Anti-caries synergistic composition for oral care

Abstract

The present invention relates to the combination of guava leaf extract, naringenin and an enzyme selected from lysozyme, labiase or a combination thereof for use in a dental or oral care product.

Description

Technical Field

[0001] The present invention relates to the fields of cosmetics, dentistry and hygiene, in particular to dental care products and oral care products that provide a high level of resistance to microbial proliferation and that favorably influence the hydration level of the oral mucosa. The compositions can be used as health or therapeutic products in human and veterinary medicine. Background Art

[0002] Despite significant efforts by global health systems to make dental clinics accessible to as large a population as possible and the widespread availability of a variety of dental care and oral care products on the global market, decay of permanent teeth (dental caries) remains one of the most prevalent health problems.

[0003] Two bacterial species, Streptococcus mutans and Streptococcus sobrinus, have been identified as the primary microorganisms responsible for the development of dental caries (Mathur and Dhillon, 2018). In conditions such as poor oral care, excessive consumption of sweets, or other factors, these strains of bacteria gain a significant selective advantage over the rest of the oral microbiome and proliferate in dense populations. The adhesion of these microorganisms to tooth surfaces leads to the formation of a biofilm (dental plaque), which protects the bacterial colony from the effects of components of the human immune system, antimicrobial agents, and other adverse factors (Sims et al., 2020; Lemos et al., 2019; Yue et al., 2018; Pleszczyńska et al., 2016; Bhagavathy et al., 2019). Like other microorganisms colonizing tooth surfaces, S. mutans forms biofilms using a nonspecific and sugar-dependent adhesion mechanism (Lemos et al., 2019; Yue et al., 2018). The nonspecific mechanism is achieved through hydrophobic and hydrophilic interactions and hydrogen bond formation between the superficial structures of the bacteria and the pellicle (a layer composed of salivary proteins and sulcular fluid on the teeth). Sugar-dependent adhesion is mediated by insoluble polysaccharides called glucans, which have a branched structure and are synthesized by bacterial enzymes called glycosyltransferases (GTFs) (Lemos et al., 2019; Yue et al., 2018; Cai et al., 2018). Polysaccharides promote the attachment of Streptococci to the tooth surface and also promote the formation of aggregates composed of Streptococci and other bacteria. The life cycle of cariogenic species involves the cleavage of easily fermentable carbohydrates into lactic acid, which causes focal demineralization and destruction of tooth enamel, leading to the formation of deep cavities (Mathur and Dhillon, 2018; Al-Ansari et al., 2021; Cai et al., 2018; Bhagavathy et al., 2019).

[0004] Therefore, preventive measures aimed at minimizing the risk of developing dental caries mainly focus on reducing the S. mutans and S. sobrinus populations in the oral cavity and preventing biofilm formation on tooth surfaces.

[0005] In the claimed invention, aimed at controlling Streptococcus mutans and Streptococcus sobrinus and destroying the biofilms formed by these species, two bioactive ingredients were selected: naringenin and the decomposing enzymes labiase and / or lysozyme. The authors also demonstrated that the use of guava as a supplementary ingredient can enhance the efficacy of the above agents.

[0006] Naringenin is a flavonoid that exhibits antibiofilm, antibacterial, antioxidant, anti-inflammatory, and antitumor properties (Wilcox et al., 1999; Yue et al., 2018). Yue et al. comprehensively investigated the mechanisms of naringenin and demonstrated its ability to shut down the expression of the genes encoding glycosyltransferases gtfB and gtfC in Streptococcus mutans (Yue et al., 2018). Consequently, the microorganism loses the ability to produce these enzymes and, consequently, the ability to synthesize glucans, which are crucial for the sugar-dependent mechanism of adhesion to tooth surfaces and biofilm formation. Furthermore, naringenin exhibits adverse effects on the nonspecific adhesion phase of bacteria.

[0007] Several authors have reported high antimicrobial activity of naringenin against Streptococcus mutans: the minimum inhibitory concentration (MIC) of naringenin against this agent ranged from 100 to 200 micrograms per milliliter (μg / mL). Naringenin at a concentration of 200 μg / mL almost completely inhibited biofilm formation.

[0008] Advantages of using naringenin include its ready availability and simple production from extracts of citrus plants, most commonly grapefruit (Citrus paradisi) and lime (Citrus aurantium).

[0009] The decomposing enzymes - lysozyme and / or labiase - are the second component of the composition and exhibit a biocidal action manifested in the destruction of bacterial cell walls.

[0010] Lysozyme is a hydrolase that cleaves β-(1→4)-glycosidic bonds in peptidoglycan molecules present in the cell walls of Gram-positive bacteria (Pleszczyńska et al., 2016). Furthermore, experiments have shown that lysozyme can protect the oral cavity from harmful microbial effects by cation-dependent activation of autolysins, inhibiting bacterial adhesion to tooth surfaces, and inhibiting glucose metabolism, thereby reducing the synthesis of acids that erode tooth enamel (Tenovuo, 2002; Niwa et al., 2005; Pleszczyńska et al., 2016). Lysozyme is commonly used as an active ingredient to control the cariogenic bacterium Streptococcus mutans in toothpastes, mouthwashes, chewing gums, hydrating gels, and sprays manufactured by manufacturers such as Biotene, BioXtra, Oralbalance, and Zendium Saliva (Tenovuo, 2002; Pleszczyńska et al., 2016). It has been determined that supplementing these products with lysozyme reduces dry mouth in patients with xerostomia and improves the body's natural defenses against dental caries. The antimicrobial activity of lysozyme can be enhanced if it is combined with lactoferrin and peroxidase (Pleszczyńska et al., 2016).

[0011] The advantage of lysozyme is its potential to maintain long-term activity in oral care products (Tenovuo, 2002).

[0012] Although naringenin has been reported in the literature to inhibit lysozyme, the authors unexpectedly discovered a synergistic effect between these components. For example, Ashrafi et al. showed that naringenin inhibits the enzymatic activity of lysozyme and has an affinity for the lysozyme active center. Based on the findings of a comprehensive study, the authors of this study showed that the interaction between naringenin and lysozyme leads to a change in the lysozyme conformation and a significant reduction in lysozyme activity (Ashrafi et al., 2022). Despite this, the authors of the present invention found that naringenin and lysozyme exhibit synergistic antibacterial activity.

[0013] Labiase is an enzyme with N-acetylglucosaminidase (disrupting bacterial wall synthesis) and mureinase (cleaving the peptidoglycan layer of the cell wall) activities (Ohbuchi K. et al., 2001). Labiase has been shown to rapidly and efficiently cleave the cell walls of many Gram-positive bacteria, including Streptococcus mutans (Tenovuo, 2002; Niwa et al., 2005).

[0014] One of the advantages of labiase is its high antibacterial efficacy against lysozyme-resistant strains (Niwa et al., 2005). However, to date, labiase has only been used for cell wall lysis for the purpose of further DNA and RNA isolation (Oana et al., 2009; Ishii et al., 2017).

[0015] Guava (Psidium guajava) leaf extract contains a wide range of bioactive compounds, including alkaloids, flavonoids, polyphenols, phenols, tannins, saponins, glycosides, and sterols (Garode and Waghode, 2014; Bhagavathy et al., 2019; Shaheena S. et al., 2019). These compounds exhibit significant antibacterial activity against a wide range of Gram-positive and Gram-negative bacteria (Nayak N. et al., 2019). In particular, tannins dissolve lipids in bacterial cell walls, leading to osmotic disruption and cell death (Netrusov and Kotova, 2018; Bhagavathy et al., 2019). Polyphenols inhibit the activity of several bacterial virulence factors, including lipopolysaccharides and various bacterial enzymes (Bhagavathy et al., 2019; Chen et al., 2005). In 2014, Garode and Waghode extracted bioactive components from guava leaves using solvents such as acetone, methanol, benzene, petroleum ether, chloroform, and ethanol, and then used the agar diffusion method to evaluate the biocidal efficacy of the extracts in a model strain of Streptococcus mutans (Garode and Waghode, 2014). These authors noted that solutions of guava extract in acetone, methanol, and petroleum ether exhibited antimicrobial activity comparable to that of the antibiotic ampicillin.

[0016] A 2019 study confirmed the anti-biofilm activity of a methanol extract of guava leaves (Bhagavathy et al., 2019). Some components of the extract appear to interact with the active site of glucosyltransferase (GTF), acting as competitors for the substrate (sucrose) and inhibiting the synthesis of glucans, a major biofilm component.

[0017] In the opinion of most authors, the advantages of guava leaf extract include: easy availability of raw materials, easy extraction process and environmental friendliness. The authors of the present invention conducted several experiments, which showed that guava can further enhance the antibacterial activity of the composition containing naringenin and lysozyme.

[0018] The above-mentioned components are used separately in various oral care products and dental care products. However, their synergistic effects have not been previously disclosed.

[0019] Toothpastes containing lysozyme and an extract from the purple goose down plant (Gynura procumbens (Lour.) Merr.) have been previously described (see patent CN105534827B, published on February 23, 2018; IPCs A61K8 / 97, A61K8 / 66, A61Q11 / 00, etc.). This toothpaste contains lysozyme and the purple goose down plant extract in a weight ratio of 1:10 to 10:1. The manufacturer reports that this toothpaste effectively cleans teeth and the oral cavity against pathogenic bacteria without developing microorganisms resistant to the toothpaste's active ingredients.

[0020] A drawback of the toothpaste is that it contains only one active ingredient—lysozyme—which is intended to inhibit bacterial proliferation and disrupt biofilms. In contrast, the purple velvet plant extract possesses anti-inflammatory, hemostatic, and wound-healing activities. To date, at least three mechanisms have been identified in bacteria that overcome the biocidal effects of lysozyme (Ragland and Criss, 2017). Therefore, the accumulation of lysozyme-resistant bacteria in the oral cavity would ultimately lead to a reduction in the efficacy of the proposed toothpaste.

[0021] ROCS "Balsam For Gums" toothpaste is manufactured by The Commercial and Industrial Group of Companies (DRC). The product's composition includes asparagine tanning agent, sodium bicarbonate, xylitol (6%), benzoic acid, salvinyl glycosides, salicin glycosides, the enzyme salicase, the flavonoids naringenin and sacuranten, coumarin, and the saturated fatty acids palmitic, behenic, lignoceric, and cerotic. However, the toothpaste was not developed for caries prevention, but rather for the prevention of inflammatory diseases of the oral cavity, as the listed ingredients primarily exhibit anti-inflammatory, decongestant, and healing effects.

[0022] "Guava Herbal Toothpaste," manufactured by Beautecrets (Thailand), contains guava extract. The toothpaste's ingredients include extracts of the tropical guava (Psidium guajava) and the Morinda citrifolia tree, along with Syzygium aromaticum leaf oil and Mentha piperíta oil. These ingredients are known for their anti-inflammatory, anesthetic, antiviral, and antimicrobial properties, with the latter also being used as a deodorant. However, due to its weak antimicrobial properties, the manufacturer markets the product as a freshening toothpaste.

[0023] Another whitening herbal toothpaste known as "HERBAL CLOVE TOOTHPASTE," manufactured by 5Star Cosmetic (Thailand), contains guava and clove extracts. The toothpaste's ingredients are: calcite, borneol, menthol, lauryl sulfate, Stebelius leaf extract, guava leaf extract, camphor, bamboo salt, and clove extract. It should be noted that locating the original information, manufacturer, and composition of this product is quite challenging. The product description states that these ingredients have a disinfecting effect, remove plaque, and prevent caries (https: / / www.japshop.ru / good / otbelivayuschie-udalenie-kamnya-naleta-10003485 / 100081-25- / ?ymclid=16014590748368498010500004), which is consistent with numerous scientific studies on guava leaf extract. However, it is known that the antibacterial effect is directly dependent on the concentration and extraction method of guava leaves (Nair and Chanda, 2007; Garode and Waghode, 2014). The absence of a specific guava extract or the percentage of active ingredient in the toothpaste's composition raises some questions about the claimed properties of this ingredient. The ingredient "CLOVE" in the toothpaste's name suggests that clove extract is the primary ingredient and is responsible for the product's health benefits. However, the percentage of clove extract in the product's composition, a key indicator of antibacterial activity, is not stated (Kaur & Chandrul, 2017). Therefore, the active ingredient listed in the toothpaste likely serves a marketing purpose.

[0024] Another known composition contains polyphenols / flavonoids and their derivatives for treating or preventing dental caries, plaque formation, gingivitis, periodontitis, candidiasis, denture stomatitis, and fungal infections (see US20140314686A1, published on October 23, 2014; IPCA61K8 / 66, A61Q11 / 00, A61Q17 / 005, A61P1 / 02, etc.). This composition contains various combinations of aglycone flavonoids, proanthocyanidins, dihydroflavonols, flavonols, and flavanols, including naringenin, added at therapeutically effective concentrations. When introduced into the oral cavity, this composition inhibits the activity of glucosyltransferases on the tooth surface and also suppresses the growth and proliferation of microorganisms, particularly those that produce glucosyltransferases. However, this composition does not contain the enzymes lysozyme and / or labiase.

[0025] Another known antimicrobial combination composition contains one or more glycerol derivatives selected from 3-[(2-ethylhexyl)oxy]-1,2-propanediol, 3-[(n-octyl)oxy]-1,2-propanediol, and 3-[(2-octyl)oxy]-1,2-propanediol], and one or more bicyclic compounds selected from naringenin, quercetin, hesperetin, and eriodictyol (see patent CA3104962A1, published on January 9, 2020; IPC A61Q17 / 00, etc.). This composition is designed to be added to toothpaste, mouthwash, and other hygiene products. This composition also does not contain lysozyme and / or labiase.

[0026] The limitations of the above compositions arise from the fact that they contain components that have the same antimicrobial mechanism as naringenin, thus providing conditions for the development of bacterial resistance to the products upon long-term use.

[0027] In terms of technical substance, the prior art closest to the claimed invention is an additive composition containing lysozyme and plant extracts (see patent CN110237241A; published on September 17, 2019; IPC A61Q11 / 00, A61K38 / 47, A61K8 / 66, A61P31 / 04, etc.). The plant extracts primarily exhibit anti-inflammatory and wound healing activities, and include Centella asiatica (Gotu Kola PE) extract, Polygonum japonicum Meissn. root extract, Scutellaria baicalensis extract, common licorice (Glycyrrhiza glabra) extract, German wallweed (Matricaria recutita) flower extract, Camellia sinensis leaf extract, and dwarf rosemary (Rosmarinus officinalis) extract, or extracts of their respective varieties. These extracts contain various alkaloids, essential oils, flavonoids, polysaccharides, organic acids, tannins, and other active ingredients with varying antimicrobial potency. Some flavonoids exhibit properties similar to naringenin. In particular, apigenin, found in extracts of German wallwort, dwarf rosemary, and common licorice, is produced in plants by the enzyme flavanone synthase, which oxidizes naringenin.

[0028] A disadvantage of such compositions is the excessive use of large quantities of plant extracts exhibiting similar biological activities. The complexity of the composition inevitably complicates the manufacturing process and increases financial and time expenditures. Furthermore, standardizing the chemical composition of the plant extracts is a complex process, and as can be seen from the specifications, their use as oral care additives provides low antibacterial effects.

[0029] The present invention overcomes all the above-mentioned disadvantages by combining in one composition several (two to four) components with essentially different mechanisms of biocidal activity, thereby enabling a synergistic improvement of their antimicrobial and anticaries efficacy. In general, the two strategies of the effect achieved by the claimed composition can be specified as follows:

[0030] Eradication of biofilms, achieved through targeted exposure to naringenin and, in addition, the effect of guava leaf extract on the synthesis of glucan, an important matrix component. Specifically, naringenin disrupts glycosyltransferase synthesis at the genetic level, thereby inhibiting the adhesion of S. mutans to tooth enamel. Guava leaf extract binds to the active site of the enzyme, glycosyltransferase, and also inhibits glucan synthesis.

[0031] • Bacterial populations are reduced because decomposing enzymes (lysozyme and / or labiase) kill bacterial cells by destroying the cell wall.

[0032] Because the mechanisms of action of the composition's components are not species-specific, its antibacterial and antibiofilm activities are also applicable to Streptococcus sobrinus, which is closely related to Streptococcus mutans. It is reasonable to assume that the claimed composition will not induce bacterial resistance, a significant advantage that allows for its routine use as an oral care product. Summary of the Invention

[0033] The present invention relates to a combination of naringenin and an enzyme selected from lysozyme, labiase or a combination thereof for use in dental care products and / or oral care products.

[0034] The combination of the present invention may be characterized in that it additionally contains guava leaf extract.

[0035] The combination of the present invention may be characterized in that the guava leaf extract is a guava leaf CO2 extract.

[0036] The combination of the invention may be characterized in that the guava leaf extract is a Psidium guajava leaf extract.

[0037] The combination of the present invention may be characterized in that the guava leaf extract is a Psidium guajava leaf CO2 extract.

[0038] The combination of the invention may be characterized in that naringenin is obtained from a plant source.

[0039] The combination of the present invention may be characterized by containing guava leaf extract, naringenin, and enzyme in a ratio of (1.00-170.73):(1.00-235.77):(0.16-365.85), respectively.

[0040] The combination of the present invention may be characterized in that the combination is a composition.

[0041] The present invention relates to dental care products and / or oral care products comprising the combination according to the invention.

[0042] The product of the present invention may be characterized in that the weight % of the components are as follows:

[0043] Guava leaf extract 0.0615-10.5

[0044] Naringenin 0.0615-14.5

[0045] Enzyme 0.0096-22.5

[0046] The product of the invention may be characterized in that the product is a toothpaste or a mouthwash.

[0047] The present invention relates to the use of the combination of the invention for controlling Streptococcus mutans and / or Streptococcus sobrinus on tooth surfaces and / or in the oral cavity.

[0048] The present invention relates to the use of a dental care product and / or an oral care product for controlling Streptococcus mutans and / or Streptococcus sobrinus on tooth surfaces and / or in the oral cavity.

[0049] The technical results achieved by the present invention are: unexpected anti-biofilm and antibacterial synergistic effects, favorable effects on oral hydration, and effects and functions that can be understood by those skilled in the art, whether disclosed in this specification, especially in the experimental examples or directly derived therefrom. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Depicted are the results obtained from the assessment of S. sobrinus viability (in individual replicates) by the resazurin assay. DETAILED DESCRIPTION

[0051] The authors unexpectedly found that the use of naringenin and an enzyme (selected from lysozyme, labiase or a combination thereof) in combination provides a clear synergistic effect manifested in significantly enhanced anti-biofilm and antibacterial effects and also improved oral hydration.

[0052] Additional beneficial effects and functions that can be understood by those skilled in the art have been disclosed throughout this document or can be directly derived from the description of the present invention, especially from the experimental data obtained.

[0053] In a first aspect of the present invention, the present invention relates to a combination of naringenin and an enzyme selected from lysozyme, labiase or a combination thereof. Said combination can be used in dental care products and / or oral care products.

[0054] The combination may also be supplemented with guava leaf extract.Such a combination may be used in dental care products and / or oral care products.

[0055] Guava can be used as the fruit, bark or leaves of Psidium guajava. Naringenin can be obtained from any plant source.

[0056] The combination of the invention may be characterized in that the guava leaf extract is a guava CO2 extract, such as a Psidium guajava CO2 extract.

[0057] In this combination, the weight ratios of guava extract, naringenin, and enzyme are (1.00-170.73):(1.00-235.77):(0.16-365.85), respectively, wherein the ranges include the boundary values ​​and all values ​​within the range, as if each such value were expressly indicated. In other words, such a record discloses the boundary values, all values ​​between the boundary values, and any range consisting of the boundary values ​​and each value within the range.

[0058] The combination of the present invention may be characterized as a composition. Thus, a combination may not involve a mixture of substances, but rather encompasses, for example, concomitant and non-fixed administration of the components, whereas a composition is a special case of a combination involving a mixture of the components.

[0059] The present invention also relates to a dental care product and / or oral care product containing the combination according to the invention, such as, for example, a toothpaste or a mouthwash, having the following composition (in % by weight):

[0060] Guava leaf extract 0.0615-10.5,

[0061] Naringenin 0.0615-14.5,

[0062] Enzyme 0.0096-22.5.

[0063] Furthermore, the present invention encompasses the use of the combination of the present invention or the dental and / or oral care product of the present invention for controlling Streptococcus mutans and / or Streptococcus sobrinus on tooth surfaces and / or in the oral cavity.

[0064] Example

[0065] Group of Example Nos. 1 to 3. The antimicrobial activity of the substances or substance combinations can be assessed in terms of minimum inhibitory concentration (MIC) according to the recommendations of MUK (Methodological Guidelines) 4.2.1890-04 and Leclercq et al., 2013 and from the growth pattern in liquid nutrient medium.

[0066] Reference strains Streptococcus mutans and Streptococcus sobrinus were used as bacterial models.

[0067] The guava leaf extract (Psidium guajava leaf extract) used in all examples was obtained via modifier-enhanced supercritical CO extraction. The finished samples of the raw material contained at least 50% extract (other components included water (up to 100%), glycerin (up to 30%), and sodium benzoate (0.01%). The naringenin in all samples was isolated from grapefruit extract. The naringenin concentration in the finished raw material was 98%. The labiase used in all tests was a pure β-N-acetyl-D-glucosaminidase enzyme isolated from Streptomyces. The lysozyme used in all tests was 99% anhydrous lysozyme hydrochloride isolated from egg yolk.

[0068] Example No. 1. The following procedure was used to determine the MIC of the individual composition components, namely naringenin, guava leaf CO2 extract, and the enzymes lysozyme and labiase:

[0069] 1) Prepare liquid nutrient medium LB, which consists of: H2O 蒸馏 1 L of culture medium, 1.0% tripton; 0.5% yeast extract; 0.5% NaCl. Adjust the pH to 8.0. Autoclave the medium at 121°C for 20 minutes. Then, add sterile glucose solution to a final concentration of 1% and bovine serum to a final concentration of 5%.

[0070] 2) Using a microbiological loop, prepare suspensions (at a concentration of 1.5·10 turbidimetric units) from 24-hour agar cultures of Streptococcus mutans and Streptococcus sobrinus in sterile buffered saline (BS) using a McFarland standard 0.5 under visual control. 8 The obtained suspension was diluted 1:100 in sterile nutrient medium LB to obtain 1.5·10 6 Final cell concentration in CFU / mL.

[0071] 3) 100 μL of the test substance at a concentration twice the maximum concentration was added to well 1 of a sterile 96-well microtiter plate (Medpolymer, RF) and serially diluted two-fold to the minimum concentration with sterile distilled water. Then, 100 μL of the prepared inoculum was added to each well, and the bacterial suspension density was 10 6 CFU / 1 mL of sterile nutrient medium LB, thereby diluting the test compound two-fold.

[0072] 4) Prepare the following controls: a culture growth control (3 wells in which the liquid nutrient medium LB was diluted 2-fold with sterile distilled water, and bacterial culture was added, but no test substance was added); a nutrient medium purity control (3 wells in which the liquid nutrient medium LB was diluted 2-fold with sterile distilled water, but no bacterial culture was added); and a solvent control (3 wells in which the liquid nutrient medium LB was diluted 2-fold with sterile distilled water, and the test substance or test composition was added, but no bacterial culture was added).

[0073] 5) Incubate the plate with shaking at 37°C in an orbital shaker for 24 hours.

[0074] 6) The viability of bacterial planktonic cell cultures was assessed using the resazurin assay [Taneja NK, Tyagi J.S. Resazurin reduction assays for screening of anti-tubercular compounds against dormant and actively growing Mycobacterium tuberculosis, Mycobacterium bovis BCG and Myco-bacterium smegmatis. JAntimicrob Chemother. 2007; 60(2): 288-293]. For this purpose, after the incubation was complete, 10 μL of a 0.01% aqueous resazurin solution (Sigma-Aldrich, USA) was added to each well of the plate and the solution was allowed to develop color for 5 minutes under the same conditions.

[0075] 7) This test is based on a colorimetric measurement of cellular metabolic activity: in the presence of living cells, blue resazurin is reduced to pink resorufin. A pink resazurin solution indicates that the tested concentration is insufficient to completely inhibit bacterial metabolic activity. Therefore, the first well in which the solution does not turn pink is considered the minimum inhibitory concentration (MIC) of the test substance. Because a serial dilution method with a two-fold dilution step is used to determine the MIC, differences between adjacent dilutions are not considered significant.

[0076] 8) The following concentrations were tested: naringenin and guava leaf extract at 1.6, 0.8, 0.4, 0.2, 0.1, and 0.05 mg / mL; lysozyme and labiase at 4.0, 2.0, 1.0, 0.5, 0.25, and 0.125 mg / mL. Well-known antimicrobial components were used as controls for antimicrobial activity: chlorhexidine digluconate at 64, 32, 16, 8, and 4 μg / mL; and zinc gluconate at 5.0, 2.5, 1.25, 0.6, 0.3, and 0.15 mg / mL.

[0077] The MIC was defined as the median inhibitory concentration (MIC) obtained from five independent biological replicates.

[0078] The results of the antimicrobial activity evaluation of the test components are summarized in Table 1 .

[0079] Table 1 - Minimum concentration (MIC) of substances that inhibit the growth of bacterial species Streptococcus mutans and Streptococcus sobrinus

[0080]

[0081] As is apparent from Table 1, the test strain Streptococcus mutans is more sensitive to the antibacterial action of zinc gluconate than Streptococcus sobrinus. However, this strain is much more resistant to chlorhexidine digluconate.

[0082] Naringenin and lysozyme at concentrations of 0.8 and 4.0 mg / L, respectively, successfully inhibited the growth of the strain S. mutans.

[0083] In the experiments with S. sobrinus, lysozyme was the only component that showed growth inhibition at a concentration of 4.0 mg / L.

[0084] Thus, two closely related cariogenic species, S. mutans and S. sobrinus, show different levels of susceptibility to various antimicrobial agents. Based on these findings, it is of great interest and rational to include substances that exhibit different sets of biocidal mechanisms in antimicrobial compositions.

[0085] Example 2. The synergistic antimicrobial effect of a composition containing naringenin, guava leaf extract, and a degrading enzyme (specifically, lysozyme and / or labiase) was evaluated by MIC assay. All steps for the MIC assay were similar to those described in Example 1, except that 100 μL of the "naringenin + guava leaf extract + enzyme" composition was added to the wells of the plate instead of adding the individual substances. Naringenin and guava leaf extract were used in the composition at a constant concentration of 0.8 mg / mL. The concentrations of lysozyme and labiase in the composition were varied: 1.0, 0.5, and 0.25 mg / mL. The final composition containing naringenin + guava leaf extract + enzyme was also diluted 1:1, 1:2, and 1:4, and the MICs of the resulting dilutions were determined.

[0086] The MIC was defined as the median inhibitory concentration value measured in four independent biological replicates. The synergistic effect of the resulting mixture against cariogenic bacteria was determined by evaluating the enzyme concentration required to inhibit bacterial growth at different dilutions of the final composition when used as a single component and in combination with guava leaf extract and naringenin.

[0087] Table 2 shows the results of evaluating the antibacterial activity of the components used in the "naringenin (0.8 mg / mL) + guava leaf extract (0.8 mg / mL) + enzyme (1.0-0.25 mg / mL)" composition against the cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus when the composition was tested at concentrations of 100%, 50%, and 25%.

[0088] Table 2 - Differences in the minimum concentration (MIC) of lysozyme or labiase in the combination of "naringenin + guava leaf extract + enzyme" to inhibit the growth of Streptococcus mutans and Streptococcus sobrinus

[0089]

[0090] For both bacterial strains, all components were employed in the compositions at concentrations well below the previously determined MICs (Table 1).

[0091] As is apparent from Table 2, in the presence of naringenin 0.8 mg / mL and guava leaf extract 0.8 mg / mL, the MICs of lysozyme against S. mutans and S. sobrinus were reduced by 4-fold and 24-fold, respectively. The MIC of labiase against S. sobrinus was reduced by 32-fold, while the antibacterial activity of labiase against S. mutans remained unchanged.

[0092] Therefore, it was concluded that the antibacterial effect of the "naringenin + guava leaf extract + enzyme" combination against both cariogenic bacteria was increased due to a synergistic effect, which was reflected by the decrease in the MIC of the decomposing enzymes in the system compared to the MIC of the individual agents (Table 1).

[0093] Example No. 3. The antibacterial activity of naringenin, guava leaf extract, and lysozyme was evaluated based on the growth pattern of the reference strain Streptococcus mutans No. 22081 (UNU Collection of the Mechnikov Research Institute of Vaccines and Serum) in liquid nutrient medium according to the following procedure:

[0094] 1) Prepare Heart-Brain Broth (DM106D, Mast Group Ltd., United Kingdom) and Columbia Agar supplemented with defibrinated blood (OOO "GEM", catalog number BP-023) according to the manufacturer's instructions.

[0095] 2) Using a microbiological loop, prepare a suspension of a 24-hour agar culture of Streptococcus mutans in sterile buffered saline (BS) (at a concentration of 1.5·10 8The obtained suspension was diluted in BS to a concentration of 10 6 CFU / mL.

[0096] 3) Add 240 μL of heart-brain broth and 30 μL of 10 6 A suspension of Streptococcus mutans at 100 CFU / mL and 30 μL of the test substance from naringenin, guava leaf extract, or lysozyme at the test concentrations were prepared. A composition of 0.33 mg / mL naringenin, 0.33 mg / mL lysozyme, and 0.33 mg / mL guava leaf extract was prepared.

[0097] 4) The plate with the samples was placed in an incubator with a 5% CO2 atmosphere and incubated at 37°C for 5 hours on a shaking device.

[0098] 5) After incubation for 5 hours, the planktonic mutans Streptococcus culture was re-inoculated into the agar nutrient medium to count the CFU per 1 mL of the medium.

[0099] 6) Optical density (OD) measurements were performed using a multi-mode microplate reader at a wavelength of 620 nm before incubation (OD0) and then after the culture had grown for 5 hours (OD t＝5 In the calculation, the average optical density obtained during the baseline measurement was subtracted from the average optical density obtained to offset the inherent color of the test sample (OD t＝5 –OD0).

[0100] The level of inhibition (%) of the growth of mutans Streptococcus culture was calculated from the following formula:

[0101] %OD = OD 物质 (OD t＝5 –OD0) / OD 对照 (OD t＝5 –OD0)×100–100,(%) (2)

[0103] 7) The test substance was measured three times for each concentration; the control sample was measured 12 times.

[0104] The results of the evaluation of the antibacterial activities of naringenin, guava leaf extract, and lysozyme against Streptococcus mutans in liquid nutrient medium are shown in Table 3.

[0105] Table 3 - Effects on the growth of planktonic cultures of Streptococcus mutans in liquid nutrient medium

[0106] Active ingredient ∑CFU,mL % of CFU obtained for the control comparison <![CDATA[9х10 8 ]]> 100 Lysozyme (1 mg / mL) <![CDATA[4.5х10 7 ]]> 5 Naringenin (1 mg / mL) <![CDATA[4.3х10 7 ]]> 4.8 Guava extract (1mg / mL) <![CDATA[4.6х10 7 ]]> 5.1 Composition (1 mg / mL) <![CDATA[2.1х10 7 ]]> 2.3

[0107] As is apparent from Table 3, all the test substances greatly affected the growth of planktonic cultures of S. mutans in liquid nutrient medium. The composition containing lysozyme, guava leaf extract and naringenin at a final concentration of 1 mg / mL showed the highest inhibitory effect.

[0108] The combination exhibits a synergistic effect on the growth of planktonic cultures of S. mutans compared to the effects of the individual components.

[0109] Groups of Examples No. 4 to 6. The antibiofilm activity of the substances or combinations of substances was assessed from the minimum biofilm inhibitory concentration (MBIC) and from the rate of biofilm formation on hydroxyapatite disks.

[0110] Reference strains Streptococcus mutans and Streptococcus sobrinus were used as bacterial models.

[0111] Example No. 4. As a measure of the effect of a substance on bacterial biofilms, MBIC is equal to the minimum concentration that inhibits biofilm formation or disrupts an already formed biofilm during a 24-hour incubation period.

[0112] The MBIC of each component of the composition (specifically, naringenin, guava leaf CO2 extract, and the enzymes - lysozyme and labiase) can be determined according to the following procedure:

[0113] 1) All stages of nutrient medium preparation, preparation and incubation of bacterial cultures in 96-well microtiter plates with the test substance composition added to the nutrient medium were as described in Example 1.

[0114] 2) The controls for culture growth, nutrient medium purity, and solvent were all as described in Example 1.

[0115] 3) Incubate the plate at 37°C for 24 ± 4 hours without using an orbital shaker.

[0116] 4) The bacterial biofilm accumulated on the bottom of the plate wells was stained with crystal violet. For this purpose, the planktonic culture was removed from the plate wells after 24 hours of incubation and washed with two portions of phosphate buffered saline (PBS, pH 7.4) to remove the remaining planktonic culture. The plate was then dried for 12-15 hours. 100 μL of a 0.5% crystal violet solution (Sigma-Aldrich, USA) dissolved in 96% ethanol was added and incubated for 20 minutes. After removing the crystal violet solution from the wells, the plate was washed with 3-4 portions of PBS. After drying, 1 mL of 96% ethanol was added to the wells to elute the crystal violet that had been bound to the biofilm.

[0117] 5) The plate was shaken on an orbital shaker at 37°C for 2 minutes, and then the optical density (OD) was measured at a wavelength of 550 nm using a multimode microplate reader compared to a blank (i.e., a well in which sterile LB culture medium was incubated and then exposed to all staining procedures). Since a serial dilution method using a 2-fold dilution step was used to determine the MBIC, the differences between adjacent dilutions were considered insignificant, and the MBIC was defined as the median value calculated for 5 independent biological replicates.

[0118] 6) The following concentrations were tested: naringenin and guava leaf extract at 1.6, 0.8, 0.4, 0.2, 0.1, and 0.05 mg / mL; lysozyme and labiase at 4.0, 2.0, 1.0, 0.5, 0.25, and 0.125 mg / mL. Well-known antimicrobial components were used as controls: chlorhexidine digluconate at 64, 32, 16, 8, and 4 μg / mL; and zinc gluconate at 5.0, 2.5, 1.25, 0.6, 0.3, and 0.15 mg / mL.

[0119] The results of the evaluation of the antibiofilm activity of the test components against biofilms produced by cariogenic species are summarized in Table 4.

[0120] Table 4 - Minimum biofilm inhibitory concentration (MBIC) of substances against bacterial species Streptococcus mutans and Streptococcus sobrinus

[0121]

[0122] As is evident from Table 4, the test strain S. mutans produced less biofilm in the presence of zinc gluconate than did S. sobrinus. However, this strain was much more resistant to chlorhexidine digluconate.

[0123] Naringenin and lysozyme at concentrations of 0.8 and 4.0 mg / L, respectively, successfully inhibited biofilm formation of the strain S. mutans.

[0124] Lysozyme at a concentration of 4.0 mg / L was also able to prevent biofilm formation by the strain S. sobrinus.

[0125] Thus, two closely related cariogenic species, S. mutans and S. sobrinus , showed different levels of susceptibility to different mechanisms of antibiofilm action of the test agents.

[0126] Example No. 5. Evaluation of the synergistic antibiofilm effect of a composition containing naringenin, guava leaf extract, and a degradative enzyme (specifically, lysozyme and / or labiase) by MBIC determination. The MBIC determination used a method similar to that described in Example 4 in all stages, except that 100 μL of a "naringenin + guava leaf extract + enzyme" composition was added to the plate wells instead of adding the individual substances. Naringenin and guava leaf extract were used in the composition at a constant concentration of 0.8 mg / mL. The concentrations of lysozyme and labiase in the composition were varied: 1.0, 0.5, and 0.25 mg / mL. The final composition containing naringenin + guava leaf extract + enzyme was also diluted 1:1, 1:2, and 1:4, and the MBIC of the resulting mixture was determined.

[0127] MBIC was defined as the median inhibitory concentration value measured in four independent biological replicates. The synergistic effect of the resulting mixture against cariogenic bacteria was determined by evaluating the enzyme concentrations required to inhibit bacterial biofilm at different dilutions of the final composition when used as single components and in combination with guava leaf extract and naringenin.

[0128] Table 5 summarizes the results of the evaluation of the synergistic antibiofilm activity of the components used in the composition of "naringenin (0.8 mg / mL) + guava leaf extract (0.8 mg / mL) + enzymes (1.0-0.25 mg / mL)" against the cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus.

[0129] Table 5 - Differences in Minimum Biofilm Inhibitory Concentration (MBIC) of Lysozyme or Labiase in the Composition of “Naringenin + Guava Leaf Extract + Enzyme” Against S. mutans and S. sobrinus

[0130]

[0131]

[0132] As is apparent from Table 5, naringenin 0.8 mg / mL and the combination of naringenin and guava leaf extract 0.8 mg / mL provided a 32-fold reduction in the lysozyme and labiase inhibitory concentrations against biofilm formation by S. mutans; and a 16-fold and 12-fold reduction in the lysozyme and labiase inhibitory concentrations against biofilm formation by S. sobrinus, respectively.

[0133] Thus, a composition containing naringenin (0.8 mg / mL), guava leaf extract (0.8 mg / mL), and a degradative enzyme selected from lysozyme and labiase (1.0-0.25 mg / mL) provided enhanced anti-biofilm effects against both cariogenic species of Streptococcus. A strong synergistic activity of this system was observed, as the desired effects were achieved at significantly lower concentrations of naringenin and guava leaf extract, along with labiase and lysozyme, when used as a combination, compared to the MBIC of each substance alone (Table 4).

[0134] Example No. 6. The anti-biofilm activity of a substance selected from naringenin, guava leaf extract, and lysozyme, or a combination thereof, was evaluated from the rate of biofilm formation on hydroxyapatite disks using the following procedure:

[0135] 1) Prepare Heart-Brain Broth (DM106D, Mast Group Ltd., United Kingdom) and Columbia Agar supplemented with defibrinated blood (OOO "GEM", catalog number BP-023) according to the manufacturer's instructions.

[0136] 2) All stages of the preparation of the bacterial culture suspension were similar to those described in Example 1.

[0137] 3) Preparation of saliva, which is essential for biofilm formation and facilitates bacterial adhesion to the hydroxyapatite disc surface. Saliva was collected from 15 healthy, non-smoking adult volunteers who had not taken antibiotics within the past three months no earlier than one hour after eating or brushing their teeth into sterile, cooled tubes. The collected saliva was centrifuged at 12,000 rpm for 30 minutes at 4°C. The supernatant was collected into a clean tube and pasteurized at 65°C for 30 minutes, followed by another centrifugation.

[0138] The efficacy of saliva pasteurization was assessed by direct plating on Columbia blood agar. The plates were incubated at 37°C for 72 hours under aerobic and anaerobic conditions. If no growth of bacterial and fungal colonies was observed during the 72-hour observation period, the saliva was considered sterile.

[0139] The saliva was stored at -80°C before use.

[0140] 4) To form a saliva film on the surface of the hydroxyapatite disk, three sterile disks (autoclaved at 132°C for 20 minutes) were placed in each of nine 40 mm diameter Petri dishes and incubated in 3 mL of pasteurized saliva at room temperature for 4 hours while gently shaking on an orbital shaker. The saliva was then removed and 3 mL of a nutrient medium containing 1.2 mL of heart-brain broth, 1.2 mL of pasteurized saliva, and 0.6 mL of a 10% sucrose solution was added to the Petri dish. 6The mutans S. biofilm was formed on the dish surface by gentle shaking on an orbital shaker at 37° C. in an ambient 5% CO 2 atmosphere for 16 hours.

[0141] 5) After 16 hours of incubation, remove the old nutrient medium from the culture dish and add a fresh portion of heart-brain broth containing a suspension of mutans Streptococcus and pasteurized saliva. Add an active agent selected from naringenin, guava leaf extract, and lysozyme, or a combination thereof, to a final concentration of 0.1 or 1.0 mg / mL. Repeat the medium exchange procedure twice daily within 4 to 8 hours after the first exchange.

[0142] 6) To remove the biofilm formed on the dish surface, remove the dish from the Petri dish after 2 days of incubation and rinse in a large amount of PBS (pH 7.2). The dish is then placed in a sterile Petri dish containing 3 mL of PBS (pH 7.2) and shaken vigorously on an orbital shaker for 30 minutes. The resulting mutans S. mutans suspension is transferred to a labeled sterile test tube and the CFU / mL is determined by inoculation of nutrient agar medium.

[0143] Table 6 summarizes the results of the inoculations, wherein the biofilm was washed off the disk surface after 64 hours of incubation in the presence of the active substance alone or the combination of substances.

[0144] Table 6 - Quantitative comparison of CFU in resuspended S. mutans biofilms formed on the surface of hydroxyapatite disks during 64 hours of incubation in the presence of naringenin, guava leaf extract, lysozyme, or a combination thereof

[0145]

[0146] Notes: (*) – Control. C 物质 – concentration of the test substance; N 变异链球菌 - CFU of mutans S. on the plate after 5 hours of incubation in the presence of the substance; % N 对照 – Percentage of S. mutans CFU on the plates in the control experiment.

[0147] In this experiment, inoculation was performed using S. mutans biofilms that had been partially disrupted by shaking. Under these conditions, each biofilm fragment formed a single S. mutans colony on the nutrient agar medium. It should be noted that the more active the destructive processes in the biofilm are during incubation when exposed to active substances, the more pronounced the breakdown of chemical bonds between the components of the biofilm matrix, the greater the number of biofilm fragments formed during shaking, and ultimately, the higher the number of colony-forming units (CFUs).

[0148] Table 6 shows that exposure to guava leaf extract at a concentration of 1.0 mg / mL was associated with significant disruption of the biofilm. However, a 10-fold reduction in the concentration of active substance in the culture medium resulted in a substantial reduction in the level of disruption.

[0149] In addition to their antibiofilm activity, naringenin and lysozyme also demonstrated high antimicrobial activity against S. mutans (Tables 1, 2, and 3). This may explain the modest CFU / mL values ​​compared to the control. These substances maintained their biocidal potential down to a concentration of 0.1 mg / mL—the levels of viable cells in suspension were 66.7% and 88.1%, respectively, compared to the control.

[0150] The greatest disruption of S. mutans biofilms was observed following exposure to the 1.0 mg / mL "naringenin + guava leaf extract + lysozyme" combination. Compared to the control, the level of colony-forming fragments of the biofilm reached 262%. Due to the synergistic effect of several disruptive mechanisms, the combination maintained high anti-biofilm activity even at a 10-fold reduction in concentration.

[0151] Example No. 7. Evaluation of the effect of the active composition "naringenin + guava leaf extract + enzyme" on oral hydration (saliva secretion).

[0152] equipment:

[0153] Containers for saliva collection, such as (Sarstedt Inc., Germany), which contains a cotton swab without any reagent and is fixed in a special holder on the upper part of the container.

[0154] Analytical balance with an accuracy of m ± 0.0001г and a weighing limit of at least 300g;

[0155] Professional complete toothbrush of medium hardness (SPLAT).

[0156] The compositions of the two toothpastes tested in the experiment are shown in Table 7.

[0157] Table 7 - Comparative properties of toothpaste chemical compositions

[0158]

[0159]

[0160] Test procedure:

[0161] 1) Container for saliva collection Mark them well, then weigh them three times on an electronic balance, calculate the arithmetic mean weight of each container and name it "Weight 1.1".

[0162] 2) Ten volunteers were randomly assigned to Group A to test toothpaste without an active composition (5 subjects) or to Group B to test toothpaste containing an active composition (5 subjects).

[0163] 3) All volunteers used a toothbrush with a test toothpaste sample (paste weight ~2 mg) to clean their teeth for 2 minutes.

[0164] 4) Then, remove the swab from the labeled container and place it in each subject's mouth. After two minutes, use tweezers to return the swab to its respective container.

[0165] 5) The container with the collected saliva was then weighed three times, and the arithmetic mean of the three measurements was calculated and designated as "Weight 1.2".

[0166] 6) The weight of the collected saliva is calculated using the following formula:

[0167] Saliva weight = weight 1.2 - weight 1.1 (2).

[0168] 7) The test was repeated twice, with subjects testing the toothpaste samples in a random order. The weight of the saliva collected each time was calculated using formula (2).

[0169] 8) After the test was completed, the arithmetic mean weight of saliva collected from each subject during the test period of the toothpaste containing no active composition (Group A) or containing the active composition (Group B) was calculated.

[0170] 9) Significant differences between groups were established using the Mann-Whitney U test (nonparametric analysis) or the Student's t test for comparison of unrelated populations (parametric analysis).

[0171] The test results are shown in Table 8.

[0172] Table 8 - Determination of statistically significant differences in saliva weight among subjects testing the two types of toothpaste

[0173]

[0174] Notes : (*) - arithmetic mean calculated from three measurements. Group A - toothpaste without active composition tested; Group B - toothpaste with active composition tested. Mann-Whitney U-test critical value = 23 for the specified statistic of the compared groups.

[0175] As is apparent from Table 8, all subjects showed differences in saliva secretion levels after using toothpaste containing or not containing the active composition. It was established that the toothpaste containing the active composition statistically significantly increased the saliva secretion levels in the subjects (Group B) compared to the toothpaste without the active composition (Group A).

[0176] Example 8. Evaluation of the Compatibility of Lysozyme and Naringenin. The potential effect of naringenin on lysozyme activity was evaluated by measuring lysozyme activity according to the method described in patent RU 2294373C2. To determine lysozyme activity, the optical density values ​​obtained for control wells were subtracted from those obtained for wells incubated in the presence of lysozyme. An amount of bacterial cells, Micrococcus lysodeikticus, corresponding to an optical density of 0.6 was mixed with an equal volume of buffer containing lysozyme, naringenin, and guava leaf CO2 extract, while no lysozyme was added to control wells. The plates were then incubated at 37°C for 30, 60, and 120 minutes, and the optical density was measured. A decrease in optical density indicates lysozyme activity. The final density value was then subtracted from the initial density value and normalized for incubation time and enzyme concentration according to the procedure described in RU 2294373C2.

[0177] Figure 1 The results showed that the lysozyme activity values ​​remained the same (accounting for statistical error) regardless of whether naringenin and / or guava extract were added to the buffered medium. These results indicate that naringenin and guava extract neither affect nor reduce lysozyme activity, thus supporting the synergistic effect of the combination of these components on cariogenic bacteria and biofilms.

[0178] Figure 1 Lysozyme activity values ​​in specific activity units (sau) are shown.

[0179] References

[0180] 1.Global Burden of Disease Study 2019(GBD 2019).Seattle: Institute of Health Metrics and Evaluation(IHME); 2020.Available at http: / / ghdx.healthdata.org / gbd-results-tool.

[0181] 2.Microbiology:theory and practice.In 2parts.Part 1:Textbook for Baccalaureate and Master's Course / Α.I.Netrusov,IBKotova.—M.:UrightPublishing Company,2018.—315с.—Series:Bachelor and Master.AcademicCourse.

[0182] 3.Kuleshiva Svetlana Ivanovna.Determination of antibiotic activity byagar diffusion method / / Journal of Scientific Center For Expert Evaluation ofMedical Products.2015.№3.

[0183] 4.Al-Ansari,M.M.,Al-Dahmash,N.D.,&Ranjitsingh,A.J.A.(2021).Synthesisof silver nanoparticles using gum Arabic:Evaluation of its inhibitory actionon Streptococcus mutans causing dental caries and endocarditis.Journal ofInfection and Public Health,14(3),324-330.

[0184] 5.Bhagavathy,S.,Mahendiran,C.,&Kanchana,R.(2019).Identification ofglucosyl transferase inhibitors from Psidium guajava against Streptococcusmutans in dental caries.Journal of traditional and complementary medicine,9(2),124-137.

[0185] 6.Shaheena S.,Chintagunta A.D.,Dirisala V.R.,Sampath KumarN.S.Extraction of bioactive compounds from Psidium guajava and theirapplication in dentistry.AMB Express.2019.V.9,№1.Ρ.208.doi:10.1186 / s13568-019-0935-x.

[0186] 7.Nayak N.,Varghese J.,Shetty S.,Bhat V.,Durgekar T.,Lobo R.,NayakU.Y.,Vishwanath U.Evaluation of a mouthrinse containing guava leaf extract aspart of comprehensive oral care regimen-a randomized placebo-controlledclinical trial.BMC Complement Altern Med.2019.V.19,№1.Ρ.327.doi:10.1186 / s12906-019-2745-8.

[0187] 8.de Soet JJ,van Loveren C,Lammens AJ,Pavicic MJ,Homburg CH,ten CateJM,et al.Differences in cariogenicity between fresh isolates of Streptococcussobrinus and Streptococcus mutans.Caries Res.1991;25:116–22.

[0188] 9.Okada M,Kawamura M,Oda Y,Yasuda R,Kojima T,Kurihara H.Cariesprevalence associated with Streptococcus mutans and Streptococcus sobrinus inJapanese schoolchildren.Int J Paediatr Dent.2012;22:342–8.

[0189] 10.Okada M,Soda Y,Hayashi F,Doi T,Suzuki J,Miura K,et al.Longitudinalstudy of dental caries incidence associated with Streptococcus mutans andStreptococcus sobrinus in pre-school children.J Med Microbiol.2005;54:661–5.

[0190] 11.Chen,L.,Yang,J.,Yu,J.,Yao,Z.,Sun,L.,Shen,Y.,&Jin,Q.(2005).VFDB:areference database for bacterial virulence factors.Nucleic acids research,33(suppl_1),D325-D328.

[0191] 12.Garode,A.M.,&Waghode,S.M.(2014).Antibacterial activity of guavaleaves extracts against S.mutans.Inter J Bioassays,3(10),3370-2.

[0192] 13.Ibrahim,M.S.,Ibrahim,A.S.,Balhaddad,A.A.,Weir,M.D.,Lin,N.J.,Tay,F.R.,...&Melo,M.A.S.(2019).A novel dental sealant containingdimethylaminohexadecyl methacrylate suppresses the cariogenic pathogenicityof Streptococcus mutans biofilms.International journal of molecular sciences,20(14),3491.

[0193] 14.Ishii,S.,Fukui,K.,Yokoshima,S.et al.High-throughput Screening ofSmall Molecule Inhibitors of the Streptococcus Quorum-sensing SignalPathway.Sci Rep 7,4029(2017). https: / / doi.org / 10.1038 / s41598-017-03567-2 .

[0194] 15.Jian-Na Cai,Ji-Eun Jung,Min-Ho Lee,Hyeon-Mi Choi,Jae-Gyu Jeon,Sucrose challenges to Streptococcus mutans biofilms and the curve fitting forthe biofilm changes,FEMS Microbiology Ecology,Volume 94,Issue 7,July 2018,fiy091, https: / / doi.org / 10.1093 / femsec / fiy091 .

[0195] 16.Jun,J.-Y.;Jung,M.-J.;Jeong,I.-H.;Yamazaki,K.;Kawai,Y.;Kim,B.-M.Antimicrobial and Antibiofilm Activities of Sulfated Polysaccharides fromMarine Algae against Dental Plaque Bacteria.Mar.Drugs 2018,16,301. https: / / doi.org / 10.3390 / md16090301 .

[0196] 17.Kassebaum NJ,BernabéE,Dahiya M,Bhandari B,Murray CJ,MarcenesW.Global burden of untreated caries:a systematic review and metaregression.JDent Res.2015 May;94(5):650-8.doi:10.1177 / 0022034515573272.Epub 2015 Mar4.PMID:25740856.

[0197] 18.Kaur,D.,&Chandrul,K.K.(2017).Syzygium aromaticum L.(Clove):A vitalherbal drug used in periodontal disease.Indian Journal of Pharmaceutical andBiological Research,5(02),45-51.

[0198] 19.Lemos,J.A.,Palmer,S.R.,Zeng,L.,Wen,Z.T.,Kajfasz,J.K.,Freires,I.A.,Abranches,J.,&Brady,L.J.(2019).The Biology of Streptococcusmutans.Microbiology spectrum,7(1),10.1128 / microbiolspec.GPP3-0051-2018.

[0199] https: / / doi.org / 10.1128 / microbiolspec.GPP3-0051-2018.

[0200] 20.Mathur,V.P.,Dhillon,J.K.Dental Caries:A Disease Which NeedsAttention.Indian J Pediatr 85,202–206(2018). https: / / doi.org / 10.1007 / s12098- 017-2381-6 .

[0201] 21.Nair,R.,&Chanda,S.(2007).In-vitro antimicrobial activity ofPsidium guajava L.leaf extracts against clinically important pathogenicmicrobial strains.Brazilian Journal of Microbiology,38,452-458.

[0202] 22.Niwa,T.,Kawamura,Y.,Katagiri,Y.,&Ezaki,T.(2005).Lytic enzyme,labiase for a broad range of Gram-positive bacteria and its application toanalyze functional DNA / RNA.Journal of microbiological methods,61(2),251-260.

[0203] 23.Nomura,R.,Matayoshi,S.,Otsugu,M.,Kitamura,T.,Teramoto,N.,&Nakano,K.(2020).Contribution of severe dental caries induced by Streptococcus mutansto the pathogenicity of infective endocarditis.Infection and immunity,88(7),e00897-19.

[0204] 24.Oana,K.,Kawakami,Y.,Hayashi,T.,&Ohnishi,M.(2009).Simple broad-spectrum protocol using labiase for bacterial cell lysis to prepare genomicDNA for pulsed-field gel electrophoresis analysis.Microbiology andimmunology,53(1),45-48.

[0205] 25.Philip,N.,Suneja,B.&Walsh,L.Beyond Streptococcus mutans:clinicalimplications of the evolving dental caries aetiological paradigms and itsassociated microbiome.Br Dent J 224,219–225(2018). https: / / doi.org / 10.1038 / sj.bdj.2018.81 .

[0206] 26.Pitts,N.,Zero,D.,Marsh,P.et al.Dental caries.Nat Rev Dis Primers3,17030(2017). https: / / doi.org / 10.1038 / nrdp.2017.30 .

[0207] 27.Pleszczyńska, Wiater,Adrian;Bachanek,Teresa;Szczodrak,Janusz(2016).Enzymes in therapy of biofilm-related oraldiseases.Biotechnology and Applied Biochemistry,(),n / a–n / a.doi:10.1002 / bab.1490

[0208] 28.Sims Jr,K.R.,Maceren,J.P.,Liu,Y.,Rocha,G.R.,Koo,H.,&Benoit,D.S.(2020).Dual antibacterial drug-loaded nanoparticles synergistically improvetreatment of Streptococcus mutans biofilms.Acta Biomaterialia,115,418-431.

[0209] 29.Schleifer,K.H.,&Kandler,O.(1972).Peptidoglycan types of bacterialcell walls and their taxonomic implications.Bacteriological reviews,36(4),407-477.

[0210] 30.Tang,X.,Kudo,Y.,Baker,J.L.,LaBonte,S.,Jordan,P.A.,McKinnie,S.M.,...&Edlund,A.(2020).Cariogenic Streptococcus mutans produces tetramicacid strain-specific antibiotics that impair commensal colonization.ACSinfectious diseases,6(4),563-571.

[0211] 31.Tenovuo,J.(2002).Clinical applications of antimicrobial hostproteins lactoperoxidase,lysozyme and lactoferrin in xerostomia:efficacy andsafety.Oral diseases,8(1),23-29.

[0212] 32.Valm A.M.The structure of dental plaque microbial communities inthe transition from health to dental caries and periodontal disease / / Journalof molecular biology.–2019.–Τ.431.–№.16.–С.2957-2969.

[0213] 33.Yue,J.,Yang,H.,Liu,S.,Song,F.,Guo,J.,&Huang,C.(2018).Influence ofnaringenin on the biofilm formation of Streptococcus mutans.Journal ofdentistry,76,24-31.

[0214] 34.Wilcox,L.J.,Borradaile,N.M.,&Huff,M.W.(1999).Antiatherogenicproperties of naringenin,a citrus flavonoid.Cardiovascular drug reviews,17(2),160-178.

Claims

1. A composition comprising the following combination: i) naringenin, ii) an enzyme selected from lysozyme, labiase, or a combination thereof, and iii) Guava leaf extract.

2. The composition according to claim 1, wherein the guava leaf extract is guava leaf CO2 extract.

3. The composition of claim 1, wherein the guava leaf extract is a Psidium guajava leaf extract.

4. The composition according to claim 3, wherein the guava leaf extract is a Psidium guajava leaf CO2 extract.

5. The composition of any one of the preceding claims, wherein the naringenin is obtained from a plant source.

6. The composition according to any one of the preceding claims, wherein the guava leaf extract, naringenin and enzyme are contained in a weight ratio of (1.00-170.73):(1.00-235.77):(0.16-365.85), respectively.

7. The composition according to any one of the preceding claims, wherein the composition is a dental care product and / or an oral care product.

8. The composition of claim 7, wherein the weight % of the components are as follows: Guava leaf extract 0.0615-10.5, Naringenin 0.0615-14.5, Enzyme 0.0096-22.

5.

9. The composition according to claim 8, wherein the composition is a toothpaste or a mouthwash.

10. A composition according to any one of claims 1 to 9 for use in controlling Streptococcus mutans and / or Streptococcus sobrinus on tooth surfaces and / or in the oral cavity.

11. The composition for use according to claim 10, wherein the controlling comprises inhibiting the proliferation and / or growth of the Streptococcus mutans and / or Streptococcus sobrinus on the tooth surface and / or in the oral cavity.

12. The composition according to any one of claims 1 to 9, which is used for the prevention or treatment of dental caries.

Images