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Antimicrobial and Physical Properties of Experimental Endodontic Sealers Containing Vegetable Extracts

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www.nature.com/scientificreports OPEN Antimicrobial and physical properties of experimental endodontic sealers containing vegetable extracts Daniela Coelho dos Santos 1, Andressa da Silva Barboza 2, Lara Rodrigues Schneider 1, Carlos Enrique Cuevas‑Suárez 3, Juliana Silva Ribeiro 2, Melissa Feres Damian 2, Angela Diniz Campos 4 & Rafael Guerra Lund 1,2,5* To assess the antimicrobial activity and the physical properties of resin‑based experimental endodontic sealers with the incorporation of vegetable extracts obtained from Bixa orellana, Mentha piperita, and Tagetes minuta species. The extracts were obtained and characterized by gas chromatography‑mass spectrometry (GC–MS), and minimum inhibitory concentration (MIC) against Streptococcus mutans, Enterococcus faecalis, and Candida albicans. The extracts were individually incorporated into a dual‑cure experimental sealer at a mass concentration of 0.5%. A commercial reference RealSeal was used. The sealers were evaluated by measuring the setting time, degree of conversion, dimensional stability, radiopacity, fow, and flm thickness of these materials, also and its antimicrobial efect was evaluated using the direct contact test. Data were statistically analyzed by analysis of variance and Tukey’s post‑hoc test at α = 0.05 signifcance level. The physical properties were not infuenced by the addition of the vegetable extracts (p > 0.05). For S. mutans, only T. minuta and B. orellana groups presented antibacterial activity after 24 h of contact (p < 0.05). All extracts evidenced an antibacterial efect against E. faecalis (p < 0.05). The experimental sealers hold promise as a novel vegetable sealer with great antimicrobial activity and also great physical–mechanical properties. Nonetheless, more studies are needed. Endodontic treatment has the main objective to eliminate the bacteria and their products from the root canal system to prevent reinfection1–3. To achieve this, a clinical protocol including chemomechanical preparation and adequate flling of root canal is performed4. Although these procedures allow a substantial microbial reduction, evidence suggests that persistent microorganisms afer endodontic treatment can survive, interfering with the healing and repair processes2,5. Despite the importance of clinical restorative protocol and material selection in the success of the endodontic treatment, the use of root canal sealers with antibacterial properties has been suggested for the reduction and elimination of persistent microorganisms3. In this context, plants with various chemical constituents ofer certain promising sources of new antimicrobial agents with general as well as specifc activities 1,3,6,7. For example, peppermint oil derived from Mentha piperita L. (Lamiaceae) has been commonly used in several pharmaceutical and industrial products due to its wide range of demonstrated pharmacological properties including antioxidant, antitumor, antiallergenic, antiviral, fungicide, insecticide, and antibacterial8,9. Furthermore, the powders and extracts of Tagetes species (Asteraceae) present insecticidal, antioxidant, and antibacterial properties 10,11. Another example is found in B. orellana, which 1Post-Graduate Program in Biochemistry and Bioprospecting, Federal University of Pelotas, Eliseu Maciel Avenue, Building 31, Pelotas, RS 96010-900, Brazil. 2Post-Graduate Program in Dentistry, Pelotas Dental School, Federal University of Pelotas, 457 Gonçalves Chaves, Room 503, Pelotas, RS 96015-560, Brazil. 3Dental Materials Laboratory, Academic Area of Dentistry, Autonomous University of Hidalgo State, Circuito Ex Hacienda La Concepción S/N, 42160 San Agustín Tlaxiaca, Hidalgo, Mexico. 4Brazilian Agricultural Research Corporation, Embrapa Temperate Climate, Highway BR-392, 78th km, 9th District, Monte Bonito, Pelotas, RS 96010-971, Brazil. 5Laboratory of Oral Microbiology, Department of Restorative Dentistry, Pelotas Dental School, Federal University of Pelotas, Gonçalves Chaves Street, 457/Rm 702-3, Pelotas, RS 96015-560, Brazil. *email: [email protected] Scientifc Reports | (2021) 11:6450 | https://doi.org/10.1038/s41598-021-85609-4 1 Vol.:(0123456789) www.nature.com/scientificreports/ possesses antimicrobial, antioxidant, antidiabetic, anticonvulsant, and cardio-protective activity12,13. Essential oils and plant tinctures are examples of vegetable extracts obtained from diferent processes of chemical extraction of bioactive agents from plants. Te essential oil of herbs is traditionally obtained by hydrodistillation or solvent extraction. Tinctures or also popularly known as simply ‘extracts’ are obtained through cold-pressing or soaking a plant (typically in water, oil, or alcohol) to create a tincture-type liquid 14,15. Considering the antibacterial potential of these species, this study aimed to evaluate the antimicrobial activ- ity of essential oils from M. piperita and T. minuta, and the tincture of B. orellana by incorporating them into experimental root canal sealers in order to develop new products of dental use with antimicrobial activity against microorganisms that cause infections of the canal root system. Tus, it is extremely important to validate that the addition of the essential oils and tincture to root canal sealers neither damage the integrity of the root fll- ing nor cause deterioration of the endodontic sealer. Te null hypothesis is that the incorporation of vegetable extracts into the experimental root canal sealers will neither present antibacterial activity nor physicochemical changes in the endodontic sealers. Materials and methods Experimental design. Tis in vitro study was divided in two parts: (1) selection and characterization of essential oils from M. piperita and T. minuta and ethanolic tincture from seeds of Bixa orellana was performed to determine their essential constituents and minimum inhibitory concentration (MIC) against, E. faecalis, S. mutans and C. albicans; and (2) characterization of the resin-based experimental endodontic sealers afer incor- poration of the vegetable extracts throughout the direct contact test, fow, flm thickness, dimensional stability, setting time, degree of conversion, and radiopacity. Selection and characterization of essential oils and tincture. Plant material. Aerial parts of M. piperita, T. minuta, and seeds of B. orellana were collected from the Brazilian Agricultural Research Corporation, Embrapa Temperate Climate, Monte Bonito, RS, Brazil. Te essential oils from M. piperita and T. minuta were collected according to the National Sanitary Surveillance Agency-Anvisa16, using a Clevenger type apparatus for 3 h. Te tincture of the annatto seeds from B. orellana was obtained via the technique described by Lorenzi and Matos17, with modifcations, where 100 g of seeds were placed in 1000 mL of ethanol at 70% v/v. Afer 15 days, the tincture was fltered to separate the supernatant from the residue, which was then lyophilized for obtaining the dried extract. Te tincture then was stored in a desiccator protected from light until its further use. Determination of the core constituents of essential oils and tincture. Identifcation of the T. minuta and M. piper- ita essential oils compounds was performed using a gas chromatograph-mass spectrometer GC/MS-QP2010 SE (Shimadzu, Japan). Separation of the compounds was done on a RTX-5MS capillary column (30 mm × 0.25 mm, 0.25 μm; Restek, USA). Te compounds were quantifed by their normalized area and were identifed by the mass spectra using the GC Solution Program and the NIST 8 library. Te B. orellana tincture compounds were identifed according to Swain and Hillis 18, with minor modifcations. A UV/Vis spectrophotometer (JENWAY 6705, Cole-Parmer, UK) at 725 nm was used for the quantifcation of the phenolic compounds. A standard curve prepared with gallic acid was used, and the results were expressed in milligrams of gallic acid equivalent. For determining the carotenoids, the method described by Rodriguez- Amaya19, was followed with minor modifcations, the reading was carried out in a UV/Vis spectrophotometer (JENWAY) at 470 nm, and the results were expressed in microgram of sample lycopene/g. Antimicrobial assay. Te reference strains used in this study were selected based on their pathological efects on dentistry. Te strains were E. faecalis American Type Culture Collection (ATCC) 4083, S. mutans ATCC 25175, and C. albicans ATCC 62342. Te microorganisms used in this study were collected from the Research Labora- tory of Microbiology of Dentistry School, Federal University of Pelotas (UFPel). Determination of MIC. Tis test evaluated the antimicrobial activity from the essential oils of T. minuta and M. piperita and the ethanolic tincture from seeds of B. orellana. Te MIC was determined in triplicate by the broth microdilution technique (MIC) using a modifed version of the reference documents M27-A320, and M7-A721, adapted for use with vegetable extracts. In order to standardize the inoculum, culture suspensions were pre- pared, diluted in 0.9% saline using a 0.5 MacFarland scale to obtain approximately 1.5 × ­108 Colony Forming Units (UFC/mL) for bacteria and 2.0 × ­106 CFU/mL for yeast. Te susceptibility test was performed on 96-well microplates; essential oils and tincture were dissolved in ethanol at 0.5 g/mL concentration. Te solution was placed into 96-well plates comprising the culture medium (RPMI for fungi and Mueller-Hilton for bacteria) in concentrations ranging from 0.39 to 50 mg/mL (B. orellana tincture) and from 0.10 to 25 μL/mL (T. minuta or M. piperita essential
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