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.

Brazilian Copaifera Species: Antifungal Activity against Clinically Relevant Candida Species, Cellular Target, and In Vivo Toxicity.

Plants belonging to the genus Copaifera are widely used in Brazil due to their antimicrobial properties, among others. The re-emergence of classic fungal diseases as a consequence of antifungal resistance to available drugs has stimulated the search for plant-based compounds with antifungal activity, especially against Candida. The Candida-infected Caenorhabditis elegans model was used to evaluate the in vitro antifungal potential of Copaifera leaf extracts and trunk oleoresins against Candida species. The Copaifera leaf extracts exhibited good antifungal activity against all Candida species, with MIC values ranging from 5.86 to 93.75 µg/mL. Both the Copaifera paupera and Copaifera reticulata leaf extracts at 46.87 µg/mL inhibited Candida glabrata biofilm formation and showed no toxicity to C. elegans. The survival of C. glabrata-infected nematodes increased at all the tested extract concentrations. Exposure to Copaifera leaf extracts markedly increased C. glabrata cell vacuolization and cell membrane damage. Therefore, Copaifera leaf extracts are potential candidates for the development of new and safe antifungal agents.

Investigation of Copaifera genus as a new source of antimycobaterial agents.

AIM: This paper reports on the antimycobacterial activity of the oleoresins and extracts obtained from Copaifera spp. MATERIALS & METHODS: The minimum inhibitory concentration (MIC) and fractional inhibitory concentration index techniques helped to evaluate the effect of these oleoresins and extracts against six strains of mycobacteria that cause tuberculosis. RESULTS & CONCLUSION: Among the assayed oleoresins and plant extracts, the Copaifera langsdorffii, Copaifera duckei, Copaifera reticulata and Copaifera trapezifolia oleoresins provided the lowest MIC values against some of the tested strains. The combination of Copaifera spp. samples with isoniazid did not evidence any synergistic action. Some Copaifera spp. oleoresins may represent a future source for the discovery of new antimycobacterial drugs due to their low MIC values.

Potential antibacterial and anti-halitosis activity of medicinal plants against oral bacteria.

This study aimed to evaluate the in vitro activity of the crude extracts obtained from Caesalpinia ferrea Mart. (Jucá), Cinnamomum cassia B. (Cinnamon), Mallow sylvestris L. (Mallow), Punica granatum L. (Pomegranate), Rosmarinus officinalis L. (Rosemary), Aeolanthus suaveolens (Als.) Spreng. (Macassá), Sysygium aromaticum L. (Clove), and Tamarindus indica L. (Tamarind) against oral microorganisms (e.g., Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, and Parvimonas micra) that produce volatile sulfur compounds (VSC). The pure extracts were placed in culture medium for the diffusion test in agar. The Minimum Inhibitory Concentration (MIC) was determined by the microdilution method, and microbial growth was assayed with resazurin. Total polyphenols in the extracts were measured by using the Prussian Blue Method. For the salivary sediment test, the sediments were exposed to the Jucá and Pomegranate extracts, which was followed by incubation and organoleptic measurements with a monitor (Halimeter®) at 1-, 2-, 4-, and 24 -h intervals. The diffusion test revealed mixed results for the extracts. When the zone of inhibition was present, it ranged from 1.6-10.3 mm. The Pomegranate extract was the only extract that inhibited all the evaluated microorganisms; the MIC values ranged from 50 to 400 µg/mL. The Pomegranate and Jucá extracts presented higher levels of polyphenols, 7.3 % and 3.9 %, respectively, and less VSC formation as compared to the negative control. In conclusion, the extracts display antimicrobial activity against the tested microorganisms. The investigated plants have the potential to reduce the main substances related to halitosis of oral origin.

Antibacterial and Cytotoxic Activities of Pinus tropicalis and Pinus elliottii Resins and of the Diterpene Dehydroabietic Acid Against Bacteria That Cause Dental Caries.

Considering the impact of dental caries on public health and the wide biological potential described for species belonging to the genus Pinus, here we investigate the antibacterial activity of the P. elliottii and P. tropicalis resins and of the diterpene dehydroabietic acid (DHA) against cariogenic bacteria. For this purpose, we have determined the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the resins and of the diterpene. We have also investigated the biofilm inhibition ability (through MBIC50 determination), as well as the synergistic effect (through fractional inhibitory concentration index) and the cytotoxic potential (through XTT assay) of the P. elliottii and P. tropicalis resins. The resins gave promising MIC and MBC values, which ranged from 12.5 to 400 µg/mL; DHA provided MIC and MBC values ranging from 25 to 400 µg/mL. The MICB50 values ranged from 0.78 to 400 µg/mL for the resins. Neither additive nor synergistic effects emerged for the combinations of one of the resins with chlorhexidine. The cytotoxic activity was ≥312.5 and ≥156.3 µg/mL for the P. elliottii and P. tropicalis resins, respectively. The resins showed antibacterial activity against planktonic and sessile cariogenic bacteria. These data are relevant and encourage further research into these plants, which may contribute to the discovery of new substances that can inhibit the growth of cariogenic microorganisms and reduce the incidence of dental caries.

Kaurenoic acid and its sodium salt derivative: antibacterial activity against Porphyromonas gingivalis and their mechanism of action.

AIM: To evaluate the antibacterial activity of 12 kaurane-type diterpenes against a panel of bacteria that cause endodontic infection. METHODS & MATERIALS: We conducted tests against bacteria in the planktonic or in the sessile mode, cytotoxic assays for the most promising compounds against human normal lung fibroblast cells, and Porphyromonas gingivalis (ATCC 33277) proteomic analysis. RESULTS & CONCLUSION: Kaurenoic acid and its salt exhibited satisfactory antibacterial action against the evaluated bacteria. Proteomic analysis suggested that these compounds might interfere in bacterial metabolism and virulence factor expression. Kaurane-type diterpenes are an important class of natural products and should be considered in the search for new irrigating solutions to treat endodontic infections.

Antibacterial, Preservative, and Mutagenic Potential of Copaifera spp. Oleoresins Against Causative Agents of Foodborne Diseases.

Foodborne diseases (FBDs) are a serious public health concern worldwide. In this scenario, preservatives based on natural products, especially plants, have attracted researchers' attention because they offer potential antimicrobial action as well as reduced health impact. The genus Copaifera spp., which is native of tropical South America and West Africa, contains several species for which pharmacological activities, including antibacterial effects, have been described. On the basis of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), antibiofilm activity (inhibition and eradication), preservative capacity, and Ames test, we evaluated the antibacterial, preservative, and mutagenic potential of Copaifera spp. oleoresins against the causative agents of FBDs. The Copaifera duckei, Copaifera reticulata, Copaifera paupera, and Copaifera pubiflora oleoresins displayed promising MIC/MBC values-from 12.5 to 100 µg/mL-against Staphylococcus aureus (American Type Culture Collection [ATCC] 29213), Listeria monocytogenes (ATCC 15313), and Bacillus cereus (ATCC 14579). C. duckei, C. reticulata, C. paupera, and C. pubiflora oleoresin concentrations ranging from 25 to 200 µg/mL and from 100 to 400 µg/mL inhibited biofilm formation and eradicated biofilms, respectively. The oleoresins did not exert mutagenic effects and had superior food preservative action to sodium benzoate (positive control). In conclusion, Copaifera oleoresins exhibit potential antibacterial activity and are not mutagenic, which makes them a promising source to develop novel natural food preservatives to inhibit foodborne pathogens.

ent-Copalic acid antibacterial and anti-biofilm properties against Actinomyces naeslundii and Peptostreptococcus anaerobius.

Diterpenes are an important class of plant metabolites that can be used in the search for new antibacterial agents. ent-Copalic acid (CA), the major diterpene in Copaifera species exudates, displays several pharmacological properties. This study evaluates the CA antibacterial potential against the anaerobic bacteria Peptostreptococcus anaerobius and Actinomyces naeslundii. Antimicrobial assays included time-kill and biofilm inhibition and eradication assays. Time-kill assays conducted for CA concentrations between 6.25 and 12.5 µg/mL evidenced bactericidal activity within 72 h. CA combined with chlorhexidine dihydrochloride (CHD) exhibited bactericidal action against P. anaerobius within 6 h of incubation. As for A. naeslundii, the same combination reduced the number of microorganisms by over 3 log10 at 24 h and exerted a bactericidal effect at 48 h of incubation. CA at 500 and 2000 µg/mL inhibited P. anaerobius and A. naeslundii biofilm formation by at least 50%, respectively. CA at 62.5 and 1.000 µg/mL eradicated 99.9% of pre-formed P. anaerobius and A. naeslundii biofilms, respectively. These results indicated that CA presents in vitro antibacterial activity and is a potential biofilm inhibitory agent. This diterpene may play an important role in the search for novel sources of agents that can act against anaerobic bacteria.

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.

Candida/Candida biofilms. First description of dual-species Candida albicans/C. rugosa biofilm.

Denture liners have physical properties that favour plaque accumulation and colonization by Candida species, irritating oral tissues and causing denture stomatitis. To isolate and determine the incidence of oral Candida species in dental prostheses, oral swabs were collected from the dental prostheses of 66 patients. All the strains were screened for their ability to form biofilms; both monospecies and dual-species combinations were tested. Candida albicans (63 %) was the most frequently isolated microorganism; Candida tropicalis (14 %), Candida glabrata (13 %), Candida rugosa (5 %), Candida parapsilosis (3 %), and Candida krusei (2 %) were also detected. The XTT assay showed that C. albicans SC5314 possessed a biofilm-forming ability significantly higher (p < 0.001) than non-albicans Candida strains, after 6 h 37 °C. The total C. albicans CFU from a dual-species biofilm was less than the total CFU of a monospecies C. albicans biofilm. In contrast to the profuse hyphae verified in monospecies C. albicans biofilms, micrographies showed that the C. albicans/non-albicans Candida biofilms consisted of sparse yeast forms and profuse budding yeast cells that generated a network. These results suggested that C. albicans and the tested Candida species could co-exist in biofilms displaying apparent antagonism. The study provide the first description of C. albicans/C. rugosa mixed biofilm.

Copaifera langsdorffii oleoresin and its isolated compounds: antibacterial effect and antiproliferative activity in cancer cell lines.

BACKGROUND: Natural products display numerous therapeutic properties (e.g., antibacterial activity), providing the population with countless benefits. Therefore, the search for novel biologically active, naturally occurring compounds is extremely important. The present paper describes the antibacterial action of the Copaifera langsdorffii oleoresin and ten compounds isolated from this oleoresin against multiresistant bacteria; it also reports the antiproliferative activity of the Copaifera langsdorffii oleoresin and (-)-copalic acid. METHODS: MICs and MBCs were used to determine the antibacterial activity. Time-kill curve assays provided the time that was necessary for the bacteria to die. The Minimum Inhbitory Concentration of Biofilm (CIMB50) of the compounds that displayed the best results was calculated. Cytotoxicity was measured by using the XTT assay. RESULTS: The diterpene (-)-copalic acid was the most active antibacterial and afforded promising Minimum Inhibitory Concentration (MIC) values for most of the tested strains. Determination of the bactericidal kinetics against some bacteria revealed that the bactericidal effect emerged within six hours of incubation for Streptococcus pneumoniae. Concerning the antibiofilm action of this diterpene, its MICB50 was twofold larger than its CBM against S. capitis and S. pneumoniae. The XTT assay helped to evaluate the cytotoxic effect; results are expressed as IC50. The most pronounced antiproliferative effect arose in tumor cell lines treated with (-)-copalic acid; the lowest IC50 value was found for the human glioblastoma cell line. CONCLUSIONS: The diterpene (-)-copalic acid is a potential lead for the development of new selective antimicrobial agents to treat infections caused by Gram-positive multiresistant microorganisms, in both the sessile and planktonic mode. This diterpene is also a good candidate to develop anticancer drugs.