Antimicrobial Activity on Streptococcus mutans and Enterococcus faecalis of Cyperus articulatus Ethanolic Extracts

Oral diseases are one of the biggest public health problems worldwide, caused by opportunistic pathogens such as Streptococcus mutans and Enterococcus faecalis. Cyperus articulatus (priprioca) is a plant conventionally used in traditional medicine in the Amazon region. However, little is known about the possible dentistry-related uses of extracts from the rhizomes and solid waste generated by the extraction of essential oils from this vegetable. This study aimed to investigate the chemical composition of volatile compounds and antimicrobial activity through the Minimum Inhibitory Concentration test (MIC and assessment of the toxicity by Hens Egg Test-Chorion Allantoic Membrane (HET-CAM) of the ethanolic extracts from Cyperus articulatus intact rhizomes and solid waste. We identified sesquiterpenes as the main constituents, strong antimicrobial activity of the ethanolic extract of intact rhizomes against S. mutans (MIC = 0.29 mg/mL), moderate antimicrobial activity against E. faecalis of the extract obtained from the solid waste (MIC = 1.17 mg/mL), and absence of toxicity for both tested extracts. The absence of irritation and the antibacterial activity of the ethanolic extract from C. articulatus rhizomes and solid waste reveal its potential for use in the alternative control of bacteria that cause oral infections and may present economic viability as a raw material for dental products.


Introduction
Oral diseases remain one of the largest public health problems worldwide, impacting over 3.5 billion individuals [1].In 2010, untreated caries in permanent teeth emerged as the predominant condition, affecting 2.4 billion people [2].Recent data from Brazil's national survey (SB Brazil 2010) showed a declining trend in caries prevalence among 12-year-olds and adolescents compared to the 2003 survey.Specifically, for individuals aged 15-19 years, the caries index reduced by 35%, and in 12-year-old children it reduced by 26.2%.Conversely, adults had only a slight (17%) decrease and in elderly the prevalence remained unchanged [3].
The SB Brazil 2010 findings suggest that the country shifted from an average prevalence of caries in 2003 (CPO-index of decayed and filled teeth-between 2.7 and 4.4) to a low prevalence condition in 2010 (CPO index between 1.2 and 2.6), according to WHO's classification [4].Nonetheless, regional disparities in oral health observed since the 1986 survey perpetuate marked inequalities, particularly among children and adolescents.The north and northeast regions consistently present the worst indicators of oral health, with a CPO of 3.2 and 2.7, respectively [3].In addition to having the highest caries prevalence values, the northern region also exhibits the highest proportion of decayed teeth (yet to be treated) compared to filled and missing teeth.This highlights the pronounced dental caries disparities in Brazil, with higher oral health impact in the poorest Brazilian regions [5].
Dental caries is a multifactorial pathology involving a susceptible host, microbial biofilm, and cariogenic diet.Among the microorganisms involved, Streptococcus mutans stands out as a pivotal initiator of biofilm formation [6].This microorganism is known for its high cariogenic potential, acidogenicity, and acid tolerance.When exposed to a diet rich in sugar, it metabolizes carbohydrates and produces acids that demineralize the tooth structure [7].If left untreated, carious lesions penetrate the dentin and reach the pulp, leading to endodontic infections that cause pulpal and periapical alterations.The etiology of these infections is directly linked to microorganisms, with Enterococcus faecalis being particularly prominent [8].Both S. mutans and E. faecalis are opportunistic pathogens and belong to the group of microorganisms that exhibit high resistance in the oral cavity.
Oral diseases often result in persistent pain, sepsis, reduced quality of life, missed school days, disruptions in family life, and decreased work productivity [9].Considering these consequences, it is crucial to prioritize the implementation of preventive measures, as the costs associated with treating oral diseases place a substantial economic burden on families and healthcare systems.The most effective approach to controlling dental biofilms is through proper hygiene practices such as brushing and flossing (mechanical methods).However, this method alone may not be fully effective as it relies on the ability and manual dexterity of individuals to perform quality tooth brushing and interdental cleaning.Therefore, it is essential to combine chemical procedures to control biofilm formation [10].
In 2006, medicinal plants and phytotherapy were incorporated into the Unified Health System through the National Policy on Integrative and Complementary Practices [11].Despite the regulation of phytotherapy, a lack of knowledge regarding the indications and precautions for the use of medicinal plants persists among health professionals.In dentistry, the utilization of medicinal plants for treating oral pathologies, or systemic diseases with oral manifestations, remains relatively unexplored [12].
The highly biodiverse Amazon region harbors approximately 55,000 plant species.Of these, 10,000 may possess medicinal properties.These plants, steeped in centuriesold cultural practices, offer a rich reservoir for therapeutic exploration, requiring the need for rigorous pharmacological evaluation [13,14].In general, these natural products may be a source for the development of herbal formulations for the treatment of various illnesses [15][16][17].One common plant in this region is Cyperus articulatus (Cyperaceae), an aromatic plant popularly known as priprioca.In the state of Pará, priprioca essential oil has been utilized as a raw material for the cosmetic industry for over 30 years [18,19].
Like many plants, priprioca has a low essential oil yield.The extraction process from the rhizomes of C. articulatus generate solid waste, which is often discarded.However, this by-product harbors bioactive compounds, spotlighting its potential as a sustainable resource [27,28].
To date, no study has reported the antimicrobial activity of ethanolic extracts derived from C. articulatus rhizomes and its solid waste on S. mutans or any other bacteria commonly associated with oral diseases.Therefore, this study aims to elucidate the chemical composition of priprioca ethanolic extracts, assess their toxicity, and evaluate their antimicrobial efficacy against S. mutans and E. faecalis, providing insights into potential therapeutic applications.

Ethics Committee Approval
This research was approved by the Animal Ethics Committee of the Federal University of Western Pará (UFOPA) under protocol number 1120220228.All procedures were performed in accordance with the guidelines of the National Council for Ethics in Animal Care (CONCEA) and Law No. 11.794/2008 for the care and use of experimental animals.

Plant Material
Cyperus articulatus was grown on a small scale in a rural property (54 • 43 00.10 W; 02 • 37 41.10 S) in the municipality of Santarém, Pará, Brazil.The botanical material was identified by Dr. Antônio Elielson Sousa da Rocha, and a voucher specimen was deposited under registration number MG-207174 in the herbarium of the Museu Paraense Emilio Goeldi (MPEG) (Belém, Pará, Brazil).

Ethanolic Extract from C. articulatus Rhizomes
Immediately after harvesting from a mature plant, 85 g of rhizomes were crushed and subjected to an 8 h extraction process using 96% ethanol in a Soxhlet apparatus.The resulting extract was then concentrated using a rotary evaporator under reduced pressure at a temperature of 50 • C, until the complete evaporation of the solvent.Finally, the extract was carefully packaged in sterile amber bottles and stored refrigerated at 10 • C.

Ethanolic Extract from the Solid Waste of C. articulatus
To obtain the residual essential oil, 300 g of rhizomes underwent hydrodistillation using a Clevenger apparatus.Following the oil extraction process, 62.75 g of the resulting solid waste was subjected to Soxhlet extraction using the same methodology as described for the rhizome extract.The extract obtained from the solid waste was carefully stored in sterile amber bottles and refrigerated at 10 • C.

GC-MS Analysis of Extracts
The volatile compounds present in the ethanolic extracts were analyzed using an Agilent gas chromatograph model 5977E MSD + 7820A gas chromatography-mass spectrometry (GC-MS) system, equipped with an HP-5MS capillary column (30 m × 0.25 mm × 0.25 µm).The analysis was performed under the following conditions: injector temperature of 270 • C, initial temperature of 60 • C, temperature ramp of 3 • C/min, reaching a final temperature of 240 • C, and maintaining it for 60 min (total run time).Helium gas was used as the carrier gas at a flow rate of 1 mL/min.The selective mass detector operated at 70 eV and a m/z range of 50-400.To enhance the analysis, the extracts underwent a methylation sample derivatization process.The preparation involved the addition of 0.2 g of sample to 5 mL of hexane, the mixture was vortexed for 5 min, centrifuged for 5 min, then the supernatant phase was added with 0.5 µL of methanol and a solution of sodium hydroxide (0.5 L).The identified compounds were compared with electronic libraries of the equipment, including NIST, KAAPI, and FFNSC, according to methodology already described in the literature [27,29].

Antimicrobial Evaluation 2.6.1. Microorganisms and Culture Conditions
Lyophilized reference strains of S. mutans (ATCC 25175) and E. faecalis (ATCC 29212) were reactivated by culturing them in BHI broth (Brain Heart Infusion, DIFCO) to obtain bacterial suspensions.The S. mutans and E. faecalis suspensions were incubated under aerobic and anaerobic conditions, respectively, for 48 h at 37 • C. The resulting cultures were then transferred to plates containing BHI agar.Inocula were prepared by directly inoculating colonies into 1 mL of sterile saline solution and adjusting them to a standard of 0.5 on the McFarland scale, corresponding to 1.5 × 10 8 CFU/mL (NCCLS/CLSI, 2006).The turbidity of the suspension was measured using a turbidimeter (MS Tecnopon Instrumentação, Piracicaba, SP, Brazil) to ensure the desired standard on the McFarland scale.

Determination of the Minimum Inhibitory Concentration (MIC)
The antibacterial activity was assessed using the broth microdilution method as recommended by the National Committee for Clinical Laboratory Standards [28].The initial concentration of the ethanolic extract from the C. articulatus rhizomes and the ethanolic extract from the solid waste of C. articulatus was 150 mg/mL, dissolved in propylene glycol as the solvent.Serial dilutions were prepared (1:1 dilution factor) using BHI as the diluent to obtain different concentrations.Concurrently, microbial suspensions were adjusted to a standard of 0.5 on the McFarland scale and further diluted in sterile saline solution (1:10 dilution factor) to achieve a final concentration of 1.5 × 10 4 CFU/mL.The tests were conducted in 96-well plates, with each well containing 90 µL of the specific extract concentration, 90 µL of sterile BHI, and 10 µL of the microbial inoculum.As a positive control, 0.12% chlorhexidine gluconate (PerioGard, Colgate, Campo, SP, Brazil) was used at an initial concentration of 0.6 mg/mL.The viability of the strains, sterility of the medium, and solvent activity was simultaneously monitored.The microplates were incubated in a bacteriological oven at a controlled temperature of 37 ± 2 • C for 24 h.After the incubation period, the inhibition of microbial growth was assessed by adding a 20 µL aqueous solution of resazurin dye (Sigma-Merck, Darmstadt, Germany) (0.02% w/v), followed by an additional 3 h incubation.MIC, defined as the lowest concentration capable of inhibiting microbial growth, was determined by the presence of a persistent blue color in the wells.A visible color change from blue to pink (indicating the reduction of resazurin to rephazurin) indicated the growth of viable cells.The experiments were performed in triplicate.

Toxicity Assessment: Hens Egg Test-Chorion Allantoic Membrane (HET-CAM)
The HET-CAM test (Hens Egg Test-Chorion Allantoic Membrane) was performed according to the protocol described by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) [30].
The study utilized fertilized chicken eggs (Gallus gallus) that were incubated for a period of nine days.This timeframe was chosen because it falls within a stage of development where the embryo has not yet formed nervous tissues and does not possess the ability to perceive pain.Following the incubation period, the shell surrounding the air chamber was carefully removed, revealing a moistened internal membrane soaked in saline solution.This membrane was then delicately extracted to expose the chorioallantoic membrane.Next, 0.3 mL of the test formulations were directly applied to the CAM, and the resulting reactions were observed for a duration of 300 s.The time taken for the occurrence of specific events (endpoints) was carefully recorded, monitored, and filmed in seconds.
The following groups were included in the testing: a negative control of 0.9% NaCl, a solvent control of 1% propylene glycol, a positive control of 0.1 mol/L NaOH, 4.68 mg/mL of ethanolic extract from C. articulatus rhizomes, and 4.68 mg/mL of ethanolic extract from solid waste of C. articulatus.To ensure consistent and reliable results, reference photographs of all the endpoints were used.The three endpoints that were monitored consisted of hemorrhage (bleeding from vessels), vascular lysis (disintegration of blood vessels), and coagulation (denaturation of intra-and extravascular proteins).The concentration of the plant extracts used in the toxicity test was two times higher than the minimal concentration considered to have antimicrobial activity.
The assessment of the test formulations was based on the occurrence of each of the three HET-CAM endpoints at specific time intervals: 30 s, 2 min, and 5 min.A specific score was assigned to the time at which each event took place, reflecting the occurrence of lysis, bleeding, and clotting.Table 1 provides a summary of this relationship.The scores were then summed to obtain a single numerical value, indicating the irritation capacity of the tested formulation on a common scale ranging from 0 to 21, as shown in Table 2 [30].The test was performed in triplicate and with the results obtained, an average (between 0 and 21) was determined, indicated for the irritant and non-irritant categories (Table 2) [30].

Scoring Range
Irritation Category 0 to 0.9 not labeled 1 to 4.9 slight irritation 5 to 8.9 moderate irritation 9 to 21 severe irritation HET-CAM: Hens Egg test Chorion Allantoic Membrane.Test according to the ICCVAM protocol [30].

Statistical Analysis
The results obtained from the toxicity test (HET-CAM) were expressed as the mean ± standard error of the mean.Analysis of variance (ANOVA) was employed to compare the experimental data, while Tukey's test was utilized to identify differences at a significance level of p < 0.05.

Analysis of the Chemical Composition of Volatile Compounds in the Extracts
The ethanolic extract from C. articulatus solid waste yield was 21%.Oxygenated sesquiterpenes (53.59%) and fat acids (33.58%) were predominant in the extract, followed by a minor number of oxygenated monoterpenes (3.69%).Eight components were identified after derivatization of solid waste ethanolic extract, representing 90.86% of the composition, with mustakone being the primary constituent (17.38%) (Table 3).

Antimicrobial Evaluation
In our study we have established as having potential antimicrobial activity only the extracts presenting a MIC below 2.0 mg/mL.Therefore, chlorhexidine exhibited antimicrobial activity against both microorganisms tested in lower concentrations (MIC of 0.004 mg/mL against S. mutans and 0.002 against E. faecalis).When we compare the extracts, we observe that the ethanolic extract from the rhizomes of C. articulatus was able to inhibit the growth of S. mutans in a lower concentration with values of MIC 0.29 mg/mL; on the other hand, the ethanolic extract of the solid waste showed more expressive MIC values against E. faecalis, inhibiting the growth of this bacteria at a concentration of 1.17 mg/mL.Table 5 presents the minimum inhibitory concentrations (MIC), expressed in mg/mL, required to inhibit bacterial growth.

Antimicrobial Evaluation
In our study we have established as having potential antimicrobial activity only the extracts presenting a MIC below 2.0 mg/mL.Therefore, chlorhexidine exhibited antimicrobial activity against both microorganisms tested in lower concentrations (MIC of 0.004 mg/mL against S. mutans and 0.002 against E. faecalis).When we compare the extracts, we observe that the ethanolic extract from the rhizomes of C. articulatus was able to inhibit the growth of S. mutans in a lower concentration with values of MIC 0.29 mg/mL; on the other hand, the ethanolic extract of the solid waste showed more expressive MIC values against E. faecalis, inhibiting the growth of this bacteria at a concentration of 1.17 mg/mL.Table 5 presents the minimum inhibitory concentrations (MIC), expressed in mg/mL, required to inhibit bacterial growth.

Toxicity Assessment: HET CAM
The analyzed test substances, including the ethanolic extract from C. articulatus rhizomes, the ethanolic extract from C. articulatus solid waste, and propylene glycol 1%, demonstrated an absence of vascular alterations in the chorioallantoic membrane.This represents the first alterations associated with skin and mucosa irritation and contrasts with the negative control of NaCl 0.9%.Consequently, these substance extracts and solvent did not demonstrate any irritant potential or toxicity and received an "unlabeled" irritation index (II = 0), with no statistical difference when compared to the control group (Na Cl 0.9%).In contrast, sodium hydroxide (NaOH) at 0.1 mol/L was classified as "Serious irritation" due to the appearance of the three endpoints (lysis, hemorrhage, and coagulation) after its contact with the chorioallantoic membrane of embryonated chicken eggs, obtaining an irritation index of 17.67, with statistical difference compared to the control group (p ≤ 0.0001) (Table 6).

Discussion
In this research, we analyzed two plant materials sourced from priprioca (C.articulatus).The ethanolic extract from the plant's rhizomes showcased a phytochemical profile of volatile compounds consistent with findings from other studies in the Amazon region [27][28][29].Rhizomes serve as a raw material used for extracting priprioca essential oil, which holds substantial commercial value and economic interest, particularly in the perfume industry [18,19].Numerous studies have highlighted the therapeutic benefits of priprioca essential oil, including antimicrobial, anticonvulsant, antiparasitic, antioxidant, and anti-inflammatory properties, and potent bactericidal and fungicidal activities.Additionally, its effectiveness in controlling oral biofilm formation has been recorded [20,21,26].
Priprioca yields a low essential oil content of approximately 0.45% The processing associated with essential oil extraction results in a substantial volume of plant waste that often goes unused [28].Preliminary research suggests that this priprioca solid waste contains bioactive compounds with antifungal properties and potential anticancer effects [27,28].Therefore, in parallel with the evaluation of the plant's raw materials (rhizomes), we also examined the ethanolic extract obtained from the solid waste generated after extracting the essential oil from C. articulatus.
Our results of chemical analyses of volatile compounds in C. articulatus extracts align with prior investigations.Corimbolone and mustakone was reported previously as primary sesquiterpenes present in both the rhizome and solid waste extracts derived from C. articulatus [22,27,31].Additionally, cyperotundone, previously detected in the ethanolic extract of the solid waste of C. articulatus [27], was found to be an important constituent of the ethanolic extract derived from C. articulatus rhizomes [32].Oleic acid was found in this study to be a major constituent of the ethanolic extract obtained from the solid waste, and it was also identified at a lower concentration in a similar study [29].
Our results agree with previous research, in which C. articulatus oil analyzed by GC-MS showed the presence of alpha-guaiene, β-selinene [26], alpha-calacorene, pogostol, cyperotundone, spathulenol, and thujopsenal [34].This is similar to our findings, where those compounds were found as constituents of the ethanolic extract of C. articulatus rhizome.
Previous reports regarding ethanolic extract from solid waste of C. articulatus from Santarém, PA, showed corimbolone as the main compound found (14.25%) [29].This compound was also present in minor relative amounts within the rhizome ethanolic extract used in this study (3.94%).
One limitation of the present study was the use of GC-MS analysis for ethanolic extracts.GC-MS is a technique that relies on separating substances from a sample based on their distribution between the stationary (solid or liquid) and mobile (gas) phases.It is most commonly employed for the analysis of volatile gases or substances.Although an important portion of the volatile compounds are removed via distillation of the essential oil, there are still a considerable amount of volatile compounds present in the ethanolic extract, mainly certain volatile sesquiterpenes, that can be detected using GC-MS [27,29].
S. mutans and E. faecalis are microorganisms found in the oral cavity that are closely linked to the development of dental caries [6] and endodontic infections [8].To the best of our knowledge, no studies have yet demonstrated the effects of C. articulatus extracts on bacteria associated with oral diseases.
Studies have indicated that the chemical compounds α-pinene and β-pinene, present in the essential oil of C. articulatus, exhibit antimicrobial activity against gram-positive bacteria and yeasts.These compounds have the potential to impact the viability and/or matrix of the oral biofilm [35].In our study α-pinene and β-pinene were not found in extracts, suggesting the antimicrobial activity was due to the presence of other chemical compounds.Furthermore, previous research showed strong antimicrobial activity in the essential oil from C. articulatus against the oral pathogens Candida albicans, Fusobacterium nucleatum, Porphyromonas gingivalis, Streptococcus sanguis, and Streptococcus mitis, and chemical analysis of the oil revealed the presence of the sesquiterpene mustakone as the main component, with the presence of alpha-copaene, alpha-guaiene, and β-selinene compounds also recorded in the ethanolic extract from C. articulatus rhizomes used in this study [26].
Preliminary studies evaluating the C. articulatus extract also identified high concentrations of corimbolone and mustakone and showed in vitro antiplasmodial activity of these substances [29,31].Similarly, linoleic acid-found in this study to be one of the major compounds in the ethanolic extract of intact rhizomes of C. articulatus-was isolated and associated with the death of worms and microfilariae of the bovine parasite Onchocerca ochengi [22].
Citronellal, a representative acyclic monoterpene compound, showed antibacterial activity against E. faecalis (ATCC 29212) in a previous study [36].The presence of this compound present in the ethanolic extract from C. articulatus solid waste may be an explanation for the results found in this study, where the MIC of E. faecalis decreased in waste materials compared to plant roots.
The antimicrobial activity of essential oils is not fully understood, but it is thought to be due to the complex mix of components in oils, and not the result of the influence of a single chemical.It has been suggested that the combination of components within the essential oils have either a synergistic or antagonistic relationship, which may cause differences in their antimicrobial properties [36].Thus, the mechanism of antimicrobial action of the ethanolic extracts from C. articulatus rhizomes and solid waste tested in this study on the bacteria that cause oral infections may be related to the synergism of these chemical compounds.
Our research is one of the first reports to investigate the antimicrobial effect of C. articulatus extracts against oral bacteria.The strength of the antimicrobial activity of a plant material can be determined based on the MIC values, which corresponds to the lowest concentration of an antibiotic necessary to inhibit the growth of a specific microorganism, essential for developing effective antimicrobial therapies [33].When MIC values are up to 0.5 mg/mL, the antimicrobial activity is considered strong, while values between 0.6 and 1.5 mg/mL indicate moderate activity [33,37].Thus, both the ethanolic extract from C. articulatus rhizomes and chlorhexidine (positive control) demonstrated strong antimicrobial activity against S. mutans, with MIC values below 0.5 mg/mL.Additionally, the ethanolic extract from C. articulatus solid waste displayed moderate antimicrobial activity against E. faecalis, with a MIC value of 1.17 mg/mL [28,37].
Mustakone, the main compound of the essential oil from C. articulatus, was also identified in the ethanolic extract of the rhizomes, as well as in the ethanolic extract obtained from solid waste derived from oil extraction.However, there were variations in the percentages [22,27,31].Other studies have identified saponins, flavonoids, terpenes, tannins, and sugars in the extract from the decoction of priprioca rhizomes [25].The presence of these bioactive substances may be related to the antimicrobial activity observed in this study against S. mutans and E. faecalis.
The ethanolic extracts from both the rhizomes and the solid waste from C. articulatus did not present toxic activity.In a previous study using the MTT test, the ethanolic extract from C. articulatus solid waste did not demonstrate cytotoxic activity in macrophages at concentrations of 12.5, 25, and 50 mg/mL [27].However, no previous studies have assessed the toxicity of C. articulatus extracts using the method employed in this research (HET-CAM).
Given the absence of toxicity, clinical applications of the ethanolic extract of C. articulatus rhizomes and ethanolic extract of C. articulatus solid waste at a concentration of 4.8 mg/mL are possible and not risky.

Conclusions
Our study demonstrated that the ethanolic extract from C. articulatus rhizomes and its solid waste contained sesquiterpenes, with mustakone being the primary constituent.The ethanolic extract from rhizomes had strong antimicrobial activity against S. mutans, while the extract from the solid waste presented moderate antimicrobial activity against E. faecalis.The ethanolic extracts from both the rhizomes and the solid waste of C. articulatus were safe; they did not present toxic activity.Our results suggest that the Amazon plant priprioca Plants 2024, 13, x FOR PEER REVIEW 8 of 13(1)

Table 1 .
[30]ing criteria for irritation test with the HET-CAM.Hens Egg test Chorion Allantoic Membrane.Test according to the ICCVAM protocol[30].

Table 2 .
Irritation category according to the HET-CAM score range.

Table 3 .
Constituents identified in the ethanolic extract from C. articulatus solid waste.byGC-MS and relative amounts.
RT: retention time per minute; %: relative amount of constituents in the extract fraction.

Table 4 .
Constituents identified in the ethanolic extract from C. articulatus rhizomes by GC-MS and relative amounts.
RT: retention time per minute; %: relative amount of constituents in the extract fraction.

Table 5 .
Minimum Inhibitory Concentration in mg/mL of the ethanolic extract from C. articulatus solid waste, ethanolic extract from C. articulatus rhizomes and chlorhexidine gluconate against the

Table 5 .
Minimum Inhibitory Concentration in mg/mL of the ethanolic extract from C. articulatus solid waste, ethanolic extract from C. articulatus rhizomes and chlorhexidine gluconate against the bacteria S. mutans and E. faecalis.Extr EtOH C. articulatus waste: ethanolic extract from C. articulatus solid waste; Extr EtOH C. articulatus rizhomes: ethanolic extract from C. articulatus rhizomes; MIC: Minimum Inhibitory Concentration expressed in mg/mL.The initial concentrations of the extract (150 mg/mL) and chlorhexidine (0.6 mg/mL).