Background
Dental caries is still the most prevalent oral condition associated with biofilm in the world [
1‐
3], which harms the quality of life of millions of people [
1‐
3]. This ubiquitous disease results from complex interactions between specific oral organisms, host factors, and diet, which promote the transition from a healthy biofilm to a pathogenic one on the surface of the teeth [
4,
5]. Therefore, approaches to hinder and control the formation of pathogenic biofilms can be a strategy for the prevention of dental caries.
Streptococcus mutans plays a key role in the modulation and transition from non-pathogenic form to highly cariogenic biofilms [
6], although additional organisms may be associated [
7‐
9]. This species is highly acidogenic and aciduric and is the main producer of the extracellular matrix in dental biofilms [
5]. What makes
S. mutans the leading producer of exopolysaccharides is that it encodes multiple exoenzymes, mainly glycosyltransferases (Gtfs) [
5,
10] that are secreted into the extracellular medium and use dietary sucrose as the substrate to synthesize glucans. Gtfs become constituents of the salivary pellicle and are also adsorbed to the surfaces of
S. mutans and other microorganisms, such as
Candida albicans, maintaining its enzymatic activity [
5,
11,
12]. Glucan synthesis in the pellicle provides additional microbial binding sites, while the polymers on the surface of microorganisms increase the cohesion between organisms [
5,
11,
12].
C. albicans is the fungus most commonly found on human mucosal surfaces and frequently participates in the formation of polymicrobial biofilms on biotic and abiotic surfaces [
13,
14], especially in the presence of dietary sucrose [
15]. This fungus has an extraordinary acidogenic capacity and acid tolerance, and its association with
S. mutans results in increased exopolysaccharides formation, enhancing biofilm cariogenicity [
16‐
18]. In addition,
C. albicans produces and secretes exoenzymes capable of degrading dentin collagen under acidic conditions, contributing to biofilm virulence and cariogenicity [
19,
20].
Preventing the formation of this biofilm is essential to avoid the occurrence of dental caries. Chlorhexidine (a broad-spectrum antimicrobial agent) and fluoride are considered gold standard in dentistry for biofilm treatment and caries prevention, respectively. However, chlorhexidine suppresses the buccal microbiota [
21] and is not suitable for daily and continuous preventive and / or therapeutic use due to its side effects [
21], while fluoride provides incomplete protection against disease and has no antimicrobial effect under the conditions clinically used [
22].
Therefore, it is necessary to search for strategies to control and/or modulate cariogenic biofilms and, at the same time, do not cause toxicity to the human organism. One attractive approach would be the use and/or inclusion of bioactive agents that affect the virulence of pathogenic microorganisms without unbalancing the normal microbiota of the mouth. Consequently, there is a growing interest of researchers and industry in the development of new therapies based on natural products [
23]. These products could be used to reduce biofilm pathogenicity as an adjunct of fluoride for caries prevention.
Natural products have a wide range of activities and functions and have a rich history of use in traditional medicine. The prospection of compounds extracted from plant extracts with antimicrobial properties and antibiofilm is a relevant strategy for dentistry and other areas.
Casearia sylvestris Swartz (Salicaceae) is a plant that is distributed in the tropical and subtropical regions of Brazil, and other countries of South America and Asia. It has a huge pharmacological and cytotoxic arsenal, anti-inflammatory, antiplasmodial, and anti-ulcer properties [
24].
C. sylvestris (“guaçatonga”) is part of popular/traditional use in Brazil [
24,
25]. This plant is cited in the “National List of Medicinal Plants of Interest to SUS” (RENISUS), which contains 71 species that could treat the diseases with a high incidence in Brazil [
25]. Indigenous tribes use the macerated bark of
C. sylvestris to treat gastrointestinal disorders (i.e., diarrhea), leprosy, snake bites, and to heal wounds [
24], while decocted bark is used as an anti-inflammatory [
26], and for snake bite where the bark is infused for on-site application [
27]. In addition, Brazilian natives use leaves of
C. sylvestris as tonic and antispasmodic, for fever, syphilis, herpes, and snake bites [
26,
28]. The usual administration is oral, and the most common form of preparation is decoction.
However, there is little elucidation about its antimicrobial activity [
29,
30], while its anti-cariogenic biofilm effect is non-existent. The chemical profile of leaf extracts of
C. sylvestris var.
sylvestris (from Atlantic Forest) presents a rich phytochemical composition, with abundant diterpenes, considered taxonomic markers for this genus [
31] while phenolic compounds (flavonoids) predominate in var.
lingua [
32,
33].
Hence, C. sylvestris provides a rich source of molecules that may exhibit antimicrobial and antibiofilm properties. Therefore, the current study evaluated the antimicrobial and antibiofilm activities of C. sylvestris leaves extracts and fractions from distinct Brazilian biomes, belonging to varieties lingua, sylvestris, and intermediate. The extracts that presented an antibiofilm response against S. mutans were used to verify the effect on the adhesion of this microorganism to the salivary pellicle and the initial matrix (glucans) formed on hydroxyapatite surface.
Discussion
The results obtained in this study indicate that certain extracts and fractions of
C. sylvestris exhibit potent antimicrobial and antibiofilm effects against two of the main microorganisms related to the pathogenesis of dental caries. Previous studies that performed systematic screenings of
C. sylvestris demonstrated a varied phytochemical composition for leaf extracts [
32,
33]. This varied phytochemical composition (presence of clerodan diterpenes and glycosylated flavonoids) provides a rich source of molecules that may exhibit cariostatic properties, and their potential must be explored. Also, the natural origin of these extracts allows them to be more easily accepted for the long-term control of biofilm-mediated diseases, such as dental caries, in addition to being less costly [
49].
Here, the tested samples of C. sylvestris extracts and fractions were from three distinct varieties (lingua, intermediate, and sylvestris) that cover the five Brazilian biomes. The relevance of screening samples of different varieties and biomes is due to the variability of chemical composition concerning secondary metabolites. The chemical composition of C. sylvestris is related and/or conditioned by the biomes, and mainly associated with the respective predominant morphotypes and, therefore, under strong genetic control. The different origin biomes influence the production of secondary metabolites in the C. sylvestris varieties and, therefore, also associated with the respective predominant morphotypes. There was evidence of the differential production of flavonoids and clerodanic diterpenes by the var. lingua and sylvestris, respectively, which was constant throughout the circadian cycle and had modulations only in the reproductive period (Bueno PCP, unpublished observation). This variability should, therefore, be considered for the biological activity of natural products derived from C. sylvestris.
Although Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal and Fungicidal Concentration (MBC/MFC) tests are widely used to determine the activity of new compounds [
37,
48,
50], the number of extracts and fractions evaluated here has limited the use of that methodology. Therefore, based on concentrations reported in the literature [
34,
35], an antimicrobial activity screening was performed using a single concentration of each extract. This screening model allowed a larger number of samples to be evaluated in a single experiment and identified extracts/fractions with potential inhibitory effects. Nevertheless, the antimicrobial assay data obtained showed that the concentration of 0.50 mg/mL of specific extracts inhibited
> 50% of the viable counts of the microbial population for both the bacteria and the fungus (IC
50 or
> 3 logs) [
48]. For
S. mutans, the extracts that inhibit
> 3 logs were FLO/SC, GUA/CE, and PRE/SP; while both PAC/CE and PRE/SP reduced
> 3 logs of
C. albicans population. Therefore, the four extracts FLO/SC, GUA/CE, PAC/CE, and PRE/SP could be used to obtain the MIC and MBC data in a future study. Similarly, among the 12 AcOEt fractions at 0.25 mg/mL, seven decreased
> 3 logs of
S. mutans (FLO/SC, GUA/CE, CAM/SP, CAC/MT, CAR/CE, SRM/MG, and ARA/SP), and could also be used for MIC evaluation in the future. Thus, the current findings will direct studies for optimization of extract/fraction concentration (or extract/fraction combination) in more complex in vitro (e.g., microcosm) and in vivo models.
The single-species biofilm model used in this study has been recommended for initial screenings of new anti-caries agents [
51] and, although it does not mimic the complexity of the microbiota of the teeth' coronal surfaces, expresses a critical virulence characteristic of the biofilm, the polysaccharide matrix [
52]. The advantages of this model include high reproducibility of biofilm formation; allows high throughput screening of multiple compounds and concentrations in a single experiment [
38,
53]; the treatment steps can be consistently controlled [
54,
55].
In addition, biofilms using a single microorganism are advantageous for the analysis of the mechanisms of action of therapeutic agents, especially in the glucan-mediated processes involved in the formation of the polysaccharide matrix in the
S. mutans biofilm [
52]. Single-species model is also resourceful for the formation of hyphae, a critical virulence factor of
C. albicans. This fungus has also been chosen for the initial screening of extracts as it is one of the microorganisms frequently detected in biofilms (dental plaque) of children affected by early childhood caries [
56] and provides an increase in the binding sites of
S. mutans derived Gtfs [
57]. The results of antimicrobial and antibiofilm tests (biomass and viable population data) indicate that FLO/SC, GUA/CE, PAC/CE, and PRE/SP made the most significant reductions for
S. mutans. These four extracts are from the Atlantic Forest, var.
sylvestris.
Among these extracts, PAC/CE and PRE/CE presented a concomitant reduction of
C. albicans viable counts in both antimicrobial and antibiofilm evaluations. These findings can be attributed to the extracts’ phytochemical composition, which is marked by the simultaneous presence of phenolic compounds (glycosylated flavonoids) and clerodane-type diterpenes. The presence of both plant metabolites in these extracts can be particularly advantageous over the other extracts because flavonoids and terpenoids belong to the classes of compounds reported to be effective for controlling virulence factors of cariogenic microorganisms [
58]. Thus, the best biological activity observed for the four extracts (FLO/SC, GUA/CE, PAC/CE, and PRE/SP) can be attributed to a potential synergism between the plant metabolites, which promotes multi-target effects. This hypothesis is because the other extracts evaluated present in their composition the predominance of only one of the metabolite classes, and none of them produced significant inhibitory effects.
The current study aimed not to isolate bioactive compounds from extracts and fractions and to identify their possible targets of action, but to perform a systematic screening to identify which ones have a potential biological activity to control/prevent cariogenic biofilm. However, knowledge of the secondary metabolites found in
C. sylvestris leaf extracts provided the basis for the interpretation of the results found.
C. sylvestris has secondary metabolites that confer different pharmacological properties to the plant and justify its use in folk medicine. A previous phytochemical study provided valuable information about the phenolic components from the leaves of the ARA/SP extract, following the same extraction method as the extracts tested here [
33,
47]. Fourteen glycosylated flavonoids and one catechin were isolated and identified, as follows: (+)-catechin, quercetin-3-O-α-L-rhamnopyranosyl-(1 → 6)-ß-D glucopyranoside (rutin), isorhamnetin-3-O-α-L-rhamnopyranosyl-(1 → 2)-ß-Dglucopyranoside (isorhamnetin-3-O-neo-hesperidoside, isorhamnetin-3-O-α-Lrhamnopyranosyl-(1 → 6)-ß -D-glucopyranoside (narcissin) e isorhamnetin-3-O-α-Lrhamnopyranosyl-(1 → 2)-α -L-arabinopyranoside [
47]. A previous study [
33] with ARA/SP, CAM/SP, MOG/SP, and PRE/SP samples selected two peaks from the chromatograms and included the clerodane-type diterpenes (casearins) detected at 235 nm. Other chemical investigations have isolated 41 clerodane-type diterpenes, including casearins [
59‐
64] and casearvestrins [
65].
Flavonoids have different pharmacological properties, including antimicrobial and antioxidant activities [
66,
67]. These compounds may be complexed with extracellular and soluble proteins as well as with bacterial cell wall and can also inhibit Gtfs activity [
66,
67]. In contrast, terpenoids can promote rupture of the microbial cell membrane by their lipophilic characteristics [
68]. Therefore, the activity of these compounds may justify the antimicrobial activity against
S. mutans (≥50% reduction of viable bacterial counts) caused by Atlantic Forest extracts, var
. sylvetris. Further investigation is needed to corroborate these effects of
C. sylvestris.
The
S. mutans viable counts data after treatment with ARA/SP, CAR/CE, and SRM/MG ethyl acetate fractions confirm a significant reduction in the bacterium viable population ≥ 80%, in contrast to the other fractions. The fractions were fractionated from extracts with a higher content of glycosylated flavonoids. Although ethyl acetate fractions are fractionated from extracts with higher content of glycosylated flavonoids, the extractive method used results in fractions with higher content of diterpenes in relation to flavonoids, since ethyl acetate is a good solvent to extract clerodan diterpenes (specifically casearins) due to their polarity, so even in extracts with low diterpene content, the content that is present will be extracted, varying only the yield between each resulting fraction (Bueno PCP, unpublished observation). The terpenes may affect the virulence and could be responsible for the bactericidal effect found [
67,
69,
70]. Thus, they should now be considered for future studies concerning activity against oral biofilms and anti-Gtfs activity, in addition to the paramount importance of isolating and identifying bioactive compounds from fractions.
Among the mapped phenolic compounds identified in ARA/SP [
33], two of them (catechins and quercetins) affect the activity of
S. mutans Gtf enzymes [
71], interfering with the synthesis of soluble and insoluble glucans in biofilms [
72,
73]. Moreover, quercetin has inhibitory activity against bacterial acid production [
74,
75]. This anti-Gtfs action described for the phenols also found in
C. sylvestris may be indirectly responsible for the biomass decrease observed for treated
S. mutans biofilms. Here, the extracts with promising antimicrobial and antibiofilm activities against
S. mutans also inhibited GtfB activity. The anti-Gtfs effect may reduce the quantity of exopolysaccharides and may cause both the reduction of the overall biofilm volume and affect bacterial biomass, as observed here. This cascade of events would limit the binding sites available for adhesion and bacterial co-aggregation, interfering in the initial stage of biofilm formation. Another effect on Gtfs activity would be the modification of glucans produced, in which the type of glycosidic linkage could be affected. Changes in the type of and proportions of glycosidic linkages and ramifications can hinder biofilm build-up by weakening the binding of microbial cells and the overall tridimensional structure of biofilms [
5].
The simultaneous presence of flavonoids and diterpenes compounds in FLO/SC, GUA/SP, PAC/CE, and PRE/SP can be particularly advantageous concerning the other extracts, as previous studies show that phenols and terpenes are effective against
S. mutans [
58,
60,
61]. Thus, here, FLO/SC, GUA/CE, and PRE/SP affected the activity of GtfB and caused a decrease in the amount of glucans produced. However, FLO/SC, PAC/CE, and PRE/SP extracts may have also affected the quality of glucans formed by GtfB, since a larger number of
S. mutans cells were removed after adhesion to glucans when these extracts were present during their synthesis. Therefore, the four extracts from the Atlantic Forest, var.
sylvestris could modify the glucans formed on the sHA surfaces, and this modification could weaken the adhesion of
S. mutans to the initial glucan matrix. Insoluble glucans are virulence markers of cariogenic biofilms [
5,
56]; thus, interfering with this trait is an approach to prevent dental caries. This behavior is clinically desirable because it could facilitate the mechanical removal of cells when using a formulation with these extracts in the future. In contrast, these four extracts did not modify the salivary pellicle, since no extract significantly affected the removal of cells adhered to the salivary pellicle.
Clerodane-type diterpenes are secondary metabolites of the terpene class, a class to which the diterpenes of the extracts belong. This class of compounds has been identified as responsible for the activity on
C. albicans. Terpenes act on the permeability of fungal cells causing changes in membrane properties and their functions [
76‐
79], cell morphology, and inhibit the growth of this fungus. Tannins (phenolic substance) are bioactive compounds of
C. sylvestris [
80] that inhibited in vitro yeast growth and inactivated the fungal cell membrane by precipitating proteins [
81]. These bioactive compounds may be related to the results obtained after treatments of
C. albicans with PAC/CE and PRE/SP. In addition, although RIO/RS and SRM/MG showed a statistically significant reduction in the biofilm population of
C. albicans, how far this reduction is biologically significant needs to be investigated because of the data high variability. Both RIO/RS and SRM/MG presented predominantly glycosylated flavonoids, suggesting that these phenolic compound act better when in association with diterpene compounds in the extract, as found for PAC/CE and PRE/SP, probably due to an action of some component(s) that increases the stability or bioavailability of the other or increasing its metabolism [
82]. Moreover, the lack of effect of extracts on biomass reduction of
C. albicans biofilms can be attributed to the morphological difference between the tested microorganisms, since one is a Gram-positive bacterium while the other is a fungus.
Here, extracts of
C. sylvestris were more active against
S. mutans compared to the effects against
C. albicans, both in antimicrobial and antibiofilm analyses.
C. albicans has several virulence factors, such as polymorphism [influenced by quorum sensing signaling molecules tyrosol, a phenylethanoid (yeast to hyphae) and farnesol, a sesquiterpene (hyphae to yeast)], biofilm formation (presence of extracellular matrix), and control of nutrient competition [
83,
84]. These are the main factors associated with resistance and drug tolerance and may cause a less pronounced effect of the extracts for this microorganism. In addition, the biomass of
C. albicans after the treatments showed high variability and this behavior was not reported in previous studies, but it could be related both to the virulence factors of the microorganism and to the variability of the antifungal activity within the same extract [
84].
The extracts PRE/SP, GUA/CE, MOG/SP, SRM/MG, ARA/SP, and V control inhibited about 25% of cell viability, thus, exerting slight cytotoxicity to the oral keratinocytes. However, two extracts caused moderate cytotoxicity (PAC/CE and FLO/SC). Nevertheless, because all extract treatments and V caused cell death, the cytotoxic effect observed is mostly associated with the concentration of ethanol and DMSO used in the vehicle. Therefore, the reduction of the concentration of ethanol and DMSO should be considered for further studies. Furthermore, the two treatments that were moderately cytotoxic (PAC/CE and FLO/SC) could have antimicrobial and antibiofilm activities because of the toxicity observed in the cytotoxicity assay. Also, the methodology of MTT has its limitations [
85], because the oral mucosa is a tissue with three-dimensional organization and not monolayer cells, and therefore toxicity studies should be performed using in vivo models and even more than one assay should be used to determine cell viability in vitro studies, as this would increase the reliability of the results obtained. Moreover, a reduction in the cytotoxicity of these extracts could be achieved by isolating the active fraction from the crude extract, which should be better evaluated, and/or decreasing the concentration of EtOH and DMSO in the vehicle.
Caution is required in interpreting the data presented to avoid overestimation of the effects for the control of cariogenic biofilms. Here, simple models were used to verify which extracts have potential antimicrobial and/or antibiofilm activities (which usually employ an exposure time of 24 h during screenings [
38]) and evaluated those that had an inhibitory outcome also for cytotoxic effect against oral keratinocytes organized in monolayers after 1 h exposure (which do not mimic the complex organization of the oral mucosa tissue). Moreover, to test further the effect of selected extracts with ‘promising’ antibiofilm activity, we used models for GtfB activity and
S. mutans adhesion to treated pellicle with exposure time of 30 min, while for microbial adhesion to glucan matrix, the treatments were present for 4 h (which are the exposure times established in the literature) [
11,
12,
39,
40]. As described above, the next step of this line of research, in addition to using optimized concentrations of the extracts, will be to evaluate the extracts and AcOEt fractions using more complex models with shorter exposure time to mimic a clinical application.
The content of flavonoids and diterpenes could be standardized in extracts and promising fractions, overcoming problems of compositional variation associated with phytochemical extraction and geographic or seasonal influences. It is noteworthy that because
C. sylvestris flourishes and grows fruit in the second year of life [
86] and lives at least 20 years [
87], cultivation is favorable for therapeutic purposes. Also, its wood can be used as fuel and for the construction of fences, posts, stakes, rustic carpentry, and cable tools [
87]. Therefore, the controlled cultivation of
C. sylvestris may contribute to the development of new agents for the control of cariogenic biofilm, which reduces the final cost when compared to the purified compounds. Although this study used simplistic in vitro models, the results substantiate the ethnobotanical use of extracts and fractions of specific biomes and varieties for the prevention of oral cariogenic biofilms. However, in vitro data from more complex (e.g., microcosm) and in vivo models may be useful in determining the potential utility of this plant.