Oleoresins and naturally occurring compounds of Copaifera genus as antibacterial and antivirulence agents against periodontal pathogens

Invasion of periodontal tissues by Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans can be associated with aggressive forms of periodontitis. Oleoresins from different copaifera species and their compounds display various pharmacological properties. The present study evaluates the antibacterial and antivirulence activity of oleoresins obtained from different copaifera species and of ten isolated compounds against two causative agents of periodontitis. The following assays were performed: determination of the minimum inhibitory concentration (MIC), determination of the minimum bactericidal concentration (MBC), and determination of the antibiofilm activity by inhibition of biofilm formation and biofilm eradication tests. The antivirulence activity was assessed by hemagglutination, P. gingivalis Arg-X and Lis-X cysteine protease inhibition assay, and A. actinomycetemcomitans leukotoxin inhibition assay. The MIC and MBC of the oleoresins and isolated compounds 1, 2, and 3 ranged from 1.59 to 50 μg/mL against P. gingivalis (ATCC 33277) and clinical isolates and from 6.25 to 400 μg/mL against A. actinomycetemcomitans (ATCC 43717) and clinical isolates. About the antibiofilm activity, the oleoresins and isolated compounds 1, 2, and 3 inhibited biofilm formation by at least 50% and eradicated pre-formed P. gingivalis and A. actinomycetemcomitans biofilms in the monospecies and multispecies modes. A promising activity concerning cysteine protease and leucotoxin inhibition was also evident. In addition, molecular docking analysis was performed. The investigated oleoresins and their compounds may play an important role in the search for novel sources of agents that can act against periodontal pathogens.

The inclusion criteria were as follows: patients aged over 18 years, of both genders, who had indication for periodontal treatment with over 5-mm deep gingival pockets in at least one of the four facets of the analyzed tooth, and who were examined a dental professional. The patient needed to have at least four teeth.
The exclusion criteria were patients with a history of systemic disease such as rheumatic fever, coronary heart disease, and respiratory diseases; patients that had received systemic or local antibiotic therapy in the six previous months; patients that had used antibiotics or anticoagulants; and patients that had undergone periodontal treatment or dental prophylaxis in the previous six months.
The clinical isolates were collected from patients after approval of the Research Ethics Committee of the University of Franca-CAAE 41530915. 9.0000.5495, in accordance with international protection guidelines and the Helsinki Declaration. All the patients signed an informed consent for their participation in this research.
The P. gingivalis and A. actinomycetemcomitans species were isolated according to the methodology of Esfahani et al. 35 . The species were obtained from patients with chronic and aggressive periodontitis. Ten strains were selected from the total number of isolated and identified bacteria, namely five P. gingivalis strains and five A. actinomycetemcomitans strains. The Polymerase Chain Reaction (PCR) technique was used to detect the P. gingivalis 16SrDNA and A. actinomycetemcomitans 16SrDNA genes. DNA was extracted from the strains by employing the Pure Link Microbiome DNA purification kit (Invitrogen, Carlsbad, California, USA); the manufacturer's instructions were followed. The PCR technique was conducted as described by Wu et al. 36 .
The Minimum Inhibitory Concentration (MIC; the lowest concentration of the test compound that can inhibit microorganism growth) and the Minimum Bactericidal Concentration (MBC; the lowest concentration of the test compound at which no bacterial growth occurs) were determined as described by Abrão et al. 29 .
The antibiofilm activity was investigated by using two distinct methodologies, in two modes, monospecies and multispecies biofilm. In the first methodology, the Minimum Inhibitory Concentration of Biofilm (MICB 50 ) of the most promising metabolites against the bacteria evaluated in this study was determined according to the methodology described by Abrão et al. 29 , on the basis of the minimum concentration of antimicrobial agent that inhibited biofilm formation by at least 50%. In the second methodology, the Minimum Concentration of Biofilm Eradication (MCBE), defined as the lowest concentration that reduced the number of biofilm cells by at least 99.9%, was determined as described by Souza et al. 31 .
Antivirulence assays. The oleoresins and isolated compounds were used to inhibit virulence factors. The antivirulence assays included assessment of the P. gingivalis and A. actinomycetemcomitans hemagglutination activity, P. gingivalis gingipaine inhibition, and A. actinomycetemcomitans leukotoxin inhibition.
Hemagglutination assay. To perform the hemagglutination assay, which allowed fimbriae to be detected, the methodology of Kikuchi et al. 37 was applied. P. gingivalis and A. actinomycetemcomitans colonies that had been grown for five days and 24 h, respectively, were suspended in PBS buffer (Sigma) pH 7.4 and adjusted to an absorbance of 2.0 at 660 nm. The oleoresins and isolated compounds were tested at subinhibitory concentrations (½ MIC). To this end, 150 μL of an oleoresin or isolated compound was mixed with 150 μL of the bacterial suspension, which was followed by incubation at appropriate temperature and atmosphere for each strain (anaerobic chamber at 36 °C for P. gingivalis strains and CO 2 chamber at 36 °C for A. actinomycetemcomitans strains) in sterile Eppendorf tubes. After incubation, the bacteria were centrifuged at 6000× g for 5 min, resuspended with 500 μL of PBS buffer (Sigma) pH 7.4, and diluted to 1/32 in 96-well plates. A 50-μL aliquot of each dilution was homogenized with 50 μL of a 2% fresh red blood cell suspension in PBS (Sigma) pH 7.4 and incubated in appropriate atmosphere for 3 h. Hemagglutination was visually assessed after incubation.
The P. gingivalis strains were grown in Brucella broth in anaerobic chamber at 36 °C for 72 h. Then, the bacteria were centrifuged at 6000× g for 5 min and resuspended with PBS (Sigma) pH 7.4 buffer containing 1 mM L-cysteine, to adjust the absorbance at 660 nm to 0.26, and the resulting suspensions were added to the 96-well microplate. The oleoresins and isolated compounds were tested against the P. gingivalis strains at subinhibitory concentrations (½ MIC).
The Arg-X activity was assayed in 100 μL of PBS containing 1 mM L-cysteine, 100 μL of substrate, and 5 μL of bacterial suspension. The Lis-X activity was tested in 100 μL of PBS containing 1 mM L-cysteine, 100 μL of substrate, and 50 μL of bacterial suspension. The 7-starch-4-methylcoumarin that was released due to cleavage of the substrates was measured on a fluorimeter (Ascent FL, Thermoscientific, Waltham, MA, USA) upon excitation at 365 nm and emission at 460 nm. The proteolytic activity was compared to the untreated control 40 . Leukotoxin inhibition assay. This test was carried out by following the methodology described by de Lima et al. 41  www.nature.com/scientificreports/ blood leukocytes by using Histopaque 1077 (Sigma-Aldrich); the manufacturer's instructions were followed. The LMNS-containing fraction was collected, and the cells were washed three times with PBS (1000 rpm for 5 min). The cell pellet was resuspended to a concentration of 5 × 10 6 cells/mL in RPMI culture medium containing L-glutamine, 10% fetal bovine serum, and penicillin-streptomycin (Sigma-Aldrich). The leukocyte fraction was mixed with an oleoresin or isolated compound at a ratio of 10 6 cells to 100 μL of oleoresin or isolated compound in sterile tubes. Triton X100 at 0.1% was the positive control. The A. actinomycetemcomitans strains were cultivated in TSA broth in a CO 2 chamber at 36 °C for 24 h. After incubation, the strains were centrifuged at 9000 rpm and 4 °C for 15 min, and the pellet was sonicated (16 kHz at 200 W) to obtain the proteins. After sonication, the supernatant was separated into a sterile tube, and the amount of protein was dosed with the Bicinchoninic Acid Kit for Protein Determination, Sigma-Aldrich). An amount of 500 μg/mL protein was added to sterile tubes containing leukocyte and an oleoresin or isolated compound and incubated in the CO 2 chamber at 37 °C for 1 h. After incubation, the previously prepared trypan blue solution was added to a concentration of 1.6 mg/mL and kept in the CO 2 chamber for seven minutes. After that, the dead cells were counted with the aid of the Neubauer chamber, and the results were graphically expressed. Statistical analysis. Statistical analysis of the data obtained from the gingipain inhibition assay and leukotoxin inhibition assay was accomplished by One Way ANOVA analysis and Tukey's test with the aid of the software GraphPad Prism version 5.00.

Results and discussion
MIC and MBC. We used strains that were isolated from patients diagnosed with chronic and aggressive periodontitis. More specifically, we detected five P. gingivalis strains and five A. actinomycetemcomitans strains and named them PG01 to PG05 and AA01 to AA05, respectively. PCR with the 16srDNA gene helped to confirm the strains. We evaluated the C. paupera, C. pubiflora, and C. reticulata oleoresins and isolated compounds against the P. gingivalis and A. actinomycetemcomitans strains isolated from patients with periodontitis and against standard strains. Table 1 summarizes the results of these assays. Regarding the tested P. gingivalis strains, the MIC and MBC values of the C. reticulata oleoresin ranged from 3.12 to 12.5 μg/mL. The MBC results revealed a bactericidal effect against all the strains. The exception was P. gingivalis ATCC 33277, against which the C. reticulata oleoresin exerted a bacteriostatic action. The C. paupera oleoresin provided MIC and MBC values between 3.12 and 50 μg/mL and had a bactericidal effect against all the strains. The C. pubiflora oleoresin exhibited MIC and MBC values between 3.12 and 50 μg/mL and was bacteriostatic only against P. gingivalis (ATCC 33277). Polyalthic acid (1) afforded MIC and MBC values between 3.12 and 12.5 μg/mL and displayed a bactericidal effect against all the strains. Kaurenoic acid (2) provided MIC and MBC values of 6.25 μg/mL. Hardwickiic acid (3) exhibited MIC and MBC results ranging from 1.59 to 25 μg/ mL and exhibited bactericidal activity against PG03, PG04, PG05, and P. gingivalis (ATCC 33277).
Concerning the assayed A. actinomycetemcomitans strains, the C. reticulata oleoresin afforded MIC and MBC results between 25 and 100 μg/mL and exerted a bacteriostatic effect only against A. actinomycetemcomitans (ATCC 43717). The MIC and MBC values of the C. paupera oleoresin varied from 12.5 to 100 μg/mL, and this oleoresin had a bacteriostatic effect against AA04 and A. actinomycetemcomitans (ATCC 43717). The C. pubiflora oleoresin exhibited MIC and MBC results between 6.25 and 50 μg/mL and displayed a bactericidal effect against AA01, AA02, and AA04. This same oleoresin exhibited a bacteriostatic effect against AA03, AA05, and A. actinomycetemcomitans (ATCC 43717). Isolated compound 1 afforded MIC and MBC results between 6.25 and 50 μg/mL and had a bacteriostatic effect against AA03 and AA05. Isolated compound 2 provided MIC and MBC values between 25 and 400 μg/mL and exerted a bactericidal effect against all the A. actinomycetemcomitans strains except AA02. Isolated compound 3 exhibited MIC and MBC results between 6.25 and 25 μg/mL and presented a bacteriostatic effect against AA02 and AA04.
As for isolated compounds 4-10, they afforded MIC values higher than 10 μg/mL against all the tested P. gingivalis and A. actinomycetemcomitans strains. According to Ríos and Récio 44 and Gibbons 45 , only crude plant extracts with MIC values lower than 100 μg/mL and isolated substances with MIC values lower than 10 μg/mL are considered as promising sources of antibacterial agents.
Few articles have addressed the antibacterial activity of the oleoresins and isolated compounds investigated in this study. Tincusi et al. 46 evaluated the antibacterial activity of terpenoids isolated from the C. paupera oleoresin against Gram-positive strains. The authors highlighted the results they achieved with copalic acid, polyalthic acid, www.nature.com/scientificreports/ and kaurenoic acid (MIC lower than 10 μg/mL) against Gram-positive strains and the antibacterial potential of the C. paupera oleoresin, used in traditional Peruvian medicine. In a recent work, Furtado et al. 30 evaluated the cytotoxic and genotoxic activity of oleoresins obtained from six Copaifera species, including C. reticulata, C. paupera, and C. pubiflora. The authors employed the clonogenic efficiency assay to determine the cytotoxicity of the oleoresins extracted from the different species. On the basis of the cytotoxicity results, they selected C. paupera and C. pubiflora for the genotoxicity assay. Testing with V79 cells (Chinese hamster fibroblasts) revealed that all the oleoresins are cytotoxic: IC 50 values range from 9.8 to 99.2 μg/mL, but none of the oleoresins is cytotoxic at 2000 mg/kg. Compared to the negative control, V79 cell cultures treated with the Copaifera oleoresins do not have significantly different frequency of micronuclei, demonstrating the absence of genotoxic effect. Here, the C. reticulata, C. paupera, and C. pubiflora oleoresins showed cytotoxicity: their IC 50 values were 60.8, 40.6, and 54.0 μg/mL, respectively. Comparison of these IC 50 values with the MIC results (from 3.12 to 12.5 μg/mL for C. reticulata, from 3.12 to 50 μg/mL for C. paupera, and from 3.12 to 25 μg/mL for C. pubiflora against the evaluated P. gingivalis strains) showed that microorganism inhibition was due to the oleoresin antibacterial activity and not to the cytotoxic action. With respect to the tested A. actinomycetemcomitans strains, the MIC values of the oleoresin were higher than the IC 50 values found by Furtado et al. 30 , but we also attributed microorganism inhibition to the antibacterial activity and not to the cytotoxicity of the oleoresins.
According to Bakri and Douglas 47 , for new therapeutic agents to translate into effective in vivo therapies for periodontitis, they must be active against biofilms rather than just planktonic cells. Alves et al. 48 stated that antibiofilm compounds act in distinct ways: they can have a preventive effect through inhibition of biofilm formation or a therapeutic effect by acting on already established biofilms. Therefore, we decided to evaluate the two possible antibiofilm activities by conducting biofilm inhibition and eradication assays. Antibiofilm assays. To carry out the biofilm inhibition and eradication tests on the P. gingivalis and A.
actinomycetemcomitans strains, first we had to verify the ability of the analyzed strains to grow in the sessile mode. All the strains formed monospecies and multispecies biofilms. In the antibiofilm activity assay, we evaluated concentrations from 0.78 to 1600 µg/mL ( Fig. 2A-F). To determine the anti-biofilm activity of the oleoresins and isolated compounds, we used two techniques: optical density reading (which evaluates the presence of biofilm mass) at 570 nm and microorganism count expressed in log 10 and colony forming units per milliliter (CFU/mL).
Concerning the inhibitory activity of the oleoresins against the monospecies biofilms, the C. paupera oleoresin exhibited IC 50 and MICB 50 of 95.37 and 25.0 µg/mL, respectively, against PG03 ( Fig. 2A). The C. pubiflora oleoresin provided IC 50 and MICB 50 of 186.1 and 12.5 µg/mL, respectively, against AA04 (Fig. 2C). In the case of the multispecies mode, the C. pubiflora oleoresin at 800 and 1600 µg/mL displayed high antibacterial activity, with IC 50 of 60.17 µg/mL and MICB 50 of 400 µg/mL against a combination of AA04 and PG03 (Fig. 2E). www.nature.com/scientificreports/ According to Fux et al. 49 , the drug concentration that is required to kill bacteria in the sessile mode may be 10 to 1000 times higher than the concentration that is necessary to kill bacteria in the planktonic mode. Here, the MICB 50 values of the evaluated oleoresins and isolated compounds against the monospecies mode ranged from 12.5 to 100 μg/mL ( Fig. 2A-D). For the tested P. gingivalis strains, the MICB 50 values were 8 to 251 times higher than the MIC values, corroborating the data established by Fux et al. 49 . With respect to the assayed A. actinomycetemcomitans strains, the MICB 50 values were 2 to 16 times higher than the MIC values and are considered promising for anti-biofilm activity.
Abrão et al. 29 assessed the anti-biofilm activity of C. duckei and its isolated compound polyalthic acid against P. gingivalis (ATCC 33277) and found MICB 50 of 200 μg/mL for the oleoresin and 6.25 μg/mL for polyalthic acid. In our studies, the C. pubiflora and C. paupera oleoresins exhibited MICB 50 of 12.5 and 25 μg/mL against the A. actinomycetemcomitans and P. gingivalis clinical isolates, respectively ( Fig. 2A,C). Regarding isolated compound 1, which exhibited MICB 50 of 50 μg/mL against PG03 (Fig. 2B), which was lower than the result found in the studies by Abrão et al. 29 . These results confirmed the potential antibiofilm action of the Copaifera species oleoresins and  www.nature.com/scientificreports/ their isolated compounds and reaffirmed the selectivity toward more resistant strains in the bacterial biofilm of patients with periodontal pouch 50,51 . Graphs in Fig. 3 illustrate the biofilm eradication activity of the investigated oleoresins and isolated compounds against the tested bacteria in the monospecies and multispecies modes. In the monospecies mode, isolated compound 3 provided the best activity against PG03 (IC 50 of 55.79 µg/mL) (Fig. 3B), whereas the C. paupera oleoresin exhibited the best result against A. actinomycetemcomitans (ATCC 43717) (IC 50 of 58.66 µg/ mL) (Fig. 3C). With respect to the multispecies mode, isolated compound 3 eradicated the combination of AA03 and PG03 with IC 50 of 278.7 µg/mL (Fig. 3E).
Souza et al. 31 evaluated the biofilm eradication activity of ent-copalic acid against A. naeslundii and P. anaerobius biofilms. These authors calculated the Minimum Inhibitory Concentration of Biofilm Eradication, which is defined as the concentration of the substance that is able to reduce biofilm cells by 99.9%. The ent-copalic acid at 1000 and 62.5 μg/mL eradicates A. naeslundii biofilm cells and P. anaerobius biofilm cells, respectively. We determined the biofilm eradication activity on the basis of IC 50 , which is defined as the concentration of an inhibitor that results in half the inhibition of a response as compared to the control group 52 . In the case of the P. gingivalis clinical isolates, the IC 50 of the evaluated oleoresins and isolated compounds ranged from 94.02 www.nature.com/scientificreports/ to 282.3 μg/mL and from 55.79 to 419.6 μg/mL, respectively (Fig. 3A,B). For A. actinomycetemcomitans, the oleoresins and isolated compounds exhibited IC 50 from 58.66 to 189.4 μg/mL and from 95.68 to 265.6 μg/mL, respectively (Fig. 3C,D). These results were more promising than the values reported by Souza et al. 31 .
In their natural environments, most biofilms probably consist of a consortium of species that influence each other synergistically or antagonistically. However, there is little knowledge of their structure, characteristics (including community dynamics), and response to antimicrobial agents 53 .While monospecies biofilms have been extensively studied, little is known about multispecies biofilms and their interactions 53 . Interactions between microorganisms are complex and play an important role in the pathogenesis of infections. These interactions may range from fierce competition for nutrients and niches to highly evolved cooperative mechanisms between different species that support their mutual growth 54 .
Hemagglutination assay. Herein, we evaluated the oleoresins and isolated compounds at sub-inhibitory concentrations. Concerning the A. actinomycetemcomitans strains, the oleoresins and isolated compounds inhibited the hemagglutination activity at all the assayed dilutions. Controls were performed by assessing the ability of all the strains to hemagglutinate blood suspension. All the P. gingivalis strains were capable of hemagglutination, and the oleoresins and isolated compounds inhibited their hemagglutination activity at all the tested dilutions. In P. gingivalis, the hemagglutination capacity is associated with the process of adhesion to gum cells and later lysis of red blood cells for iron capture [55][56][57][58] .
A. actinomycetemcomitans exhibits many virulence factors (fimbriae, hemagglutinin, capsule, lipopolysaccharide, outer membrane vesicles, and enzymatic activities) that can disrupt host defense mechanisms and initiate tissue destruction; however, no specific invasion mechanism has been identified 59 . For Nakagawa et al. 60 , the A. actinomycetemcomitans cellular invasion rate is low as compared to P. gingivalis. In the studies by Meyer et al. 61 , invasion of human cells by A. actinomycetemcomitans was limited to some bacterial strains, approximately 25% of invasive A. actinomycetemcomitans. Löhr et al. 62 evaluated the ability of polyphenols obtained from Myrothamnus flabellifolia to inhibit hemagglutination in the case of P. gingivalis, and they found that the polyphenols at 1000, 500, 100, 50, and 1 μg/mL inhibit this activity at all the tested dilutions (1 to 1:64). In the present study, we examined subinhibitory concentrations of the oleoresins and isolated compounds, from 0.79 to 3.12 μg/mL, and verified the promising potential of the C. paupera, C. pubiflora, and C. reticulata oleoresins and their isolated compounds 1, 2, and 3 to inhibit the P. gingivalis and A. actinomycetemcomitans hemagglutination activity.
Gingipain inhibition assay. We evaluated the ability of the C. paupera, C. pubiflora, and C. reticulata oleoresins and isolated compounds 1, 2, and 3 to inhibit the gingipain activity of the P. gingivalis strains PG01 and PG03. The tested concentrations were sub-inhibitory, and the results are shown in Fig. 4A,B.
Löhr et al. 62 investigated the ability of Myrothamnus flabellifolia Welw polyphenols to inhibit the Arg-X and Lis-X enzymatic activity. After contact with 1 μg/mL polyphenols for 1 min, inhibition is 50%. Prolonged incubation does not increase the anti-protease effects. Polyphenols at 50 and 100 μg/mL inhibit Arg-gingipain by 70-80% and by about 80%, respectively. As for Lys-gingipain, inhibition is only 50%. The authors indicated that M. flabellifolia polyphenol is a potent inhibitor of Arg-X activity.
Here, we evaluated the antivirulence activity of the oleoresins obtained from the Copaifera species and isolated compounds by the Arg-X and Lis-X enzyme inhibition assay. The oleoresins and isolated compounds inhibited at least 50% of the Arg-X and Lys-X enzymatic activity (Fig. 4).
The structures of Arg-X and Lis-X gingipaines are described as resembling a tooth 65,66 , with a crown encompassing the N-terminal subdomain (NSD) and C-terminal subdomain (CSD), see Supplementary Fig. 1. The catalytic site is part of the CSD. The "root" is composed of a IgSF. The hydrolase activities (Cys-proteinase, EC 3.4.22.47) of Arg-X and Lis-X gingipaines are related to different sets of catalytic residues. For Arg-X, H444 and C477 form the the catalytic dyad, while for the Lis-X enzyme the catalytic triad is composed by H444, C477 and D388, as shown in recent studies 66 . Table 2. GoldScore fitness results obtained for the binding of the isolated compounds polyalthic, kaurenoic acid and ( +)-hardwickiic acid into the active sites of Arg-X and Lys-X gingipaines.

Isolated compound
Arg-X Lys-X www.nature.com/scientificreports/  www.nature.com/scientificreports/ To obtain further information on the mechanism of inhibition of Arg-X and Lis-X gingipain, the GOLD molecular docking suite was used 42,43 . The isolated compounds polyalthic acid, kaurenoic acid, and hardwickiic acid were docked into the active sites of the enzymes Arg-X (PDB ID 1CVR) and Lis-X gingipain (PDB ID 6I9A) from P. gingivalis (Fig. 5). The results obtained here, in combination with the ligand interaction diagrams, demonstrate that all three free compounds can interact with the active sites of both enzymes. Interestingly, the compounds endowed with the furan-containing flexible "arm" (polyalthic acid and hardwickiic acid) could be expected to bind more tightly with the active site binding pocket. This trend is observed in the GoldScore Fitness scores. For Lys-X, polyalthic acid and hardwickiic generated posed with better interaction with the active site of the enzyme than kaurenoic acid (See Table 2). For Arg-X, on the other hand, the only compound that was predicted to bind tightly with the active site was polyalthic acid (see Table 2).
The molecular docking analysis indicates that, although some of the compounds are predicted to have the potential to bind to the active sites of both Arg-X and Lys-X, gingipaines, this factor alone does not account to for inhibitory potencies observed experimentally in vitro. It suggests that other forms of inhibition are taking place experimentally. Allosteric inhibition, for example, is not uncommon for proteases 67 . Leukotoxin inhibition assay. We exposed different concentrations of the C. paupera, C. pubiflora, and C. reticulata oleoresins and isolated compounds 1, 2, and 3 (MIC, ½ MIC, and 2 × MIC) to 10 6 cells/mL of LMNS and 500 μg/mL of A. actinomycetemcomitans proteins. The results in Fig. 6A-F showed that the oleoresins and the isolated compounds inhibited A. actinomycetemcomitans leucotoxins: they reduced the leukocyte mortality percentage to less than 50%.
The ltxA gene encodes leukotoxin; the ltxB and ltxD genes encode proteins required for toxin secretion; and the ltxC gene encodes acyl transferase production, which underlies toxin transformation from protoxin to the active form 51,68 . The presence of leukotoxin has been associated with the A. actinomycetemcomitans ability to escape the main line of defense in the periodontal pocket and contributes to the pathogenesis of periodontal disease 51,69 . Leukotoxic activity is determined by a cytolytic action that kills human polymorphonuclear leukocytes, T lymphocytes, and macrophages. In contrast, epithelial and endothelial cells, fibroblasts, and platelets are resistant to this action 51,69,70 .
In the present study, we assessed the A. actinomycetemcomitans leukotoxin inhibition by counting the number of viable cells by Trypan Blue staining. This methodology is based on the observation that viable cells are impermeable to the dye, whereas nonviable cells are permeable to Trypan Blue, which enters pores in the cell membrane 71 . Here, exposure of the A. actinomycetemcomitans strains to the C. paupera, C. pubiflora, and C. reticulata reduced leukocyte mortality to between 15.93 and 31.19% (Fig. 6A,C,E). Isolated compounds 1, 2, and 3 diminished leukocyte mortality to between 11.44 and 39.83% (Fig. 6B,D,F). There are no literature data on the inhibition of A. actinomycetemcomitans leukotoxins by compounds isolated from natural products, so we could not compare the efficiency of the oleoresins and isolated compounds evaluated in the present study with literature results. Compared to the control group, the oleoresins and isolated compounds abated the effect of leukotoxins and decreased leukocyte mortality to below 50%.

Conclusion
The C. paupera, C. pubiflora, and C. reticulata oleoresins and the isolated compounds polyalthic acid, kaurenoic acid, and hardwickiic acid have promising antibacterial activity and monospecies and multispecies antibiofilm activity against major pathogens that cause periodontitis, namely A. actinomycetemcomitans and P. gingivalis. Promising antivirulence activity was evident in the hemagglutination assays, P. gingivalis cysteine protease inhibition (Arg-X and Lys-X) assays, and A. actinomycetemcomitans leukotoxin inhibition assays. The mechanism of inhibition of Arg-X and Lys-X by the isolated compounds was further studies by molecular docking. Direct binding of the small molecules to the active site of the gingipaines does not completely explain the potent inhibitory activities of the compounds observed experimentally (> 74% for kaurenoic acid, for example). Therefore, allosteric inhibition is proposed.