Effect of flavonoids from grape seed and cranberry extracts on the microbiological activity of Streptococcus mutans: a systematic review of in vitro studies

Objective To provide an overview of the available scientific evidence from in vitro studies regarding the effect induced by the flavonoids contained in grape seed extracts (GSE) and cranberry on the microbiological activity of Streptococcus mutans (S. mutans). Methods This systematic review was performed following the parameters of the PRISMA statement (Preferred Reporting Items for Systematic Reviews and Meta-Analysis). Electronic and manual searches were conducted using PubMed, ScienceDirect, Web of Science, EBSCO, and Cochrane databases. Reference lists of selected articles were reviewed to identify relevant studies. The search was not limited by year and was conducted solely in English. Eligible studies comprised publications describing in vitro studies that evaluated the effect of flavonoids derived from GSE and cranberry extracts on the microbiological activity of S. mutans. Common variables were identified to consolidate the data. Authors of this review independently screened search results, extracted data, and assessed the risk of bias. Results Of the 420 studies identified from the different databases, 22 publications were finally selected for review. The risk of bias was low in 13 articles and moderate in 9. The studies analyzed in this review revealed that cranberry extract has an inhibitory effect on the bacterial growth of S. mutans in ranges from 0.5 mg/mL to 25 mg/mL, and GSE exerts a similar effect from 0.5 mg/mL to 250 mg/mL. Additionally, the extracts or their fractions showed reduced biofilm formation capacity, decreased polymicrobial biofilm biomass, deregulation of glycosyltransferases (Gtf) B and C expression, and buffering of pH drop. In addition to adequate antioxidant activity related to polyphenol content. Conclusions The overall results showed that the extracts of cranberry and grape seed were effective in reducing the virulence factors of the oral pathogen. According to the data, proanthocyanidins are the active components in cranberry and grape seed that effectively resist S. mutans. They can inhibit the formation of insoluble polysaccharides in the extracellular matrix and prevent glycan-mediated adhesion, cohesion, and aggregation of the proteins in S. mutans. This suggests that these natural extracts could play an important role in the prevention of cariogenic bacterial colonization, as well as induce a decrease in their microbiological activity. Supplementary Information The online version contains supplementary material available at 10.1186/s12903-024-04263-0.


Introduction
Dental caries is a biofilm-mediated disease that has been described as a highly dynamic, multimicrobial, dietinduced disease process [1].Carious biofilms develop because pathogens accumulate on tooth surfaces, forming highly structured microbial communities that adhere tightly and organize in an extracellular matrix [2].Streptococcus mutans (S. mutans) is a Gram-positive, facultative anaerobic, catalase-negative bacterium that produces lactic acid and can reduce the pH of the environment from 7 to 4.2 in approximately 24 h.Additionally, it is capable of fermenting and producing acids from carbohydrates such as glucose, lactose, and raffinose, among others, which is why it is implicated as the key pathogen in the development of dental caries.S. mutans is capable not only of carbohydrate breakdown, but also of forming glucans that are important for interacting with tooth structure [3].By metabolizing carbohydrates such as sucrose, intracellular (IPS), and extracellular (EPS) polysaccharides are synthesized.On the one hand, IPS provides bacteria with an endogenous nutrient source of carbohydrates, during periods when they are deprived of nutrients, to continue acid formation.On the other hand, the synthesis of EPS promotes the adhesion and accumulation of bacteria on the tooth surface and causes structural changes such as increased porosity in dental biofilms [2,3].
The EPS matrix creates cohesive biofilms that adhere to surfaces, while protecting enclosed pathogens from antimicrobials, making them difficult to treat or remove [3,4].The virulence of S. mutans is related to the expression of Glucosyltransferases (Gtf ), enzymes that are responsible for the synthesis of extracellular glucan polymers from sucrose.These enzymes cover the dental structure with glucans to promote adhesion, by means of surface proteins that contain glucan-binding properties and allow the formation of an extracellular matrix that has the function of anchoring the biofilm [5].In order to bind with the dental structure, the surface adhesins of S. mutans, called antigens I and II, interact with salivary proteins to form a biofilm, which will be anchored to the dental structure [6].Once the dental biofilm composed of S. mutans is formed, due to a prolonged intake of carbohydrates and the porosity caused by the EPS, the sugars penetrate the deepest parts of the biofilms.This allows the microorganism to induce an acidic extracellular environment that helps to reduce its vulnerability, given its virulence factors.This process is the basis of its cariogenic action since it gives rise to an imbalance that initiates the demineralization process, with a reduction in the concentrations of calcium (Ca), inorganic phosphorus (Pi) and fluorine (F), associated with the dissolution of mineral deposits or the inhibition of their storage [3].
Inadequate oral health is particularly predictive of multiple pathological outcomes, including dental caries [1,7].Caries is a highly predictable and avoidable condition; however, it remains the most prevalent chronic disease in children and adults worldwide [8,9].It is estimated that three billion people have dental caries in the world, and that between 60 and 100% of children and adults suffer from caries at some point in their lives [10].For this reason, it is necessary to develop and refine different strategies to prevent and solve it.The most used prevention method is the application of different forms of fluoride; however, exploring new alternatives to prevent, stop and restore caries with the help of natural agents can help to reduce caries rates in the population.Biofilm control approaches that disrupt the EPS production and thereby compromise the ability of S. mutans to assemble and maintain biofilms on tooth surfaces could be potentially effective alternatives to antimicrobials [11].
Grape seed extract (GSE) and cranberry (Vaccinium macrocarpon) are readily available plant-based supplements.Their extracts are rich in polyphenols comprising flavonoids such as proanthocyanidins (PACs) and they have promising biological properties, such as antioxidant and antimicrobial action.The association between PACs and oral health has been demonstrated in the literature through their antimicrobial and antiadherent action against the pathogen S. mutans, which leads to reduction in the formation of insoluble polysaccharides in the matrix.
For this reason, the objective of this review is to collect and analyze the available evidence regarding the effect of the flavonoids contained in grape seed and cranberry extracts on the microbiological activity of S. mutans, for the prevention of dental caries.

Methods
This systematic review was performed following the parameters of the PRISMA statement (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) [12].Based on the study population, intervention, comparison, and outcome, the research question was posed as follows: What is the effect generated by the extracts of grape seeds and cranberry on the microbiological activity of S. mutans?

Eligibility criteria
The criteria for inclusion in this study were as follows: (1) publications of in vitro studies that evaluated the effect of flavonoids from grape seed and cranberry extracts on the microbiological activity of S. mutans; (2) in vitro studies that evaluated the growth, coaggregation, and formation of biofilms with S. mutans by exposure to flavonoids contained in GSE and cranberry extracts; (3) in vitro studies comparing the effect of flavonoids from GSE and cranberry extracts with the effect of other antimicrobial agents on the activity of S. mutans.
The criteria for exclusion from the study were as follows: (1) literature review studies, systematic reviews, meta-analyses, and case reports; (2) studies that reported the crosslinking effect of flavonoids on dentine collagen or their ability to inhibit matrix metalloproteinases to stabilize the hybrid layer; (3) publications that did not report the number of replicates per experiment or that reported a number of replicates less than 3 per test group; (4) studies that reported the remineralizing effect of flavonoids without reporting their effect on the microbiological activity of S. mutans.

Outcomes of interest
The outcomes of interest in the present study were: (1) bacterial proliferation of S. mutans in samples exposed to GSE and cranberry extract; (2) the impact of exposure to GSE and cranberry on biofilm formation with S. mutans; (3) concentrations and effective times of GSE and cranberry for the inhibition of the microbiological activity of S. mutans.

Data sources and search strategy
An electronic search was performed in the following databases: PubMed, Elsevier, Scopus, ScienceDirect, Web of Science, EBSCO, and Cochrane.For searching the articles, there was no year restriction, and the search was limited to articles written in the English language.The latest search was performed to include studies published up to December 30, 2023.Keywords and their combinations used in the searches included: Streptococcus mutans, biofilm, dental caries, proanthocyanidins, grape seed extract, lingonberry extract, and cranberry extract (Table 1 and Additional file 1).In addition, to determine the relevance of other articles that met the inclusion criteria, the references of all included articles were also searched.To find unpublished data, we also searched the database listing unpublished studies (OpenGray).

Selection and data extraction
Potential primary studies were identified by examining the titles and abstracts of the investigations.The manuscripts that met the eligibility criteria were fully read.Two authors (JC, PB) reviewed the titles and abstracts to assess compliance with the inclusion criteria.Subsequently, only the abstracts were independently reviewed by two authors to reach a consensus on compliance with the inclusion criteria (JC, PB).In those summaries which led to doubts between the authors, a third evaluator (DB) conducted an additional evaluation.Full-text articles were obtained and reviewed by the two authors (JC, PB), and the final inclusion was made in consensus with the Language: English Scopus ( ( TITLE-ABS-KEY ( ( "streptococcus mutans" OR "s.mutans" OR "biofilm" OR "biofilms" OR "dental plaque" OR "dental biofilms" OR "dental caries"))) AND ( TITLE-ABS-KEY ( ( "cranberry" OR "lingonberry" OR "lingonberry extract*" OR "cranberry extract*" OR "grape seed*" OR "grape seed extract")))) AND ( TITLE-ABS-KEY ( "microbiological activity" OR "minimum bactericidal concentration" OR "microbial sensitivity tests" OR "minimum inhibitory concentration" OR "microbial sensitivity tests" OR "concentration minimum inhibitory")) Science Direct ("streptococcus mutans" OR Biofilm OR "dental caries") AND (proanthocyanidins OR "grape seed extract" OR "lingonberry extract" OR "cranberry extract") Research Article Thematic area: medicine and dentistry.Immunology and Microbiology Web of Science (AB = (("streptococcus mutans" OR Biofilm OR "dental caries") AND (proanthocyanidins OR "grape seed extract" OR "lingonberry extract" OR "cranberry extract"))) Advanced search Language: English EBSCO ("streptococcus mutans" OR Biofilm OR "dental caries") AND (proanthocyanidins OR "grape seed extract" OR "lingonberry extract" OR "cranberry extract")

Assessment of the risk of bias and quality of included studies
The methodological quality of the studies was determined using a tool to assess the risk of bias, adapted by de Almeida et al. [13] based on the methodology used by Cericato et al. [14] with some modifications.The parameters considered are described in Table 2.A score was assigned to each study, classifying them according to their quality as: low quality (0-9 points), moderate quality (10-14 points), or high quality (15-18 points).

Data analysis
The data extracted from the full-text articles are recorded in Tables 3, 4, 5, 6, 7, 8, 9 and 10, which includes the methodology of the included studies.Identification of the common variables in the different articles was made to facilitate the consolidation of the data.A descriptive summary of the variables considered (bacterial inhibition, biofilm formation, antioxidant capacity, expression of GtfB and C, and F-ATPase, morphological changes, pH changes, adhesion, induction of cell death and cytotoxicity) was compiled.

Search results
A total of 420 studies were identified from the different databases, out of which 295 duplicate reports were eliminated; of 125 studies reviewed, 93 studies were excluded due to the title and abstract.Accordingly, 32 publications were selected for full-text reading.Subsequently, ten articles were eliminated for not meeting the criteria of reporting a minimum of three replications per experiment [15][16][17][18][19][20][21][22][23] and, finally, 22 publications were selected for our review (Fig. 1).

Risk of bias and quality assessment
The risk of bias of the 22 included studies is presented in Tables 11, 12, 13, 14 and 15. 13 of them presented low risk [59%], 9 studies presented moderate risk (41%), and no study was classified as high risk [0%].Bias evaluation was performed according to the parameters established in Table 2.The main risk of bias was found in that study limitations were either not reported in the study report [24-26, 29, 36, 37, 39, 41-43] or were described briefly [27, 28, 31-34, 40, 44].Secondly, there was a failure to include relevant ethical aspects such as institutional ethics committee approval, or patient informed consent for saliva sample collection was not clearly detailed in the article text, or a declaration of project financing and conflict of interest was absent [27-29, 31-37, 39-42, 44].
Thirdly, there was no clear and precise description of the study objective regarding which specific aspects were to be evaluated regarding the effect of the extracts on antimicrobial activity or biofilm formation [7,24,26,27,35,37,38,40,42].

Specimens and bacterial culture
The reported sample size was determined for each study method.In evaluating the biofilm formation, all studies reported having performed the trials at least in triplicate.
All studies evaluated the activity of PAC on S. mutans [7, 24-44], and ten studies evaluated its activity on two or more bacterial strains [7,24,27,29,30,36,[41][42][43][44].Several studies have used certified American Type Culture Collection (ATCC) bacterial strains of S. mutans to assess the antibacterial activity of test substances.These strains meet quality criteria such as: i) they were obtained from a certified collection, ii) they were isolated from cultures of the reference strains, and iii) they are identical strains obtained from a subculture of the reference strains.The most used strain was S. mutans ATCC 25175 [26,31,34,35,38,44].The second-most used strain was S. mutans UA159 [28,32,33,37,40], which is a naturally competent strain and contains all essential genes for competition and quorum sensing [45].Kokubu et al. [24] and Yamanaka et al. [42] used S. mutans MT8148R15; Daglia et al. [31] used the strain S. mutans 9102 in addition to ATCC 25175, Ikai et al. [36] used strain S. mutans JCM 5705, and Smullen et al. [41] used the S. mutans 10,499 strain.For their part, Duarte et al. [32] and Gregoire et al. [39] used S. mutans WHB 410.Some studies do not specifically report the strain used [29,43] or biofilm formation was performed by collecting saliva from healthy donors [7,30].

Extracts used for microbiological tests
For the evaluation of the antimicrobial effect, the studies used different sources of flavonoids.On the one hand, some studies started with fresh cranberry extracts [27-30, 32, 37, 39, 43], from commercially obtained cranberry juice concentrates [7,24,25,30,35,38,[40][41][42], or from samples of red wine and concentrated or distilled white wine [41] or dealcoholized red wine [31].In contrast, other studies used GSE from fresh red grapes [26] or extracts of GSE obtained from the seeds of Vitis vinifera in commercial presentation [26,33,44], or the previously purified PACs in commercial presentations [36].

Obtaining and characterizing the extracts and fractions rich in polyphenols
For the separation of the fraction rich in polyphenols, they used chromatography [24,28] and the total polyphenols in each fraction were determined by the Folin-Ciocalteu Method [24,40].Greene et al. [25] microencapsulated concentrated cranberry extract in poly(lactic-co-glycolic) (PLGA) using a double emulsion manufacturing procedure.Swadas et al. [26] and Singhal et al. [27] used the maceration method of the previously dried grape seeds or cranberry fruits, as did Abu-obaid et al. [30]; the preparation was carried out in a hydroalcoholic solvent with a ratio of ethanol (70%): water (30%) [26,27] and subsequent filtration.Sumati et al. prepared the extract of fresh cranberries with methanol.Koo et al. [28], Duarte et al. [32], and Feng et al. [37] determined the composition of PAC by high-performance liquid chromatography (HPLC)-mass spectrometry (MS) with photodiode/electrochemical detection and matrixassisted laser desorption/ionization (MALDI) time-offlight (TOF) and was confirmed using LC-MS-MS in product ion scanning mode followed by multiple reaction monitoring (MRM) scanning.Gregoire et al. [39] isolated phenolic acids, flavonols and PACs by semi-preparative HPLC and the isolated compounds were characterized by HPLC-MS.Philip et al. [7], Zhao et al. [33], Furiga et al. [34], Prince et al. [35], and Smullen et al. studies [41] were based on manufacturer-supplied information on polyphenol concentrations in cranberry extracts [7,35] or GSE [33,34,41].Daglia et al. [31] performed dealcoholization of red wine and fractionation of dealcoholized red wine components such as PACs by Solid Phase Extraction and Gel Filtration Chromatography.Yamanaka et al. [42] dialyzed powdered cranberry juice against distilled water with a molecular mass cut-off point of 14,000.Elgamily et al. [43] full scan chromatograms with subsequent hydrolysis of the samples were used to measure ms ms spectra.Nowaczyk et al. [44] used a constant stirring method with a rotary stirrer followed by centrifugation, decantation and rotary evaporation.

Determination of antimicrobial activity
The Minimum Inhibitory Concentration (MIC) was defined by the authors [7,26,27,29,31,33,34,36,39,41,43,44] as the minimum concentration of the antimicrobial substance that inhibits the visible growth of the bacteria after incubation.The antibacterial assay was generally conducted using the macrodilution method with agar in petri dishes [26, 30, 34-36, 39, 41, 44], or the microdilution method [39] in liquid medium such as trypticase soy broth [29] or BHI [27,35,40] or Mueller-Hinton broth [41] in a 96-well titre plate, and the MIC was evaluated by counting colony-forming units (CFU/ mL).The two controls used in the studies to determine the MIC were positive control (MH broth with bacterial suspension) and negative control (MH broth without antimicrobial and without bacterial suspension).The Minimum Bactericidal Concentration was defined as the minimum antimicrobial concentration that eliminated more than 99.9% of viable microorganisms after a given incubation time.Sumati et al. [29] evaluated the ability of cranberry extract to induce cytotoxicity against microorganisms by means of the MTT assay that is based on mitochondrial metabolism.Philip et al. [7] evaluated the ecological effects of extracts on polymicrobial biofilms using real-time quantitative polymerase chain reaction (qPCR) assay to determine the bacterial load, considering 14 bacterial species of interest.Prince et al. [35] performed a checkerboard assay to assess zones of inhibition to determine bacterial growth.Finally, Net et al. [40] determined the inhibition capacity of cranberry fractions in influenza-induced hemagglutination.

Effect on biofilm adhesion
The most widely used method to evaluate the effect of test substances on stable biofilm formation was culturing S. mutans strains on plastic [24,27,31,40], glass (42), or on hydroxyapatite surfaces (sHA) coated with human saliva obtained from healthy donors [7,25,28,30,32,37,38,42].Several studies have used the crystal violet staining method for culture or biofilm staining, with different concentrations and time incubation: 0.1% for 15 min [7,24,25,31], 0.5% for 30 min [27,29], and 45 min without reporting concentration [40], staining with ethanol [25,27,29], acetic acid [7,40], or ethanol/acetone [31] and the absorbance reading was measured by spectrophotometry.Greene et al. [25] performed the evaluation through optical microscopy and Daglia et al. [31] used a stereomicroscope with radiolabeled strains [31] through a scintillation counter assay, as well as Duarte et al. [32] and Feng et al. [37].Kokubu et al. [24] used an ATP bioluminescence assay.Zhao et al. [33] induced caries lesion formation in vitro by S. mutans biofilm and determined lesion depth and relative optical density using polarized light microscopy and confocal laser scanning microscopy to assess lesion progression.Furiga et al. [34] used a Gram stain to confirm species identity.Philip et al. [38] determined the effects of extracts on S. mutans biofilms by evaluating the metabolic activity of biofilm microorganisms with the XTT reduction assay.Yamanaka et al. [42] used direct scintillation counting of the number of attached bacterial cells.

Morphological evaluation of biofilms treated with the test substances
To examine the effects of treatments on S. mutans biofilms, structural organization, EPS formation, and microbial biovolumes were analyzed by confocal microscopy [7,34,37,38] or by scanning electron microscopy [27,44].

Determination of the effect on microbial glycosyltransferase enzymes (Gtf)
The importance of determining the effect of flavonoids on microbial glycosyltransferase enzymes (Gtf ) lies in the fact that they are the enzymes responsible for synthesizing glycans.These glycans are polysaccharides that form the biofilm matrixes.Glycans synthesized by surface-adsorbed GtfB and GtfC provide specific binding sites for bacterial colonization on the tooth surface and with each other.The glycosyltransferase enzymes expression was evaluated by Feng [37] using the RT-qPCR technique and its activity was measured by Duarte et al. [32] and Gregoire et al. [39] by incorporating (14C) glucose into labeled sucrose in the glycans.Koo et al. [28] determined it by means of the enzymes in solution or by adsorbing it on saliva-coated hydroxyapatite beads free of Gtf activity, in the presence of test agents, and Furiga et al. [34] determined it by measuring the amount of reducing sugars released by a dinitrosalicylic acid assay, with fructose as a standard, and by the amount of insoluble glycan synthesized.

Effects of the extracts on the drop in glycolytic pH by S. mutans
Koo et al. [28] and Duarte et al. [32] evaluated the effects of cranberry PAC on glycolysis by standard pH drop using suspensions of cells exposed to test agents spiked with glucose and assessed for pH drop by electrode pH glass.Philip et al. [7,38] measured acidogenicity using the lactate dehydrogenase assay to determine the concentrations of lactic acid formed by biofilms.

Effect of extracts on bacterial growth inhibition (MIC, MBC)
Kokubu et al. [24] showed a reduction in the bioactivity of S. mutans at concentrations of 0.5.1.0 and 2.0 mg/mL of cranberry extract.Swadas et al. [26] reported an inhibition of bacterial activity at concentrations of 250 and 500 mg/mL of GSE, while the concentration of 125 mg/ mL did not show significant antibacterial activity.Zhao et al. [33] demonstrated bacterial inhibition at a concentration of 4 mg/mL of GSE.According to Ikai et al. [36] 2 mg/mL PACs from cranberry extract significantly increased the bactericidal activity of photoirradiated H 2 O 2 at concentrations of 80 and 320 nM.Elgamily et al. [43] showed an inhibition of bacterial growth at concentrations of 0.5 and 0.25 mL/mL of cranberry juice and at concentrations of 0.50 mL/mL of functional cranberry drink.
Singhal et al. [27] reported an MIC of 12.5 mg/ dL for the ethanolic extract of cranberry, and a MBC of 25 mg/dL.Sumati et al. [29] determined a MIC of 60 mg/mL for cranberry extract, which was characterized as having a bactericidal effect.Abu-obaid et al. [30] showed that the effect of the MIC and MBC of cranberry extract was like that of chlorhexidine and that when these two substances were combined, its effect increased twice and was even superior to that of treatment with chlorhexidine plus alcohol.Daglia et al. [31] reported an MIC of 19.07 mg/mL for GSE dealcoholized wine and 2.53 mg/mL for polymeric PAC eluted with 15 mL of methanol.Furiga et al. [34] for their part, recorded a MIC of 1000 μg/mL of GSE, a concentration at which no bacterial growth was shown after 24 h, and a MBC of 4000 μg/mL, a concentration at which reduction of 99.9% bacterial count was observed.Prince et al. [35] demonstrated by means of the checkerboard test, that the MIC of cranberry extract in subcritical water of presscake with tannase and in resin with tannase was 0.75 mg/mL, while the resin extract was 0.75 mg/mL to 0.5 mg/mL.Smullen et al. [41] determined an MIC for the red GSE extract of 0.5 mg/mL and 1 mg/mL for inhibition of a procyanidin polymer fraction.

Effect of extracts on biofilm formation
Kokubu et al. [24] reported a significant decrease in biofilm formation at concentrations of 0.5 and 1.0 mg/mL of cranberry extract in a 1000 mL dilution of 70% ethanol.Greene et al. [25] found that at concentrations of 0.5 and 5 mg/mL of cranberry extract alone, or + C 18 H 36 O 2 + polyvinylpyrrolidone, or + C 18 H 36 O 2 + ethyl lauroyl arginate, a reduction in the formation of biofilms.Singhal et al. [27] indicated that after 24 h of treatment, 16.67 mg/dL and 20.83 mg/dL of cranberry extract reduced 50% and 70% of the preformed biofilm, respectively.According to Sumathi et al. [29], 50 mg/mL of cranberry extract acted as a potent antimicrobial agent by inhibiting biofilm formation by 95.2% after 24 h of treatment.Philip et al. [7] demonstrated a significant decrease (51%) in the biofilm formation at 500 μg/mL of cranberry extract.Daglia et al. [31] on the other hand, stated that the polymeric PAC of the GSE extract with 15 mL of methanol had the ability to reduce biofilm formation by 89%, while dealcoholized wine reduced it by 79%.Zhao et al. [33] reported that 4 mg/mL of GSE extract had the ability to inhibit biofilm formation.
Furiga et al. [34] found that 2000 μg/mL GSE + 10.2 mg/ mL Fluorinol contributed to decrease the number of microcolonies.According to Ikai et al. [36], there was a significant decrease in colony count when cultures were treated with 8 mg/mL PAC and 500 nM H 2 O 2 .Feng et al. [37] demonstrated a decrease in the biofilm of 4.3 μm 3 for the treatment group, compared to 28.9 μm 3 for the control group.Philip et al. [38] evaluated different concentrations, thus, at 500 μg/mL, a 32% biofilm reduction is generated, at 250 μg/mL 29%, while at 125 μg/mL the reduction is minimal (14%).Net et al. [40] found biofilm reduction at concentrations as low as 80 μg/mL with non-dialyzable cranberry plus acetone, while non-dialyzable cranberry only produced a significant decrease at a concentration of 320 μg/mL.Smullen et al. [41] reported that GSE decreases biofilm formation at 30 min of treatment.Yamanaka et al. [42] indicated an inhibition in the formation of biofilms at concentrations of 100 μg/ mL and 500 μg/mL at 25% KCl.Finally, Nowaczyk et al. [44] found a 68% decrease in bacterial colony formation at concentrations of 50 μg/2 ml of probiotic candy-GSE with distilled wáter.

Antioxidant capacity of the test extracts
Furiga et al. [34] in their study of the equivalent antioxidant capacity of Trolox (TEAC), demonstrate that GSE was the group with the highest antioxidant capacity (6.99 μg/mL), compared to Eludril Daily (6.95 μg/mL), ascorbic acid (5.70 μg/mL) and fluorinol (0.01 μg/mL), which offers a high degree of protection to the organism against the damage of free radicals.Additionally, Neto et al. [40] demonstrated that polyphenols in the studied cranberry fractions of non-dialyzed material, were able to exhibit antioxidant properties.These experiments indicated that the NDMac fraction (eluted in acetone) was about twice as rich in polyphenols and showed twice as higher antioxidant activity in solution or when bound to bacterial surfaces as the NDMet fraction (eluted in ethanol).

Effect of the extracts on the expression of GtfB and C. F-ATPase
Koo et al. [28] demonstrated a reduction in the number of extracellular EPSs from 35 to 40%.Additionally, the PACs inhibited the activity of the absorbed GtfB and reduced it to between 40 and 70%.Philip et al. [7] showed a significant decrease in EPS formation with the cranberry extract treatment (1.14 μm 3 /μm 2 ) compared to the control (2.64 μm 3 /μm 2 ).Duarte et al. [32] reported that treatment with anthocyanidin + flavonol inhibited F-ATPase activity by 85%, and this treatment Fig. 1 Identification of studies via databases caused the greatest inhibition of GtfB activity (70%), followed by treatment with PACs + anthocyanidin (50%) and PACs + flavonol (45%).Furiga et al. [34] showed Gtfs inhibition of 43.9% with GSE treatment, with 65.7% inhibition for GSE combined with fluorinol.Feng et al. [37] reported a decrease of 19.8 μm 3 of EPS, unlike the control group, which showed a decrease of 68.1 μm 3 .On the one hand, Gregoire et al. [39] reported an inhibition of the enzymatic activity of the proton-translocating F-ATPase at 500 μmol from 18 to 33%, with myricetin and procyanidin A2 being the most inhibitory.On the other hand, it showed a 20% to 35% reduction of glycan synthesis by GftB at a concentration of 500 μmol, with procyanidin A2 being the most effective.

Morphological changes associated with exposure to flavonoids
Singhal et al. [27] reported that with the increase in cranberry concentration, the integrity of the biofilm is altered, an effect characterized by agglutination and cell degradation at high concentrations.Philip et al. [7] found inhibition of polymicrobial biofilm biomass at 96 h, with a reduction of 38% compared to the control group.Furiga et al. [34] reported that with 2000 μg/mL of GSE there was a decrease in the number of microcolonies and in the thickness of biofilms.Feng et al. [37] reported defective biofilm accumulation and altered architecture, as well as impaired EPS matrix development with cranberry treatment.Philip et al. [38] stated that cranberry treatment resulted in reduced biovolume and less compaction of exposed biofilms.Finally, Nowaczyk et al. [44] report a significantly altered structure of the bacteria cells with a concentration of 50 μg / 2 ml of probiotic candy-GSE.

Effects of extracts on pH change
Koo et al. [28] showed that after 4 h of incubation with PAC treatment of cranberry extract, the pH remained slightly higher (5.6) than that of the group containing no extract (5.2).Philip et al. [7] found a significant difference in the production of lactic acid in the control group without extract, which was 11.2 mM/L between the experimental group treated with cranberry extract, in which it was 6.2 mM/L, showing a 44% reduction.Duarte et al. [32] reported a final pH of 4.7 to 4.9 with the PAC of cranberry extract, which was different from the control group without extract, and was 3.7.Philip et al. [38] showed that at a concentration of 500 μg/mL of cranberry extract, there was a 40% reduction in lactic acid production by S. mutans.Finally, Gregoire et al. [39] stated that cranberry extract flavonoids, such as

Effect of extracts on bacterial adhesion
Greene et al. [25] evaluated the adhesion of microencapsulated mouthwashes to hydroxyapatite (HA), formulations with cranberry extract were able to adhere to the HA disks after 24 h of incubation and prevent the biofilm formation.Daglia et al. [31] demonstrated inhibition of S. mutans adhesion to hydroxyapatite of 81.5% with GSE at a concentration of 0.125 mg/mL with an elution of polymeric proanthocyanidin with 15 mL of methanol (SPE F4), while dealcoholized wine inhibited 25% adhesion at a concentration of 0.25 mg/mL.Feng et al. [37] for their part, reported an inhibition of bacterial adhesion due to the reduction in EPS synthesis, associated with the inhibition of the enzymatic activity of GtfB and GtfC generated by the PAC extract.Finally, Smullen et al. [41] reported that at an MIC of 0.5 mg/mL, the adhesion capacity of S. mutans is decreased.Yamanaka et al. [42] showed a significant reduction of initial colonization by S. mutans of 40-60%, the inhibitory activity was related to the reduction of the hydrophobicity.

Induction of cell death and cytotoxicity
Singhal et al. [27] found a reduction in the bacterial count that was dependent on time and extract concentration.After 10 h, the reduction was 50%.Furiga et al. [34]  showed a very low level of cell damage (< 1%), with few visible dead cells.

Summary of the main results
The studies included in this review demonstrated an inhibitory effect on the bacterial growth of S. mutans, of cranberry extract in ranges from 0.5 mg/mL to 25 mg/ mL, and of GSE from 0.5 mg/mL to 250 mg/mL.Additionally, a reduction capacity of the extracts or their fractions in the formation of biofilms, decrease in the biomass of the polymicrobial biofilm, deregulation of the expression of GtfB and C, and buffering of the drop in pH were observed, in addition to an adequate antioxidant activity associated with the content of polyphenols.
These positive effects have been related to the decrease in virulence factors of the oral pathogen, and the inhibition of the formation of insoluble polysaccharides in the extracellular matrix, which prevents glycan-mediated adhesion, cohesion, and aggregation of S. mutans [28].This may suggest that these natural extracts could play an important role in the prevention of cariogenic bacterial colonization, as well as induce a decrease in their microbiological activity.

Quality of the evidence, limitations, and potential biases in the review
The results of the studies included in this review correspond to those of in vitro studies, which must be interpreted with caution, since they cannot fully reflect all the circumstances of a clinical situation.Additionally, the differences in the methodologies used and variations in the characteristics of the laboratory tests (type of extracts, methods of obtaining, separation or not of fractions, concentrations, etc.) can yield heterogeneous results, so only one test was performed according to individual analysis of studies.According to the bias assessment, all the articles (100%) had a medium and low risk of bias (Table 3), which allowed for a compilation of reliable results that may be useful to guide in vivo studies and improve research methodologies for the analysis of new alternatives for microbiological control in the formation of dental biofilms.However, it is important to recognize that while the studied compounds may have demonstrated effects on S. mutans, dental caries is a polymicrobial disease.In addition to being biofilm-mediated, it is also a diet-modulated pathology that is multifactorial, noncommunicable, and dynamic.Therefore, it is necessary to consider the analysis of other additional factors for the control of dental caries, as it is determined by biological, behavioral, psychosocial, and environmental factors [1].This recognition underscores the complexity of dental caries etiology and emphasizes the need for comprehensive approaches that address multiple contributing factors beyond the singular focus on specific bacterial strains.

Agreements and disagreements with other studies or reviews
Biofilms are the natural habitat of S. mutans, with their maturation, the bacteria are provided with greater anchorage and protection, and play a biologically active role that allows them to retain nutrients and water for their metabolism [46].According to the analyzed studies, the above-mentioned biofilm is affected by an inhibition of 34% to 51% in considerable concentrations of PAC, as well as a reduction in the structural organization of already formed biofilms.In addition, morphological alterations such as a decrease in the thickness of the biofilm, defective architecture, agglutination, and interruption in integrity were reported, which consequently result in a disorganized biofilm that is susceptible to degradation and with little response capacity.
One of the factors that the authors have given high relevance to in the analysis of the effectiveness of the extracts has been their effect on changes in pH since these have managed to create an adequate cellular environment to inhibit bacterial proliferation.The flavonoids contained in the extracts such as PACs increase the pH from 4.7 to 5.6.This generates a propitious effect for the inhibition of bacterial growth since it has been previously reported that environments with a pH lower than 5.0 are suitable for bacterial proliferation, and that the pathogen can even keep its cytoplasm alkaline at lower pH (2.5 to 3.0) [47].Additionally, it has been reported that raising the pH value will result in the bacteria not having the ability to induce repeated changes in the fatty acids of their membrane [48].
To make the environment conducive to bacterial proliferation, the action of F-ATPase is essential.This enzyme plays an important role in the production of ATP in S. mutans, for the protection and survival of the pathogen in conditions of sudden change to the acidic environment, inhibition of ATP reduces its resistance and, once again, conditions cause decrease in its proliferation [3].One study reported the inhibition of this proton-translocating enzyme between two complexes, the hydrophobic proton-conducting F0 and the F1 consisting of the water-soluble catalytic site, with PAC treatment, by binding to the F1 complex [32].The inhibition effect by exposure to PAC in the different studies ranges between 18 and 85%.
Another important factor is the effect on Gtfs and bacterial adhesion.A decrease in the expression of GtfB between 40 and 70% was reported, which produced an impact on the reduction of 35 to 40% in the number of EPS generated for biofilm formation.This effect has been associated with a decrease in bacterial colonization capacity, since the GtfB enzyme, capable of synthesizing glucans that are insoluble in water, has the vital function of interacting with other bacteria of the same species, which produces environments rich in glucan that allow the adhesion of more pathogens [49].The studies reported that the decrease in the expression of GtfC leads to a decrease in the primary colonization of S. mutans, since this enzyme synthesizes soluble and insoluble glucans that are rich in glucose, presenting the highest affinity for hydroxyapatite crystals of the three types of Gtfs [50].Likewise, this adhesion to the hydroxyapatite crystals was altered in in vitro studies by an inhibition of 81.5% by GSE, and by a decrease in the direct production of glucans.
The relationship between the structure of the compound and its antimicrobial activity has been investigated.Core structures with 3,4,5-trihydroxyphenyl groups found in epigallocatechin, epigallocatechin-3-ogallate, castalagin, and prodelphinidin may be important for antibacterial activity.It indicated that the number of hydroxyls and the degree of polymerization could be fundamental for the antimicrobial activity of phenolic compounds.Regarding the antimicrobial activity of flavonoids, the importance of the epicatechin subunit and the presence of orthohydroxyl groups in the B ring for radical scavenging and modulation of the immune response have been reported.Different concentrations of anthocyanins and flavonols markedly decreased the activity of Gtfs B and C in S. mutans cultures [51].The results suggest that the conjugation of phenolic compounds and proteins in microorganisms, especially key enzymes for their proliferation and adhesion, could be an important way to inhibit the growth of microorganisms.

Implications for practice
Although this is a systematic review of in vitro studies, it is expected that the compilation of scientific evidence will contribute to developing novel alternatives such as antimicrobials of natural origin, by incorporating agents rich in polyphenols extracted from fruits such as cranberries and the grape seeds.This can help to reduce the virulence factors of S. mutans, to generate a favorable environment that inhibits its primary colonization capacity and subsequent formation of cariogenic multispecies biofilms.These alternatives, in addition to demonstrating their high effectiveness, have few side effects and promote the use of natural resources for human health.

Implications for future research
Despite the number of in vitro studies included in this review, the need for more such studies to evaluate the antimicrobial effects of flavonoids, is evident.Corroborate the results already exposed to carry out the transfer of its use to the clinic.Additionally, the effects of the individual active compounds or fractions of the extracts could be further explored.Finally, it is necessary to develop more efficient extraction processes for natural compounds and to insist on incorporating them into daily oral hygiene methods.

Conclusions
The overall results demonstrated that cranberry and grape seed extracts or their respective fractions were effective in altering the virulence properties of S. mutans.The data showed that PACs are the components of cranberry and grape seeds that are active against S. mutans.
The main routes through which these substances affect the virulence of S. mutans may be the inhibition of the synthesis of insoluble glucans by GTF B and C adsorbed on the surface, inhibition of proton-translocating F-ATPase activity, and disruption of acidogenesis.This suggests the antimicrobial potential of the polyphenols contained in these fruits, as agents capable of modulating the pathogenicity of cariogenic biofilms.

Table 2
Risk of bias evaluation criteria and the quality of the selected studies

Table 3
Characteristics and methodology of 22 articles included in the review C 18 H 36 O 2 Stearic acid, PVP Polyvinylpyrrolidone, LAE Ethyl lauroyl arginate * Saliva coated hydroxyapatite **Brain Heart Infusion

Table 4
Characteristics and methodology of 22 articles included in the review MIC Minimum inhibitory concentration, MBC Minimum bactericidal concentration, Gtfs Glycosyltransferases

Table 5
Characteristics and methodology of 22 articles included in the review

Table 6
Characteristics and methodology of 22 articles included in the reviewMIC Minimum inhibitory concentration, Gtfs Glycosyltransferases, BHIB brain heart infusion broth, BHIA Brain heart infusion agar, GSE Grape seed extract, DRW

Table 7
Characteristics and methodology of 22 articles included in the review MIC Minimum inhibitory concentration, MBC Minimum bactericidal concentration, GSE Grape seed extract *Saliva coated hydroxyapatite **Brain heart infusion

Table 8
Characteristics and methodology of 22 articles included in the review

Table 9
Characteristics and methodology of 22 articles included in the review

Table 10
Characteristics and methodology of 22 articles included in the review GSE Grape Seed Extract, TSB Tryptic soy brothhi, KCl Potassium chloride

Table 12
Effects induced by cranberry and grape seed extracts on S. mutans

Table 15
Effects induced by cranberry and grape seed extracts on S. mutans G1, G2 experimental groups, Gtfs Glucosyltransferases