Antibacterial Effect of Copaifera duckei Dwyer Oleoresin and Its Main Diterpenes against Oral Pathogens and Their Cytotoxic Effect

This study evaluates the antibacterial activity of the Copaifera duckei Dwyer oleoresin and two isolated compounds [eperu-8(20)-15,18-dioic acid and polyalthic acid] against bacteria involved in primary endodontic infections and dental caries and assesses the cytotoxic effect of these substances against a normal cell line. MIC and MBC assays pointed out the most promising metabolites for further studies on bactericidal kinetics, antibiofilm activity, and synergistic antibacterial action. The oleoresin and polyalthic acid but not eperu-8(20)-15,18-dioic provided encouraging MIC and MBC results at concentrations lower than 100 μg mL−1. The oleoresin and polyalthic acid activities depended on the evaluated strain. A bactericidal effect on Lactobacillus casei (ATCC 11578 and clinical isolate) emerged before 8 h of incubation. For all the tested bacteria, the oleoresin and polyalthic acid inhibited biofilm formation by at least 50%. The oleoresin and polyalthic acid gave the best activity against Actinomyces naeslundii (ATCC 19039) and L. casei (ATCC 11578), respectively. The synergistic assays combining the oleoresin or polyalthic acid with chlorhexidine did not afford interesting results. We examined the cytotoxicity of C. duckei oleoresin, eperu-8(20)-15,18-dioic acid, and polyalthic acid against Chinese hamster lung fibroblasts. The oleoresin and polyalthic acid were cytotoxic at concentrations above 78.1 μg mL−1, whereas eperu-8(20)-15,18-dioic displayed cytotoxicity at concentrations above 312.5 μg mL−1. In conclusion, the oleoresin and polyalthic acid are potential sources of antibacterial agents against bacteria involved in primary endodontic infections and dental caries in both the sessile and the planktonic modes at concentrations that do not cause cytotoxicity.


INTRODUCTION
The oral bacterial microbiome encompasses ∼700 commonly occurring phylotypes, about half of which can be present at any time in any individual. Oral bacteria are inseparably intertwined with diseases, such as gingivitis, periodontal diseases, endodontic infections, and dental caries, which will impact every human at some point in their lives (Palmer, 2013).
Dental caries is one of the most common biofilm-dependent oral diseases among humans (Bowen, 2002). Colonization of the tooth surface by cariogenic microorganisms, like Streptococcus mutans, Streptococcus sobrinus, and Lactobacillus spp., can destroy the tooth structure (Gross et al., 2012). S. mutans has been implicated as the primary etiological agent of dental caries and plays a decisive role in dental plaque formation, known as biofilm, and in dental caries development (Hamada et al., 1984;Kuramitsu and Trapa, 1984;Loesche, 1986;Rozen et al., 2001;Banas, 2004). The key to preventing such diseases is to control these cariogenic bacteria effectively. However, eliminating bacteria is a difficult task because biofilm may emerge, which enhances bacterial resistance to antimicrobial agents (Watnick and Kolter, 2000;Ding et al., 2014). Endodontic infections have a polymicrobial nature, with obligate anaerobic bacteria conspicuously dominating the microbiota in primary infections (Narayanan and Vaishnavi, 2010). Microorganisms and their products play an essential part in the development of pulp and periapical diseases and account for endodontic treatment failure (Guerreiro-Tanomaru et al., 2015). Chemomechanical preparation of the infected root canal with antimicrobial agents, followed by obturation and coronal restoration, provides a favorable outcome (Narayanan and Vaishnavi, 2010). Nevertheless, root canal treatment sometimes fails due to persistent or secondary intraradicular infection (Siqueira, 2001;Nair, 2006;Narayanan and Vaishnavi, 2010). Although chlorhexidine is usually employed as an active ingredient in mouthwash to inhibit or diminish oral bacteria, adverse reactions including bitter taste and tooth staining have limited its clinical application. Therefore, the search for alternative antibacterial agents without or with few side effects is urgent (Peng et al., 2013).
Brazil is a continental country that is recognized for housing one of the greatest plant diversities in the world. In each Brazilian region, the population uses plants according to their cultural traditions and to the types of vegetation growing therein (Brandão et al., 2013). Plants continue to be an important source of new bioactive substances, and the economic interest in prospecting them for drug discovery remains high. At least 25% of all modern medicines are estimated to derive from medicinal plants either directly or indirectly (Newman and Cragg, 2012). The oleoresin obtained by tapping the trunk of trees belonging to the Copaifera genus is widely used in Brazilian folk medicine under the name "oleo de copaiba" (copaiba balsam), which acts mainly as a healing, antiseptic, and antiinflammatory agent (Cascon and Gilbert, 2000;Veiga and Pinto, 2002). The Copaifera duckei Dwyer oleoresin exhibits biological activities such as antiproliferative, antimutagenic, embryotoxic, anti-inflammatory, and analgesic actions (Castro-e-Silva et al., 2004;Carvalho et al., 2005;Maistro et al., 2005;Lima et Leandro et al., 2012). Recently, Borges et al. (2016) evaluated the in vitro schistosomicidal effects of the C. duckei oleoresin and its major secondary metabolite, (-)-polyalthic acid, to demonstrate that these substances are active against Schistosoma mansoni and may be employed for further investigations into compounds that can combat this parasite. Santos et al. (2013) assessed the antibacterial activity of the C. duckei oleoresin against bacteria of clinical and food interest, to verify that the oleoresin showed good activity against Gram-positive bacteria and acted on the bacterial cell wall of Bacillus cereus, affecting the cell-division process. The authors suggested that the oleoresin has a potential antibacterial effect.
This study examines the antibacterial activity of the C. duckei Dwyer oleoresin and its secondary metabolites against bacteria involved in primary endodontic infections and dental caries in both the planktonic mode and the sessile mode.

Bacterial Strains and Antimicrobial Assays
The Minimum Inhibitory Concentration (MIC; the lowest concentration of the test compound that is capable of inhibiting microorganism growth) and the Minimum Bactericidal Concentration (MBC; defined as the lowest concentration of the test compound at which no bacterial growth occurs) of the oleoresin and the pure metabolites were determined in triplicate; the microdilution broth method in 96-well microplates was employed. The following culture media were used for the cariogenic strains: Tryptic Soy Broth-TSB (Difco, Kansas City, MO, USA) and Tryptic Soy Agar-TSA (Difco) mixed with sheep blood (5%) (Nassar et al., 2012;Krzyściak et al., 2017). The culture media employed for the representative strains of endodontic infections were Schadler broth or Schadler agar (Difco), both supplemented with hemin (5.0 µg mL −1 , Sigma, St. Louis, MO, USA), vitamin K1 (10 µg mL −1 , Sigma), and sheep blood (5%, Bio Boa Vista, Valinhos, SP, Brazil), as recommended by CLSI (2007). Samples were dissolved in dimethyl sulfoxide (DMSO) 1.0 mg mL −1 and diluted in the desired broth. The concentrations ranged from 0.195 to 400 µg mL −1 . The final DMSO content was 5% (v/v), and this solution was used as negative control. Chlorhexidine dihydrochloride (CDH) and metronidazole (Sigma) were used as positive controls for aerobic/anaerobic facultative and anaerobic bacteria, respectively. The inoculum was adjusted for each organism, to yield a cell concentration of 5 × 10 5 colony forming units (CFU) mL −1 for the aerobic and anaerobic facultative strains and 5 × 10 6 CFU mL −1 for the anaerobic strains according to a previous standardization by the Clinical Laboratory Standards Institute (CLSI, 2007(CLSI, , 2009). The anaerobic strains were incubated in an anaerobic chamber (Don Whitley Scientific, Bradford, UK) for 72 h, under atmosphere containing 5-10% H 2 , 10% CO 2 , and 80-85% N 2 . The anaerobic facultative strains were incubated in a microaerophilic jar system for 24 h, except for the E. faecalis (ATCC and clinical isolate) and S. salivarius (ATCC and clinical isolate) strains, which were incubated aerobically at 37 • C for 24 h.
After incubation, 30 µL of an aqueous resazurin (Sigma) solution (0.02%) was added to the microplates to observe bacterial growth. Development of a blue and pink color indicated absence and presence of bacterial growth, respectively. To determine the MBC, an aliquot of the inoculum was removed from each well prior to addition of resazurin (Sigma) and seeded in an appropriate culture medium.

Time-Kill Curves
Time-kill assays against the anaerobic strains P. gingivalis (ATCC 33277) and P. micros (clinical isolate) and the microaerophilic strains S. mutans (ATCC 25275), S. sobrinus (ATCC 33478), and L. casei (ATCC 11578 and clinical isolate) were performed in triplicate, as described by D'arrigo et al. (2010). All the results are expressed as the mean ± S.E.M. Tubes containing the most promising metabolites at final concentrations equal to the MBC values for the respective strains were inoculated with the target microorganism at an initial bacterial density of 5 × 10 5 CFU mL −1 for the anaerobic facultative strains and 5 × 10 6 CFU mL −1 for the anaerobic strains, followed by anaerobic or microaerophilic incubation conditions. To count viable colonies, aliquots were removed at 0 min and 30 min and at 6, 12, 18, and 24 h for microaerophilic bacteria, and at 0 min and 30 min and at 6, 12, 18, 24, 48, and 72 h for anaerobic bacteria. The diluted samples (50 µL) were spread onto appropriate agar, incubated at 37 • C under appropriate atmosphere, and counted after the growth period. Time-kill curves were constructed by plotting log 10 CFU mL −1 vs. time on the Graphpad Prism (version 5.0) software. Promising metabolites at their MBC and a suspension of bacteria without the added metabolites were used as the positive and the negative control, respectively.

Antibiofilm Activity Evaluation
The Minimum Inhibitory Concentration of Biofilm (MICB 50 ) of the most promising metabolites against the bacteria evaluated in this study was determined on the basis of the minimum concentration of antimicrobial agent that was able to inhibit biofilm formation by at least 50% (Wei et al., 2006). For this purpose, a microdilution plate assay was used according to the CLSI guidelines (CLSI, 2007(CLSI, , 2009, with some modifications. This method was similar to the MIC assay conducted for planktonic cells except that the inoculum was adjusted at a higher concentration so that it could adhere to the microplate to form the biofilm. Two-fold serial dilutions of each sample were prepared in the wells of a 96-well polystyrene tissue culture plate (TPP, Trasadingen, Switzerland) containing appropriate medium at a volume of 200 µL per well. The final concentrations of the most promising metabolites ranged from 0.195 to 400 µg mL −1 . Chlorhexidine dichlorohydrate (Sigma) at a concentration between 0.115 and 59 µg mL −1 was assessed as negative control; the bacterial strains in the absence of the antibacterial agent were used as positive controls, and the inoculum was adjusted to give a cell concentration of 1 × 10 6 CFU mL −1 for all the bacteria. P. gingivalis (ATCC 33277) and P. micros (clinical isolate) were incubated in an anaerobic chamber, and the microaerophilic strains S. mutans (ATCC 25275), S. sobrinus (ATCC 33478), and L. casei (ATCC 11578 and clinical isolate) were incubated in a microaerophilic jar system. Biofilm formation was quantified, and the number of microorganisms was counted by using the methodology described by da Silva et al. (2014), with some modifications.

Synergistic Antimicrobial Activity
Checkerboard assays were performed according to the protocol previously described by White et al. (1996) to investigate the in vitro antimicrobial efficacy of the combination of the oleoresin or (-)-polyalthic acid with chlorhexidine (Sigma). The synergy tests were carried out in triplicate, and concentrations of each compound were combined by using a standard MIC format against 5 × 10 5 CFU mL −1 of the microaerophilic strain and 5 × 10 6 CFU mL −1 of the anaerobic strain. To evaluate the synergistic effect of the most promising metabolites and chlorhexidine, the fractional inhibitory concentration (FIC) index values were calculated on the basis of the equation previously established in the literature (White et al., 1996). Synergy was defined as FIC ≤ 0.5, and additivity was defined as FIC > 0.5 but <1. Indifference was defined as FIC ≥ 1 but <4, whereas antagonism was defined as FIC ≥ 4 (Lewis et al., 2002).
Cytotoxicity was measured by using the in vitro Toxicology Colorimetric Assay Kit (XTT; Roche Diagnostics, Indianapolis, Indiana, EUA) according to the manufacturer's instructions. For these experiments, 1 × 10 4 cells were plated onto 96-well microplates. Each well received 100 µL of HAM-F10/DMEM or DMEM containing the C. duckei oleoresin, polyalthic acid, or eperu-8(20)-15,18-dioic acid at concentrations ranging from 2.43 to 5,000 µg mL −1 . The negative (without treatment), solvent (Tween 80 0.25%), and positive (doxorubicin, DXR, Zodiac, Pindamonhangaba, SP, Brazil) controls were included. After incubation at 37 • C for 24 h, the medium was removed; the cells were washed twice with 100 µL of phosphate buffered saline (PBS) and exposed to 100 µL of HAM-F10 medium without phenol red. Then, 50 µL of XTT was added to each well. The microplates were covered and incubated at 37 • C for 17 h. The absorbance of the samples was determined by using a multiplate reader (ELISA, Tecan-SW Magellan vs. 5.03 STD 2PC) at a test wavelength of 492 nm and a reference wavelength of 690 nm (Roehn et al., 1991). The experiments were conducted in triplicate, and the antiproliferative activity was assessed by using the parameter of 50% inhibition of cell growth (IC 50 ) with the aid of GraphPad Prism 5.0. Figure 1 illustrates the chemical structures of the secondary compounds obtained from C. duckei and evaluated herein. According to Rios and Recio (2005) and Gibbons (2008), a promising plant extract must have MIC lower than 100 µg mL −1 , whilst pure compounds must display MIC values lower than 10 µg mL −1 . Polyalthic acid gave MIC values ranging between 12.5 and 100 µg mL −1 for the cariogenic strains. Table 1 summarizes the MIC and MBC values for the assessed bacteria involved in endodontic infections and dental caries. The oleoresin Shigella sonnei, which were both clinical isolates. The oleoresin was active against nine of the 11 tested bacterial strains. B. cereus was the most sensitive: the oleoresin MIC was 31.25 µg mL −1 , which denoted bactericidal action. The authors verified that the C. duckei oleoresin is a potential antibacterial agent and suggested that this oil can be used as a therapeutic alternative, mainly against B. cereus (ATCC 25922). Here, the oleoresin gave MIC values of 25 µg mL −1 against most cariogenic strains, and it was the most promising against P. gingivalis (ATCC 33277), P. micros (clinical isolate), and A. naeslundii (ATCC 19039), with MIC values of 6.25, 25, and 12.5 µg mL −1 , respectively. These results attested to the antibacterial potential of the C. duckei oleoresin. Moraes et al. (2016) studied the antibacterial activity of the C. oblongifolia oleoresin against bacteria involved in caries and endodontic infections, to achieve promising MIC and MBC values spanning from 25 to 200 µg mL −1 as well as encouraging MIC values against S. sanguinis (ATCC 10556 and clinical isolate), S. mutans (ATCC 25175), S. mitis (ATCC 49456), L. casei (ATCC 11578 and clinical isolate strains), P. gingivalis (ATCC 33277), P. micros (clinical isolate), and A. actinomycetemcomitans (ATCC 43717). In our study, the C. duckei oleoresin displayed good results against the same bacteria evaluated by Moraes et al. (2016), with MIC values ranging from 6.25 to 50 µg mL −1 , which constituted a bactericidal effect. The exception was A. actinomycetemcomitans, against which the oleoresin was bacteriostatic. Bardají et al. (2016) assessed the Copaifera reticulata oleoresin against the causative agents of tooth decay and periodontitis, to obtain the best result against P. gingivalis (ATCC 33277), with MIC value of 6.25 µg mL −1 . In the present work, the C. duckei oleoresin provided the same result against P. gingivalis (ATCC 33277), which corresponded to bactericidal action. Polyalthic acid also afforded good results for both groups of bacteria tested  herein, with MIC values lower than 10 µg mL −1 for the anaerobic strains P. gingivalis (ATCC 33277) and P. micros (clinical isolate). There are no reports on the use of pure compounds of the C. duckei oleoresin against bacteria. However, our research group has already obtained pure substances from C. langsdorffii and found good results for copalic acid against cariogenic bacteria (Souza et al., 2011a) and periodontal anaerobic bacteria (Souza et al., 2011b).

RESULTS AND DISCUSSION
Based on our promising MIC results, we examined the bacterial death kinetics (time-kill assays), the in vitro antibiofilm activity (MICB 50 ), and the synergistic effect of the C. duckei oleoresin and polyalthic acid associated with chlorhexidine.
We accomplished the time-kill curve assay (Figure 2) against two anaerobic strains and four microaerophilic strains, which best represented endodontic and cariogenic infections and provided the greatest results in the MIC and MBC assays. In this assay, the bactericidal effect of the oleoresin and polyalthic acid varied. We highlight the results obtained against L. casei (ATCC 11578 and clinical isolate), which had inferior bactericidal effect after incubation for 8 h. According to Petersen et al. (2004), bactericidal activity corresponds to a reduction of >3 log10 CFU mL −1 in the original inoculum, whereas bacteriostatic activity refers to maintenance of the original inoculum concentration or reduction of <3 log 10 CFU mL −1 in the original inoculum. Santos et al. (2013) reported the time-kill assay of the C. duckei oleoresin at 15.62, 31.25, 62.5, and 125 µg mL −1 against B. cereus (ATCC 25922). The oleoresin exerted bactericidal effect on B. cereus in <4 h, at concentrations ranging from 31.25 to 125 µg mL −1 (1-4 times the MIC value). We also achieved similar results with the oleoresin against L. casei (ATCC 11578 and clinical isolate) and with polyalthic acid against L. casei (clinical isolate), which afforded bactericidal action after incubation for 4 h. Souza et al. (2011a) tested C. langsdorffii copalic acid against S. mutans (ATCC 25275), to find that copalic acid only inhibited inoculum growth during the first 12 h. The authors concluded that copalic acid displayed a bacteriostatic Frontiers in Microbiology | www.frontiersin.org effect during this time, but its bactericidal action was clearly noted thereafter (between 12 and 24 h). Souza et al. (2011b) also investigated C. langsdorffii copalic acid against P. gingivalis (ATCC 33277) in a time-kill curve assay in which this compound was tested at 3.1, 6.2, and 12.4 µg mL −1 (one, two, and three times its MBC, respectively); chlorhexidine at its MBC value (0.9 µg mL −1 ) was the positive control. Copalic acid 3.1 µg mL −1 completely killed P. gingivalis after incubation for only 24 h. However, the data suggested that copalic acid only inhibited inoculum growth during the first 12 h. Therefore, copalic acid displayed a bacteriostatic effect during this time, but its bactericidal action was clearly noted thereafter (between 12 and 24 h). In our study, P. gingivalis (ATCC 33277) behaved similarly. It was killed within 24 and 48 h of exposure to the C. duckei oleoresin and to polyalthic acid, respectively. Leandro et al. (2016) conducted a time-kill assay of the hydroalcoholic extract from C. trapezifolia leaves at 100 µg mL −1 against P. gingivalis (ATCC 33277) and P. micros (clinical isolate) and detected bactericidal activity within 72 h. Moraes et al. (2016) accomplished a time-kill assay for the C. oblongifolia oleoresin at 100 mg mL −1 , to find that this oleoresin exerted a bactericidal effect against L. casei (ATCC) FIGURE 3 | Antibiofilm activity of the Copaifera duckei Dwyer oleoresin and (-)-polyalthic acid as demonstrated by optical density (A 570 ) and number of microorganisms (Log 10 CFU mL −1 ) against cariogenic bacteria. The experiments were performed in triplicate and statistical significance was examined by Student's t-test. Results are indicated as means ± SDs. *Significantly different from the negative control group (P < 0.05). Filled bars correspond to MICB 50 concentration. and A. actinomycetemcomitans within 24 h. In addition, these authors tested the same C. oblongifolia oleoresin at 25 mg mL −1 against P. micros (clinical isolate), to verify that the number of microorganisms decreased by over 3 log 10 CFU mL −1 after 48 h, and that bactericidal activity emerged at 72 h of incubation. In the present study, both the C. duckei oleoresin and polyalthic acid reduced the number of microorganisms by at least 3 log 10 CFU mL −1 at 48 h of incubation for all the evaluated anaerobic strains.
According to Stewart and Costerton (2001), biofilms are more resistant to antimicrobial agents as compared to planktonic cells. During MICB 50 evaluation, the oleoresin and polyalthic acid displayed promising results against all the tested bacteria. We highlight the results found for L. casei (ATCC 11578) exposed to polyalthic acid, which displayed MICB 50 of 3.12 µg mL −1 (Figures 3, 4). Fux et al. (2003) affirmed that the concentration of a drug required to eliminate sessile bacteria can vary from 10-to 1,000-fold when it comes to eliminating planktonic bacteria.  Most of the evaluated strains showed MICB 50 values lower than the MIC values. However, cell counting demonstrated that at all the concentrations that represented MICB 50 , there still were living cells. According to Wei et al. (2006), avoiding biofilm formation is more important than destroying the fully developed biofilm. Spectrophotometric readings (O.D.) and microorganism count (log 10 CFU mL −1 ) can show the ability of antimicrobial agents to inhibit biofilm formation (antibiofilm activity). The existing methods have limitations such as long processing time, incompatibility with screening techniques, expensive reagents, and measurement of mass instead of cell viability. Despite these limitations, the combination of both techniques provides reliable results concerning biofilm activity (Kharazmi et al., 1999;Polonio et al., 2001;Walters et al., 2003;da Silva et al., 2014).
According to our study, the C. duckei oleoresin at concentrations of 200 and 6.25 µg mL −1 inhibited at least 50% of biofilm formation in the case of P. gingivalis (ATCC 33277) and P. micros (clinical isolate), respectively (Figure 4). The pure compound (-)-polyalthic acid at a concentration of 6.25 µg mL −1 inhibited at least 50% of biofilm formation of P. gingivalis (ATCC 33277) and P. micros (clinical isolate). Moraes et al. (2016) investigated the ability of the C. oblongifolia oleoresin to inhibit biofilm formation. They found MICB 50 of 400 µg mL −1 for L. casei and P. micros, 200 µg mL −1 for S. mutans and A. actinomycetemcomitans, and 100 µg mL −1 for S. mitis and P. gingivalis. Bardají et al. (2016) evaluated the MICB 50 of the C. reticulata oleoresin. At 50, 100, and 200 µg mL −1 , this oleoresin inhibited biofilm formation by at least 50% in the case of L. casei, and S. salivarius, and S. mitis, respectively. Compared to the results of Bardají et al. (2016), in this work inhibition of biofilm formation by cariogenic strains provided by the C. duckei oleoresin and (-)-polyalthic acid was more promising: from 3.12 to 12.5 µg mL −1 and from 12.5 to 50.0 µg mL −1 , respectively (Figure 3).
We also evaluated the synergistic effect of chlorhexidine and the C. duckei oleoresin or polyalthic acid against some of the assayed bacteria ( Table 2). The checkerboard methodology described by Lewis et al. (2002) did not reveal any synergistic effects for the tested combinations. The FICI results only evidenced additive and indifferent interactions. Bardají et al. (2016) studied the combination of chlorhexidine with the C. reticulata oleoresin, to find an additive effect for S. mutans (ATCC 25175) and S. mitis (ATCC 49456). Moraes et al. (2016) also detected an additive effect for the combination of chlorhexidine with the C. oblongifolia oleoresin against S. mitis (ATCC 49456) and A. actinomycetemcomitans (ATCC 43717). In turn, Leandro et al. (2016) did not verify any synergistic effect for the combination of the hydroalcoholic extract from C. trapezifolia leaves with chlorhexidine. These results corroborate with our present findings.
Finally, we investigated the cytotoxic potential of the C. duckei oleoresin, polyalthic acid, and eperu-8(20)-15,18-dioic acid ( Figure 5). The oleoresin and polyalthic acid afforded IC 50 values of 777.4 ± 8.3 and 127.3 ± 10.97 µg mL −1 , respectively. Eperu-8(20)-15,18-dioic acid showed IC 50 values of 1441.33 ± 13.43 µg mL −1 . In conclusion, eperu-8(20)-15,18-dioic acid was not cytotoxic to the V79 cell line, and it did not display antibacterial activity at MIC and MBC. The oleoresin and polyalthic acid did not present cytotoxicity at the MIC and MBC concentrations. These results suggested that these natural products could be safely applied to treat oral diseases. Leandro et al. (2016) also evaluated the cytotoxicity of the hydroalcoholic extract from C. trapezifolia leaves against the V79 cell line and found cytotoxicity at concentrations above 156 µg mL −1 . As reported by Moraes et al. (2016), the C. oblongifolia oleoresin was cytotoxic activity against the V79 cell line at concentrations ≥625 µg mL −1 . Bardají et al. (2016) treated GM07492-A cells with the C. reticulata oleoresin, to demonstrate that concentrations up to 39 µg mL −1 significantly reduced cell viability as compared to the negative control; IC 50 was equal to 51.85 ± 5.4 µg mL −1 .