Diterpenes of the pimarane type isolated from Viguiera arenaria : Promising in vitro biological potential as therapeutic agents for endodontics

Viguiera arenaria, family Asteraceae, is a plant that contains diterpenoids, which make this species potentially applicable in endodontics. More specifically, V. arenaria contains diterpenes of the pimarane type, which display various classic biological activities. This study evaluates the antibiofilm activity, the time-kill curve, and the inhibitory concentration index of diterpenes of the pimarane type (entpimara-8(14),15-dien-19-oic acid, ent-8(14),15-pimaradien-3β-ol, and ent-8(14),15-pimaradien-3β-19-oic acid sodium salt, designated diterpenes I, II, and III, respectively) toward nine anaerobic bacteria commonly found in endodontic infections; this study also assesses the cytotoxic activity of these diterpenes against human fibroblasts. According to the antibiofilm assay, diterpenes I, II, and III inhibit at least 50% of all the bacteria. On the basis of the time-kill curve experiments, the behavior of these diterpenes depends on the tested bacteria, diterpene concentration, and microorganism sensitivity. Synergism of diterpenes I and II with chlorhexidine (CDH) was higher against P. gingivalis (clinical isolate) and Aggregatibacter actinomycetemcomitans (ATCC). As for diterpene III, synergism with CDH is higher against P. micros. As revealed by the XTT assay, none of the diterpenes of the pimarane type tested here are cytotoxic. Hence, diterpenes I, II, and III are promising biomolecules and may provide therapeutic solutions in the field of endodontics.

the basis for the development of new chemicals for pharmaceutical products (Palombo, 2011).In this scenario, professionals in the area of dentistry cannot overlook the current perspective of treatment based on compounds originating from novel plant-derived bioactive molecules (Palombo, 2011).
Primary endodontic infection is the infection of the necrotic root canal and is the prime cause of apical periodontitis (Siqueira et al., 2002;Roças et al., 2011).This infection is chacacterized by anaerobic bacteria that constitute a readily mixed biofilm (Roças et al., 2011;Ricucci and Siqueira, 2010) of polymicrobial origin.
Gram-negative bacteria are the main bacteria causing this infection (Fabricius et al., 1982;Caetano da Silva et al., 2014).Despite the promising results regarding the action of diterpenes of the pimarane type against some potentially pathogenic bacteria in the oral cavity, their antimicrobial effects on bacteria that cause endodontic infections are little known.Biological assays on diterpenes of the pimarane type could provide useful information for the development of new biocampatible materials that can inhibit the growth of bacterial biofilms responsible for endodontic infections.
Notwithstanding the extensive literature on diterpenes, there are not many studies on the activity of these compounds.Therefore, the present study aimed to investigate the antibacterial and antibiofilm activity of diterpenes of the pimarane type against bacterial strains that cause endodontic infections.This study also evaluates diterpene cytotoxicity to ensure that these compounds are safe for subsequent application.

MATERIALS AND METHODS
Obtaining ent-pimarane diterpenes from Viguiera arenaria  were isolated from V. arenaria.,15-dien-19-oic acid sodium salt (III) was obtained by semi synthesis from I. All the isolation, identification, and semi synthetic procedures are detailed in Carvalho et al. (2011).Figure 1 shows the chemical structures of compounds I-III.Carvalho et al. (2011) reported the in vitro antibacterial activity of diterpenes obtained from V. arenaria.Here, we decided to expand the in vitro assays to confirm the potential action of said diterpenes.
The clinical isolates were isolated from patients with primary endodontic infections and had been obtained during a previous research work conducted in the dental clinic of the University of Franca.The clinical isolates were isolated, identified, and kept at LaPeMA/UNIFRAN and at the State University of Campinas (UNICAMP), Brazil.

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determination
The microdilution method, as recommended by the Clinical and Laboratory Standards Institute (CLSI, 2007), was used to determine the MIC.The experiments were conducted in triplicate.The diterpenes were dissolved in dimethyl sulfoxide (DMSO) at 1.0 mg/mL, followed by dilution in Brucella broth (DIFCO, Kansas City, MO, USA) supplemented with hemin (5.0 mg/mL, Sigma, St. Louis, MO, USA) and menadione (1 mg/mL, Sigma) for anaerobic bacteria; and A. actinomycetemcomitans were diluted in Brain Heart Infusion broth (BHI; Difco Labs, Detroit, MI, USA).Then, 12 diterpene concentrations ranging from 0.25 to 80 μg/mL were tested.
The inocula were adjusted to give a cell concentration of 5 x 10 5 CFU/mL for A. actinomycetemcomitans (CLSI, 2009) and 1 x 10 6 CFU/mL for anaerobic bacteria (CLSI, 2007).DMSO 5% (v/v) was used as the negative control, and chlorhexidine dihydrochloride (CDH) was used as the positive control.An inoculum was included to monitor ground for bacterial growth.The 96-well microplate containing A. actinomycetemcomitans was incubated in microaerophilic conditions at 37°C for 24 h, and the plates containing anaerobic microorganisms were incubated at 36°C for 72 h in an anaerobic chamber containing 5-10% H2, 10% CO2, and 80-85% N2 (Don Whitley Scientific, Bradford, UK).After incubation, 30 μL of a 0.02% aqueous resazurin (Sigma) solution was added to each well.Resazurin is an oxireduction probe that allows immediate microbial growth observation.The blue and red colors represent microbial growth absence and presence, respectively (Sarker et al., 2007).
To determine the minimum bactericidal concentration (MBC), a 10-μL aliquot of the inoculum was removed from each well before resazurin (Sigma) was added, and the aliquot was seeded in blood agar supplemented with 5% defibrinated sheep blood for microaerophilic strains.For anaerobic bacteria, Schaedler agar (Difco) supplemented with hemin (5 mg/mL, Sigma), menadione (1 mg/mL, Sigma) and 5% defibrinated sheep blood (Bio Boa Vista, Valinhos, SP, Brazil) was used.The agar plates were incubated under gaseous conditions for an appropriate time.After incubation, MBC was defined as the lowest diterpene concentration that killed > 99.9% of the initial bacterial population, where no visible bacterial growth was observed after the subculture in Schadler agar plates.The MBC assays were performed in triplicate.

Biofilm formation inhibition as assessed by minimum inhibitory concentration of biofilm (MICB50)
The minimum inhibitory concentration of biofilm (MICB50) is defined as the minimum antibacterial agent concentration that is able to inhibit 50% or more of biofilm formation (Wei et al., 2006).MICB50 was determined as described in international guidelines (CLSI, 2007) with some modifications.To determine MICB50 of the three diterpenes, serial dilutions were accomplished in a polystyrene tissue 96-well plate (TPP, Trasadingen, Switzerland) containing Brucella (Difco) broth supplemented with hemin (5 mg/mL, Sigma, St. Louis, MO, USA) and menadione (1 mg/mL, Sigma) for the anaerobic bacteria (medium 1), and Brain Heart Infusion (BHI, medium 2) broth for A. actinomycetemcomitans.The final diterpene concentrations varied from 0.195 to 400 µg/mL.Chlorhexidine dichlorohydrate (CDH, Sigma) was used as positive control, at concentrations ranging from 0.115 to 59 µg/mL.Bacterial strains in the absence of antimicrobials were used as negative controls.
After incubation at 36ºC for 72 h in anaerobic chamber containing 5-10% H2, 10% CO2, and 80-85% N2 (atmosphere 1) for the anaerobic bacteria, and at 37°C for 24 h for A. actinomycetemcomitans in microaerophilia (atmosphere 2), the well contents were removed.Then, each well was washed three times with 200 µL of sterile Milli Q water and fixed with 200 µL of methanol for 15 min.MICB50 was determined in triplicate.
Following the procedures described by Sandberg et al. (2008), optical density (OD) quantification in the biofilm was conducted by adding 200 µL of crystal violet (0.1%) to the microplate wells.After 10 min at room temperature, excess dye was removed with tap water and dried in air at room temperature.Next, 200 µL of ethanol 95% was slowly added to each well, to re-solubilize the dye bound to the cells.The microtitration plate was covered with a lid and kept at room temperature for at least 30 min, to minimize evaporation.The OD of each well was measured at 595 nm with a microtitration plate reader, and the inhibition percentage was calculated by using the equation (Wei et al., 2006): (1 -At595 /Ac595) × 100 Where, At595 nm and Ac595 nm are the absorbances of the well treated with a diterpene and the control, respectively.
The best incubation time and inoculum concentration for the antibiofilm activity assay were selected by standardizing biofilm formation (data not presented).

Diterpenes of the pimarane type bactericidal kinetics ("time-kill curve")
The time-kill curves were constructed, in triplicate, to determine the time that was necessary for the diterpenes and CDH to inhibit bacterial growth completely (D'Arrigo et al., 2010), as previously described by Moraes et al. (2016).The tubes containing 1 mL of medium 1 for the anaerobic bacteria or 1 mL of medium 2 for the microaerophilic bacterium and one of the diterpenes at their minimum bactericidal concentration (MBC, from 1.0 to 60.0 µg/mL) were inoculated with the assayed microorganisms and incubated in atmosphere 1 or 2 for 72 or 24 h in the case of anaerobic and microaerophilic bacteria, respectively.After incubation, 100-µL aliquots were removed from the tubes at zero, 6, 24, 48, and 72 h for the anaerobic bacteria and at zero, 30 min, and 6, 12, and 24 h for A. actinomycetemcomitans.
Seven decimal serial dilutions were then prepared, and 50 µL of each concentration was spread on supplemented Schaedler agar and blood agar for the anaerobic and microaerophilic bacteria, respectively.After the incubation time, the viable colonies were counted, and time-kill curves were constructed by graphical representation of Log10 CFU/mL as a function of time, with the aid of the software Prism (versão 5.0; GraphPadV).CDH at concentrations ranging from 0.922 to 7.38 µg/mL was added as positive control.

Fraction inhibitory concentration index (FICI) determination for CDH and diterpenes of the pimarane type
The checkerboard assay was derived from the standard procedure established by the CLSI ( 2007) to investigate the antimicrobial efficacy of the in vitro association of the diterpenes with CDH that provided the most satisfactory results.The assays were performed according to the protocol previously described by Chaturvedi et al. (2008).The synergy tests were evaluated in triplicate, and the concentrations of diterpenes I, II, and III and CDH were combined in the MIC standard format.
To evaluate the synergism effect (FIC), index values were calculated as previously established in the literature, according to Chaturvedi et al. (2008).The combination was algebraically calculated to determine the fractional inhibitory concentration (FIC) index.FICA was calculated as the MIC of drug A in the combination/MIC of drug A alone; FICB was determined as the MIC of drug B in the combination/ MIC of drug B alone.The FIC summation (ƩFIC) was calculated as ƩFIC index= FICA + FICB (Chaturvedi et al., 2008).

Cytoxic activity of diterpenes of the pimarane type to human fibroblasts as assessed by the XTT assay
The cell line GM0749-A, corresponding to normal human lung fibroblasts, was employed in this assay.The GM0749-A cells were stored in liquid nitrogen at -195°C.The aliquots contained 1 x 10 6 cells /mL of a freezing solution consisting of 50% culture medium (HAM F10 + DMEM at 1:1 ratio, Sigma), 40% fetal bovine serum (Nutricell), and 10% DMSO.
Cell monolayers were cultured in 10 mL of culture medium by using disposable flasks with 25-cm 2 area (Corning) at 37 °C in a BOD incubator.Every 2-3 days, the cells were sub-cultured by using PBS (for washing) and ATV (Instituto Adolfo Lutz, at ATV/PBS 1:1 ratio) to release the cells adhered to the culture flask internal surface.After the cells were released, about 1.5 mL of complete culture medium was added to the flask to inactivate ATV, which was followed by homogenization.A small amount of the cells was then placed in culture flasks containing 5 mL of culture medium and incubated at 37°C.The cells were sub-cultivated, and approximately 10 4 cells were seeded in microplates containing 100 µL of culture medium (HAM F10 + DMEM at 1:1 ratio).The cells were treated with diterpene I or II at concentrations varying from 0.3125 to 640 µg/mL, or with diterpene III at concentrations ranging from 0.218 to 448 µg/mL.They were then dissolved in DMSO (0.25%) and incubated at 37ºC for 24 h.The negative control (culture medium only), solvent control (DMSO 0.25%), and positive control (DMSO 25%) were included.After treatment, the cells were washed with PBS, which was followed by addition of 100 µL of medium culture HAM F10 without phenol red and 25 µL of XTT (sodium 3′-[1-[(phenylamino)carbony]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate).After incubation at 37ºC for 17 h, the absorbances were read at 450 and 620 nm on a microplate reader.The cytotoxic activity was evaluated by using the inhibition parameter of 50% cell line growth (IC50).To compare the different treatments, statitstical analyses of the results were accomplished by analysis of variance (ANOVA) followed by the Tukey test.

Minimum Inhibitory Concentration (MIC) and minimum bactericidal concentration (MBC) determination
The MIC and MBC values obtained for diterpenes I, II, and III against the tested bacteria varied from 0.5 to 10, 2.5 to 60, and 1.0 to 14 μg/mL, respectively (Table 1).

Diterpenes of the pimarane type bactericidal kinetics ("time-kill curve")
Figure 2A to I illustrates the bactericidal kinetics for the evaluated bacteria considering colony forming units (CFU/mL) as a function of time.
We considered that the antibacterial action corresponded to a decrease of over 3 Log 10 in the number of microorganisms.Diterpenes III, III, and I provided a decrease of over 3 Log 10 in the case of P. gingivalis (clinical isolate; Figure 2B), P. nigrescens (Figure 2C), and A. naeslundii (Figure 2G), respectively, after 6 h of incubation.Diterpene II led to a decrease of over 3 Log 10 for A. naeslundii (Figure 2G) and P. micros (Figure 2H) after 24 and 48 h of incubation, respectively.
Diterpenes I and III gave a decrease of over 3 Log 10 in the case of P. nigrescens (Figure 2C) and P. micros (Figure 2H) after 24 and 48 h of incubation, respectively.

Fraction inhibitory concentration index (FICI) determination for CDH and diterpenes of the pimarane type
Regarding the combination of CDH with diterpenes I, II, or III against P. gingivalis (clinical isolate), the effects were synergic, synergic, and additive, respectively (Figure 3B).
As for the combination of CDH with diterpenes II and III against A. naeslundii (Figure 3G), the effects were additive in both cases.CDH combined with diterpene II or III against P. micros (Figure 3H) elicited additive and synergic effects, respectively.
Finally, the effect synergic was obtained for A. actinomycetemcomitans (Figure 3I).For the other bacteria, combination of CDH with diterpenes I, II or III promoted indifferent or antagonistic actions.

Cytoxic activity of diterpenes of the pimarane type to human fibroblasts as assessed by the XTT assay
We evaluated the antiproliferative effects of diterpenes I, II, and III, at different concentrations, by using the GM0749-A cell line and the XTT assay.None of the diterpenes were cytotoxic at the assayed concentrations (Figure 3A to I).
Only at higher concentrations (Table 3, IC 50 values of 321.60, 202.30, and 61.26 µg/mL for diterpenes I, II, and III, respectively) were the diterpenes cytotoxic, especially diterpene III, which reduced GM0749-A cell growth during the XTT assay.

DISCUSSION
The results reported by Carvalho et al. (2011)     According to the criteria of Rios and Recio (2005), compounds with such MIC values should be promising antibacterials.The plant kingdom is an important source of new and effective antimicrobial agents because plants have the ability to produce natural products for chemical defense against microorganisms present in their own environment (Gibbons, 2004;Hemaiswarya et al., 2008;Gibbons, 2008).Ambrosio et al. (2006) stated that medicinal plant derivatives like diterpenes of the pimarane type could serve as lead compounds, drugs, or linking structures during the development of synthetic molecules.Furthermore, according to Bakri and Douglas (2005), a new drug can only be considered a therapeutic agent for oral infections if it is active against biofilms.
As part of this scenario, the present paper points out the significant antimicrobial activity displayed by diterpenes isolated from V. arenaria against bacteria that cause endodontic infection.Moreti et al. (2017) evaluated ent-kaurenoic acid against endodontic pathogens, to find MIC and MBC values ranging from 3.12 to 400 g/mL.Souza et al. (2011) investigated copalic acid, a labdane-type diterpene isolated from the Copaifera langsdorffii oleoresin, against anaerobic bacteria, to obtain MIC values spanning from 3.1 to 200 µg/mL.In our study, diterpenes I, II, and III afforded MIC values between 0.5 and 10 µg/mL, which agreed with the claim by Rios and Recio (2005) that compounds that can inhibit microorganisms at concentrations close to 10 μg/mL are promising antimicrobials.
Literature reports have described that biofilms can be ten times more resistant to antimicrobials than planktonic cells (Gursoy et al., 2009).According to Signoretto et al. (2011), bacteria in the planktonic and biofilm modes have remarkably different behavior, a fact that must be born in mind during laboratory tests.Moreover, depending on the growth phase, bacterial biofilms respond distinctly to antimicrobial concentration and exposure time (Gursoy et al., 2009;La et al., 2010;Nwaokorie et al., 2010).
The larger bacterial resistance to the diterpenes evaluated herein (MICB 50 values were higher than MIC values) may have been due to the presence of the extracellular polysaccharide (EPS) matrix, which serves the strains, as in the case of P. gingivalis (ATCC 33277 and clinical isolate).Indeed, combination of CDH with one of the diterpenes was indifferent against the P. gingivalis standard strain (ATCC 33277) but synergistic (diterpenes I or III) or additive (diterpene II) against theclinical isolate.Irrespective of the associations tested herein, antagonism was more frequent than synergism for all the diterpenes, indicating that their combination with CDH could in fact limit their action and culminate in undesirable effects.
The results provided by the cytotoxic activity investigation might be as clinically relevant as previous findings for other dose-and time-dependent compounds like CDH (Lessa et al., 2010) and sodium hypochlorite (Pashely et al., 1985;Sabala and Powell, 1989).The in vitro cytotoxicity of diterpenes I, II, and III against human fibroblasts might not be the same as the cytotoxicity of these compounds in dental materials or in other experimental conditions, which would require additional in vitro and in vivo assays.The cytotoxicity of plants, extracts, fractions, and isolated compounds has often been reported to depend on concentration and contact time (Gursoy et al., 2009;Vargas et al., 2010).This toxicity is many times moderate (More et al., 2008;Signoretto et al., 2011) or even uncapable of causing deleterious effects (Seneviratne et al., 2011), but further drug safety assessment is essential before diterpenes of the pimarane type are introduced into the market for use in humans.

Conclusion
Our MIC and MBC results confirmed the data reported by Carvalho et al. (2011).On the basis of the antibiofilm action of diterpenes I, II, and III, these compounds can inhibit at least 50% of the tested bacteria.
The bactericidal kinetic assay evidenced that the behavior of diterpenes I, II, and III against the assayed bacteria varied as a function of diterpene concentration and microorganism sensitivity.Antagonism was the most frequent interaction between CDH and diterpenes of the pimarane type, followed by indifferent, synergistic, and additive interactions.
The cytotoxicity assays demonstrated that diterpenes I, II, and III at different concentrations were not cytotoxic.Diterpene III displayed the most pronounced antiproliferative effect, but this effect emerged at higher concentrations than the ones used during evaluation of the antimicrobial activity of this same diterpene.In conclusion, diterpenes of the pimarane type isolated from plants native to the Brazilian savannah are promising biomolecules and may provide innovative therapeutic solutions in the field of endodontics.

Figure 3 .
Figure 3. Results for the combination of diterpenes I, II, or III with CDH by the fraction inhibitory concentration index (FICI) method.

Table 2 .
Minimum inhibitory concentration of biofilm (MICB50) of the diterpenes against the assayed bacteria.

Table 3 .
IC50 values obtained for the GM0749-A cell line after 24 h of incubation with different concentrations of diterpenes I, II, and III.