An Acad Bras Cienc (2020) 92(4): e20200408 DOI 10.1590/0001-3765202020200408 Anais da Academia Brasileira de Ciências | Annals of the Brazilian Academy of Sciences Printed ISSN 0001-3765 I Online ISSN 1678-2690 www.scielo.br/aabc | www.fb.com/aabcjournal HEALTH SCIENCES Schinopsis brasiliensis Engl. to combat the Running title: S. brasiliensis to biofilm-dependents diseases in vitro combat biofilm oral diseases Academy Section: Health Sciences PEDRO HENRIQUE SETTE-DE-SOUZA, CLEILDO P. DE SANTANA, ILZA MARIA O. SOUSA, MARY ANN FOGLIO, FRANCINALVA D. DE MEDEIROS & ANA CLÁUDIA D. DE MEDEIROS e20200408 Abstract: Dental caries and periodontal disease are the most prevalent of the biofilm- dependent diseases. With numerous side effects on the use of chlorhexidine, the 92 (4) search for new safe therapeutic alternatives for microorganisms involved with these 92(4) diseases increases every day. This study aimed to evaluate the antimicrobial activity and cytotoxicity of extracts made from the bark of Schinopsis brasiliensis Engl. against five oral microorganisms and analyze their phytochemical and thermal degradation profile. The liquid-liquid partition was performed with hexane, chloroform and ethyl acetate. The identification and quantification of the chemical marker was done. Antimicrobial activity was evaluated based on the minimum inhibitory concentration. The cytotoxicity was analyzed based on the hemolysing potential of the samples. The thermal degradation profile was performed by two different methods. Gallic acid was identified as the main compound of the samples and showed the highest amount in the chloroform fraction. All samples were able to inhibit the growth of the microorganisms tested and showed no cytotoxicity. The ethanol extract absorbs less heat than the fractions. All samples exhibited exothermic peak consistent with degradation of gallic acid. Based on the results, the samples used are potential candidates for use in dental formulations for biofilm control. Key words: antibacterial agents, natural products, oral diseases, Schinopsis brasiliensis. INTRODUCTION Dental caries is a widely prevalent disease problem globally (Bagramian et al. 2009). It is a The resistance of microorganisms to antibiotics multifactorial and prevalent chronic infectious has become a big problem for medicine, seeking resulting from tooth-adherent specific bacteria, to develop new antimicrobial therapies with primarily Streptococcus spp, that metabolize high resistance to microorganisms (Pereira et sugars to produce acid, which over time, al. 2011). Therefore, research on herbal products demineralizes tooth structure (Abranches et is growing gradually. In dentistry, these studies al. 2018). Streptococcus mitis, S. oralis, and S. mainly examine the effect of bacteria and fungi salivarius are associated with an initial bacteria on the most prevalent oral diseases such as tooth-adhesion, which can be used as a bridge dental caries and periodontal disease, which to other oral microbiota members such as other are considered biofilm-dependent (Lima et al. streptococci and periodontopathogens (Butler 2014, Santamaria Junior et al. 2014, Sponchiado et al. 2017). Furthermore, there are reports of et al. 2014). meningitis caused by S. salivarius (Falomir et al. An Acad Bras Cienc (2020) 92(4) PEDRO HENRIQUE SETTE-DE-SOUZA et al. S. brasiliensis TO COMBAT BIOFILM ORAL DISEASES 2014, Suy et al. 2013, Vignier et al. 2014); whereas MATERIALS AND METHODS S. oralis and S. mitis can increase virulence Collection of Plant material and obtaining of factors of Candida albicans (Palma et al. 2019) the extract and cause bloodstream infections in pediatric The bark of Schinopsis brasiliensis Engl. was patients (Basaranoglu et al. 2019). The S. mutans collected from the semi-arid region in the State is the most frequently associated microorganism of Paraíba, Brazil. A voucher specimen was with caries development, being responsible prepared and identified in the herbarium of for initiated tooth demineralization acidifying Professor Jayme Coelho de Morais, at the Federal microenvironment and tooth surface (Abranches University of Paraíba and given the number et al. 2018; Baker et al. 2016). Moreover, S. EAN-14049. mutans is also associated with bacteremia and The plant material was dried at 40 ± 1 °C. endocarditis (Nakano et al. 2005, 2009). So, it is The ethanolic extract (EE) was obtained by crucial to research solutions to minimize these percolating bark powder, using ethanol (Merck - infections. Darmstadt, Hessen, Germany) as a solvent for five Therefore, many studies using bioactive days in three cycles at 30 °C, and concentrated compounds and plants have been done (Lima in a vacuum rotary evaporator (40 °C, 55 rpm) et al. 2014, Macedo-Costa et al. 2017, Veloso et until complete solvent removal was achieved. al. 2020, Sette-de-Souza et al. 2014, Silva et al. 2012). In this context, the Schinopsis brasiliensis Partition of the ethanolic extract Engl., is used in folk medicine to combat various The EE was subjected to liquid-liquid partition. diseases, including toothache, diarrhea, flu, and The EE was then solubilized in a solvent system inflammations (Agra et al. 2006, Albuquerque of ethanol:water (80:20, v/v) in a 1:1 (v/v). EE 2006, Albuquerque et al. 2007, 2012). The bark of solution was put in a separatory funnel and the S. brasiliensis presents polyphenols, flavonoids, solvents [hexane - Hex, chloroform – Chlo, and tannins, and saponins (Chaves et al. 2015, ethyl acetate – EtAc - (Merck - Darmstadt, Hessen, Fernandes et al. 2015) and a lot of compounds Germany)] were sequentially added in a 1:1 (v/v) with biological activities have been isolated and stirred. The funnel was allowed to stand (Cardoso et al. 2005, 2015, Santos et al. 2017). In for about 10 minutes so that there was phase this way, Santos et al (2014) detected flavonoids separation. The process was repeated three and tannins in the hydroalcoholic extract of the times for each solvent to reach the appropriate bark of S. brasiliensis, along with the absence amount of each fraction. Subsequently, each of toxicity. These phenolic compounds in the fraction was concentrated in a vacuum rotary bark of this plant are degraded in temperatures evaporator (40 °C, 55 rpm) until complete above 125 °C (Fernandes et al. 2013). solvent removal was achieved. The objective of this study was to evaluate the antimicrobial activity against oral bacteria Minimum Inhibitory Concentration and the cytotoxicity of extract and fractions For this stage the standard strains used were produced from the bark of S. brasiliensis Engl. the American Type Culture Collection (ATCC) and analyze its phytochemical and thermal Streptococcus mutans (25175), Streptococcus degradation profile. oralis (10557), Streptococcus mitis (903) and Streptococcus salivarius (7073), which were An Acad Bras Cienc (2020) 92(4) e20200408 2 | 11 PEDRO HENRIQUE SETTE-DE-SOUZA et al. S. brasiliensis TO COMBAT BIOFILM ORAL DISEASES provided by the Oswaldo Cruz Foundation was washed three times with 1% saline solution. (FIOCRUZ-RJ). The erythrocytes were re-suspended in the same The minimum inhibitory concentration (MIC) solution, and the volume was adjusted to 5%. determination was performed as recommended Then 1.5 mL of 5% erythrocyte suspension was by the Clinical and Laboratory Standards Institute added together with 1.5 mL of the test solution (CLSI 2009). The inoculum was standardized at concentrations of 1.0 mg/mL, 2.5 mg/mL, and in tubes containing 5 mL of 0.9% sterile saline 5.0 mg/mL in tubes, remaining for 1 hour at room solution. The microbial suspension was adjusted temperature for the hemolysis to occur. After using a spectrophotometer (Shimadzu UV-mini this period, each tube was centrifuged at 2500 1240 – São Paulo, Brazil) at a wavelength of rpm for 10 minutes, and the supernatant was 625 nm, equivalent to 106 Colony Forming Units removed for reading in a spectrophotometer per mL (CFU/mL). For this step, the extract and (Shimadzu UV-mini - 1240) at a wavelength of the fractions were solubilized in 10% dimethyl 540 nm (Cruz-Silva et al. 2001). The positive sulfoxide (DMSO - Merck - Darmstadt, Hessen, control used was Turk’s solution 1% negative and Germany). One hundred microliters of each 1% saline solution. The analysis was performed fraction or extract at a concentration of 500 mg/ in triplicate. The calculation of the potential mL was serially diluted in brain heart infusion hemolyzing substances followed the following broth (BHI – Difco - Detroit, MI, USA) in a 96-well equation: plate (TPP - Trasadingen, Switzerland) along with Ae− Ab Hp= x100 the positive control (0.12% Chlorhexidine - Sigma- At (1) Aldrich - St. Louis, MO, USA). Ten microliters of inoculation with the microorganism were added Where: to the wells. The plates were incubated at 37 ± Hp = Hemolyzing potential (in percentage) 0.5 °C for 24 hours. The bacterial growth was Ae = Absorbance of the test sample indicated by the addition of 20 µL of aqueous Ab = Absorbance of the Negative Control resazurin (Sigma-Aldrich - St. Louis, MO, USA) At = Absorbance of the Positive Control at 0.01% with subsequent incubation at 37 ± From the data generated from each sample’s 0.5 °C for two hours. Viable bacteria reduce linearity, it was possible to determine the the dye, changing its color to blue, and the MIC appropriate concentration of each extract that was defined as the lowest concentration of test would cause hemolysis of red blood cells by substance that inhibited the change of color of 50% (IC50). These data were used to determine resazurin. the selectivity index (SI) of each extract for the bacteria tested, and enabled the calculation Cytotoxicity and Selectivity Index according to the methodology proposed by The red blood cells’ preparation for the Protopopova et al. (2005) using the equation: cytotoxicity assay followed the method described IC SI 50 by Cruz-Silva et al. (2000). Whole blood from a = MIC (2) voluntary individual working in our laboratory, type O+, was collected and placed in a tube Where: containing heparin. The plasma was removed SI = Selectivity Index after centrifugation at 2500 rpm for 5 minutes.