Antibacterial activity of salvia officinalis L. against periodontopathogens: An in vitro study.

Being aware of the remarkable antimicrobial potential of S. officinalis L., we aimed to evaluate the antimicrobial activity of the S. officinalis dichloromethane crude extract (SOD), dichloromethane-soluble fractions (SODH and SODD), SODD subfractions (SODD1 and SODD2), and pure substances (manool, salvigenin, and viridiflorol) against periodontopathogens. This bioassay-guided study comprises five antimicrobial tests-determination of the Minimum Inhibitory Concentration (MIC), determination of the Minimum Bactericidal Concentration (MBC), determination of the antibiofilm activity, construction of the Time-kill curve (determination of Bactericidal Kinetics), and determination of the Fractional Inhibitory Concentration Index-on six clinical bacterial isolates and three standard bacterial strains involved in periodontal disease. SOD has moderate activity against most of the tested bacteria, whereas SODD1, SODH1, SODH3, and manool afford the lowest results. The Porphyromonas gingivalis (ATTC and clinical isolate) biofilm is considerably resistant to all the samples. In association with chlorhexidine gluconate, only SODH1 exerts additive action against P. gingivalis (clinical isolate). Therefore, SODH1 and manool are promising antibacterial agents and may provide therapeutic solutions for periodontal infections.

Mikania glomerata Sprengel extract and its major compound ent-kaurenoic acid display activity against bacteria present in endodontic infections.

The search for new, effective and safe antimicrobial compounds from plant sources has continued to play an important role in the maintenance of human health since ancient times. Such compounds can be used to help to eradicate microorganisms from the root canal system, preventing/healing periapical diseases. Mikania glomerata (Spreng.), commonly known as "guaco," is a native climbing plant from Brazil that displays a wide range of pharmacological properties. Many of its activities have been attributed to its phytochemical composition, which is mainly composed of diterpenes, such as ent-kaurenoic acid (KA). The present study evaluated the potential activity of an ent-kaurenoic-rich (KA) extract from Mikania glomerata (i.e. Mikania glomerata extract/MGE) and its major compound KA against bacteria that can cause endodontic infections. Time-kill assays were conducted and the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), anti-biofilm activity, and synergistic antimicrobial activity of MGE and KA were determined. The MGE exhibited MIC and MBC values, which ranged from 6.25 to 100 µg/mL and 12.5 to 200 µg/mL respectively. The MIC and MBC results obtained for the KA, ranged from 3.12 to 100 µg/mL and 3.12 to 200 µg/mL respectively. Time-kill and anti-biofilm activity assays conducted for KA at concentrations between 3.12 and 12.5 µg/mL exhibited bactericidal activity between 6 and 72 h of incubation and 50% inhibition of biofilm formation for Porphyromonas gingivalis (clinical isolate), Propionibacterium acnes (ATCC 6919), Prevotella nigrescens (ATCC 33563), P. melaninogenica (ATCC 25845), Aggregatibacter actinomycetemcomitans (ATCC 43717). For synergistic antimicrobial activity, KA combined with chlorhexidine dichlorohydrate (CHD) had an additive effect with increased efficacy against P. gingivalis (clinical isolate) compared to CHD alone. It was concluded that M. glomerata extract and its major compound ent-kaurenoic acid (KA) showed in vitro antibacterial activity, the latter being a potential biofilm inhibitory agent. They may play important roles in the search for novel sources of agents that can act against bacteria present in endodontic infections.

Copaifera reticulata oleoresin: Chemical characterization and antibacterial properties against oral pathogens.

Oral infections such as periodontitis and tooth decay are the most common diseases of humankind. Oleoresins from different copaifera species display antimicrobial and anti-inflammatory activities. Copaifera reticulata is the commonest tree of this genus and grows abundantly in several Brazilian states, such as Pará, Amazonas, and Ceará. The present study has evaluated the chemical composition and antimicrobial potential of the Copaifera reticulata oleoresin (CRO) against the causative agents of tooth decay and periodontitis and has assessed the CRO cytotoxic potential. Cutting edge analytical techniques (GC-MS and LC-MS) aided the chemical characterization of CRO. Antimicrobial assays included determination of the Minimum Inhibitory Concentration (MIC), determination of the Minimum Bactericidal Concentration (MBC), determination of the Minimum Inhibitory Concentration of Biofilm (MICB50), Time Kill Assay, and Checkerboard Dilution. Conduction of XTT assays on human lung fibroblasts (GM07492-A cells) helped to examine the CRO cytotoxic potential. Chromatographic analyses revealed that the major constituents of CRO were ß-bisabolene, trans-α-bergamotene, ß-selinene, α-selinene, and the terpene acids ent-agathic-15-methyl ester, ent-copalic acid, and ent-polyalthic acid. MIC and MBC results ranged from 6.25 to 200 µg/mL against the tested bacteria. The time-kill assay conducted with CRO at concentrations between 50 and 100 µg/mL showed bactericidal activity against Fusobacterium nucleatum (ATCC 25586) and Streptococcus mitis (ATCC 49456) after 4 h, Prevotella nigrescens (ATCC 33563) after 6 h, Porphyromonas gingivalis (ATCC 33277) and Lactobacillus casei (clinical isolate) after 12 h, and Streptococcus salivarius (ATCC 25975) and Streptococcus mutans (ATCC 25175) after 18 h. The fractional inhibitory concentration indexes (FICIs) revealed antagonistic interaction for Lactobacillus casei (clinical isolate), indifferent effect for Porphyromonas gingivalis (ATCC 33277), Fusobacterium nucleatum (ATCC 25586), Prevotella nigrescens (ATCC 33563), and Streptococcus salivarius (ATCC 25975), and additive effect for Streptococcus mutans (ATCC 25175) and Streptococcus mitis (ATCC 49456). Treatment of GM07492-A cells with CRO demonstrated that concentrations up to 39 µg/mL significantly reduced cell viability as compared to the negative control, being IC50 equal to 51.85 ± 5.4 µg/mL. These results indicated that CRO plays an important part in the search for novel sources of agents that can act against oral pathogens.

Antibacterial activity of Pinus elliottii against anaerobic bacteria present in primary endodontic infections.

Endodontic infections have a polymicrobial nature, but anaerobic bacteria prevail among the infectious microbes. Considering that it is easy to eliminate planktonic bacteria, biofilm-forming bacteria still challenge clinicians during the fight against endodontic diseases. The chemical constituents of the oleoresin of Pinus elliottii, a plant belonging to the family Pinaceae, stand out in the search for biologically active compounds based on natural products with potential application in the treatment of endodontic infections. Indeed, plant oleoresins are an abundant natural source of diterpenes that display significant and well-defined biological activities as well as potential antimicrobial action. In this context, this study aimed to (1) evaluate the in vitro antibacterial activity of the oleoresin, fractions, and subfractions of P. elliottii as well as the action of dehydroabietic acid against 11 anaerobic bacteria that cause endodontic infection in both their planktonic and biofilm forms and (2) assess the in vitro antibiofilm activity of dehydroabietic acid against the same group of bacteria. The broth microdilution technique helped to determine the minimum inhibitory concentration (MIC) of the oleoresin and fractions. This same technique aided determination of the MIC values of nine subfractions of Fraction 1, the most active fraction. The MIC, minimum bactericidal concentration, and antibiofilm activity of dehydroabietic acid against the tested anaerobic bacteria were also examined. The oleoresin and fractions, especially fraction PE1, afforded promising MIC values, which ranged from 0.4 to 50 µg/mL. Concerning the nine evaluated subfractions, PE1.3 and PE1.4 furnished the most noteworthy MIC values, between 6.2 and 100 µg/mL. Dehydroabietic acid displayed antibacterial activity, with MIC values lying from 6.2 to 50 µg/mL, as well as bactericidal effect for all the investigated bacteria, except for Prevotella nigrescens. Assessment of the antibiofilm activity revealed significant results--MICB50 lay between 7.8 and 62.5 µg/mL, and dehydroabietic acid prevented all the evaluated bacteria from forming a biofilm. Hence, the chemical constituents of P. elliottii are promising biomolecules to develop novel therapeutic strategies to fight against endodontic infections.

Assessing the efficacy of gutiferone E in photodynamic therapy for oral candidiasis.

The rise in antifungal resistance and side effects of conventional treatments drive the search for innovative therapies like Photodynamic Therapy (PDT). This study explored the efficacy of PDT mediated by gutiferone, an isolated compound from red propolis, for candidiasis treatment. Multiple evaluation methods were employed, including determining the minimum inhibitory concentration (MIC) via broth microdilution, quantifying biomass using crystal violet detachment, and cell counting through total plate count. PDT mediated by gutiferone was also assessed in five groups of mice, followed by histopathological examination and agar plating of lingual tissue samples. Among the seven Candida species tested, gutiferone displayed efficacy against C. albicans, C. glabrata, and C. tropicalis, with MIC values of 1000 µg/mL. In C. tropicalis biofilms, exposure to gutiferone led to a reduction of 1.61 Log10 CFU/mL. PDT mediated by gutiferone achieved an average reduction of 3.68 Log10 CFU/mL in C. tropicalis biofilm cells, underscoring its potent fungicidal activity. Histopathological analysis revealed fungal structures, such as pseudohyphae and hyphae, in infected groups (G2) and irradiated mice. In contrast, groups treated with gutiferone or subjected to gutiferone-assisted PDT (G5) exhibited only few blastoconidia. Furthermore, CFU/mL assessments in lingual tissue post-treatment demonstrated a significantly lower count (0.30 Log10 CFU/mL) in the G5 group compared to G2 (2.43 Log10 CFU/mL). These findings highlight the potential of PDT mediated by gutiferone as a promising alternative for managing denture stomatitis. Future research and clinical investigations offer the promise of validating its clinical applicability and improving outcomes in the treatment of oral candidiasis.

Polyhexamethylene guanidine hydrochloride as promising active ingredient for oral antiseptic products to eliminate microorganisms threatening the health of endangered wild cats: a comparative study with chlorhexidine digluconate.

The aim of this study was to evaluate the antimicrobial efficacy of polyhexamethylene hydrochloride guanidine (PHMGH) compared to chlorhexidine digluconate (CLX) for use as an oral antiseptic during dental procedures in wild cats. This research is crucial due to limited information on the diversity of oral microorganisms in wild cats and the detrimental local and systemic effects of oral diseases, which highlights the importance of improving prevention and treatment strategies. Samples were collected from the oral cavities of four Puma concolor, one Panthera onca, and one Panthera leo, and the number of colony-forming units per milliliter (CFU/mL) was counted and semi-automatically identified. The antimicrobial susceptibility profile of bacterial isolates was determined using minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and time-kill kinetics of PHMGH and CLX. A total of 16 bacterial isolates were identified, consisting of six Gram-positive and 10 Gram-negative. PHMGH displayed MIC and MBC from 0.24 to 125.00 µg/mL, lower than those of CLX against three isolates. Time-kill kinetics showed that PHMGH reduced the microbial load by over 90% for all microorganisms within 30 min, whereas CLX did not. Only two Gram-positive isolates exposed to the polymer showed incomplete elimination after 60 min of contact. The results could aid in the development of effective prevention and treatment strategies for oral diseases in large felids. PHMGH showed promising potential at low concentrations and short contact times compared to the commercial product CLX, making it a possible active ingredient in oral antiseptic products for veterinary use in the future.

Antibacterial activity of Brazilian red propolis and in vitro evaluation of free radical production.

OBJECTIVE: This study aimed to evaluate the antibacterial activity of crude Brazilian red propolis (BRP) extract against anaerobic bacteria involved in primary endodontic infection. Additionally, we evaluate the cell viability and free radical production of human dental pulp fibroblasts (HDPF) in direct contact with mineral trioxide aggregate (MTA) and BRP. DESIGN: The Minimum Inhibitory Concentration, Minimum Bactericidal Concentration (MIC, MBC) and Minimum Inhibitory Concentration of Biofilm (MICB50) of BRP against anaerobic endodontic pathogens were determined. HDPF were exposed to BRP10 (10 µg/mL), BRP50 (50 µg/mL), MTA extract (1:1, 1:2, 1:4 e 1:8), dimethyl sulfoxide 0.5% (DMSO), and cell culture medium (DMEM). The groups were tested for cell viability (MTT assay), and free radical production (reactive oxygen species - ROS, DCFH-DA probe and nitric oxide - NO, Griess reagent). The one-way ANOVA and Tukey's tests were employed at a significance level of 5%. RESULTS: MIC/MBC values of BRP performed antibacterial activity for Parvimonas micra (6.25/6.25 µg/mL), Fusobacterium nucleatum (25/25 µg/mL), Prevotella melaninogenica (50/100 µg/mL), Prevotella nigrescens (50/100 µg/mL), Prevotella intermedia (50/100 µg/mL), and Porphyromonas gingivalis (50/200 µg/mL). The MICB50 values ranged from 1.56 to 50 µg/mL. BRP and MTA stimulated cell viability, emphasizing BRP10 (p = 0.007). Furthermore, it was observed that MTA 1:1, MTA 1:2, and BRP50 slightly increased ROS (p < 0.001) and NO production (p = 0.008, p = 0.007, and p < 0.001 respectively) compared to DMEM group. CONCLUSIONS: BRP exhibits good antibacterial activity against endodontic pathogens, and both BRP and MTA promote the viability of HDPF without increasing NO and ROS production.

Antimicrobial evaluation of diterpenes from Copaifera langsdorffii oleoresin against periodontal anaerobic bacteria.

The antimicrobial activity of four labdane-type diterpenes isolated from the oleoresin of Copaifera langsdorffii as well as of two commercially available diterpenes (sclareol and manool) was investigated against a representative panel of microorganisms responsible for periodontitis. Among all the evaluated compounds, (-)-copalic acid (CA) was the most active, displaying a very promising MIC value (3.1 µg mL-1; 10.2 µM) against the key pathogen (Porphyromonas gingivalis) involved in this infectious disease. Moreover, CA did not exhibit cytotoxicity when tested in human fibroblasts. Time-kill curve assays performed with CA against P. gingivalis revealed that this compound only inhibited the growth of the inoculums in the first 12 h (bacteriostatic effect). However, its bactericidal effect was clearly noted thereafter (between 12 and 24 h). It was also possible to verify an additive effect when CA and chlorhexidine dihydrochloride (CHD, positive control) were associated at their MBC values. The time curve profile resulting from this combination showed that this association needed only six hours for the bactericidal effect to be noted. In summary, CA has shown to be an important metabolite for the control of periodontal diseases. Moreover, the use of standardized extracts based on copaiba oleoresin with high CA contents can be an important strategy in the development of novel oral care products.

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.

Assessment of the antibacterial, antivirulence, and action mechanism of Copaifera pubiflora oleoresin and isolated compounds against oral bacteria.

The microorganisms that constitute the oral microbiome can cause oral diseases, including dental caries and endodontic infections. The use of natural products could help to overcome bacterial resistance to the antimicrobials that are currently employed in clinical therapy. This study assessed the antimicrobial activity of the Copaifera pubiflora oleoresin and of the compounds isolated from this resin against oral bacteria. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays provided values ranging from 6.25 to > 400 µg/mL for the C. pubiflora oleoresin and its isolated compounds. The fractional inhibitory concentration index (FICI) assay showed that the oleoresin and chlorhexidine did not act synergistically. All the tested bacterial strains formed biofilms. MICB50 determination revealed inhibitory action: values varied from 3.12-25 µg/mL for the oleoresin, and from 0.78 to 25 µg/mL for the ent-hardwickiic acid. Concerning biofilm eradication, the C. pubiflora oleoresin and hardwickiic acid eradicated 99.9 % of some bacterial biofilms. Acid resistance determination showed that S. mutans was resistant to acid in the presence of the oleoresin and ent-hardwickiic acid at pH 4.0, 4.5, and 5.0 at all the tested concentrations. Analysis of DNA/RNA and protein release by the cell membrane demonstrated that the oleoresin and hardwiickic acid damaged the bacterial membrane irreversibly, which affected membrane integrity. Therefore, the C. pubiflora oleoresin and ent-hardwickiic acid have potential antibacterial effect and can be used as new therapeutic alternatives to treat oral diseases such as dental caries and endodontic infections.

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.

ent-Kaurenoic acid-rich extract from Mikania glomerata: In vitro activity against bacteria responsible for dental caries.

Many studies have reported that medicinal plant extracts can inhibit oral pathogen growth or adhesion to surfaces and therefore reduce dental caries formation. The addition of these extracts to oral products like mouthwashes and dentifrices is considered an important strategy in caries control. In this sense, we have developed a Mikania glomerata extract with high ent-kaurenoic acid content (KAMg). So, this work describes the preparation of such extract and the development of a validated HPLC-DAD method to determine its ent-kaurenoic acid (KA) content. Herein it is also described the KAMg in vitro antibacterial evaluation against several cariogenic bacteria in comparison with KA and the investigation of further aspects of the KAMg activity. Toxicological aspects of the developed extract were evaluated by assessing its cytotoxicity and genotoxicity. KA and a KA-rich extract like KAMg showed to inhibit the growth of microorganisms responsible for dental caries at relatively low MIC (Minimum inhibitory concentration) values, albeit not as low as the MIC value obtained for chlorhexidine digluconate (CHD), the golden anticariogenic standard approved by the American Dental Association Council on Dental Therapeutics. However, KAMg was more effective to inhibit the formation of a Streptococcus mutans biofilm with four times lower MICB50 (minimum inhibitory concentration that reduces 50% of the biofilm) value as compared with CHD. Taking into account all these data and considering the absence of genotoxic and cytotoxic activity under the tested conditions, it is suggested that KAMg is a natural product to be considered as active ingredient in oral care products.