyde was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., batch number: C10105443, ≥95%;

[0048] 4. Porphyromonas gingivalis (Pg) (standard strain, ATCC 33277) was purchased from Beijing Biotechnology Collection Center; Bacteroides fragilis (ATCC 25285) was purchased from Guangdong Microbial Culture Collection Center.

[0049] 5. Preparation method of Polygonum cuspidatum extract: Weigh 200 g of coarse powder of Polygonum cuspidatum medicinal material, add 50% ethanol 8 times the weight of the medicinal material, soak for 30 minutes, extract in a 60°C water bath for 1 hour, extract twice, combine the filtrate and concentrate to obtain 200 mL of concentrated solution, which is the Polygonum cuspidatum extract, and place it in a 4°C refrigerator away from light for later use. Calculated according to the amount of crude drug, the concentration of the Polygonum cuspidatum extract is c=200g / 200mL=1g / mL.

[0050] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0051] Example 1: Inhibitory effects of Polygonum cuspidatum extract, carvacrol, and cinnamaldehyde on planktonic bacteria 1. Experimental methods

[0052] The MICs of Polygonum cuspidatum extract, carvacrol, and cinnamaldehyde were determined using the broth microdilution method of the M11-A7 protocol developed by CLSI. The specific steps are as follows:

[0053] (1) The drugs were diluted with BHI liquid culture medium to obtain 5120 μg / mL of carvacrol, 5120 μg / mL of cinnamaldehyde, 2560 μg / mL of chlorhexidine, and 800 mg / mL of Polygonum cuspidatum extract.

[0054] (2) Add 100 μL of BHI liquid culture medium to rows AG and columns 2-11 of the 96-well plate, and add 200 μL of BHI liquid culture medium to column 12. Add 200 μL of the above solution to column 1 and dilute the solution by two-fold dilution. At this time, the volume of each well is 100 μL. Then add the bacterial solution to columns 1-11. At this time, the final volume of each well is 200 μL. Column 11 is the growth control well, and column 12 is the blank control well. Place in a constant temperature incubator and culture anaerobically at 37°C for 24 to 48 hours. Read and record the results.

[0055] (3) Result evaluation standard: The drug concentration corresponding to the microwells where no bacterial growth is observed with the naked eye is the MIC.

[0056] 2. Experimental results

[0057] The test results of Example 1 are shown in Table 1.

[0058] Table 1 MIC results of in vitro drug susceptibility test of Porphyromonas gingivalis (n=6)

[0059] Strain\Sample Polygonum cuspidatum extract Carvacrol Cinnamaldehyde Chlorhexidine Pg 110.00mg / mL 533.33 μg / mL 640.00μg / mL 1.94 μg / mL

[0060] As can be seen from Table 1, polygonum cuspidatum extract, carvacrol, cinnamaldehyde and chlorhexidine all have inhibitory effects on porphyromonas gingivalis (Pg). Among them, the minimum inhibitory concentration (MIC) of polygonum cuspidatum extract against Pg is 110.00 mg / ml, the MIC of carvacrol against Pg is 533.33 μg / mL, the MIC of cinnamaldehyde against Pg is 640.00 μg / mL, and the MIC of the positive drug chlorhexidine against Pg is 1.94 μg / mL.

[0061] Example 2: Inhibitory effects of composition A and composition B on planktonic bacteria

[0062] 1. Experimental method

[0063] In this example, the combination of polygonum cuspidatum extract and carvacrol is used as composition A, and the combination of polygonum cuspidatum extract and cinnamaldehyde is used as composition B. The microdilution checkerboard method is used to determine the antibacterial activities of composition A and composition B respectively. The specific experimental method is as follows:

[0064] (1) According to the MIC results determined by the microbroth dilution method in Example 1, the drugs are diluted with BHI liquid medium to obtain carvacrol at 4×MIC concentration, cinnamaldehyde at 4×MIC concentration, and polygonum cuspidatum extract at 4×MIC concentration.

[0065] (2) Add 50 μL of BHI liquid medium to rows A-G and columns 2-10 of a 96-well plate. Take the above 4×MIC carvacrol and add 100 μL to columns A1-G1 respectively, and dilute it twofold horizontally to the 6th column. At this time, the volume of each well is 50 μL. Add 50 μL of polygonum cuspidatum extract with a series of concentrations to rows A–G. In addition, set up a growth control well and a blank control well. Except for the blank control well which is added with BHI medium, the rest are added with 100 μL of bacterial suspension, and the final volume of all wells is 200 μL. The experimental operation of the combination of cinnamaldehyde and polygonum cuspidatum extract is the same as above. Place it in an anaerobic incubator at 37°C for 24-48 h. Compared with the growth control well, the micro-well without the growth of the test bacteria observed with the naked eye is its MIC combined value, and the experimental operation of each strain is repeated in parallel 3 times.

[0066] (3) Result evaluation criteria: Use FICI as the judgment basis for the combined drug sensitivity test: FICI ≤ 0.5 is a synergistic effect; 0.5 < FICI ≤ 1 is an additive effect; 1 < FICI ≤ 2 is an irrelevant effect; FICI > 2 is an antagonistic effect; among them:

[0067] FICI = MIC of drug A in combination / MIC of drug A used alone + MIC of drug B in combination / MIC of drug B used alone.

[0068] 2. Experimental results

[0069] (1) Experimental results of composition A

[0070] Table 2 Results of drug sensitivity test of composition A (combination of Polygonum cuspidatum extract and carvacrol) (n=6)

[0071]

[0072] It is known that the smaller the MIC value, the stronger the antibacterial effect. As shown in Table 2, after the combination of Polygonum cuspidatum extract and carvacrol, the MIC of Polygonum cuspidatum extract against Pg decreased from 110.00 mg / mL to 6.25 mg / mL, and the MIC value of the combination of Polygonum cuspidatum extract was about 1 / 17 to 1 / 18 of the MIC value of the single use. The MIC of carvacrol against Pg decreased from 533.33 μg / mL to 160.00 μg / mL, and the MIC value of the combination of carvacrol was about 1 / 3 to 1 / 4 of the MIC value of the single use. According to the formula FICI = MIC A drug combination / MIC A drug alone + MIC B drug combination / MIC B drug alone, the calculated FICI after combination is <0.5, indicating that the Polygonum cuspidatum extract and carvacrol of composition A have a synergistic antibacterial effect on Porphyromonas gingivalis; at the same time, composition A enhances the inhibitory effect of Polygonum cuspidatum extract or carvacrol on Porphyromonas gingivalis.

[0073] (2) Experimental results of composition B

[0074] Table 3 Results of drug sensitivity test of composition B (combination of Polygonum cuspidatum extract and cinnamaldehyde) (n=6)

[0075]

[0076] It is known that the smaller the MIC value, the stronger the antibacterial effect. As shown in Table 3, after the combination of Polygonum cuspidatum extract and cinnamaldehyde, the MIC of Polygonum cuspidatum extract against Pg decreased from 110.00 mg / mL to 12.50 mg / mL, and the MIC value of the combination of Polygonum cuspidatum extract was about 1 / 8 to 1 / 9 of the MIC value of the single use. The MIC of cinnamaldehyde against Pg decreased from 640.00 μg / mL to 160.00 μg / mL, and the MIC value of the combination of cinnamaldehyde was about 1 / 4 of the MIC value of the single use. According to the formula FICI = MIC A drug combination / MIC A drug alone + MIC B drug combination / MIC B drug alone, the calculated FICI after combination is <0.5, indicating that the Polygonum cuspidatum extract and cinnamaldehyde in composition B have a synergistic antibacterial effect on Porphyromonas gingivalis; at the same time, composition B enhances the inhibitory effect of Polygonum cuspidatum extract or cinnamaldehyde on Porphyromonas gingivalis.

[0077] Example 3: Inhibitory effects of Polygonum cuspidatum extract, carvacrol, cinnamaldehyde and their combination on biofilm bacteria

[0078] 1. Experimental Methods

[0079] (1) Inoculate 200 μL of Pg bacterial suspension into each well of a 96-well plate and incubate at 37°C for 48 to 72 hours until a biofilm is formed. Rinse with sterile PBS three times to remove the culture medium and floating bacteria in each well. Take 200 μL of each of Polygonum cuspidatum extract, carvacrol, cinnamaldehyde, chlorhexidine, etc. and add them to the 96-well plate. Set the 11th column as the growth control well and the 12th column as the blank control well. Continue anaerobic incubation at 37°C for 24 hours and observe the results. Result judgment standard: The lowest drug concentration at which no turbidity is observed by the naked eye is the minimum inhibitory concentration (MBIC).

[0080] (2) 200 μL of the Pg bacterial suspension was inoculated into each well of a 96-well plate and incubated anaerobically at 37°C for 72 h. The wells were rinsed three times with sterile PBS to remove the culture medium and floating bacteria, and the plates were set aside.

[0081] (3) According to the detected MBIC value, samples were prepared using BHI liquid culture medium to obtain 4×MBIC to 1 / 8×MBIC concentrations of Polygonum cuspidatum extract, carvacrol, cinnamaldehyde, chlorhexidine, etc. (4) 100 μL of each concentration of Polygonum cuspidatum extract was added horizontally to the 1st to 6th columns of the 96-well plate prepared with the biofilm, and 100 μL of a series of concentrations of carvacrol were added vertically from rows AG. Place in a 37°C incubator and continue anaerobic culture for 24 hours. The combined MBIC detection method of Polygonum cuspidatum extract and cinnamaldehyde is the same as above. When observing the results, the microwells without growth of the test bacteria are used as their combined MBIC values, and the experimental operation is repeated 3 times in parallel.

[0082] 2. Experimental results

[0083] (1) Experimental results of composition A

[0084] Table 4 Minimum inhibitory concentration (MBIC) of composition A for Pg (n=6)

[0085]

[0086] It is known that the smaller the MBIC value, the stronger the effect of inhibiting bacterial biofilm. As shown in Table 4, after the combination of Polygonum cuspidatum extract and carvacrol, the MBIC of Polygonum cuspidatum extract on Pg decreased from 200.00 mg / mL to 58.33 mg / mL, and the combined MBIC of Polygonum cuspidatum extract was about 1 / 3 to 1 / 4 of the MBIC value of single use. The MBIC of carvacrol on Pg decreased from 1066.67 μg / mL to 373.33 μg / mL, and the combined MBIC of carvacrol was about 1 / 3 of the MBIC value of single use. This shows that composition A enhances the inhibitory effect of Polygonum cuspidatum extract or carvacrol on Porphyromonas gingivalis biofilm.

[0087] (2) Experimental results of composition B

[0088] Table 5 Minimum inhibitory concentration (MBIC) of composition B for Pg (n=6)

[0089]

[0090] It is known that the smaller the MBIC value, the stronger the effect of inhibiting bacterial biofilm. As can be seen from Table 5, after the combination of Polygonum cuspidatum extract and cinnamaldehyde, the MBIC of Polygonum cuspidatum extract on Pg decreased from 200.00 mg / mL to 66.67 mg / mL, and the combined MBIC of Polygonum cuspidatum extract was about 1 / 3 of the MBIC value of single use. The MBIC of cinnamaldehyde on Pg decreased from 1280.00 μg / mL to 426.67 μg / mL, and the combined MBIC of cinnamaldehyde was about 1 / 3 of the MBIC value of single use. This shows that composition B enhances the inhibitory effect of Polygonum cuspidatum extract or cinnamaldehyde on Porphyromonas gingivalis biofilm.

[0091] Example 4: Inhibitory effects of composition A and composition B on biofilm growth metabolic activity

[0092] 1. Experimental Methods

[0093] (1) Prepare composition A and composition B samples. The concentration of composition A is 58.33 mg / ml of Polygonum cuspidatum extract MBIC + 373.33 μg / ml of carvacrol MBIC. The concentration of composition B is 66.67 mg / ml of Polygonum cuspidatum extract MBIC + 426.67 μg / ml of cinnamaldehyde MBIC.

[0094] (2) 200 μL of Pg bacterial suspension was inoculated into each well of a 96-well plate. Pg was cultured anaerobically at 37°C for 36 h, 60 h, and 84 h. The culture was completed at the corresponding time points and the culture medium and floating bacteria in each well were removed by washing with sterile PBS three times. 200 μL of each drug was added and cultured for another 24 h. The wells were gently washed with PBS three times. After drying naturally, 100 μL of BHI culture medium and 10 μL of CCK-8 solution were added to each well in the dark according to the instructions of the CCK-8 kit. The wells were mixed and incubated anaerobically at 37°C in the dark for 1 h. After the reaction was completed, the absorbance value at OD450nm was measured with an ELISA reader.

[0095] 2. Experimental results

[0096] (1) Experimental results of composition A

[0097] The experimental results of composition A are shown in Table 6 and Figure 1 shown.

[0098] Table 6 Inhibitory effect of composition A on the metabolic activity of Pg biofilm growth at various stages (n=3)

[0099]

[0100]

[0101] 36h (adhesion stage): compared with the Control group, Figure 1 As shown in (A1), carvacrol, chlorhexidine and composition A can effectively reduce the metabolic activity of Pg biofilm in the adhesion period (all groups are P < 0.0001). Specifically, as shown in Table 6, compared with the Control group, the inhibitory effects of Polygonum cuspidatum extract, carvacrol, composition A and chlorhexidine on the metabolic activity of Pg biofilm in the adhesion period increased by 4.15%, 53.59%, 87.76% and 87.78%, respectively. The experimental results show that the most obvious effect is the composition A and chlorhexidine group, and the effects of the two are similar.

[0102] 36h (adhesion phase): Compared with knotweed extract or carvacrol, Figure 1 As shown in (A1), composition A can effectively reduce the metabolic activity of Pg biofilm during the adhesion period (all groups are P < 0.0001). Specifically, as shown in Table 6, compared with the extract of Polygonum cuspidatum, the inhibitory effect of composition A on the metabolic activity of Pg biofilm during the adhesion period increased by 87.23%; compared with carvacrol, the inhibitory effect of composition A on the metabolic activity of Pg biofilm during the adhesion period increased by 73.64%. This shows that the effect of composition A is better than that of Polygonum cuspidatum extract or carvacrol.

[0103] 36h (adhesion stage): compared with the chlorhexidine group, Figure 1 As shown in (A1), there was no significant difference in reducing the metabolic activity of Pg biofilm in the adhesion period by composition A (P>0.05), indicating that there was no statistical difference in the effects of composition A and chlorhexidine, and the effects were similar.

[0104] In summary, at 36 h (adhesion stage), composition A had a better inhibitory effect on the metabolic activity of Pg biofilm than knotweed extract or carvacrol, that is, compared with single use, composition A increased by 87.23% and 73.64%, respectively, and the effect of inhibiting the metabolic activity of biofilm was close to that of the positive drug chlorhexidine.

[0105] 60h (aggregation phase): compared with the Control group, Figure 1 As shown in (B1), carvacrol, chlorhexidine and composition A can effectively reduce the metabolic activity of Pg biofilm (all groups are P < 0.0001). Specifically, as shown in Table 6, compared with the Control group, the inhibitory effects of Polygonum cuspidatum extract, carvacrol, composition A and chlorhexidine on the metabolic activity of Pg biofilm increased by 6.63%, 48.79%, 91.86% and 80.68%, respectively. The experimental results show that the most obvious effect is the composition A and chlorhexidine group, and the composition A 91.86%> chlorhexidine 80.68%.

[0106] 60h (aggregation phase): Compared with knotweed extract or carvacrol, Figure 1 As shown in (B1), composition A can effectively reduce the metabolic activity of Pg biofilm (all groups are P < 0.0001). Specifically, as shown in Table 6, compared with the extract of Polygonum cuspidatum, the inhibitory effect of composition A on the metabolic activity of Pg biofilm increased by 91.28%; compared with carvacrol, the inhibitory effect of composition A on the metabolic activity of Pg biofilm increased by 84.10%. This shows that the effect of composition A is better than that of Polygonum cuspidatum extract or carvacrol.

[0107] 60h (aggregation phase): Compared with chlorhexidine, Figure 1 As shown in (B1), there was no significant difference in reducing the metabolic activity of Pg biofilm in the adhesion period by composition A (P>0.05). Specifically, as shown in Table 6, compared with chlorhexidine, the inhibitory effect of composition A on the metabolic activity of Pg biofilm increased by 57.87%.

[0108] In summary, at 60h (aggregation stage), compared with the Control group, the inhibition of biofilm metabolic activity was improved by Composition A 91.86%>Chlorhexidine 80.68%. Composition A had a better inhibitory effect on Pg biofilm metabolic activity than Polygonum cuspidatum extract or carvacrol, that is, compared with single use, Composition A increased by 91.28% and 84.10%, respectively. The inhibitory effect of Composition A on biofilm metabolic activity was close to that of the positive drug chlorhexidine, and compared with chlorhexidine, the inhibitory effect was increased by 57.87%. 84h (maturation stage): Compared with the Control group, Figure 1 As shown in (C1), carvacrol (P < 0.05), chlorhexidine (P < 0.0001) and composition A (P < 0.0001) can effectively reduce the metabolic activity of Pg biofilm. Specifically, as shown in Table 6, compared with the Control group, the inhibitory effects of Polygonum cuspidatum extract, carvacrol, composition A and chlorhexidine on the metabolic activity of Pg biofilm increased by 3.82%, 23.01%, 90.11% and 66.19%, respectively. The experimental results show that the most obvious effect is the composition A and chlorhexidine group, and the composition A 90.11%> chlorhexidine 66.19%.

[0109] 84h (mature stage): Compared with knotweed extract or carvacrol, Figure 1 As shown in (C1), composition A can effectively reduce the metabolic activity of Pg biofilm (all groups are P < 0.0001). Specifically, as shown in Table 6, compared with the extract of Polygonum cuspidatum, the inhibitory effect of composition A on the metabolic activity of Pg biofilm increased by 89.71%; compared with carvacrol, the inhibitory effect of composition A on the metabolic activity of Pg biofilm increased by 87.15%. This shows that the effect of composition A is better than that of Polygonum cuspidatum extract or carvacrol.

[0110] 84h (mature stage): Compared with chlorhexidine, Figure 1 (C1) shows that composition A can effectively reduce the metabolic activity of Pg biofilm (P < 0.01). Specifically, as shown in Table 6, compared with chlorhexidine, the inhibitory effect of composition A on the metabolic activity of Pg biofilm increased by 70.74%. In summary, at 84h (mature stage), compared with the Control group, the inhibition of biofilm metabolic activity increased by 90.11% for composition A> 66.19% for chlorhexidine. The inhibitory effect of composition A on the metabolic activity of Pg biofilm is better than that of knotweed extract or carvacrol, that is, compared with single use, composition A increased by 89.71% and 87.15%, respectively. The effect of composition A in inhibiting the metabolic activity of biofilm is better than that of the positive drug chlorhexidine (P < 0.01). Specifically, compared with chlorhexidine, the inhibitory effect increased by 70.74%.

[0111] (2) Experimental results of composition B

[0112] The experimental results of composition B are shown in Table 7 and Figure 2 shown.

[0113] Table 7 Inhibitory effect of composition B on the metabolic activity of Pg biofilm growth at various stages (n=3)

[0114]

[0115] 36h (adhesion stage): compared with the Control group, Figure 2 As shown in (A2), cinnamaldehyde, composition B and chlorhexidine can significantly inhibit the metabolic activity of biofilm (all groups are P < 0.0001). Specifically, as shown in Table 7, compared with the Control group, the inhibitory effects of Polygonum cuspidatum extract, cinnamaldehyde, composition B and chlorhexidine on the metabolic activity of Pg biofilm increased by 1.55%, 52.36%, 87.17% and 87.63%, respectively. The experimental results show that the most obvious effect is the combination B and chlorhexidine group, and the effects of the two are similar.

[0116] 36h (adhesion stage): Compared with Knotweed extract or cinnamaldehyde, Figure 2 As shown in (A2), composition B can effectively reduce the metabolic activity of Pg biofilm (all groups are P < 0.0001). Specifically, as shown in Table 7, compared with the Polygonum cuspidatum extract, the inhibitory effect of composition B on the metabolic activity of Pg biofilm increased by 86.96%; compared with cinnamaldehyde, the inhibitory effect of composition B on the metabolic activity of Pg biofilm increased by 73.06%. This shows that the effect of composition B is better than that of Polygonum cuspidatum extract or cinnamaldehyde.

[0117] 36h (adhesion stage): Compared with chlorhexidine, Figure 2As shown in (A2), there was no significant difference in the inhibition of Pg biofilm metabolic activity by composition B (P>0.05), indicating that there was no statistical difference in the effects of composition B and chlorhexidine, and the effects were similar.

[0118] In summary, at 36 h (adhesion stage), the inhibitory effect of composition B on the metabolic activity of Pg biofilm was better than that of knotweed extract or cinnamaldehyde, that is, compared with single use, composition B increased by 86.96% and 73.06%, respectively, and the effect of inhibiting the metabolic activity of biofilm was close to that of the positive drug chlorhexidine.

[0119] 60h (aggregation phase): compared with the Control group, Figure 2 As shown in (B2), cinnamaldehyde, composition B and chlorhexidine can significantly inhibit the metabolic activity of biofilm (all groups are P < 0.0001). Specifically, as shown in Table 7, compared with the Control group, the inhibitory effects of Polygonum cuspidatum extract, cinnamaldehyde, composition B and chlorhexidine on the metabolic activity of Pg biofilm increased by 4.02%, 46.43%, 91.65% and 80.29%, respectively. The experimental results show that the most obvious effect is the composition B and chlorhexidine group, and the composition B 91.65%> chlorhexidine 80.29%.

[0120] 60h (aggregation stage): Compared with Polygonum cuspidatum extract or cinnamaldehyde, Figure 2 As shown in (B2), composition B can effectively reduce the metabolic activity of Pg biofilm (all groups are P < 0.0001). Specifically, as shown in Table 7, compared with the Polygonum cuspidatum extract, the inhibitory effect of composition B on the metabolic activity of Pg biofilm increased by 91.30%; compared with cinnamaldehyde, the inhibitory effect of composition B on the metabolic activity of Pg biofilm increased by 84.41%. This shows that the effect of composition B is better than that of Polygonum cuspidatum extract or cinnamaldehyde.

[0121] 60h (aggregation phase): Compared with chlorhexidine, Figure 2 (B2) shows that there is no significant difference in the inhibition of Pg biofilm metabolic activity by composition B (P>0.05). Specifically, as shown in Table 7, compared with chlorhexidine, the inhibitory effect of composition B on Pg biofilm metabolism is increased by 57.63%. In summary, at 60h (aggregation stage), compared with the Control group, the inhibition of biofilm metabolic activity was improved by composition B 91.65%> chlorhexidine 80.29%; the inhibitory effect of composition B on Pg biofilm metabolic activity was better than that of knotweed extract or cinnamaldehyde, that is, compared with single use, composition B increased by 91.30% and 84.41%, respectively; the inhibitory effect of composition B on biofilm metabolic activity was close to that of the positive drug chlorhexidine, and compared with chlorhexidine, the inhibitory effect was increased by 57.63%. 84h (maturation stage): Compared with the Control group, Figure 2As shown in (C2), both composition B and chlorhexidine can significantly inhibit the metabolic activity of biofilm (all groups are P < 0.0001). Specifically, as shown in Table 7, compared with the Control group, the inhibitory effects of Polygonum cuspidatum extract, cinnamaldehyde, composition B and chlorhexidine on the metabolic activity of Pg biofilm increased by 2.02%, 17.05%, 89.70% and 66.01%, respectively. The experimental results show that the most obvious effects are in the composition B and chlorhexidine groups, and composition B 89.70%>chlorhexidine 66.01%.

[0122] 84h (mature stage): Compared with knotweed extract or cinnamaldehyde, Figure 2 As shown in (C2), composition B can effectively reduce the metabolic activity of Pg biofilm (all groups are P < 0.0001). Specifically, as shown in Table 7, compared with the Polygonum cuspidatum extract, the inhibitory effect of composition B on the metabolic activity of Pg biofilm increased by 89.49%; compared with cinnamaldehyde, the inhibitory effect of composition B on the metabolic activity of Pg biofilm increased by 87.58%, indicating that the effect of composition B is better than that of Polygonum cuspidatum extract or cinnamaldehyde.

[0123] 84h (mature stage): Compared with chlorhexidine, Figure 2 (C2) shows that composition B can effectively reduce the metabolic activity of Pg biofilm (P < 0.01). Specifically, as shown in Table 7, compared with chlorhexidine, the inhibitory effect of composition B on the metabolic activity of Pg biofilm increased by 69.70%. In summary, at 84h (mature stage), compared with the Control group, the inhibition of biofilm metabolic activity increased as composition B 89.70%> chlorhexidine 66.01%. The effect of composition B is better than that of Polygonum cuspidatum extract or cinnamaldehyde, that is, compared with single use, composition B increased by 89.49% and 89.49%, respectively. The effect of composition B in inhibiting biofilm metabolic activity is better than that of the positive drug chlorhexidine (P < 0.01). Specifically, compared with chlorhexidine, the inhibitory effect increased by 69.70%.

[0124] Example 5: Inhibitory effects of composition A and composition B on extracellular polysaccharides of biofilm 1. Experimental methods

[0125] (1) The EPSs of the biofilm of the test bacteria were extracted by heat treatment. The EPSs of the biofilm were used to determine the extracellular polysaccharides and extracellular proteins of the biofilm. The specific preparation process is as follows: 1 mL of the bacterial suspension was inoculated into each well of a 24-well plate and cultured anaerobically at 37°C for 48 to 72 hours. Rinse gently with sterile PBS three times. Add 1 mL of each group of drugs prepared in advance, set up a growth control group, continue to culture anaerobically at 37°C for 24 hours, and then rinse with PBS three times. Add sterile PBS to resuspend the biofilm and adjust to OD600nm = 0.40. Take 1 mL of the resuspended solution and place it in an 80°C water bath for 30 minutes, centrifuge at 5000 r / min for 15 minutes, collect the supernatant and filter it with a 0.22μm filter membrane. The obtained liquid is the EPSs extract and is stored at 4°C in the dark for later use.

[0126] (2) The method for determining the extracellular polysaccharide content of the biofilm is as follows. First, the glucose standard curve is determined by the phenol-sulfuric acid method, and then the extracellular polysaccharide content of the biofilm of the sample is determined. Specifically, 1 mL of the test bacteria biofilm EPSs extract of each group is aspirated, 1 mL of 5% phenol solution is added and mixed, 5 mL of 98% concentrated sulfuric acid is added, and the mixture is incubated in a 90°C dark-proof water bath for 5 minutes. After cooling to room temperature, 200 μL are aspirated into a 96-well plate, and the absorbance at a wavelength of 490 nm is measured using an enzyme reader. Finally, the test results are substituted into the glucose standard curve to calculate the corresponding polysaccharide content. The experiment was repeated 3 times.

[0127] 2. Experimental results

[0128] (1) Experimental results of composition A

[0129] The experimental results of composition A are shown in Table 8 and Figure 3 shown.

[0130] Table 8 Inhibitory effect of composition A on the content of polysaccharide outside Pg biofilm (n=3)

[0131]

[0132] Compared with the Control group, Figure 3 As shown in Table 8, both chlorhexidine and composition A can effectively inhibit Pg biofilm exopolysaccharides (all groups are P < 0.001). Specifically, as shown in Table 8, compared with the Control group, the inhibitory effects of Polygonum cuspidatum extract, carvacrol, composition A and chlorhexidine on Pg biofilm exopolysaccharides were increased by 27.30%, 35.01%, 86.08% and 86.18%, respectively. The experimental results show that the most obvious effects are in the composition A and chlorhexidine groups, and the effects of the two groups are similar.

[0133] Compared with knotweed extract or carvacrol, e.g. Figure 3As shown in Table 8, composition A can significantly inhibit the exopolysaccharide of Pg biofilm (P<0.01 for the group of Polygonum cuspidatum extract and P<0.05 for the group of Carvacrol). Specifically, as shown in Table 8, compared with the Polygonum cuspidatum extract, the inhibitory effect of composition A on the exopolysaccharide of Pg biofilm increased by 80.86%; compared with Carvacrol, the inhibitory effect of composition A on the exopolysaccharide of Pg biofilm increased by 78.59%. This shows that the effect of composition A is better than that of Polygonum cuspidatum extract or Carvacrol.

[0134] Compared with chlorhexidine, Figure 3 As shown, there was no significant difference in the inhibition of Pg biofilm exopolysaccharide by composition A (P>0.05), indicating that there was no statistical difference between the effects of composition A and chlorhexidine, and the effects were similar.

[0135] In summary, the inhibitory effect of composition A on Pg biofilm exopolysaccharides was better than that of knotweed extract or carvacrol, that is, compared with single use, composition A increased by 80.86% and 78.59%, respectively, and the effect of inhibiting biofilm metabolic activity was not statistically different from that of the positive drug chlorhexidine (P>0.05).

[0136] (2) Experimental results of composition B

[0137] The experimental results of composition B are shown in Table 9 and Figure 4 shown.

[0138] Table 9 Inhibitory effect of composition B on the content of polysaccharide outside the Pg biofilm (n=3)

[0139]

[0140] Compared with the Control group, Figure 4 As shown in Table 9, both chlorhexidine and composition B can effectively inhibit Pg biofilm exopolysaccharides (all groups are P < 0.001). Specifically, as shown in Table 9, compared with the Control group, the inhibitory effects of Polygonum cuspidatum extract, cinnamaldehyde, composition B and chlorhexidine on Pg biofilm exopolysaccharides were increased by 28.67%, 35.89%, 85.22% and 86.04%. The experimental results show that the most obvious effect is the composition A and chlorhexidine group, and the effects of the two are similar.

[0141] Compared with knotweed extract or cinnamaldehyde, e.g. Figure 4 As shown in Table 9, composition B can significantly inhibit the exopolysaccharide of Pg biofilm (P<0.01 for the group of Polygonum cuspidatum extract and P<0.05 for the group of Cinnamaldehyde). Specifically, as shown in Table 9, compared with Polygonum cuspidatum extract, the inhibitory effect of composition B on the exopolysaccharide of Pg biofilm is increased by 79.28%; compared with Cinnamaldehyde, the inhibitory effect of composition B on the exopolysaccharide of Pg biofilm is increased by 76.95%, indicating that the effect of composition B is better than that of Polygonum cuspidatum extract or Cinnamaldehyde.

[0142] Compared with chlorhexidine, Figure 4 As shown, there was no significant difference in the inhibition of Pg biofilm exopolysaccharide by composition B (P>0.05), indicating that there was no statistical difference in the effects of composition B and chlorhexidine, and the effects were similar.

[0143] In summary, the inhibitory effect of composition B on Pg biofilm exopolysaccharides was better than that of knotweed extract or cinnamaldehyde, that is, compared with single use, composition A increased by 79.28% and 76.95%, respectively, and the effect of inhibiting biofilm metabolic activity was not statistically different from that of the positive drug chlorhexidine (P>0.05).

[0144] Example 6: Inhibitory effects of composition A and composition B on extracellular proteins of biofilm 1. Experimental methods

[0145] According to the instructions of the BCA kit, 20 μL of the EPSs extract of each group of test bacteria biofilm prepared in advance was aspirated into a 96-well plate, and the blank control group was added with an equal amount of PBS, and 200 μL of the working solution was added, and the reaction was carried out at 37°C for 30 minutes. The OD595nm value of the solution in each well was detected by an ELISA instrument, and the extracellular protein content of the biofilm was calculated according to the OD value. The experiment was repeated 3 times.

[0146] 2. Experimental results

[0147] (1) Experimental results of composition A

[0148] The experimental results of composition A are shown in Table 10 and Figure 5 shown.

[0149] Table 10 Inhibitory effect of composition A on the content of extracellular protein in Pg biofilm (n=3)

[0150]

[0151] Compared with the Control group, Figure 5 As shown in the results, composition A can significantly inhibit biofilm extracellular protein (P < 0.05), and chlorhexidine can significantly inhibit biofilm extracellular protein (P < 0.01); specifically, as shown in Table 10, compared with the Control group, the inhibitory effects of composition A and chlorhexidine on biofilm extracellular protein were increased by 23.87% and 30.00%, respectively.

[0152] Compared with the Polygonum cuspidatum extract, as shown in Table 10, the inhibitory effect of composition A on biofilm extraprotein was increased by 3.32%, and compared with carvacrol, the inhibitory effect of composition A on biofilm extraprotein was increased by 13.77%, indicating that composition A was better than Polygonum cuspidatum extract or carvacrol in inhibiting biofilm extraprotein.

[0153] Compared with chlorhexidine, Figure 5As shown, there was no significant difference in the inhibition of biofilm extracellular proteins by composition A (P>0.05).

[0154] In summary, composition A inhibited biofilm extracellular protein by 23.87% compared with the Control group, 3.32% compared with Polygonum cuspidatum extract, 13.77% compared with carvacrol, and had no statistical difference compared with chlorhexidine (P>0.05).

[0155] (2) Experimental results of composition B

[0156] The experimental results of composition B are shown in Table 11 and Figure 6 shown.

[0157] Table 11 Inhibitory effect of composition B on Pg biomembrane extracellular protein (n=3)

[0158]

[0159] Compared with the Control group, Figure 6 As shown, composition B can significantly inhibit biofilm extracellular protein (P < 0.05), and chlorhexidine can significantly inhibit biofilm extracellular protein (P < 0.01). Specifically, as shown in Table 11, compared with the Control group, the inhibitory effects of composition B and chlorhexidine on biofilm extracellular protein were increased by 24.12% and 31.20%, respectively.

[0160] Compared with the Polygonum cuspidatum extract, as shown in Table 11, the inhibitory effect of composition B on biofilm extraprotein was increased by 3.91%, and compared with cinnamaldehyde, the inhibitory effect of composition B on biofilm extraprotein was increased by 14.16%, indicating that the inhibitory effect of composition B on biofilm extraprotein was better than that of Polygonum cuspidatum extract or cinnamaldehyde.

[0161] Compared with chlorhexidine, Figure 6 As shown, there was no significant difference in the inhibition of biofilm extracellular proteins by composition B (P>0.05).

[0162] In summary, composition B inhibited biofilm extracellular protein by 24.12% compared with the Control group, 3.91% compared with Polygonum cuspidatum extract, 14.16% compared with carvacrol, and there was no statistical difference compared with chlorhexidine (P>0.05).

[0163] Example 7: Inhibitory effects of composition A and composition B on extracellular DNA in biofilm 1. Experimental methods

[0164] (1) Preparation of biofilm eDNA solution. Take 1 mL of bacterial suspension per well and inoculate it into a 24-well plate. Culture it anaerobically at 37°C for 48 to 72 hours. Rinse it gently with sterile PBS three times. Add 1 mL of each group of drugs prepared in advance, set up a growth control group, continue to culture it anaerobically at 37°C for 24 hours, and then rinse it with PBS three times. Add TEN buffer to resuspend the biofilm and make a bacterial suspension with OD600nm=0.4. Take 1 mL respectively and follow the instructions of the bacterial genomic DNA extraction kit to extract a high-purity biofilm eDNA solution of the test bacteria. (2) Determine the content of biofilm eDNA in the sample. Determine the nucleic acid concentration value of each group of eDNA solution using an ultra-micro UV spectrophotometer. Repeat the experiment three times.

[0165] 2. Experimental results

[0166] (1) Experimental results of composition A

[0167] The experimental results of composition A are shown in Table 12 and Figure 7 shown.

[0168] Table 12 Inhibitory effect of composition A on DNA outside Pg biofilm (n=3)

[0169]

[0170] Compared with the Control group, Figure 7 As shown, both composition A and chlorhexidine can significantly inhibit the DNA outside the biofilm (all groups are P < 0.001). Specifically, as shown in Table 12, compared with the Control group, the inhibitory effects of composition A and chlorhexidine on the DNA outside the biofilm increased by 48.04% and 60.62%, respectively.

[0171] Compared with knotweed extract or carvacrol, e.g. Figure 7 As shown, composition A significantly inhibited the extrabiofilm DNA (all groups were P < 0.01). Specifically, as shown in Table 12, compared with the knotweed extract, the inhibitory effect of composition A on the extrabiofilm DNA was increased by 42.70%; compared with carvacrol, the inhibitory effect of composition A on the extrabiofilm DNA was increased by 42.60%.

[0172] Compared with chlorhexidine, Figure 7 As shown, composition A had no significant difference in inhibiting the biofilm extracellular DNA (P>0.05).

[0173] In summary, composition A inhibited extrabiofilm DNA by 48.04% compared with the Control group, 42.70% compared with Polygonum cuspidatum extract, and 42.60% compared with carvacrol, but there was no statistical difference compared with chlorhexidine (P>0.05).

[0174] (2) Experimental results of composition B

[0175] The experimental results of composition B are shown in Table 13 and Figure 8 shown.

[0176] Table 13 Inhibitory effect of composition B on DNA outside Pg biofilm (n=3)

[0177]

[0178] Compared with the Control group, Figure 8 As shown in Table 13, the inhibitory effects of composition B and chlorhexidine on biofilm extracellular DNA were increased by 42.77% and 53.69%, respectively, compared with the Control group.

[0179] Compared with knotweed extract or cinnamaldehyde, Figure 8 As shown in Table 13, composition B can significantly inhibit DNA outside biofilm (all groups are P < 0.05). Specifically, as shown in Table 13, compared with the Polygonum cuspidatum extract, composition B has an inhibitory effect on DNA outside biofilm of 37.62%, and compared with carvacrol, composition B has an inhibitory effect on DNA outside biofilm of 40.67%.

[0180] Compared with chlorhexidine, Figure 8 As shown, there was no significant difference in the inhibition of DNA outside the biofilm by composition B (P>0.05).

[0181] In summary, composition B inhibited extrabiofilm DNA by 42.77% compared with the Control group, 37.62% compared with Polygonum cuspidatum extract, 40.67% compared with carvacrol, and there was no significant difference compared with chlorhexidine (P>0.05).

[0182] In summary, although the present invention has been disclosed as above in terms of preferred embodiments, the above preferred embodiments are not intended to limit the present invention. A person skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be based on the scope defined in the claims.

Claims

1. A composition for inhibiting oral malodor-causing bacteria, characterized in that: The composition comprises a knotweed extract and carvacrol; in the composition, the concentration of the knotweed extract is ≥6.25 mg / mL, and the concentration of carvacrol is ≥160.00 μg / mL.

2. A composition for inhibiting oral malodor-causing bacteria according to claim 1, characterized in that: In the composition, the concentration of the knotweed extract is ≥58.33 mg / mL, and the concentration of carvacrol is ≥373.33 μg / mL.

3. A composition for inhibiting oral malodor-causing bacteria according to claim 1, characterized in that: The extraction method of the polygonum cuspidatum extract is as follows: placing polygonum cuspidatum medicinal material powder in an alcohol solution of 5 to 10 times its weight, soaking for 5 to 60 minutes; then heating and refluxing extraction at 50° C. to 80° C. for 1 to 3 hours, extracting 1 to 3 times, combining the filtrates, and obtaining the polygonum cuspidatum extract.

4. Use of the composition according to any one of claims 1 to 3 in the preparation of a medicament for inhibiting oral malodor-causing bacteria and the biofilm formed thereby.

5. The use according to claim 4, characterized in that: The oral malodor-causing bacteria include Porphyromonas gingivalis.

6. An oral care product, characterized in that: The oral care product comprises the composition according to any one of claims 1 to 3.

7. An oral care product according to claim 6, characterized in that: The oral care products include toothpaste, tooth powder, mouthwash and breath freshener.

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